Tieu Luan DNA Tai to Hop-Ngoc Tuan

I HC NNG

TRNG I HC BCH KHOA

(((TIU LUN

CNG NGH DNA TI T HP ti: Sn xut vaccine cm gia cm bng k thut DNA ti t hp

Gio vin hng dn: GS. NGUYN HONG LC

Sinh vin thc hin: H NGC TUN

Lp:Cao hc K29

Ngnh:

Cng ngh sinh hc

NNG THNG 3/2015

MC LC3LI M U

4I. Tnh hnh nghin cu cm A/H5N1 Vit Nam v trn th gii:

6II. Gii thiu virus cm A/H5N1:

62.1. Cu to:

72.2. Tnh thch ng a vt ch ca virus cm A/H5N1:

82.3. Kh nng gy bnh ca virus cm A/H5N1:

92.4. C ch xm nhim gy bnh ca virus cm A trong t bo vt ch:

10III. Mc tiu v cch tip cn mi trong sn xut vaccine cm:

12IV. Khng nguyn HA:

13V. K thut chuyn gen thng qua A.rhizogens:

135.1. Gii thiu vi khun A.rhizogens:

155.2. H vector nh th:

165.3. ng dng nui cy r t trong sn xut dc phm sinh hc ti t hp:

175.4. To vector chuyn gen bng k thut Gateway:

185.5. Bin np vector chuyn gen vo A. rhizogenes bng xung in :

185.6. Chuyn cu trc mang gen m ha protein v virus vo thuc l thng qua vi khun A. Rhizogenes:

20KT LUN

21TI LIU THAM KHO

LI M US lng tng ln cc bo co v s nh hng trc tip ca virus cm gia cm n con ngi nhn mnh nhu cu cho chin lc kim sot ngn nga i dch cm gia cm. Dch cm gia cm gy ra bi virus cm A/H5N1 v nhng nh hng ca dch ny n i sng kinh t x hi l mt trong nhng vn c quan tm hng u ca nhiu quc gia trn th gii c bit l nhng nc nm trong vng dch. Ch trong nm 2003 v 2004, chng virus cm A/H5N1 ly lan nhanh trong trong n gia cm nhiu nc chu v qua ly truyn sang ngi.T thng 2 nm 2003, hng triu gia cm v khong 360 ngi nhim bnh vi khong 230 ngi cht 12 nc Chu Phi, Chu v c Chu u. iu ng ch l virus H5N1 vn tip tc tin ha v s gy nh hng nghim trng n sc khe cng ng nu chng c kh nng ly truyn trc tip t ngi sang ngi v gy ra i dch.V nhng l do , vic to ra mt loi vaccine hiu qu bo v ngi v gia cm chng li virus cm l mt trong nhng mi quan tm hng u trong cng tc phng chng cm, nht l khi chng ta ang ng trc nguy c bng pht i dch. Hin nay, sn xut vaccine n c t thc vt ang l hng i ph hp vi cc nc ang pht trin nh Vit Nam do t bo thc vt c u th nui cy d dng, mi trng nui cy n gin, r tin, d dng sn xut mt lng sinh khi ln trong khong thi gian ngn v quan trng hn c l t bo thc vt nui cy in vitro khng mang cc mm bnh cho ngi.I. Tnh hnh nghin cu cm A/H5N1 Vit Nam v trn th gii:Chng virus cm A/H5N1 c pht hin ln u tin gy bnh dch trn g ti Scotland vo nm 1959. C th gi cm A/H5N1 phn lp nm 1959 ti Scotland l virus cm A/H5N1 c in. T cho n nay, H5 v N1 c thay i ln xt v cu trc thnh phn gen v khng nguyn min dch. Sau gn 40 nm khng pht hin, cm A/H5N1 xut hin ti Qung ng (1996), v Hng Kng (1997) vi bin i su sc, khng nhng gy cht gia cm m cn thch ng v gy cht ngi bnh. Cm A/H5N1 giai on 2003 n nay, c bn v cu trc vn nh trc , nhng xt v c lc, loi vt ch nhim bnh, tnh khng nguyn - min dch v mc truyn ly c nhiu nt c trng hn v khc vi nhiu bin chng H5N1 trc y.

