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ginh git s sng con ngi tm mi bin php nhm hn ch tc ng c hi ca cc i tng gy bnh.

h. Vacxin c coi l mt tin b y hc quan trng nht ca th k XX Ngy nay cng vi s pht trin Click cng ngh sinh hc khng ngng cato edit Master subtitle style con ngi tm ra c vaccine hu hiu bo v chnh mnh vi s lng nhiu, an ton v nhanh chng.

5/31/12

Tn tai

CAC PHNG PHAP SAN XUT VACCINE. CNVS VI SAN XUT VACCINE TH H MI
GVHD: Trn Thanh Thuy SVTH: Nguyn Thanh Thao
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Nisadung I. C s sinh hoa cua cng ngh n xut vaccine


II. S lc v vaccine
1.

Vi nt v lch s vaccine Khi nim v vaccine Thanh phn cua vaccine c tinh c ban cua vaccine C ch hoat ng cua vaccine Phn loai vaccine

2.

3.

4.

5.

6.

III. Phng phap san xut vaccine


1.

Phng phap san xut truyn thng


1.1. Qui trinh san xut

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I.C s sinh ha ca cng ngh sn xut vacxin


q

H thng min dch ca c th

Phn loai MD Cac t bao tham gia vao h thng MD

q q

Tnh cht ca min dch C ch bao v c th cua MD c hiu

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H thng min dch ca c th Phn loai MD

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Cac t bao tham gia vao h thng MD


q

T bao limpho :

T bao B:tng tac vi KN va tao khang th T bao T: tng tac c hiu vi KN


TH:kich thich t bao B san xut nhiu KT TD:hoat hoa cac t bao khng c hiu (ai thc bao) Tc:tng tac va pha huy trc tip cac t bao co KN trn b mt Ts:iu hoa ap ng MD, c ch tac ng cua cac t bao MD(t bao B)

ai thc bao: bt gi, nut, pha huy KN, hp tac vi t bao


limpho sx KT c hiu
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Tnh cht ca min dch:


q

Tnh c hiu: Mi KN ch
c th kt hp va khp vi mt loi KT c hiu do chnh n kch thch to thnh.

Tnh ghi nh: Sau khi tip


xc vi KN, c th hnh thnh p ng min dch nh. Nu ln sau c dp tip xc vi khng nguyn th c th s to mt p 5/31/12 ng min dch nhanh

Tnh c hiu KN - KT

Mi quan h gia MD t bao va MD dich th KN + APC Cac t bao T


MD t bao MD dich th

T bao T qua mn chm (TD) Kich thich ngoai Vim

T bao git t nhin (NK)

T bao T c (TC)

T bao T tr giup (TH)

Kich thich t bao B Git t bao Tng hp KT

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MIN DICH DICH TH


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MIN DICH T BAO

C CH BAO V C TH CUA MD C HIU

iI. S lc v vaccine

1.Vi nt v lch s vaccine


Edward Jenner -mt bc s ngi Anh -c cng nhn l ngi u tin dng vc-xin nga bnh cho con ngi ngay t khi ngi ta cn cha bit bn cht ca cc tc nhn gy bnh. Nm 1796, chu u ang c dch u ma, Jenner thc hin thnh cng th nghim vaccine nga cn bnh ny Edward Jenner (1749-1823) 5/31/12

Louis Pasteur la ngi m ra ky nguyn u tranh chng cac bnh nhim trung bng vic tim chung d phong.

4/7/1885 ln u tin Pasteur va cng s a tin

hanh tim chung 13 ln virus dai ly t tuy sng tho vi c tinh tng dn cho cu be 9 tui, Joseph Meister a bi cho dai cn n 14 vt thng va cu be a thoat khoi ban tay t thn.

14/10/1885, vin Pasteur c thanh lp, chi trong

vong 2 nm vin nay a iu tri cho trn 5000 ngi.

Louis Pasteur (1822-1895)


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Hin nay ,nhiu loai vaccine truyn thng a c cai tin va phat trin qui m cng nghip, phuc vu cho chng trinh tim chung m rng, lam giam c ban ti l t vong tre em i vi nhiu bnh nguy him. 1978 bnh u mua a vinh vin bi xoa bo trn pham vi toan cu va sp ti la bnh bai lit.

nh chp nm 1977, Ali Maow Maalin, ngi Somalia, c xem l bnh nhn cui cng mc bnh u ma 5/31/12

2. Khi nim v vaccine


Vaccine 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.

