TAO K H A N G T H t DA D O N G K H A N G VUNG CTD C O A PROTEIN BcIA V 6 B A O Tlir VI K H U A N BACILLUS ANTHRACIS VA (fHG DUNG T R E N QUE THCK NHANH
Hoing T h i Ydn\ Dinh Duy Khdng*, BiJi Phuwng T h u | ^ ^ Nguyln Thi N9a\ Tran Minh Tri', Nguyln Thanh 0?t\
Phiing Huyin Nhung' LS Tr^ng VSn'*
Wiin Kf thugt Hda sinh vi Tai lieu nghiip v(j, Bd Cdng an
^W$n Cdng nghi sinh hgc, Vi^n Hin lim Khoa hgc va Cdng nghi ^iit Warn
^Tnrdng D^i hgc Khoa hgc T\f nhiin. Dai hgc Qu6c gia Hi ngi
T 6 M TAT
Protein Bel A li mpt glycoprotein chilm ty If ldn trfin vd bio tii vi khuin than, nim d dau ngoai cOng, tren lonp bio tii vi kJiuin, Chiing thvc hifn mpt chfte nang quan ti|mg lim tSng cudng khi nang khing nhi|t, khing protease, khi nang gin ket cila bio tii vii ti bio vat chii. Protein BclA cung dugc xic djnh gay dip ihig mien dieh manh vdi te bio vat chi. Vi?c tinh chl protein tv ntufin vd biio tiJ vi idiuln gip nhirai khd khan nm nhi^u nghifin ciru da sii dyng protein tii td hgp (rBclA) thay thi trong vific giy dip ling miSn djch t90 khang thi don vi da ddng. Trong nejufin cuu niy chiing toi di sii d\mg viing CTD tii to hpp cua protein BclA gay dip ting miSn djch trfin chugt BALB/c 6 - 8 tuin tuoi. Sau 60 ngay gay dip ting miln djch, hifu gii khin_g huylt thanh d?t tJ 1§ pha loang 1/5000. Khing thi da ddng tinh chl d^t nong dd 2 mg/ml. Chiing toi da kiem tra khi nang lifin kfit cua khing the vdi protem tii tfi hijp vi djch phi bio tii vi khuin (protein tp nhifin) bing ky thu&t ELISA va Western bloL Vi#c thii nghiem trfin que diii dpi kit qui tit, khing the da ddng c$ng hpp h^t ving 40 nm di phin img vdi khing nguyfin tii tfi hpp vi protein t^r nhifin tren que thii t?o thinh hai vaeh test line (Tl) vi (T2), do dd cd thfi iing dyng trfin que thii dang lateral flow test eho m^c dich pliit lii§n bio tii vi khuan than.
TH kkoa: que thii nhanh, khing the da ddng, rBclA, vach ddi chiing (control line), vjich thii nghiem (test line), vi khuan than, MdDAU
Vi khuSn than Bacillus anthrads \i tn/c khuin gram duo'ng. cd kh3 nfing sinh bao ti>, ky khf tu^ ti$n. BSo ti> c£ia chOng c6 thi tin tgi Idu tnang mfli tnrflmg (Mock ef ai. 2001). Bdo ti> vl khuSn than \i tdc nhSn sinh hpc nguy hilm trong khung b l bang vO khi sinh hgc (Hawley et ai., 2001; Fennelly et ai 2004).
Protein BclA (Bacillus collagen like protein of anthracis) dvtrjo ma h6a b*i gene bcIA \i thanh phin dilnh ciia v l bdo fi (Brian ef al., 2008). Cdc protein cOa Iftp ngodi ndy la thdnh phin diu tien cCia bdo ti> lu'O'ng tdc tri,fc tilp vdi h$ &i6ng miln djch trong ca thi vdt chu, Idm tang khd ndng bdm dinh ISn b l mdt t l bdo v$t chO, vl v^y ddy Id mOt trong nhOng protein cd tlnh khdng nguySn rat cao (Sylvestre et ai, 2002; Canaline ef ai, 2004; Trupti ef ai, 2007). Vide linh che protein 6 d^ng t^ nhiSn g^p nhieu khd l^dn vl clu tnic bin vQ'ng cCia v6 bdo tir.
