Tgp chi Cong nghe Sinh hoc 10(2): 225-231, 2012
NGHIEN CUU PHAT TRIEN KY THUAT DOT BLOT DE CHAN DOAN VIRUS VIEM GAN B TRONG HUYET THANH BENH NHAN TREN CO SO KHANG NGUYEN HBcAg TAI TO HOP
O^ng v a n Quyfn, P h a m Minh Tuiin, Le Phuong iliing, ninh Duy Khilng Vien Cong nghe sinh lux. Vien Khoa hpc vii C 'dug iigli^' Vi('t Nam
TOM T A T
Khang nguyen loi (HBcAg) l.i mpt trong nhitngkh.ing nguyen quan trgng cua virus viem gan li (Hcpulilis B virus-HBV), la \icn g^ich c.iu moncn vocapsid chira \al chaldi truyen cua virus Nhicu nghien cim dii chi ra rang,_ HBcAe co tinh sinh mii-n d|ch lal m^inh. khiing the khang HBcAg (anIi-HBc) xual hi?n lit rat srrm va ton tai §an nhu suot cupc diri ngircri benh. Vi \ay. HBcAg va khang the khang HBcAg dugc xcm nhu la dau an huyet thanh quan trgng cua qua trinh lay nhieni virus cCinj; nhu tinh irangb^nh. Do vai tro ihiet yeu uong vong doi cua HBV va img dung trong chan doan stVm ,sg lay nhiem virus, HBcAg da Iro Ihanh tam diem nghien ciru ciia nhieu phong thi nghiem tren the gioi. Hnjn Iren Ihi truong da c6 nhieu I091 kit chan doan HBV khac nhau nhu ELIS A, Western blot, PCR, luy nhien gia thanh kh;i cao va phitc tap trong thao tac cung nhu doi hoi nhieu thai gian. Vdi muc dich phat trien dugc bp kii chan doan HBV nhanh, nhgy va dc dang cho nguoi su dyng, tring cong irinh nay chiing toi nghien ciiu bieu hi^n va tinh s^ich gen ma hoa khang nguyen 16i cua HBV o trgc k'luan E. coli va sir dung prolein lai to hgp de chan doan HBV bang k j thu^t Dot bloi HBcAg lai 16 bgp co do li lb sach cao, phan img dac hieu voi anti-HBc trong huyet thanh b?nh nhan, Cac thu nghiem ban dau tren 40 mau benh pham cua benh nhan HBV cho thay, bg Dot blot co dg nhay va do dac hieu tuong duong voi bg kit ELISA phat hien anti-HBc ciia Bio-Rad. Chiing toi dang nghien ciru voi so lugng mau nhieu ban va toi uu hoa cac dieu kien phan img de hoan thien bo kit chan doan HBV bang ky thuat Dot blot.
Tir khoa: Diagnostics, kit chan doan Dot blol. HBcAg lai ii >. E. coll expression. Hepatitis B Vi
DAT VAN DE
Viem gan virus B la mot benh truyen nhilm nguy hiem gay nhiem triing cho gan. Nguai bi nhiem virus viem gan B (Hepatitis B virus-HBV) co nguy ca cao ehuyen thanh man tinh va c6 the phat Wien thanh xa gan va ung thu gan (Almeida et al., 1 9 7 1 ; Beasley, 1 9 8 8 ; C h i e s a et al.. 2 0 0 0 ) . Cac s6 li?u thong ke gan day cho thay, tren the gioi hien co khoang 350 trieu nguai dang song chung vai HBV va hang nam khoang 2 Uieu nguoi chet lien quan true tiep den virus nay (Raimondo et al, 2008). Hi?n dS CO mot so thuoc dieu tri benh viem gan virus B, tuy nhien hieu qua ciia cac thuoc nay chua cao, vi the tiem chiing vaccine viem gan B van la phuong phap hull hieu nhit d6 phong tranh can b?nh nguy hiSm nay. Mac dii vay, vaccine chi co tac dgng phong ngira su gay nhiem cua virus. Doi voi nhung truong hgp da nhilm HBV, c^n phai co bien phap phong franh su lay lan rgng ra cong dong.
