Final Thesis

PSZ 19:16 (Pind. 1/07)

UNIVERSITI TEKNOLOGI MALAYSIA

DECLARATION OF THESIS / UNDERGRADUATE PROJECT PAPER AND COPYRIGHT

Author’s full name :

Date of birth :

Title :

Academic Session:

I declare that this thesis is classified as:

I acknowledged that Universiti Teknologi Malaysia reserves the right as follows:

1. The thesis is the property of Universiti Teknologi Malaysia.

2. The Library of Universiti Teknologi Malaysia has the right to make copies for the purpose

of research only.

3. The Library has the right to make copies of the thesis for academic exchange.

Certified by:

SIGNATURE SIGNATURE OF SUPERVISOR

(NEW IC NO. /PASSPORT NO.) NAME OF SUPERVISOR

Date : Date :

NOTES : * If the thesis is CONFIDENTAL or RESTRICTED, please attach with the letter from

the organization with period and reasons for confidentiality or restriction.

√

CONFIDENTIAL (Contains confidential information under the Official Secret

Act 1972)*

RESTRICTED (Contains restricted information as specified by the

organization where research was done)*

OPEN ACCESS I agree that my thesis to be published as online open access

(full text)

ALI ADEL DAWOOD

1st NOVEMBER 1966

CLONING OF INFLUENZA B NS1 GENE IN Escherichia

THE EFFECT OF ETHNO/LINGO DIVERSITY ON coli

2009/2010

S2925947 DR. CHAN GIEK FAR

3rd

DECEMBER 2010 3rd

DECEMBER 2010

“I hereby declare that I have read this thesis and in my/our

opinion this thesis is sufficient in terms of scope and quality for the

award of the degree of Master of Science (Biotechnology)”.

Signature : .....................................................................

Name of Supervisor : Dr. CHAN GIEK FAR

Date : 3rd DECEMBER 2010

i

CLONING OF INFLUENZA B NS1 GENE IN Escherichia coli

ALI ADEL DAWOOD

A dissertation submitted in partial fulfillment of the

requirements for the award of the degree of

Master of Science (Biotechnology)

FACULTY OF BIOSCIENCES AND BIOENGINEERING

UNIVERSITI TEKNOLOGI MALAYSIA

NOVEMBER 2010

ii

Hereby, I declare that this thesis entitled “Cloning of influenza B NS1 gene in

Escherichia coli” is the result of my own research except as cited in the

references. The thesis has not been accepted for any degree and is not

concurrently submitted in candidature of any other degree.

Signature : ................................

Name : Ali Adel Dawood

Date : 3rd

DECEMBER 2010

iii

To my beloved parents& my wife;

Thanks for the dim of light when all I see were darkness…

Thanks for giving me the best things in my life …and finally

Thanks for your sacrifices to make me a better person each day….

iv

ACKNOWLEDGEMENT

In the Name of Allah, the Most Benevolent, Most Merciful

Firstly, I thank Allah for giving me the patience, persistency and his

blessings throughout my completing this project. I would like to express my

utmost gratitude and special appreciation to my supervisor Dr. Chan Giek Far,

for encouragement, guidance, support, advices and care helped me in all the

time of research and writing of this thesis.

I am deeply indebted and grateful to my family especially my mother,

father and my wife for their love, support and praying for my success in every

time, their patience and kindness in helping and guiding me in every part of this

project. No word can express my appreciation for their love.

Special thanks to all the staffs of the Faculty of Biosciences and

Bioengineering and members of the labs for not only helping me with my study

but also making my stay in the lab a very pleasurable and memorable one.

Sincere appreciation and thanks are also extended to all staff of Mosul

College of Medicine especially members of the Department of Anatomy for

their encouragement, guidance, advices even in some words.

Last but not least, I would like to thank to people and everyone who has

helped me direct or indirectly towards completing this research project.

v

ABSTRACT

The non-structural NS1 protein of influenza B virus is a multi-

functional virulence protein which is involved in the transport of viral RNA.

Inside the host cell, NS1B antagonizes and inhibits the α/β interferon system

which is induced as host antiviral response. Moreover, it prevents the activation

of double-stranded-RNA activated protein kinase (PKR) by binding to dsRNA

and inhibits the maturation of GAS8 (gene of tumor suppressor). NS1 protein is

utilized as a target for diagnostic of influenza viruses in infected animals.

pUC57 carrying NS1 synthetic gene of influenza B was attempted to be

transformed into Escherichia coli strain BL21(DE3). NS1B was extracted and

attempted to be cloned into prokaryotic expression vectors pET-32b, pET-32a,

pQE-81L and pQE-80L, respectively using restriction digestion enzymes (SacI,

PstI and HindIII). Then, recombinant DNA was attempted to be transformed

into Escherichia coli strains BL21(DE3) and DH5α.

vi

ABSTRAK

Protein non-struktur NS1 virus influenza B adalah virulensi pelbagai

fungsi dan terlibat dalam pengangkutan RNA virus. Di dalam sel perumah,

NS1B menghalang sistem interferon α/β yang menyebabkan tindakbalas

antivirus oleh perumah. Selain itu, protein NS1 mencegah pengaktifan double-

stranded RNA-aktif protein kinase (PKR) dengan cara mengikat dsRNA dan

menghalang pematangan GAS8 (gen tumor supresor). NS1 protein digunakan

sebagai target untuk diagnostik terhadap virus influenza pada haiwan yang

dijangkiti. pUC57 membawa gen NS1 sintetik influenza B diklonkan ke dalam

Escherichia coli strain BL21(DE3). NS1B diekstraksi dan diklon ke vektor

ekspresi prokariotik pET-32b, PET-32a, pQE-81L dan pQE-80L masing-

masing dengan menggunakan enzim pembatas (SacI, PstI dan HindIII).

Kemudian DNA rekombinan ditransformasikan ke dalam strain Escherichia coli

BL21 (DE3) dan DH5α.