Phdn to BclA cd 3 vCing: Vung tdn cung N (NTD: N-terminal domain), viing collagen like repeat (CLR) chi>a cdc do^n ISp ddc bvng; vd vung tdn ciing diu C (CTD: C- terminal domain) Id vOng cd khd nang khdng nhilu lo^l protease, cd cau tnic I n djnh, Viing CTD hlnh thdnh clu tnic xoln bdc 3 tirerng dli bin vu-ng vcri nhl#t dO, viing ndy ndm ngodl bl mSt bdo ti> Id mdt phin ouan trgng cOa BclA. Clu tnic viing CTD bao gim nhieu sg-i beta xIp l^i vd'i nhau d l ddng hlnh thdnh cdc ^ m tinh the 16n (Jeremy ef ai. 2005).
TrSn t h i gidi, cd nhilu cdng blnh nghiSn cO'U t^o khdng t h i do'n dong, da ddng khdng Igi rBcIA vdi nhilu mgc dich khic nhau (Christopher et ai, 2003; Trupti ef ai. 2007; Jenemy ef af., 2005). Trong nghiSn cii'u ndy chOng tdi tgo khdng thi da ddng khdng Igi viing CTD tai to hgp ciia protein BclA nhim i>ng di,ing tren que thi> phdt hiSn bdo tir vi khuin S.
antiiracis.
NGUYEN LI$U VA PHlfONG P H A P Nguyen lieu
Khdng nguySn SLF dyng Id protein tdi t l hg'p viing CTD cd kich thu'dc khodng 14 kDa kem dudi Trx khodng 20 kDa &\st}c bilu hiSn vd tinh sgch ti> chiing vi khuan E. coli BL21 (DE3) tgi phdng Sinh hpc nghiSp vy - ViSn ky thudt Hda sinh vd TSI li$u nghldp vy - Tong eye IV - Bg COng an. Protein tdi t l hg'p ndy da dyi^c xdc nhdn trong cdc thi nghiem txirdc ddy cua phdng (kit qud khdng trlnh bdy),
ChuOt gdy miln dich: chugt cdi BALB/c 6-8 tuIn tuoi du'g'c mua cOa Vi§n kilm djnh Qulc gia Vac xin vd Sinh phim yti- Bdo ti> vi khuin 6. anthracis chiing nhu'g'c dgc cd nguln glc tic Vign vd smh dich t l Trung u'O'ng. Cdt tinh chl HiTrapTM Protein G HP duwc mua cua hang GE. Td du'g'c day du (CAF), td du'gc khdng dly dii (lAF) - Sigma; Protein marker BenchMark™ Pre-Stained Protein Ladder - Invitrogen.
Hda chit cho Westem blot: Khdng t h i dS khdng IgG chudt cdng hg'p alkaline phosphatase, co chit BCIP (5-Bromo-4- chloro-3-lndolyl phosphate disodium salt)/ NBT (nitrotetrazoiium blue chlonde) cCa Sigma. Hda chit cho EUSA: anti- mouse IgG (whole molecule)- peroxidase antibody du'gc sdn xuit tif di; ca chat TMB (3,3',5.5'-tetramethylbenzidine) cua Sigma.
Mdng nitrocellulose, mdng chipa ehit cgng hap. mdng hiit miu, mang hiit trSn, tam d l - Hang Aristablologycal (MJ).
Phu>o>ng phdp nghiSn cuu GiymlSn djch tren chudt
? C C 6 N G N G H E S I N H H Q C T O A N Q U O C 2 0 1 3
C h u ^ c d i d d n g B A L B / c t t H i l n c h i i n g , tir 6 - 8 t u I n t u l i , k h o e m g n h Ouffc gdy m i l n d j c h vdri k h d n g n g u y e n tdi t l h g p vOng C T D c i i a protein B d A d u g c tinh c h l b i n g c d t N i k e n , ^ k i e m tra d g t m h s g c h . v o i t i l u tiSm 100 n l / l l n tiSm, t ^ n g c d c h
^ n dirCTi d a or 4 vi tri d g c ttieo s d n g l u i i g . S a u Od. chuOt d u g c g d y miSn dich n h i c Igi s a u 1 0 ngdy. S a u khi g d y m i l n
^. ffinh ky l a y m d u 5 n g d y mO* l l n s a u tiSm, k i l m tra k h a n d n g d d p i>ng m i e n d i c h b a n g E U S A . S a u 10 n g d y k l b> l l n hSm thir 4 c h u g t d u g c t i e m n h I c lgi l l n c u l i vdfi k h a i g n ^ i y S n p r o t ^ tinh c h l t n x i g P B S n h i m g k h d n g t r d n v d i t d chSt v a o v e n d u o i . S a u 3 d i n 4 n g d y k l tir l l n tiSm c u l i c w i g . l l y m a u t i r h i c m d t v d t a c h h u y l t t h a n h . k i l m tra khd n d n g d d p u n g m i e n djch b i n g E U S A . D e n n g a y t h i i 6 0 t h i ^ c h u p t v d t h u t h u y l t t h a n h .