Viec xet nghi6m sam, chinh xac su lay nhilm HBV sg gop phSn quan trcsig Uong cong tac dg phong va diSu tri benh. Nhtrng nghien ciru ve HBcAg cho thSy co m6i lien quan mat thilt giua su
xuat hien ciia HBcAg va khang the khang lai no (Asim et al, 2010). Khi ca th^ bj nhilm HBV, khang the khang HBcAg (anti-HBc) bat dau dugc san xuat de chong lai su xam nhap va nhan len ciia virus Anti-HBc dugc phat hien khi co the bi nhilm virus viem gan B cap ciing vai Anti-HBs va van tiep tuc ton tai trong co the nguai benh sau khi b?nh da thuyen giam (Stief et al., 2010; Hooftiagle el al.
1973). O nhimg nguoi co mien dich tg nhien, anti- HBc thuong hien dien ciing vai anti-HBs. Doi vai nhung nguoi da tiem chiing va chua timg nhiem HBV, sg chi CO anti-HBs ton tai. Su xuit hien anti- HBc 6 nguai dugc tiem chiing cho thay ca the truac day da bi nhiem virus nay Nhung ngucri co anti-HBc trong khi khong co HBsAg hoac anti-HBs, la nhChig nguoi da timg bi nhiem HBV, nhung nguoi nay co the da CO miln dich hay nhiem virus man miirc dg thip (Barker ef a/,, 1973).
Thai gian tir khi ca the bi nhiem virus den khi co dap ling mien dich dau tien doi voi HBV trong khoang tir 2 den 26 tuan. HBcAg khong dugc phat hi^n trong mau nhimg khang the IgM khang HBcAg (IgM anti-HBc) lai xuat hi$n sam ngay sau HBsAg va HBeAg. DNA cua virus viem gan B co the dugc 225
D6ng V3n Qtiyen el al.
phiil hiv'ii bSng pliumig pluip VC\< mnVc khi xul^l hiv'il IIHsAg v4 llik-\:.: h i \ nbieii Ir.Hig khoang lhi>i gian ur Ihiing ihii lu den thiing (hit 11A111, irong miiu kii khiing 11h.1i hn-n dugc diiu hi<;u \i\ cua IIHSALI Limg nhu khang ihc khiing Igi no. hi ihiin^
ihu n.im n o di. khani; thi- khiing 1IBS\L> miVi \ual hivn. Trong khi do kluini: the kluing IIIUAj: \u;ii liii,-ii lu uil siVm (lu duVi k\ I\M\ U bi;nh) I lieu du d m lh.i> kh.mg the kli.mg I Ilk \ g thii<)c hVp IgM la dau ,in hu\ei ihanh quan trgng ui.i sg l,i\ nliicin IIDV (I looliiiigle <•/.'/ 197v l.ennelle. Schmidt, 1''^'')
\ lei \.iin la iiitoc coll K' 115:1101 nliiem l l i W can nh^l ihe uun \.i virus iia> time dugc nil nhicu phong thi nghiijni troiii; iiiroc quiin liiin iiiihicii LUU Irong ciic niLhicn ciru truac d.i\. chung loi d,i tiich dong.
guii trinh tg \.i bieu hii;n c;n. kliaiij.' ngii\eii quan iioni; UI.I H i n (Dinh \\\\ Kli.niij ct al IW('. f ^ n g Irmmg Minh cl al 2IH)1. DOIIL; V^n (,)u>eii el al.
1999. 2003 \.i 2005: Nguven h e n Minh a al 2i)0S) de nghien cim djnh u p e cac chung HBV dang luu hanh irong nuac dong thin thu nhiin nguon khiing nguyen tai to hgp phgc vg cho \ iC'c che t^o cac bg kit chan doan HBV.
Voi muc dich phat trien bg kit chiin doan HBV CO do nhay, dg dac hieu cao \ a lian gian cho nguiri su dung, trong cong trinh nay chiing toi nghien bieu hien \ a tinh sach gen ma hoa HBcAg cua MTUS viem gan B a Ecoli va su dung protein tai to hgp de phat hien aiiti-HBc trong huyet thanh b?nh nhan b5ng k\
thuat dot blot.