vii

TABLE OF CONTENTS

CHAPTER TITLE PAGE

DECLARATION ii

DEDICATION iii

ACKNOWLEDGEMENTS iv

ABSTRACT v

ABSTRAK vi

TABLE OF CONTENTS vii

LIST OF TABLES xii

LIST OF FIGURES xiv

LIST OF SYMBOLS/ABBREVIATIONS xvi

LIST OF APPENDICES

xix

1 INTRODUCTION 1

1.1 Influenza 1

1.2 Influenza virus 1

1.3 Problem statement of the study 2

1.4 Objective of the study 2

1.5 Scope of the study 3

1.6 Significant of the study 3

2 LITERATURE REVIEW 4

2.1 Overview of influenza 4

2.1.1 Influenza infection 4

viii

2.1.2 Treatment 6

2.1.3 Preventative measures for human

Influenza virus 7

2.1.4 Isolation of influenza virus 7

2.2 Influenza virus 8

2.2.1 Viral genome 8

2.2.2 Types of influenza virus 8

2.2.3 Lifecycle of influenza virus 10

2.3 Influenza B virus 11

2.4 Nonstructural (NS1) protein 14

2.4.1 Nonstructural protein NS1 of

Influenza B virus 15

2.4.2 function and influences of NS1

protein 17

2.4.2.1 NS1B binds to ISG-15

protein 18

2.4.2.2 NS1B inhibits and antagoni-

-sts α/β interferon system 19

2.4.2.3 NS1 inhibits nuclear export

of mRNA 21

2.4.2.4 NS1 protein interacts with

GAS8 22

2.4.2.5 NS1 protein inhibits pre-

mRNA 23

2.4.2.6 NS1 protein prevents

activation of PKR 24

2.5 Previous studies of cloning and expression

of NS1 gene 25

3 MATERIALS AND METHODS 29

3.1 Experimental Design 29

ix

3.2 Materials 35

3.2.1 Bacterial strains 35

3.2.2 Chemicals 35

3.2.3 pET vectors 35

3.2.4 pQE vectors 37

3.2.5 NS1B Synthetic Gene 38

3.3 Methods 40

3.3.1 Liquid and solid media preparation 40

3.3.1.1 Luria Bertani agar and

broth 40

3.3.1.2 Preparation of ampicillin

stock solution 40

3.3.2 Preparation of competent cells 41

3.4 Transformation pUC57-NS1B into E. coli

BL21(DE3) competent cells 41

3.5 Isolation of pUC57-NS1B plasmid 42

3.6 Agarose gel electrophoresis 43

3.7 Isolation of pET-32b plasmid 44

3.8 Single digestion 44

3.9 DNA Extraction 44

3.10 Determination of DNA concentration 45

3.11 Ligation NS1B with pET-32b 46

3.12 Transformation of recombinant product 46

3.13 Isolation of pQE-81L plasmid 47

3.14 Amplification of NS1B gene using

Polymerase Chain Reaction (PCR) 47

3.14.1 Primer design 47

3.14.2 Polymerase chain reaction 48

3.15 Double digestion using PstI and HindIII

restriction enzymes in one step 49

3.16 Double digestion using restriction

x

enzymes in two steps 50

3.17 Ligation and transformation into DH5α 51

3.18 Screening of positive colonies 52

3.18.1 Chelex 100 extraction method 52

3.18.2 PCR amplification and gel

electrophoresis to detect NS1B 53

3.19 Isolation of pET-32a plasmid 53

3.20 Amplification of NS1B gene using

Polymerase Chain Reaction (PCR) 54

3.20.1 Primer design 54

3.21 Double digestion using SacI and HindIII

restriction enzymes in one step 55

3.22 Double digestion using SacI and HindIII

restriction enzymes in two steps 55

3.23 Ligation, transformation into E. coli BL21

(DE3) and and screening of colonies 57

3.24 Isolation plasmid (pQE-80L) 57

3.25 PCR amplification, double digestion,

ligation, transformation into DH5α and

screening of colonies 58

4 RESULTS AND DISCUSSION 59

4.1 Transformation of pUC57-NS1B into

E. coli BL21(DE3)

4.2 Cloning of pET-32b-NS1B into E. coli

59

BL21(DE3) 59

4.3 Cloning of pQE-81L-NS1B into E. coli

DH5α 61

4.4 Cloning of pET-32a-NS1B into E. coli

BL21(DE3) 65

4.5 Cloning of pQE-80L-NS1B into E. coli

xi

DH5α 68

4.6 Confirmation of ligation 69

5 CONCLUSION AND FUTURE WORKS 71

5.1 Conclusion 71

5.2 Future works 72

REFERENCES 74

Appendices A-B 79-80

xii

LIST OF TABLES

TABLE NO. TITLE PAGE

3.1 Single digestion components in PCR tube

using HindIII 44

3.2 Ligation mix components of pET-32b and

NS1B 46

3.3 The pUC57 primers for PCR of NS1B gene

from pUC57-NS1B 48

3.4 PCR reaction components of pUC57-NS1B 48

3.5 PCR cycle 49

3.6 Double digestion components in PCR tube

using PstI and HindIII 50

3.7 Double digestion components in PCR tube

using HindIII(1st step) 50

3.8 Double digestion components in PCR tube using

PstI(2nd

step) 51

3.9 The pQE primers for PCR 53

3.10 Primers containing SacI and HindIII restriction

enzyme sites used to amplify NS1B gene 54

3.11 Double digestion components in PCR tube

using SacI and HindIII 55

3.12 Double digestion components in PCR tube using

HindIII(1st step) 56

xiii

3.13 Double digestion components in PCR tube using

SacI(2nd

step) 56

3.14 Primers containing SacI and HindIII restriction

enzyme sites used to amplify NS1B gene 57

xiv

LIST OF FIGURES

FIGURE NO. TITLE PAGE

2.1 Life cycle of influenza virus 10

2.2 Influenza virus structure 11

2.3 Electron microscope image of influenza B 13

2.4 Influenza virus particles on cells lining 14

2.5 Homology modeling of RNA binding domain

of NS1B 16

3.1 Flow chart of experimental design 30

3.2 Cloning of pET-32b-NS1B into E. coli

BL21(DE3) 31

3.3 Cloning of pQE-81L-NS1B into E. coli DH5α 32

3.4 Cloning of pET-32a-NS1B into E. coli

BL21(DE3) 33

3.5 Cloning of pQE-80L-NS1B into E. coli DH5α 34

3.6 pET-32 vectors 36

3.7 pQE-80L vector 37

3.8 pQE-81L vector 38

3.9 pUC57-NS1B gene construct 39

4.1 Restriction digestion by HindIII. 60

4.2 Transformation of pET-32b-NS1B into

competent E. coli BL21(DE3) 61

4.3 Plasmid isolation and digestion of pUC57-

xv

NS1B and pQE-81L 62

4.4 PCR amplification of NS1B 63

4.5 Transformation of pQE-81L-NS1B into

competent DH5α 64

4.6 PCR of recombinant pQE-81L-NS1B 65

4.7 Miniprep of pUC57-NS1B and PCR

amplification of NS1B 66

4.8 Transformation of pET-32a-NS1B into

competent E. coli BL2(DE3) 67

4.9 PCR of recombinant pET-32a-NS1B 67

4.10 Transformation of pQE-80L-NS1B into

competent DH5α 68

4.11 PCR of recombinant pQE-80L-NS1B 69

4.12 PCR amplification 70

xvi

LIST OF SYMBOLS/ ABBREVIATIONS

AMP - Adenosine monophosphate

ATP - Adenosine triphosphate

bp - Base pairs

BSA - Bovine Serum Albumin

α/β - Alfa / Beta interferon

˚C - Degree Celsius

cDNA - Clone deoxyribonucleic acid

del NS1 - Deletion nonstructural 1

dH2O - Deionized water

dNTP - Deoxynucleoside triphosphate

DNA - Deoxyribonucleic acid

dsRNA - double-stranded RNA

E. coli - Escherichia coli

EDTA - Ethylene diamenetetraacetate

ELISA - Enzyme linked immunosorbent assay

g - Gram

GAS - Growth arrest specific gene

GST - Glutathione S-transferase

HA - Hemagglutinine

His-Tag - Histidine tagged

IFN - Interferon

IPTG - Isopropyl β-D-1-thiogalactopyranoside

IRF - Interferon regulator factor

xvii

ISG - Interferon stimulate gene

kb

KDa

-

-

Kilo base

Kilo dalton

LB - Luria Bertani

LPAI - Low pathogenicity avian influenza

M - Molar

mM - Milmolar

MCS - Multi cloning site

ml - Milliliter

mg - Milligram

min. - Minutes

µg - Microgram

µl - Microliter

µm - Micromter

MgCl2 - Magnesium chloride

mRNA - Messenger ribonucleic acid

NA - Neuraminidase

NEP - Nuclear export protein

NES - Nuclear export sequence

ng - Nanogram

NLS - Nuclear localization sequence

NMR - Nuclear Magnetic Resonance

NS1 - Nonstructural 1

OD - Optical density

PACT - Protein activator of the interferon-induced protein

kinase

PCR - Polymerase chain reaction

PKR

RNA

-

-

Protein kinase

Ribonucleic acid

xviii

RNP - Ribonucleoprotein

rpm - Rotation per minute

RT-PCR - Reverse transcription polymerase chain reaction

SDS-PAGE - Sodium Dodecyl Sulphate- Polyacrilamide Gel

Electrophoresis

sec. - Seconds

SIV - Simian immunodeficiency virus

ssRNA - Single strand RNA

TAE - Tris-acetate-EDTA

Tris

U6 SnRNA

-

-

2-hydroxymethyl-2-methyl-1,3-propanediol

U6 small nuclear ribonucleoprotein

UV - Ultraviolet

V - Volts

xix

LIST OF APPENDICES

APPENDEX TITLE PAGE

A NS1B gene sequence (870 bp) of Influenza B virus

(B/Taiwan/45/2007)

79

B NS1B protein sequence (281 amino acids)

80

1

CHAPTER 1

INTRODUCTION

1.1 Influenza

Influenza is a contagious respiratory viral illness of global importance.

The disease was caused by influenza viruses known as flu. The most common

symptoms of the flu are chills, fever, sore throat, muscle pains, severe headache,

coughing, weakness and general discomfort. Some influenza viruses can cause

more severe diseases than the common cold like pneumonia. Influenza viruses

spread around the world and can be transmitted through the air by coughs,

sneezes, creating aerosols containing the virus. This can also be transmitted by

direct contact with infected animals or humans (Metreveli et al., 2006; Spickler et

al., 2009).

1.2 Influenza virus

Influenza viruses have unique features of reverse sense single strand

RNA. They have been classified into three distinct types: A, B and C. Influenza B

viruses are mainly found in humans. These viruses can cause epidemics in human

populations, but have not been responsible for pandemics. Influenza B viruses

2

comprised of single group of hemagglutinin and neuraminidase antigens since

their first isolation in 1940 (Nerome et al., 1998). Influenza B viruses are

categorized into lineages rather than subtypes and are also classified into strains.

Influenza B viruses undergo antigenic drift, though it occurs more slowly than in

influenza A viruses (Metreveli el al., 2006; Spickler et al., 2009). Twelve

antigenic variants were distinguished by a panel of monoclonal antibodies

appeared to circulate in the 1981–1982 epidemic season in Japan. The

evolutionary lineages of influenza B viruses since 1988 have been represented by

two epidemic strains B/Victoria/2/87 and B/Yamagata/16/88 (Nerome et al.,

1998).

1.3 Problem statements of the study

The main problem of this study is to clone NS1B gene in pET-32b, pET-

32a, pQE-81L, and pQE-80L vectors for transformation into Escherichia coli

BL21(DE3) and DH5α.

1.4 Objective of the study

The objective of this research was to clone of NS1B synthetic gene into

pET-32b, pET-32a, pQE-81L, and pQE-80L vectors. The recombinant constructs

were transformed into E. coli hosts.

3

1.5 Scope of the study

The scope of this study encompassed the cloning of NS1B gene of

influenza B into pET-32b, pET-32a, pQE-81L, and pQE-80L vectors, which are

subsequently transformed into competent E. coli BL21(DE3) and DH5α.

1.6 Significant of the study

Recombinant NS1 fusion protein of high purity is more significant for

detection of antigenicity. Successful cloning and overexpression of NS1 gene are

useful for specific diagnostic and further applications. This reverse genetic system

will allow studies to explore the functions of NS1B domains during the

replication cycle and to assess their contributions to the pathogenesis and

virulence of influenza B virus.

4

CHAPTER 2

LITERATURE REVIEW

2.1 Overview of influenza

2.1.1 Influenza infection

Historically, Influenza A is responsible for occasional pandemics

affecting millions of people worldwide. It’s often associated with considerable

morbidity and mortality. During the years 1918-1920, influenza virus caused one

of the most destructive disease outbreaks in world history and later become

known as the Spanish flu pandemic. It resulted in the death of an approximately

50-100 million people thus, this influenza pandemic killed more people than the

World War 1 (Metreveli et al., 2006).

Since, the diseases occurred in the United States in 1924-1925, and then

emerged in 1929 in a much milder form. This virus continued to circulate in

humans until 1957. It was then replaced by another human influenza virus called

the Asian flu virus. Pandemic influenza A viruses emerged three times during the

last century: in 1918 (H1N1 subtype), in 1957 (H2N2), and in 1968 (H3N2).

5

Currently both H3N2 and H1N1, a late fifties variants that re-appeared in 1977and

remained co-circulate in humans (Metreveli et al., 2006).

Influenza B can also cause similar symptoms as Influenza A, but

generally in a milder form. Influenza C viruses infect mammals only and

generally without cause disease. They are genetically distinct from A and B types

(Metreveli et al., 2006).

The human influenza viruses are transmitted from person to person.

Infected adults usually begin to shed influenza A viruses the day before the

symptoms appear, and are infectious for 3-5 days after initial signs. Young

children can shed virus up to six days before, and 10 days or more after they

become ill. Severely immunocompromised individuals may remain infectious for

weeks or months. Humans can transmit influenza viruses to ferrets, and

occasionally to swine and rare cases from person-to-person spread, including a

localized outbreak among recruits at a military base which have been reported in

humans infected with swine influenza viruses. No cases of sustained transmission

have been reported in humans infected with the avian influenza viruses. Fecal

shedding of the avian H5N1 virus has been documented in a child with diarrhea.

Transmission of this virus across the placenta may also be possible (Spickler et

al., 2009).

In poultry, there are two forms of disease. Low pathogenicity avian

influenza (LPAI) viruses generally cause asymptomatic infections and mild

respiratory. High pathogenicity avian influenza (HPAI) viruses cause severe

disease that can kill up to 90-100% of a poultry flock. The severity of zoonotic

avian influenza varies with the virus. Generally, avian influenza viruses do not

spread efficiently in mammals, and infections remain limited to individual

animals or small groups (Spickler et al., 2009).

6

Other avian influenza viruses can also undergo cross-species

transmission. LPAI H9N2 viruses have become endemic in poultry in parts of

Asia and the Middle East may be of particular concern. Recently, they were found

in pigs with respiratory disease and fatal paralysis in China. In addition, H9N2

viruses have been infected humans. As of January 2009, human H9N2 infections

have been significantly less severe than those caused by avian HPAI H5N1

viruses. Epidemics occur every few years, due to small changes in the influenza

viruses. Human pandemics, resulting from antigenic shifts, were most recently

reported in 1918, 1957 and 1968 (Spickler et al., 2009).

2.1.2 Treatment

Antiviral drugs are available for influenza treatment in the United States.

Amantadine and rimantadine (adamantanes) are active against human influenza A

viruses, if treatment begun within the first 48 hours. Zanamivir and oseltamivir

are used effective for both influenza A and influenza B. Treatment usually results

in milder symptoms and recovery one day sooner. Side effects including

neuropsychiatric events may appear. Drug resistance develops rapidly in viruses

exposed to amantadine or rimantadine and may emerge during treatment. For

example, during the 2006-2008 flu seasons, human influenza viruses circulating

in the United States and Canada exhibited high resistance to amantadine and

rimantadine. Laboratory studies have shown that influenza viruses can also

become resistant to zanamivir and oseltamivir. However, this appears to be less

common than resistance to adamantanes (Spickler et al., 2009).

7

2.1.3 Preventative measures for human influenza viruses

An annual vaccine is available for influenza A and B. Both inactivated

(injected) and live (intranasal) vaccines may available. The vaccine is given in the

fall before the flu season. It contains the viral strains which are most likely to

produce epidemics during the following winter, and is updated annually (Spickler

et al., 2009).

2.1.4 Isolation of influenza virus

Human influenza A and influenza B infections can be diagnosed by virus

isolation, detection of antigens or nucleic acids, or retrospectively by serological

test. The viruses can be isolated in cell lines or chicken embryos, and also can be

identified by hemagglutination inhibition tests. Antigens can be detected in

respiratory secretions by immunofluorescence or enzyme-linked immunosorbent

assays (ELISA). Commercial rapid diagnostic test kits such as (Directigen® Flu A

test) can provide a diagnosis within 30 minutes. Reverse transcription polymerase

chain reaction (RT-PCR) techniques are also available; besides the serological

tests which include complement fixation, hemagglutination inhibition and

immunodiffusion. A rising titer is necessary to diagnose human influenza when

using serological tests. RT-PCR can be used for the diagnosis of influenza C and

avian influenza viruses (Spickler et al., 2009).

8

2.2 Influenza virus

2.2.1 Viral genome

The viral genome consists of eight segments of reverse single stranded

RNA or called negative polarity. A total of 11 known proteins are encoded by the

genome. The three largest segments (1, 2 and 3) encode polymerase proteins,

PB1, PB2 and PA that are responsible for RNA synthesis. Segment numbers 4 and

6 encode surface proteins which are hemagglutinin (HA) and neuraminidase (NA)

that are involved in attachment to the host cell and fusion between the viral

envelope and cellular membrane, and release of virus particles. The 5th segment

encodes a nucleoprotein (NP) that protects the viral RNA, but also has other

functions. The 7th segment encodes the M1 and M2 proteins. M1 is a matrix

protein that covers the inner surface of the viral membrane. The last segment

encodes two proteins: non-structural protein 1 (NS1) and nuclear export protein

(NEP). NS1 and NEP are involved in various aspects in the process of taking over

the host cell (Metreveli et al., 2006).

2.2.2 Types of influenza virus

Influenza viruses have been classified into three distinct types A, B and

C. Influenza viruses are found in a number of species including birds, swine,

horses, dogs beside humans. Influenza A viruses include the avian, swine, equine

and canine influenza viruses, as well as the human influenza A viruses. Influenza

A viruses were classified into subtypes based on two surface antigens, the

hemagglutinin (H) and neuraminidase (N) proteins. There are 16 hemagglutinin

antigens (H1 to H16) and nine neuraminidase antigens (N1 to N9). These two

9

proteins are involved in cell attachment and release virions from cells, and are

also major targets for the immune response. Limited subtypes are found in each

species of mammal. Influenza A viruses were also classified into strains. Strains

of influenza viruses are described as their types, hosts, place of first isolation,

strain number, year of isolation, and antigenic subtype. For example, the

prototype strain of the H7N7 subtype of equine influenza virus which first was

isolated in Czechoslovakia in 1956 is A/eq/Prague/56 (H7N7) (Spickler et al.,

2009).

