cong nghe vacxin - tieu luan cnst

Tiu lun Cng ngh sinh hoc

GVHD: PGS-TS. Nguyn Ba Lc

M UTim chng l mt trong nhng trang b ti cn thit mt a tr ln ln an ton v kho mnh, tuy nhin bt k mt a tr no, (v ngay nhng tr ln) u rt s ch cn ngh n vic phai tim thuc. V ng tim thng gy cam giac au cho ngi s dng, cng thm s lng virt gy bnh nguy him ngy cng nhiu ko theo s lng mi tim cng tng ln v mi ln tim vo mi ch khc nhau ca c th . Cha k n vic bo qun, vn chuyn vi iu kin nghim ngt i vi cc Vc-xin. Gii quyt ht tt c nhng vn ny cc nh khoa hc ca chng ta to ra mt ci gi l Vc-xin n. No co th dung thay th cho vic tim nhng hiu qua min dich vn c am bao. nc ta, Vc-xin n 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, bi t nhng nm u thp nin 90 ngi ta to thnh cng nhng cy Vc-xin n u tin. Tuy nhin cho n nay vic Nghin cu v pht trin Vc-xin n vn cn ang l mt 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. Nghin cu v pht trin Vc-xin n cn s kt hp ng thi gia lnh vc min dch hc v thc vt hc. Do vy vic nghin cu, pht trin v th nghim Vc-xin n thng qua cy trng chuyn gen vn cn nhiu hn ch, tuy nhin cng thu c nhng nhiu thnh tu to ln. Qua bai tiu lun nay ti mun cung cp thm mt s thng tin c ban v linh vc mi nay, nhm giup nhng ngi quan tm co th tham khao thm. Chinh vi vy ti chon tai Cng ngh san xut Vc-xin n c nhm ap ng nhng nhu cu trn.

HV: Vo Quang Trung TVH K20

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Tiu lun Cng ngh sinh hocI. NH NGHA 1. Vc-xin


NI DUNG

Vc-xin l ch phm c tnh khng nguyn dng to min dch c hiu ch ng, nhm tng sc khng ca c th i vi mt (s) tc nhn gy bnh c th. Cc nghin cu mi cn m ra hng dng vc-xin iu tr mt s bnh (vc-xin liu php, mt hng trong cc min dch liu php). Thut ng vc-xin xut pht t vaccinia, loi virus gy bnh u b nhng khi em chng cho ngi li gip nga c bnh u ma (ting Latinh vacca ngha l "con b ci"). Vic dng vc-xin phng bnh gi chung l chng nga hay tim phng hoc tim chng, mc d vc-xin khng nhng c cy (chng), tim m cn c th c a vo c th qua ng ming. * C ch hot ng ca vc-xin H min dch nhn din vc-xin l vt l nn hy dit chng v "ghi nh" chng. V sau, khi tc nhn gy bnh thc th xm nhp c th, h min dch t th sn sng tn cng tc nhn gy bnh nhanh chng hn v hu hiu hn (bng cch huy ng nhiu thnh phn ca h min dch, c bit l nh thc cc t bo lympho nh). y chnh l cc u im ca p ng min dch c hiu. * Cc loi vc-xin Vc-xin c th l cc virus hoc vi khun sng, gim c lc, khi a vo c th khng gy bnh hoc gy bnh rt nh. Vc-xin cng c th l cc vi sinh vt b bt hot, cht hoc ch l nhng sn phm tinh ch t vi sinh vt. * Ba loi vc-xin kinh in Nui cy virus cm (chng gy i dch nm 1918) phc v nghin cu v sn xut vc-xin: - Vc-xin bt hot l cc vi sinh vt c hi b git bng ha cht hoc bng nhit. Th d: cc vc-xin chng cm, t, dch hch v vim gan siu vi A. Hu ht cc vc-xin loi ny ch gy p ng min dch khng hon ton v ngn hn, cn phi tim nhc nhiu ln. - Vc-xin sng, gim c lc l cc vi sinh vt c nui cy di nhng iu kin c bit nhm lm gim c tnh c hi ca chng. Vc-xin in hnh loi ny thng gy c p ng min dch di hn v l loi vc-xin c a chung dnh cho ngi ln khe mnh. Cc vc-xin nga bnh st vng, si, bnh ban o v quai b u thuc loi ny.


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Cc "toxoid" l cc hp cht c b bt hot trch t cc vi sinh vt (trong trng hp chnh cc c cht ny l phng tin gy bnh ca vi sinh vt). Th d: cc vc-xin nga un vn v bch hu. - Vc-xin sng nga bnh lao khng phi l dng vi khun lao gy bnh, m l mt dng ln cn c gi l BCG. * Mt s loi vc-xin mi ang nghin cu Cc vc-xin ny cn c xem l vc-xin ca tng lai, c 6 hng pht trin chnh hin nay: - S dng cc ph gia (adjuvant) mi, nhm gy ra loi p ng min dch mong mun. Th d, cht nhm phosphate v cc oligonucleotide cha CpG demethyl ha a vo vc-xin khin p ng min dch pht trin theo hng dch th (to khng th) thay v t bo. - Vc-xin khm: s dng mt sinh th quen bit hn ch hin tng "phn tc dng", th d dng virus vaccinia mang mt s yu t ca virus vim gan B hay virus di. - Vc-xin polypeptidique: tng cng tnh sinh min dch nh lin kt tt hn vi cc phn t MHC: peptide nhn to 1/2 ging virus, 1/2 kia gn MHC; on peptide m phng 1 quyt nh khng nguyn (epitope). - Anti-idiotype: idiotype l cu trc khng gian ca khng th ti v tr gn khng nguyn, c hiu vi khng nguyn tng ng. Anti-idiotype l cc khng th c hiu i vi idiotype, do anti-idiotype xt v mt c hiu li tng t vi khng nguyn. Vy, thay v dng khng nguyn X lm vc-xin, ngi ta dng idiotype anti-anti-X. - Vc-xin DNA: DNA ca tc nhn gy bnh s c biu hin bi t bo ngi c chng nga. Li th ca DNA l r, bn, d sn xut ra s lng ln nn thch hp cho nhng chng trnh tim chng rng ri. Ngoi ra, vc-xin DNA cn gip nh hng p ng min dch: tc nhn gy bnh ngoi bo c trnh din qua MHC loi II, dn n p ng CD4 (dch th v t bo). Khi khng nguyn ca tc nhn c chnh c th ngi biu hin, n s c trnh din qua MHC loi I, lc ny p ng min dch t bo qua CD8 c kch thch. Tuy nhin phng php ny l con dao hai li bi l t bo mang DNA l c nguy c b nhn din l "khng ta", sinh ra bnh t min. - S dng vc-t ti t hp dng cc vi khun thun tnh hoc cc t bo trnh din khng nguyn nh t bo tua c chuyn gen biu hin khng nguyn mong mun. * Vc-xin dng iu tr


