IC CONG NtJMI;: SINH MyU I UAN UUUU ZU13
SO S A N H TiNH SINH M I E N DICH CAC EPITOPE BAO T 6 N O A D u ' a c DU- OOAN TU" K H A N G NGUYEN HA VIRUS CClM A/H5N1 DUNG HOP VA K H O N G DUNG HQP vCf\ K H A N G NGUYEN ROI C O A SALMONELLA TYPHIMURIUM
Trin Thi Hong Kim'', Trin Linh Thu*c^
2 J y ? ^ ^ ^ ' ' ' ^ '^°^ ^^ Ty " " ' 6 " . ^?' ^9C Quoc gia Tp. Ho Chl Minh
Phdng thi nghidm Cdng ngh$ Sinh hgc phdn lu; Tnrdng Bai hgc Khoa hgc Tg- nhien, Tp. H6 Chi Minh
T 6 M TAT
Mpt trong iJiihig li€p c|n de phdt triln vaccine co hipi liic In dinh doi voi vims dl biln dli A/H5Ni la dya vio cic epitope bao tin dugc dp d o ^ in sUico nho su hd tro ciia tin sinh hpc. BSng cich tiqi c ^ nay, chung toi da d\f doin duoc cic epitope nh|n di§n boi tl bio B va te bio T tu vilng bao tfln tren khing nguyen HA ciia vims ciim A/H5NI. Dl ting cuimg tinh sinh mien djch dac higu ciia cac epitope niy, chiing tfli dfi tong hgp cac epitope tai to hop 6 d ^ g dung hgp vfli khang nguySn roi H: 1.2 ciia Salmonella Typhimunum bSng i ^ thu|t tii td hgp gen trong te bio vi khuin Escherichia coli. N ^ e n cuu niy bio cio kit qui so sanh tinh sinh nuen d|ch d^c hi?u cua cic epitope dung hgp va khflng dung hop vfli khang nguyen roi H:l,2 cua Salmonella Typhimurium Bing phuong phip uc che ngung ket hong cau, chiing tfli da chiing minh dugc rang khang the hien difn trong khang huylt tiianh chugt tiem khang nguvfin epitope tfl bio B dung hgp H: 1,2 uc chl hicu qua ngung ket hflng cJu ciia khing nguyen vims ciim A/H5N1 cao hon 2 dea 2,3 iSn so voi hoat tinh niy ciia kfaing till chuflt tiem khing nguyen epitope tl bio B khflng dung hgp H: 1,2 doi vfli chiing A/H5Nl/Chickenl3Vietnam/LA/2006 vi 1,5 din 1,8 liin dli voi chiing AA'ietnam/CL26/2004(H5Nl).
Djch cum A/H5N1 Id mOt b^nh djch c6 kha nSng lay lan rdng vd gay thigt hai nghiem trpng & gia c^m cung nhir dnti hwng den sire khde con ngtfdrj (Lewris, 2006). Tgi Vi^t Nam, d(Ch aim do vims cum A/H5N1 bCing phdt ti> nam 2003 den nay da c6 121 ca mac b#nh ciim gia c^m A/H5N1, trong dd c6 61 tm-d-ng hg-p tCr vong tgi 30 dja phuang. Cho den nay, nhilu phu-ong phdp ngSn ngi>a vd dilu trj b#nh djch H5N1 da dirg'c phat triSn nhir cdc loai vaccine vd thu6c i>c ch^
pmtein cua virus. Tuy rihi§n, virus cdm co khd nang biln doi khang nguyen mOt cdch li§n hie vd nhanh chdng, tgo ra nhieu chiJng miri c6 the khdng lgi cdc logi vaccine dang iim hdnh. Do dd, cdc phu'O'ng phdp mir\ d l phdt triln vaccine van tilp tyc dirge nghiSn cu-u nhdm cdi thi?n cdc iogi vaccine hi§n cd, khic phyc vln d l hdng ndm phdi tgo vaccine m»i cho cdc ChCing vims cCim md'i, dong thd-i nhdm nghi&n ci>u tgo ra mgt logi vaccine da trj c6 khd ndng phdng ngi>a dmTC nhilu Chung vims cum khdc nhau (Gerhard etal., 2006; Stanekovd va Vareckovd 2010; Steel, 2010).
Mgt trong nh&ng hifdng nghign ci>u triln vgng d l tgo vaccine phdng b&nh do vims cum A/H5N1 id sir dgng cdc epitope bao t i n b cdc chiing vims thuOc cdc subtype khdc nhau cua vims cum A (Ben-Yedidia vd Amon, 2005; Stanekovd vd Varedkovd, 2010). Epitope Id mOt vung nho djnh vj tren be mgt c l u trtic pnatetn vd cd khd nang gdy ddp i>ng miln djdi.