V.AT LIEU V \ PIH 0 \ ( . PHAP NCiHIEN ( ULl Chung vi khuan va hoa ehat
Chiing t i bao E. coli BL21 Star™ (DE3) (Invitrogen, My) dugc dimg lam te bao chu de bieu hien gen dich. IPTG mua tit hang Fermentas (M\) ProBond™ Nickel-Chelaltng Resin lu Invitrogen (My), Mang PVDF tir Millipore (My) SO'a gay (skim milk), cong hgp khang IgG ngucri gSn HRP tir Sigma (My)
Bleu hien v^ xde djnh trang th^i cua protein tiii td hqp Gen ma hoa HBcAj^ dugc chimg toi tach dong va thi^t k i vao vector bieu hj?n pET28a+ (pEHBc) a E.coli nhu mo ta chi tiet trong nghien ciru truAc day (Nguyen Tien Minh el ai, 2005). D i biiu hien, chiing E coli BL21 Star™ (DE3) mang vector pEHBc dugc nuoi lac a 37°C trong khoang 3 gia den
kill ()D„m ii{[\ 0,6-1 thi cam img bSng IPTG, De toi uu mire dii bieu hiC'ii protein tai to hgp. mgt so dieu kiC'ii nuoi cay dugc liiay din nhu nhiC'l dp (37, 30. 25 va 2(rC), nimg di. I I ' K . (1.0. 0.5 \ a 0.2 mM) va ibiTi giim ihu 111.111 sail cam ung ( I , 2. 3 va 4 gicr), 11,111(1 Ihiil bii;uhi?n eua protein tai to hgp dugc kiem ira li.ini; cich' ly lain thu c^n tO bao tir 5 ml djch nuoi tav sail cam imp I P l d va hoa Igi trong 0.3 ml d?m pha li-bao (M) I,iM Ins-IK I pH 8,0; 100 mM \a(_l.
0,1 iiiM I'MSF), Pha li- b^o bSng m i y sieu am trong ihiri gian 5 phiil. L\ tam UK dg 12.000 vong/phtJt trong 15 phiit a 4"( de tach djch nin khoi ciln, s a u d i hoa l^i i.riii Uong 0.3 ml d^in phii le biio. Kiem tra protein iiii 16 hgp ca trong djch noi va c^n bingdi^n di gel pol\;ii.i>l;imKle 12.5"o.
l i n h sach piolein l:ii to hgp bSma, cgl ProbontT^
Ni.kd-< hchilin^Kesin
Priiteii) HBcAg tii to hgp bieu hi^n dudi d ^ g dung hgp co giin ilieni 6 His o dau N. do do chiing toi da linh sach King cgt sdc ky in Igc ProBond Nickcl-t helaniii; Kesin (Invitrogen). Phuong phap tinh sach dugc ilurc lucn theo huong dan cua nha san
\iiiii Mo la vfin l^l nhu sau: te bao thu dirge sau khi l> tam dugc hoa trong S ml d^m Guanidme (Gu HCI (1 M. 20 m \ I Sodium Phosphate pH ^.^. 500 m\l NaCI), Pha te bao bing may ap Igc French pressure vai ap Igc 25000 psi. Dtch phii dugc ly tam \6i 16c dp 15,000 vong/phiit trong thin gian 15 phiil a 4 C de I091 \.ic te bao. Phan d|ch noi co chira prolein tai to hgp dugc dtta len cgt ProBond Nickel-Cheladag.
Resin da chuan bj iruoc Cac protein kliong co ai lye vol Ni" bj day ra khoi cgl bang each rua cgt bang 5 lan the iich cgt lan lugt voi cac d^in Denaturing binding bulTer (8 M Urea. 20 mM Sodium i*os[*att pH 7.8'^, 500 mM NaCl). Denaluiing wash buffCT (8 M Urea, 20 mM Sodium phosphate pH 6,0; 500 mM NaCl), N:iii%e «ash buffer (50 mM NaH;PO. pH 8,0; 500 mM NaCI; 20 niM Imidazole). Cu6i cimg, protein tai to hgp dugc d.i> ra khoi cgt bJng dem Native elution buffer (50 mM NaH;P04 pH 8,0; 500 mM NaCI; 250 mM Imidazole) Dg tinh s?ch ciia protein dugc kiem tra bang phuong phap dien di tren gel polyacrylamide. Ham lugng protein dugc xac djnh bang phuong phfip Bradford (1976).