Nhm ngn chn dch bnh ly lan, trn th gii c hng trm triu gia cm b tiu hy, gy thit hi nng n cho ngnh chn nui v kinh t. c bit, s ngi nhim v t vong do virus cm A/H5N1, mi nm mt cao hn, theo thng k s ngi b nhim cm gia cm H5N1 bo co vi T chc Y t th gii, t nm 2003 n thng 6/2008, c ti 385 trng hp mc cm A/H5N1, trong , 243 trng hp t vong chim ti 63,11%. Vit Nam v Indonesia l 2 quc gia c s ngi nhim v t vong cao nht do virus cm A/H5N1 trn th gii. Tnh gy bnh ca A/H5N1 th c lc cao khng ch gii hn chc nng im ct protease ca HA v hot tnh ca NA, m l hiu ng ca sn phm a gen v kh nng ti t hp to virus mi vi c tnh gy bnh v c lc khc nhau l vn cn tnh n. Hng ngn cng trnh nghin cu v cm A ni chung v cm A/H5N1 ni ring, trong c pht trin cng ngh v cc loi vaccine gy min dch cho gia cm v chun b i ph vi i dch c th xy ra ngi.Nghin cu nh type, bin i di truyn v gen hc tin ha ca virus cm A/H5N1 c cc c quan nghin cu ca Vit Nam tin hnh ngay t nhng thng u tin xy ra dch cm gia cm cui nm 2003. Nhng chui gen gip xc nh phn type H5, phn type N1 v cc gen cu trc c Vin Cng ngh Sinh hc, Vin Pasteur TP H Ch Minh, Vin V sinh dch t trung ng, Vin Th y gii m v cng b trn Ngn hng gen. Trn c s phn tch trnh t gen khng nguyn H5 v N1, cc tc gi khng nh ngun gc ca virus cm A gy bnh trn gia cm v ngi ti Vit Nam cng nhm vi virus H5N1 phn lp ti Trung Quc. Cc bin chng H5N1 ca Hng Kng, Trung Quc phn lp nhng nm 1997 - 2001 v Hn Quc, i Loan (phn lp nm 2003) u c ngun gc t chim ct v ngng (A/Goose/Guandong/1/96) vng Qung ng (Trung Quc), l cc bin chng thuc dng Qung ng. Nh vy, virus cm gia cm gy bnh gia cm v ngi ti Vit Nam l cm H5N1 type A thuc th h mi c bin i c bn v gen H5 v gen N1, nhng vn c cng ngun gc vi H5N1 t vng a l Nam Trung Quc v Hng Kng. Cc chng phn lp nhng nm 2004-2006 c nghin cu kh chi tit v gc gen hc v quan h phn t vi cc chng trong vng v th gii, kt qu khng nh virus H5N1 vng Nam v ng Nam thuc nhm di truyn VTM (vit tt: Vietnam-Thailand-Malaysia), c nhng c tnh sinh hc nht nh khc vi cc nhm vng Trung Quc v Hng Kng. Nm 2007, xut hin thm bin chng H5N1 di dng Phc Kin ti Vit Nam, v ang lm phc tp thm vn dch t hc v quan h khng nguyn v min dch, do t l tng ng khng nguyn HA(H5) v NA(N1) thp so vi cc chng phn dng Qung ng, tuy nhin vn cn c kh nng bo h min dch.

Vn chn on v xy dng phng php pht hin nhanh v phn bit cm A vi cc tc nhn gy triu chng h hp khc, cng nh phn bit cc phn type HA v NA c cc nh khoa hc Vit Nam quan tm, kt hp nghin cu vi cc t chc th gii. Pht hin nhanh H5N1 v cc phn type khc bao gm vic s dng khng nguyn hoc khng th, hoc sinh hc phn t c xy dng thnh phng php. Nghin cu vaccine v min dch, cc nh khoa hc Vit Nam cng c nhng ng gp nht nh v to ch phm khng nguyn, to vaccine di truyn ngc hoc vector ti t hp trn nn virus cm A/H5N1 ca Vit Nam.