3. Thanh phn cua vaccine


Khang nguyn :La c cht hoa hoc co kha nng gy ra trong c th s tra li min dich

vaccine
Cht b tr vaccine 5/31/12 :L nhng cht c b sung vo vaccine, c kh nng kch thch sinh min dch khng c hiu nhm nng cao hiu lc v di min dch ca vaccine

4. c tinh c ban cua vaccine:

An toan:
V trng, thun khit, khng c.

Hiu lc :
Vacxin c hiu lc ln l vacxin gy c min dch mc cao v tn ti trong mt thi gian di.

Tnh khng nguyn


Khng nguyn mnh l khng nguyn khi a vo c th mt ln sinh ra nhiu khng th. khng nguyn yu l nhng cht phi a vo nhiu hoc phi km theo mt t dc mi sinh c mt t khng th .

Tnh min dch

5/31/12 To ra hiu qu khng cho c th v sau khi tc nhn gy bnh xm nhp

5. C ch hoat ng cua vaccine


MD ln 1

vaccine

C th

TB nh

MD ln 2
Tac nhn gy bnh thc s

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6. Phn loai vaccine


Da vo thnh phn khng nguyn h I vaccineton khun (bao gm khng nguyn thn, vaccineth

v bc v c t ) Vaccineth h II: Trong vacxin ch cha mt s thnh phn gy bnh cDa a vobhot tnh ca mm bnh mm nh

Vaccineth h III-Vaccineti t hp :vaccinec sn xut bng ngh gen

vaccinev hot vaccine

Vaccinev hot khng c b tr :vaccinecha khng nguyn c

v hot bng cc yu t vt l (Nhit , tia Vaccinev hot m); ng cc t ngoi, sng siu c b b ha cht (Cc loi(keo c nhum,cc axit, trvaccine thu phn, phn formol v.v..) chua )

vaccinenhc :Vaccine vaccine cha mm bnh c lm nhc c hoc v c, nhng c


5/31/12 vn bo ton tnh khang nguyn.

II.1. Phng phapsan xut vaccine Phng phap sn xut truyn thng
1.1. Qui trinh san xut vaccine truyn thng
Nui cy
To sinh khi

Lm bt hot

Vacxin vi khun cht: dung cc tc nhn dit khun nh cc ha cht (formalin, alcool, aceton), tia cc tm, siu m Vacxin t vi khun sng gim c: dng phng php cy chuyn vi khun nhiu ln trong mi trng nui cy

Tinh khit ha ng kh ng gi Kim tra sn phm


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Sn xut ra ch phm v trng Nng

1.2. Cac vaccine san xut theo phng phap truyn thng (vaccine truyn thng)
a. Vaccine bt hot b. Vaccine giam c lc c. Vaccine gii c t d. Vaccine khng khng th idiotyp

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Nhc im cua vaccine truyn thng

Chi mt s it vaccine c san xut do khng phai moi tac nhn gy bnh u co th c nhn thanh s lng ln u san xut vaccine.

Cn phai nui t bao ngi hoc ng vt rt tn kem san xut vaccine t virus ngi va ng vt.

Cn co bin phap an toan khi thao tac vi s lng ln tac nhn gy bnh Cac chung nhc c co th tr lai trang thai cng c. Vic bt hoat co th khng hu hiu gy lan truyn bnh bi vaccine. Thi han s dung ngn va cn iu kin bao quan lanh. 5/31/12

2. Phng phap san xut vaccine bng ky thut AND tai t hp


2.1. Qui trinh san xut B1: La chon KN ich B2: Phn tch v xc nh gen ch B3: nhn dong gen B4: Biu hin gen trong t bao chu B5: Nui cy va thu nhn protein B6: Kim tra cht lng va tinh sachnprotein B7: Sa xut ch phm B8: Kim tra san phm
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B2: Phn tch v xc nh gen ch


Tach chit mARN Gn ui poly A Tao cADN mach n

ct oligo T- cellulose

Tinh ch

Tach ring tng mARN

Tng hp cADN kep

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B3: nhn dong gen


cADN kep Tao AND tai t hp T bao chu Thu nhn khun lac cha cADN Xac inh dong cn
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Bin nap E.coli Cht khang sinh Lai axit nucleic vi vic s dung mu do anh du phong xa

Thu nhn dong cn

Muc ich:s dung b may di truyn cua t bao chu nhn ban AND tai t hp thanh mt lng ln ban sao.

