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Tieu Luan Viem Gan B

Apr 07, 2018

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Park Young Hyuk
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    TIU LU N: CNSX VacxinVa khang sinh Tai: CNSX Vacxin vim gan B

    GV hng dn:GV Nguyn Th Thng ThngNhm SV thc hin :Trn Trung Hiu (Leader)036Nguyn Quang Huy 042

    Cn Th Khnh Huyn 044Hong Th Hng 049Hong Th Yn 098

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    Mc lc

    Gii thiu v bnh vim gan B

    Virus vim gan B

    Giithiuv vaccine Quy trnh snxut vaccine vim gan B ti

    thp

    Quy trnh sn xut vaccime vim gan Bt huyt tng ngi lnh

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    Bnh vim gan Bla gi?

    Vim gan siu vi B l bnh gan himngho thng gp nht trn th gii do siu

    vi khun B gy ra. Vit Nam l mt trongnhng quc gia c t l b nhim siu vi B caonht th gii. nc ta c khong 10 triungi ang b nhim cn bnh ny.

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    Ti sao vim gan B c coi l cn bnhnguy him? V y l cnbnh ly lan lng l, tin trin m thm. a s

    ngibnhim siu vi B u khngbit l mnhbnhim v cth v tnh ly sang ngi khc qua ng mu. asngiln

    bnhimu c thloitr siu vi B d dng. Tuy nhin astrs sinh v tr em b nhim u khng th loi trc siu vikhun ny v strthnh ngi mang siu vi B mn tnh.

    C 4 ngi mang siu vi B mn tnh s c 1 ngibthitmngdo xgan v ung th gan trong sutcucicah. Siu vi B cththmlngtn cng gan trong nhiunm mbnh nhn khngh hay bit. n khi bnh nhn cm thycni khm bnh ththngbnh vo giai oncui.Nhiubnh nhn cht doung th gan vn khngbitthphm chnh l siu vi khun B.

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    Bnh vim gan siu vi B c th pht hinsm mt cch d dng nh vo cc xt nghimmu n gin. Vic pht hin sm vim gan B

    mn tnh gip gia tng hy vng ngn nga binchng x gan v ung th gan bng cch khmsc khe nh k v iu tr vi nhng loi

    thuc c tr mi.

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    Tnh hnh nhim HBV trn th gii

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    Virus vim gan B

    Virus vim gan B( HBV)thuc h Hepadnaviridae lnhng virus c kch thcnh.

    C 3 loi tiu th: hnh cunh (d=22nm), hnh cu ln(d=42-45nm) v hnh cunh v hnh ng.

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    B gene gm mt DNA c phngp i, khong 3.2 kilo cp

    base, to nn cc antigen:

    1. HBsAg (khng nguyn bmt): thuc lp v ca HBV -dng trong xt nghim mu

    bit c HBV trong c th.

    2. HBcAg (khng nguyn li):

    thuc lp li ca HBV - dng bit HBV ang pht trin.

    3. HBeAg (khng nguyn nisinh): nu c trong mu bnh

    nhn ang c kh nng ly rtcao.

    4. gen X : c th l nguyn nhnto ung th gan.

    5. gen P.

    Cu trcb gene vim gan B

  • 8/4/2019 Tieu Luan Viem Gan B

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    Cau tao bo gen HBV

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    S xm nhp vo t bo

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    Vacine l g ?

    Vaccine: l chphmc tnh khng nguyn

    dng tomindch

    c hiu ch ng,nhm tng sc khng ca c th i

    vi mt s tc nhngybnhcth.

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    Cc dng vaccine

    Phn loi theo ngun gc:

    Vaccine truyn thng

    Vaccine ti t hp

    Vc-xin kinh in

    Vc-xin bt hotVc-xin sng, gim c lc

    Cc "toxoid

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    Vaccine DNA

    Loi k thut c s dng bo vsinh vt chng libnhbng cch tim

    trctip DNA vo cth sinh vt To ra mtpngmindch

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    DNA ti t hp tSaccharomyces cervisiae

    Bc 1: Gii trnh t chui, la chn v

    thitk vector.

    Bc 2: Nhn dng plasmide v biuhin

    cu trc vector.

    Bc 3: Phn tch v xc nh gen ch.

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    Bc 4: Bin tnh t bo v a plasmide ti thp vo.

    Bc 5: T bo sinh ra Protein, chit tchprotein.

    Bc 6: Kim tra chtlng protein.