II. Gii thiu virus cm A/H5N1:2.1. Cu to:

Cm gia cm (Avian Influenza) l bnh truyn nhim cp tnh ca gia cm, do nhm virus cm A, thuc h Orthomyxoviridae gy ra. y l nhm virus c bin ch rng, c phn chia thnh nhiu phn type khc nhau da trn khng nguyn HA v NA c trn b mt v ca ht virus. Cc ht virus cm A (virion) c hnh cu hoc hnh khi a din, ng knh 80 -120 nm, i khi cng c dng hnh si, khi lng phn t khong 250 triu Dal. Phn tch thnh phn ha hc mt virion c cha khong 0,8 - 1,1% RNA; 70 - 75% l protein; 20 - 24% lipid v 5 - 8% l carbonhydrate. Ht virus c cu to n gin gm v (capsid), v bc ngoi (envelope) v li l RNA si n m - negative single strand.

Hnh 1: Cu trc virus cm. Ghi ch: Hemagglutinin: phn t khng nguyn HA, Neuraminidase: phn t khng nguyn NA; PB2, PB1, PA: ba di n v phc hp enzyme polymerase ca virus. (Ngun: http://www.nature.com/nri/journal/v7/n4/fig_tab/nri2054_F1.html

V virus c chc nng bao bc v bo v vt cht di truyn RNA ca virus, bn cht cu to l mng lipid kp, c ngun gc t mng t bo nhim c c hiu ha gn cc protein mng ca virus. Trn b mt c khong 500 gai mu nh ra v phn b dy c, mi gai mu di khong 10 - 14 nm c ng knh 4 - 6 nm, l nhng khng nguyn b mt v virus, bn cht cu to l glycoprotein gm: HA, NA, MA v cc du n khc ca virus. Nhm virus cm A c 16 phn type HA (t H1 n H16) v 9 phn type NA (t N1 n N9), v s ti t hp (reassortment) gia cc phn type HA v NA, v mt l thuyt, s to ra nhiu phn type khc nhau v c tnh v kh nng gy bnh. 2.2. Tnh thch ng a vt ch ca virus cm A/H5N1:Vt ch t nhin ca tt c cc chng virus cm A/H5N1 l chim hoang d (ch yu l vt tri), y l nguyn nhn lan truyn virus trong t nhin rt kh kim sot. Virus cm A c kh nng gia tng bin vt ch ca chng trong qu trnh ly truyn t nhin.Nh c tnh lun thay i khng nguyn trong t nhin, virus cm A c kh nng xm nhim nhiu loi vt ch trung gian khc nhau nh gia cm, mt s loi ng vt c v (hi cu, c voi, nga, ln) v c ngi, to nn tnh thch ng lan truyn ni loi nh g - g, hay ngoi loi nh g - ln; g - ln - ngi. Vt (vt tri) v mt s loi thu cm khc (ngng) lun lun l vt ch tng tr ngun virus gy nhim. c im thch ng vt ch ny l iu kin thun li cho virus cm A trao i, ti t hp cc phn on gen, c bit l cc phn on gen khng nguyn (gen c HA v NA) gia cc chng, to ra mt chng virus cm mi c kh nng thch ng xm nhim loi vt ch mi ca chng c bit khi chng vt qua c ro cn loi d dng thch ng ly nhim gy bnh t gia cm sang ngi v gia ngi vi ngi.

Hnh 2: Mi quan h ly nhim v thch ng cc loi vt ch ca virus cm A (Ngun:http://www.impe-qn.org.vn/impe-qn/vn/portal/InfoDetail.jsp?area=58&cat=1101&ID=3194)

2.3. Kh nng gy bnh ca virus cm A/H5N1:Virus cm A c tnh thch ng ly nhim cao vi biu m ng h hp, gy bnh ch yu ng h hp, v cng c th tc ng gy tn thng nhiu c quan khc trong c th ca cc ng vt cm nhim, do cn c gi l virus hng a ph tng. Kh nng gy bnh ca virus cm A ph thuc vo c lc v tnh thch nghi vt ch ca tng chng virus. Thng thng chng khng gy bnh hoc ch gy bnh nh gii hn ng h hp ca chim hoang d v gia cm nhim, nhng mt s chng cng c (H5, H7, v H1, H2, H3) c th gy bnh nng hu ht cc c quan trong c th, gy nn dch cm gia cm v ngi, c l do tnh thch ng th th sialic ca chng. Hu ht cc chng virus cm A nhn ln rt tt trong phi g sau ln cy truyn th nht, tuy nhin cc chng cng c phn type H5, H7 gy cht phi g ngay sau vi gi, c khi hm lng virus rt thp cha c nhn ln nhiu, v c th gy bnh cm thc nghim trn chut lang, chut Hamster, chn t.

