B GIO DC V O TO TRNG H K THUT CNG NGH TP.

HCM KHOA MI TRNG V CNG NGH SINH HC

NG DNG CA VACXIN DNA TRONG NUI TRNG THY SN

GVHD: Ths. Nguyn Minh Nht Nhm: Bi Th Anh Th
L Minh V Nguyn Khnh Ngn Nguyn Vi Tho

1.Gii thiu

Vacxin c hiu qu cao nht trong vic kim sot mm bnh ly nhim gip ch cho vic phng nga 1 s bnh c tm gp phn tng nng sut p ng nhu cu ca ngi tiu dng. Vacxin DNA c v nh nhng vacxin khng nguyn truyn thng thnh cng trn nhiu vt nui khc. Ngoi vic ti u ha hiu qu ca vc-xin DNA, cc vn quan trng khc, chng hn nh an ton v chi ph sn xut s l yu t quyt nh cho cc ng dng tim nng ca cng ngh ny trong cc trang tri nui c.

Hnh 1. liu vaccine

1.1 Nhu cu DNA vaccine cho c

c tnh c khong 10% cc loi thy hi sn b cht do mc bnh truyn nhim. Dch bnh c th gy ra thit hi nghim trng cho nng dn v trong nhiu trng hp khng c cc bin php phng v iu tr sn c. Tc dng ph ca cc thuc ha cht lm nh hng khng tt n thy hi sn. Vacxin chng minh c kh nng phng bnh ca n trn hu ht gia sc v vt nui, nhng c v thy sn th vn cn nhiu hn ch.

Hnh 2. C hi thng phm

1.2 u im ca Vaccine DNA

Vc-xin DNA mang li mt s li th so vi vc-xin khng nguyn c in (tc l gim c lc, gy cht ton b v vacxin tiu n v). Chi ph thp, d dng sn xut trn quy m cng nghip, d dng bo qun lu tr cng nh vn chuyn. p ng min dch c th lu di v mnh m, cng ging nh cc vacxin truyn thng khc nhng vacxin DNA th khng gy nguy c nhim trng c hi. c, cc p ng min dch t c bit n sau khi tim plasmid, nhng khng c biu hin no cho thy n khc bit vi nhng ghi nhn ng vt khc.

2. Cu trc Plasmid 2.1 Kim sot cc yu t

Trnh t iu khin phin m trong cc vect tiu chun (promoter, enhancer, intron, tn hiu b sung, vv) dng nh lm vic hiu qu ng vt thy sn. Cc promoter khc nhau quyt nh kh nng iu khin biu hin ca gen l trong cc m c. (bng 1).

Bng 1. Cc promoter th nghim trn m c

Hiu qu ca chng khc nhau ng k, nhng ni chung cc cytomegalovirus (CMV) ngay lp tc cho kt qu tt nht. Cc yu t phin m khc trong vc-xin DNA khng c gi tr trong c, nhng c nhng du hiu cho thy trong nghin cu in vitro intron ng vt c v khng phi lc no cng c th p dng cho t bo c. Cc tnh nng ty chn ca cc khung vector nh epitope Th khng lin quan n vic b sung T , qu trnh min dch CpG, hoc cho nhng gen th hin tng ng nh cc cytokine hoc cc phn t costimulatory, cui cng c b sung vo vc-xin DNA. Trnh t kim sot phin m trong cc vect tiu chun (promoter, enhancer, intron, tn hiu b sung, vv) dng nh c hiu qu thy sn./

2.2 M ha gen khng nguyn

Bt k gen m ha cho mt loi protein ca mm bnh gy ra mt p ng min dch bo v vt ch c th c s dng trong cc vc-xin DNA, min l vic s dng codon ca gen cho php th hin trong cc t bo c. to ra cc phn ng min dch, u tin khng nguyn t bo B phi c kch hot. V vy, cc hot cht tit ra ca gen ngoi lai c th nh hng n vic sn xut khng th. Tuy nhin, phn ng min dch l c th ngay c khi khng nguyn khng c tit ra. Trong trng hp khng nguyn khng tit ra, t bo B-khng c kch hot y , tr khi khng nguyn c gii phng t cc t bo c bin np. Chng hn nh phn gii bi khng nguyn gy c t bo t bo lympho T (CTL) Do vc-xin DNA c th bao gm mt peptide tn hiu tit trc tip khng nguyn c m ha, nhng qu trnh khng xy ra tuyt i.

