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Mc lc
Gii thiu v bnh vim gan B Virus vim gan B Gii thiu v vaccine Quy trnh sn xut vaccine vim gan B ti t hp Quy trinh sn xut vaccime vim gan B t huyt tng ngi lanh
Bnh vim gan siu vi B c th phat hin sm mt cach d dang nh vao cac xt nghim mau n gin. Vic phat hin sm vim gan B mn tnh gip gia tng hy vng ngn nga bin chng x gan va ung th gan bng cach kham sc khe inh k va iu tri vi nhng loi thuc c tri mi.
B gene gm mt DNA c phn gp i, khong 3.2 kilo cp base, to nn cac antigen: 1. HBsAg (khang nguyn b mt): thuc lp v ca HBV dng trong xt nghim mau bit c HBV trong c th. 2. HBcAg (khng nguyn li): thuc lp li ca HBV - dng bit HBV ang phat trin. 3. HBeAg (khang nguyn ni sinh): nu c trong mau bnh nhn ang c kh nng ly rt cao. Cu trc b gene vim gan B 4. gen X : c th la nguyn nhn to ung th gan. 5. gen P.
Hepatitis B virus
Vacine l g ?
Vaccine: l ch phm c tnh khng nguyn dng to min dich c hiu ch ng, nhm tng sc khng ca c th i vi mt s tc nhn gy bnh c th.
Cc dng vaccine
Phn loi theo ngun gc: Vaccine truyn thng Vaccine tai t hp Vc-xin kinh in
Vc-xin bt hot Vc-xin sng, gim c lc Cac "toxoid
Vaccine DNA
Loi k thut c s dng bo v sinh vt chng li bnh bng cch tim trc tip DNA vo c th sinh vt To ra mt ap ng min dich
thit k vector.
Bc 2: Nhn dng plasmide v biu hin
cu trc vector.
Bc 3: Phn tch v xc inh gen ch.
. in bin np + y la k thut hiu qu nht bin np vi khun. - Hai thng s quan trng ca phng thc ny l Loi t bao vi khun Tn s xung in cn thit.
Phng phap in bin np c mt s u im sau: Hiu sut bin np cao. C th dng mt th tch dich t bao nh. Th tch dich t bao khong 20 L C th cho hiu sut khong 109 th bin np. Nhc im ca phng phap nay la i hi thit bi bin np t tin.
Ha bin np y la phng thc kinh in bin np plasmid vao t bao E. coli. Cac t bao c trong dung dich CaCl2 tr thanh t bao kh bin gip cho chng d tip nhn plasmid.
Gm 2 bc : Chun bi t bao kh bin Cy plasmid vao bng shock nhit
Chuyn 1 mL dch nui sang 100 mL mi trng LB lng mi, tip tc nui lc 37 0C, 200 vng/pht Sau 2 3 gi, khi OD600 t 0,375
Dch t bo sau khi ly tm 6000 vng/pht 40C trong 5 pht c ra 2 ln bng 3 mL CaCl2 60mM sau dch ha tan c gi trong a 30 pht.
Bin np DNA plasmid vao t bao E. coli JM109 bng phng phap sc nhit
B sung 5 L DNA vector PMD 18 T vao ng eppendorf cha 50 L t bao kh bin va gi trn a 20 pht
T bao sau bin np c nui trong 600 L mi trng LB (lc 200 vng/pht 370C, 60 pht)
Tach chit va tinh khit ADN ca plasmid (plasmid DNA extraction and purification)
Tin hnh a.
Cho vo ni nhit 42oC trong vng 30s Sau a trong vng 20 pht.
Ht sephadex
Thnh phn:
Mt liu 1ml bao gm:
- Khang nguyn b mt virt vim gan B tinh khit..............20 g - Hydroxyt nhm ...................................0,5 mg - Thimerosal ................................0,01% (W/V)
Bc 1. thu thp huyt tng ngi mang HbsAg c hiu gia cao (>104-105). Bc 2. to huyt thanh t huyt tng (ta vi (NH4)2S04 30% nng cui), nhit phng 30 pht, ly tm 5000v/pht trong 20 pht/40c. b cn, ly dich ni. Bc 3. ta vi sulphatamol 50% (nng cui). qua m nhit phng, ly tm nh trn. B nc ni, cn
Bc 4. ha cn vi KBr, khuy u t nht 1 gi nhit phng, lng. loi b cn bng phu lc ht chn khng. Bc 5. iu chnh dich c t trng 1,32g/cm3 (thm bt KBr) Bc 6. chun bi my siu li tm HITACH55pp v onal rotor RPZ35T.
Bc 7. cho cac dung dich vao rotor o Nc ct d=1: 500ml o Dung dich KBr d = 1,3g/cm3: 400ml o Dung dich mu d=1,32g/cm3: 1000ml o Ly tm 25000 v/pht trong 24 gi/20oc. Ly mu tng o Phn on 30ml va xac inh HsbAg trong chng. Trn cac phn on c HsbAg dng tnh cao. o Siu li tm 3 ln. Bc 8. c c n th tch 300ml.
Bc 9. siu li tm tng vng vi sacaroza graddient (2 ln). o Dung dich Nacl 1 M :500ml o Dungdich mu :300ml o Dung dich sacaroza 10% : 300ml o Dung dich sacaroza 20% : 300ml o Dung dich sacaroza 30% : 300ml o Dung dich sacaroza 40% : 300mk o Li tm 25000 v/pht trong 20 gi/10oc. B qua 300 ml u, ly mu tng phn on 30 ml va xac inh HsbAg trong chng. Trn cac phn on HsbAg c hiu gia cao.
Bc 10. c c dich (V=300ml) Bc 11. bt hot HsbAg tinh khit 60oc/10 gi. Lc qua mang lc 8um v 0,45um. Bt hot tip vi formaldehyt (nng cui 1:2000) trong 96 gi/37oc. Bc 12. c c va thm tch loi formaldehyt (bng Pellicon cassette). Kim tra tinh sch ca HbsAg (in di, knh hin vi in t). kim tra HBV-ADD tn d (PCA) Bc 13. kim tra ham lng HbsAg (o mt quang va ELISA)
Bc 14. lc v trng qua mang lc 0.22um Bc 15. hp ph HbsAg vao gel Al(OH)3 va pha vacxin vi ham lng HbsAg 20ug/ml. nng cui ca nhm: 300ug/ml. thm cht bo qun thimerosal nng 1:10000 Bc 16. lc u cac chai trong 1 tun, 2 ln/tun
Bc 17. ly mu ban thanh phm kim tra v khun, an toan, cng hiu, cht gy st va cac ch s ha hc. Bc 18. ng l, dan nhn, ng gi Bc 19. ly mu kim tra cht lng vacxinvoo khun, an toan, cng hiu, cht gy st va cac thanh phn ha hc. Bc 20. xuaatss xng vacxin.
San phm vaccine vim gan B c iu ch t huyt tng ngi lnh mang khng nguyn b mt virt vim gan B (HBsAg) khng c triu chng lm sng.
Thnh phn: Mt liu 1ml bao gm: - Khang nguyn b mt virt vim gan B tinh khit........20 g - Hydroxyt nhm ..................0,5 mg - Thimerosal ...............0,01% (W/V)
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