Dung VK E.coli lam t bao chu: d thao tac, it tn kem lai sinh san nhanh, tao dong AND tai t hp nhanh.

Hin nay nhn dong gen co th s dung phan ng PCR


E.coli

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B4: Biu hin gen trong t bao chu:

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B 5,6: Nui cy ,thu nhn protein ,tinh sach protein


Nui cy

Thu nhn protein

C c

Phng phap vi loc tua B1:Trao i ion

Tinh sach protein

Phng phap sc ki co phn giai cao

B2: Loc gel B3: Sc ki tng tac ki nc hay sc ki ai lc

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u im cua vaccin tai t hp


Nng cao nng sut sn xut vacxin To ra sn phm vacxin vi nhiu u im: Vacxin ti t hp rt an ton v khng s dung trc tip vi sinh vt gy bnh. Gia thanh ha vi khng cn phai nui virus trn phi ga hay cac t chc m cua ng vt vn rt phc tap va tn kem, giam bt chi phi vn chuyn va chi phi kim tra tinh an toan trc khi dung, khng cn bao quan lanh)=> ph hp vi iu kin s dng nhng nc ang pht trin
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1.2. Cac vaccine san xut bng ky thut AND tai t hp


v

Vaccine sng gim ng lc sx bng lc bng k thut di truyn Vaccine cha KN sn xut bng phng php ti t hp Vaccine vector Vaccine ADN Vacxin da trn c th chuyn gen. Vaccine bo t

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III. NG DUNG cua vacine th h mi

i vi con ngi: To "l chn" cho c th


Theo cc nghin cu ca Gio s Daniel Kaufman : i vi bnh tiu ng tp I : cn bnh ny tin trin khi h min dch ca

ngi bnh "tn cng" loi protein c tn l GAD (nm trong cc t bo tuyn ty)cc t bo ny c chc nng gii phng ra insulin. Khi c tim vc-xin th h mi vo c th trit tiu qu trnh ny ngn chn vic gii phng cc khng th min dch tiu dit cc t bo c cha loi protein GAD. Ngoi ra, loi vc-xin mi ny cn c tc dng iu chnh cc hot ng ca h min dch, ngn chn n khng tn cng cc t bo nguyn vn ca cc 5/31/12

vaccine thc phm

Nguyn l c bn ca m hnh vaccine thc phm:


Khi nhng KN ny i vo c th ngi thng qua n ung h thng min

Chuyn mt loi gen c bit t bo thc vt sinh ra protein KN.

dch ca ngi s t ng sinh ra khng th chng li khng nguyn.

u im : gi thnh r, n nh, d sn xut trn quy m ln, d qun l, khng

cn tinh sch, bo qun lu v d vn chuyn, phng thc a vaccine vo c th li n gin,KN c sx trong thc vt khng mang ngun bnh n cho con ngi, nhng c t trong h thng vaccine c gim thiu.

VD: Vaccine thc phm iu tr bnh vim gan B. Loi cy trng c s

dng chuyn gen vim gan B l khoai ty (Solanum tuberosum), Bnh than,

Bnh dch hch 5/31/12

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Vaccine khi phc tr nh

Gio s Tadeusz Parnowski, thuc Vin Tm thn v Thn kinh Ba Lan tng phat biu : Vc xin th h mi tr thnh nim hy vng duy nht cho nhng ngi mc cn bnh Alzheimer " .

Nguyn nhn chnh gy ra tnh trng mt tr nh ca con ngi :protein thoai hoa bi kt tua trong nao ngi.
Loi vc-xin mi, do Cng ty Elan Irlandia bo ch, khi c tim vo ngi bnh, c tc dng ngn chn mt cch hiu qu qu trnh "kt ta" ca protein beta-amyloid
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i vi vt nui:

DNA vaccine c nhng thnh cng nht nh trong vic ngn chn nhiu bnh trn ng vt a s l do virus gy ra. in hnh nh: + Bnh m trng tm do virus WSSV gy ra + Chng li thnh cng virus West Nile (WNV) trn nga (2005) + Ngn chn IHNV hoi t c quan to mu trong c hi (2005) + Bnh hoi t tuyn ty do IPNV c
5/31/12 San xut vaccine DNA cho ca

Kt lun

S ra i ca vacxin c ngha v cng to ln i vi nhn loi. N

gip cho loi ngi thot khi nhiu i dch him ngho (u ma, bi lit; si, un vn v.v.