Limited information is available on the subtypes found in other species

of birds. Subtypes that have been found in ratites include H3N2, H4N2, H4N6,

H5N1 H5N2, H5N9, H7N1, H7N3, H9N2, H10N4 and H10N7. Isolates from

cage birds usually contain H3 or H4. However, infections with high pathogenicity

subtypes containing H7 or H5 can also occur (Spickler et al., 2009).

Human influenza A viruses are mainly found in humans, and they can

also infect ferrets and sometimes swine. Experimental infections have been

reported in raccoons. Human viruses can also replicate in the nasal epithelium of

experimentally infected horses. H1N1, H1N2 and H3N2 viruses are currently in

general circulation in humans. The H1N2 viruses appeared most recently. These

viruses were first seen in human populations in 2001, probably as a result of

genetic reassortment between the H3N2 and H1N1 viruses. H2N2 viruses

circulated in the human population between 1957 and 1968 (Spickler et al., 2009).

Influenza C viruses are not classified into subtypes, but are classified

into strains. Each strain is antigenically stable, and accumulates few changes over

time (Spickler et al., 2009).

10

2.2.3 Lifecycle of influenza virus

Life cycle of influenza virus is started through attachment to sialic acid

receptor of the host cell. However, strains vary in their affinities for different

sialyloligosaccharides. Hemagglutinin is a viral glycoprotein binds to the cell

receptor sialic acid. The virus receptor bound is taken into the cell by endocytosis.

Viral envelope fuses with the lipid bilayer of the vesicle and releasing the viral

ribonucleoprotein (RNP) into the cell cytoplasm. Then RNP is transported into the

nucleus. New viral proteins are translated from transcribed mRNA and the viral

RNA is replicated from the negative stranded templates. The new viral RNA is

encapsidated by the nucleoprotein and transported to the cell surface where

envelope hemagglutinin and neuraminidase components are incorporated from the

cell membrane. Progeny virion is formed and then released from the cell by

budding (Metreveli el al., 2006).

Figure 2.1 Life cycle of influenza virus (Metreveli el al., 2006)

11

2.3 Influenza B virus

Influenza B virus is an envelope virus, belonging to the family

Orthomyxoviridae. It contains 8 segments of negative sense single-stranded RNA

virion (Palese et al., 2007). Until now, it is still unknown why influenza B does

not have many natural hosts like influenza A and can be antigenily drifted (Hai et

al., 2008). Influenza B viruses have only one subtype and evolve under three

major lineages: B/Lee/40virus like variants, B/Victoria/2/87- like variants and

B/Yamagata/16/88- like strains (Nerome et al., 1998; Hai et al., 2008).

Figure 2.2 Influenza virus structure

(http://micro.magnet.fsu.edu/cells/viruses/influenzavirus.html)

Influenza B viruses comprised of a single group of hemagglutinin and

neuraminidase antigens since the first isolation in 1940. For this difference, it has

been shown that numerous influenza A viruses circulate in wild and domestic

12

animals such as wide variety of bird species, swine, equine, seals, whales and

mink. Indeed, since has reported in early 1970s. This is in sharp contrast that

influenza B viruses have not been isolated from animals other than humans. These

differences in epizootic background may lead to evidence that influenza B viruses

have no antigenic shift observed (Nerome et al., 1998).

Despite the lack of antigenic shift, a number of antigenic variants of B

viruses have been isolated during periods of widespread influenza virus activity

since its first isolation in 1940. Particularly, it is noteworthy that a higher number

of monoclonal variants of influenza B viruses have been detected in the same

epidemic season than influenza A viruses. For example, twelve antigenic variants

distinguished by a panel of monoclonal antibodies appeared to circulate in the

1981–1982 epidemic seasons in Japan. Thus, evolution of influenza B virus is

definitely characterized by coexistence of several antigenic variants in the same

epidemic period of influenza A virus in which a single dominant virus

predominates in a single epidemic period (Nerome et al., 1998).

The first demonstrated of influenza B viruses have evolved in multiple

lineages which can cocirculate for considerable periods of time. The continuing

analysis of recent epidemic viruses from evolutionary point of view showed that

there have been two distinct lineages of influenza B viruses since 1988, being

represented by the two epidemic strains B/Victoria/2/87 and B/Yamagata/16/88,

respectively. The evolutionary rate of influenza B viruses was reported to be

lower than of human influenza A viruses. It is still uncertain why the former virus

is able to survive for a long period of time without antigenic drift in the

hemagglutinin glycoprotein (Nerome et al., 1998).

Common cold is caused by influenza B and is milder than type A

viruses. However, some cases lead to severe diseases but rarely morbidity

(Spickler et al., 2009). Most virion segments encode structural protein from

reverse RNA. On the other hand, the segment number 8 encodes nonstructural

13

proteins typically 890 nucleotides which encode for two different proteins:

nonstructural protein NS1 (26 KDa) and nuclear export protein NEP (11 KDa)

which was previously known as NS2 (Spickler et al., 2009, Palese et al., 2007).

Until now, it is still uncertain why former virus is able to survive for a

long period time without antigenic drift. Recently, antigenic analysis along with

investigation of genomic structure, systematic deletion and insertion mutations

have occurred in the same nucleotide regions of hemagglutinin molecule of

influenza B viruses (Nerome et al., 1998).

Figure 2.3 Electron microscope image of influenza B

(http://www.wellcome.ac.uk/Education-resources/Teaching-and-education/Big-

Picture/All- issues/Epidemics/WTD028128.htm)

14

Figure 2.4 Influenza virus particles on cells lining

(http://www.wellcome.ac.uk/Education-resources/Teaching-and-education/Big-

Picture/All- issues/Epidemics/WTD028128.htm)

2.4 Nonstructural (NS1) protein

Influenza NS1A protein consists of 230-237 amino acids, which are

divided into two major domains: N-terminal RNA-binding domain, which

comprises the first 73 amino acids residues forming symmetric homodimer

insolution. On the contrary, the influenza NS1B protein comprises of 281 amino

acids. The first 93 amino acids is N-terminus. This domain is responsible for

15

binding with nucleolin. Other domain is located in C-terminal of protein body.

This domain is known as an effector domain which interacts with many viral and

cellular factors. It involves in translation and post-transcriptional processing of

RNA (Manasatienkij et al., 2008).

NS1 protein persists only in the infected animal cells and not in

vaccinated animals, so that, these vaccinated animals are not producing NS1

protein specific antibodies and this is allows differentiation between vaccinated

and natural infected animals (Fang-kun et al ., 2008). Hence, this may path the

way of utilizing NS1 protein as a target for diagnostic of influenza viruses.

The crystal structure of the NS1B protein has not yet been solved.

Computer modeling based on the crystal structure of the N-terminal domain of the

NS1A protein has been allowed alignment of the N-terminal RNA-binding

domains of the two proteins, which are thought to possess similar α-helical

structures (Donelan et al., 2004).

2.4.1 Nonstructural protein NS1 of influenza B virus

The RNA-binding domain (N-terminus domain) of the NS1B protein is

93 amino acids in length and thus 20 amino acids larger than that of the NS1A

protein. The RNA binding domain has a molecular weight of approximately 22.7

KDa and the three-dimensional structure has not yet been determined.

Nonetheless, sequence alignment of the RNA-binding domains of the NS1A and

NS1B proteins suggested that the RNA binding domain of the NS1B protein

exhibits a dimeric six-helical chain fold similar to that of the NS1A protein,

except that there are large loops between the three α- helices in each chain (Yuan

et al., 2002).

16

The three-dimensional structure of the NS1B (1-93) RNA-binding

domain is based on NMR solution structure of the NS1A (1-73) RNA-binding

domain and a sequence alignment of NS1A. The large surface loops between the

α-helices in the NS1B model contain 21 amino acids residues. Because these

loops appear as insertions in the sequence alignment, these amino acids are left

unconstrained in the structure calculations, so that homology modeling approach

does not predict a specific structure for these loops. The ribbon representations of

the predicted structure of NS1B (1-93) were shown in (Fig. 2.5). The loop 1

(residues 24–36) of polypeptide chain 1 is located between the N and C-terminus

domain respectively, of the chain 2. Loop 2 (residues 64–71) of polypeptide chain

1 is located near the N-terminus of the same chain, and by symmetry loop 2 of

chain 2 is located near the N-terminus of chain 2 (Yuan et al., 2002).

Figure 2.5: Homology modeling of RNA binding domain of NS1B

R53, R50, R53׳and R50׳ are the RNA binding residues (Yuan et al., 2002)

17

2.4.2 Functions and influences of NS1 protein

Previous studies suggested two nonexclusive explanations for the strong

general impact of the NS1B gene deletion on viral replication. First, the NS1B

protein may play an important role in counteracting the activity of antiviral gene

products that are expressed in Vero cells in an IFN-independent manner. A second

possible explanation for restriction of the NS1B virus in Vero cells might be a

critical contribution of the NS1B protein to the principal viral replication process

rather than an effect on the host cell (Dauber et al., 2003).

The nonstructural NS1A protein (26 KDa) of influenza virus has been

shown to enter and accumulate in the nucleus of virus-infected cells

independently of any other influenza viral protein (Greenspan and Palese, 1988).

NS1A contains two nuclear localization sequences (NLS1 and NLS2), and a

nuclear export sequence (NES). A nucleolar localization sequence (NoLS) has

been reported for some strains, and is concomitant with NLS2 (Hale et al., 2008).

NLS mediate the active nuclear import of NS1 via binding to cellular

importin-α. Translocation of NS1 into the nucleus is extremely rapid. NLS1 is

highly conserved, monopartite, and involves three residues also involved in

binding dsRNA. NLS2 is absent from the NS1 proteins of a large number of virus

strains. It is difficult to describe a function to this sequence with regard to viral

replication. Concurrent with NLS2 is a functional nucleolar localization signal

(NoLS), which includes additional basic residues. Despite this, the nucleolar

function of NS1 is unknown (Hale et al., 2008).

The NLSs of the influenza B virus NS1 proteins are located within the

RNA-binding domains at the N-terminal 93 amino acids. NLSs overlapping with

an RNA-binding domain have been described for other viral regulatory proteins,

including UL69 of human cytomegalovirus and human immunodeficiency virus

that promote nuclear export of the respective viral mRNAs. RNA binding and

18

nuclear imports are mutually exclusive in the Rev protein, thereby ensuring that

exported viral mRNAs do not return immediately to the nucleus. Although, it has

not yet been determined whether, the NS1B protein is involved in the transport of

viral RNA. It is possible that this overlap indicates a similar regulatory

mechanism. The NLS of the NS1B protein was necessary but not sufficient to

mediate speckle association, which is required the whole N-terminal domain

(Schneider et al., 2008).

At the early infection, NS1B protein revealed a highly dynamic

intracellular localization and accumulates in nuclear speckle which is an important

for efficient virus replication. At the late stage of infection, NS1B protein

accumulates in the cytoplasm which depends on the interaction with another

transported proteins and RNAs which could be regulated by an exposed export

signal (Schneider et al., 2008).

2.4.2.1 NS1B binds to ISG-15 protein

The RNA-binding domain at the N-terminal residue and the adjacent

94–103 regions are required for the binding of interferon stimulate gene (ISG-15)

(Yuan et al., 2001 and Krug et al., 2003). This site is located in one of the surface

loops of this domain. The NS1B proteins are required for dsRNA binding (Yuan

et al., 2002). However, ISG-15 and dsRNA can bind to the N-terminal 103 amino

acid long fragment of the NS1B protein. ISG-15 protein does not have detectable

effect on the binding of dsRNA. The N-terminal region of the NS1B protein binds

not only dsRNA but also a specific cellular protein (Yuan et al., 2002). Unique

biological activities of the NS1B protein are indicated by its deficiency to inhibit

pre- mRNA processing, which has ability to bind to the antiviral response gene

product ISG-15 and to inhibit its conjugation to cellular proteins (Yuan et al.,

2001).

19

NS1B protein has prevented the conjugation of the ubiquitin-like ISG-15

protein to cellular target proteins, which may additionally benefit the activation of

such antiviral reactions (Donelan et al., 2004). Moreover, NS1 protein was

showed bind non-specifically to all ssRNAs and has greater binding activity

(Hatada et al., 1992).

The major goals is to determine the role of the N-terminal, 93 amino

acid RNA-binding domain in ISG-15 binding, whether it is required because it

maintains the dimerization, and hence proper folding of the NS1B protein, or

whether it contains part of the binding site for the ISG-15 protein. However, the

binding dsRNA to the N-terminal domains of the NS1A and NS1B proteins are

well established in vitro (Hatada et al., 1992, Lu et al., 1994). The specific

function of this dsRNA binding during virus infection remains elusive. The NS1

proteins are at a distinct disadvantage in competing with these cellular proteins for

dsRNA (Yuan et al., 2002).