2.4. C ch xm nhim gy bnh ca virus cm A trong t bo vt ch:

Qu trnh xm nhim ca virus cm A c m u bng s kt hp ca HA v th th thch ng ca n trn b mt cc t bo ny, v cui cng l gii phng h gen ca virus vo trong bo tng ca t bo nhim.

Qu trnh nhn ln ca RNA virus cm A ch xy ra trong nhn ca t bo, y l c im khc bit so vi cc virus khc (qu trnh ny xy ra trong nguyn sinh cht), v cui cng l gii phng cc ht virus ra khi t bo nhim nh vai tr ca enzyme neuraminidase. Thi gian mt chu trnh xm nhim v gii phng cc ht virus mi ca virus cm ch khong vi gi. S to thnh cc ht virus mi khng ph tan t bo nhim, nhng cc t bo ny b ri lon h thng tng hp cc i phn t, v ri vo qu trnh cht theo chng trnh (apoptosis) lm tn thng m ca c th vt ch.

Sau khi c gii phng vo trong bo tng t bo nhim, h gen ca virus s dng b my sinh hc ca t bo tng hp cc protein ca virus v cc RNA vn chuyn. Phc hp protein RNA ca virus c vn chuyn vo trong nhn t bo.

Trong nhn t bo cc RNA h gen ca virus tng hp nn cc si dng t khun l si m ca h gen virus, t cc si dng ny chng tng hp nn RNA h gen ca virus mi nh RNA-polymerase. Cc si ny khng c Adenine ha (gn thm cc Adenine - gn m) u 5- v 3-, chng kt hp vi nucleoprotein (NP) to thnh phc hp ribonucleoprotein hon chnh v c vn chuyn ra bo tng t bo. ng thi, cc RNA thng tin ca virus cng sao chp nh h thng enzyme tng phn on gen ca virus, v c enzyme PB2 gn thm 10 - 12 nucleotide Adenin u 5, sau c vn chuyn ra bo tng v dch m ti li ni bo c ht tng hp nn cc protein ca virus.

Cc phn t NA v HA ca virus sau khi tng hp c vn chuyn gn ln mt ngoi ca mng t bo nhim nh b my Golgi, gi l hin tng ny chi ca virus. NP sau khi tng hp c vn chuyn tr li nhn t bo kt hp vi RNA thnh RNP ca virus. Sau cng cc RNP ca virus c hp nht vi vng ny chi, to thnh cc chi virus gn cht vo mng t bo ch bi lin kt gia HA vi th th cha sialic acid. Cc NA phn ct cc lin kt ny v gii phng cc ht virus trng thnh tip tc xm nhim cc t bo khc.III. Mc tiu v cch tip cn mi trong sn xut vaccine cm:Bi v khng th d on cc dng virus cm gia cm c th gy i dch tip theo cho con ngi cho nn cn mt vaccine l tng c th gy p ng min dich bo v vt ch t s tim nhim vi s lng ln cc loi virus cm t nhng dng cng hoc khc nhau. Glycoprotein HA v NA ca virus cm gia cm tri qua cc bin th di truyn v khng nguyn thot khi phn ng min dch. S hin din ca cc khng th trung ha c hiu cho glycoprotein HA ti cc v tr nim mc v h thng b xm nhim cung cp s bo v kp thi chng li virus cm, trong khi khong h ca virus cm ngi ph thuc ch yu vo min dch trung gian. Mc d khng th c hiu cho glycoprotein NA khng trung ha c s xm nhim, nhng chng hn ch s sao chp virus bng cch ngn cn s phng thch cc ht virus mi, mt qu trnh i hi protein NA ca virus. Do , khng th c hiu cho NA c th lm gim tnh nghim trng ca bnh. Cc epitop c nhn bit bi c lc t bo lympho T hin din trn protein NP, PB2 v PA ca virus cm gia cm trn ngi.Nguyn tc nm bn di cc vaccine hin ang c cp php chng li virus cm gia cm trn ngi l cm ng cc khng th bo v c hiu i vi cc glycoprotein HA ca chng bnh dch d on. Nng ca glycoprotein HA trong vaccine virus bt hot c cp php cho bnh cm theo ma c tiu chun ha, nhng nng glycoprotein NA th khng c tiu chun ha.