2.3 DNA l mt phn t gy kch thch interleukin-2 p ng min dch mnh cytokin cho c :

Phn ng min dch mnh gy ra bi chng nga DNA khng ph thuc hon ton vo biu hin ca khng nguyn. Trong thc t, n l cc trnh t ADN ngn c cha dinucleotides CpG unmethylated trong iu kin c th c th trc tip kch thch t bo h min dch nh n bo, t bo B, i thc bo v t bo ui gai(dendritic te bao mien dich).

Hnh 3. p ng ch tit cytokine ca i thc bo v chc nng ca cc cytokine do i thc bo ch tit

Nhng hot cht tit ra kch thch h min dch c gi l CpG-S. trong iu kin bt li, cc hat CpG khc trung ha hoc chn kch hot min dch bi cc CpG-S motifs c gi l cc CpG-N motifs. Vect DNA plasmid c s dng tim chng cho c chc chn cha CpG-S v cc CpG-N trong chui. N c th l mt khung ti u ha CpG , c cha mt s lng ti u ca cc CpG-S c hiu cho c v s gim st ca cc m-tp-N CpG c th ci thin p ng min dch c, v c th cho php qun l vi lng nh hn ca DNA.

3. Phng php

3.1 Tim 3.1.1 V tr tim

Plasmid DNA c tinh sch trong mt th tch nh ca nc mui hoc dung dch m

c s dng tim c bn sn, bn di hoc gn vy lng.

Tim vo mang kt qu cho thy khng thy hot ng ca gene ch th , trong khi n tn ti phc mc .

V d :

chut: Tim trc tip vo c bp (IM) mi tim s gy ra p ng ch yu loi Thl, vi t l IgG2a : IgGl cao, interferon IFN- v mc thp interleukin IL(-4). Tim vo gene biu b s gy ra p ng dng Th2, biu hin u th ca IgG1 i vi IgG2a, km hn IFN- v cao hn IL-4. Tim DNA vo trong da (ID) c th l biu hin loi Th1 hoc l Th2.

c, hu ht khng c thng tin tn ti trn nhng tc ng khc nhau ca phn ng min dch,do nghin cu hn ch v c tnh tng i km ca h min dch ca c.

3.1.2 Liu lng v th tch:

Liu lng ln thng lm s gim hot ng ca gene ch th, nhng t c s bin i gia nhng c th c. Liu in hnh cho c : 1-50 g DNA Trong th tch khong : 10-50 l DNA Tuy nhin, vi liu lng thp hn nhiu cng c hiu qu. Liu lng ti u ca DNA c th thay i ty theo loi v kch thc ca ng vt, nhng n khng t l thun i vi trng lng ca c. Ngoi ra, tnh trng sinh l ca ng vt c nh hng n mc biu hin. VD: Nhng con c chp con giai on pht trin nhanh th mc biu hin hot ng ca Chloramphenycol acetyl transferase (CAT) cao hn rt nhiu so vi c trng thnh.

iu th v l, biu hin ca gene ch th Luciferase (c iu khin bi promoter CMV ) hot ng c cao hn trong c bp chut s dng vi cng 1 liu lng DNA. Khng bit iu ny c phi do s tng ln n nh ca cc protein luciferase ca c so vi t bo ng vt c v, ti mt mc biu hin cao hn, hoc mt hiu qu bin np tt hn .
Trong thc t, liu lng thp nh 0.1 g DNA cho mi con c c hiu qu nh 10 g gy bo v min dch. iu bo trc tim nng ca ng dng vaccine DNA trong tri c.

Bng 3. PP tim v liu DNA th nghim trn m c

3.1.3 V tr biu hin ca gene l

C s khc bit r rng trong cc ti liu lin quan ti v tr biu hin ca gene l sau khi tim vo c th vector plasmid.
nhng loi c ln, biu hin ca gene ch th dng nh ch yu gii hn v tr tim. Bin np vo t bo khng phi t bo c, c bit l t bo trnh din khng nguyn chuyn nghip c th gp phn hon thin p ng min dch. i vi c nh, biu hin ca gene ch th luciferase c tim vo c bp c pht hin trong cc c quan khc nhau,c gan, l lch v thn nhng mc cao nht l trong c.

S khc nhau hu ht gia c nh v c ln c kh nng l do s pht tn nhanh chng cc DNA c tim vo c c nh, nhng c th c nhng bin th trong Protocol , nhy ca th nghim v mu ng vt s dng.
Ngoi ra cc promoter c th nh hng ti v tr biu hin gene l, nh promoter CMV c s dng trong cc nghin cu ny.