2.4.2.2 NS1B inhibits and antagonists α/β interferon system

NS1 protein is involved in virulence and inhibition of the α/β interferon

system during virus infection (Donelan et al., 2004; Garcia-Sastre et al., 1998).

Efficient replication of influenza viruses and most other viruses necessitates are

suppressed of antiviral responses mediated by the α/β interferon system, which is

an important part of the immune responses of vertebrates (Dauber et al., 2006).

The NS1B protein is identified as a viral factor that antagonizes

interferon induction and boosts viral replication (Dauber et al., 2003). On the

contrary, Dauber and his coworkers found that analysis of these viral mutants

demonstrated inhibition of α/β interferon production is largely independent of the

dsRNA binding activity of NS1 but critically relies on the presence of the C-

20

terminal part of the protein. The dsRNA shielding does not appear to be sufficient

to prevent IFN induction by influenza B virus (Dauber et al., 2006).

The roles of dsRNA binding by NS1B protein are to promote viral

replication and to inhibit of antiviral responses. Dauber (2003) has generated a set

of isogenic influenza B viruses with mutations that either affected NS1 dsRNA

binding or introduced a large C-terminal truncation. By using in vitro binding and

reporter gene assays, it was established in previous studies that the larger C-

terminal part (amino acid 94 to 281) of NS1B protein contains can suppress

activation of the IFN-β promoter by Sendai virus infection (Donelan et al., 2004).

The observed lack of activity of the N-terminal domain of NS1 towards

IFN inhibition in influenza B virus-infected cells underscores the important role

of the C-terminal part of the protein in this process, which presumably involves an

RNA-independent mechanism. Further analyses of the intracellular RNA and

protein targets of the NS1 domains appear to be promising strategy not only to

learn more about details of the IFN suppressive activities of influenza viruses but

also about the factors and mechanisms that drive cellular responses to virus

infections in general (Dauber et al., 2006).

NS1B protein is not related to its ability to antagonize the interferon

(IFN) induced host antiviral response, since NS1/B/Lee virus is significantly

impaired in its ability to replicate even in IFN-deficient systems, such as VERO

cells and 6-day-old eggs (Dauber et al., 2003). Deletion of the truncated NS1B

gene residues from (1-93) protein showed that RNA-binding activity correlated

with β interferon promoter inhibition. In addition, a recombinant influenza B virus

with NS1 deleted induces higher levels of IRF-3 activation. NS1B protein

prevents the nuclear translocation of interferon regulator factor (IRF-3) and

especially β interferon induction in virus-infected cells. It was shown that, the

expression of the NS1B protein complements the growth of influenza A virus

with NS1A deleted (Donelan et al., 2004).

21

Inhibition of IFN- β synthesis correlates with the inhibition of IRF-3

nuclear translocation. This study found that the C-terminal domain of the NS1B

protein possesses IFN antagonist activity, so that deletion of this region would

result in a virus that is more attenuated than the wild-type virus. Viruses

containing mutations within the NS1B N-terminal domain that adversely affect

binding to dsRNA may also prove to be promising live attenuated vaccine

candidates (Donelan et al., 2004).

On the other hand, the role of dsRNA binding by the influenza B virus

NS1 protein is to promote viral replication and to inhibit of antiviral responses.

However, dsRNA binding was necessary for inhibiting protein kinase (PKR)

activation and enable efficient viral replication. The blockade of PKR activities by

both of the NS1 A and B is a common theme in many virus families, highlighting

that this enzyme is a key factor of the cellular antiviral response (Dauber et al.,

2006).

2.4.2.3 NS1 inhibits nuclear export of mRNA

The NS1 protein also functions inhibition rather than facilitation of

nuclear export of mRNA (Qian et al., 1993). The RNA-binding domain binds to

the poly (A) sequence in mRNAs. The effector domain and RNA binding domain

of NS1 protein represses transcription. Interactions between proteins that regulate

nuclear mRNA export and their nuclear targets will be varied and complex (Qian

et al., 1993).

Cytoplasmic as well as nuclear localization of the NSI protein occurred

only when the effector domain was present and was inactivated by a point

mutation. A possible explanation from the observation even wild-type NSI protein

has some affinity for one or more cytoplasmic targets. Specifically, the small

22

amount of the wild-type NS1 protein that is present in the cytoplasm has been

shown to be associated with ribosomes and polysomes. It has not been established

whether this ribosome associated NS1 protein has any function. This study

showed that interactions between proteins that regulate nuclear mRNA export and

their nuclear targets will be varied and complex depending on the interaction of

transcription factors with their protein targets (Qian et al., 1993).

2.4.2.4 NS1 protein interacts with GAS8

Growth arrest-specific (GAS) genes are expressed preferentially in

cultured cells that have entered a quiescent state following serum deprivation or

growth. Eleven GAS genes have been identified from a variety of biological

functions, including the control of microfilament organization, nerve cell growth

or differentiation, apoptosis, tyrosine kinase receptor activity, and negative and

positive control of the cell cycle. GAS8 is located at 16q24.3 and was found to be

a common deletion present in breast and prostate carcinomas. It was shown as a

potential tumor suppressor gene. The GAS8 gene consists of 11 exons spanning

approximately 25 kb. GAS8 protein associates with microtubules in vitro and in

vivo (Zhao et al., 2009).

Zhao and coworkers (2009) found that neither RNA binding domain nor

the effector domain of NS1 protein interacts with GAS8. Deletion analysis

revealed that the N-terminal 260 amino acids of GAS8 were able to interact with

NS1. Examination of the localization of GAS8 protein in cells revealed that two

types of localization existed: Golgi and cytoplasmic. This phenomenon was even

observed in the same types of cells. The Golgi apparatus localization is dependent

on intact microtubules and in cell-cycle regulated. Although, the function of GAS8

has not yet been determined, the effect of the association between GAS8 and NS1

on virus infection is unknown. The mammalian GAS8 gene is a possible tumor

23

suppressor that was previously identified as one of several genes that are up-

regulated upon growth arrest. GAS8 was expressed in HeLa, CV-1, A549, and

DU145 cells. NS1 inhibited the maturation of GAS8 mRNA. This is consistent

with the fact that transient expression of NS1 in mammalian cells leads to

retention of polyadenylation RNA in the nucleus and inhibition of pre-mRNA

splicing (Zhao et al., 2009).

2.4.2.5 NS1 protein inhibits pre-mRNA

Lu and coworkers (1994) found the NS1 protein inhibits pre-mRNA

splicing both in vitro and in vivo by associating with the spliceosomes that are

formed from pre-mRNA. He found that NS1 protein associates with U6 snRNA

(one of the basic protein of spliceosome) in infected cells. A fraction of NS1 was

also detected in association with ribosomes and polysomes in cytoplasmic

fractions of infected cells (Lu et al., 1994).

The nuclear export and splicing of pre-mRNAs are competing

processes. Genetics of these processes have been best studied in yeast by the

isolation and characterization of pre-mRNA processing. These studies found that

some of the protein factors involved in the splicing reaction might act as RNA

helicases and suggesting that dsRNA unwinding processes are necessary for

spliceosome assembly and release of the mRNA from the splicing complex. NS1

mRNA is poorly spliced to yield NS2 mRNA. However, inactivation of the NS1

gene led to a substantial increase in the splicing efficiency, as shown by the

relative accumulations of NS1 and NS2 mRNAs (Fortes et al., 1994).

24

2.4.2.6 NS1 protein prevents activation of PKR

NS1 protein prevents the activation of double-stranded-RNA (dsRNA) -

activated protein kinase (PKR) by binding to dsRNA. Activation of PKR results

in down regulation of cellular translation and is believed to be part of an antiviral

defense strategy in mammalian cells. Host proteins interacting with the NS1

protein during infection have been characterized. The identification of such

proteins is of great interest. Hence, they may influence the host range and

virulence of influenza virus strains. Such a hypothesis is supported by studies of

influenza viruses with mutated or heterologous NS1 alleles which showed altered

growth characteristics in different cells. By using the yeast interaction trap, it has

identified NS1-I by its ability to bind to NS1. It was demonstrated the

conservation of the interaction of NS1-I with six different human and avian

influenza virus NS1 proteins, suggesting that there is a role for the NS1–NS1-I

interaction during the virus life cycle. Overexpression of a truncated of NS1-I

protein would affect the normal course of infection and detailed mapping of the

interacting domains of NS1. NS1-I interaction should reveal whether one or more

of the temperature- sensitive mutations and identified NS1 protein can be

correlated with reduced ability to bind NS1-I (Wolf et al., 1996).

PKR needs to be activated via a conformational change that is brought

about by binding to either of its activators, double-stranded RNA (dsRNA) or the

PACT protein. Other studies were carried out a series of in vitro experiments to

determine whether the NS1 protein could be utilized a common mechanism to

inhibit PKR activation by both PACT and dsRNA, despite their different modes

of activation. They demonstrated that the direct binding of the NS1A protein to

the N-terminal region of PKR can serve as such a common mechanism and this

binding does not require the RNA binding activity of the NS1A protein (Li et al.,

2006).

25

2.5 Previous studies of cloning and expression of NS1 gene

NS1 gene from various influenza viruses had been cloned. This gene

was successfully expressed in Escherichia coli (Binns et al., 1996, Young et al.,

1983, Hatada et al., 1992). Antibody of NS1 was detected in serum sample from

ponies’ experimental infected cell with influenza virus without the animals being

vaccinated with whole inactivated virion. While determination of NS1 antigens

were appeared to be located in the C-terminal half of the protein. However, NS1

protein appears only in infected cells. Using NS1 protein as a diagnostic marker

for influenza virus infections in the presence of high levels of antibodies of

influenza hemagglutinin generated by recent vaccination is an attractive

alternative. NS1 protein from equine influenza virus its entirety or in part as a

fusion protein with glutathione S-transferase (GST) in a diagnostic tests for

detection this type of virus (Binns et al., 1996).

Electrophoresis in slab gels (12.5%) in the presence of SDS was used to

observed NS1 protein. ELISA was carried out on sera experimental samples to

test for the presence of anti-NS1 antibodies. ELISA results on control animals’

positive showed 60% of detection level which were similar to the 79% of

detection for naive animals, while in vaccinated animals was 26% (Binns et al.,

1996).

NS1 gene was isolated from equine influenza virus A/equine 2/Suffolk

/89(H3N8), cloned into pGEX-3X vector and transformed in E. coli TG1

competent cell. Recombinant protein was observed by SDS-PAGE and

immunoblot analysis was carried out to detect the antigens. ELISA diagnostic was

usefulness depending upon duration of the antibodies response to NS1 following

the initial infection. Detection efficiency of about 70% would be useful for batch

testing training yards (Birch-Machin et al., 1997).

26

The antigenic determinants of NS1 protein are situated in C-terminal

domain. Highly homologous regions are present at the position of the Miami/63

and Suffolk /89 NS1 proteins and clearly indicate that these proteins should be

capable of eliciting a similar immune response. Combination antibody response

with a cytotoxic T –cell response if elicited would be indicated that NS1 protein

could be an important component of future vaccines in equines and other species

(Birch-Machin et al., 1997).

pET-28 vector was employed to express the NS1 gene under the control

of the T7 promoter and fused with His·Tag sequences. NS1 gene from Influenza

A (H9N2) was cloned and highly expressed to about 37.4% of the total cellular

protein, and nearly 95% after purification. Utilizing IPTG inducer besides lactose

inducer had no effect on the growth of host cells (Fang-kun et al., 2008). The

antigenicity of the recombinant protein was demonstrated in Western-blotting test

by using positive sera from pigs. This way was not only laying the foundation for

development of ELISA antibody test for differentiation diagnosis between

vaccinated and naturally infected pigs, but also facilitating the monitoring and

eradication of SIV. This procedure focused on the antibody titer in pigs infected

with influenza virus without interference by vaccinated antibody titers and this

allows the differentiation between the vaccinated and the naturally infected pigs

(Fang-kun et al .,2008).

pQE-80L, the other type of vector with 6xHis-tag, was used to express

NS1 protein of influenza A/Chicken/TH/KU14/04 (H5N1). The NS1 gene was

inserted into pQE-80L using T4 DNA ligase. Ligation product was transformed

into subcloning efficiency DH5α competent cell by heating shock of the cells at

42°C for 20 seconds. The transformed E. coli was plated on LB agar containing

100μg/ml ampicillin. The transformants were screened by PCR using specific

primers. IPTG was used to induce E. coli. The recombinant NS1 proteins were

purified by precipitation and affinity chromatography and characterized by SDS-

PAGE and western blot analysis. The amount of NS1 protein purified by the

27

precipitation method was less than that purified under denatured conditions and

contained a small amount of E. coli protein. To confirm the expression, the fusion

protein was reacted with anti-histidine monoclonal antibodies and was indicated

specifically with antibodies raised against the NS1 protein (Manasatienkij et al.,

2008). The recombinant proteins produced in E. coli were not post-translationaly

modified. However, in some cases, it may be more effective antigens than the

recombinant proteins produced in insect cell systems (Manasatienkij et al., 2008,

Spencer et al., 2007). It was unknown whether the lack of modification in the NS1

protein would enhance its serological activities (Manasatienkij et al., 2008).