    Bc 7: Phn loi v tinh sch protein

    Bc 8: Thu nhn thnh cng protein ch.

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    Gii trnh t chui

    Thc hin PCR Ly mu v bo qun

    Ly trch v tinh sch

    Khuch i DNA bng phnng PCR trong my lunnhit

    Kim tra v pht hin sn

    phm khuch i trn gelAgarose

    Thc hin phn ng ct

    in di v phn tch kt qu

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    . in bin np

    + y l k thut hiu qu nht bin np vikhun.

    - Hai thng s quan trng ca phng thc

    ny lLoi t bo vi khun

    Tn s xung in cn thit.

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    Phng php in bin np c mt s u im

    sau: Hiu sut bin np cao.

    C th dng mt th tch dch t bo nh. Th

    tch dch t bo khong 20 L C th cho hiu sut khong 109th bin np.

    Nhc im ca phng php ny l i hi

    thit b bin np t tin.

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    Ha bin np

    y l phng thc kinh in bin npplasmid vo t boE. coli. Cc t boc trong dung dch CaCl2 tr

    thnh t bo kh bin gip cho chng dtip nhn plasmid.Gm 2bc :

    Chun b t bo kh bin Cy plasmid vo bng shock nhit

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    Chun b t bo kh bin

    ly mt khun lc n, nui trong 3 mLmi trng LB lng, (lc qua m 37 0C,200 vng/pht)

    Cy ria trn mi trng LB(Luria-Bertani) c (nui qua m, 370C)

    Chuyn 1 mL dch nui sang 100 mLmi trng LB lng mi, tip tc nui lc 37 0C, 200vng/pht Sau 2 3 gi, khi OD600t 0,375

    Chuyn dch sang 6 ng ly tm 15 mL c gi lnh trong

    B phn ni, ha tan phn cn trong 300 L CaCl2

    T bo kh bin cchia vo cc ng eppendorf, mi ng 100 Lem bo qun nhit -800C

    Dch t bo sau khi ly tm 6000 vng/pht 40C trong 5 phtc ra 2 ln bng 3 mL CaCl260mM sau dch ha tan c gi trong 30 pht.

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    Bin np DNA plasmid vo t bo E. coliJM109bng phng php sc nhit

    T bo c sc nhit 420C trong 30 giy sau gi trn 2 pht

    B sung 5 L DNA vectorPMD 18 Tvo ng eppendorf cha 50 L tbo kh bin v gi trn 20 pht

    T bo sau bin np c nui trong 600 L mi trng LB(lc 200 vng/pht

    370C, 60 pht)

    Cy tri 100 L dch ln mi trng LB rn c b sung ampicillin(100mg/L) (370C, qua m)

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    Thu nhan dong can

    Cat plasmide

    Chuyen gen vao plasmide

    bieu hien (pcDNA 3.1)

    ien di

    Ket qua

    Hind IIIEcoR1

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    Tch chit, thu nhn DNA ca plasmidt E.coli

    Ph v mng t bo (lysis method)

    Ly tm (centrifugation)

    vi enzym Hind III v EcoR1 Tch chit v tinh khit ADN ca

    plasmid (plasmid DNA extraction and

    purification)

    http://www.bbioo.com/bio101/UploadFiles/200805/2008052206590823.jpghttp://www.bbioo.com/bio101/UploadFiles/200805/2008052206590823.jpghttp://www.bbioo.com/bio101/UploadFiles/200805/2008052206590823.jpghttp://www.bbioo.com/bio101/UploadFiles/200805/2008052206590823.jpghttp://www.bbioo.com/bio101/UploadFiles/200805/2008052206590823.jpghttp://www.bbioo.com/bio101/UploadFiles/200805/2008052206590823.jpghttp://www.bbioo.com/bio101/UploadFiles/200805/2008052206590823.jpghttp://www.bbioo.com/bio101/UploadFiles/200805/2008052206590823.jpghttp://www.bbioo.com/bio101/UploadFiles/200805/2008052206590823.jpghttp://www.bbioo.com/bio101/UploadFiles/200805/2008052206590823.jpghttp://www.bbioo.com/bio101/UploadFiles/200805/2008052206590823.jpghttp://www.bbioo.com/bio101/UploadFiles/200805/2008052206590823.jpghttp://www.bbioo.com/bio101/UploadFiles/200805/2008052206590823.jpghttp://www.bbioo.com/bio101/UploadFiles/200805/2008052206590823.jpghttp://www.bbioo.com/bio101/UploadFiles/200805/2008052206590823.jpghttp://www.bbioo.com/bio101/UploadFiles/200805/2008052206590823.jpg
  • 8/4/2019 Tieu Luan Viem Gan B

    32/49

  • 8/4/2019 Tieu Luan Viem Gan B

    33/49

    a Plasmid vo Saccharomycescerevisiae

    To mi trng khbin:Mi trng LB. Cho dung dch CaCl2

    vo.

