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vn
LI M U........................................................................................................................2
PHN 1 TNG QUAN TI LIU....................................................................................................................................3 PHN 2 KT QU NGHIN CU..............................................................................................................................31 PHN 3 KT LUN........................................................................................................................................................33 TI LIU THAM KHO................................................................................................................................................36 6. PHAN KIM NGC V PHM VN PHC (2007),CNSH TRN NGI V NG VT, NXB GIO DC, TR 643........................................................................................................................................................................36 12. YASMIN THANAVALA V CS (2005), IMMUNOGENICITY IN HUMANS OF AN EDIBLE VACCINE FOR HEPATITIS B, PNAS VOL. 102 _ NO. 9, P3378-3382.....................................................................................37

Li m u
Vaccine thc phm (edible vaccine) l mt trong nhng sn phm cng ngh sinh hc hin i, ch mi xut hin cch y khong 2 thp k tr li, l s kt hp gia sinh hc phn t, nui cy m v min dch hc. Cc loi vaccine tim hin nay c gi thnh rt cao v ngi tim yu cu phi c k nng, hn na vic dng li kim tim mt s ni kh khn c th ly truyn virut nh vim gan B, C, HIVVaccine tim cng yu cu bo qun lnh, c bit c nhiu ni vng su, vng xa mng in li cha n th vaccine cng khng n c Cho n thi gian gn y ngi ta vn s dng vaccine sng nhc c lm khng nguyn kch thch to khng th cn thit trong c th ngi v vt nui. Vaccine kiu ny c mt s hn ch nh: c kh nng quay tr li dng c hoc hot lc ca n gim kh nhanh trong c th ngi v vt nui. Hin nay, nh cng ngh DNA ti t hp ngi ta sn xut c protein v ca mt s loi virus nh virus bnh l mm long mng, bnh
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di v vim gan B. Tuy nhin, vaccine c sn xut theo cc phng php trn c gi thnh cao, iu kin bo qun v vn chuyn nghim ngt, cn c k thut vin tin hnh tim chng [3]. Vaccine thc phm l mt m hnh l tng v n gip khc phc c cc kh khn ni trn ca vaccine c sn xut theo phng php truyn thng hoc vaccine DNA ti t hp. Nguyn l c bn ca qu trnh ny l chuyn mt loi gen c bit vo t bo thc vt. Loi gen ny hot ng trong c th thc vt, s bin thnh ni sinh ra protein khng nguyn. Khi nhng khng nguyn ny i vo c th ngi thng qua n ung (di dng ti sng khng nu chn, nu khng s lm mt hot tnh khng nguyn), h thng min dch ca ngi s t ng sinh ra khng th chng li khng nguyn. Nh vy l thay vic tim chng vaccine bng vic n nhng hoa qu hoc rau xanh c khng nguyn [3]. nc ta Vaccin thc phm c l l mt cm t kh mi m nhng i vi cc nc pht trin trn th gii th n khng c g xa l lm, bi t nhng nm u thp nin 90 ngi ta to thnh cng nhng cy Vaccine n u tin, t bng ln mt chin dch nghin cu vaccine n c trong thc vt ca gii khoa hc khp ni trn th gii, v thu c nhng thnh cng ng k: - Sn xut vaccine chng bnh infectious bursan desease virus (IBDV) g trong c rabidopsis chuyn gen. - Chuyn gen orf2 ca virus gy bnh vim gan E vo cy c chua, v cy Pichia pastoris. - Sn xut vaccine vim gan B trong cy chui chuyn gen, cy Physalis ixocarpa, u lupin vng, rau dip v c chua. - Chuyn gen LTB ca E. coli (B subunit of E. coli heat-labile enterotoxin) gy bnh ng rut vo khoai ty. - Chuyn gen CTB (cholera toxin B subunit) gy bnh t ca vi khun Vibrio cholerae v gen LTB vo cy thuc l Tuy nhin cho n nay vic nghin cu v pht trin vaccine n vn cn ang l mt
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phng hng nghin cu rt mi ca cng ngh sinh hc, c bit l nhng nc ang pht trin v nhng nc ngho, ni m vn min dch thng l mi quan tm ln. Xut pht t nhng l do trn nn ti chn ti : Vaccine thc phm

PHN 1 TNG QUAN TI LIU

1.1
. nh ngha Vaccine thc phm Vaccine thc phm: L loi vaccine th h mi c to ra bng cch chuyn mt gen khng nguyn vo thc vt. Gen ny khi hot ng trong c th thc vt s sinh ra protein khng nguyn tng ng. Khi nhng khng nguyn ny i vo c th ngi thng qua n ung (di dng ti sng), h thng min dch ca ngi s t ng sinh ra khng th chng li khng nguyn . Nh vy l thay vic tim chng vaccine bng vic n nhng hoa qu hoc rau xanh c khng nguyn [3]. Theo Mason v cng s nh ngha: Vaccine thc phm l vaccine tho mn: T bo thc vt c kh nng tng hp chnh xc v tch lu protein gy min dch; vic n thc vtchuyn gen biu hin khng nguyn vaccine c th kch thch p ng min dch c th [10].

1.2

. C s khoa hc v vaccine thc phm Chuyn gen l k thut s dng DNA tinh khit a vo c th hay t bo khc

1.2.1. V sinh hc phn t nhm to tnh trng di truyn mi do DNA ny biu hin trong i tng ch. T khi ra i n nay, k thut chuyn gen mang li cho nhn loi nhng thnh qu to ln trong nhiu lnh vc, c bit l trong nng nghip v y hc. Ngi ta thng k c hn 2/3 din tch nng nghip th gii s dng cy trng chuyn gen vi cc mc ch khc nhau nh tng nng sut, ci thin ging, thu sn phm tri hay c tnh chng chu iu kin no , trong s ny tiu biu l cc ging Bt biu
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hin c t ca Bacillus thuringiensis (Bt) to ra tnh khng i vi cc cn trng nhm nhai- nghin (chewing insects) v ging cy trng chng chu cht dit c. Y hc cng l mnh t mu m cho dng cc sn phm bin i di truyn trn c 3 i tng vi sinh vt, thc vt v ng vt. Ph bin hin nay l cc dc liu t nhin c sn xut nh cc t bo thc vt. Thc vt l ngun cung cp cc hp cht ha hc dng lm dc liu rt c gi tr. Nhng sn phm ny, c bit nh l cc cht trao i th cp (secondary metabolites), thng c sn xut vi mt lng rt nh (dng vt) trong thc vt v khng c chc nng trao i cht r rng. Chng dng nh l sn phm ca cc phn ng ha hc ca thc vt vi mi trng chung quanh, l s thch nghi vi stress ca mi trng hoc l s bo v ha hc chng li vi sinh vt v ng vt. sn xut cc sn phm th cp t thc vt, cc m thc vt ngoi sinh t cy hon chnh c nui cy dch huyn ph (suspension culture) trong iu kin v trng (Hnh 1.1). C s ca k thut nui cy m thc vt da trn tnh ton th ha sinh (biochemical totipotency) duy nht ca t bo thc vt. Nhiu sn phm trao i cht c th c sn xut t nui cy dch huyn ph c cht lng cao hn trong cy hon chnh [3]. Dng sn phm y dc bin i di truyn c quan tm na l vaccine thc phm. Trong k thut ny, ngi ta sn xut vaccine bng cch chuyn cc gen sn xut protein khng nguyn ca tc nhn gy bnh vo c th thc vt. Sn phm thu c l l, c, hay qu ca cy chuyn gen chnh l sn phm vaccine cn thit, khi n cc b phn ny ca cy s kch thch c th p ng min dch vi tc nhn gy bnh khi chng xm nhp c th. Vi s tin b ca cng ngh sinh hc hin i, c bit l cc k thut di truyn phn t cho php chng ta to ra v sn xut lng ln bt k protein no c thy trn b mt ca tc nhn gy bnh, nhng protein ni trn c th c s dng nh l vaccine tiu phn(subunit vaccine) hay lm khng nguyn a vo c th cn kch thch min dch. to ra khng th chng li tc nhn gy bnh . iu ny thc vt nhng h gi thnhL phm khng trong h ln men 100 sn
Hnh 1.1. Nui cy t bo dch huyn ph

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vaccine m cn loi b c nguy c ly lan cc bnh c th gy truyn nhim qua ng tim hay qua huyt thanh. Trc kia cc nh khoa hc thng sn xut cc loi protein tiu phn ny nh cc nh my vi sinh vt, vi sinh vt c la chn ph hp s c chuyn gen sn xut ra protein khng nguyn ca tc nhn gy bnh vi hiu sut cao nht nh 1 vector c thit k c bit, vi sinh vt c bin i gen ny s c nhn ln vi s lng ln thu c sn phm cn thit qua nhiu bc tinh ch, tinh sch trung gian. i vi vaccine thc phm cng vi c s l thuyt tng t nhng ngi ta s dng i tng chuyn gen l thc vt. Mun a mt gen vo t bo thc vt trc tin n phi c gn vo 1 vector y l nhn t gip mang gen, nhn bn v i khi biu hin mt trnh t DNA trong t bo ch

Mt s c tnh m 1 vector cn c.