The plasmid pGEX- NS1B encodes a protein consisting of GST fused to

the NS1 protein of influenza B/Yamagata/1/73 virus. The wild type NS1 cDNA

was subcloned into the pGEX-5X-1 vector between the XhoI and EcoRI

restriction sites. The pGEX-NS1B (1-93) plasmid was constructed by in frame

insertion of the GST ORF in front of the coding region from 1 to 93 amino acids

of the NS1B protein. Similarly, pGEX-NS1 (94-281) was constructed by fusing

the GST ORF to a cDNA encoding from 94 to 281 amino acids of the NS1B

protein. Fusion proteins were expressed in E. coli BL21 and purified using

glutathione-sepharose beads. NS1B mutant cDNAs were also cloned into

pCAGGS vectors for expression in mammalian cells. On the other hand, NS1A

was expressed in trans by transfecting MDCK cells with plasmid pCAGGS as a

result, these cells support del NS1A virus replication. In order to determine

whether expression of the NS1B protein complements the growth of del NS1A

virus, del NS1A transfected MDCK cells with pCAGGS-NS1B plasmid.

Expression of NS1B protein was able to complement the growth of the del NS1A

virus to levels slightly lower than those seen when the NS1A protein was

expressed in trans. Hence, the results indicated that the NS1 protein of influenza

B virus can functionally replace the NS1 protein of influenza A virus in this assay

(Donelan et al., 2004).

28

Other rapid diagnostic method for detection of antigens in nasal swabs

of test animals and immune-PCR were established. It is a highly sensitive way to

detect NS1 protein (Ozaki et al., 2001).

29

CHAPTER 3

MATERIALS AND METHODS

3.1 Experimental Design

pUC57 plasmid carrying NS1B synthetic gene was cloned and

transformed into competent E. coli strain BL21(DE3). Figure 3.1 shows the

general flow chart of experimental design for each vector that started from

plasmid isolation, restriction digestion, agarose gel electrophoresis, ligation,

transformation and screening of colonies. In the first attempt (Figure 3.2), pET-

32b was amplified in E. coli strain BL21(DE3). Plasmid miniprep was done for

pUC57-NS1B and pET-32b. Then, single digestion was done using HindIII

enzyme. Ligation, transformation and screening colonies were done on clones of

BL21(DE3). In the second attempt (Figure 3.3), plasmid miniprep was done for

pUC57-NS1B and pQE-81L, which was amplified in E. coli strain DH5α. Double

digestion, was done using PstI and HindIII enzymes. Ligation, transformation and

screening colonies were done on clones of DH5α. In the third attempt (Figure

3.4), plasmid miniprep was done for pUC57-NS1B and pET-32a. NS1B gene

containing SacI and HindIII restriction enzyme sites were PCR amplified from

pUC57-NS1B. Double digestion was done using SacI and HindIII enzymes.

Ligation, transformation and screening colonies were done on clones of

BL21(DE3). In the fourth attempt (Figure 3.5), plasmid miniprep was done for

pUC57-NS1B and pQE-80L. Double digestion was done using SacI and HindIII

30

enzymes. Ligation, transformation and screening colonies were done on clones of

DH5α.

Restriction digestion using Hind III

Figure 3.1 Flow chart of experimental design

Isolation of pUC57-NS1B and expression vectors from E.

coli strains

Restriction digestion using SacI, PstI and HindIII enzymes

DNA agarose gel electrophoresis and extraction

Ligation of NS1B gene with vectors and transformation into

E. coli competent strains

Screening of colonies and PCR amplification

Plasmid isolation, digestion, gel electrophoresis

31

pET-32b pUC57-NS1B

Single digestion with HindIII Single digestion with HindIII

pET-32b vector (5899bp) NS1B gene (870bp)

Ligation

pET-32b – NS1B

Screening of colonies

Figure 3.2 Cloning of pET-32b-NS1B into E. coli BL21(DE3)

NS1B gene

HindIII HindIII

T7 promoter

HindIII

T7 promoter

NS1B

HindII

I HindII

I

Transformation into E. coli BL21(DE3)

32

pQE-81L

Double digestion with PstI and HindIII

pQE-81L vector (4753bp) NS1B gene (870bp)

Ligation

pQE-81L – NS1B

Screening of colonies

Figure 3.3 Cloning of pQE-81L-NS1B into E. coli DH5α

HindIII PstI

pQE for

NS1B

PstI HindIII

Transformation into E. coli DH5α

pQE for

PCR amplification of NS1B gene using

pUCfor and pUCrev primers

NS1B gene

HindIII PstI

33

Double digestion with SacI and HindIII

pET-32a vector (5900bp) NS1B gene (870bp)

Ligation

pET-32a – NS1B

Screening of colonies

Figure 3.4 Cloning of pET-32a-NS1B into E. coli BL21(DE3)

pET-32a PCR amplification of NS1B gene using

NS1BforSacI and NS1BrevHindIII primers

NS1B

T7 promoter

SacI

HindIII

Transformation into E. coli BL21(DE3)

NS1B gene

HindIII SacI

T7 promoter

HindIII

SacI

34

pQE-80L

Double digestion with SacI and HindIII

pQE-80L vector (4751bp) NS1B gene (870bp)

Ligation

NS1B

pQE-80L – NS1B

Screening of colonies

Figure 3.5 Cloning of pQE-80L-NS1B into E. coli DH5α

HindIII SacI

pQE for

pQE for

Transformation into E. coli DH5α

SacI HindIII

PCR amplification of NS1B gene using

NS1BforSacI and NS1BrevHindIII primers

NS1B gene

HindIII SacI

35

3.2 Materials

3.2.1 Bacterial strains

Two strains of Escherchia coli were used in this study - BL21(DE3) and

DH5α. These strains are commonly used as a host for cloning and protein

expression in molecular biology labs because they have the simplest genomic and

foreign DNA can be transformed easily. On the other hand, use of E. coli as a host

enables large quantity production of recombinant proteins (Leonhartsberger,

2006).

3.2.2 Chemicals

Most chemicals were of analytical and molecular biology grade obtained

from Sigma-Aldrich, Merck, GenScript, Promega, QIAGEN, Yeastern Biotech

and GeneAll. All restriction enzymes, DNA polymerase were purchased from

Promega. Ligase enzyme was obtained from Yeastern Biotech. Primers used for

PCR amplification were synthesized by 1st Base Laboratories Sdn. Bhd. Plasmid

miniprep and gel extraction kits were purchased from GeneAll and QIAGEN. The

pET and pQE vectors were ordered from Novagen and QIAGEN, respectively.

3.2.3 pET vectors

pET is a bacterial plasmid designed to enable quick high quantity

production of desired protein. pET-32a (5900bp), and pET-32b (5899bp) contain

36

several elements like: LacI gene, T7 promoter which is specific to T7 RNA

polymerase, Lac operator which is poly linker, f1 origin of replication, ampicillin

resistance gene and colE1 origin of replication (Blaber et al.,1998).

pET-32a is designed for cloning and high-level expression of peptide

sequences comprise of 109 amino acids Trx• Tag™ thioredoxin protein. Cloning

sites are available for cloning and producing fusion proteins. The plasmid also

contains cleavable His• Tag® and S• Tag™ sequences for detection and

purification (LaVallie et al., 1993).

Figure 3.6 pET-32 vectors

(http://www.lablife.org/p?a=vdb_view&id=g2.nm7d74YBP4q6Utqf6tNu_Ho-).

37

3.2.4 pQE vectors

The pQE series have been designed to allow tightly regulated 6xHis-

tagged protein expression in any E. coli host strain. The vectors are based on the

pQE-30 series and include a lacIq repressor gene. pQE-80L and pQE-81L vectors

have a cis-lacIq gene that overexpresses the lac repressor, strongly suppressing

protein expression from the lac promoter before induction with IPTG (QIAGEN,

2001).

Figure 3.7 pQE-80L vector

(ttp://www.lablife.org/p?a=vdb_view&id=g2%2eL3YawzFqJIzZU2TasKbNJ)

38

Figure 3.8 pQE-81L vector

(http://www.lablife.org/p?a=vdb_view&id=g2%2eL3YawzFqJIzZU2TasKbNJ)

3.2.5 NS1B Synthetic Gene

The synthetic NS1B gene was installed from synthetic oligonucleotides

and PCR products by GenScript (USA). Fragment was cloned into pUC57 into

EcoRV site.

39

MCS of pUC57

Figure 3.9 pUC57-NS1B gene construct (GenScript, 2009)

40

3.3 Methods

3.3.1 Liquid and solid media preparation

3.3.1.1 Luria Bertani agar and broth

LB medium was prepared by dissolving tryptone (4g), yeast extract (2g)

and sodium chloride (4g) in deionized water and it was made up to 400 ml. For

LB agar, 2% of agar was added. The contents were mixed and autoclaved at 121

°C for 20 minutes. For LB broth or agar containing 50µg/ml ampicillin, 10 ml of

ampicillin from stock solution of 2mg/ml was added to the media. For LB broth or

agar containing 100µg/ml ampicillin, 20 ml of ampicillin from stock solution of

2mg/ml was added to the media. The ampicillin solution was added to the

medium after it was cooled to 55°C.

3.3.1.2 Preparation of ampicillin stock solution

Ampicilin powder (Sigma-Aldrich) of 0.1g was dissolved in 50 ml of

sterile deionized water and filtered by using Sartobind nylon filter of size 0.2µm.

41

3.3.2 Preparation of competent cells

DNA is very hydrophilic molecule that does not normally pass through a

bacterial cell membrane. Bacteria need to be made "competent" in order to uptake

foreign DNA. Two strains of E. coli BL21(DE3) and DH5α hosts were used in

transformation. E. coli was grown overnight in 10ml of LB broth at 37°C with

shaking at 200rpm. The cells were chilled for 10 minutes on ice. Then, the cells

were centrifuged at 4°C for 10 minutes at 3000 rpm. After that, the supernatant

was discarded and the pellet was gently resuspended in 10ml of 0.1M calcium

chloride. Then, cells were incubated for 20 minutes on ice. The cells were spun

again at 4°C for 10 minutes at 3000 rpm. For long term storage, the cells were

resuspended in 1ml of 0.1M calcium chloride plus 15% glycerol and aliquoted

into 0.2ml suspension and stored at -80°C. It is the best to use fresh competent

cell for transformation.

3.4 Transformation pUC57-NS1B into E. coli BL21(DE3) competent

cells

pUC57-NS1B (2µl) was added to competent cells and kept on ice for

approximately 20 minutes and this was heat shocked at 42°C for 45seconds.

Immediately, the tube was placed back on ice for at least 2 minutes. Then, 1ml of

LB broth was added and incubated with agitation at 37°C for 1 hour at 200rpm.

The cells were very briefly spun and 700μl of supernatant was removed. The cells

were resuspended and plated out evenly using a sterile glass spreader at 100µl on

LB agar containing 50µg/ml ampicillin. The plates were incubated overnight at

37°C. Each colony was streaked on LB agar containing 100µg/ml ampicillin and

the plates were incubated overnight at 37°C.

42

3.5 Isolation of pUC57-NS1B plasmid

E. coli BL21(DE3) containing pUC57-NS1B was cultured overnight in

LB broth containing 100µg/ml ampicillin at 37°C with shaking at 200rpm. The

plasmids were isolated using plasmid miniprep kit from GeneAll. The cells were

harvested by centrifuging at 4000rpm for 5 minutes. The supernatant was

removed and the pellet was resuspended by pipetting thoroughly in 250µl of

buffer S1. The solution was transformed to a new 1.5ml microcentrifuge tube.

Then, 250µl of S2 buffer was added. The solution was gently mix by inverting

tube for 10 times and incubated for 2 minutes until the cell suspension became

clear. 350µl of buffer S3 was added to the solution and immediately inverted

gently for 10 times before centrifuged for 10 minutes at 14000rpm. PD column

was placed in a collection tube. Clear solution was transferred to the PD column

by pipetting while white pellet formed remained in the microcentrifuge tube. The

PD column was spun at 8000rpm for 30 seconds at room temperature. The flow-

through was discarded and the PD column was placed back in the collection tube.

