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8/4/2019 Tieu Luan Viem Gan B
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TIU LU N: CNSX VacxinVa khang sinh Tai: CNSX Vacxin vim gan B
GV hng dn:GV Nguyn Th Thng ThngNhm SV thc hin :Trn Trung Hiu (Leader)036Nguyn Quang Huy 042
Cn Th Khnh Huyn 044Hong Th Hng 049Hong Th Yn 098
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Mc lc
Gii thiu v bnh vim gan B
Virus vim gan B
Giithiuv vaccine Quy trnh snxut vaccine vim gan B ti
thp
Quy trnh sn xut vaccime vim gan Bt huyt tng ngi lnh
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Bnh vim gan Bla gi?
Vim gan siu vi B l bnh gan himngho thng gp nht trn th gii do siu
vi khun B gy ra. Vit Nam l mt trongnhng quc gia c t l b nhim siu vi B caonht th gii. nc ta c khong 10 triungi ang b nhim cn bnh ny.
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Ti sao vim gan B c coi l cn bnhnguy him? V y l cnbnh ly lan lng l, tin trin m thm. a s
ngibnhim siu vi B u khngbit l mnhbnhim v cth v tnh ly sang ngi khc qua ng mu. asngiln
bnhimu c thloitr siu vi B d dng. Tuy nhin astrs sinh v tr em b nhim u khng th loi trc siu vikhun ny v strthnh ngi mang siu vi B mn tnh.
C 4 ngi mang siu vi B mn tnh s c 1 ngibthitmngdo xgan v ung th gan trong sutcucicah. Siu vi B cththmlngtn cng gan trong nhiunm mbnh nhn khngh hay bit. n khi bnh nhn cm thycni khm bnh ththngbnh vo giai oncui.Nhiubnh nhn cht doung th gan vn khngbitthphm chnh l siu vi khun B.
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Bnh vim gan siu vi B c th pht hinsm mt cch d dng nh vo cc xt nghimmu n gin. Vic pht hin sm vim gan B
mn tnh gip gia tng hy vng ngn nga binchng x gan v ung th gan bng cch khmsc khe nh k v iu tr vi nhng loi
thuc c tr mi.
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Tnh hnh nhim HBV trn th gii
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Virus vim gan B
Virus vim gan B( HBV)thuc h Hepadnaviridae lnhng virus c kch thcnh.
C 3 loi tiu th: hnh cunh (d=22nm), hnh cu ln(d=42-45nm) v hnh cunh v hnh ng.
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B gene gm mt DNA c phngp i, khong 3.2 kilo cp
base, to nn cc antigen:
1. HBsAg (khng nguyn bmt): thuc lp v ca HBV -dng trong xt nghim mu
bit c HBV trong c th.
2. HBcAg (khng nguyn li):
thuc lp li ca HBV - dng bit HBV ang pht trin.
3. HBeAg (khng nguyn nisinh): nu c trong mu bnh
nhn ang c kh nng ly rtcao.
4. gen X : c th l nguyn nhnto ung th gan.
5. gen P.
Cu trcb gene vim gan B
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Cau tao bo gen HBV
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S xm nhp vo t bo
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Vacine l g ?
Vaccine: l chphmc tnh khng nguyn
dng tomindch
c hiu ch ng,nhm tng sc khng ca c th i
vi mt s tc nhngybnhcth.
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Cc dng vaccine
Phn loi theo ngun gc:
Vaccine truyn thng
Vaccine ti t hp
Vc-xin kinh in
Vc-xin bt hotVc-xin sng, gim c lc
Cc "toxoid
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Vaccine DNA
Loi k thut c s dng bo vsinh vt chng libnhbng cch tim
trctip DNA vo cth sinh vt To ra mtpngmindch
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DNA ti t hp tSaccharomyces cervisiae
Bc 1: Gii trnh t chui, la chn v
thitk vector.
Bc 2: Nhn dng plasmide v biuhin
cu trc vector.
Bc 3: Phn tch v xc nh gen ch.
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Bc 4: Bin tnh t bo v a plasmide ti thp vo.
Bc 5: T bo sinh ra Protein, chit tchprotein.
Bc 6: Kim tra chtlng protein.
Bc 7: Phn loi v tinh sch protein
Bc 8: Thu nhn thnh cng protein ch.
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Gii trnh t chui
Thc hin PCR Ly mu v bo qun
Ly trch v tinh sch
Khuch i DNA bng phnng PCR trong my lunnhit
Kim tra v pht hin sn
phm khuch i trn gelAgarose
Thc hin phn ng ct
in di v phn tch kt qu
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. in bin np
+ y l k thut hiu qu nht bin np vikhun.
- Hai thng s quan trng ca phng thc
ny lLoi t bo vi khun
Tn s xung in cn thit.