Dua trdn co chl gdy ddp Crng miln dich, epitope duvc phan thdnh epitope t l bdo T (nhdn dign bdi te bdo T) vd epitope te bdo B (nh$n di#n biri t l bdo B). Lfu dilm chinh cCia vaccine dga trdn epitope id khd ndng gay ddp irng miln djch vd'i clu tnJc khdng nguySn t l i thieu; do vdy, neu chi>a dly du cdc epitope cdn thiet, vaccine se kich thich phdn i>ng miln d|ch chuySn bi|t trong khi trdnh difoc nhijng dnh huo-ng khdng mong muon. Nhu'g'c dilm cua vaccine epitope Id tinh smh mien djch ttilp tuy nhien dgc tinh ndy co the khdc phuc bing cdch tdng cudng tinh sinh miln djch cua cac khdng nguySn epitope thdng qua vigc gan epitope vdi m$t pmtein mang (Ben-Yedidia vd Amon, 2005; Cuadras et al., 2004).
Cac nghiSn cuu cho thiy flagellin (H:1,2) cua vi khuan Salmonella Typhimurium cd vai trd hogt hda hg miln djch cua vgt chu do SM- hogt hda thg the TLR-5 (Toll-like receptor 5) hi^n di$n tren cdc t l bao trlnh diSn khdng nguySn. Khdng nguyen flagellljn la thdnh phin cau tgo thdnh long mi, giup vi khuin di dgng vd Id mgt protein cd vai trd tmng dOc IM^C cua vi khuin nay (Cuadros et al. 2004). BSn cgnh higu qua gdy ra cdc dap ung viem khi dug'C du'a vdo co' the v$t chCi, flagellin cd thi duoc si> dung nhu mOt td dtro'c nhilu tilm nang do khd ndng gdy ddp dng miln djch dgc hiSu vdi khdng nguyen muc tidu khi chung cung hien dlSn frong cung mgt phdn tO protein dung hg'p. 0 dgng ndy, ftageilin tn,rc tilp gdy ra s^
trudng thdnh cdc DC (dendritic cells), kfch hogt sy dilu chmh tang cudng cdc tfn hieu dong kfch thich vd cdc phdn ti>
trinh diSn khdng nguySn GD80, C083, CD86, MHC Idp II. TNFa, IL-^, IL-ip, CCL2, CCL5. I^dt s6 nghien cuu cho thiy rang cdc vaccine tdi t l hg'p khdng nguySn-flagellin tgo ra khdng thi bao vg ching Igi Yersinia pestis, vims West Nile, vims ddu bd vd Pseudomonas aeruginosa (Steinhagen, 2011).
Tnfdc day, bing cdng cu tin smh hpc, chung toi da di^ doan dirge cdc epitope nh§n di$n bdi t l bdo B va te bdo T tu khdng nguySn HA cua vims cum A/H5N1 (LS, 2010). Cdc epitope nay lln iugl dirg'c tong hg'p bdng phuo'ng phdp hda hgc vd bdng kj? ttiudt tdi to hg'p gen trong t l bdo vi khuin E.coli. D l tang cudng tinh sinh miln djch dgc hiSu cua cdc epitope nay, chCing tSi da tong hp'p cdc epitope tai t l hg'p d dgng dung t\gp vdi khdng nguySn roi H:1,2 cOa S.
Typhimurium bing ky thudt tdi t l hg'p gen trong te bdo vi khuin £.coA'. Nghien cuu nay nham so sdnh tinh sinh miln dich d$c hiSu cua cdc epitope dung hep vd khdng dung hg'p vdi khdng nguySn roi H:1,2 cCia S Typhimurium. Day Id mgt budc quan trgng tao tiln d l cho cdc nghiSn cuu tang cudng tinh sinh miln djch cua cdc khang nguyen epitope hudng din nghien cdu phdt triln vaccine cum gia cam H5N1 pho rong.
NGUYEN LI$U VA PHU'aNG P H A P Vgt li$u sinh hgc
Vector pVFT dug'C si> dgng cho myc dich bieu hi§n epitope tdi to hg'p, c6 kich thudc 6023 bp, mang gen ma hda cho glutathione S tranferase (GST) nam ngay sau promoter T7 duoc dieu khiln bang each cdm ung vdi IPTG. pVFT Id dan
HOI NGH! KHOA HOC CONG NGHE SINH H y u I C . , ^ . ^ ^ , ^ i & i l f K i P i ^ ^
xuit cua vector bilu hign pET-28a(*) (Novagen) dug'C cai b i ^ ^ P ^ " J : ; - ^ , ' ^ , £ ^ " ^ ; : ! f " ^ „ ^ " ^ ^ ^ ^ ^ GST vd trinh tv pepbde nhdn dign bdi bdi TEV protease. Chung chu E. coli BL21(DE3) (F- dcm onipT hsdSB (r-B m-B) gal MDE3)) d u ^ s u dung d l bilu h i ^ epitope tdi to hcrp.