Phinrng p h d p dot blot
Mang PVDF dugc c6 dinh bang Methanol 100%
va riia l^i bang dem TBS IX. N h o (100 ng) protein t^i t6 hgp len mang, de kho o nhiet dg phong. Phu
Tap chi Cong ngh? Smh hoc 10(2): 225-231, 2012
mang qua dem 6 4''C bang BSA 5% pha trong dem TTBS IX, Rira mang ba i k bang dgni TTBS IX.
moi lan 5 phut, sau do u vai khang the 1 la huyil thanh benh nhan pha loang 500 i k trong gelatin l"-«.
lac a nhiet dg phong trong 2 gitv Rira mang ba Irin bang dem TTBS IX, ni6i lan 5 phiit. siiu do ii vcri khang the hai (cong hgp khiing IgG nguai giin HRP) pha loang 10.000 lan trong jielatin \"'o. lae nh? a nhiet dp phong trong 2 gitr. Tiep theo niiing dugc rii'a hai lan lan lugt bang dem TTBS I \ \ .i TBS IX, moi lan 5 phiit Bo sung djch hien mau. sau do ii 10 phitt a nhiet dg phong va doc ket qua.
KET QUA VA THAO LUAN
Trong nghien ciru gan day. y nghia cua \iec xet nghiem anti-HBc de kiem tra lai ket qua cua cac kii chan doan HBV phat hien HBsAsi \ a anti-HBs bang ky thuat EIA hoac ELISA mpt lan nira dugc khang dinh (Chen. Oon, 2000). Nghien ciru dirge lien hanh Uen 78 benh nhan nguiri Singapore (63 nguoi Ion \ii 15 tre em duoi 15 tuoi da dugc tiem chung vaccine viem gan B) truac do da dugc chan doan la am tinh voi HBsAg Ket qua nghien ciru cho thay, tat ca cac truong hgp n;i\ deu cho ket qua duong tinh voi anii- HBc, Nghien ciru sau han ve DNA cua HBV tir cac benh nhan nay cho thay, tren viing ua nuoc quyet dinh tinh sinh mien dich eiia HBV nam a \ i Ui acid amin tir 110 den 160 tren HBsAg co mang cac dot bien diem d ^ den thay doi cau true khang nguyen ciia HBsAg lam cho cac kit chan doan mien dich hien co khong phat hien dugc su c6 in|t cua HBV (Chen, Oon, 2000). Phat hien na\ da chi ra su c k thiit phai kit hgp nhieu phuong phap chan doan khac nhau nham tranh truong hgp am tinh gia va tang do chinh xac uong xet nghiem HBV noi rieng va cac benh do virus noi chung Hay noi each khac la viec phat u i i n kit c h k doan HBV tren ca so phat hien anti-HBc co y nghia thuc tien Ion.
B i i u hien \ a c dinh t r a n g thai ton tai ciia HBcAg tai to hgp
Chiing E. cob BL21 /pEHBc dugc n uoi bieu hien a 3 7 ^ va cam ung bang I mM IPTG. Theo thiit ke HBcAg (20 kDa) dugc bieu hien duai dang protein dung hgp vai thioredoxin (Trx,I4 kDa) va 6-His a d§u N, CO kich thuoc klioang 35 kDa, D I lira chon phuong phap tinh chi phii hgp, chiing toi tien hanh k i i m Ua trang thai bieu hien ciia protein tai to hgp a trong t i bao E. coli theo phuong phap da mo ta 6 tren, cac pha can va dich noi dugc dien di tren gel
polyacrylamide de phat hien sg co mat ciia prolein tai to hgp.
Ket qua dien di (Hinh 1) cho Ih^y hh.\i hit lirgng prolem tai to hgp thay xuat hien o pha c?n. Nlur vay.
prolein lai to hgji dugc bieu hien duiri dang the viii (inclusion bodies), Hien tugng nay xay ra kliii pho bien doi vai ciic gen dugc bieu hien qua mgnh o trong E coll. dan den cac san pham protein t?o ra khiing k|p gap ncp (folding) iheo diing voi cau hinh tg nhien cua diimg va lap irung vai nhau trong nguyen sinh chat cua le bao vi khuan dum dang cac hat the viii.