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Phng php in bin np c mt s u im
sau: Hiu sut bin np cao.
C th dng mt th tch dch t bo nh. Th
tch dch t bo khong 20 L C th cho hiu sut khong 109th bin np.
Nhc im ca phng php ny l i hi
thit b bin np t tin.
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Ha bin np
y l phng thc kinh in bin npplasmid vo t boE. coli. Cc t boc trong dung dch CaCl2 tr
thnh t bo kh bin gip cho chng dtip nhn plasmid.Gm 2bc :
Chun b t bo kh bin Cy plasmid vo bng shock nhit
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Chun b t bo kh bin
ly mt khun lc n, nui trong 3 mLmi trng LB lng, (lc qua m 37 0C,200 vng/pht)
Cy ria trn mi trng LB(Luria-Bertani) c (nui qua m, 370C)
Chuyn 1 mL dch nui sang 100 mLmi trng LB lng mi, tip tc nui lc 37 0C, 200vng/pht Sau 2 3 gi, khi OD600t 0,375
Chuyn dch sang 6 ng ly tm 15 mL c gi lnh trong
B phn ni, ha tan phn cn trong 300 L CaCl2
T bo kh bin cchia vo cc ng eppendorf, mi ng 100 Lem bo qun nhit -800C
Dch t bo sau khi ly tm 6000 vng/pht 40C trong 5 phtc ra 2 ln bng 3 mL CaCl260mM sau dch ha tan c gi trong 30 pht.
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Bin np DNA plasmid vo t bo E. coliJM109bng phng php sc nhit
T bo c sc nhit 420C trong 30 giy sau gi trn 2 pht
B sung 5 L DNA vectorPMD 18 Tvo ng eppendorf cha 50 L tbo kh bin v gi trn 20 pht
T bo sau bin np c nui trong 600 L mi trng LB(lc 200 vng/pht
370C, 60 pht)
Cy tri 100 L dch ln mi trng LB rn c b sung ampicillin(100mg/L) (370C, qua m)
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Thu nhan dong can
Cat plasmide
Chuyen gen vao plasmide
bieu hien (pcDNA 3.1)
ien di
Ket qua
Hind IIIEcoR1
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Tch chit, thu nhn DNA ca plasmidt E.coli
Ph v mng t bo (lysis method)
Ly tm (centrifugation)
vi enzym Hind III v EcoR1 Tch chit v tinh khit ADN ca
plasmid (plasmid DNA extraction and
purification)
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a Plasmid vo Saccharomycescerevisiae
To mi trng khbin:Mi trng LB. Cho dung dch CaCl2
vo.
Nui cy Saccharomycescerevisiaetrn mitrng.
Chi Saccharomyces
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34/49
Mi trng LB khbint trong lnh.
Cho plasmid pcDNA 3.1+ vo mi trng.
Tin hnh .
Cho vo ninhit42oC trong vng 30s
Sau trong vng 20 pht.
Bin np bng phng php sc nhit
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Nui cy Saccharomyces cerevisiae
Nui cy nm men trong mi
trng250C lc 250 rmp.
Khi o OD600 khong 0.4 - 0.6
dnglc
Cho dung dch SDS.
Ly tm 2500 rmp trong 5 pht,
40C.
Lc tch kt ta thu c dch
protein.
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Tch v tinh sch protein
Mun thu nhn c cc protein nguyn th tcl protein c tt c
tnh cht t nhin c trng ca n, cn sdng cc bin php khc nhau.
-Nhit
-Nng proton (pH)-Tc nhn ha hc
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Tinh sch protein
1.Loibtpcht-Phng php thm tch (dialysis)
-Phng phpbin tnh chnlc
- Phng php ktta phn onbngmui trung
tnh hoc cc dung mi huc,-Cc phng php sc k trao i ion, in di,
phng php lc gel...
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Phng php sc k lc gel
Cc phn t ln khng thmvo trong ht sephadex
Ht sephadex Cc phn t nh thmvo trong c ht
sephadex
Tch cc phn t theo kch thc bng sc k lc gel.Hot ng ca lc phn t sephadex.