Hnh 3: Cc bc trong qu trnh sn xut vaccine dng protein ti t hp (Bng A), cc phn ging virus (Bng B), vector virus (Bng C), phng php da vo DNA (Bng D); on gen ca virus cm c xc nh. Cng ngh DNA ti t hp c th to nhanh vaccine cm bng cch s dng dy cc chin lc sn xut sau.

bng A, gen HA c nhn dng vo mt vector, n c biu din nh protein HA ti t hp trong t bo nhim, sau protein c tinh sch. Bng B, gy nhim t bo vo cc vector ring cha cc gen HA, NA v M1, ln lt, kt qu l thu c cc thnh phn ging virus m bao gm cc protein NA v HA trn b mt ca chng nhng thiu cc thnh phn ca gen cm. bng C, protein HA c biu din trn b mt ca virus mang lm cho n ch c mt trong min dch h thng ch khng gy bnh. bng D, vaccine DNA bao gm mt plasmid vi 1 hoc nhiu on gen c ci vo.

IV. Khng nguyn HA:

Hemagglutinin hay Haemagglutinin l glycoprotein khng nguyn tm thy trn b mt ca virus cm cng nh nhiu vi khun v virus khc. N c tc dng gn virus vo t bo b nhim. Tn gi ca HA bt ngun t vic protein HA c kh nng ngng kt hng cu.16 loi khng nguyn HA khc nhau c nh du t 1 n 16. H16 mi c pht hin virus phn lp t mng bin u en ti Thu in v Na Uy nm 2005. Ba loi protein HA u tin (H1, H2 v H3) c tm thy ph bin virus cm ngi. 4.1. Cu trc:

HA l mt glycoprotein mng to thnh t 3 tiu phn ging ht nhau. N c hnh tr, di khong 135 . 3 monomer to thnh li xon alpha gia; 3 u hnh cu cha v tr gn sialic acid. Nhng monomer HA c tng hp, glycosyl ho, ri ct thnh hai chui polypeptide nh hn: HA1 v HA2. Mi monomer bao gm mt chui xon di HA2 neo bm vo mng virus v mt ht HA1 ln u.

4.2. Chc nng:

Cho php nhn bit t bo ch ca ng vt c xng sng v hon thnh qu trnh nhn bit bng cch gn kt vi th th cha sialic acid ca nhng t bo ny; v cho php a b gen ca virus vo t bo ch bng cch hp nht mng endopisome ca t bo ch vi v ngoi virus.

4.3. C ch hot ng:

HA gn kt vi phn t glycoprotein trn b mt ca t bo ch, dn n cc phn t virus kt dnh vi t bo vt ch. Mng t bo s bao bc ly virus v phn mng ny s tch ra hnh thnh mt mng mi nm trong t bo gi l endosome, endosome cha virus c bao gi. T bo sau bt u c gng phn gii nhng thnh phn bn trong endosome bng cch acid ho phn bn trong n v bin n thnh mt lysosome (th sinh tan). Tuy nhin, ngay khi pH trong endosome xung 6.0, cu trc vn c gp li ca phn t HA tr nn khng bn, m ra mt phn v gii phng mt chui peptide rt k nc nm trong phn t protein. Chui fusion protein ny hot ng nh mt m neo, gn vo mng ca endosome v kho li. Sau , pH thp hn, phn cn li ca HA s gp li hnh thnh mt cu trc mi, rt m neo li v ko mng endosome vo st mng virus ri hp nht hai mng ny. Ngay lc ny, cc thnh phn ca virus, bao gm RNA genome, t do ho vo t bo cht.V. K thut chuyn gen thng qua A.rhizogens:5.1. Gii thiu vi khun A.rhizogens:Agrobacterium l cc loi vi khun t, thuc nhm Gram (-), ym kh, khi xm nhp qua vt thng, cc loi vi khun ny gy ra cc triu chng bnh trn thc vt nh to khi u hay lng r. Agrobacterium thuc h Rhizobiaceae, chi Agrobacterium c 4 loi chnh: Agrobacterium tumefaciens, Agrobacterium rhizogens, Agrobacterium radiobacter, Agrobacterium rubi. Trong A. tumefaciens v A. rhizogenes l hai loi c nghin cu nhiu nht l vi khun gy ra bnh khi u (crown gall) v bnh lng r (hairy root) cc v tr tn thng ca thc vt hai l mm.