Kch thc ca vector cng nh cng tt, v d xm nhp vo t bo, ng thi thun Vector phi c kh nng ti bn, nh DNA trn vector mi c th sao chp v Mang nhng c tnh cho php pht hin d dng, c m ho bi cc gen ch th

li trong qu trnh sao chp. c duy tr trong qun th t bo chn lc, nh cha gen ch th thao tc trong E.coli v gen ch th khc tin hnh chn lc thc vt. Thng thng l cc gen khng vi khng sinh, nh c th la chn c t bo mang gen chuyn (vector) t qun th ln cc t bo

Cha t nht 1 v tr nhn bit ca enzyme gii hn s dng lm v tr ghp DNA Vi khun v mt s vi sinh vt khc c cha cc phn t DNA dng vng, tng i

trong to th ti t hp. nh, tch bit vi nhim sc th vi khun v sao chp c lp. Nhng phn t ny c gi l Plasmid v ni chung khng thit yu i vi s sinh trng ca vi sinh vt, chng thng cung cp li th chn lc i vi sinh vt ch nh tnh khng khng sinh. V nhng c tnh ny cc Plasmid c s dng rng ri lm vector c bt trong xy dng cc phn t ti t hp phc tp.
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Ngoi cc vi khun cc vi rt cng c s dng lm vector v chng c th hot ng nh nhng phn t vn chuyn i vi cc on DNA tng i ln [7]. 1.2.2. V min dch hc Hu ht cc vi sinh vt gy bnh u xm nhp vo c th qua b mt nhy trong ng tiu ho, h hp v ng tit niu. Khi vaccine n vo c th theo ng ming n s cm ng h thng min dch th dch sn xut cc khng th chng li vi sinh vt gy bnh, tip h thng th dch li tc ng vo h thng min dch ca t bo, to ra cc globulin min dch tng cng kh nng bo v sm v hiu qu cho c th. Khi tiu ho vaccine n c, khng nguyn c gii phong trong rut non [5]. 1.2.2.2. Khng nguyn nh ngha: Nhng cht khi vo c th s kch thch c th gy ra hin tng min dch c gi l khng nguyn. iu kin 1 cht c xc nh l khng nguyn:

Tnh l: C th s khng bao gi p ng bo v i vi nhng cht c sn hoc

nhng cht ging nh cc cht c sn trong c th. C th ch p ng bo v khi cht a vo phi khc hon ton vi cc cht c sn trong c th, nhng cht khi vo c th c bn cht cng khc vi c th bao nhiu th cng tng nhanh v tng mnh kh nng min dch ca c th by nhiu.

Trng lng phn t ln: Khng nguyn phi c trng lng phn t ln hn

10.000 dalton (n v o trng lng, tng ng trng lng nguyn t hir). Cu trc phn t phc tp: Cu trc phn t cng phc tp th kh nng gy min dch cng cao. C khng nguyn ng loi (alloantigen hay isoantigen) v khng nguyn a loi. Khng nguyn ta quan tm l khng nguyn a loi: tn ti trn b mt t bo m ng vt, vi sinh vt (virut v vi khun) Ngi ta chia khng nguyn vi sinh vt ra cc loi sau: -Khng nguyn vi khun gm: Khng nguyn vi khun ho tan l cc enzyme ngoi bo.
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Khng nguyn vi khun khng ho tan l thnh phn t bo ch yu nm trn b mt t bo, 2 loi c quan tm nht l khng nguyn lng (k hiu H), v khng nguyn thn (k hiu O) Cc cht c (toxin) khi b mt c tnh tr thnh gii c t (toxoid) c tnh khng nguyn nhng khng gy c cng l mt khng nguyn cn quan tm. -Khng nguyn virut gm: Khng nguyn V: bao gm 1 phn ca virut hoc c virut Khng nguyn S: l khng nguyn ho tan c th l v glycoprotein hay nucleic acid ca virut [5]. 1.2.2.2. Khng th (antibody) Khng th l cc globulin c trong huyt thanh ca ng vt c to ra do qu trnh kch thch ca khng nguyn v c kh nng lin kt c hiu vi khng nguyn. Khng th c to ra theo c ch ny c gi l khng th c hiu hay khng th min dch, c k hiu l Ig. Ngi ta thng tm thy khng th ny trong huyt thanh ca ng vt nn huyt thanh cha khng th c gi l khng huyt thanh. C 4 loi khng th: IgG, IgM, IgD, IgA. L lch l c quan chnh to ra khng th [5]. 1.3. Phng Php chuyn gen Chuyn gen hay bin np thng tin di truyn l k thut s dng DNA tinh khit a vo c th hay t bo khc v theo di biu hin ca thng tin di truyn mi ny. bin np hiu qu nguyn liu di truyn vo t bo vt ch, cc cu trc di truyn cn c thit k thch hp cho s hp nht v biu hin ca cc gen ngoi lai. Cu trc di truyn phi mang 1 gen ch th chn lc (selectable marker gen: gen m ho 1 protein kh c ca ho cht b sung trong mi trng nui cy, cho php sinh trng u tin ca cc t bo c DNA c hp nht) hoc sng lc (screenable marker gen: gen m ho 1 protein cho kt qu trong sn phm sng st nh c th xc nh t bo bin np th hin gen) nhn bit hiu qu bin np gen. Mt cu trc di truyn c trng bao gm: gen khi u (promoter), gen m ho (coding gen), v gen kt thc (terminator) [2].
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Cc phng php chuyn gen c th b hoc khng b gii hn bi cc genotype khc nhau ca thc vt. Tu thuc vo mc ch ng dng, i tng, c th thit k mt phng thc bin np thch hp cho tng genotype khc nhau. Trong cc nghin cu c bn, ngi ta thng tp trung nghin cu v cu trc v chc nng ca cc gen bin np, kho st cc promoter v cc c ch phn t thc vt c th chuyn gen thnh cng vo cc loi khc nhau [2]. Cc cy chuyn gen ang c thng mi ha hin nay l th h u tin ca cc cy trng chuyn gen, v ba th h cy trng chuyn gen c th c d on trc l [3] - Th h th nht. Cc tnh trng sn xut (v d chng chu cht dit c, khng bnh/cn trng). - Th h th hai. Cc gen xp thnh chng cho nhiu tnh trng (v d t hp ca cc gen khng bnh cng vi cc tnh trng cht lng). - Th h th ba. Cc tnh trng khc nhau c p ng cho vic s dng c bit cui cng (v d thc phm, si, nhin liu, du nhn, nha, dc phm v cc nguyn liu th cho cc qu trnh cng nghip). Cng ngh chuyn gen (bin np gen) thc hin vic chuyn cc gen ngoi lai vo t bo v m thc vt c th dng cc phng php sau 1.3.1. Chuyn gen gin tip bng vi khun Agrobacterium Agrobacterium tumefaciens v Agrobacterium rhizogenes l hai loi vi khun gy bnh cho thc vt, c s dng nh cc vector t nhin mang cc gen ngoi lai vo m t bo thc vt. A. tumefaciens c cha mt plasmid ln kch thc khong 200 kb gi l Tiplasmid (tumor inducing plasmid) chnh l tc nhn gy bnh cho cy. Khi cy b nhim A.tumefaciens qua cc vt thng, biu hin bnh r nht l cc khi u c hnh thnh ngay ch ly nhim, s hnh thnh khi u sau c th tip tc m khng cn thit phi c s hin din ca vi khun. Kh nng ny c c do A. tumefacien chuyn 1 on DNA ca Ti-plasmid (T-DNA) xm nhp vo h gen ca cy b bnh [2].
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C ch ly nhim ca Agrobacterium rhizogenes i vi cy 2 l mm cng tng t, nhng trong vng T-DNA ca A.rhizogenes ch c gen sn sinh ra auxin, v th s thay i hnh thi chnh ca thc vt l to ra nhiu r t (hairy roots) khi b nhim bnh [2]. T-DNA l on DNA c kch thc 25 Kb trong c cha gen m ho cho sinh tng hp auxin, xytokinin, opin v cc gen gy khi u. Trong Ti- plasmid, v tr ca T-DNA c gii hn bi RB v LB. Ngoi T-DNA, trn Ti- plasmid cn c cc vng DNA m ho cho vic ti sinh plasmid, cho kh nng ly nhim v tip hp (vng vir), cho vic tiu ho opine [2]. Vng vir ph trch kh nng ly nhim, sn phm ca gen nm trong vng vir di tc ng ca cc hp cht phenol tit ra t vt thng l mt lot cc protein c hiu nh virE2, virB, virD, ... cc protein ny nhn bit cc vt thng cc cy ch thch hp, kch thch sn sinh ra cc on T-DNA, bao bc che ch cc on DNA ny v gip chng tip cn vi h gen ca cy ch mt cch an ton [2].