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Mt trong nhng hng nghin cu mi l min dch liu php, bao gm min dch liu php th ng v ch ng (tc vc-xin liu php). Ngi ta hy vng l phng php ny s cha c nhng bnh nh ung th, AIDS v bnh Alzheimer. * Hn ch ca vc-xin Nhng hn ch ca vc-xin tp trung thnh hai nhm chnh: hiu qu km v cc tai bin i km. * Hn ch v hiu qu - Mt s vc-xin rt c hiu qu, khng k vc-xin u ma ni ting, th d vc-xin nga bnh un vn, si v.v. Mt s vc-xin khc c hiu qu va phi (hiu qu ca BCG ch vo khong 50%). Ngc li, c nhng bnh n u th k 21 vn cha c vc-xin thch hp (AIDS, st rt v.v.). Do vy, vc-xin cha phi l v kh vn nng i ph vi bnh tt. - Hiu qu ca vc-xin cng kh nh gi chnh xc. Kt qu nghin cu trn ng vt khng th p dng 100% cho loi ngi, v nhng c im ring ca tng loi. Trn l thuyt, phng php duy nht chng minh hiu qu l ly 2 nhm ngi, mt nhm c tim chng, mt nhm khng ri truyn mm bnh cho c hai nhm xem kt qu. D nhin phng php ny khng th s dng c v tri o c. Do , ngi ta bin ha i mt cht, cng chia ra 2 nhm c chng v khng c chng nh trn nhng khng truyn bnh m ch quan st s nhim bnh qua cc ng thng thng. Hn ch ca phng php ny l nu mt vc-xin t ra c hiu qu, ngi ta khng th trin khai nghin cu trn quy m rng tnh chnh xc hiu qu v nh th mt s ln qun chng s b thit thi do khng c bo v. Bi vy, khi mt vc-xin c xem l c hiu qu, ngi ta em tim chng cho mi ngi v quan st s gim s ngi mc bnh. Tuy nhin, ngay c khi mt bnh c chiu hng gim xung, ngi ta cng khng bit vai tr tht s ca vc-xin, th d tn sut bnh lao gim rt nhiu, nhng vai tr ca cc bin php v sinh, cch ly ngun ly cng rt ng k. ( hiu r hn cch nh gi hiu qu, xem thm bi khoa hc thng k.) Tnh km hiu qu ca vc-xin c th biu hin v mt cht (p ng min dch khng thch hp) hoc v mt lng (khng c p ng min dch). * Nguyn nhn gy km hiu qu v lng: Cc "l hng" trong kho tng min dch: trn l thuyt, cc t bo lympho B c th


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to ra hn 1012 loi khng th c hiu [1], cn lympho T c th nhn din trn 1015 khng nguyn khc nhau , nhng con s ny tuy rt ln nhng khng phi l v hn, h min dch khng th chng li mi th. Hiu qu ca vc-xin cn ty thuc vo thi gian bo v: tr nh min dch c th tn ti sut i nhng s sn xut khng th th khng nu khng c ti kch thch. t bin ca tc nhn gy bnh: y l c ch sinh tn ca cc tc nhn gy bnh. t bin y h min dch vo mt cuc rt ui trng k. Tiu biu cho c ch ny l HIV, virus st xut huyt, virus cm vi nguy c i dch cm gia cm hin nay. * Nguyn nhn gy km hiu qu v cht: Vai tr ca ph gia: gim tc dng khng mong mun ca vc-xin, ngi ta thng tinh lc cc ch phm, nhng c nhng vc-xin qu tinh khit li tr nn km hiu qu. l do h min dch mun c kch hot, phi nhn c mt tn hiu bo nguy, tn hiu ny thng khng phi l khng nguyn dng lm vc-xin. khc phc, ngi ta dng mt s loi ph gia trong ch phm vc-xin. Th d ph gia Freund, nhm hyrxt, nhm phosphate hoc trn ln cc vc-xin vi nhau. Loi phn ng min dch v hin tng chuyn hng min dch: i vi cc tc nhn gy bnh ngoi bo, p ng min dch dch th l thch hp (loi p ng ny c s h tr ca cc t bo lympho Th1). Ngc li, p ng min dch t bo (cn s h tr ca lympho Th2) li hu hiu cho cc tc nhn gy bnh ni bo. Do , nu vc-xin gy c p ng min dch nhng khng ng loi p ng nn c, hiu qu cng khng c bo m. Th1 v Th2 c xu hng khc ch ln nhau. Vc-xin kinh in c xu hng to p ng Th1. Do i vi nhng bnh do tc nhn ni bo nh nhim leishmania, min dch c hiu sau lnh bnh li tt hn vc-xin, v vc-xin li gy hiu qu ngc, kim hm phn ng bo v. * Tai bin khi dng vc-xin C hai loi tai bin: nhim bnh v cc bnh min dch. Nhim bnh Vc-xin sng, gim c lc c th gy bnh cho ngi b suy gim min dch. Nguy c hi phc ca tc nhn vi sinh: mt tc nhn b lm gim c lc tm li c c tnh ca mnh. Nguy c ny vc-xin nga bi lit l 10-7, ngha l c 10 triu tr em ung vc-xin Sabin th c 1 em b tai nn loi ny. iu khng may ny khng ngn cn c vic s dng vc-xin ny bi l t l c xem l chp nhn c.