Peptide HeBc (FG>*WGF/£GGWQGMVDGlVy) va HeBdc (NDAAEQTKLYCU4PT) ^ ^ ^ ^ ^ bdi ^Jj/J^
Bioscience. Tmng Qulc c6 dg tinh sach 95 - 98%. Bdy Id 2 epitope te bdo B ^en tyc vd khong Wn ^ic bdo t6n fld duoc d u ^ n bing phuang phap tin sinh hgc tif khdng nguySn HA vims cum A/H5N1, du^c su dyng d l gdy ddp ung mAi djch trdn ChuOt Protease TEV dugc cung d p bdi Cong ty TNHH Cdng nghg Smh hgc HT.
Vic xin cum gia d m subtype H5N1 - vd hogrt nhd diu NAVET-VIFLUVAC duo'c cung d p bdi bdi C6ng ty thulc Thu y Tmng uemg 2 du^c diing bdm cho lfl chugt doi chung duong.
Cdc khdng nguyen vims cum H5N1 dang bit hogt (AftH5N1/Chicten13\^tnamAJV2006)^ vd AAfietnam/CL26«004(H5N1) (Babakir-Mina 2009) da dug'C phan lap tu b#nh phdm ga vd ngu<n ^ i Vi|t Nam (duvc cung d p bdi Dtm vi NghiSn cdu lam sdng Dgi hoc Oxfoni - Benh viSn Bgnh Nhi#t doi Tp. Hd Chi Mmh).
Chudt nhlt tring (Mus muscuHis var. albino) cdi. 6-7 tudn hwi,. tn?ng lugng 2 0 - ^ g {ViSn Pasteur Jp.HCM) diA« dimg d l bSm epitope tdi lfi hgp va cdc epitope long hgp hda hgc, thu nhgn khdng huylt thanh vd kiem chung khd ndng nh$n di$n vd gin khdng t h i trong khang huylt thanh vdi khdng nguyen chuyen bigt
Tgo cdc ddng E. coli b i l u hign protein tai to hgp GST-H:1,2. GST-H:1,2-HeBc vd GST-H:1,2-HeBdc
Cdc plasmid tdi t l hgp pVFT-fljB-hebc vd pVFT-fljB-hebdc dugc biln ngp vdo chung chO E. ccrii Bt.21(DE3) d l tgo cdc ddng E coli Bl^1(DE3V pVFT-fJjB-hebc va E. coli BL21(DE3)/ pVFT-fljB-hebdc cd khd ndng bilu hiSn epitope Id bdo B llSn tyc HeBc vd epitope te bdo B khflng liSn tyc HeBdc d dang dung hgp vdi GST vd H:1,2 Id GST-H:1 >HeBc vd GST- H-1 2-HeBdc Sdng lgc cdc the biln nap bing mra tnfdng LB-Kan 30. TuySn chgn cdc ddng E cofr BL21(DE3) chda plasmkl tdi t l hgp bang PCR khuin lgc nhd cdc cap mli fijB-F, flj&« vd fljB-F, hebc-R hay hebdc-R (Trdn Thi Hing Kim, 2012).
Cdm i>ng vd bilu hi$n vd thu nhgn cdc protein tdi t l hgp GST-H:1,2-HeBc vd GST-H:1,2-HeBdc
Nudi d y cdc ddng E. coli BL21(DE3) diua plasmid tdi t l hgp btjng 200 mi mfli bufimg LB-Kan 30, lie 250 vdng^ihOt trang 4 gid, 37'C vd cdm ung ting hgp protein tai to hgu (GST-H:1,2-HeBc hogc GST-H:1,2-HeBdc) bing IPTG 0,1 mM. Sinh khli t l bdo dugc huyen phii hda trong 20 ml dung djch PBS IX (140 mM NaCI, 2,7 mM KCI, lOmM Na2HP04 1,8 mM KH2P04, pH 7,3). Bo sung lysozyme Ipg/ml, u 37°C trong 30 phiit b l sung 0,1 mM PMSF (phenvl methyl sulphonyl fluoride). Pha te bdo bdng sdng sidu dm 12W trong 30 gidy, nghi 30 gidy, ldp Igi 20 lln. Thu djch nli sau ty tdm (13.000 vdng/phCit trong 10 phiit d 4°C). Nap mlu vdo cOt GSTrap FF 5 ml (GE healthcaiB), rtra cdt bing 25 ml dung djch PBS IX, ly gidi protein tdi to hgp bang 20 ml dung djch ly gidi pH 8 (SO mM Tris-HCI, 10 mM glutathkme khir). Thu nhdn cdc phdn dogn ly giai va Idlm tra protein tdi t l hgp (GST-H:1,2-HeBc/ GST-H:1.2-HeBdc) bing SDS- PAGE vd lai westem vdi khdng t h i khdng GST.