500µl of buffer AW was added to the PD column and spun at 8000rpm for 30

seconds. The flow-through was discarded and the PD column was placed back in

the collection tube. Then the PD column was washed by adding 700µl of buffer

PW. The column was then centrifuged at 8000rpm for 30 seconds. The flow-

through was discarded and the PD column was placed back in the collection tube.

Additional centrifugation was done at 14000rpm for 1 minute. Next, PD column

was transferred into a new 1.5ml of microcentrifuge tube.

DNA was eluted by adding 50µl of elution buffer to the centre of the PD

column. After 2-minutes stand at room temperature, the tube was centrifuged at

14000rpm for 1 minute. The product was run on 1% agarose gel to check

whether the plasmid DNA is successfully isolated from E. coli before being

kept at -20°C.

43

3.6 Agarose gel electrophoresis

Agarose gel electrophoresis is widely used in molecular biology labs to

separate DNA strands by size, and to estimate the size of the separated strand by

comparing with the known fragments (DNA ladder). This is achieved by pulling

negatively charged DNA molecules through an agarose matrix with an electric

field. The concentration of the agarose depends on the size of the DNA fragments

to be separated.

50X of TAE buffer was prepared from EDTA (186g, pH 8), Tris base

(242g), and glacial acetic acid (57.1ml), and the solution was made up to 1000ml

with deionized water. The final pH was 8.5 and autoclaved at 121°C for 20

minutes. 1X TAE buffer was diluted from 50X TAE buffer by mixing 20ml of

50X TAE buffer with 980ml of distilled water. 0.5g of agarose powder was added

to 50ml of 1X TAE buffer and was boiled in microwave for 1 minute and then left

to cool down. 5µl of ethidium bromide was added to the agarose gel. The gel

casting tray was assembled in gel tank and well forming comb was inserted into

slots on casting tray. The agarose gel was poured into the gel casting tray. After

the gel was solidified in 30 minutes, the comb was removed carefully. The tray

containing agarose gel was placed into electrophoresis chamber and 1X TAE

buffer was added to cover the gel.

To prepare sample, 5µl of the DNA was mixed with 1µl of 6X loading

dye. 6µl of 1kb DNA ladder was used as a marker. The sample and the marker

were loaded separately into wells and the gel was run at 100V for 50 minutes.

After gel running, power supply was turn off. The gel was removed from the

casting tray and was viewed on a UV transilluminator and documented by using

GeneFlash.

44

3.7 Isolation of pET-32b plasmid

The same protocol as described in section 3.5 for plasmid miniprep was

used to isolate pET-32b from E. coli BL21(DE3).

3.8 Single digestion

pUC57-NS1B and pET-32b were digested using HindIII and the

following components were mixed in PCR tube:

Table 3.1: Single digestion components in PCR tube using HindIII

pUC57-NS1B 16.8µl pET-32b 16.8µl

Acetylated BSA 0.2µl Acetylated BSA 0.2µl

Buffer E 2µl Buffer E 2µl

HindIII enzyme 1µl HindIII enzyme 1µl

Total 20µl 20µl

The reactions were incubated at 37°C for 5 hours. The digestion

products were then purified by gel extraction to remove the buffer and restriction

enzymes.

3.9 DNA Extraction

The digestion product needs to be purified before further use. Following

electrophoresis, the NS1B band with the size of 870bp and pET-32b at size nearly

6000bp were cut out with a clean sharp scalpel from the gel. The gel slices were

45

separately put inside 2ml microcentrifuge tube. The tubes were weighed before

and after putting in the gel. Three times volume of QG solution buffer was added

to one volume of gel as mentioned in the protocol of QIAGEN gel extraction kit.

The solution was incubated at 50ºC for 10 minutes and vortexed at each 2

minutes. One volume of isopropanol was added to the sample and mixed. The

sample was applied to the QIAquick column and centrifuged at 13000rpm for 1

minute. The flow-through was discarded and the column was placed back. This

step was repeated till all the sample was passed through the column. Then, 0.5ml

of QG buffer was added to column and centrifuged at 13000rpm for 1 minute. The

flow-through was discarded. Then, the column was placed back, washed by

adding 0.7ml of PE buffer and centrifuged at 13000rpm for 1 minute. The flow-

through was discarded and centrifuged again to remove all PE buffer. The column

was transferred to a new 1.5ml microcentrifuge tube and 50µl of elution buffer

was added to the centre of column. It was stood at room temperature for 2-5

minutes and then centrifuged at 13000rpm for 1 minute. The product was kept in -

20ºC.

3.10 Determination of DNA concentration

The measuring of DNA concentration was carried out by using Nanodrop

Spectrophotometer. The DNA concentration and purity was measured by using 1

μl of the sample. An OD260: OD280 ratio of purified plasmid is to be found in

the expected range of 1.8 and 2.0. 1 µl of distilled water was used to initialize the

instrument and 1µl of elution buffer was used as a blank.

46

3.11 Ligation NS1B with pET-32b

T4 DNA Ligase was used in ligating the vector and the insert gene.

DNA ligases catalyze the formation of a phosphodiester bond between adjacent

nucleotide with the hydrolysis of ATP to AMP and inorganic phosphate. NS1B

and digested pET-32b with HindIII were ligated and the following components

were mixed in PCR tube:

Table 3.2: Ligation mix components of pET-32b and NS1B

1st mix 2

nd mix

pET-32b 1µl 2µl

NS1B 3µl 4µl

10X ligation buffer A 1µl 1µl

10X ligation buffer B 1µl 1µl

YEA T4 DNA ligase 1µl 1µl

dH2O 3µl 1µl

Total 10µl 10µl

The reactions were incubated in thermocycler at 22°C for 20 minutes

followed by inactivation at 65°C for 10 minutes. The ligation mix was kept at 4°C

for transformation. It is better to directly transform the ligation mix into

competent cells.

3.12 Transformation of recombinant product

The constructed recombinant plasmid prepared as described in section

3.11 was transformed into E. coli BL21(DE3) competent cells. The ligation mix

(10μl) was added to 100µl of competent cells and incubated on ice for 20 minutes.

47

The cells were heat shocked at 42°C for 45 seconds. Tube was immediately

placed back on ice for at least 2 minutes. 1ml of LB broth was added to the tube.

Then, tube was incubated at 37°C for 1 hour with shaking at 200rpm. Cells were

collected by centrifugation at 10,000 rpm for 30 seconds. 0.7ml was removed

from supernatant and the cells were resuspended. The cells (100µl) were spread

out on LB agar containing 50µg/ml ampicillin. IPTG (5µl) and X-gal (10µl) were

spread on the plates for 1 hour prior to use. Some cells were plated out on LB agar

as a control. The plates were incubated at 37°C for overnight.

3.13 Isolation of pQE-81L plasmid

The same protocol as described in section 3.5 for plasmid miniprep was

used to isolate pQE-81L from E. coli DH5α.

3.14 Amplification of NS1B gene using Polymerase Chain Reaction

(PCR)

3.14.1 Primer design

Primers were designed based on the upstream and downstream region of

NS1B gene of pUC57 vector. The characteristics of the primers are as described

in Table 3.3.

48

Table 3.3: The pUC57 primers for PCR of NS1B gene from pUC57-NS1B

Primers Sequences (5׳3-׳) Length(bp) % of GC Content Melting

Temp.(°C)

pUC for 5׳-

GTAAAACGACGG

CCAGTGA-3׳

19 52-6 60.2

pUC rev 5׳-

CAGGAAACAGCT

ATGACC-3׳

18 50.0 57.6

3.14.2 Polymerase Chain Reaction (PCR)

Using micropipette, a PCR reaction was prepared by pipetting the

following reagents into PCR tube:

Table 3.4: PCR reaction components of pUC57-NS1B

Go Taq DNA polymerase 0.25µl

5X Green buffer 10µl

dNTP 1µl

MgCl2 5µl

Forward primer (pUCfor) 0.2µl

Reverse primer (pUCrev) 0.2µl

dH2O 32.35µl

pUC57- NS1B 1µl

Total 50µl

49

The PCR process was initiated with initial denaturation step at 94ºC for

5 minutes. The process continued with the denaturation, annealing and extension

steps. The reaction conditions for each step in PCR cycle were as follows:

Table 3.5: PCR cycle

Steps Temperature Time

Denaturation 94ºC 30 sec.

Annealing 50ºC 30 sec.

Extension 72ºC 1 min.

The above cycle was run for 25 cycles. Then the cycle was continued by

further extension at 72ºC for 7 minutes. Then, the sample was run on gel

electrophoresis as described in section 3.6. The PCR products were purified by

using the protocols as mentioned in section 3.9 prior to digestion with PstI and

HindIII restriction enzymes.

3.15 Double digestion using PstI and HindIII restriction enzymes in one

step

NS1B amplified gene and pQE-81L were digested using PstI and

HindIII restriction enzymes and the following components were mixed in a PCR

tube:

50

Table 3.6: Double digestion components in PCR tube using PstI and HindIII

pQE-81L or NS1B gene 15.8µl

Acetylated BSA 0.2µl

10X Multicore enzyme buffer 2µl

HindIII enzyme 1µl

PstI enzyme 1µl

Total 20µl

The reactions were incubated at 37ºC for 5 hours. Then, gel

electrophoresis was done and extracted using the same protocols mentioned in

sections 3.6 and 3.9. The extracted product was used for ligation reaction.

3.16 Double digestion using restriction enzymes in two steps

The following components were mixed in PCR tube for the first

digestion for NS1B amplified gene and pQE-81L:

Table 3.7: Double digestion components in PCR tube using HindIII(1st step)

pQE-81L or NS1B gene 15.8µl

Acetylated BSA 0.2µl

Buffer E 2µl

HindIII enzyme 2µl

Total 20µl

The reaction was incubated at 37ºC for 5 hours. Then, the reaction was

observed by gel electrophoresis and extracted using the same protocols as

51

mentioned in sections 3.6 and 3.9. The extracted product was used for the second

digestion. The following components were mixed in PCR tube:

Table 3.8: Double digestion components in PCR tube using PstI(2nd

step)

First digestion product 15.8µl

Acetylated BSA 0.2µl

Buffer H 2µl

PstI enzyme 2µl

Total 20µl

The reaction was incubated at 37ºC for 5 hours. Then, the reaction was

observed by gel electrophoresis and extracted using the same protocols as

mentioned in sections 3.6 and 3.9. The extracted product was used for ligation

reaction.

3.17 Ligation and transformation into DH5α

Ligation and transformation of pQE-81L-NS1B into E. coli DH5α

competent cells were done using the same protocol as mentioned in sections 3.11

and 3.12.

52

3.18 Screening of positive colonies

Colonies grown on LB agar containing 30µg/ml ampicillin were isolated

and further streaked on LB agar containing 100µg/ml ampicillin. The plates were

incubated at 37°C for overnight. Colony PCR was used as the screening method.

3.18.1 Chelex 100 extraction method

Chelex 100 resin is highly purified, and most suitable for DNA

applications. It is composed of styrene divinylbenzene copolymers with paired

iminodiacetate ions. Chelex 100 is very effective in binding metal contaminants

with a high selectivity for divalent ions, without altering the concentration on non-

metal ions. One benefit of using the Chelex extraction is that divalent heavy

metals which introduce DNA damage at high temperature can be removed. The

extraction was set up under aqueous alkaline conditions, as the Chelex 100

suspension was prepared in TE buffer

(http://www.nfstc.org/pdi/Subject03/pdi_s03_m03_01.htm).

10% Chelex 100 was used to extract DNA. 100µl of chelex solution was

added into 1.5ml microcentrifuge tube and the number of tubes was prepared

according to the numbers of colonies to be screened. Each tube was labeled as the

number of colonies. A scrap of each colony was removed using toothpick,

resuspended and vortexed with the Chelex suspension for 5 to 10 seconds. The

samples were incubated at 95ºC for 5 minutes with agitation 400rpm. Then, the

samples were centrifuged at 12000rpm for 5 minutes. The supernatant was used

for PCR amplification. The samples were kept in 4ºC for further studies.

53

3.18.2 PCR amplification and gel electrophoresis to detect NS1B

For screening of colonies, sequencing primers of pQE-81L vector were

used the characteristics of the primers used are as in Table 3.9. The PCR

amplification and gel electrophoresis were as mentioned in sections 3.14.2 and

3.6.

Table 3.9: The pQE primers for PCR

Primers Sequences (5׳3-׳) Length(bp) % of GC Content Melting

Temp.(°C)

pQE for 5׳-

CGGATAACAATTTC

ACACAG-3׳

20 40.0 56.3

pQE rev 5׳-

GTTCTGAGGTCATT

ACTGG-3׳

19 47.4 58.0

3.19 Isolation of pET-32a plasmid

The same protocol as described in section 3.5 for plasmid miniprep was

used to isolate pET-32a from E. coli BL21(DE3).