nuang phap EUSA
^^t ' i ^ ' ^ " ^ ' " ^ ^ ^ n g u y e n d i f g c pha frong dSm g d n bffli (coating buffer 0.05 M p H 9,6; t h a n h p h a n Na2C03 1.59 g; N a H C O a 2 . 9 3 g ; NaNa 0,1 g b l s u n g d u 1 lit nud-c d t v d t r i k i g ) d e dgt n o n g d f i 5 0 (igftrt. S a u d d . moi g i l r ^ frSn d i a E L I S A d i r g e p h i i v d i 100 nl d u n g djch k h a n g nguySn. u 4 ° C q u a dSm. DTa E L I S A flucrc n>a 3 lan b a n g P B S - T ( p h o s p h a t e buffer s a l i n e - t w e e n 2 0 0.05%), m i i lan 2 0 0 n l . B o s u n g 2 0 0 td d i m p h u g i i n g (blocking solution) ( 2 % B S A ' ™ * ^ ^ A ^ ^ ^ " ^ ^ ^ ^'^^ " ° ' ^ ^ ^ P ^ * ^ ' ™ ^ ^ 9 * * - ^ " * ' ^ " ^ ' * ^ " 9 ™ ^ ' ' ' ^ ' ^ " ' ' " ^ ' ^ " 2 ™ ^^• ^ "
d u g c b d s u n g huySt t h a n h 100 p l / g i i n g (pha k i a n g 6' n i n g d d thfch h g p ) trong b k w k i n g solution, u 3 0 phOt. 37*C. B d n d u g c n r a b a n g P B S - T 3 l l n , m i l l l n 2 0 0 pi. K h d n g t h i 2 d i f g c b l s u n g v d i l u g n g 100 pl/gieng (pha loang a n i n g dO 1/10.000) t r o n g b l o c k i n g s o l u t i o n , u 1 gidr. 3 7 ' C . B d n d i r t r c r i r a b a n g P B S - T 3 l l n . m f i i ldn 200 j J . H i g n m d u b a n g vige b l s u n g 100 pi T W B w w W n g s o l u t i o n , a l 5 phut nhigt d d p h d n g . P h d n i>ng d u g c d i m g b a n g 5 0 ^^^ H2S04 1 N . X d c i^nh dO h a p t h y ven OD450 n m b a n g m a y d g c E U S A reader.
T i n h sach khang the t>mg M T r a p T M t*rotein G HP
K h a n g I h l d u g c tinh s g c h b i n g cdt ai I g c HFTrapTM Protem G H P d g a vdo k h d n a n g BSn ket ^ c hiSu o i a protein G v d i v u n g Fc c u a I J d n g t h i I g G . C h u i n bj cdc d u n g djch dSm v d hda c h i t d l tinh s g c h IgG c h u d t b a n g cot sdc k^ d i l y e protein G . HuySt t h a n h d u g c hoa Iang frong dSm P B S p H 7,4 v d i t J I f 1.5 nti h u y l t ttianh v d i 2,5 m l d d m COt d u g c c d n b d n g v d i d | m P B S p H 7 . 4 t 6 c d d d d n g 1 m l / p h u L M i u h u y l t t h a n h drnTC ISn cOt v d n>a d l logi c d c protein liSn k i t k h d n g ddc hiSu b d n g d d m P B S p H 7,4. K h a n g ttil I g G g i n fren c g t d i f g c ttiu b ^ d $ m d l y ^ y d n - H C I p H 2.7 v d i t i c d d d d n g 1 ml/phuL D j c h d l y d u g c frung hda bang d d m T r i s - H Q p H 9 v d i ty 1# 1 ml d u n g d j c h d l y v d i 2 0 0 pi dSm Tris-HCI.