3.1.4 Thi gian biu hin v hiu qu trn m c

Tim vo c bp IM c hiu qu trong thi gian di v mc biu hin gene tn ti trong nhiu nm (bng 3).

Bng 3. thi gian v hiu qu hoat ng ca gene ch th trn m c

Nghin cu biu hin ca luciferase

Vi lucifer gene, Anderson v cng s thy c s gim bt hot tnh ca cc emzyne sau 7 ngy, nhng iu ny khng ng cc enzyme khc. chut,nhng t bo bin np luciferase khng b ph hy bi CTL bi v khng c ( hoc rt t) p ng min dch sinh ra chng li protein luciferase do s kch thch min dch km , biu hin c nhn thy trong 2 nm gn nht.

Nghin cu biu hin ca gen lacZ

chut, hu ht cc t bo c c biu hin ca protein min dch u ph hy trong khong thi gian t ngy 10 v 20.

c,qu trnh loi b ny cng xy ra nhng chm hn. S lng galactosida biu hin trong t bo c c gim t gia ngy 21 v 70 sau khi tim gene lacZ, nhng vic s dng pp ng tim (Coinjection) vi gene GM-CSF ca chut chng minh c s nh du trn t l bin mt ca cc si c c bin np.

Nghin cu m hc vi gene ch th lacZ cho thy gii php tim DNA tinh khit vo c c hi vn khng lm tn thng m vnh vin. Xut hin vim ch tim sau khi tim, nhng khng c du hiu b hoi t, v cc m c ti sinh m khng li so vo 28 ngy sau khi tim.

3.1.5 La chn thay th kim tim

Vic thay th kim tim, gene s c chuyn bng cch s dng ht bn ph, tuy nhin, kt qu khng tt nh s dng kim tim c hai mc v t l biu hin trn gene ch th ng vt.
Ngay c khi hiu qu k thut c ci thin, cng khng chc l cng ngh ny s c s dng rng ri trong nhng tri c bi v n c gi thnh cao v hn ch v tnh thit thc. ( VD: DNA b ri ra khi cc ht vng nano trong MT m).

3.2 Cho n vaccine v ngm vaccine

C li th min dch lin quan n pp cho n vaccine , nh biu hin min dch ca h nim mc.
u im: D lm v r tin Chng khng gy stress cho vt nui. Khuyt im: Khng th xc nh c lng DNA em hiu qu thp hn so vi tim hoc ngm trong vic nga bnh c.

Ngm vaccine

PP ngm vaccine : vt nui c nhng vo hoc c phun vi dung dch vaccine m c. u im: So vi pp tim, s dng t thao tc v dng ngay c cho c rt nh Nhc im: Cn nhiu dd vaccine hn , c bit l i vi c ln.

So snh gia pp cho n v ngm vaccine

Hiu qu ln hn ca pp ngm vaccine so vi cho n l do khng th c hp thu qua da hoc qua mang tt hn so vi rut, cng nh l tht thot lng vaccine qua qu trnh tiu ha. Mt s th nghim gn y ch ra rng c trn da v mang u tham gia hp thu trong pp ngm, da( phm vi hot ng) c u th hn. Ti thi im bi bo co ny c xem xt, khng c ghi nhn vaccine DNA no dng cho c qua pp cho n hoc ngm. Bng vic s dng cc gene ch th, chng ti khng bao gi quan st thy cc mc biu hin ca gene sau khi ngm hay qua vic cho n cc plasmid DNA tinh sch c nh. Mc d, s kt hp ca DNA vi tc nhn bin np nh l liposomes hoc microcarries c th mang li kt qu trong bin np ca c.[Heppel et el.,kt qu cha c cng b]. Gi thuyt rng hai pp ngm hay cho n DNA vaccine c th kh thi v c hiu qu.

4. p ng min dch vi DNA vaccines

Trc ht, l kh nng min dch khng c hiu
B mt nim mc v da, s a dng ca bch cu (thc bo/ i thc bo, bch cu ht v nhng t bo gy c khng c hiu) v c cht khng c hiu ( nh lysozyme, b th, interferon, protein C-ceative, transferring v protein ngng kt t bo ). H thng min dch bm sinh c vai tr rt quan trng c.