Tc nhn gy bnh ca hai loi vi khun trn c xc nh l do s c mt ca Ti (tumour inducing) A. tumefaciens v Ri (root-inducing) A. rhizogenes. Khi t bo thc vt b thng, s tit ra cc polyphenol hp dn cc vi khun, ti y n chuyn on T-DNA (transfer DNA) t T-plasmid hay R-plasmid vo h gen ca t bo vt ch. Nhn chung, c ch ca qu trnh xm nhim v c ch phn t ca qu trnh vn chuyn T-DNA vo t bo vt ch ca hai loi vi khun ny c chnh minh l tng t nhau. Tuy nhin, s hnh thnh khi u vi khun A. tumefaciens do gen m ha sinh tng hp auxin quy nh. Trong khi cc gen m ha sinh tng hp auxin vi khun A. rhizogenes c vai tr rt nh trong qu trnh hnh thnh r t thc vt.

Ging nh Ti-plasmid, Ri-plasmid l phn t DNA mch vng, si kp, c trng lng phn t ln, t 200 - 800 kb, gm hai vng chnh l T-DNA v vir (virulence). Da vo kh nng sinh tng hp cc opine r t - ngun cacbon v nit cho vi khun. Ri-plasmid c chia lm hai nhm chnh: agropine v mannopine v mt s nhm ph c tm thy v phn loi sau ny nh: cucumopine v mikimopine. T-DNA Ri-plasmid ca nhm agropine bao gm hai vng chnh l vng bin tri TL-DNA v bin phi TR-DNA. Hai vng ny u c kch thc khong 15 - 20 kb v c xen k bi mt on DNA, on DNA ny s khng c chuyn vo h gen ca t bo vt ch. Vng TR-DNA mang cc gen m ha tng hp DNA (tms1 v tms2), vng TL-DNA bao gm 18 khung c (ORFs) trong c bn loci 10, 11, 12 v 15 m ha cho rol A, B, C v D (root locus). Cc Ri-plasmid ca cc chng A.rhizogenes thuc nhm mannopine, cucumopine v mikimopine cha vng T-DNA n, c cu trc ging nh vng TL-DNA ca cc chng thuc nhm agropine nhng khuyt gen rolD 20[], 24[].

Cc gen rolA, rolB and rolC ng vai tr quan trng trong qu trnh cm ng to r t m t bo thc vt. S biu hin ng thi ca ba gen ny gy nn kiu hnh r t m t bo thc vt b xm nhim. Cc r t c im sinh trng, phn nhnh mnh v pht trin nhanh hn rt nhiu so vi r bnh thng. Trong 3 gen rol trn, gen rolB ng vai tr quan trng hn c, khi gen rolB c biu hin, m t bo thc vt cm ng to kiu hnh r t, trong khi khi bt hot gen rolB khng to c kiu hnh r t.5.2. H vector nh th:

Kch thc ln ca Ri-plasmid v Ti-plasmid gy nhiu kh khn trong qu trnh chuyn gen thng qua Agrobacterium v trong cc thao tc s dng thit k gen vo cc vector ny. Mt khc cc enzyme gii hn c th ct DNA ca Ti-plasmid nhiu ch khc nhau, trong khi cng ngh gen li cn nhng v tr ct duy nht cho hot ng ca mt s enzyme gii hn. Thm vo , A.tumefaciens, mt s gen m ha sinh tng hp auxin c s dng lm ch th chn lc c tnh tri nhng li cn tr qu trnh ti sinh bnh thng thc vt. Vi cc l do trn y, cc nh khoa hc ci tin Ti-plasmid thnh mt h vector nh th gm c vector chuyn gen (mang cu trc T-DNA) v vector b tr (mang cc gen vir) m nhim chc nng vn chuyn T-DNA vo t bo thc vt.