Thong thuimg protein bieu hiv'n a the vin ihuimg niiii liogc giam hoat tinh/chirc nang tu nhien cua chiing, protein sau khi tinh $^c\\ pliiii lai gap nep (refolding), Viyc tim ra dieu kien thieh hgp cho qua trinh lai gap nep la rat phirc t^p. Do do, chimg toi da tien hanh toi uu hoa cac dieu kien bieu hien nhu thay doi nhiet dg, nong dg chat cam ting IPTG va thai gian thu mau sau ciim img de thu dugc prolein tai to hgp bieu hien a dang hoa tan. Kel qua cho thay, protein tai to hgp luon bieu hien 6 dang the viii va bieu hien manh nhat o 37°C, 1 mM IPTG va sau 3 gio cam ling (ket qua khong trinh bay a day) Do do chiing toi chgn dieu kien bieu hien nay cho cac nghien ciiu tiep theo.
Tinh sach HBcAg tai to hgp
No luc bieu hien HBcAg trong vi khuan a dang hoa tan nhu diay doi nhiet do. giam nong do chat cam img IPTG deu kliong thanh cong, Hau het protein tai to hgp dugc bieu hien a E coli a dang khong tan (Hinh 1). E>o do chiing toi tien hanh tinh sach protein tai to hgp bang phuong phap bien tinh nhu da trinh bay a tren. Cac phan doan sau khi tinh sach chiia protein tai to hgp duac kiem Ua bang dien di tren gel polyacrylamide. Ket qua cho thay HBcAg tai to hgp da dugc tinh sgch, kliong lan nhieu protein cua E.coh va CO the dimg cho cac nghiencim tiep iheo (Hinh 2)
Muc dich cuoi ciing cua cong uinh nay la su dyng protein tai to hap tinh sach de phat trien bg kit chan doan HBV bang ky thuat Dot blot, vi viiy yeu to quan trgng nhat la khang nguyen tinh sach phai gio dugc dac tinh khang nguyen hay noi each khiic la phai phan ung dac hieu voi khang the kliiing HBV.
Tinh dac hieu cua khang nguyen HBcAg lai i6 hgp dugc kiem tra bang ky thuat Western blot sii dgng khang the khang HBV tu nhien iiong hii\ii ihanh benh nhan. Ket qua phan tich cho ili.n. Hii> - to hgp chi phan ung vai anti-HBe (Hinh 2b. 1) the hien bai mpt bang co kich thuac -35 kDa, trong
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D6ng van Quyen et ai
khi do a m^u HBV iim linh hoiin toAn khi hien bang phiin img iniv (Hinh 2b. ginig 2). Nhu
v^y, kh^ng nguyen HBcAg tdi to hgp sau tinh sach dii liSu chuAn cho c'dc nghiSn ciru tiep theo.
Hinh 1 Ki4m tra trgng Ihfii biiu hi^n cua HBcAg Idl l6 hgp bSng dl$n di tr6n gel polyacrylamide 12,5%. M thang prolein chuAn, 1 prolein t6ng s6, 2 prolein Ihu dugc 6 pha c^n, 3' protein Ihu dugc tj pha nii, 4 m3u dAi chu'ng trirdc cim i>ng IPTG Mui I6n chi v| tri bSog prolein Idi t6 hgp
Hinh 2. A. Kiem tra dg tinh s^ch cua HBcAg tai t6 hgp bSng di^n di tr§n gel polyacrylamide 12,5% (M): thang protein chuan, (1) protein tong so irifdc khi tinh sgch, (2-6) cSc phSn dogn sau khi linh sgch B. Ki^m Ira phin img cua HBcAg \A\
tohgrp vol anti-HBe trong huyll thanh b^nh nhan b^ng kylhu^lt Western blol (M) thang protein chuSn, (1)ki6mfravcnmiu huyet thanh du'crng tinh va m§u huy^t thanh Sm tinh (2) Mui ISn chi bSng HBcAg tii lA hop,
^ 2 3 4 5 6 7 8 9 10
° 1 1 1 1 1 1 1 I I M
paaoQ ayy
Hinh 3 Kilm Ira phan img cua HBcAg tSi t6 hgp win anti-HBc trwig huy^t thanh b^nh nhan bSng dot blot Bang nhOng (A.