Hnh 1.2 C ch cm ng tn thng r ca Agrobacterium

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Hnh 1.3 Ti- plasmid

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1.3.2. Chuyn gen trc tip

Trong phng php chuyn gen trc tip thnh t bo cellulose kh vng chc l ro cn ngn chn s hp th DNA vo t bo thc vt, v vy tt c cc phng php chuyn gen trc tip u phi gii quyyt vn ny u tin. Thng thng ngi ta c th dng phng php enzyme(x l m thc vt bng enzyme peclinase v cellulose, di tc dng ca 2 enzyme ny thnh t bo thc vt b phn hu to ra protoplast (t bo trn), t bo trn c th hp th DNA trc tip, sau s to iu kin t bo tng hp thnh t bo ri tin hnh ti sinh cy hon chnh [2]. 1.3.2.1. Chuyn gen bng sng bn gen Sng bn gen (Gene gun) l mt thit b s dng a thng tin di truyn vo t bo, c thit k u tin cho bin np DNA ngoi lai vo t bo thc vt v c pht trin vo u thp nin 1980 do cc nh thc vt hc i hc Corrnell cng vi cc nh nghin cu Corrnell Nanofabrication Facility, Newyork, USA.

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Hnh 1.4 Chuyn gen nh vi khun Agrobacterium

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Hnh 1.6 S nguyn l hot ng

Sng bn gen c bn trn th trng vo nm 1990. n s dng cho loi sng ny l cc ht kim loi nng c bn c bao bc DNA. Tn chnh xc v y ca sng bn gen l h thng phn phi ht biolistics (biolistic particle delivery system) v k thut ny thng c gi mt cch n gin l biolistics (s kt hp gia hai thut ng biology (sinh hc) v ballistics (s bn tung)). Mc d c nhiu thit k k thut khc nhau nhng

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nguyn l chung ca phng php ny l s dng p lc xung ca kh helium gia tc cc ht. Sng bn gen bao gm hai bung bng thp khng g, kch thc 6x7x10 ni vi hai bm chn khng. DNA ngoi lai c gn vo cc ht tungsten c ng knh rt nh, khong 1m (cc kim loi nng khc nh vng v bc cng c s dng nhng khng thng xuyn do gi c Hnh 1.5 Shthot ng ca sng bn genci a mt bn trong ca t). Cc ny c t trn mt sng. S bng n kh helium 1000psi lm cho ci a bn v pha trc vi tc 1300 food/s, tng ng vi tc khi mt vin n ri khi nng sng. Mt tm chn lm dng a li v cc ht vng hay tungsten c phng v pha cc t bo ch. Chng xuyn qua vch t bo v phng thch cc phn t DNA (Hnh 1.6). Sng bn gen s dng k thut DNA ti t hp hp nht s biu hin cc gen phn phi. Cc t bo bin i di truyn c th c s dng to thc vt bao gm c s sa i di truyn mong mun trong tt c cc t bo ca chng (Voiland, 1999). Mc tiu ca sng bn gen thng l callus ca cc t bo thc vt ging nhau sinh trng trong mi trng gel trn a petri. Sau khi cc ht tungsten va chm vo a, gel v callus b ph v nhiu. Tuy nhin mt s t bo khng b ph v khi va chm mnh v tip nhn cc ht tungsten c bao bc DNA v cui cng cc phn t khng ngoi lai xm nhp v hp nht bung chn DNA vo nhim sc th thc vt. Cc t bo t a petri c tp hp li v chn lc cc t bo hp nht thnh cng v biu hin DNA ngoi lai bng cc k thut ha sinh hin i nh s dng gen chn lc ni tip v Northern blots.
Hnh 1.8. Sng bn gen cm tay Hnh 1.7. Sng bn gen c

Cc t bo n chn lc t callus c th c x l vi mt s hormone thc vt nh auxin, gibberelin v mi mt t bo c th phn chia, bit ha thnh cc t bo m, c

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Hnh 1.9. Thao tc chuyn gen bng phng thc di bom

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quan, t bo chuyn ha ca ton b cy. Cy mi c ngun gc t mt t bo ny mm thnh cng c th mang cc c tnh di truyn mi. Chuyn gen bng phng thc di bom cng l mt k thut pht trin t sng bn gen ch khc v thit b cn nguyn l th vn tng t nh m t trn. Phng php ny c u im l thao tc d dng, c th chuyn gen vo nhiu loi t bo v m, cc t bo c bin np c t l sng st cao, cho php a cc gen vo t bo v tr mong mun....Do vy n c s dng rng ri trong nhiu lnh vc [1]. 1.3.2.2. Dng ho cht- Hp th DNA trc tip vo t bo trn phng php ny, thnh t bo c loi b bng phng php enzyme, to ra protoplast (t bo trn). T bo trn c th hp th DNA, tng hp thnh t bo sau ti sinh cy hon chnh K thut calcium phosphate K thut calcium phosphate (calcium phosphate technique) c pht trin u tin l xc nh s ly nhim ca DNA virus (Graham,1973) v hin nay c s dng rng ri th nghim hot ng bin np ca DNA virus cng nh DNA tch chit t cc t bo eukaryote (Wigler, 1978; Pellicer, 1980). K thut ny yu cu cc t bo nhn vi cc cht ng kt ta DNA v calcium phosphat (Hnh 1.10). Kt ta ny bm vo t bo v sau s hp th vo t bo qua qu trnh m bo (Loyter, 1982). Trong t bo, cc phn t DNA DNA i n nhn v hp nht vo genome ch Cho n nay, y l k thut v cng c gi tr i vi cc nghin cu chuyn gen vo cc t bo soma nui cy v ang c s dng nhiu chuyn cc dng genome vo t bo ch. T l cc t bo c bin np n nh ca k thut ny l tng ng vi
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Hnh 1.10. Phc hp DNA-calcium phosphat

Graham, 1979;

ngoi lai nm trong khng bo c to thnh do m bo v lysosome th hai nhng rt t

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phng php vi tim nhng khc vi vi tim l nhiu t bo c bin np cng mt ln. Phng php ny c s dng ph bin bi v n gin protocol d thc hin, t tn km, s t bo cht sau bin np khng ng k, s biu hin gen c th l nht thi hoc n nh v quan trng trong vic thit k vector virus ti t hp. Tuy nhin hiu qu bin np v mc biu hin ca gen chuyn thp [3].
Chuyn gen qua liposome