    Nui cy Saccharomycescerevisiaetrn mitrng.

    Chi Saccharomyces

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  • 8/4/2019 Tieu Luan Viem Gan B

    34/49

    Mi trng LB khbint trong lnh.

    Cho plasmid pcDNA 3.1+ vo mi trng.

    Tin hnh .

    Cho vo ninhit42oC trong vng 30s

    Sau trong vng 20 pht.

    Bin np bng phng php sc nhit

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  • 8/4/2019 Tieu Luan Viem Gan B

    35/49

    Nui cy Saccharomyces cerevisiae

    Nui cy nm men trong mi

    trng250C lc 250 rmp.

    Khi o OD600 khong 0.4 - 0.6

    dnglc

    Cho dung dch SDS.

    Ly tm 2500 rmp trong 5 pht,

    40C.

    Lc tch kt ta thu c dch

    protein.

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  • 8/4/2019 Tieu Luan Viem Gan B

    36/49

    Tch v tinh sch protein

    Mun thu nhn c cc protein nguyn th tcl protein c tt c

    tnh cht t nhin c trng ca n, cn sdng cc bin php khc nhau.

    -Nhit

    -Nng proton (pH)-Tc nhn ha hc

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  • 8/4/2019 Tieu Luan Viem Gan B

    37/49

    Tinh sch protein

    1.Loibtpcht-Phng php thm tch (dialysis)

    -Phng phpbin tnh chnlc

    - Phng php ktta phn onbngmui trung

    tnh hoc cc dung mi huc,-Cc phng php sc k trao i ion, in di,

    phng php lc gel...

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  • 8/4/2019 Tieu Luan Viem Gan B

    38/49

    Phng php sc k lc gel

    Cc phn t ln khng thmvo trong ht sephadex

    Ht sephadex Cc phn t nh thmvo trong c ht

    sephadex

    Tch cc phn t theo kch thc bng sc k lc gel.Hot ng ca lc phn t sephadex.

    http://www.bbioo.com/bio101/UploadFiles/200805/2008052206590823.jpghttp://www.bbioo.com/bio101/UploadFiles/200805/2008052206590823.jpghttp://www.bbioo.com/bio101/UploadFiles/200805/2008052206590823.jpghttp://www.bbioo.com/bio101/UploadFiles/200805/2008052206590823.jpghttp://www.bbioo.com/bio101/UploadFiles/200805/2008052206590823.jpghttp://www.bbioo.com/bio101/UploadFiles/200805/2008052206590823.jpghttp://www.bbioo.com/bio101/UploadFiles/200805/2008052206590823.jpghttp://www.bbioo.com/bio101/UploadFiles/200805/2008052206590823.jpghttp://www.bbioo.com/bio101/UploadFiles/200805/2008052206590823.jpghttp://www.bbioo.com/bio101/UploadFiles/200805/2008052206590823.jpghttp://www.bbioo.com/bio101/UploadFiles/200805/2008052206590823.jpghttp://www.bbioo.com/bio101/UploadFiles/200805/2008052206590823.jpghttp://www.bbioo.com/bio101/UploadFiles/200805/2008052206590823.jpghttp://www.bbioo.com/bio101/UploadFiles/200805/2008052206590823.jpg
  • 8/4/2019 Tieu Luan Viem Gan B

    39/49

    Thnh phn:

    Mt liu 1ml bao gm:

    - Khng nguyn b mt virt vim gan B tinhkhit..............20 g- Hydroxyt nhm ...................................0,5 mg

    - Thimerosal ................................0,01% (W/V)

    Sn phm vacxin ti t hp HBsAg

    Q i h i i b h

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  • 8/4/2019 Tieu Luan Viem Gan B

    40/49

    Qui trnh sn xut vacxin vim gan b t huyttng ngi

    Vacxin ny thuc loi vacxin di n v.phn khng nguyn c tnh sinh min dch lprotein v ca virus vim gan b ( HbsAg) c

    tch v tinh ch t huyt tng ca nhngngi mang virus vim gan B nh cc k thuttch thng thng nh kt ta, c c, ly

    tmvi 20bc nh sau:

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  • 8/4/2019 Tieu Luan Viem Gan B

    41/49

    Bc 1. thu thp huyt tng ngi mang

    HbsAg c hiu gi cao (>104-105).Bc 2. to huyt thanh t huyt tng (ta

    vi (NH4)2S04 30% nng cui), nhit

    phng 30 pht, ly tm 5000v/pht trong 20pht/40c. b cn, ly dch ni.