Tuy nhin, nhiu bnh vn cn ang thch thc con ngi, cha c

vaccine no hiu qu ngn nga:bnh do k sinh trng (st rt, giun, sn), vi khun (lao), virus (cm, st xut huyt, AIDS v.v.) Mt s l do c th l cc tc nhn gy bnh bin i thng xuyn khin cho min dch khng cn hu hiu hoc thm ch tn cng ngay vo h min dch nh trng hp ca HIV v.v

C th ni, nhng thnh tu t khoa hc k thut c bit l cng ngh

sinh hc s m ra nhiu hng nghin cu mi v hiu qu trong cng tc iu tr bnh . 5/31/12

Tai liu tham khao

Trn Thi Thanh, Cng ngh vi sinh, NXB Giao duc GS.TS. Pham Vn Ty,TS. Vu Nguyn Thanh, Cng ngh sinh hoc (Tp 5), NXB Giao duc

Mt s trang web

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a. Vaccine bt hot

La vaccine cha cac tac nhn gy bnh a bi git cht bng hoa cht, hoc un nong. vacxin bai lit Salk (IPV) dung tim.

u im: an toan Khuyt im: -Gy p ng min dch khng hon ton v ngn hn, phai tim nhc lai nhiu ln .

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vaccine cum (Vaxigrip)

b. Vaccine giam c lc
La vaccine ch t tac nhn gy bnh a lam giam tinh c bng cac tac nhn vt ly, hoa hoc,hoc bng cac phng phap khac khng gy bnh, nhng vn con kha nng sinh san va kich thich c th tng hp khang th bao v. u im: gy c p ng min dch di hn, khng cn tim nhiu ln Khuyt im: C th gy bnh nh, c th chuyn thnh dng gy bnh,virus c th b cht, cn bo qun lnh,c th gy bnh ung th

Vaccine si
vaccine bai lit Sabin V(OPV)

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c. Vaccine gii c t

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. Co th dung nhit hoc formaldehyt lam bin i hoa

hoc mt tinh c, nhng vn con tinh khang nguyn Cung nh vacxin cht, mun tao MD co hiu qua

cn phai tim nhc lai nhiu ln.

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d. Vaccine khng khng th idiotype

KT 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. Vd : KN:Salmonella KT :anti-Salmonella KN: anti-Salmonella KT:anti- anti-Salmonella dung anti-anti-Salmonella lam khang nguyn thi se an toan hn. 5/31/12 Antiidiotype

a.

Vaccine sng gim ng lc san xut bng k thut di truyn

Tach genom khoi capsid

enzyme gii Loai bo cac gen hac n


virus khng con gen c vaccine

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b. Vaccine cha KN sn xut bng phng php ti t hp


Virus vim gan B Gen ma hoa cho KN b mt HBs Ag Khuch ai PCR

ADNligas plasmid e Bin nap

S.cerevisae

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Nui nm men trong ni ln men nhn KN dung cho san xut vacxin.

c. Vaccine vector

Vacxin sng tai t hp. Vaccine vector: gn gen ma hoa cho KN vao vector la t bao (salmonella, E.coli..) hoc virus (vaccinia, pox, adeno) tao KN va dung toan b tai t hp o lam vaccine.

Tach AND ma hoa cho KN TB tng hp KN Virus hay TB trinh din KN

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S san xut vaccine vector nh virus u bo


Vi tri ct cua enzim gii han
Gen t tac nhn gy bnh

AND ma hoa cho KN tach t tac nhn gy bnh


TB m nui cy khng co TK

Tai t hp tng ng

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d. Vaccine ADN

S dng AND (AND ma hoa cho KN)

nh mt loi vaccine bng cch a trc tip vo sinh vt . Gen ma hoa cho KN c gn vao

plasmid a trc tip vao t bao qua da nh sung bn gen cac plasmid chui vao nhn tin hanh sao chep, phin ma ri dich ma tng hp protein KN..