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Nguy c nhim cc tc nhn gy bnh khc vo trong ch phm vc-xin. iu ny c th hn ch bng cc quy trnh sn xut, bo qun v s dng cht ch. Bnh min dch Th nghim vc-xin phng bnh di trn cu cho thy c xc sut gy EAE, mt bnh t min trn h thn kinh khong 1/3000-1/1000.L do c th l vc-xin chit xut t no ch mang theo c nhng mu protein ca t bo thn kinh, khi to min dch, c th (c tim) to ra c khng th chng li cu trc thn kinh ca mnh. Vc-xin nga ho g c th gy sc km di chng thn kinh vi xc sut 10-4-10-6. Vic tinh lc vc-xin ny lm tng mc an ton nhng mt ln na, gim hiu qu. * Chng nga - Chng nga l cho vc-xin tip xc vi h min dch. Ty bn cht k sinh, bnh sinh ca tc nhn gy bnh cng nh ca ch phm vc-xin m ngi ta dng cc phng php khc nhau nhm t hiu qu cao nht. - Chng l cch to mt vt rch trn da (cho rm mu) ri cho tip xc vi vc-xin. Phng php ny trc y c dng cho vc-xin u ma v lao. - Tim di da, trong da v.v. l phng php ph thng nht hin nay, k c vc-xin BCG phng lao. Khng c tim vo mch mu. - Ung vc-xin l phng php dng cho vc-xin Sabin nga bnh bi lit. * nh gi hiu qu v theo di Trong mt s trng hp, th d sau khi tim vc-xin nga vim gan siu vi B, ngi ta cn lm xt nghim huyt thanh tm hiu gi khng th qua nh gi hiu qu ca vc-xin trn c th ngi c tim (c to c p ng min dch hu hiu khng). * Trin khai T chc Sc khe Th gii (WHO) h tr nhiu chng trnh tim chng m rng trn phm vi ton cu trong k hoch chm sc sc khe ban u. Trong khun kh cc chng trnh ny, ngi ta xut cc lch tim chng i vi mt s bnh truyn nhim nguy him thng gp. Mt trong nhng mc tiu ca y sinh hc hin nay l y mnh nghin cu nhm tm ra cc vc-xin mi, hiu qu vi gi thnh ph hp cho mc tiu ph cp cho mi ngi, nht l i vi nhng bnh gy cht ngi nhiu nh st rt hay nhng bnh nan y nh ung th, AIDS. Hin nay, Vit Nam sn xut c 9/10 vaccine phc v chng trnh tim chng


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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. Bn cnh , cng vi s pht trin ca cng ngh sinh hc thc vt trn th gii, cc nh khoa hc Vit Nam t c nhiu thnh cng trong vic nng cao gi tr v phm cht cy bng nhiu phng php khc nhau nh chuyn gen Bt khng su vo bng, gen khng o n vo la.. Tuy nhin, cha c bo co no v nghin cu sn xut khng nguyn trong thc vt, nht l cy n c. 2. Vc-xin n c Vc-xin n c cn l Vc-xin tiu phn bao gm mt hoc nhiu chui polypeptit ca protein khng nguyn trong vi sinh vt gy bnh. Ngi ta chn lc nhng gen m ho cho cc thnh phn ny, a vo vect, da vo h thng di truyn thc vt khuych i gen v biu hin thnh cng khng nguyn protein mong mun trong cc b phn n c ca thc vt, loi vccine ny c c th chp nhn v n bn vng trong dch tiu ho i qua ng tiu ho m khng b phn hu. Vc-xin n c c hot tnh tng t nh Vc-xin thng thng, ch khc l Vcxin ny c thc vt sn xut trong nhng phn n c nh l, c, qu, ht. N lc sn xut Vc-xin u tin t thc vt c ghi nhn vo nm 1990 khi cng trnh nghin cu biu hin protein khng nguyn b mt A ca vi khun Streptococus mutans cy thuc l. II. C S KHOA HC Vi cc tin b khoa hc hin nay trong vic to cy trng chuyn gen cho php to cy trng chuyn gen c cha Vc-xin n c vi cc bc: - Chn la v nhn bn on gen khng nguyn ca vi khun v vi rt gy bnh. - Th nghim thnh cng cc vect biu hin gen ti t hp. - Chuyn thnh cng gen khng nguyn vo nhiu loi i tng thc vt. - Gia tng tc v khi lng protein ti t hp c c sn sinh trong cy trng . Vc-xin n c c nhng u im ni tri: - D dng tng qui m sn xut v d thu sinh khi. - Tnh n nh cao,d bo qun v s dng 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


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dng bo qun v s dng (ngay trong thc vt) m khng cn gi lnh nh cc Vc-xin tim. Trong qu trnh sn xut Vc-xin n c ngi ta ch cn vn chuyn v s dng ngay b phn thc vt cha Vc-xin Tnh n c:

Loi Vc-xin trong thc vt ny c chnh m trong thc vt bao bc, hn ch c s phn hu ca dch tiu ho ng rut v n nh, bn vng trong c thnn Vc-xin ny c th n ti (qu, l) hoc nu chn (ht, c). Nhiu nghin cu cho thy nhiu khng nguyn Vc-xin c biu hin hiu qu rau dip c (l), khoai ty (c), c chua (qu) v ng (ht). Tnh An ton: V Vc-xin c sn xut trong thc vt l Vc-xin di n v s dng gen m ho cho mt phn protein v virus m khng cn n virus sng nh Vc-xin gim c lc hay virus cht nh Vc-xin bt hot. Do Vc-xin 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 Vc-xin. Do , khng cn tch chit v tinh sch khng nguyn Vc-xin

Vc-xin n c kch thch sn xut khng th ca h thng min dch hiu

qu hn Vc-xin 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 Vc-xin 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 Vc-xin n c, khng nguyn c gii phong trong rut non. Nhng nghin cu bo v khng nguyn lm Vc-xin n c trc tc ng ca dch tiu ho, c bit ca c th con ngi khng nh gi tr thc tin ca Vc-xin n c sn xut nh thc vt chuyn gen Vi nhng u im ni bt ca Vc-xin n th vic sn xut Vc-xin n uc xem l h thng sn xut Vc-xin l tng n gin v gi thnh thp thnh cng v c ng k bo h sng ch. Sau nhiu thnh cng khc v Vc-xin thc vt cng oc cng b trn nhiu loi cy khc nhau nh thuc l, rau dip, c chua, khoai tyS lng nghin cu v Vc-xin n c c gia tng chng t tnh u vit ca thc vt nh mt h thng biu hin hiu qu cao, chi ph sn xut thp, an ton v mt sinh hc, s dng v


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Tiu lun Cng ngh sinh hocbo qun d dng khng cn gi lnh


III. NGUYN L SN XUT VC-XIN N C Quy trnh sn xut vaccie n c:

- La chn gen cn c biu hin (gen quan tm) v a vo mt vector thch hp.- La chn i tng thc vt thch hp chuyn gen; - Chuyn vector ti t hp mang gen quan tm vo thc vt la chn bng cc phng php chuyn gen khc nhau; - Kim tra biu hin ca gen quan tm trong nhng b phn n c ca thc vt;

- Th nghim kh nng p ng min dch ca Vc-xin sn xut t thc vt; - S dng Vc-xin th nghim thnh cng bng cch n ti di dng thc n ch bin.Gen ly t ngun bnh ngi c chuyn vo vi khun gy nhim thc vt Vi khun c nhim vo cc mu l khoai ty mm to oc t cc mu l mang gen bnh ngi Khi n khoai ty gy ra phn ng min dch mm bnh