    Bc 3. ta vi sulphatamol 50% (nng

    cui). qua m nhit phng, ly tmnh trn. B nc ni, cn

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  • 8/4/2019 Tieu Luan Viem Gan B

    42/49

    Bc 4. ha cn vi KBr, khuy u t nht 1gi nhit phng, lng. loi b cnbng phu lc ht chn khng.

    Bc 5. iu chnh dch c t trng1,32g/cm3 (thm bt KBr)

    Bc 6. chun b my siu li tm HITACH-

    55pp v onal rotor RPZ35T.

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  • 8/4/2019 Tieu Luan Viem Gan B

    43/49

    Bc 7. cho cc dung dch vo rotor

    o Nc ct d=1: 500ml

    o Dung dch KBr d = 1,3g/cm3: 400ml

    o Dung dch mu d=1,32g/cm3: 1000ml

    o Ly tm 25000 v/pht trong 24 gi/20oc.

    Ly mu tngo Phn on 30ml v xc nh HsbAg trong

    chng. Trn cc

    phn on c HsbAg dng tnh cao.o Siu li tm 3 ln.

    Bc 8. c c n th tch 300ml.

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  • 8/4/2019 Tieu Luan Viem Gan B

    44/49

    Bc 9. siu li tm tng vng vi sacaroza graddient(2 ln).

    o Dung dch Nacl 1 M :500ml

    o Dungdch mu :300ml

    o Dung dch sacaroza 10% : 300ml

    o Dung dch sacaroza 20% : 300ml

    o Dung dch sacaroza 30% : 300ml

    o Dung dch sacaroza 40% : 300mk

    o Li tm 25000 v/pht trong 20 gi/10oc. B qua 300

    ml u, ly mu tng phn on 30 ml v xc nhHsbAg trong chng. Trn cc phn on HsbAg chiu gi cao.

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  • 8/4/2019 Tieu Luan Viem Gan B

    45/49

    Bc 10. c c dch (V=300ml)

    Bc 11. bt hot HsbAg tinh khit 60oc/10

    gi. Lc qua mng lc 8um v 0,45um. Bthot tip vi formaldehyt (nng cui1:2000) trong 96 gi/37oc.

    Bc 12. c c v thm tch loi formaldehyt(bng Pellicon cassette). Kim tra tinh schca HbsAg (in di, knh hin vi in t).

    kim tra HBV-ADD tn d (PCA)Bc 13. kim tra hm lng HbsAg (o mt

    quang v ELISA)

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  • 8/4/2019 Tieu Luan Viem Gan B

    46/49

    Bc 14. lc v trng quamng lc 0.22um

    Bc 15. hp ph HbsAg vo

    gel Al(OH)3 v pha vacxin vihm lng HbsAg 20ug/ml.nng cui ca nhm:300ug/ml. thm cht bo qunthimerosal nng 1:10000

    Bc 16. lc u cc chaitrong 1 tun, 2 ln/tun

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  • 8/4/2019 Tieu Luan Viem Gan B

    47/49

    Bc 17. ly mu bn thnh phm kim tra v

    khun, an ton, cng hiu, cht gy st v ccch s ha hc.

    Bc 18. ng l, dn nhn, ng gi

    Bc 19. ly mu kim tra cht lngvacxinvoo khun, an ton, cng hiu, cht gyst v cc thnh phn ha hc.

    Bc 20. xuaatss xng vacxin.

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  • 8/4/2019 Tieu Luan Viem Gan B

    48/49

    Sn phm vaccine vim gan B c iu ch t huyt tngngi lnh mang khng nguyn b mt virt vim gan B

    (HBsAg) khng c triu chng lm sng.

    Thnh phn:

    Mt liu 1ml bao gm:

    - Khng nguyn b mt virtvim gan B tinh

    khit........20 g

    - Hydroxyt nhm

    ..................0,5 mg

    - Thimerosal ...............0,01%

    (W/V)

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  • 8/4/2019 Tieu Luan Viem Gan B

    49/49

    THE END

    THANK YOU FOR LISTENING

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