K y t f i u l f W e s t e n M o t
Protein t i n g s l d u g c p h a n I d c h b i n g d i g n di fren gel S D S - P A G E sau d d d u g c c h u y e n ISn m d n g P V D F b i n g p h u o m g phdp didn c h u y i n t r o n g d f i m T r i s - g t y d n e c 6 2 0 % m e t t i a n o l . M a n g PNTOF d u g c c a t v d i kich t h u d c b i n g v d i kich ttiudc b a n g e l v d d u g c » > l y v d i m e t t i a n o l 1 0 0 % trong 5 p h i i L G i i y c h u y i n d u g c n g d m u d t d i m frong d g m T B S fTris-HCl 10 m M p H 7.5, 1 5 0 m M N a C l ) . D g t 3 t l m g i i y c h u y i n trong k h a y c 6 c h d a dSm T B S , d d t m d n g P V D F d a d u g c xij Yj frong methanol l d n t r d n ( I h e o c h i l u m a t bdng x u I n g d u d i ) , l i y g e l dd di#n di ddt len frdn m d n g , t i l p t y c ddt 3 l d p g i i y c h u y i n dd tam T B S ISn frSn. D i S n c h u y e n trSn m d y T E 7 0 P W R ( A m e r s h a m ) . 2 5 m A frong 1 g i d . M d n g d u g c p h d b d n g d u n g djch T B S - T ( 0 . 1 % h « e e n 2 0 ) c d c h d a 1 % BSA,lac nhg 6 0 p h Q t M d n g 8 l p tgc d u g c it v d i k h d n g t h e 1 Id IgG c h u d t gdy midn d j c h . l i e n h g 1 g i d nhi$t d d p h d n g . M d n g d u g c n>a b d n g d d m T B S - T 3 l l n n i d i l i n 10 phut, s a u d d d u g c t i l p b^c u v d i k h d n g t h i 2 Id k h d n g t h i dS k h d n g I g G c h u d t e d n g h g p alkaline phosptiatase. M d n g s a u d d d u g c r u a 3 l l n m o i t i n 10 p h i i t b a n g d S m T B S - T . H i g n m d u b i n g d u n g djch p-nibo blutetraBum c h k x i d e l 5-1jfDmo-4-chtoro-3indoly( (rfiosphate ( N B T / B C I P ) trong d g m T B S p H 9.0 v a 5 m M M g C l j . K h i cac b d n g htSn ro, p h d n u n g ducrc d d n g b d n g d g m T B S 2 0 m M EDTA.
K y tfiu$rp/idn img miSn dfch tren que thtr nhanh
ok l u l m tra k h a n d n g u n g d y n g frdn q u e t h u n h a n h d g n g lateral flow test c h i i n g t d i tien h d n h frdi cdc h d a chat ISn frSn m d n g n i t n x ^ l l u l o s e ttieo c d n g nghS cOa H a n g Arista - My. V g c h confrtri ducrc tral sinh p h i m goat anti-mouse c u a h a n g M s t a b i o l o g y c a l . V g c h l e s t l i n e d u g c trdi c a c sinh p h i m protein tdi to h g p va djeh p h d bdo t u v i k h u i n . IQidng ttie d a ddng d u g c c d n g h g p v d n g v a c h g y trdn n^dng nittocellukjse khi d l q u a v i i n g test line s e bat g i u l ^ d n g nguySn. T g o r a v g c h m d u tgi test line. P t i l n c d n lgi s e di c h u y e n t i l p v a t>i bat g i u tgi control line.
KfeT Q U A V A T H A O L U A N G d y m i e n d j c h v d tinh C I K k h a n g t h e
K l h o g c h g a y m i l n d i c h c a n d u g c c h u i n bj k y l u d n g v d frian t h i i chgt che t h e o q u y trinh c d v l t h d i gian vd l i i u l u g n g khdng n g u y i n .