Hinh 1: Quy trinh san xut vaccine n c


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Tiu lun Cng ngh sinh hocThit k vector biu hin


im quan trng nht trong thit k vector biu hin l promoter, y phi l promoter kho, c i lc mnh vi RNApolymerase ca vt ch v hot ng ca promoter c iu ho mt cch d dng. Trong nhiu nghin cu gn y, vi mc ch biu hin khng nguyn Vc-xin trong cc b phn n c ca thc vt, ngi ta thit k promoter c hiu m thc vt, v d promoter c hiu m c hoc m ht th protein s c sn xut trong c hoc ht.Hnh 1.1 :To thc vt chuyn gen bng phng php chuyn gen gin tip nh Agrobacterium


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IV. CC PHNG PHP CHUYN GEN VO THC VT Cc phng php biu hin gen da trn thc vt c pht trin t cui nhng nm 1970 u 1980. Hin nay, c th xp nhng phng php ny vo hai nhm chnh sau: Chuyn gen n nh tc l gen quan tm c bo tn qua nhiu th h do gn vo h gen vt ch (chuyn gen vo nhn hoc plastid) v biu hin gen tm thi da trn Agrobacterium v vector virus thc vt, theo nguyn tc c th s dng bt k phng php chuyn gen vo thc vt no cng c th to ra thc vt cha Vc-xin n c, tuy nhin hin nay ngi ta ch mi to thnh cng Vc-xin n nh sng bn gen v nh vi khun Agrobacterium. 1. Phng php chuyn gen gin tip nh Agrobacterium Cy chuyn gen u tin c to ra nm 1983 s dng vi khun Agrobacterium tumefaciens. y l loi vi khun sng trong t, gy bnh cho cy bng cch gn cc on gen vo h gen ca t bo ch v sinh ra u nh mt loi plasmid ca vi khun ny, plasmid Ti. Ngi ta li dng c im ca vi khun Agrobacterium chuyn gen mong mun vo thc vt, trong plasmid Ti b bt hot, n ch cn kh nng gn DNA vo t bo v mt kh nng gy bnh. Trong sn xut Vc-xin n c, ngi ta thit k mt vector gm hai gen: Mt gen m ho cho khng nguyn virus v mt gen khng khng sinh. Do , trong mi trng c khng sinh, nhng t bo thc vt khng mang gen chuyn s b cht, tri li t bo mang gen s hnh thnh callus, t to thnh cy hon chnh. Phng php ny c mt s bt li: plasmid Ti gn gen ngu nhin vo h gen thc vt, lm tng tnh khng ng u v mc biu hin khng nguyn trong cy chuyn gen. Ngoi ra cch gn gen ny c th ph v biu hin gen dn n sinh trng bt thng ca cy chuyn gen. Mc d h thng chuyn gen gin tip nh Agrobacterium l c hiu qu i vi mt s loi nhng khng phi tt c thc vt c th c bin np bng con ng ny. c bit, lp mt l mm bao gm cc cy ng cc chnh trn th gii nh la, la m v ng l khng c bin np d dng nh A. tumefaciens. khai thc v s dng A. tumefaciens nh l mt vector chuyn gen cc nh khoa hc loi b cc gen gy khi u v gen m ho opine ca T - DNA v thay th vo l cc marker chn lc, trong khi vn duy tr cc vng b phi v b tri ca T-DNA v cc gen vir. Gen chuyn c xen vo gia cc vng b ca T-DNA. N s c chuyn vo


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t bo v tr nn hp nht vi nhim sc th t bo thc vt. Phng php chuyn gen gin tip nh Agrobacterium c kim tra i vi s xm nhp bn vng, s biu hin v s di truyn ca cc gen chuyn c bit. Tuy nhin, mt vi yu t nh hng n hiu qu bin np l loi m c bin np, giai on pht trin ca m, mc khi u ca vi khun A. tumefaciens s dng, mi trng nui cy m sau khi bin np, marker c s dng chn lc th bin np, loi vector s dng v kiu gen ca thc vt. 2. Chuyn gen n nh: Chuyn gen vo nhn L phng php chuyn gen n nh do gn gen quan tm vo nhim sc th thc vt c ng dng ph bin trong sn xut protein chc nng. Ngoi ra, c th a gen vo lc lp. Lc lp l c quan t ca thc vt c ngun gc t vi khun cng sinh trong thc vt v c c th di truyn rt ging vi cc plasmid vi khun. Ngi ta tnh rng trong t bo l trng thnh c ti 100 lc lp, mi lc lp c cha 100 bn sao DNA vng, v th mc biu hin gen rt cao, c th ti 35% protein tng s. Tuy nhin, protein c biu hin thng khng c chc nng y do b my di truyn ca lc lp mc c quan t nn kh c th m bo cc bin i sau dch m. Chuyn gen trc tip vo protoplast DNA d xm nhp c vo t bo thc vt, phi loi b vch t bo to protoplast. Protoplast c th c duy tr trong mi trng nui cy nh cc t bo sinh trng mt cch c lp hoc vi mt mi trng c hiu, vch t bo c th c to thnh v ton b cc cy c th c ti sinh t cc t bo ny. Qu trnh chuyn gen nh th ny c thc hin mt cch trc tip bng mt c ch vt l n gin, khng cn c vector. nng cao hiu qu bin np, ngi ta x l protoplast vi PGE (polyethylene glycol) hoc bng xung in. Phng php chuyn gen ny rt c hiu qu, c bit i vi nhng loi thc vt m phng php chuyn gen gin tip nh Agrobacterium khng th thc hin c. Vi phng php ny, cc nh khoa hc chuyn gen thnh cng vo mt s loi cy mt l mm nh loi la ph Japonica (Datta, 1990), ng (Doon, 1990), la m (Vassil, 1992). Chuyn gen bng k thut xung in


12



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 2.14), 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.