Vo thp nin 1980, liposome nhn to c s dng a DNA vo t bo. Lipid vi ton b li tch in dng pH sinh l l thnh phn lipid tng hp ph bin nht ca liposome c pht trin cho chuyn gen. Thng th lipid cation c trn vi mt lipid trung tnh nh L-dioleoyl phosphatidy l-ethanolamine (DOPE) . Phn cation ca phn t lipid kt hp vi DNA tch in m v kt qu l cha y DNA trong phc hp liposome-DNA (Hnh 1.11). i vi cc t bo nui cy, ton b li tch in dng ca phc hp liposome-DNA ni chung l gy ra hiu qu chuyn gen cao hn bi v n cho php phc hp ny kt hp vi mng t bo tch in m bn hn. Nh c ch nhp bo, cc phc hp xut hin trong endosome v sau i vo nhn. Cha r DNA c phng thch t endosome v i qua mng nhn nh th no. DOPE c xem l mt lipid kch thch s dung hp v vai tr ca n l phng thch cc phc hp ny t endosome cng nh lm cho s dung hp ca mng t bo pha ngoi vi phc hp liposome-DNA xy ra d dng. Trong phng php ny, cc i phn t trc ht c a vo trong cc ti phospholipid. Cc loi ti khc nhau c m t, nhng ti mt lp mng l thch hp nht cho chuyn gen v chng c t l khong trng cha nc bn trong tng i cao i vi mi n v lipid v bi v chng c t l phn phi cao hn. S dung hp ca liposome vi mng plasma l mt s kin
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Hnh 1.11. Phc hp liposome-DNA

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him. Hiu qu bin np ca phng php ny thp hn so vi phng php vi tim vo tin nhn. Cc n lc nghin cu ang c tin hnh tm ra cc iu kin th nghim m c th lm tng s phng thch cc phn t kt nang t con ng m bo. Liposome c s dng a protein, lipid v cc phn t nh vo nhiu loi t bo nui cy, tuy nhin hiu qu thp hn vi tim i vi RNA hoc protein. Cng nh th, chuyn gen qua liposome v s biu hin ca gen chuyn l khng vt qua c cc phng php chuyn gen thng thng (nh h thng virus), s biu hin gen chuyn thng nht thi, s c ch bi cc thnh phn ca huyt thanh c th xy ra. Bn cnh , k thut ny c nhiu u im l gen chuyn s khng hp nht vo genome ch, c hiu qu tt i vi c t bo in vitro v in vivo, c th mang c cc DNA c kch thc rt ln, tinh khit cao, khng gy min dch, c th s dng vi cc t bo m bin np bng k thut calcium phosphat khng c hiu qu [3]. 1.3.2.3 Chuyn DNA trc tip bng xung in K thut xung in (electroporation) l mt phng php c hc c s dng a cc phn t phn cc vo trong t bo ch qua mng t bo. Trong phng php ny, mt xung in cao th trong khonh khc (vi phn nghn giy) c kh nng lm ri lon cu trc mng kp phospholipid (hnh 1.12), to ra cc l thng tm thi cho php cc phn t DNA ngoi lai t mi trng xm nhp vo bn trong t bo. Nhiu k thut nghin cu trong sinh hc phn t yu cu a gen hoc protein ngoi lai vo trong t bo ch. V lp phospholipid kp ca mng sinh cht c mt u a nc pha ngoi v mt u a nc pha trong , nn bt k phn t phn cc no, bao gm c DNA v protein, u khng c kh nng i qua mng mt cch t do. S bn cho thy cc thnh phn ha hc ca mng sinh cht. Cc u a nc phn cc hng v pha ngoi trong khi cc
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Hnh 1.12. S mng phospholipid kp

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ui k nc hng v pha trong v tng tc vi ui k nc khc cng bm gi mng. Cc phn t phn cc khng th i qua mng ny nu nh khng c s h tr bn ngoi.

Hnh 1.13. S b tr mch c bn ca my xung in

Hnh 1.14. :My xung gen (Gene pulser) (Hng Biorad)

K thut xung in da trn trng thi tng i yu ca cc tng tc k nc ca phospholipid kp v kh nng tp hp li mt cch t ng ca n sau khi b ri lon (Purves, 2001). V vy, mt xung in chp nhong c th gy ra ri lon cc v tr ca mng mt cch nht thi, lm cho cc phn t phn cc c th i qua, nhng sau mng c th ng kn li nhanh chng v t bo khng b nh hng g c. Cc t bo ch v DNA ngoi lai c to thnh dch huyn ph v cho vo trong mt cuvette nha c in cc (hnh 1.15) gi l my xung gen (gene pulser). (hnh 1.14) Qu trnh c bn din ra bn trong my ny c th c trnh by bng s (hnh 1.13)
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Hnh 1.15. Cuvette nha c in cc

to ra xung in cao th trong mt thi gian ngn ngi ta s dng mt thit b

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S ny cho thy mch in c bn cung cp in cho k thut xung in. Khi cng tc th nht ng, t in np in vo v tch mt in p cao. Khi cng tc th hai ng, in p ny phng qua dch huyn ph t bo. Mt xung in cn thit cho k thut ny thng l khong 10.000-100.000 v/cm (thay i ty theo kch thc ca t bo) trong vi phn triu giy n mt phn ngn giy. Xung in ny lm ri lon phospholipid kp ca mng t bo v to ra cc l tm thi. Kh nng in qua mng t bo cng lc tng ln 0,5-1,0 v v vy cc phn t c np in ny i qua mng t bo thng qua cc l bng cch thc tng t nh in di (Hnh 1.16). Li DNA i vo t bo khng th quan st thy di knh hin vi, nhng hnh v ny cho thy khi nim c bn ca s to thnh cc l trn mng m DNA c th i qua. Khi cc ion np in v cc phn t i qua cc l, mng t bo phng in v cc trn mng bo cht l ny ng li mt cch nhanh chng v phospolipid kp phc hi li cu trc c (Weaver, 1995). Lc ny cc phn t mong mun trong t bo v chng c s dng cho cc nghin cu tip theo. Phng php ny c th s dng i vi gn nh tt c cc loi t bo ca cc loi. Lc u phng php ny c s dng chuyn gen vo cc t bo ng vt c v, v sau cho c t bo thc vt dng protoplast... Vi mt s cy mt l mm quan trng (loi la ph Japonica, ng, la m) m khng th thc hin c bng phng php chuyn gen gin tip nh Agrobacterium th ngi ta thnh cng vi phng php ny. Hiu qu bin np cao. Trong mt nghin cu E.coli, 80% s t bo nhn c DNA ngoi lai (Miller v Nickoloff, 1995). Lng DNA ngoi lai cn thit l t hn so vi cc phng php khc (Withers, 1995). Phng php ny c th thc hin vi cc m in vivo cn nguyn vn (Weaver, 1995). on DNA ngoi lai c bin np c kch thc ln.
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Hnh 1.16. S plasmid cha DNA ngoi lai i qua cc l tm thi

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Tuy nhin nu cc xung in c cng v chiu di khng ng th mt s l ca t bo s tr nn qu ln hoc b hng khng th ng li sau khi t bo phng in, lm cho t bo b tn thng hoc b thng (Weaver, 1995). Mt hn ch na l s vn chuyn DNA ngoi lai vo v ra khi t bo trong sut thi gian in bin np l tng i khng c hiu. iu ny dn n kt qu l khng cn bng ion m sau s lm ri lon chc nng ca t bo v t bo cht (Weaver, 1995). K thut xung in c s dng rng ri trong nhiu lnh vc khc nhau ca sinh hc phn t v y hc. Cc ng dng ca k thut xung in bao gm Bin np DNA: cc gen c hiu c th c to dng trong plamid v sau plasmid ny c a vo t bo ch nghin cu cu trc v chc nng ca gen v protein. Dung hp t bo kch thch s to thnh cc l thng trn mng xy ra do xung in chp nhong to ra cho thy kch thch s dung hp t bo (Weber v Berrg, 1995) [3]. 1.3.2.4 Chuyn gen DNA trc tip bng vi tim T bo c gi trong mt ng thu tinh bng 1 lc ht yu v DNA c tim trc tip vo nhn t bo qua pipet thu tinh rt nh. Qu trnh vi tim cn s h tr ca knh hin vi v thit b vi thao tc nn i hi thit b c chnh xc cao, k thut phc tp

Vi tim (microinjection). K thut avo tin vo nhn hoc vo t bo cht ca t Hnh 1.17. Vi tim gen ngoi lai DNA nhn ca trng th tinh bo bng kim mao dn v bm p lc. Ton b thao tc c tin hnh trn knh hin vi ngc pha (inverted microscope) [14].

