TIU LUN: CNSX Vacxin Va khang sinh Tai: CNSX Vacxin vim gan B

GV hng dn: GV Nguyn Thi Thng Thng Nhm SV thc hin : Trn Trung Hiu (Leader)036 Nguyn Quang Huy 042 Cn Thi Khanh Huyn 044 Hoang Thi Hng 049 Hoang Thi Yn 098

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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 B la gi?

Vim gan siu vi B l bnh gan him ngho thng gp nht trn th gii do siu vi khun B gy ra. Vit Nam l mt trong nhng quc gia c t l b nhim siu vi B cao nht th gii. nc ta c khong 10 triu ngi ang b nhim cn bnh ny.

Ti sao vim gan B c coi l cn bnh nguy him?

V y l cn bnh ly lan lng l, tin trin m thm. a s ngi bi nhim siu vi B u khng bit l mnh bi nhim v c th v tnh ly sang ngi khc qua ng mu. a s ngi ln bi nhim u c th loi tr siu vi B d dng. Tuy nhin a s tr s sinh v tr em bi nhim u khng th loi tr c siu vi khun ny v s tr thnh ngi mang siu vi B mn tnh. C 4 ngi mang siu vi B mn tnh s c 1 ngi bi thit mng do x gan v ung th gan trong sut cuc i ca h. Siu vi B c th thm lng tn cng gan trong nhiu nm m bnh nhn khng h hay bit. n khi bnh nhn cm thy cn i khm bnh th thng bnh vo giai on cui. Nhiu bnh nhn cht do ung th gan vn khng bit th phm chnh l siu vi khun B.

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.

Tinh hinh nhim HBV trn th gii

Virus vim gan B

Virus vim gan B( HBV) thuc h Hepadnaviridae la nhng virus c kch thc nh. C 3 loi tiu th: hinh cu nh (d=22nm), hinh cu ln (d=42-45nm) va hinh cu nh va hinh ng.

 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

Cau tao bo gen HBV

S xm nhp vao t bao

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

DNA tai t hp t Saccharomyces cervisiae

Bc 1: Gii trnh t chui, la chn v

thit k vector.
Bc 2: Nhn dng plasmide v biu hin

cu trc vector.
Bc 3: Phn tch v xc inh gen ch.

 Bc 4: Bin tnh t bao va a plasmide tai t

hp vao. Bc 5: T bao sinh ra Protein, chit tach protein.

Bc 6: Kim tra cht lng protein. Bc 7: Phn loi v tinh sch protein

Bc 8: Thu nhn thnh cng protein ch.

Gii trinh t chui

Thc hin PCR
Ly mu va bo qun Ly trch va tinh sch Khuch i DNA bng phn ng PCR trong may lun nhit Kim tra va phat hin sn phm khuch i trn gel Agarose Thc hin phn ng ct in di va phn tch kt qu

. 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

Chun bi t bao kh bin

Cy ria trn mi trng LB(Luria-Bertani) c (nui qua m, 370C)
ly mt khun lc n, nui trong 3 mL mi trng LB lng, (lc qua m 37 0C, 200 vng/pht)

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

Chuyn dch sang 6 ng ly tm 15 mL c gi lnh trong a

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.

B phn ni, ha tan phn cn trong 300 L CaCl2

T bao kh bin c chia vo cc ng eppendorf, mi ng 100 L em bo qun nhit -800C

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 c sc nhit 420C trong 30 giy sau gi trn a 2 pht

T bao sau bin np c nui trong 600 L mi trng LB (lc 200 vng/pht 370C, 60 pht)

Cy tri 100 L dich ln mi trng LB rn c b sung ampicillin (100mg/L) (370C, qua m)

Thu nhan dong can

Hind III EcoR1 Cat plasmide Chuyen gen vao plasmide bieu hien (pcDNA 3.1) ien di Ket qua

Tach chit, thu nhn DNA ca plasmid t E.coli

Pha v mang t bao (lysis method) Ly tm (centrifugation) vi enzym Hind III v EcoR1

Tach chit va tinh khit ADN ca plasmid (plasmid DNA extraction and purification)

a Plasmid vao Saccharomyces cerevisiae

To mi trng kh bin:
Mi trng LB. Cho dung dich CaCl2 vo. Nui cy Saccharomyces cerevisiae trn mi trng.
Chi Saccharomyces

Bin np bng phng phap sc nhit Mi trng LB kh bin t trong a lnh.

Cho plasmid pcDNA 3.1+ vo mi trng.

Tin hnh a.
Cho vo ni nhit 42oC trong vng 30s Sau a trong vng 20 pht.

Nui cy Saccharomyces cerevisiae

Nui cy nm men trong mi trng 250C lc 250 rmp. Khi o OD600 khong 0.4 - 0.6 dng lc Cho dung dich SDS. Ly tm 2500 rmp trong 5 pht, 40C. Lc tch kt ta thu c dich protein.

Tach va tinh sch protein

Mun thu nhn c cac protein nguyn th tc la protein c tt c tnh cht t nhin c trng ca n, cn s dng cac bin phap khac nhau. -Nhit -Nng proton (pH) -Tc nhn ha hc

Tinh sch protein

1.Loi b tp cht

-Phng php thm tch (dialysis)

-Phng php bin tnh chn lc

- Phng php kt ta phn on bng mui trung

tnh hoc cc dung mi hu c, -Cc phng php sc k trao i ion, in di, phng php lc gel...

Phng phap sc k lc gel

Cc phn t ln khng thm vo trong ht sephadex

Ht sephadex

Cc phn t nh thm vo trong c ht sephadex

Hot ng ca lc phn t sephadex.

Tch cc phn t theo kch thc bng sc k lc gel.

Sn phm vacxin ti t hp HBsAg

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)

Qui trinh sn xut vacxin vim gan b t huyt tng ngi

Vacxin nay thuc loi vacxin di n vi. phn khang nguyn c tnh sinh min dich la protein v ca virus vim gan b ( HbsAg) c tach va tinh ch t huyt tng ca nhng ngi mang virus vim gan B nh cac k thut tach thng thng nh kt ta, c c, ly tmvi 20 bc nh sau:

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)

THE END
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