Hnh

2.14:S

mng

phospholipid kp

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 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. Nhiu phng php c pht trin vt qua ro cn ny, cho php a DNA v cc k thut xung in. 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 mtHnh 2.16 :My xung gen (Gene pulser) (Hng Biorad)

Hnh 2.15: Cuvette nha c in cc

phn t khc vo trong t bo c nghin cu. Mt trong nhng phng php ny l


13

Tiu lun Cng ngh sinh hoccuvette nha c in cc (hnh 2.15)


to ra xung in cao th trong mt thi gian ngn ngi ta s dng mt thit b gi l my xung gen (gene pulser). (hnh 2.16) Qu trnh c bn din ra bn trong my ny c th c trnh by bng s (hnh 2.17) 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 khongHnh 2.17: S b tr mch c bn ca my xung in

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 0,5-1,0V 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 2.18). 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 trnHnh 2.18: S plasmid cha DNA 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 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 trn mng bo chtngoi lai i qua cc l tm thi


14



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. 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 xungin chp nhong to ra cho thy kch thch s dung hp t bo (Weber v Berrg, 1995). 3.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. 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 htSng bn gen


15Hnh 2.21:S nguyn l hot ng ca sng bn gen



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 nguyn l chung ca phng php ny l s dng p lc xung ca kh helium gai 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 t). Cc ht ny c t trn mt ci a mt bn trong ca 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 2.21). 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 DNA ngoi lai xm nhp v hp nht 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. 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 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. 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. 4. K thut calcium phosphate


16



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; Graham, 1979; Pellicer, 1980). K thut ny yu cu cc t bo nhn vi cc cht ng kt ta DNA v calcium phosphat (Hnh 2.23). Kt ta ny bm vo tHnh 2.23: Phc hp DNA-calcium phosphat

bo v sau s hp th vo t bo qua qu trnh m bo (Loyter, 1982). Trong t bo, cc phn t DNA ngoi lai nm trong khng bo c to thnh do m bo v lysosome th hai nhng rt t 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 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. 5. Chuyn gen qua liposome

Hnh 2.24: Cu trc tng qut ca lipid cation tng hp


17



Hnh 2.25: Cu trc ca DOPE (L-diolecyl

phosphatidylethanolamine) Vo thp nin 1980, liposome

Hnh 2.26: Phc hp liposome-DNA

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 (Hnh 2.24). Thng th lipid cation c trn vi mt lipid trung tnh nh Ldioleoyl phosphatidyl-ethanolamine (DOPE) (Hnh 2.25). 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 2.26). 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 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


18

Tiu lun Cng ngh sinh hockt 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..

V. NH HNG V THNH TU1. Trn th gii:Nhng thnh cng ca cc nhm nghin cu trn th gii chng t vic sn xut v a sn phm Vc-xin trong thc vt ra th trng s tr thnh hin thc trong mt tng lai khng xa. Nhng tin b m cc nh cng ngh sinh hc thc vt trn th gii t c tp trung vo mt s vn sau: *Tng cng mc biu hin khng nguyn phn trn, chng ta bit nhiu k thut c s dng ci bin di truyn thc vt, tuy nhin hu ht cc bo co hin nay v sn xut Vc-xin n c u lin quan n phng php chuyn gen bng Agrobacterium v promoter ph bin nht trong thit k gen biu hin l CaMV 35S (promoter ca vi khun khm sp l), mt promoter kho cho php biu hin gen mc cao. Bn cnh , nhiu h thng vector khc cng c s dng biu hin khng nguyn. Vic nghin cu sn xut khng nguyn di n v B ca c t km bn nhit (LT-B) E. coli, c iu khin bng promoter c hiu v tr (c hiu ht), lm bng mc biu hin LT-B ti 1,8% protein ho tan tng s. ng thi, vic p dng hai phng php lai to ging ng khc nhau nng thnh phn khng nguyn gp 5 v 10 ln. Chikwamba v ng tc gi cng biu hin LT-B thnh cng ng, y cng l bo co u tin s dng sng bn gen sn xut Vc-xin trong thc vt. Tuy nhin hin nay mc biu hin khng nguyn thc vt cn thp l tr ngi chnh trong vic pht trin Vc-xin ny. Trong n lc tm kim gii php cho vn ny, Gleba v ng tc gi (2005) thuc cng ty Genetics (c) cng b phng php mi


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nng cao mc biu hin ca khng nguyn Vc-xin trong thc vt. Phng php ny, gi l magnifection kt hp c nhng u im ca ba h thng sinh hc l tnh hiu qu v kh nng ly nhim h thng ca Agrobacterium, tc v mc biu hin cao ca virus, kh nng ci bin sau dch m v gi thnh sn xut thp ca thc vt. Nguyn tc ca phng php ny l li dng s ly nhim ca Agrobacterium vn chuyn v pht tn vector virus vo thc vt, sau vector mang gen quan tm ny s tin hnh sao chp, nhn ln v ly nhim cho ton b t bo. S dng phng php ny trn cy thuc l Nicotiana benthamiana v c ci n c, nhm nghin cu trn thu kt qu rt kh quan. Tc sn xut khng nguyn rt nhanh, vi mg-g trong 3-4 tun, c th tng sn lng ti 100kg/nm. Mc biu hin rt cao, t 5g protein ti t hp trn 1kg l ti tng ng 80% protein ho tan tng s, gp hn 10 ln so vi cc phng php biu hin thng thng. Nhm tc gi cng ch ra hn ch ca phng php ny nh biu hin hn ch cc oligopeptide a thnh phn. Tuy nhin vi nhng u im vt tri, phng php ny t ra rt hiu qu v c tim nng ng dng cao sn xut khng nguyn Vc-xin thc vt gi r an ton v mt sinh hc. Tregoning v cng s (2004) biu hin khng nguyn Vc-xin mc cao trong lc lp thuc l vi khng nguyn m hnh l Tet C (khng nguyn vi khun un vn). Trong nghin cu ny, tc gi thu c mc biu hin khc nhau khi thay i hai yu t duy tr tnh n nh ca promoter (vng iu khin gen) l cch s dng m b ba (codon usage) v vng trnh t khng dch m 5 (5 UTR). V d, khi tng gp i cc codon giu A-T trong gen, mc biu hin cng tng gp i t 10 n 20% protein ho tan tng s. Hoc khi thay i 5 UTR t rbcl UTR thnh T 7gen 10 5 UTR cng lm tng gp i mc biu hin ca khng nguyn TetC. Nh vy, c th thy pht hin ny rt quan trng trong vic chun ho cc c im ca cy chuyn gen v mc biu hin gen chuyn qua cao c th gy hi cho cy. *La chn i tng thc vt n nm 2000 c 5 khng nguyn c biu hin thnh cng rau qu. Trong di n v B ca ni c t km bn nhit E. coli (LT-B), di n v B ca c t t (CL-B), protein v capsid virus Norwalk v khng nguyn b mt virus vim gan B u c sn xut khoai ty. Ring protein G ca virus di c biu hin c chua. Nm 2005, trong mt bo co mi nht v Vc-xin n c trong thc vt, c nhng phn tch su sc v cc i tng thc vt c s dng sn xut Vc-xin, c