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Hnh 1.18. M hnh vi tim gen ngoi lai vo t bo

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1.4 . Phng php to Vaccine thc phm

Bng cc k thut di truyn phn t khc nhau ngi ta s a gen sn xut khng nguyn ca mm bnh vo t bo thc vt. Kim tra biu hin gen nh cc gen ch th, nu chuyn gen thnh cng s tin hnh nhn dng bng cc k thut nui cy m nh to m so, nhn nhanh, ti sinh cy hon chnhTu theo tng tnh cht cy, khng nguyn to ra u th b phn, giai on pht trin ca i tng iu khin s sinh trng v pht trin thch hp nht, to ra sn phm nng sut v cht lng cao nht. Tuy nhin, a gen m ho protein khng nguyn vo thc vt hin nay ta thng dng phng php chuyn gen gin tip nh Agrobacterium, v cc bo co khoa hc thnh cng trong ti ny hu ht l s dng vi khun Agrobacterium, di y l mt s cng trnh nghin cu vaccine thc phm ca mt s nh khoa hc t cc quc gia khc nhau trn th gii 1.4.1. Mt s quy trnh sn xut vaccine thc phm trn th gii 1.4.1.1. Vaccine vim gan B . Tinh. Vim gan B (HBV) l mt trong nhng cn bnh ph bin v ly lan nhanh hn c HIV. Hng nm c khong 200 triu ngi b vim gan B. Vit Nam, t l ngi lnh mang trng kh cao t 15-25%. Virut vim gan B thuc nhm virut Hepadna, l tc nhn gy vim gan virut B [19]. Hin nay c vaccine vim gan B nhc c, kch thch min dch qua ng tim, tuy nhin gi thnh hi cao v phi tim nhc li nhiu ln tu theo loi vaccine.
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y l mt bnh ph bin trn th gii nht l khu vc chu , chu Phi v M La

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S gii m ton b h gen ca virut vim gan B xc nh c ni khu tr ca cc gen m ho nhng khng nguyn khc nhau ca virut, trong s c khng nguyn HbsAg l khng nguyn quan trng nht hnh thnh v bao ngoi ca virut v c s dng lm vaccine to khng th chng li s xm nhim ca virut vim gan B. Nu tch chit v tinh sch ngun khng nguyn b mt virut vim gan B trong huyt thanh ngi b ly nhim th rt gii hn v s lng v gi thnh cao. Nu khng tinh sch th vic dng ngun khng nguyn ny s dn ti s ly nhim cc loi bnh khc c th pha tp trong ch phm nh HIV chng hn [10]. Khng nguyn b mt gy vim gan B ti t hp (recombinant hepatitis B surface antigen_rHBsAg) l vaccine tiu phn u tin c chp nhn v ng dng(1986). Khi c khng nguyn l xm nhp cc t bo limpho B s bit ho thnh cc tng bo(plasma cells) v sn xut khng th c hiu khng li khng nguyn. Do mi khng nguyn thng mang nhiu yu t quyt nh tnh khng nguyn (epitope) nn s c nhiu loi khng th to ra l khng th a dng [6]. Vaccine vim gan B trong khoai ty Thng 7 nm 2005, Yasmin Thanavala v cng s tin hnh th nghim vaccine vim gan B sn xut trong cy khoai ty bin i gen, b khng nh kh nng min dch ca vaccine thc phm- vaccine vim gan B trong khoai ty l c ngha thc tin v c th b sung vo chin dch phng chng vim gan B ton cu. Trong nghin cu ny gen sn xut khng nguyn HbsAg t dng gy bnh pMTSA trong c th 1 ngi mang bnh vim gan B, c chn vo vector plasmid pHB114, sau pHB114 c chuyn vo Agrobacterium tumefaciens (LBA 4401). Vi khun cha gen chuyn HbsAg s chuyn gen tng hp HbsAg vo cy khoai ty FL1607, loi vi khun khi mu, kim tra gen chuyn, nhn dng nhng t bo cha gen tng hp HbsAg, la chn nhng dng c mc sinh khng nguyn cao k hiu FL 1607 HB114-16, ti sinh cy nhn ln vi s lng ln, a ra nh trng, trng song song cc cy khoai ty bnh thng i chng.

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Trong th nghim ny, nhng tnh nguyn vin c cho n s liu vaccine khc nhau, mi liu l 100g c khoai ty sng chuyn gen (cha 8,5 % protein khng nguyn ca virut vim gan B (HBV). Kt qu thu c l s ngi n 3 liu vaccine th c 62,5% ngi sinh huyt thanh khng virut HBV, nhng ngi dng 2 liu th s liu ny l 52,9%, cn nhng ngi n khoai ty bnh thng th khng ai sinh khng th chng HBV [11]. mt th nghim mi y trn 42 ngi tnh nguyn ca gio s Artzen v cng s thc hin, khong 60% c th tnh nguyn vin cho thy c nhng du hiu chng li virus vim gan B, sau khi n nhng ming khoai ty sng bin i di truyn, c gt sch v (do v c cht gy cho cc tnh nguyn vin c triu chng bun nn v c mi kh chu). Cc vaccin HBV truyn thng, c mt t sut min nhim cao n 90%. Nhng Artzen v ng nghip vn hy vng s tng thm phn ng min nhim v s nng cp k thut c vaccine tt nht. H lm ng lnh kh (freeze dried) tng lt khoai ty chuyn gen v tn nh chng thnh bt, to ra mt cht trch m hiu lc c th o lng c ri ng gi thnh nhng vin nang. Dng vin nang s gip iu chnh lng nng khng nguyn ng nht t nghin cu lng vaccine thng nht trong mi liu v ngn nga cc di gen n c (edible transenic) khng may th phn cho vi cc cy ci xung quanh, c th ln vo cc cy thc phm. Arntzen ni: y l mt sch lc mi sn xut v phn phi vaccin n nhng ni xa xi, ho lnh [13]. Vacccine vim gan B rau dip v cy u lupin Nghin cu ca J. KAPUSTA v cng s thuc hc vin sinh ha t nhin Balan v vaccine thc phm trong cy rau dip v cy u lupin c ng trn tp ch FASAB vol 13 ra thng 10 nm 1999 [9], vi cc bc tin hnh nh sau
1.

Nhn dng DNA Dng plastmid pROK2S lm vector mang trnh t gen m ho HbsAg (trnh t gen m ho protein khng nguyn b mt c tch t virut HBV-pHB614). Plastmid pROK2S (hnh 1.19) c chuyn vo A. tumefaciens C58 v LBA4404

2.

Chuyn gen vo cy
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Hnh1.19. Vector mang trnh t gen m ho HbsAg nm trn plasmid pROK2S

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Ht u Lupinnus vng 4 ngy tui c s dng nh mu ban u . Sau khi ct b nhng phn xa chi, nhng l mm c dng gy nhim bi A. tumefaciens C58 (pROK2S). Sau 2 ngy nui cy trn mi trng MS ci bin (T. Pniewski, J. Kapusta,and A. B. Legocki, Kt qu khng c cng b) c khng sinh t do (antibiotic-free), mu c chuyn sang mi trng c bit pht sinh callus. Dng gen chuyn c chuyn sang mi trng chn lc c b sung khng sinh kanamicin v carbenicillin (T. Pniewski, J. Kapusta, and A. B. Legocki, khng cng b kt qu), mu chu c khng sinh s c tch ra dng tip tc th nghim. Pht trin callus. L mm ca rau dip gieo bng ht c gy nhim vi A. tumefaciens LBA4404 (pROK2S) vi cc bc nh i vi u lupin .
3.

Tch chit v phn tch Protein M nui cy th nghim trn c em li tm vi tc 30000 vng trong 15 pht loi b nhng mnh vn t bo khng ng nht. Khng nguyn HbsAg trong dch ni c nh lng bng kit chn on n dng Auszyme (Auszyme monoclonal diagnostic kit -Abbott Lab., North Chicago, Ill.).

4.

Xc nh hm lng khng nguyn Hm lng khng nguyn tch c xc nh da trn ng chun vi cc nng khc nhau ca HbsAg tinh khit. Bng k thut Elisa s hin din ca khng th c hiu vi khng nguyn HbsAg c kim tra trong huyt thanh ca chut v ngi.

5.