Vector chuyn gen trong h vector nh th A.tumefaciens c th s dng chuyn gen thng qua A.rhizogenes. Khi nhim A.rhizogenes vi thc vt s lm chuyn T-DNA t vector chuyn gen cng vi T-DNA ca A.rhizogenes vo genome thc vt.

S ti sinh thnh cy chuyn gen thng qua vi khun A.rhizogenes c th xy ra thng qua s pht trin phi soma hoc s pht sinh c quan t r. Tuy nhin, thc vt ti sinh t lng r thng b bin i hnh thi: l b gp np, h thng r n nghing, sinh trng cn ci v kh nng sinh sn km.

5.3. ng dng nui cy r t trong sn xut dc phm sinh hc ti t hp:

T bo thc vt ni chung v r t ni ring vi u th nui cy d dng, mi trng nui cy n gin, r tin, d dng sn xut mt lng sinh khi ln trong khong thi gian ngn v quan trng hn c t bo thc vt khng mang cc mm bnh cho ngi c xem l mt trong nhng xu hng hin nay c s dng biu hin cc dc phm sinh hc ti t hp. c bit r t c th sinh trng, pht trin tt trn mi trng khng cn b sung cc cht iu ha sinh trng v th loi b c d lng ca cc cht ny trong sn phm to ra. Hn na cc r t c th c nui cy to sinh khi lin tc, iu ny c ngha trong dy chuyn sn xut cc cht th cp hay cc dc phm sinh hc ti t hp. Thm vo , so snh vi thc vt chuyn gen, nui cy m t bo thc vt c u im ln trong cng tc sn xut cc dc phm sinh hc ti t hp l khng i hi din tch canh tc, khng chu nh hng vo kh hu, ma v, sn phm thu c khng mang cc yu t c hi t thuc tr su, thuc dit c v rt ngn c rt nhiu thi gian trong trng trt. c bit hn c h thng nui cy lng cc m t bo thc vt vic biu hin dc phm sinh hc ti t hp ra ngoi mi trng nui cy gip lm n gin hn rt nhiu trong qu trnh tinh sch cc dc phm sinh hc ny.

Cho ti nay, c nhiu nghin cu sn xut thnh cng cc protein ti t hp hay cc dc phm sinh hc ti t hp t h thng nui cy r t nh SEAP (human secreted alkaline phosphatase), protein hunh quang kt hp vi protein ricin B (GFP-ricin B), fungal phytase, v c bit hn c l cc khng th v cc chui nng, chui nh ca khng th.

5.4. To vector chuyn gen bng k thut Gateway:K thut Gateway l mt k thut dng ha ph bin, n mang li hiu qu cao v nhanh chng khi phn tch chc nng, biu hin protein v dng ha on DNA. Da vo im c hiu trong h thng ti t hp ca phage I. K thut Gateway cho php chuyn on DNA gia cc vector tch dng khc nhau trong khi vn duy tr nh hng chnh v cu trc c, thay th vic s dng enzym ct gii hn v enzym ni hiu qu. K thut ny l mt phng php c hiu qu cao cho vic tch dng c nh hng ca cc sn phm PCR.

K thut ny gm c hai loi phn ng LR v BP (Invitrogen). Trong nghin cu ny phn ng LR c thc hin gia vector tip nhn v vector ch pK7WG2D (1) di s xc tc bi hn hp enzym LR ClonaseTM II . on gen cn quan tm sau khi a vo vector tip nhn (v d, pENTR/HA1), bn sn c hai im ti t hp (attL1 v attL2). Chuyn on gen quan tm vo vector ch (v d, pK7WG2D (1)), vector ny cha tt c trnh t cn biu hin v cng cha hai im ti t hp (attR1 v attR2) gia hai im c on gen chn lc m tnh ccdB. Trn hai plasmid, att1 v att2 l hai im nh hng v c hiu ti t hp, mi mt im trn vector ny ch ti t hp vi mt im tng ng trn vector kia m khng kt hp vi v tr khc, nhng n s kt hp vi nhng im xc nh theo th t phn t., v vy, attL1 phn ng vi attR1, cn attL2 phn ng vi attR2. Hai s ti t hp ny to ra dng biu hin, mt l gia attL1 v attR1 th hai l attL2 v attR2. Sn phm ca hai s ti t hp s to ra mt plasmid nh mong mun v cc sn phm ph. Plasmid mi c hai s la chn: Khng khng sinh v chn lc m tnh. V vy, t bo sau khi c bin np sn phm LR u l nhng dng khun lc dng tnh trn mi trng c khng sinh chn lc.