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e. Vacxin da trn c th chuyn gen. Sn xut cy chuyn gen nh Agrobacter tumefaciens


Gen ma hoa cho KN

a plasmid vao VK nh tip hp Agrobacter tumefaciens

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T bao thc vt Cy hoan chinh Phn tich mc biu hin Phn tich kha nng sinh

f. Vaccine bo t

Bao t co cu truc c bit gm nhiu lp mang day kho thm bao boc, co

kha nng khang cao vi cac tac nhn vt ly, hoa hoc va c bit co kha nng chiu nhit (cha canxi dipicolinat). Bao t vi khun ln u tin c L.H.c va Simong M. Cutting s dung

lam vt tai vacxin. Ho tin hanh gn khang nguyn lam manh C cua c t un van TTFC (tetanus toxin fragment C) vao vo bao t Bacillus subtilis gy min dich theo ng ung va ng h hp (hit) cho chut va a tao c ap ng min dich nim mac va min dich h thng. u im cua bao t Bacillus la d nhn ln, bao quan lu dai, thao tac di

truyn n gian nn se tr thanh mt loai cht tai vacxin rt hp dn. 5/31/12

NUI CY
Trc khi nui cy kim tra cc chng VSV v tinh khit. Nui cy c thc hin trong cc ni nui cy c bit, c cc thit b kim sot n qu trnh tng trng ca vi sinh vt. Nui cy trong mi trng thch hp t c mt s lng ln sinh khi hoc sn phm ca chng VD: san xut c t un van dung mi trng Mueller va Miller at hiu gia 50-90 Lf/ml

5/31/12

(1) La chon KN ich cho san xut vaccine

Mt hay nhiu phn khac nhau cua protein b mt cua virus gy bnh. 5/31/12

KN bn trong co vai tro tao KT bao v chng lai s ly nhim bnh, tao kha nng min dich tt hn, lu dai hn.

La san phm protein cua gen iu hoa trong t bao.

Protein cu truc gy ap ng tao khang th trung hoa tao vaccine


Qua trinh trung hoa cac virus c thc hin bi s lin kt cua khang th vi mt hay nhiu phn khac nhau cua protein b mt cua virus gy bnh.
VD: virus bnh si co cac vi tri lin kt khang th trn 2 loai protein HA va Protein F KN ich tao vaccine si cn co ca hai loai protein HA va F

Hinh cu truc virus si


5/31/12

Protein cu truc gy ap ng tao khang th khng trung hoa tao vaccine

VD: Khi nghin cu ap ng min dich chng lai s ly nhim virus vim gan B.

KT trung hoa c tao ra trc tin ap ng min dich vi KN loi bn trong

chng lai KN b mt HBsAg.

HBcAg co tac dung ngn chn s gy nhim va phat trin cua virus vim gan B.

Hinh cu truc virus vim gan B


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Protein khng cu truc gy ap ng tao khang th tao vaccine La san phm protein cua gen iu hoa trong t bao. S sao chep , nhn i cua virus cn oi hoi san phm iu hoa cua gen iu hoa . Do vy nhng KT ngn can , chng lai cac san phm iu hoa nay co th ngn can s nhn i cua virus.

5/31/12

T bao chu sinh vt nhn s:thich hp cho cac gen la khi cac gen nay khng co qua trinh bin i sau dich ma.

E. coli
u im: d dang nui cy, phat trin nhanh, c im di truyn c biu hin kha ro va cac quy trinh chun, tin cy cho cac thao tac di truyn t bao E. coli a c phat trin. Khuyt im: thiu kha nng glycosylate hoa cac protein
E.coli

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Nm men :co u im sau:

D nui cy vi quy m ln thu sinh khi t bo. C kh nng thc hin cc bin i sau dch m nh ng ha, phosphoryl ha prtin c y cc hot tnh sinh hc t tng hp prtin ca bn thn nn khi a gen l tng hp prtein mi th sn phm d tinh sch. L VSV an tan, hu nh khng to ra c t. H gen khang 1.35x107 cp baze, c kch thc ln hn VK E.coli khang 3.5 ln.

Nm men (Saccharomyces cerevisiae)

T bao nui cua ng vt co vu: kiu ng hoa cua cac protein c san xut theo h thng nay tng t vi kiu ng hoa trong c th ngi.
5/31/12

b. Vaccine di n v (vaccine thnh phn)


Loai vacxin nay khng dung toan b t bao vi khun hay toan b virus ma chi dung mt s thanh phn co tinh khang nguyn cua chung.

Vi du HBsAg cua virus vim gan B, hay hemaglutinin cua virus cum. Vacxin nay co tinh an toan nhng tinh cng hiu khng cao

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Cu truc cua virus vim gan B

Cu truc cua virus cum

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