http://www.bbioo.com/bio101/UploadFiles/200805/2008052206590823.jpghttp://www.bbioo.com/bio101/UploadFiles/200805/2008052206590823.jpghttp://www.bbioo.com/bio101/UploadFiles/200805/2008052206590823.jpghttp://www.bbioo.com/bio101/UploadFiles/200805/2008052206590823.jpghttp://www.bbioo.com/bio101/UploadFiles/200805/2008052206590823.jpghttp://www.bbioo.com/bio101/UploadFiles/200805/2008052206590823.jpghttp://www.bbioo.com/bio101/UploadFiles/200805/2008052206590823.jpghttp://www.bbioo.com/bio101/UploadFiles/200805/2008052206590823.jpghttp://www.bbioo.com/bio101/UploadFiles/200805/2008052206590823.jpghttp://www.bbioo.com/bio101/UploadFiles/200805/2008052206590823.jpghttp://www.bbioo.com/bio101/UploadFiles/200805/2008052206590823.jpghttp://www.bbioo.com/bio101/UploadFiles/200805/2008052206590823.jpghttp://www.bbioo.com/bio101/UploadFiles/200805/2008052206590823.jpghttp://www.bbioo.com/bio101/UploadFiles/200805/2008052206590823.jpg8/4/2019 Tieu Luan Viem Gan B
39/49
Thnh phn:
Mt liu 1ml bao gm:
- Khng nguyn b mt virt vim gan B tinhkhit..............20 g- Hydroxyt nhm ...................................0,5 mg
- Thimerosal ................................0,01% (W/V)
Sn phm vacxin ti t hp HBsAg
Q i h i i b h
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Qui trnh sn xut vacxin vim gan b t huyttng ngi
Vacxin ny thuc loi vacxin di n v.phn khng nguyn c tnh sinh min dch lprotein v ca virus vim gan b ( HbsAg) c
tch v tinh ch t huyt tng ca nhngngi mang virus vim gan B nh cc k thuttch thng thng nh kt ta, c c, ly
tmvi 20bc nh sau:
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41/49
Bc 1. thu thp huyt tng ngi mang
HbsAg c hiu gi 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 20pht/40c. b cn, ly dch ni.
Bc 3. ta vi sulphatamol 50% (nng
cui). qua m nhit phng, ly tmnh trn. B nc ni, cn
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Bc 4. ha cn vi KBr, khuy u t nht 1gi nhit phng, lng. loi b cnbng phu lc ht chn khng.
Bc 5. iu chnh dch c t trng1,32g/cm3 (thm bt KBr)
Bc 6. chun b my siu li tm HITACH-
55pp v onal rotor RPZ35T.
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43/49
Bc 7. cho cc dung dch vo rotor
o Nc ct d=1: 500ml
o Dung dch KBr d = 1,3g/cm3: 400ml
o Dung dch mu d=1,32g/cm3: 1000ml
o Ly tm 25000 v/pht trong 24 gi/20oc.
Ly mu tngo Phn on 30ml v xc nh HsbAg trong
chng. Trn cc
phn on c HsbAg dng tnh cao.o Siu li tm 3 ln.
Bc 8. c c n th tch 300ml.
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44/49
Bc 9. siu li tm tng vng vi sacaroza graddient(2 ln).
o Dung dch Nacl 1 M :500ml
o Dungdch mu :300ml
o Dung dch sacaroza 10% : 300ml
o Dung dch sacaroza 20% : 300ml
o Dung dch sacaroza 30% : 300ml
o Dung dch 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 v xc nhHsbAg trong chng. Trn cc phn on HsbAg chiu gi cao.
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45/49
Bc 10. c c dch (V=300ml)
Bc 11. bt hot HsbAg tinh khit 60oc/10
gi. Lc qua mng lc 8um v 0,45um. Bthot tip vi formaldehyt (nng cui1:2000) trong 96 gi/37oc.
Bc 12. c c v thm tch loi formaldehyt(bng Pellicon cassette). Kim tra tinh schca HbsAg (in di, knh hin vi in t).
kim tra HBV-ADD tn d (PCA)Bc 13. kim tra hm lng HbsAg (o mt
quang v ELISA)
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46/49
Bc 14. lc v trng quamng lc 0.22um
Bc 15. hp ph HbsAg vo
gel Al(OH)3 v pha vacxin vihm lng HbsAg 20ug/ml.nng cui ca nhm:300ug/ml. thm cht bo qunthimerosal nng 1:10000
Bc 16. lc u cc chaitrong 1 tun, 2 ln/tun
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47/49
Bc 17. ly mu bn thnh phm kim tra v
khun, an ton, cng hiu, cht gy st v ccch s ha hc.
Bc 18. ng l, dn nhn, ng gi
Bc 19. ly mu kim tra cht lngvacxinvoo khun, an ton, cng hiu, cht gyst v cc thnh phn ha hc.
Bc 20. xuaatss xng vacxin.
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48/49
Sn phm vaccine vim gan B c iu ch t huyt tngngi lnh mang khng nguyn b mt virt vim gan B
(HBsAg) khng c triu chng lm sng.
Thnh phn:
Mt liu 1ml bao gm:
- Khng nguyn b mt virtvim gan B tinh
khit........20 g
- Hydroxyt nhm
..................0,5 mg
- Thimerosal ...............0,01%
(W/V)
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49/49
THE END
THANK YOU FOR LISTENING
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