Thu nh|n cdc epitope dung hgp flagellin H:1,2
Thu nhdn cdc phdn dogn ly gidi c^ua protein tdi t i hgp GST-H:1,2-HeBc vd GST-H;1,2-HeBdc vd xdc <^nh hdm lugng pmtein ldi t l hgp tinh sgch bang phuong phap Bradford. Thgc hiSn phdn irng d t cdc protein tdi t l hgp ndy bing protease TEV d ning dd 0,04 mg/mt, 37°C trong 4 gid nham logi bd protein GST. Thim tich dung djch protffln tdi t l hgp trong dung djch PBS pH7,3 10°C trong 36 gid. Thu nhdn epitope dung hgp flagellin H:1,2-HeBc/ H:1,2-HeBdc bing cdch tdi ngp mlu qua cOt GSTrap FF vd thu nhdn phan dogn djch qua cOt. Protein GST dugc glO Igi trSn d t GSTrap FF sau dd dugc thu nh$n nhd dung djch ly giai.
Gdy ddp img miln djch chugt bang epitope tai t l hgp va thu nhgn khdng huylt thanh
Chudt thi nghi|m dugc chia thdnh 7 nhdm (n =5) gom (1) Nhom chung dm: chudt chi dugc tiSm PBS hodc (ii) khdng nguySn tdi t l hgp GST-H:1,2; (iii) Nhdm chuOt duoc tiem epitope khdng dung hgp HeBc ho^c (iv) HeBdc (2,5 pg/g thi trgng); (v) nhdm diuOt tiSm epitope dung hgp H:1,2-HeBc hogc (vi) H:1,2-HeBdc (2,5 pg/g t h i trgng); (vii) Nhdm chuOt dugc bdm vaccine thuong mgi (1 pl/g the trgng). Ta chit Complete Freund Adjuvant (CFA-^lfco) dugc si> dyng cho lin tifim diu tiSn (khdng nguySn: CFA = 1:1). Tiln hdnh tiSm nhic vdo cdc ngdy 28 vd 35 vdi Iilu khdng nguySn bing 14 lik khdng nguydn ban diu vdi td chit Id Incomplete Fround Adjuvant (IFA-Dlfco). Sau lan tiSm culi 10 ngdy, thu 300 pi mdu tir tTnh mgch mit chugt. Mdu dugc ly tdm 3.000 vdng/phut, 30 phut 4°C, thu khdng huyet thanh vd tai> Ignh ir -3(fC.
Kilm tra si^ hi^n dl|n ciia khang the dac higu vdn epitope HeBc/ HeBdc trong khang huyet thanh bing phuong phdp ELISA
c l djnh epitope te bdo B dung hgp vdi khang nguySn H:1,2 trSn gilng cda dfa ELISA 96 gieng (100 pl/giing). Khda cdc vj trf khing gdn khang nguySn vims bing 100 pi dung djch sira giy (PBS bo sung 5% sda tdch bdo, PBS: 14,61 g NaCI, 1 ml Tween 20) trong 1 gld. Sau budc rtra gilng bing PBS, khdng huylt thanh chuOt dugc b l sung vdo gilng (100 pl/gilng) vd u d ST'C trong 2 gid. B l sung khdng thi antJ-(lgA + IgG + IgM) chuOt cdng hgp horseradish peroxidase (HRP) (And mouse IgA+lgG+lgM-HRP; Abeam) d dp pha toing 1: 5000 (100 pl/gilng), u 37'C trong 1 gid. P h ^ imfl dugc phdt hISn bang co chat o-phenylenediamlne (OPD; Sigma) (40 pg OPD/1 ml citrate buffer pH 5), H202 0,5 pi (1M pl/gilng). Phdn ung hiSn mdu dugc ket thuc bang 100 pi H2SO4 2N sau 20 phut Ghi nhdn cudng dd mdu cda dia phfin dng d budc sdng 492 nm bing mdy doc ^ a ELiSA (Themio Multiskan Ascent).