Bdng 1 . L|ch gay m i e n d j c h tren c h u ^ t d u v c t u i n t h i i ttieo bang
Mui tiem L u g n g khang nguyen T i c h S t s u w i » „ - « - Vj t n tiem (Mfl/100 til/chuot/l^n) dung Gdy m i l n djch Idn 1 i o o CFA Ouoi da L l y mdu
Gfty m i l n (Sch n h I c lgi l l n 1 so IFA Diraida L l y mdu ^
Gfty mifin dich nhac lai lan 2 50 IFA Duwida L d y m d u
Gfly m i i n dich nhac l?i Idn 3 50 I F A Duwida L d y m d u
Gfly m i i n dich n h l c l^i ldn 4 50 I F A D U O I da L i y m i u
Nghi trudc Uil tidm mui nhIc t^l cuoi ciing Gay miln dich nhic lgi
°" tnrdc dung hi^j 60 Gilt chupt. thu huy^lhanh
Sau 60 ngdy gay miln djch, chudt dugc gtlt vd thu huylt ttianh. Huylt ttianh dugc su dung d l ddnh gid hidu gid khdng thi thdng qua ELISA vdi dg pha loang ciia huylt ttianh: 1/100; 1/500; 1/1000; 1/5000; 1/10000
Kit qud Hlnh 1A cho ttily ehugt duwc gdy miln djeh da co dap ung khd mgnh vdi khang nguySn tdi to hgp. Ty 1$ [^a loang huylt ttianh dgt 1/5000 vdri khang nguySn tdi t l hgp vd dgt 1/1000 vdi djch phd bdo tu (protein tg nhiSn). Huylt ttianh cung co phdn ung vdi djch phd bdo tu vi khuin. TheoTmpti va cgng sy (2007) tdc gid da sO- dung protein rBdA tdi t l hOT) gdy ddp dng miln djcJi trSn ttid vd cOng thu dugc ddp ung miln djch mgnh. tf Id pha loang huylt thanh dgt 1/5000. Kit qud ndy cung hfong ddng vdi Jenery vd edng sg (2005) khi sit dyng viing CTD tdi t l hgp tgo Widng the don ddng frong nghiSn cdu v l ttnh ^ u tnic. chuc nang vd tfnh on dinh cOa pnatein BclA trdn vd bdo h> vi khuin B. anthracis.
Oilu ndy chung td protein rBcIA tdi to hgp cd tinh sinh miln djch ttdn chudt. Huylt thanh ttiu dugc tir mdu chudt gdy miln djeh dii dilu kiSn d l tinh chl sir dyng cho cdc thf nghigm ttep theo.
kDa 181 —i 1/100 • 11S,5
82,2 — 1 „ 1(500 6 4 , 2 — ^ - .-
48,8—IB ^ ^ 1/1000
2 S , 9 _
t-Khang nguytnMl A hvpCTI]
O&i chLmg £,0 _ a j £ ; ; ^ . f ^ ^ ^
Hlnh 1. (A): Phdn QngELISAvdihuyllthanh pha loang dr cdc Hlnh 2. (A) Khd nang II§n ket cda khdng Ihl vdi djch phd bdo t^l$ khdc nhau (1: khdng nguySn tdi td hgp; 2: dtch phd bdo ti>); tirva khdng nguyin tdl t l hg^ (1: Djch phd vd bdo tir; 2: khdng (B) Di$n dl d l sdn phim tinh s^ch (1- Marlcer; 2- Phdn dogn nguyin tdi to hgp; 3: marker), (B) Thtr nghiem trin que mlfin tinh sgch khdng tfil da dbng) djch vdi djch phd bao tir (Contml line: goat anb-IgG chu^t; Tl:
khang nuyen, T2: djch phd bdo ti>).
Tlnh chl khdng t h i bing cgt protein G
Huylt ttianh dugc tdch tir mdu chudt gdy miln djch chgy qua cgt protein G. dly bing ddm glycin-HCt pH 2.7 vd tmng hda bdng Tris-HCI pH 9. San phim qua cot chgy diSn di SDS-PAGE 12,5% de kilm tra dg tinh sgch.
