Y HOC VIET NAM THANG 3 • SO 2/2012
NGHIEN CUU VA THIET KE VECTOR BIEU HIEN PROTEIN CAPSID CUA VIRUS CHIKUNGUNYA TREN HE E.COLI
TOM TAT
Virus Chikungunya (CHIKV) la nguyen nhan gay ra dich va cac dai dich sot Chikungunya vdi cac bieu hien sdt, phat ban va dau khdp. De phat trien ky thuat chan doan huyet thanh dac hieu CHIKV, vector bieu hien protein tai to hpp capsid ciia virus dUdc thiet ke va bieu hien tren he E.coli. U'ng dung ky thuat 5DS- PAGE va W/estern blot phan tich cho thay, protein tai to hpp capsid ciia CHIKV bieu hien tren he E.coli va co trong lUOng phan tir la 42-44 Kd.
ABSTRACT
STUDYING AND DISIGN VECTOR EXPRESSION OF CHIKUNGUNYA VIRUS GENES CODING
STRUCTURAL CAPSID PROTEIN I N ESCHERICHIA COLI SYSTEM.
Chikungunya vims (CHIKV) causes endemic or epidemic outbreaks of CHIK fever, which typically manifests as a fever, rash and arthritis. To develop a CHIKV-spedfic senDlogical diagnostic test, capsid recombinant protein expressed vector was designed and expressed on the E.coli system. SDS-PAGE and Western blot were applied and analyzed to show that the rerombinant pnjtein vras expressed and the molecular weights of structural capsid proteins was 42-44 Kd.
I.
OAT VA'N OE"Virus Chikungunya (CHIKV) la nguyen nhan gay benh sdt CHIK d ngu'di vdi cac trieu chirng dien hinh nhu* sdt, dau khdp, viem khdp va phat ban. CHIKV la mdt alphavirus thupc hp Togaviridae va du'dc lay truyen thdng qua vector truyen benh Ae. aegypti, Ae. albopictus. Genome ciia CHIKV la sdi RNA dOn cd kich thu'dc khoang 12kb ehira gen ma hda protein eau true la protein capsid, protein vd E l , E2.
Hien nay, chan doan huyet thanh hpc CHIKV sir dyng toan bp khang nguyen ciia virus nhu' ELISA, mien djch huynh quang. Bdi vay, trong nghien ciru nay chiing tdi iing dung edng nghe sinh hpc phan tir thiet ke vector bleu hien protein tai to hdp dac hieu, protein capsid, dinh hu'dng irng dyng trong chan doan huyet thanh hpc.
Vu Xuan Nghla^'^ Pham OCtc Minh*
protein capsid tai tdhgp
CHIKV-Wuel dUPc nudi cay va phan lap tir te bao mudi C6/36. Toan bp Genome ciia CHIKV du'pc tach chiet bdi kit RNeasy mini kit (Qiagen, Birc). LTng dung ky thuat RT-PCR tdng hdp cDNA ciia doan gen ma hda capsid protein. Phan u'ng RT-PCR du'pc thdc hien theo hu'dng dan ciia nha san xuat vdi mdt sd thay dd'i de tdi du hda dieu kien phan dng. Thanh phan tham gia phan irng RT-PCR bao gdm: 5x Qiagen Onestep RT-PCR buffer X 5pl, dNTP lOmM/pl x 0,4pl, cap moi lOpmol/pl X Ipl (moi primer), enzyme taq- polymerase x 2,5U, RNA ciia virus va nu'dc khir ion RNAfree vii^ du 50pl. Chu trinh nhiet dude thyc hien theo 45°C trong 45 phiit cho chuyen doi tir RNA sang cDNA. Tiep den la 95 °C trong 2 phut va 40 vdng eiia eac giai doan: duoi xoan d 94 °C trong 30 s, bam mdi 55 °C trong 1 phut, keo dai 72
°C trong 1 phut. Cuoi ciing la giai doan keo dai 72
°C trong 10 min. Sau khi nhan len, san pham PCR du'dc chay tren Agarose Gel 1,2% d lOOv va du'dc chyp tren he thong may dpc gel.
San pham PCR du'dc gan vao vector pCRII topo (Invitrogen, Germany), sau dd gen ma hda cho protein capsid6\i<^c gan vao vector bieu hien protein pET200/D (Invitrogen, Germany). Ket qua, plasmid pET200/D-/j/-ote/rt capsid du'dc sir dung bleu bien protein tai to hdp ciia virus.