M c nh$n sy hifn dign cua khang t h i dac higu virus cum A/H5N1 bang phuvng phdp irc c h l ngung k i t hing c l u
,»C C 6 N G N G H E S I N H H O C T O A N Q U O C 2013
Sg hi§n di$n cua khdng t h i dgc higu vims H5N1 dugc kilm chung bing thd n^ISm dc chl ngung kit h ^ g d u (HemagglijiinatKtn Inhibition test - HI test). Khang huyet thanh thu nh^n tir c^ugt da dugc tiSm khdng nguySn lan lugt klS"^ ^ •S'^bdng RDE (Receptor-destroying enzyme) nham loai bd cdc chit kim ham khflng dgc higu. Pha lodng bdc hai U ^ K I ^ ' tfianh da xif ly RDE trong dia 96 g i ^ ddy hinti tJiu V (25 pi/ gieng). B l sung 25 p\ khdng nguySn vims I*i m w ^ ' ' *a"*^ ^ ^ " ^ vj HA vdo cdc gllrig khdng huylt thanh, ii 30 phut d nhigt dd phdng. Bo sung 50 pi hdng d u
^ '^^' !f*^ " * " ^ ' ^ ' ^ phdng trong 1 gid. Quan sdt kit qua dga tr&n hifn tugng irc chl ngung kit hing d u t i T h / ^ ^ " 9 - Phan i>r^ durnig tinh Wit khdng t h i uc ctiA khang nguySn HA ngan khdng cho cdc phdn ti> ndy ngung fchA^*^ - ^"^ '^^ ^ ° " ^ ^^ khdng ngung ket va bj lang xudng. Ngugc Igi, phan dng dm tlnh khi khdng nguySn khflng bj uc diS vd gay ngung kit hong d u . Bern vj hi|u gid khdng t h i khdng HA la gid bi nghjch dao dg pha lodng cao nhdt cua huyll thanh v5n cdn uc c h l ngung ket hing d u (Webster, 2002)
K t r QUA VA T H A O LUAN
Bilu hign vd thu nhgn cdc epitope t l bdo B tai t l hgp
Hlnh 1 trinh bay k i t qua bieu hign protein tdi to hgp vd thu nhdn cac phdn dogn ly gidi protein tdi to hgp GST-H:1,2- HeBc (Hinh IA) vd GST-H:1,2-HeBdc (Hinh IB). Chung toi da thu nh$n dugc protein tai t l hop Id GST-'H;1.2-HeBc cd trong lugng phan td 87,3 KDa cd dd tinh sgch khodng 80% va GST-H:1,2-HeBdc frong luong phdn tCr 90,7 KDa, dd Bnh sgch khoa_ng 75%.
Nhim thu nh§n cdc epitope dung hgp H:1,2-HeBc vd H:1,2-HeBdc chdng toi tiln hdnh log! bd protein GST cua cdc protein tdi tfl hgp GST-H:1.2+leBc vd GST-H:1,2-HeBdc thdng qua phan iing d t nhd protease TEV. Hen hdnh thu nhgn cdc epitope dung hgp vdi khang nguySn flagellin H:1,2 vi khuan S. Typhimunum bing phuong phdp sic ky di Igc nhd cdt tinh che GSTrap FF. Hinh 2 cho ttily chCing tdi da logi b i dudi dung hgp GST thdnh cflng (Hinh 2C, gieng 8) vd thu nh$n dugc cdc phdn dogn epitope dung hgp flagellin Id H:1,2-HeBc cd trong lugng phdn tCr 56,3 KDa (Hinh 2B, gilng 6) vd epitope dung hgp H:1,2-Hebdc cd tnpng lugng phan tu 59,7 KDa (Hinh 2C, gilng 7)
kDa M 11 12 13 14 15 16
Hlnh 1. Ket qua thu nh$n protein tdi ^ hgp GST-H:1,2-HeBc (A) vd GST-H:1,2-HeBdc (B) bang c$t GS TrapFF. M, thang protein; 1 va 12, protein trud'c tmh chl; 3-5 vd 14, cdc phan doan rda; 2 vd 13. ph3n dogn sau khi qua c9t; 6-9 v^ 15-16, cic phdn dogn ly gidi.