20

Tiu lun Cng ngh sinh hocl: - Mc biu hin cao


bit l vim gan siu vi B. 4 tiu chun i vi h thng thc vt p ng mc ch ny,

- Mc khng nguyn ng u trong m thc vt - Nguyn liu thc vt phi n c - Khng nguyn n nh nhit phng v c th bo qun lu di. Nhiu nghin c cho thy, ht ng hi t 4 tiu chun trn ca h thng biu hin hiu qu cao, y cng l i tng m nhm ng quan tm sn xut khng nguyn Vc-xin vim gan B. Nm 2003, h biu hin thnh cng di n v B ca c t km bn nhit E.coli (LT-B). H thy rng phi mm ca ht ng chuyn gen tp chung lng khng nguyn cao nht, gp 6 ln so vi b phn khc v nhng nhn ny tng ng vi mt liu mg khng nguyn. Sau , nm 2005 nhm ny li biu hin thnh cng khng nguyn b mt chnh ca virus vim gan B trong ht ng. Mc biu hin gen trong ht thu c t cy chuyn gen th h th nht l 0,2% protein ho tan tng s, trong khng nguyn tp trung ti 20% trong cc phi mm ca ht. Korban v ng tc gi biu hin khng nguyn Vc-xin di n v virus RSV c chua, y l virus gy bnh ng h hp nghim trng tr s sinh v tr nh. Cc thit k gen (mt loi mang promoter CaMV 35 S v mt loi mang promoter c hiu qu E-8) cha gen m ho cho khng nguyn RVS-F c chuyn vo c chua thng qua phng php chuyn gen bng Agrobacterium. S dng promoter c hiu qu cho php biu hin protein ch trong qu ca tt c cc cy chuyn gen. Protein ny to p ng min dch khi c th nghim chut. * Th nghim kh nng p ng min dch ca Vc-xin n c Hu ht Vc-xin n c thc vt th nghim ng vt v giai on 1 ngi. Mt trong nhng nguyn nhn nh hng n vic th nghim quy m ln ngi l do s ngi rng Vc-xin n c b phn hu bi dch tiu ho trong ng rut. Do , kh c th thu c kt qu chnh xc do khng c phn ng sinh khng th hoc khng th rt t, khng gy p ng min dch. ng thi Vc-xin n c khng biu hin ng nht trong cc m thc vt cng gy kh khn trong vic xc nh liu lng th nghim. Th nghim lm sng u tin ngi c ghi nhn vo nm 1997 bi nhm nghin cu ca Arntzen v c s chp nhn ca C quan qun l dc phm v thc


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phm Hoa K. Trong th nghim Vc-xin di n v c t E.coli LT-B ny, 11 ngi n sng 50-100g khoai ty chuyn gen. Kt qu cho thy 10/11 ngi kim tra u to khng th chng li LT-B, lng khng th ny tng ng vi khng th o c nhng ngi nim E. coli nng 106 . Nh vy, protein LT-B ny trong cc m thc vt n c khng b phn hu trong ng tiu ho v c kh nng p ng min dch ngi. Hin nay, Thanavala v ng tc gi (2005) ang th nghim giai on I v II Vcxin vim gan B khoai ty. Khi n 2 n 3 liu, mi liu 100g khoai ty sng tng ng 1mg khng nguyn b mt virus vim gan B, 33 ngi kim tra c phn ng to khng th vi nng 10 mIU/mL. Thnh cng u tin v biu hin gen m ho cho khng nguyn v virus Staphyloccocus mutants vo nm 1990 m ra hng mi sn xut Vc-xin n c trong thc vt, thu ht s quan tm c bit ca nhiu nhm nghin cu trn th gii, trong hai nhm nghin cu vi nhiu ng gp quan trng l nhm ca Arntzen ti vin nghin cu thc vt Boyce Thomson, thuc trng i hc Cornell, Hoa K v ti ProdiGene mt cng ty t nhn v cng ngh sinh hc thc vt ca Hoa K. C th ni nhm nghin cu ca Mason l nhm tin phong trong lnh vc Vc-xin trong thc vt vi vic biu hin thnh cng khng nguyn b mt virus vim gan B (HbsAg) thuc l nm 1992 . Nhm ny c nhng ng gp to ln trong s pht trin ca Vc-xin n c nh ng gp v mt c s, nguyn l khoa hc, la chn i tng sn xut Vc-xin nh khoai ty, chui; th nghim Vc-xin ny ngi v ng vt, tin ti a sn phm thc vt mang Vc-xin n mi ngi. Hin nay, nhm ny ang thc hin d n chuyn giao cng ngh v trao i thng tin v Vc-xin n c vi cc nh khoa hc nhng nc ang pht trin. D n tr gi 58000 USD, ko di trong 3 nm do Rockefeller Foundation ti tr, thc hin u tin vi CINESTAV - mt t chc y t chnh ph Mexico nhm mc ch sn xut Vc-xin HIV n c gi r trong chui v c th s dng trn ton th gii chng li HIV/AIDS. Hin nay nhm nghin cu ca Arntzen ang trin khai d n sn xut Vc-xin trong chui. H hi vng chui s l ngun cung cp chnh Vc-xin n c, d n v gi thnh r. Th ba l tng cng tnh bn vng ca Vc-xin vi dch tiu ho trong ng rut ngi v ng vt. Ngoi ra, vic xc nh liu lng thc vt mang Vc-xin rt quan trng khi th nghim v a sn phm ra th trng. Nhm ca Streatfield v ng tc gi (2003) mc d khi u mun hn nhng c


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nhng ng gp ng k trong vic sn xut Vc-xin n c. Hin nay nhm ny ang tham gia vo cc d n Vc-xin da trn thc vt ca cng ty ProdiGene, ch yu trn cy ng, mt i tng nghin cu c xem l l tng cho sn xut Vc-xin n c. Nhm xy dng thnh cng h thng sn xut Vc-xin vim gan B ng. c th s dng di dng bnh snack (ng qua ch bin c nghin thnh bt). Cc Vc-xin sn xut trong thc vt nng cao gi tr cy trng, nht l cy chuyn gen do chng c trng v ch bin trn quy m ln, p ng nhu cu thuc v sinh dc phm. Vc-xin n c m ra mt k nguyn mi ca nng nghip, c cc nh khoa hc gi l biofarming, trong cc cy nng nghip c ci bin cht lng (tng cng gi tr dinh dng, lm thuc), trng trong cc khu vc c bit v s dng c bit nh cc nh my sn xut Vc-xin v cc tc nhn khng khun khc. Nh vy, nhng vn cn tn ti trong sn xut Vc-xin n c t thc vt m ra nhng hng nghin cu quan trng cho cc nh khoa hc trn con ng tm kim mt Vc-xin gi r, cung cp n mi ni trn th gii, c bit nhng nc ang pht trin. V r rng lnh vc mi y tim nng ny ang bc vo giai on pht trin si ng, i hi s hp tc ca nhiu nh khoa hc trn th gii cng gii quyt nhng kh khn ny. Trong , vic chuyn giao cng ngh sn xut Vc-xin n c n cc nc ang pht trin rt cn thit v y; l nhng quc gia thc s cn loi Vc-xin ny. * Mt s thnh tu Vc-xin n c 1. Vc-xin t khoai ty chuyn gien Loi khoai ty chuyn i gien (GM), cha vc-xin nga vim gan B, thc y thnh cng kh nng min dch trong cc cuc th nghim lm sng u tin. Trong nghin cu, hn 60% tnh nguyn vin n khoai ty GM, tng ng ba liu vc xin. Kt qu l c th h to thm mt lng ln khng th chng li virus. Tnh nguyn vin n khoai ty bnh thng khng sinh thm khng th. Tuy nhin, do nhng ngi n sng khoai ty GM nng min dch ca h. to vc-xin trong khoai ty, nhm nghin cu do Charles Arntzen thuc H Arizona (M) ng u b sung vo cy khoai ty thng thng mt protein ca virus vim gan B. Khi con ngi n khoai ty ny, protein s gip h min dch nhn ra v tiuKhoai ty cha vc-xin.