Th nghim trn ngi Tin hnh th nghim trn ngi cho thy c th nhng ngi sau khi n sn phm t callus u lupin hoc l rau dip cho min dch vi HBV. 1.4.1.2. Vaccine si Trong tp ch MJA vol 176 thng 6 nm 2002, Diane E Webster gio s ca trng H dc Monash v cng s a ra m hnh to vaccine si trong thc vt chuyn gen s dng Agrobacterium tumefaciens [8]. Gen tng hp protein b mt v virut si H (gi l

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MV-H: measles virus- hemagglutinin)s c chuyn vo cy nh Agrobacterium tumefaciens. Quy trnh c m hnh ho trong s di (hnh 1.20). Loi cy trng c ng dng lm th nghim thnh cng l: Thuc l, khoai ty v rau dip, c 3 i tng trn u kch thch c th p ng min dch vi virut si, tuy nhin vn cha c i tng no c a chnh thc vo thc tin vi nhiu l do. i vi cy thuc l, y l i tng chuyn gen tt, tuy nhin n li sinh ra nhng hp cht c, lm gim tc dng ca vaccine. Rau dip v khoai ty l hai i tng cy ngn ngy c kim chng ph hp vi vic to vaccine, mt i tng khc c chn na l la, thng c s dng trong thc n ca tr nh bi t gy d ng. Mt vi th nghim gn y chng minh rng protein ng vt c th biu hin cao trong go chuyn gen, mt khc, la go th d bo qun, vn chuyn, v protein biu hin trong ht go n nh nhit thng, bt go c th trn trong thc n ca tr, nc hoc sa. Theo Diane E Webster quy trnh sn xut vaccine ny ko di trong vng t 3- 9 thng tu theo i tng

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Hnh 1.20 . Quy trnh sn xut v th nghim vaccine si

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1.4.1.3. Vaccine dch t Trong tp ch PNAS ngy 26 thng 6 nm 2007, cng b nghin cu to vaccine dch t t ht go ca gio s Tomonori Nochi v cng s [11], vi cc bc sau: 1. Chn i tng Hai ging la c chn ban u l Kitaake- ging la thng v Hosetsu ging la di ln do t bin t nhin( tng hp gibberelin thp, chiu cao 20cm v c chu k sng ngn ch 3 thng). (hnh 1.21 )

Hnh 1.21. Chon ging lua Kitaake va Hosetsu biu hin gene CTB

2. Chuyn gen Gen CTB c chn vo vector pGPTV-35S-HPT, v s dng promoter biu hin gen ti u ni nh glutelin GluB-1 (hnh 1.22). Vector c chuyn vo cy la nh vi khun Agrobacterium tumefaciens.

Hnh 1.22. Gene CTB c bin i cho phu hp v c chen vao binary vector (pGPTV-35S-HPT)

3. Phn tch kt qu chuyn gen

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Hnh 1.23. Dung ki thut PCR xac nhn s sat nhp gene CTB vao DNA

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Dng k thut PCR chng minh gen CTB c chuyn thnh cng vo c hai loi la Kitaake va Hosetsu (hnh 1.23 ) WT: Wild-type lua khng chuyn gene Tg: lua chuyn gene CTB

Dung phng phap Northern blot xac nhn s biu hin cua mRNA oan ma CTB trong 2 loi la c chuyn gen (tg) v so snh vi loi la khng chuyn gen( wt) (hnh 1.24)

Dung phng phap Western blot vi gel SDS/PAGE cho thy nng cao protein CTB c biu hin (12 15 kDa) (hnh 1.25) .( Hnh u mi tn )

Hnh 1.24. Dung phng phap Northern blot xac nhn s biu hin cua mRNA oan ma CTB

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Hnh 1.25. Dung phng phap Western blot vi gel SDS/PAGE cho thy nng cao protein CTB c biu hin (12 15 kDa)

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4. Kim tra s bn vng ca khng nguyn trong ht la Ni ca ht la cha 2 loi protein c trng l PB-I v PB-II. Phn tch bng knh hin vi in t cho thy CTB kt hp vi c hai loi protein ny (hnh 1.26)

Hnh 1.26 CTB c lu tr trn b mt PB-I (u mi tn) v PB-II (mi tn).

*X l vi pepsin: Ht go dc mang x la vi pepsin, Dng k thut Western blot cho thy 90% glutelins (1 loi protein khc l thnh phn ch yu trong go) bi tiu hoa bi pepsin trong khi 75% CTB trong gao khng bi anh hng bi pepsin. iu ny chng t CTB biu hin trong ht go bn vng vi iu kin mi trng trong d dy- rut.

1.5

. Thnh tu trn th gii v trong nc

1.5.1. Th gii 1.5.1. . Cy la bin i gen to vaccine chng li giun sn Cc nh khoa hc thuc H Tokyo, H Gifu v NIAS (Japanese National Institute of Agrobiological Sciences) pht trin thnh cng ging la bin i gen tch t vaccine chng li giun sn cc mc rt c ngha. Cy la chuyn gen ny biu hin As16 mt antigen bo v chng li k sinh ca giun Ascaris suum- phi hp vi c t ca dch t thuc toxin B subunit (CTB). Con giun Ascaris l tuyn trng k sinh trong rut, d dy ngi v ng vt, bnh k sinh ny lan rng ton th gii. c t dch t c s
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dng nh mt cht ph tr c tnh nht kch hot phn ng min dch xy ra. Cc nh khoa hc ny ghi nhn mc th hin protein dung hp dng khm trong ni nh ht thc, vi gi tr trung bnh l 50 g/g ht. Cy la GM nh vy c th trn chut vi trng ca tuyn trng; chut n thc GM c trng tuyn trng xut hin phi thp hn chut i chng. H ghi nhn rng y l nghin cu u tin chng minh rng vaccine n c th hin trong cy la gip loi ngi chng li mt dng k sinh trong mt loi ng vt dng lm m hnh l chut [15] .

1.5.1.2.

Sn xut khng nguyn HIV t cy chuyn gen Vic pht trin v sn xut mt loi vc xin ph hp l mc tiu hng ti vic ngn chn s ly lan ca bnh AIDS. Mc d c trn 20 nm nghin cu nhng ngi ta vn cha pht hin ra vc xin c hiu qu trong vic chng li bnh ny. Cc nh khoa hc cho rng vc xin l tng chng li virut gy triu chng suy gim min dch ngi (HIV) s c hiu qu trong vic ngn chn vic lan truyn vi rt cng vi vic chi ph sn xut r cho php s dng rng ri cc nc ang pht trin. Cc nh nghin cu t Vin Max Planck v sinh hc phn t thc vt ti c v i hc Cambridge ti Anh quc khai thc kh nng cy transplastomic sn xut khng nguyn HIV nh l hp phn ca AIDS vaccine. H cho th hin thnh cng HIV antigens p24, l mc tiu chnh ca T-cell-phn ng min dch vi c th dng tnh HIV, v Nef trong plastids ca thuc l v khoai ty. Gene cassettes dung hp mt cch ti ho p24-Nef kch hot s tch t protein khng nguyn khong 40% protein trong l. l kt qu gp 100 ln cao hn nhng kt qu trc y sn xut p24 bng chuyn np gen vo nhn truyn thng v chng minh c tim nng to ln ca chuyn gen vo plastid sn xut protein lm dc phm trong thc vt. Cc nh khoa hc gi nh rng kiu hnh ca cy chuyn np gen th hin trong chloroplast nh tn dng c cc th rib ca n sn xut ra protein ti t hp [17].