Kit qud iXgn di thu dugc hai bdng ed kich thudc 53 kDa vd 25 kDa Id chudi ngng va ehudi nhg ciia IgG. Di^n di d l khdng thiy xuit hign cdc bang phu chimg to san phim da tinh sgch (Hinh IB). Dich day dugc xdc ^inh ning dd protein theo phugng phdp djnh luong pnatein BradfonJ (1976). Dga vdo dudng ehuIn va cde gid trj OD do dugc a cdc ndng dd, protein ttnh sgch dugc xdc djnh nong dg dgt 2 mg/ml. Ndng dd khdng t h i thu dugc phy thuOc vdo lugng khdng the sinh ra ciia qud frinh gay ddp ung mien djch vd ky thugt ttnh c h l . Vdi nong dd nay dO de sir dyng cho cac thf nghidm fiep theo.
Kha ndng lien k i t ciia khdng t h i tinh sach vd djch pha bdo tir bdng phinrng phdp Westem blot
Sau khi tinh sgch khdng the chung toi tiln hdnh thf nghiSm ddnh gid kha nang phdn irng khang nguyen- khang thi gi&a khdng ttil da ddng khdng khdng nguyen tai t l hgp vdi dich phd v6 bao td vl khuin than (BclA tg nhidn) bdng phucrng phdp Westem btoL Ddy Id mdt bud-c quan frgng d l ddnh gid tinh sinh mien djch cua protein tdi to hgp xem khdng Ihe tgo ra cd egp dugc vdi khdng nguySn tg nhien tCr vd bdo tu vi khuin S. anthracis hay khdng.
Kit qud Hinh 2A cho thiy khdng t h i da phan dng vdi khang nguydn tai t l hgp cd kich thudc khodng 34 kDa vd protein trSn vd bdo td vi khuin ed kfch thudc khodng 70 kDa. L^ gidl eho viSc sai khdc vj tri bang trSn bdng diSn di Id protein dugc sd dung ldm khdng nguyen la mgt vung (CTD- C terminal domain) cua protein BclA trong khi dd djch phd vd bdo td la mdt pnDtein ddy du, hon nOa ir trgng thdi tu nhidn protein BclA ed eiu frOe soln 3 siSu bin do dd sg di chuyin trSn ffiSndi d l ludn khdng dOng vdi trgng lugng phan ti> eCia protein. Theo Christopher vd edng sg (2003) thi cau tnic don chudi cua protein BclA cd kich ttiudc 33,3 kDa, tuy nhiSn d dgng tu nhien phdn tO" BclA ludn ir tt^ng &idi soln 3 siSu bin nSn di chuyin tren den di do xuit hidn tgi vach khoang 70ltDa. COng theo tac gia nay eiu tiiic cua vd bdo b> rit bin do dd trong qud trinh XLF ly phd vd bdo ti>, mdt lugng Idn pnstein BclA d dgng liSn kit vdi cde thdnh phin khdc s6 xuit hidn d vgch dign di co kich ttiudc > 250 kDa. Ket qua nay tuong dong vdi mgt s6 k i t qua nghiSn cdu (Christopher ef ai, 2003; Jeremy e( ai, 2005; Lashana e( al, 2005).
Thir nghiem trdn que thii' dgng lateral flovtr lest
Vdi myc dfeh kilm tra khd ndng bit giO" ciia khang the da ddng vdi khang nguySn tdi to hgp vd protein tg nhtSn fren vd bdo tu, chiing tdi tien hdnh phd vo bdo tu bdng each cho bdo tu mdt do 10* bao td/ml trong dSm PBS 7,4. SiSu Sm trdn
mdy ulfrasonic liquid processors trong dilu kiSn Ignh. sau m i i phOt ngh! 30 gidy, trong 15 phiit. Ly tdm 13000 vdnq/phut ttong 10 phut, ttiu djch n i l .
S ' f S * " h ^ ''"® "^^*]>^t g't* 1^ goat anfi-mouse 1 mg/ml; lest line (Tl) Id khdng nguyen tdi t l hgp; test line 02)- djch phd
^ i ^ ° iL "^ ^^ ?^ ^^"9 ^ " ^ ^^^ f^W v*i hgt vdng kich ttiudc 40 nm. Khang ttil da ddng dugc dich chuyin frdn mdng nifrtx»llulose d l kilm fra khd ndng bit giir tgi cac vgch test line. » v .