c tim vc-xin vim gan B thng thng nn vc-xin khoai ty ch tng cng kh


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Tiu lun Cng ngh sinh hocdit mi virus vim gan B trong tng lai.


Theo Arntzen, bin thc phm thnh ngun vc-xin r tin rt hu ch i vi cc nc ngho v khng phi b ra nhiu chi ph bo qun lnh hoc mua kim tim. Tuy nhin, iu khng may l cc nh pht trin dc phm ang t b vic bo ch vc-xin trong cc loi thc phm c bn chng hn nh chui, c chua v khoai ty. Nguyn nhn l h lo ngi kh nng thc phm cha vc-xin c th b ln vo thc phm trong siu th hoc ca hng. Nu iu ny xy ra, hu qu s khn lng. Thay vo , cc nh bo ch thuc ang tp trung vo sn xut vc-xin trong l cy n c song thc vt khng c bn lm thc phm. Nhm nghin cu ca Arntzen ang iu tra mt s thc vt v ha hn nht l Nicotiana benthamiana, h hng ca cy thuc l. L c thu hoch, ra sch, nghin ri p lnh-sy kh bo qun trc khi ng vo cc vin con nhng. p lnh- sy kh c ngha l vc-xin tn ti trong thi tit nng, khng cn bo qun lnh ging nh vc-xin thng thng. Ngoi ra, ng vc-xin thnh vin con nhng m bo liu lng thng nht. 2. Go cha Vc-xin chng dch t Nhm nghin cu do gio s Hiroshi Kiyono thuc khoa nghin cu min dch, Trng i hc Tokyo ng u, cng b vic pht trin mt loi go c cha Vc-xin cha bnh dch t. Tin b ny c th s gim bt kh khn cho vic phn phi Vc-xin nhng quc gia ang pht trin trong thi gian sp ti. Cc loi Vc-xin tim thng thng khng to c phn ng min nhim nhng ni c mng nhy trong c th. Do vy loi Vc-xin mi ny s c tc dng tt trong vic chng li tc nhn gy nhim thng thng qua mng nhy, v d virus dch t, E. coli, virus gy suy gim h min dch ngi, virus cm v SARS. Cc nh nghin cu Nht Bn c th to ra phn ng min nhim chut, ng thi trnh phn ng d ng vi chnh loi go ny. Ngoi ra, loi go chuyn ho gen ny c th c tr nhit thng thng m khng gp nguy c nhim khun.Vi mt bt cm t la chuyn gien, va no bng li va tr c bnh.


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Mc d th, vic s dng loi go c bin i to nn phn ng Vc-xin khng c ngha rng y l loi Vc-xin n c. Cc nh khoa hc khng mun cng chng ngh rng n go ny s c chng nga. Thay vo , Vc-xin ny s c cung cp di dng vin nhng hoc vin nn c cha bt go v c xem l thuc ch khng phi thc phm. 3. 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 vc-xin c t l sng rt cao sau 21 ngy, trong khi nhng con chut gi min dch (sham-immunized) u cht sau 6 ngy. 4. Vc-xin gip ngn nga ung th: Vcxin t thc vt gip ngn nga ung th c t cung 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.


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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. 5. Chui "sn xut" Vc-xin vim gan B Cc nh cng ngh sinh hc pht trin c mt loi vcxin vim gan B tng t vi vcxin truyn thng, nhng c to ra t chui vi chi ph thp. Pht trin ny c th cu sng hng trm nghn ngi. Nm 2003, c tnh c khong 300 triu ngi mang virut vim gan v mt phn s ny t vong. Alexander V. Karasev, trng nhm nghin cu vcxin ti trng i hc Thomas Jefferson Philadelphia cho bit: "Khi c pht trin y , cc loi vcxin t thc vt c th r hn rt nhiu so vi cc loi vcxin hin c. Ngy nay, chi ph min dch hon ton vim gan B cho mt ngi c th tn 450 la. Trong tng lai, nh c vcxin t thc vt, chi ph s gim v vcxin c th cho nhiu ngi. La chn chui sn xut vcxin v y l thc phm r, c nhiu ni trn th gii v l mt trong s thc n u tin ca tr em. . cc nc ang pht trin - ni m cc thit b lm lnh v kh trng khan him v t th loi vcxin t chui c th gip ngn nga t vong v y l gii php hiu qu so vi cc loi vcxin truyn thng gi cao. 6. Vcxin t c chua gip chng cc bnh v h hp C chua c th c s dng cha vcxin phng nga RSV - loi virut tim tng gy ra cc bnh h hp thng tn cng tr em. RSV khin cho gn 90.000 ngi phi nhp vin v 4.500 tr s sinh v tr em t vong mi nm. B Nng nghip M ang ti tr cho mt nhm cc nh khoa hc i hc Illinois nhm nng cao tnh nng di truyn ca c chua, thm ch l c to nhm to ra loi vcxin chng li cc cn bnh ny. Vcxin s to kh nng bo v mng nhy bng cch kch thch cc khng th khng li cc protin ca RSV khi chng xm nhp ln u vo c th qua ng mi, hng v ming. D n cn trin khai nhiu nm na song cc th nghim ban u trong PTN i vi chut ang c nhng trin vng. 7. Vc-xin cai thuc l Loi Vc-xin mi ny tn l NicVAX, c kh nng to ra khng th chng nicotin trong mu ngi nghin thuc l,