2.2. Bieu hien va phan tich protein capsid tai tdh&p
pETlOO/D-protein capsid du'dc bien nap vao BL21 star, nudi cay te bao tren dTa thach LB chira khang sinh kanamyeine (50pg/ml) qua dem. Lay mdt colony ddn le eho vao 3ml dung djch LB chii'a khang sinh kanamyeine (SOpg/ml). Lac ii qua dem d 37°C. Ngay hdm sau, lay 500yl cho vao 10ml dung dich LB chiia Kanamyeine. Lac, u d 37''C trong 2 gid den khi dat mid-log (ODeoo la 0.5-0.7). Cho IPTG vdi nong dp cudi ciing la ImM vao dung dich nudi cay. Tai cac thdi diem 0, 2, 4 gid lay Iml dung djch te bao, phat hiiy te bao bang sieu am. Djeh phan huy du'pc ly tam, phan nu'dc ndi va phan can te bao du'pc phan tich bang SDS-PAGE khi nhudm coomassie blue va Western II. VAT u l u VA PHaONG PHAP NGHIEN COU
2.1. Tao vector bieu hien CHIKV blot sir dung khang the khang HIS-Tag, 'Hoc vien Quan y; ^Dal hoc tdng hdp Wuerzburg-CHLB D&c
.jcPhan bien khoa hgc: PGS.TS. Nguyin LTnh Toan
Y HOC VIET NAM THANG 3 - SO 2/2012
III. KET QUA VA BAN LUAN
3.1. Tach dong va tao vectorp^HQOIH-protein capsid
Dua tren khuon RNA ciia virus, cDNA doan gen ma hoa protein capsid difOc tong hop bang phan
\ing RT-PCR. San pham la doan DNA co kich thirdc khoang 781bp (hinh 1).
Gen ma hoa proteiri capsid Siiac khuyech dai va gan vao vector pCRII. Trinh tif ciia gen ma hoa di/dc xac dmh qua giai trinh tu*. Sau do, gen ma hoa tiep tuc du'dc gan vao vector bieu hien pET200/D-Topo. Vector nay difdc sir dung bleu hien protein capsid tai t6' hop.
/ - • C
pET200/D-Topo
Kana
1 2 3
(A) (B) Hinh 1: (A). San pham PCR ciia gen ma hoa protein capsid; (B) sd do vector bieu hien protein
tai to hdp
3.2. Bieu hien protein tai to hdp
pEVlOO/D-protein capsid6\i^c bien nap vao E.coli BL21 star va bleu hien protein tai to hdp. Bleu llien protein du'dc phan tich bdi SDS-PAGE va Western biot {Hinh 2)
MO 2 4 S D
c-
1 2 3 4 5 6
(A) (B) Hinh 2: (A). Protein capsid du'dc phan tich bang SDS-PAGE. Lane 1: Protein marker; lane 2,3,4:
protein bleu hien sau 0, 2, 4 gid; iane 5: protein co trong nu'dc noi; lane 6: protein trong can te_^bao.
(B). Protein capsid tai to hdp du'dc phan tich bang Western blot. Lane 1, 2, 3: protein bleu hien sau 0, 2, 4 gid; lane 4: protein co trong ni/dc noi; lane 5: protein trong can te bao.
Protein tai to hdp sau khi bleu hien du'dc phan ti'ch bang SDS-PAGE nhuom Coomassle blue. Ket qua cho thay, protein tai to hdp di/dc bleu hien sau 2, 4 gid khi cho IPTG vao trong djch nuoi. NhuTig bleu hien ro nhat la sau 4 gid d lane 4(A}. Trong dich can te bao va djch noi, protein tai to hdp co nhieu trong djch can te bao,
trong khi dd trong dich noi co rat it. Tuy nhien, de khang dinh chac chan, protein tai to hdp du'dc phat hien bang phu'dng phap Western blot su' dung khang the khang HIS-Tag. Ket qua cho thay tu'dng bi nhd SDS-PAGE. Oac biet, trong dich noi phat hien rat it protein tai to hop.
Oieu nay cho thay. Protein capsid tai to hdp
Y HOC VIET NAM THANG 3 - SO 2/2012 khi du'pc bieu hien tren he E.coli la protein khdng
hda tan va bieu hien rd nhat tai thdi diem sau 4 gid gay kich boat bang IPTG.