Th hai l kh nng min dch c hiu hoc mc phi

Min dch dch th Min dch t bo

Kh nng min dch c hiu hoc mc phi

Ph thuc rt nhiu vo T v B-lymphocytes. ng vt c v, kh nng min dch dch th trong cc loi vt nui c bit lin quan n vic nhn dng v lu thng bt buc ca khng nguyn ha tan trong cc t bo B, sau p ng bng cch sn xut v tit ra khng th khng nguyn c hiu. Mt khc, kh nng min dch ca t bo c hiu gin tip lin quan ti vic nhn dng : t bo T nhn din khng nguyn hin din trn b mt t bo (trong lin kt ha hc ) gn vi cc phn t histocompatibility phc tp (MHC), khi ng qu trnh cm ng ca t bo lympho T gy c tb (CTL) v t bo T tit ra cytokines. ng vt c v, CD4 v CD8 t bo T nhn ra khng nguyn hin din bi cc phn t MHC II v MHC I tng ng. Trong trng hp ny DNA vaccine c hiu qu trong vic kch thch p ng min dch ca t bo v th dch. Nh vy, chng th hin nh l mt vaccine sng( live vaccine).

4.1.p ng khng th

DNA vaccine cho php biu hin khng nguyn ngay ti ch. Cc gene l, c bit l nhng gene c ngun gc virus, sau khi c bin i (sau dch m v qu trnh ghp v dung np vi cc khng nguyn c sn xut trong sut qu trnh truyn nhim t nhin) c c cu to thch hp sau c th c trnh din vi h thng min dch. S hin din ca cc khng nguyn c lin quan n epitop h thng min dch c th d dng t c vi DNA vaccines hn l nhng loi vaccines khc, nh l vaccines bt hot hay tiu n v, ni m cc epitop i xng c th b mt. ng vt c v, hiu qu ca cc p ng khng th c gy ra bi vic tim DNA vaccines ph thuc vo mu ng vt v khng nguyn. p ng th dch thng tn ti lu di chut, nhng xut hin l ngn hn ng vt linh trng.

Biu hin trn c

Khng th khng galactosidase c pht hin trong c vng sm nht l 7 ngy sau khi tim DNA m ha lacZ. c hi vn, khng th vi protein G ca VHSV c tm thy 23 ngy sau khi tim vo mt plasmid m ha gene G. Nng ca khng th huyt thanh t mc cao nht trong 3 n 8 tun v vn cn cao trong vi tun sau, mc d s lng cc t bo khng th hnh thnh suy gim nhanh chng . Trong thc t, Russel et al. cho thy sau khi tim IM mt vetor DNA m ha galactosidase, chuyn i huyt thanh(seroconvert) c vng thm ch cn d dng hn so vi chut.

ng thi, Lorenzen v cng s chng minh rng cng hot ng b v hiu ha huyt thanh c thu thp c hi vn c min dch vi DNA plasmid m ha gene G ca VHSV l s khc bit khng c ngha trc v sau khi th vi virus sng.

Nhng kt qu ny cho thy p ng th dch ti a gn nht l gy ra mt liu vaccine DNA duy nht.

4.2. p ng min dch t bo

Trong tng hp in vivo khng nguyn bng vic tim vaccines da trn DNA l mt c tnh mong mun bi v n bt chc mt kh nng ly nhim t nhin bng cc tc nhn gy bnh ni bo bng s trnh din khng nguyn i vi MHC lp I. S hnh thnh kh nng ghi nh p ng ca tb lympho T c chng minh vi tnh a dng ca cu trc plamid. C du hiu gin tip cho s hot ha min dch ny l mt phn ca h thng min dch c.

Hn na, s iu chnh bin hin ca MHC lp II bng vaccines DNA c th hin c, ngay ti ch tim.

4.3. Bo v chng li thch thc sng

Mc d p ng min dch DNA vaccines trn c cha c c s, n gip bo v rt tt cc th nghim quy m nh.
C hi vn c tim DNA th hin protein G ca IHNV c tr s RPS khong 75%. C hi c tim DNA khng VHSV c tr s RPS ln ti 97 v 78%. Trong thc t, , chng nga th ng c hi vn nh( khong 700 ngy tui) vi huyt thanh t c trng thnh c chng nga DNA m ha gene G ca IHNV hoc VHSV trc mang li s bo v DNA 100% .