54

3.20 Amplification of NS1B gene using Polymerase Chain Reaction

(PCR)

PCR amplification of NS1B gene from pUC57-NS1B was done using

the same protocol mentioned in section 3.14.

3.20.1 Primer design

The forward and reverse primers were designed to contain SacI and

HindIII restriction sites for endonucleases digestion. The characteristics of the

primers design are shown in Table 3.10.

Table 3.10: Primers containing SacI and HindIII restriction enzyme sites used to

amplify NS1B gene

Primers Sequences (5׳3-׳) Length(bp) % of GC Content Melting

Temp.(°C)

NS1B forSacI

GAGCTCATGGCGAA-׳5

CAAT-3׳

18 50.0 57.0

NS1B revHindIII

AAGCTTCTAATTGT-׳5

CTCC-3׳

18 38.9 53.1

55

3.21 Double digestion using SacI and HindIII restriction enzymes in one

step

NS1B amplified gene and pET-32a were digested using SacI and HindIII

and the following components were mixed in PCR tube:

Table 3.11: Double digestion components in PCR tube using SacI and HindIII

pET-32a or NS1B gene 15.8µl

Acetylated BSA 0.2µl

10X Multicore enzyme buffer 2µl

HindIII enzyme 1µl

SacI enzyme 1µl

Total 20µl

The reaction was incubated at 37ºC for 5 hours. Then, the reaction was

observed by gel electrophoresis and extracted using the same protocols as

mentioned in sections 3.6 and 3.9. The extracted products were used for ligation

reaction.

3.22 Double digestion using SacI and HindIII restriction enzymes in two

steps

The following components were mixed in PCR tube for the first

digestion for NS1B amplified gene and pET-32a:

56

Table 3.12: Double digestion components in PCR tube using HindIII(1st step)

pET-32a or NS1B gene 15.8µl

Acetylated BSA 0.2µl

Buffer E 2µl

HindIII enzyme 2µl

Total 20µl

The reaction was incubated at 37ºC for 5 hours. Then, the reaction was

observed by gel electrophoresis and extracted using the same protocols as

mentioned in sections 3.6 and 3.9. The extracted product was used for the second

digestion. The following components were mixed in PCR tube:

Table 3.13: Double digestion components in PCR tube using SacI(2nd

step)

First digestion product 15.8µl

Acetylated BSA 0.2µl

Buffer J 2µl

SacI enzyme 2µl

Total 20µl

The reaction was incubated at 37ºC for 5 hours. Then, the reaction was

observed by gel electrophoresis and extracted using the same protocols as

described in sections 3.6 and 3.9. The extracted product was used for ligation

reaction.

57

3.23 Ligation, transformation into E. coli BL21 (DE3) and screening of

colonies

Ligation, transformation into E. coli BL21(DE3) and colony screening

were done using the same protocols as mentioned in sections 3.11, 3.12 and 3.18.

The primers used for PCR were T7 promoter and T7 terminator primer. The

characteristics of the primers used are shown in Table 3.14.

Table 3.14: Primers containing SacI and HindIII restriction enzyme sites used to

amplify NS1B gene

Primers Sequences (5׳3-׳) Length(bp) % of GC Content Melting

Temp.(°C)

T7 promoter 5׳-

TAATACGACTCACA

CAAGGG-3׳

20 40.0 56.3

T7 terminator 5׳-

GCTAGTTATTGCTC

AGCGG-3׳

19 52.5 60.2

3.24 Isolation plasmid (pQE-80L)

The same protocol as described in section 3.5 for plasmid miniprep was

used to isolate pET-32a from E. coli BL21(DE3).

58

3.25 PCR amplification, double digestion, ligation, transformation into

DH5α and screening of colonies

The same protocols as described in sections 3.20, 3.21, 3.22, 3.17 and

3.18 were used.

59

CHAPTER 4

RESULTS AND DISCUSSION

4.1 Transformation of pUC57-NS1B into E. coli BL21(DE3)

Transformation of pUC57-NS1B synthetic gene construct into

competent E. coli BL21(DE3) was successfully done. The colonies were

maintained using LB agar containing 100µg/ml ampicillin and incubated

overnight at 37°C. Plasmid isolation of pUC57-NS1B from E. coli BL21(DE3)

was done using GeneAll miniprep kit and the presence of pUC57-NS1B was

verified by agarose gel electrophoresis.

4.2 Cloning of pET-32b-NS1B into E. coli BL21(DE3)

Single restriction digestion was done for pUC57-NS1B and pET-32b by

using HindIII enzyme which recognizes 5׳A▼AGCTT 3׳ sequence.

The restriction digestion of pUC57-NS1B was successful and NS1B

gene (870 bp) was separated and this is shown in Figure 4.1. pET-32b was also

linearized and its presence was verified by the band of 5.9kb in Figure 4.1.

60

Figure 4.1 Restriction digestion by HindIII.

M: 1 kb Marker, Lane 1, 2: pUC57 and NS1B gene, Lane 3: pET-32b

Subsequently, the NS1B gene was cloned into pET-32b vector and

transformed into competent E. coli BL21(DE3). After overnight incubation,

colonies were observed and this is shown in Figure 4.2.

8000

6000

5000

3000

2500

2000

1500

1000

750

pET-32b (5.9kb)

pUC57 (2.7kb)

NS1B (0.87kb)

M 1 2 3

61

Figure 4.2 Transformation of pET-32b-NS1B into competent E. coli

BL21(DE3)

Colonies were streaked on LB agar containing 100 µg/ml ampicillin and

spread with IPTG and X-gal. X-gal is used to indicate whether a cell expresses

the β-galactosidase enzyme, which is encoded by the lacZ gene, in a technique

called blue/white screening or X-gal screening. X-gal dye is cleaved by β-

galactosidase yielding galactose and 5-bromo-4-chloro-3-hydroxyindole

(http://www.fermentas.com/en/products/all/reagents/r094-xgal). All colonies were

observed to be blue in color. Hence, the insertion of NS1B gene into pET-32b

vector gave negative result. This may due to recircularization of the vector. The

attempts to increase the ratio of gene: vector insert was done. However, the

cloning steps were not successful.

4.3 Cloning of pQE-81L-NS1B into E. coli DH5α

We attempted the directional cloning approach of NS1B gene into pQE-

81L vector. Double digestion was done for pQE-81L by using PstI and HindIII

enzymes. As for pUC57-NS1B, it was first digested with HindIII enzyme and the

result is shown in Figure 4.3.

Colonies

62

Figure 4.3 Plasmid isolation and digestion of pUC57-NS1B and pQE-81L

M: 1kb Marker, Lane 1 and 2: pUC57-NS1B digested with HindIII, Lane 3:

Miniprep of pUC57-NS1B, Lane 4: Double digestion of pQE-81L, Lane 5:

Miniprep of pQE-81L.

PstI enzyme recognizes the 5׳ CTGCA▼G 3' sequence and HindIII

enzyme recognizes 5׳A▼AGCTT 3׳ sequence. Figure 4.6 shows the isolation and

digestion of the vector and gene. Lane 1 and 2 shows the successful digestion of

pUC57-NS1B with HindIII enzyme and the band of NS1B is of correct size

(870bp). Lane 4 shows the double digestion of pQE-81L by using PstI and

HindIII and the band is of correct size (4.8kb) but in this way still unknown

whether the digestion correct complete or not. Plasmid miniprep for both pUC57-

NS1B and pQE-81L were verified in lane 3 and 5.

Because the concentration of NS1B gene extracted was low (4.7 ng/µl)

and this would affect the next step of digestion, so PCR amplification was done to

increase the concentration of NS1B by using primers designed for pUC57. A band

6000

5000

3000

2500

2000

1500

1000

750

M Lane 1 2 3 4 5

pQE-81L

(4.8kb)

NS1B

(0.87kb)

63

of the correct size was shown in lane 1 of Figure 4.4, indicating successful

amplification. Then, PCR product was extracted, double digested with PstI and

HindIII and used for cloning into pQE-81L. The transformed colonies were

shown in Figure 4.5.

Figure 4.4 PCR amplification of NS1B

NS1B

1000

750

M Lane 1

Lane 2 5 6 7

64

Figure 4.5 Transformation of pQE-81L-NS1B into competent DH5α

After transformation, colony PCR was carried out with pQEfor (5׳-

CGGATAACAATTTCACACAG-3') and pQErev (5'-

GTTCTGAGGTCATTACTGG-3') primers. However, all the colonies screened

gave negative result. As shown in Figure 4.6, the size of amplified band is smaller

than 300bp, indicating that only the pQE-81L vector was transformed to cells

without the cloning of NS1B gene. The right band size to be expected is nearly

1000bp.

Colonies

65

Figure 4.6 PCR of recombinant pQE-81L-NS1B

4.4 Cloning of pET-32a-NS1B into E. coli BL21(DE3)

PCR amplification was done on pUC57-NS1B to amplify NS1B gene

with a newly designed primers NS1BforSacI (5׳-GAGCTCATGGCGAACAAT-

and NS1BrevHindIII (5'-AAGCTTCTAATTGTCTCC-3') which introduced (׳3

SacI and HindIII restriction sites to NS1B for directional cloning of the gene into

pET-32a. The amplified NS1B gene is shown in Figure 4.7.

1000

300

M clone 21 22 23 24 25 26 27 28 29 30 31 32

66

Figure 4.7 Miniprep of pUC57-NS1B and PCR amplification of NS1B

M: 1kb marker, Lane1: Miniprep of pUC57-NS1B, Lane 2: PCR amplification of

NS1B gene

The HindIII enzyme recognizes 5׳A▼AGCTT 3׳ sequence while SacI

enzyme recognizes 5׳ GAGCT▼C 3׳ sequence. The gene was extracted, double

digested with SacI and HindIII and used for cloning into pET-32a. The

transformed colonies were shown in Figure 4.8. The colonies were screened by

PCR. pET-32a vector’s universal primers, namely T7 promoter (5'-

TAATACGACTCACTATAGGG-3') and T7 terminator (5'-

GCTAGTTATTGCTCAGCGG-3'). All colonies screened gave negative result.

As shown in Figure 4.9, the size of amplified band is smaller than 750 bp,

indicating that only the pET-32a vector was transformed into the cells without the

cloning of NS1B gene. The right band size to be expected is nearly 1500 bp.

3000

2500

1000

NS1B

M Lane 1 Lane 2

3 4

67

Figure 4.8 Transformation of pET-32a-NS1B into competent E. coli

BL2(DE3)

Figure 4.9 PCR of recombinant pET-32a-NS1B

M: 1 kb Marker, Lane 1-14: Clone from 62-75

Colonies

1500

750

M 1 2 3 4 5 6 7 8 9 M 10 11 12 13 14

68

4.5 Cloning of pQE-80L-NS1B into E. coli DH5α

The amplified NS1B gene containing SacI and HindIII sites was

extracted, digested and cloned into pQE-80L vector. The transformed colonies

were shown in Figure 4.10. After transformation into DH5α, colony PCR was

done using pQEfor and pQErev primers. All screened colonies yield negative

result. The result is shown in Figure 4.11. This result showed that only the pQE-

80L vector was transformed into cells without the cloning of NS1B. The right

band size to be expected is nearly1000bp.

Figure 4.10 Transformation of pQE-80L-NS1B into competent DH5α

Colonies

69

Figure 4.11 PCR of recombinant pQE-80L-NS1B

M: 1kb Marker, Clone number from 83-92

4.6 Confirmation of ligation

Ligation and transformation of pET-32a-NS1B into E. coli BL21(DE3)

and pQE-80L-NS1B into E. coli DH5α yield negative result. Therefore, to

confirm whether ligation had happened, PCR amplification was done on the

ligation. 1µl of ligation mix for pET-32a-NS1B and pQE-80L-NS1B were

amplified. The result shown in Figure 4.12 indicated that the fragments without

insert were amplified.

300

M Clone

83 84 85 86 87 88 89 90 91 92

70

Figure 4.12 PCR amplification

M: 1kb Marker, Lane 1: fragment without gene insert from pET-32-NS1B, Lane

2: fragment without gene insert from pQE-80L-NS1B

750

300

M Lane 1 2

83 84 85 86 87 88 89

90 91 92

71

CHAPTER 5

CONCLUSION AND FUTURE WORKS

5.1 Conclusion

Nonstructural protein NS1 is used for identification and differentiation

between the vaccinated and naturally infected animals. Vaccinated animals have

not produced nonstructural protein (Fang-kun et al., 8002). The function of NS1

protein during viral multiplication remains unclear, although a possible regulatory

role in viral replication has been suggested from studies of temperature sensitive

mutant of NS1 (Binns et al., 1993). Dauber and coworkers (2003) found that

reverse genetic system studies may allow to explore the function of NS1B during

the replication cycle and to assess their contributions to the pathogenesis and

virulence of influenza B (Dauber et al., 2003).

pUC57 plasmid carrying NS1B gene was successful transformed and

isolated from E. coli BL21(DE3). Designed primers used for PCR of NS1B

showed successful amplification.