A rf"i? " i ' ^ l ! ^ ^ ^ ° ^^ ^^^"^ ^^ " ^ ''*"9 ^"^ chl da bdt giu khdng nguyen tdi to hgp (Tl) vd cung bat gid tten vgch
% r i Hiil i ! r ° i ^ r ^ l - ^ ^ " ^ ' ' " ' '^ '^^"9 "9uy6n tdi to hgp bat gid khdng thi da ddng cdng hgp vdng mgnh hon vgch iz la ajcn pna vd bdo tu. Tuy nhiSn vdi khd ndng bit giu nhu frSn. vgch T2 khd rd ndt, dii d l quan sdt nhgn bilt kit qui.
khuin t h ^ ^" ''^^"^ "^^ ^"^ '''^"9 *"*" " " ^ * ' ' *'^"9 '^*®^^' ^°^ '®^* ^^ P^^* ^'^" ^'^'^ '^®" ^° ''^° '"^ ^' K £ T LUAN
Chiing tdi da su dyng protein tdi t l hgp gdy ddp ung midn djch bdn chudt BALB/c cdi 6- 8 tuIn tuli. Sau 60 ngdy gdy
; / K ""?• T'^i?^ j f . r " ^'^ ^^"^ "^"y^* ^^^^ ^9^ ^ 'e pha loang 1/5000. Khdng ttie da ddng khdng khang nguySn tdi 10 ngp tinn cne dgt ndng dg 2 mg/ml. Chiing tdi dd kiem tra khd ndng liSn kit ciia khdng t h i vdi khdng nguySn tdi t l hgp va aicn Ppa bdo t u v i khudn (protein tg nhiSn) bing ky ttiuat ELISA vd Westem blot. Vide thir nghigm frSn que ttid dgt Ket qua ^oj'.'Viang ine da ddng cgng hgp hgt vdng 40 nm da phdn dng vdi khdng nguySn tdi t l hgp vd protein tg nhiSn trfin que thu tgo thdnh hai vgch test line (Tl) vd (T2). do dd co t h i ung dyng frSn que thd dgng lateral flow test.
TAI Lieu THAM K H A O
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GENERATING POLYCLONAL ANTIBODIES AGAINST REGION RECOMBINANT CTD OF BCLA PROTEIN AND APPLICATION ON LATERAL FLOW TEST TO DETECT BACILLUS ANTHRACIS SPORES
Hoang The Yen^', Dinh Duy Khang^ Bui Phuong Thuan^ Le Trong Van\ Nguyen Thi Nga\ Tran Minh Tri\
Nguyen Thanh Dat^, Phung Huyen Nhung\
^Institute of Chemistry-Biology and Professional Document Technique 'institute of Biotechnology, VAST
\NU University of Science
SUMMARY
BclA protein is a glycoprotein which appropriate large prapotion and location at the end of each filament of the hair-like nap on £.
anthracis spore coat. They perform an important fiinction in enhancing heat resistance to protease, enhances adhesion of spores to the host cell. BcL\ has been identified which may cause a strong immune response lo ttie host cell. The purification of natural protein of spores IS difScult, hence there are studies which have used recombinant BclA protein in generating polyclonal and monoclonal antibodies. In this smdy, we used the recombinant CTD of BclA protem to mduce an immune response in 6-8 week-old mice BALB/c. After 60 days of immunization, antibody titer by ELISA reached to 5000. Purified polyclonal antibodies concentration were 2 mg/ml. Immunogenicity of the recombmant protein was assessed by the binding ability of antibodies with natural protein hy ELISA assay and Westem blot. Polyclonal antibodies conjugated with 40 nm gold particle capture with recombinant antigens and nature
proteins to form two line (Tl) and (T2) in ttic sfrip lateral flow test. Therefore, ttiis rescardi gives a promissoig mom^y recombinant CTD region of BclA for construction of lateral flow test for B. anthracis di'
Keywords: Bacillus anthracis, control line, lateral flow test, polyclonal antibodies, tBc\A protein, test line.
• Authorforcorresspondence:Tel'+84-989321113; Email: hoanobiojaiQmail.com