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1.5.1.3
. Chuyn gien sn xut vc xin chng bnh u ma

Mt nhm cc nh khoa hc thuc Cc phng th nghim c bn v CNSH, i hc Thomas jefferson, Philadelphia, M, sn xut thnh cng mt loi vc xin chng bnh u ma t cy thuc l chuyn gien, vc xin ny c chng minh trn chut v ln con. Bnh u ma do virut Variola gy ra, l mt bnh duy nht d ly nhim m c th gy t vong cho con ngi. Nhng kt qu ny cho thy tnh kh thi trong vic sn xut cc vcxin siu phn t an ton v tit kim nh s dng cc h thng sn xut thc vt. Li th ca sn xut vc xin t thc vt bao gm chi ph sn xut gim; quy m sn xut ln; sn phm an ton; kh nng qun l qua ng ming [16]. 1.5.1.4. Thuc l chuyn gen c cha vc-xin chng dch hch Nguyn nhn gy bnh dch hch l loi vi khun c tn Yersinia pestis, hin nay bnh dch ny vn c nguy c xy ra 1 s vng chu Phi, chu , chu M v Lin X c, c bit l nhng ni con ngi sng gn vi cc loi gm nhm. Y. pestis nguy him nht khi b ht vo trong phi, v n c th ph hy phi ca ngi bnh, dn n ci cht. Khng sinh c th c s dng cha bnh dch hch, nhng cc bin php cha tr ch c hiu qu nu bnh c pht hin sm. Mt vi chng Y. pestis c th khng thuc khng sinh, nn cc nh khoa hc phi tm kim cc bin php sn xut hng lot cc loi vc-xin mi. Luca Santi v Hugh S. Mason khm ph ra loi vc-xin phng bnh dch hch trong l cy thuc l. Trong mt nghin cu gn y, cc nh khoa hc phn tch s biu th ca 2 prtin ca Y.pestis trn cy trng: khng nguyn F1, to thnh 1 phn ca v bao ngoi t bo Y.pestis, khng nguyn V, c tham gia vo qu trnh gy bnh, v hn hp ca F1 v V. Cc gen ca 2 khng nguyn ny c a vo t bo cy thuc l bng khun Agrobacterium tumefaciens. Sau cc prtin c to thnh c phn tch tnh khng nguyn v th nghim trn chut bch. Cc nh nghin cu thy rng: 1) c 3 loi khng nguyn trn u biu l mc cao trn l cy thuc l; 2) c 3 loi prtin u to ra phn ng min dch chut th nghim; 3) sau khi xt liu Y pestsis c cha 100% c t vo chut, nhng con chut c s dng

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vc-xin c t l sng rt cao sau 21 ngy, trong khi nhng con chut gi min dch (shamimmunized) u cht sau 6 ngy [18]. 1.5.1.5. Vacxin gip ngn nga ung th Cc nh khoa hc ang pht trin cc loi chui v khoai ty cha vcxin chng virut papilloma (HPV) gy ra cc cn bnh ly nhim qua tnh dc v ng thi l nguyn nhn ch yu ca cc ca ung th c t cung ph n. Cc nh nghin cu i hc Rochester ang tin hnh th nghim cc ging mi c cha vcxin v hin nay ang trong giai on th nghim lm sng. Bnh do HPV l nguyn nhn ph bin gy ung th ph n ti cc nc ang pht trin, chim ti hn 80%. Pht trin loi vcxin phng nga cn bnh ny c gi tr to ln. Cc nh khoa hc ang tin hnh cho chut n khoai ty t cng ngh sinh hc v hy vng chng s c cc phn ng min dch t bo v. 1.5.2. Tnh hnh trong nc Hin nay, Vit Nam sn xut c 9/10 vaccine phc v chng trnh tim chng m rng v c UNICEF cng nhn l quc gia th hai thanh ton xong bnh bi lit. Nhng nc ta, vaccine n c vn l vn kh mi m v cha c ti liu no cp su v loi vaccine ny. Cc nghin cu vaccine thc phm nc ta ch yu do vin CNSH thc hin, cng ang dn c nhng kt qu kh thi. Vin cng ngh sinh hc ang ch tr thc hin ti trng im ca chng trnh nghin cu khoa hc c bn vi ni dung chuyn cc gen gy bnh ca virus di vo i tng cy lc. Vin Di Truyn Nng Nghip xut v trin khai thc hin ti sn xut vaccine n c c ngun gc thc vt Wolffia (bo tm) thng qua Agrobacterium. Nhiu nghin cu v c tnh sinh hc ca bo tm cho thy y l thc vt c tc sinh trng nhanh theo kiu ny chi, c bit n c hm lng dinh dng cao, l loi thc n rt tt cho gia sc v c th lm thc n ti cho ngi mt s nc trn th gii. bo tm mang y c im cn thit ca h thng sn xut protein hiu qu cao v l i tng l tng cho nghin cu vaccine n c.
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To vaccine vim gan B n c t tri c chua Cc nh khoa hc thuc Phng Cng ngh Gen - Vin Sinh hc Nhit i TP.HCM hin ang tin hnh nghin cu vaccine nga vim gan B n c t cc b phn n ti nh qu, l, thn, c... Trong c cy c chua. Nc ta l vng lu hnh cao ca bnh vim gan do siu vi B (HBV). T l ngi mang mm bnh vo khong 10 - 15% dn s (trn di 10 triu ngi). Ngoi ra trn th gii nhiu cng b khoa hc dng cng ngh chuyn gien m ha protein HBsAg vo nhn v lc lp t bo, vo mt s cy trng, ... Qua cc kt qu trn, nhm nghin cu ca Phng Cng ngh Gen - Vin Sinh hc Nhit i nhn thy, protein khng nguyn HBsAg s dng qua ng tiu ha c kh nng to min dch tt. iu ny m ra mt trin vng nghin cu chuyn np gien ny vo cc cy trng c b phn n ti nh qu, l, thn, c... Bng phng php chuyn gien bng vi khun Agrobacterium tumefaciens, kt qu nghin cu ca Vin Sinh hc Nhit i nhn c mt s loi cy trong c cy c chua mang gien m ho khng nguyn b mt virus vim gan B. (http://sokhoahoccn.angiang.gov.vn/xemnoidung.asp?)

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Phn 2 KT QU NGHIN CU
2.1 . u im ca Vaccine thc phm
Vaccine thc phm c mt s u im sau: gi thnh r, n nh, d sn xut trn quy m ln, d qun l, khng cn tinh sch, bo qun lu, d vn chuyn

Cy chuyn gen mang gen sn xut khng nguyn cng ging nh nhng cy

trng khc nn d dng tng qui m sn xut v d thu sinh khi, do vy gi thnh ca loi vaccine ny chc chn se thp hon rt nhiu ln so vi cc loi vaccine khc.

n nh, d bo qun v s dng: Thc vt t trc n nay c dng Vaccine thc phm c to ra trong c th nhng cy thc phm(ng cc)

nhiu trong y dc, v vy n d chp nhn i vi ngi tiu dng. thng thng nn c th trng v tiu th ti a phng m khng cn vn chuyn xa, cng khng cn c thit b bo qun lnh, khng cn kim tim nn rt d s dng v phn pht. Cc khng nguyn biu hin trong thc vt n nh ngay nhit phng do chng c sn xut v c bao bc bi cc m thc vt m c th l chng c nh v trong li ni cht, th Golgi hoc b mt t bo. Nh tnh n nh ny m chng tr nn d dng bo qun v s dng (ngay trong thc vt) m khng cn gi lnh nh cc vaccine tim. Trong qu trnh sn xut vaccine n c ngi ta ch cn vn chuyn v s dng ngay b phn thc vt cha vaccine hoc ch phm thc phm cha vaccine .
c phn phi qua ng ming (delivered orally)

Nhiu nghin cu th nghim gn y cho thy vaccine trong thc vt c th n c v pht huy tc dng trong c th ngi. Do chng uc bc trong m thc vt, s c mi v v tnh cht hu nh ging hon ton vi m thc vt cha n (ngoi tr khoai ty bin i gen vn c mi v kh chu khi n sng khng gt v), iu ny hn ch c s phn hu ca dch tiu ho cho n khi khng nguyn gp th th ca h min dch c th nhn din ra chng. An ton:
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V vaccine c sn xut trong thc vt l vaccine di n v s dng gen m ho cho mt phn protein v virus m khng cn n virus sng nh vaccine gim c lc hay virus cht nh vaccine bt hot. Do vaccine ny khng tr li thnh virus gy bnh cho ngi v ng vt, ng thi n cng trnh c nguy c nhim mm bnh tim tng t vaccine. Do , khng cn tch chit v tinh sch khng nguyn vaccine. Vaccine n c kch thch sn xut khng th ca h thng th dch hiu qu hn vaccine tim Ta bit rng hu ht cc vi sinh vt gy bnh u xm nhp vo c th qua b mt nhy trong ng tiu ho, h hp v ng tit niu. Khi vaccine n vo c th theo ng ming n s cm ng h thng min dch th dch sn xut cc khng th chng li vi sinh vt gy bnh, tip h thng th dch li tc ng vo h thng min dch ca t bo, to ra cc globulin min dch tng cng kh nng bo v sm v hiu qu cho c th. Khi tiu ho vaccine n c, khng nguyn c gii phng trong rut non nh vy cc th th min dch nim mc rut s nhn ra chng v kt hp d dng, trit nht v nh vy l to ra phn ng min dch. Nhng nghin cu bo v khng nguyn lm vaccine n c trc tc ng ca dch tiu ho, c bit ca c th con ngi khng nh gi tr thc tin ca vaccine n c sn xut nh thc vt chuyn gen.