First screening of pET-32b-NS1B colonies using white/blue method

gave negative result. Similar negative results were observed when screening

colonies of pQE-81L-NS1B, pET-32a-NS1B and pQE-80L-NS1B. Screening of

colonies for pQE-81L-NS1B and pQE-80L-NS1B revealed a band under the size

72

300bp, which is not as the expected band size of 1000bp. Screening of colonies

for pET-32a-NS1B showed a band of the size nearly 750bp, which is not as the

expected band size of 1500bp. Screening of colonies for all vectors used showed

only plasmids without any insertion of the NS1B gene.

Cloning NS1B into pET-32b using single restriction digestion with

HindIII, pQE-81L using double restriction digestion with (PstI and HindIII), pET-

32a using double restriction digestion with (SacI and HindIII), pQE-80L using

double restriction digestion with (SacI and HindIII) gave unexpected result. This

result may relate to re-ligation of digested vector for single digestion and

uncompleted digestion for vectors of double restriction digestion.

5.2 Future works

A further construction of NS1 gene into pET32a, pET-32b, pQE-80L

and pQE-81L vectors should be performed with best precaution of procedures to

avoid any contamination and the use of appropriate restriction enzymes with high

efficiency to get recombinant construction. The NS1B gene can also be cloned

into other expression vectors according to the open reading frame and use other

host.

In the future, it is suggested that the overexpression of the constructed

recombinant into expression system are being studied and work on purification

can also be performed using immobilized affinity chromatography resin. NS1B

protein can be analyzed by Sodium Dodecyl Sulphate –Polyacrylamide Gel

Electrophoresis (SDS-PAGE) analysis.

73

More studies to determine immunogenicity of influenza NS1B fusion

protein by using Western blotting or Enzyme-linked immunosorbent assay

(ELISA) methods should be carried out.

74

References

Binns, Matthew, and Ely. (1996). Expression of the non-structural protein NS1

111of influenza virus and detection of anti-NS1 antibody in serum. European

111Patent Office. Application no: 96300681.2

Birch-machin, I., Rowan, A., Jane Pick, J., Mumford, J., and Binns, M. (1997).

111Expression of the non structural protein NS1 of equine influenza a virus :

111detection of Anti-NS1antibody in post infection equine sera. Journal of

111Virology methods: 255-263.

Blaber, M. (1998). Spring. Web Page for Lecture 25 of Molecular Biology and

111Biotechnology Course: Prokaryotic Expression Vectors.

Dauber, B., Heins, G., and Wolff, T. (2003). The Influenza B Virus

111Nonstructural NS1 Protein Is Essential for Efficient Viral Growth and

111Antagonizes Beta Interferon Induction. Journal of Virology: p. 1865–1872.

Dauber, B., Schnider, J., and Wolff, T. (2006). Double-Stranded RNA Binding of

111Influenza B Virus Nonstructural NS1 Protein Inhibits Protein Kinase R but

111Is Not Essential To Antagonize Production of Alpha/Beta Interferon. Journal

111of Virology: p. 11667–11677.

Donelan, N., Dauber, B., Wang, X., Basler, C., Wolff, T., and Garcı´a-Sastre, A.

111(2004). The N-and C- Terminal Domains of the NS1 protein of Influenza B

111Virus Can Independently Inhibit IRF-3 and Beta Interferon Promoter

111activation. Journal of Virology: p.11574-11582.

75

Fang-Kun, W., Xio-Fang, Y., Yi-cheng, W., Cun, Z., Li-huan, X., and Si-dang,

111L. (2008). Cloning, Prokaryotic Expression, and Antigenicity Analysis of

111NS1 Gene of H9N2 Swine Influenza Virus. gricultural Sciences in China :

1117(7): 895-900.

Fortes, P., Beloso, A., and Ortin, J. (1994). Influenza VirusNs1 protein inhibits

111pre-mRNA splicing and blocks mRNA nucleocytoplasmic transport. The

111EMBO Journal: vol.13 no.3 pp. 704-712.

Garcı´a-Sastre, A., Egorov, A., Matassov, D., Brandt, S., Levy, D., Durbin, J.,

111Palese, P., and Muster, T. (1998). Influenza A virus lacking the NS1 gene

111replicates in interferon-deficient systems. Virology 252:324–330.

Greenspan, D., Palese P., and Krystal, M. (1988). Two Nuclear Location

111Signals in the Influenza Virus NS1 Nonstructural Protein. Journal of

111Virology, p: 3020-3026.

Hai, R., Martinez-Sobrido, L., Fraser, K., Allyon, J., Garcia-Saster, A., and

111Palese, P. (2008). Influenza B Virus NS1-Truncated Mutants: Live-

111Attenuated Vaccine Approach. Journal of Virology: p. 10580–10590.

Hale, B., Randall, R., Ortin, J., and Jackson, D. (2008). The multifunctional NS1

111 protein of influenza A viruses. Journal of General Virology: p. 89, 2359-

1112376.

Hatada, E., Takezawa, T., and Fukuda, R. (1992). Specific binding of influenza

111A virus NS1protein to the virus minus-sense RNA in vitro. Journal of

111General virology:73,17-25.

76

Krug, R., Yuan, W., Noah, D., and Latham, A. (2003). Intracellular warfare

111between human influenza viruses and human cells: the roles of the viral NS1

111protein.Virology, 309:181-189.

LaVallie, R., DiBlasio, A., Kovacic, S., Grant, L., Schendel, F. and McCoy, M.

111(1993) Bio/Technology 11, 187–193.

Leonhartsberger, S. (2006). E. coli Expression System Efficiency Secretes

111Recombinant Proteins into Culture Broth. Bioprocess internantional. 4(4),

11110-14.

Li, S., Min, J., Krug, R., and Sen, G. (2006). Binding of the influenza A virus

111NS1 protein to PKR mediates the inhibition of its activation by either PACT

111or double-stranded RNA. Journal of Virology: 349, 13-21.

Lu, Y., Qian, X., and Krug, R. (1994).The influenza virus NS1 protein: a novel

111inhibitor of pre-mRNA splicing. Genes Dev: 1817-1828.

Manasatienkij, W., Lekcharoensuk, P., and Upragarin, N. (2008). Expression

111and Purification of NS1 Protein of Highly Pathogenic Avian Influenza Virus

111H5N1 in Escherichia coli. Kasetsart J. (Nat. Sci.) 42: 485 – 494.

Metreveli, G. (2006). Expression of influenza virus nonstructural protein 1

111(NS1). Swedish University of Agricultural Sciences.

Nerome, R., Hiromoto, Y., Sugita, S., Tanabe, N., Ishida, M., Mastumoto, M.,

111Lindstrom, S., Takahashi, T., and Nerome, K. (1998). Evolutionary

111characteristics of influenza B virus since its first isolation in 1940: dynamic

111circulation of deletion and insertion mechanism.Arch Virol.143:1569-1583.

77

Ozaki, H., Sugiura, T., Sugita, S., Imagawa, H., and Kida, H. (2001).Detection

111of antibodies to the nonstructural protein (NS1)of influenza A virus allows

111distinction between vaccinated and infected horses .Veterinary Microbiology

11182:111-119.

Palese, P., and Shaw, L. (2007). Orthomyxoviridae: the viruses and their

111replication, p. 1647–1689.

Qian, X., Alonso-caplen, F., and Krug, R. (1993). Two Functional Domains of

111the Influenza Virus NS1 Protein Are Required for Regulation of Nuclear

111Export of mRNA. Journal of Virology: p. 2433-2441.

Schneider, J., Dauber, B., Mele´n, K., Julkunen, I., and Wolff, T. (2008).

111Analysis of Influenza B Virus NS1 Protein Trafficking Reveals a Novel

111Interaction with Nuclear Speckle Domains. Journal of Virology; p. 701–711.

Spickler, A. (2009). FluGrippe, avian influenza, Grippe Aviair, Fowl Plaque,

111Swine influenza, Hog Flu, Pig Flu, Equine influenza, Canine influenza.

111Lowa State University, College of Veterinary Medicine, Ames, Lowa.

Wolff, T., Oneili, R., and Palese, P. (1996). Interaction Cloning of NS1-I, a

111Human Protein That Binds to the Nonstructural NS1 Proteins of Influenza A

111and B Viruses. Journal of virology; p. 5363–5372.

Young, F., Desselberger, U., Palese, P., Ferguson, B., Shatzman, A. and

111Rosenberg, M. (1983). Efficient expression of influenza virus NS1

111nonstructureal proteins in Escherichia coli. Proc. Natl. Acad. Sci. USA. 80,

1116105- 6109.

78

Yuan, W., and Krug, M. (2001). Influenza B virus NS1 protein inhibits

111conjugation of the interferon (IFN)-induced ubiquitin-like ISG15 protein.

111EMBO J. 20, 362–371.

Yuan, W., Aramini, J., Montelione, G., and Krug, R. (2002). Structural Basis

111for Ubiquitin-like ISG 15 Protein Binding to the NS1 Protein of Influenza B

111Virus: AProtein–Protein Interaction Function That Is Not Shared by the

111Corresponding N-terminal Domain of the NS1 Protein of Influenza

111AVirus.Virology 304:291-301.

Zhao, L., Xu, L., Zhou, X., Zhu, Q., Yang, Z., Zhang, C., Zhu, X., Yu, M.,

11 Zhang, Y., Zhao, X., Huang, P. (2009). Interaction of influenza virus NS1

11.protein with growth arrest-specific protein 8. Virology Journal: 6:218.

79

APPENDIX A

NS1B gene sequence (870 bp) of Influenza B virus (B/Taiwan/45/2007)

aagcttctgcag atg gcg aac aat atg acc aca aca caa att gag gtg ggt ccg gga gca acc aat

gcc acc ata aac ttt gaa gca gga att ctg gag tgc tat gaa agg ctt tca tgg caa aga gcc ctt

gac tac cct ggt caa gac cgc cta aac aga cta aag aga aaa tta gag tca aga ata aag act

cac aac aaa agt gag cct gaa agt aaa agg atg tcc ctt gaa gag aga aaa gca att gga gta

aaa atg atg aaa gta ctc cta ttt atg aat ccg tct gct gga att gaa ggg ttt gag cca tac tgt

atg aaa agt tcc tca aat agc aac tgt acg aaa tac aat tgg acc gat tac cct tca aca cca ggg

agg tac ctt gat gac ata gaa gaa gaa cca gag gat gtt gat ggc cca act gaa ata gta tta

agg gac atg aac aac aaa gat gca agg caa aag ata aag gag gaa gta aac act cag aaa gaa

ggg aag ttc cgt ttg aca ata aaa agg gat atg cgt aat gta ttg tcc ttg aga gtg ttg gta aac

gga aca ttc ctc aaa cac ccc aat gga tac aag tcc tta tca act ctg cat aga ttg aat gca tat

gac cag agt gga agg ctt gtt gct aaa ctt gtt gcc act gat gat att aca gtg gag gat gaa gaa

gat ggc cat cgg atc ctc aac tca ctc ttc gag cgt ctt aat gaa gga cat tca aag cca att cga

gca gct gaa act gcg gtg gga gtc tta tcc caa ttt ggt caa gag cac cga tta tca cca gaa

gag gga gac aat tag aagcttctgcag

a : Adenine

c : Cytosine

g : Guanine

t : Thymine

HindIII PstI

HindIII PstI

80

APPENDIX B

NS1B protein sequence (281 amino acids)

MANNMTTTQIEVGPGATNATINFEAGILECYERLSWQRALDYPGQDRLN

RLKRKLESRIKTHNKSEPESKRMSLEERKAIGVKMMKVLLFMNPSAGIEG

FEPYCMKSSSNSNCTKYNWTDYPSTPGRYLDDIEEEPEDVDGPTEIVLRD

MNNKDARQKIKEEVNTQKEGKFRLTIKRDMRNVLSLRVLVNGTFLKHPN

GYKSLSTLHRLNAYDQSGRLVAKLVATDDITVEDEEDGHRILNSLFERLN

EGHSKPIRAAETAVGVLSQFGQEHRLSPEEGDN

A : Alanine M : Methionine

C : Cysteine N : Asparagine

D : Aspartic acid P : Proline

E : Glutamic acid Q : Glutamine

F : Phenylalanine R : Arginine

G : Glycine S : Serine

H : Histidine T : Threonine

I : Isoleucine V : Valine

K : Lysine W : Tryptophan

L : Leucine Y : Tyrosine