5. S an ton ca vacxin DNA i vi c

ng vt c v , vc xin DNA c th thc y s sn xut khng th ADN i lp v tc nhn khng gy bnh. Trn thc t , phn ng t min dch chng li DNA ca vt ch khng th quan st thy cc c tnh ca vt nui. S ha hp ca plasmid DNA trong b gen vt ch cha c biu hin c , v s hin din ca sn phm gen truyn nhim hoc bn cht DNA trong tuyn sinh dc hoc trng t thuc tim c cha c c nh. S ha hp ca plasmid DNA trong b gen vt ch khng chc chn xy ra v ri ro xut hin gen t bin c kh nng xy ra , iu khng tt , n c c lng thp hn 1000 ln so vi tc t bin t bn trong.

5.1 Hu qu tt yu ca thuc tim DNA trong c

Sau khi tim DNA tip tc tn ti vo bn trong c c phn khc vi ng vt c v. Tri vi cc pht hin chut , ng vt khc plasmid DNA bt u thoi ha ch sau 1 vi gi sau khi tim vo, DNA cung cp cho c th n nh hn. Sau mi th nghim, s xm nhp ca plasmid c th c nhn thy khong 63 ngy sau khi tim . Vi phng php PCR , plasmid DNA cn c nhn ra xm nhp ti mi thi im th nghim (sau khong 70 ngy sau khi tim) nhng khng trong phn cht khc ca chui m. S tn ti lu di ca plasmid DNA nh vo lin kt vi cc biu hin lu di ca khng nguyn c m ha.

5.2 An ton cho ngi tiu dng

Ri ro tim n duy nht i vi ngi tiu dng l lng plasmid DNA vn cn tn ti trong tht c. Hu ht plasmid DNA tim vo c s suy bin trong thi gian sau khi n, vi thng hoc vi nm sau . Ngay c khi n c va c tim, s lng DNA plasmid tiu th s l rt nh so vi tng s axit nucleic t tt c cc ngun, bao gm c vi khun v virus thng n vo hng ngy. Hn na vc xin th y c cp php l sn phm ca cc tc nhn gy bnh bt hot hoc suy yu cha lng ln axit nucleic, ch yu l t b gen ca mm bnh, nh mt thnh phn bnh thng.

T thi im ny ca vc-xin DNA xem phi l khng c nguy him hn cc loi vcxin hin ang c s dng.

5.3 Nhng ri ro vi loi c khc v mi trng:

Hu ht cc plasmid tim vo c th khng vo c nhn t bo s nhanh chng b suy thoi bi nucleases trong khng gian ngoi bo v trong t bo cht. Hn na, DNA c tn ti trong cc t bo truyn nhim s b suy thoi khi cc t bo ny b ph hy bi cc CTLs khng nguyn c hiu. Tuy nhin, mt t l nh ca DNA c th tn ti trong thi gian di trong m c. Nhng ri ro tim tng i vi mi trng khi thuc b phn tn hoc thot khi trang tri l rt nh.

6. Quan im v vacxin DNA trong nui trng thy sn

Vic tim DNA c chng minh l rt hiu qu v c kh nng s l phng php u tin c pht trin cho chng nga DNA thng mi ca c. Tuy nhin cc pp khc chuyn vc-xin axit nucleic cho c rt nh bng pp cho n hoc ngm vaccine s thch hp hn trong trng hp ny.
Vacxin thng khng hot ng trc khi c c kh nng min dch, mc d vacxin DNA hot ng tt khi tim vo con non ca 1 s loi ng vt khc. Cng ngh vacxin DNA ra i cn c s h tr cng nh chp nhn ca nh nc. cng l mong mun t c s tha nhn ca cng chng v cng ngh, c bit l cc trang tri c, khch hng chnh l ngi nui c v ngi tiu dng thy hi sn.

7. Kt lun

S pht trin vaccine DNA c vn cn trong giai on u . Tuy nhin cc th nghim vi nhng khng nguyn mu cho nhng kt qu y ha hn, vi s biu hin ca c hai p ng min dch t bo v min dch dch th. Hn na, hiu qu trong nhng thch thc vi mm bnh sng c chng minh vi hai loi bnh quan trng do virus gy ra m cha tn ti dng vaccine thng thng trn th trng. Nhu cu ngy cng tng ca vaccine p ng cho thy sn v cng vi s pht trin ca nui trng thy sn l ng lc pht trin loi sn phm mi ny. DNA vaccine c th ph v nhiu hn ch lin quan n pp chng nga truyn thng, nhng cui cng, li ch ca DNA vaccine ph thuc vo s cn bng ca iu kin thun li, s an ton, v chi ph. Hai vn u tin khng quan trng i vi s pht trin ca DNA vaccine, quan trng l chi ph nht l i vi cc loi c c gi tr thng phm thp.

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