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khng cho n ln no, lm mt cm gic thch th v do ngn c vic nghin. Khng th ny c th tn ti trong mu mt vi nm. Trong thi gian ny, vic ht thuc tr li s khng lm cho ngi ht cm thy thch th. Bnh thng, v phn t nicotin (cht c ch yu ca thuc l) qu b nn h thng min dch ca c th khng nhn ra c chng li. Cc nh nghin cu lin hp nicotin vi mt protein ln hn gi l pseudomonas toxoid. Nh , h min dch nhn din c k th v sn xut ra cc khng th chng li n. Cc nh nghin cu tim cho ng vt Vc-xin ny ri cho nicotin vo ng tnh mch ca con vt . Kt qu l nhng con ny, lng nicotin b gi li trong mu cao gp 8,5 ln so vi nhng con vt khng dng Vc-xin. c bit, lng nicotin c phn b trong no chng cng t hn 64% so vi nhng con vt khng dng Vc-xin. Nh vy, NicVAX ngn cn nicotin ln no. 8. Loi go cha d ng cha Vc-xin n c: D ng l mt vn sc kho ng ch , nh hng n hn 15% tr em v ngi ln trn th gii. D ng c th xy ra da, b my h hp v rut, c th nng ln v nhiu loi tc nhn bn ngoi mi trng khc nhau, nh tip xc vi phn hoa, si, hoc mt s loi phn t nht nh c mt trong thc phm. S dng cc loi thuc tng hp l phng php ph bin nht cha tr d ng, Tuy nhin ng Hidenori Takagi Tsukuba, Nht Bn v cc ng nghip ang tm kim mt phng php cha tr khc, thng qua tm hiu: mt loi vc xin n c da trn la goRice-based edible Vc-xin nh vo vic biu th multiple T cell epitopes, kch thch chng li s c ch i vi Th2-mediated IgE responses . Nghin cu ca h c ng trn s mi nht ca K yu trc tuyn ca Vin hn lm khoa hc quc gia. Cc nh nghin cu s dng cc epitope tri tm thy trong cht gy d ng c trong phn cy tuyt tng Nht Bn. Epitopes l nhng trnh t amino acid c th kch thch mt phn ng min nhim mt cch thm lng. Ngi ta tm cch dung hp gien ny vi gien iu khin protein d tr glycinin chuyn np vo cy la, cc nh khoa hc c th to ra ging la c 0,5% prtin ca ht go c cha hn hp glycinin v epitope. Khi th nghim go chuyn gen ny trn trn chut, cc nh khoa hc thy rng cc con chut n loi go ny th hin hm lng histamine thp hn, v khng pht trin hin


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tng d ng do phn hoa. Loi prtin ny tn ti n nh trong ht go trong vng 6 thng, ngay c khi tn tr trong phng c nhit cao. 9. To la chuyn gien cha Vc-xin Cc nh khoa hc Nht Bn va to ra mt loi la chuyn gien cha Vc-xin nga bnh st ma h - mt bnh d ng do phn hoa hoc bi gy ra. Cc cuc th nghim trn chut cho thy loi Vc-xin di dng la n ny ngn c phn ng min dch gy d ng. C th l chut c n la chuyn gien ni trn ht hi t hn khi tip xc vi phn hoa, so vi nhng con khng c n la (nhm i chng). H to ra Vc-xin bng cch dng nhng mu protein nht nh. Nhng mu ny lin quan ti d ng v c tm thy trong phn hoa ca cy thng liu Nht Bn - nguyn nhn ph bin gy st ma h ti nc ny. Bng cch b sung vt liu di truyn t nhng protein ni trn vo b gien la, h to ra c loi la cha cc protein phn hoa. Sau , h cho mt nhm chut n la hng ngy, trong khong vi tun ri cho chng phi nhim vi phn hoa ca cy thng liu. Kt qu l chng to ra t histamine hn - ho cht gy cc triu chng st ma h - v ht hi t hn so vi nhm chut i chng. 1. nh hng nghin cu Vc-xin n c Vit Nam Hin nay, Vit Nam sn xut c 9/10 Vc-xin 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, Vc-xin n c vn l vn kh mi m v cha c ti liu no cp su v loi Vc-xin ny. Bn cnh , cng vi s pht trin ca cng ngh sinh hc thc vt trn th gii, cc nh khoa hc Vit Nam t c nhiu thnh cng trong vic nng cao gi tr v phm cht cy bng nhiu phng php khc nhau nh chuyn gen Bt khng su vo bng, gen khng o n vo la.. Tuy nhin, cha c bo co no v nghin cu sn xut khng nguyn trong thc vt, nht l cy n c. Hin nay, 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 Vc-xin n c c ngun gc thc vt Wolffia (bo tm) thng qua Agrobacterium. Nhiu nghin cu v c tnh sinh hc ca Wolffia cho thy y l thc vt c tc sinh trng nhanh theo kiu ny chi, c bit n c hm lng dinh dng cao,


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l loi thc n rt tt cho gia sc v c th lm thc n ti cho ngi mt s nc trn th gii. Wolffia mang y c im cn thit ca h thng sn xut protein hiu qu cao v l i tng l tng cho nghin cu Vc-xin n c. 1. To vc-xin 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 vc-xin 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). Bnh ly lan theo hng a dng v phc tp, c th dn n x gan v ung th gan. Cch tt nht l phng nga v mt trong cc bin php phng nga quan trng l chng nga. Ti Vin Sinh hc nhit i TP.HCM, thuc Vin Khoa hc v Cng ngh Vit Nam, Mt nhm nghin cu ca Phng Cng ngh Gen, ang tin hnh nghin cu vc-xin nga vim gan B n c t cc b phn n ti nh qu, l, thn, c... Trong c tri c chua. Hin nay, trn th gii cng c 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,... Theo nhm nghin cu, mt thnh phn quan trng ca HBV l protein HBsAg gip cho HBV bm dnh vo mng t bo v sau i vo t bo v huyt tng ngi bnh. Huyt tng c cha HBsAg l ngun vt liu quan trng sn xut thuc chng nga c ngun gc huyt tng. Cc nh khoa hc chit tch phn protein tinh khit v nghin cu kh nng p ng min dch c th ng vt bng cch tim chch protein tinh khit hoc n trc tip sn phm cy chuyn gien. 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 p ng 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... Vi 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 vi-rt vim gan B.


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Ti Liu tham kho:[1] Tp ch cng ngh sinh hc 2005, vol 3 [2] [3] [4] [5](TheoNewScientist,BBC http://vietnamnet.vn/khoahoc/xuhuong/2005/02/374986/ [6]http://www.isb.vt.edu/news/2006/news06.apr.htm [7] [8] [9] http://rd.bcentral.com/?(Theo agbiotech VietNam) [10] [11]VitNamNet-X l tin: Trng An


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cong nghe vacxin - tieu luan cnst

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