H H H
Hlnh 2. Phan tich ket qua tinh chl GST-41:1,2-HeBc (P^ logi bo GST nhcr protease TEV nham thu nh^n epitope dung hgp H:1 2-HeBc (A B) ^^ epitope dung hgp H:1,2-HeBdc (C). 1 va 2. E.co/y/pVFT-fljB-HeBc IPTG (-) vd IPTG {+); 3 va 5, phan dogn ly gldi GST-H: 1.2-HeBc; 4. cdt GST-H:1 .2-HGBC b4ng TEV protease; 6, H:1,2-HeBc; 7, H:1.2-HeBdc. 9. ly gidl GST Xdc dinh hi#u gid khang t h i dac higu khang nguyen epitope HeBc/ HeBdc
Khdnq huylt thanh thu nhgn td (^udt tiSm epitope HeBc/ HeBdc khflng dung hop vd dung hgp khdng nguySn H:1,2 dugc pha lodng bdc 2 tir 1/25 din 1/3200. Cac epitope dung hgp flagelin H:1,2-HeBc (Hinh 3A} ho$c H:1,2-HeBdc (Htnh 3B) dugc pha loing vd c l djnh trSn gieng vdi ham lugng 100 ng/gieng. Khdng huyet thanh cliugt tiem PBS dugc
HOI NGHj KHOA HQC C O N G N G H E SINH HQC TCV:
sd dyng Idm dli chdng dm. Kha ndng tgo khdng the a§c hiSu epitope HeBc/ HeBdc dugc xdc djnh bang phuong p h ^ djnh lugng khang t h i fing (IgA + IgG * IgM) cd trong khdng huylt thanh chudt tiSm khdng nguySn epitope nh^n di|n vA gin dac hiSu vdi khdng n^jySn efxtope dung hpp thflng qua do m§t dd quang d budc sdng 492 nm (00492) cda mAy dgc ELISA. Kit qud cho ttidy higu gia khang ttil dgc hi$u epitope HeBc Id 100 (Hlnh 3A) va epitope HeBdc Id 400 (Htnh 3B). Bilu ttid vj la ning dg khdng t h i dgc hi#u hlSn dign trong khdng huylt ttianh chupt tiSm d c epitope dung hgp flagellin H.1,2 deu cao hon ning dfl khang ttil frong huylt ttianh chupt cht dugc tiSm epitope khflng dung hgp flageliin H:1.2, cy ttil Id k h ^ g ttil long (IgA + IgG + IgM) trong khdng huylt ttianh chugt tiSm H;1,2-HeBc tang g i p 1,17 Itn so vdi khang ttil ttong khdng h u ^ t thanh chudt ch! ttem HeBc (Hinh 3A) va khdng ttie ting (IgA + IgG + IgM) trong khdng huyet ttianh chudt tiSm H:1,2-HeBdc tang gip 1,8 lln so vdi epitope HeBdc (Hinh 3B). Nhu vdy, khdng nguydn flagellin H:1,2 cd ttil lani tdng cudng tinh sinh miln djch dgc higu ciia khdng nguySn epitope khi chiing dugc btlu hidn trong ciing mdt phdn tir.
So sanh hi^u qua trung hoa khdng nguyen virus cdm A/H5N1 cua khdng huyet thanh chu^t tiem epitope dung hgp va khflng dung hgp khang nguyen flagellin H:1,2
Kit qua d hlnh 4 cho phSp kit lugn cdc khdng nguyen epitope t l bao B dung hgp vd khdng dung hgp khdng nguyln flagellin H:1,2 cd t h i tgo ddp i>ng miSn djch fren chu$t, tao khdng ttie nh|n dign vd g i n dac hi$u chung vims cum gia d m phdn l^p tCr b§nh phim gd d Viet Nam (chung /VH5N1/Chicken13ViBbiam/LA/2006) (Hinh 4A) va vdl chung virus ciim gia d m phdn l$p td tignh pham ngudi ir Viet Nam (A/Viettiam/CL26/2004(H5N1) (Hinh 4B). Hi#u gid khdng &1I Oc chl hiSu qud ngung kit hing d u cua khdng nguydn w'ros ciim A/H5N1 hiSn dien bong khang huylt thanh chult tiem khdng nguySn epitope t l bdo B dung hgp H:1,2 cao hon 2 din 2.3 lan so vdi higu gid khdng the chuOt tiSm khdng nguySn epitope t l bdo B khflng dung hgp H:1.2 doi vdi chung A/H5N1/Chicken13Viefriam/LA/2006 (Hinh 4A) vd 1,5 din 1,8 lln ddi vdi chiing AA/ietoam/CL26/20G4(H5N1) (Hlnh 4B).
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KHT(1/x)
a
25 50 100 200 400 800 1600 3200KHTdfa) Hinh 3. Phdn tich hifu gld khang t h i tong (IgA + IgG + IgM) trong cdc khang huylt thanh chu$t tiem epitope dung hgp vd Ulflng dung tigp H:1,2 nh^n difn vd gan d^c hieu vdl epitope H:1,2-HeBc (A) vk H:1,2-HeBdc (B) bdng phuvng phdp ELISA.
Mac dil Id mSt trong hai protein chu y l u trdn bl mgt vims dim A/H5N1, &iy nhifin, t h i gidi vln dil cd mdt vdi nghiSn cOu frSn protein HA dfi tgo vaccine dim vl d ^ Id protein dl bj bien dii nh^t.