1-10 vaC 1-10) va bang nhiing (B 1-10 vS D 1-10) la cSc mSu huy^t thanh da dugc x*;d|nh la duang tinh va amtinh vdfi anti-HBc thir vol bo kit ELISA cua Bio-Rad.
Tap chi Cong ngh? Smh hpc 10(2): 225-231, 2012
Ung d u n g HBcAg t^i to hgp d i ph^t hi^n HBV trong h u y i t t h a n h b^nh nhSn bfing kv thuat Dot blot
Western blot hien dang la mpt trong nhimg phuong phap pho bien trong chan doan HBV a nhiiu nuoc tren the gioi ciing nhu o Viei Nam. Mgc dii vay, viec dua k\ thuat nay \iio su dgng dai tra de chan doan HBV tai cac benh vien se co gia thanh cao va khong tien loi. boi \ i doi voi Western blot, can c6 buoc dien di phan tach cac thanh phiin protein truoc khi ehuyen sang mnng trong dien trutnig, Tuy nhien, kill su dung protein uii to hgp da dugc tinh sach thi CO the dua true tiep ien mang duoi dgng cac diem (dot) ma kliong can phai dien di phan tich tren gel, Chinh vi the chimg toi tien hanh tao cac bang nhiing Dot blot diing cho chan doiin anti-HBc dua tren nguyen ly giong nhu \\ estem blot, nhmig kliong can dien di phan tach protein nia dua diang protein da tinh che len mang PVDF,
Thir nghiem \ a i 40 mau benh pham la huyet thanh benh nhan (20 mau am tinh, 20 mau duong tinh), u u o c do da dugc xet nghiem tai benh vien Bach Mai - Ha Noi bang kit ELISA ciia hang Bio- Rad, cho thay bp Dot blot cho ket qua chan doan tuong tu nhu ket qua bg kit ciia Bio-Rad (Hinh 3).
Hai muoi mau duong tinh (Hinh 3. bang nhiing A, I - 10 va C: 21-30) cho cac bang phan ting kha ro net tren bang nhiing, co the quan sat duoc bang mat thuong, con o cac mau am tinh (bang nhiing B: I I - 20 va D: 31-40) thi bang nay khong xuat hien.
Kit qua na>; con cho thiy. HBcAg tai to hgp tinh sach sau khi c6 dinh tren mang van phan img rat manh va dac hieu voi khang the khang HBcAg trong huylt thanh benh nhan. Do dam nhat ciia cac mlu the hien tai diem (blot) phan ung giiia cac m l u co su khac nhau, mot so mau cho bang rat dam trong khi mpt s6 m l u lai cho bang nhat ban (hinh 3). DiSu nay CO t h i giai thich la ham lugng khang the khang HBcAg giiJa cac m l u la khac nhau, hay noi each khac la cac benh nhan nay co the dang o cac giai doan lay nhilm/phat trien benh khac nhau sau khi nhiem HBV.
Tieu chi quan t r ^ g nhSt ciia cac kit chan doan benh do la do nhay va dp dac hieu. Bp kit phai dam bao khong bo sot cac m l u ducmg tinh d6ng thai khong cho kk qua duong tinh/ am tinh gia so voi ket qua da dugc xet nghiem truac do bang mot phuong phap chuan hoa. So sanh voi ket qua ciia bp kit ELISA phat hi$n khang th& khang HBcAg ciia Bio- Rad cho thly, 20 mau huy^t thanh duac xac dinh la
duong linh voi anti-HBc bang kit LLISA ciia Bio- Rad cung cho k^t qua duong tinh khi kiem tra bang bg Dot blol. Tumig tg, 20 mau dugc x6l nghiem la am tinh bang kit ELISA cua Bio-Rad cung cho kk qua am tinh khi kiem tra vai bp Dot blot (hinh 3), Tir k^t qua nay chiing toi so bg ket lufin bg Dot blot co dii iiha\ va dg dac hieu la 100% so vai kit ELISA ciia Bio-Rad.