Tuy nhin, xc nh chnh xc cc plasmid ti t hp mi c to thnh ng nh mong i, cc phn ng ct bng enzym gii hn v PCR cng s c thc hin.5.5. Bin np vector chuyn gen vo A. rhizogenes bng xung in :

Di tc ng ca in trng cc ln, l mng t bo m ra, nh DNA di chuyn theo in trng d dng xm nhp vo t bo. Khi ngng xung in, cu trc mng t bo hi phc trong mi trng LB v gi DNA li trong t bo. T bo cha DNA ti t hp c kh nng biu hin cc gen khng khng sinh nn c chn lc trn mi trng cha cht khng sinh .

5.6. Chuyn cu trc mang gen m ha protein v virus vo thuc l thng qua vi khun A. Rhizogenes:

A. rhizogenes l vi khun mang thnh phn Ri plasmid c th chuyn mt gen mong mun vo genome t bo thc vt.

Chng A. rhizogenes mang vector chuyn gen nh th, vng vir c thit k sn trong t bo vi khun, trong khi T-DNA l vector chuyn gen pK7WG2D (1) mang cu trc HA1 chuyn vo t bo bng xung in.

Quy trnh chuyn gen vo ging thuc l N. tabacum K326 thng qua vi khun A. Rhizogenes c tin hnh theo phng php ca Topping c ci tin (Topping, 1998). Cc bc chnh nh sau:

- Chun b chng A. rhizogenes mang cu trc HA: mt ngy trc khi bin np, nui mt khun lc vo 5 ml YMP lng c b sung cefotaxim 500 mg/L, nui lc 200 v/p 28oC.

- Chun b mnh l thuc l c kch thc 1 cm2 c lm tn thng bng li dao ct bn cnh xung quanh, t trn mi trng WPM trong 2 ngy.

- Ht 1 ml dch khun nui lng trn cho vo 50 ml YMP lng, o OD600 = 0,7 - 1 l c th c s dng cho bin np.

- Cc mnh l sau 2 ngy nui cm ng trn mi trng WPM, c nht ra mt bnh tam gic mi c b sung 5 ml MS.

- dch khun vo bnh tam gic c cha cc mnh cy l thuc l trn.

- Sau 30 pht chuyn cc mnh cy ln giy thm kh trng, thm kh v cy ln mi trng WPM. Sau 2 ngy chuyn sang mi trng WPM c b sung khng sinh cefotaxim 500 mg/L v kanamycin 100 mg/L.

- Sau 2 - 4 tun cc mnh cy bt u cm ng to r c ct v chuyn sang mi trng WPM c b sung cefotaxim 400 mg/l v kanamycin 100 mg/l.

Hnh 7: R t cc m l chuyn gen

KT LUNChuyn gen m ha cc vaccine ti t hp vo thc vt c th l vo cc cy trng l ngun thc n chnh cho con ngi, ng vt nui l mt trong nhng hng i chnh hin nay.

thit k c vector chuyn gen vo thc vt - mang cu trc gen m ha protein v ca virus H5N1 bng k thut lai Gateway phc v cho mc ch chuyn gen to vaccine thc vt. Vector ny c bin np thnh cng vo vi khun A.rhizogenes to cc dng r t chuyn gen biu hin vaccine ti t hp HA1 dng lm dc phm sinh hc ti t hp.

TI LIU THAM KHO1.

Hnh 5: Cu trc Ri plasmid

Hnh 4:. M hnh cu trc protein 3 chiu ca HA

(Ngun:http://vi.wikipedia.org/wiki/Hemagglutinin)

Hnh 6: Hnh nh to khi u gy ra do A. tumefaciens (tri) v

r t do A. rhizogenes (phi).