Trong dd dd co mfli vaccine nhl miJi dga trSn epitope da dugc cap
« ueul",,.'!.'?*,? *i.';1„^l*" ^ i ^ ™ l " ^ * ^ ^^ ''^^ ''*'^"9 " 9 " ^ " ^'^"s '=''"1 Sia cam A/H5N1 chiing Patent so 7192595 A/H5N1/Chicken13ViebiamnJV2006 (A) vd chdng An/letnam/CL26/2004(H5N1) (B) trong cac khdng (Arnon et a!., huy4t thanh Chuot tiem epitope d«ng hgp vd kh6ng dung hgp flagellin H:1,2 bing p h « ^ g % a p i ^ ch? 2007) VatSne 1 ngung kel hing cau (hemagglutination Inhibition - Hll jiuu/j. vaccine ray
' la hon hgp bao g6m bSn epilope cua vims duvc biSu hi»n Irong he lh6ng prolan flagellin ciia Salmonella. Tnjng do. doan epitope KA
<'" =«< amin 91 -108) d w o nh^n diSn boi l i bao B IS mol trong b6n epitope do. Kit qua ttii/ nghi«m ctio thiy vaccine C f L ' S - 1 * " 1 ' ^ i;*"'?,.?" . ' ? r ^ " ' " " ' ^ " ' • " 2 . H3 Ben canh do, cSng co mOt nghien ciiu khac tap trung vSo vSnS ?hAi ,4 ™k " h - " J . u^u^S """^^ l^*' '^9"'^" •=*" "*V chi ra ring khi tigm phin than cua prolein nSy (bo vung kh6i cSu) cho chuot se lao tinh dSp ung mien d|ch t6t hon so vijl viae tiSm mgt HA v*i diy dil de dai Kit qua thirc
1?0
' IC CONG NGHE S I N H H(?C TOAN Q U 6 C 2013
nghiSm trSn v i m s m d t s o c h u n g c u m H I . H 3 cho dap i m g m i e n djch t i t . K i t qua nay lam n l n t a n g cho vigc phdt t r i l n m d t togi vaccine p h i r d n g c h o virus c d m ( A m o n etal.. 2007; Steel. 2010).
V i g c d u n g h g p c d c e p i t o p e tiao t d n vdi flagellin ciia S. Typhimurium trong nghiSn c u u nay c u n g d a m d ra h u d n g d i m d i trong vigc nghiSn c d u v a c c i n e da gid p h d n g a i m gia d m H 5 N 1 . trdnh viSc phai nghiSn c d u thay d l i v a c a n e h d n g n a m . N l u t g o d u g c k h a n g nguySn p h i r c g o m c d c epitope t l bao B v d c a c epitope t e bdo T cua c a c khdng nguyen b l m d t khdc n h a u cua virus c d m A / H 5 N 1 thi higu q u d bao v g cua hSn h g p khdng nguySn nay c d t h i d u g c tdng ISn. S i n p ttidi vide d u n g h g p v d i flagellin cua Salmonella s g Idm t a n g d a n g k l kha nang ddp d n g mien djch cua vgt c h u . C a c ket qua ndy m d r a h u d n g nghlSn c d u s u d y n g c d c khdng nguySn epitope dung h g p flagellin Idm khang nguySn thanh phan d e phdt tiien vacane p h d n g n g d a nhieu c h u n g v i n j s c i i m A / H 5 N 1 d ViSt N a m .
K£TLUAN
Chdng tdi d a c h i r n g m i n h epitope HeBc v d HeBdc khi d u t r c d u n g h g p v a khdng dung h g p v d i khdng nguyen flagellin H:1,2 c d kha n d n g sinh m i l n djch ddc h i l u trSn chuOL O i l u t h d v\ la khdng ttil tgo ra t u chugt ttSm c d c khang nguySn epitope t e bdo B d u n g h g p v d k h d n g dung h g p khdng nguySn flagellin H:1,2 d i u n h | n dign v d g i n d g c hiSu khdng nguySn H A cua m p t s o c h d n g v i n j s c d m gia d m A / H 5 N 1 phan l$p t d bSnh p h i m g d v d n g u d i d Vidt Nam (chiing A/H5N1/Chicken13Vietnam/l.A/2006 v d AAfletoam/CL26/2004(H5N1)). Ngodi ra, khang nguySn flagellin H:1,2 c d vai t r d tdng c u d n g tinh sinh m i e n dich d g c hidu cCia khdng nguySn epitope khi chung d u g c b i l u hiSn frong cdng mdt phdn t d . Ld^ c d m am
Ngki&i dm nay dupc tdi Irfr bdi Dgi hpc Quoc gia Thanh pho HS Chi Mmh (VNU-HCM) trong khuon khd di Im ma so C2013-18-01.