KET LUAN
Chiing loi da tim dugc dieu kien toi uu cho bieu hien va linh sgch gen ma hoa klidng nguyen HBcAg eiia HBV cr E coli HBcAg tai to hgp tinh sgch phan img d?ic hieu voi anti-HBc trong huyet thanh benh nhan viem gan virus B va dugc diing de phat trien bp kit chan doiin HBV bang ky thuat Dot blot Cac thii nghiem ban dau tren 40 mau benh pham cho ket qua rat kha quan. bp Dot blot co do nhay va dp d$c hieu tuong duong \ a i bo kit ELISA cua Bio-Rad, Mac du
\ a \ day mot chi la cac ket qua kliao sal ban dau. can thu nghiem tren so lugng mau nhieu hon cung nhu loi uu do nhay, do dac hieu,..de hoan thien bp kit chdn doan HBV bang Dot blot.
Loi earn on: Cong trinh duo-c Ihirc hien nha su ho Iro kinh phi cua de tai cap Vien Khoa hpc va Cong nghe Viel Nam "Nghien cuu hoan ihien cong nghe san .xual bo kii dang hang nhung de chan doan viem gan B " va CO .sir dung cac thiel bi ciia Phong thi nghiem trgng diem Cong nghe gen, Vien Cong nghe sinh hoc.
TAI LIEU THAM KHAO
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Significance of anii-HBc screening of blood donors & its association with occult hepatitis B virus infection:
Implications for blood transfusion./Hi/./A/e(^^fs 132 312- 7.
Barker LF, Chisan FV, Mc Grath PP, Dalgard DW, Kirchstein RL, Almeida JD, Edgington TS, Sharp DG and Peterson MR (1973) Transmission of type B viral hepatiiis to chimpanzees J Infect Dis 127, 648-662.
Beasley RP (1998) HepaUtis B virus. The major etiology of hepatocellular carcinoma. CancerSl. 1942-56, Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing
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D E \ E L O P M E N T O F D O T B L O T I M M l N O A S S A Y F O R D E T E C T I O N O F A N T I - H B c IN H B V - I N F E C T E D P A T E I N T S E R A I I S I N ( ; R E C O M B I N A N 1 H B c A g P R O T E I N
Dong Van Quyen*, Pham Minh Tuan. Le Phuong Hang. Dinh Uuy Khiing Inslilule of Biotechnology. Vietnam Academy of Science and Technology'
SUMMARY
Hepatitis B core antigen (HBc or HBcAg) is one of the most important antigens of Hepatitis B virus (HBV). h is a 21 kDa protein, which has die intrinsic capacity to self-assemble into capsid panicles Many studies have shown that HBcAg is exUemely immunogenic; antibody to HBcAg (anti-HBc) appears and persists many years following initial infection. Therefore, anti-HBc is an imponam serology marker of hepatitis B infection and patient follow-up. Due to the essential role of HBc in the virus life cycle and its application in early detection of HBV infection, this protein has been the focus of much research with the ultimate aim lo develop sensitive and specific HBV diagnostic kits. In order to gel HBc recombinant antigen suitable for serodiagnosis requirements with a lowest cost, in this study we have expressed the gene coding for HBcAg in Escherichia coli. The recombinant prolein was purified under denaturing condition using Ni-NTA ' Author for correspondence Tel. +84-4-7563386; Fax- +84-4-7914815: E-mail.' dvquven(a).ibt. ac. vn 230
Tap chi Cihig nghe Smh hoc 10(2) 22.^-231,2012
idlimly chromalogiaphy (Inviiioycn) Purillcd ilHcAg was then le.sied loi ils acliviiy toward anti-MBc in patient sera by Dot blol ininumoaNsay Our initial dala bii.sgd on ihe lesl of 40 clinical samples (20 negative and 20 positivc-cliniciil samples), confirmed previously by I 1 ISA kit of Bio-Rad, showed that ihe recombinant HBcAg reacted strongly and specificdiy with anli-IIBc in the scnan of ilBV-inreclcd palienls The present method displayed high SCIIMIIV U\ and specilieily as comp.iicd lo ihal of Bio-Rad kit :ind can be adapted for detection orHIW mlcclion
Key words. i>niy,noMu.\. Dot Mol .iwav, rctoiiibiiiani HIU \g I'loli cspiewum. Hcpalilis B virus