2.2 . Nhc im ca vaccine thc phm

Thc vt l sinh vt tn ti hng ngn nm qua nhiu bin i chn lc t nhin, h gen ca cy l s hon thin dn ca sinh vt qua chn lc t nhin, vy th khi b bin i gen s khng chc chn c iu g. Vaccine thc phm khng khc g nhng loi tri cy thng thng, v vy vic s Liu dng vaccine c th thay i. V d, nhng qu chui kch c khc nhau s iu ng lo ngi nht l cy trng cha vaccine c th ln, nhim vi cy thc phm,. l mt vn quan tm ln, phi cn thn i vi vic s dng thc vt chuyn
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dng qu liu cho php do s sut rt d xy ra. cha lng vaccine khc nhau.

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gen lm vaccine v lm sao hn ch c ngn chn kh nng ln tp ca chng vo cc v ma thc phm bnh thng. Khi khng th bit c lng vaccine thay i trong thc vt th khng th n ba c, hay nh khng th dng vaccine khoai ty khi khng bit c lng protein khng nguyn tch lu trong , y cng l mt vn nan gii. Tuy nhin i vi mt s loi cy nht nh nh nghin cu ngh ra 1 cch l c th thit k chng thnh nhng vin thuc hay nhng ming bnh vi lng chnh xc protein khng nguyn trong .

Phn 3 KT LUN
Vic sn xut cc cy trng chuyn gen ni chung v vaccine thc phm ni ring ang tin trin. Tuy nhin, hng ti cc li ch tim tng ca cng ngh sinh hc thc vt chuyn gen cng nh sn phm dc phm t cy trng chuyn gen c nhiu nhn t
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b sung cn xem xt bao gm: s kim sot cng ngh sinh hc, s hu tr tu, an ton thc phm, s chp nhn ca cng ng, tnh cht gy d ng, nhn hiu, s chn la, mi trng, s phn bit ca cc sn phm chuyn gen v mu dch quc t. Tm quan trng y l vic ng dng v cc li ch tim tng ca cc k thut chuyn gen. Trong khi mi k thut ang pht trin, th mc ch cn t c l ti a cc li ch v gim thiu cc ri ro. y cng l mt phn ro cn m phn ln cc loi vaccine thc phm hin nay rt kh mang ra s dng ph bin. mc d vaccine thc phm l mt la chn ti u so vi cc loi vaccine c in. Mt ngy no khi nhc n 2 t tim chng nhng tr em s khng cn phi s hi bi nhng mi tim ca vaccine tim hay nhn mt bi nhng liu vaccine ung nng mi kh chu na, ngc li s hoan h c pht cho nhng loi tri cy ngon lnh li c tc dng chng nga cc bnh truyn nhim. Vin cnh ti p s khng xa v cc nghin cu v vaccine thc phm( hay cn gi l vaccine n) ngy cng nhiu, v ang mang li nhng thnh tu to ln. Cng trnh nghin cu biu hin thnh cng vaccine thc phm u tin (protein b mt ca vi khun Streptococcus trong cy thuc l vi 0.02 % protein tng s ca l vo nm 1990. Sau l hng lot cc bo co v khng nguyn thc vt c cng b khng nh s hp dn ca ch ny:(bng 3.1) Vit Nam chng ta th cm t Vaccine thc phm vn cn mi m, ch cha k n vic ng dng n mt cch i tr trong thc tin, tuy nhin nc ta cng nh nhiu nc ngho khc li l mt th trng tim nng cho dng sn phm ny. L mt nc ngho Vit nam cng nh nhng quc gia ang pht trin khc, lun ng trc nhng dch bnh ph bin, tuy c nhng chng trnh tim chng ln cho tr em chng nhng bnh dch thng thng, nhng s lng ngi cht v cha c tim chng nga nhng bnh thng thng cng khng nh, mt phn do dn tr cha cao, nhiu ngi cha hiu c li ch ca vic tim chng nn cn ch quan, mt khc vic bo qun, vn chuyn cc loi vaccine thng thng cn iu kin nghim ngt m trang thit b

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ca ta cn thiu thn nn lng vaccine khi n vi nhng ni vng su vng xa th gim hot lc t nhiu, thm ch h hng, mt tc dng. Do vy s lng tr em t vong do cc bnh dch thng thng c th trnh c Vit Nam ni ring v th gii ni chung l rt ln. Di y l mt s con s thng k au xt khng ng c: WHO) 200-400 triu ngi nhim vim gan B, 1-2 triu ngi cht hng nm do HBV 800000 ngi cht mi nm do bnh si Nhng con s trn vn c th tip tc tng nu vic s dng vaccine khng c ci tin trong vic bo qun v phn phi, bi iu ny c quan h mt thit vi vic chng nga nhng nc ang pht trin. Vn ny s c gii quyt d dng vi vaccine thc phm, vaccine s c sn xut v tiu th ti chnh nhng vng c dch bnh. Theo Humphreys v cs vi 1 mu rung nh trng ng cc c th sn xut 1kg khng nguyn v c th bo qun nhit thng trong 5 nm. Do vy, Vaccine thc phm l mt gii php ti u, l hng pht trin y trin vng trong tng lai, mang li li ch thit thc.
Bng 3.1 : Cc khng nguyn vaccine biu hin cc h thng thc vt (tregoining et al., 2004)[5]

Hng nm trn th gii c khong 10 triu tr em cht v dch bnh hn 2 triu tr em t vong v dch t(tiu chy) mi nm( theo t chc y t th gii

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TI LIU THAM KHO

Ti liu ting Vit
1. L Trn Bnh, Cao huyn Trang (2005),Nghin cu v pht trin vaccine n c

trong thc vt, Tp ch cng ngh sinh hc 3(2):133-142

2. L Vn Hong, Cng ngh nui cy m v t bo thc vt (2007), NXB Khoa hc

v k thut.
3. Nguyn Hong Lc(2007), Nhp mn cng ngh sinh hc, i hc Hu, tr 136-137.

tr 353
4.

L nh Lng, Quyn nh Thi (2003), K thut di truyn v ng dng, NXB Nguyn c Lng(2006), Sinh hc i cng, tp 2 , NXB HQG tp H Ch Minh

HQG H Ni
5.

6. Phan Kim Ngc v Phm Vn Phc (2007),CNSH trn ngi v ng vt, NXB
Gio Dc, tr 643.
7.

V vn V, Nguyn Mng Hng, L Hng ip,(2005), Cng ngh sinh hc, tp2,

NXB Gio Dc.

Ti liu ting Anh

8. Diane E Webster v cs (2002), Appetising solutions: an edible vaccine for

measles, MJA vol 176, p 434-437

9.

KAPUSTA v cs (1999), A plant- derived edible vaccine against hepatitis B Neeraj Mishra v cs (2008), Edible vaccines: A new approach to oral

virut, The FASEB Journal, Vol. 13 October 1999, p 1796-1799

10.

immunization, Indian Journal of Biotechnology, Vol 7, July 2008, pp 283-294

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11.

Tomonori Nochi v cs (2007), Rice-based mucosal vaccine as a global

strategy for cold-chain- and needle-free vaccination, PNAS vol 104(26), p10986 10991

12.

Yasmin Thanavala v cs (2005), Immunogenicity in humans of an edible

vaccine for hepatitis B, PNAS vol. 102 _ no. 9, p3378-3382

TI LIU INTERNET
13. 14. 15. http://dhgpharma.com.vn/dhgpharma/index.php?view=news&ID=306

http://elearning.hueuni.edu.vn/file.php/27/downgtcnchuyengen/c2.pdf.
http://tinkhoahoc.blogspot.com/2008/11/cy-la-bin-i-gen-to-vaccine-chng-li-

giun.html

16. http://www.agbiotech.vn/vn/?mnu=preview&key=1554
17. http://www.agbiotech.vn/vn/?mnu=preview&key=2682

18.

http://www.isb.vt.edu/news/2006/news06.apr.htm

19. http://www.pasteur-hcm.org.vn/index.jsp

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