TAI LI^U THAM KHAO
Amon R, Ben-Yedidia T, Levi R (2007) Peptide-based vaccine for influenza: Google Patents.
Babakir-Mina M, Ciccozzi M, Ciolli M. Marcucciiii F, Baiestra E, DImonte S. Aquaro S (2009). Phyiogenetic analysis of the surface prolans of influenza A (H5N1) vimses isolated in asian and african populatkjns. New Microtrofcig/ca 32(4) 397-403.
Ben-Yedidia T, Amon R (2005). Review Towards an Epitope-Based Human Vaccine for Influenza. Human Vaccines 1:95-101.
BUI Vdn Le (2010). Bao cSo tfing hgp k i t qua d l tai khoa hpc c6ng nghS trgng dilm d p nhS nude "NgtiiSn cdu, i>ng dyng Tin smh hpc trong vi$c thilt ke phdt t r i l n vdcnn vd thuoc' (ma sfi KC.04.18/06-10). Cpc Thdng tin Khoa hoc vi Cong nghd Oii6c gia (6$ Khoa hgc vd Cdng nghd)
Cuadros C, Lopez-Hernandez FJ, Dominguez AL, McCieiiand M, Lustgarten J (2004). Flagellin fusion proteins as adjuvants or vaccines induce specific immune responses, /nfecf/on and/mmunity 72:2810-2816.
Lewis DB (2006). Avian flu to human influenza. Annu Rev Med 57:139-154.
Stanekovd Z, Vareckovd E 2010. Conserved epitopes of influenza A vims indudng protective immunity and their prospects for universal vaccine development. V;ro/J7:1-13.
Steel J , Lowen AC, Wang TT, Yondola M, Gao Q, Haye K. Palese P (2010). Influenza vims vaccine based on the consen/ed hemagglutinin stalk domain. MBio 1.1-9.
Steinhagen F, Kinjo T, Bode C, Kiinman Dlwl (2011). TLR-based immune adjuvants Vaccine 29: 3341-3355
Tran Thj Hfing Kim, T r i n Thi Nhu TliOy, T r i n Linh Thir6c (2012). Tfing hgp vd kiem chirng tinh smh m i l n d|cti cCia epitope t l bao B dircrc dg dodn bang phuong phSp tm sinh hpc tip khdng nguyen HA cua vims ciim A/H5N1. Tap chi Phdt triin lOma hoc vd Cdng ngh$
15(3): 45-55.
Webster R (2002) WHO Manual on Animal Influenza Diagnosis and Sureeillance. World Health Organization.
COMPARATIVE IMMUNOGENICITY OF IN SILICO PREDICTED-EPITOPES ON HA FROM INFLUENZA A/H5N1 VIRUS FUSED WITH OR WITHOUT FLAGELLIN ANTIGEN FROM SALMONELLA TYPHIMURIUM
T r a n T h i H o n g K i m ' * , T r a n L i n h T h u o c ^
' Faculty of Biology, University of Sdence, VNU-HCMC
' Laboratory of Molecular Biotechnology, University of Science. VNU-HCMC
SUMMARY
In order to develop vaccine with stable efficiency against easily transforming virus such as influenza H5N1 vims, bioinformatic tools were used to predici conserved epitopes from viral antigens to be used as materials for the development of polyvalent vaccine against this virus Using this approach, we have successfully predicted B-cell and T-cell epitopes on conserved domains of HA antigen from H5N1 influenza A virus. To increase the immunogenicity of these epitopes, genetic manipulating techniques have been used to prepare the recombinant epitope in E. coli as a fusion form with H:l,2 flagellin antigen from Salmonella Typhimurium The aim of this studv is evaluating the specific immunogenicity of predicted B-cell epitopes and comparative specific immunogenicity of B-cell eoitones fiiscd with or without flagellin H 1,2 from 5. Typhimunum. Using hemagglutination inhibition method, we demonstrate that ttie ^^-sera from mice immunized wilh epitopes fiised with flagellin H:I,2 could recbgnize and inhibit the agglutination of
HQI NGHI KHOA HQC C O N G NGH? SINH HQC TO. .
inactivated influenia H5N1 virus strains isolated fiom infected chicken and human in Vietnam. The HI titer of anti-sera from mice immunized with B-cell epitope fused with H. 1^ is 2 to 2,3 was found higher than that from mice immunized with B-cell epitope iir case of vinis strain of A/H5Nl/Chickenl3Vietnam/LA/2006 and 1,5 to 1,8 for those in case of virus sfrain •
A/Viebiara/CL26/2004(H5N1). ' Key words: B-cell epitope, flagellin, influenza A/H5NI virus, Saimonelta Typhi
Author of correspondence: Tel: +84.38307079. Email: [email protected]