IV. KET LUAN
Nghien eiru da thanh edng thiet ke vector bieu hien Protein capsid tai to hdp tren he E.coli BL21 Star. Vector nay bieu hien protein tai to hdp manh nhat tai thdi diem sau 4 gid kich hoat bang IPTG.
TAI U | U THAM KHAO
1. Bodenmann, P., Genton, B., 2006.
Chikungunya: an epidemic in real time. Lancet 368, 258.
2. Pialoux, G., Gauzere, B.A., Jaureguiberry, S., Strobel, M., 2007.Chikungunya, an epidemic
arbovirosis. Lancet Infect. Dis. 7, 319-327 Karabatsos, N., 197S. Antigenic relationships of groupAarbovi ruses by plaque reduction neutralization testing. Am. J. Trap. Med Hyg. 24, 527-532.
Simizu, B., Yamamoto, K., Hashimoto, K., Ogata, T., 1984. Structural proteins of Chikungunya virus. J. Virol. Sl, 254-258.
Mavalankar, D., Shastri, P., Raman, P., 2007.
Chikungunya epidemic in India: a major public- health disaster. Lancet Infert. Dis. 7, 306-307.
Litzba, N., Schuffenecker, I., Zeller, H., Drostenr C , Emmerich, P., Charrel, R., Kreher, P., Niedrig, M., 2008. Evaluation of the first commercial chikungunya virus indirect immunofluorescence test. J. Virol. Methods 149, 175-179.
NGHIEN CUIU THAI DO VA THUC HANH CUA THUYEN VIEN VIET NAM TRONG VIEC PHONG CHONG HIV/AIDS
Khiic Thj N h u n * , Pham Minh K h u e * , Ha Manh T u a n * *
TOM TAT
Chiing toi nghien ciTu thai do va thUc hanh ciia thuyen vien (TV) Viet Nam trong viec phong chong HIV/AIDS. Ket qua nhU sau: ve thai do: 97,50% TV CO thai dp dung vdi ngUdi nhiem HIV/AIDS, chi 2,5%
TV CO thai do sai. Cd 44,5% TV coi HIV chi la mot benh (diing), 55,5% TV coi HIV la te nan xa hoi (sai).
Thu'c hanh phong chdng HIV/AIDS: tir 69,00%- 91,75% TV biet can phai ap dung tot cac bien phap thUc hanh de phong chong HIV/AIDS nhu dung neng bdm kim hem (BKT) hoac khir triing dung cu tiem chich trUdc khi tiem (91,75%); sdng chung thuy (84,00%); truyen mau an toan (79%); luon diing bao cao su (BCS) khi quan he tinh due (QHTD) khong an toan (77,75%); khong smh con khi b; nhiem HIV/AIDS (69%). 18,5% TV CO QHTD v6i ban tinh khong thUdng xuyen (KTX) trong do chi co 41,89% sir dung BSC trong tat ca cac lan quan he. 35% TV co QHTD vdi gai mai dam trong dd co 82,27% TV sir dung BCS trong tat ca cac lan quan he. Nhu vay thai do va thUc hanh phong chong HIV/AIDS ciia TV Viet Nam la chUa thUc sUtot. Can tuyen truyen tot hdn niJa va phai c6 nhij'ng chinh xach hiJu hieu tao dieu kien cho TV c6 thai do va thuc hanh tot trong viec phong chong HIV/AIDS.
RESUME
Nous avons etudie le comportement et la mise en pratique des marins vietnamiens face au fait de prevention contre I'HIV/AIDS. Void le resultat obtenu:
concernant le comportement: 97,5% des manns vietnamiens ayant un comportement correct avec des sideens, seuls 2,5% ne I'avaient pas. 44,5% des manns vietnamiens interroges pensaient que I'HIV n'etait qu'une maladie (vrai) contre 55,5% la considerent un maux social (faux). A propos de la mise en pratique contre I'HIV/AIDS: de 69% a 91,75% des manns vietnamiens acceptaient la necessite de prendre des mesures preventives comme:
utiliser leurs propres seringues ou steriliser les instruments avant d'injecter (91,75%); etre fidele (84%); appliquer une transfusion sanguine saine (79%); ubliser souvent des preservatifs pendant des rapports sexuels incertains (77,75%); ne pas avoir d'enfants en cas de seropositifs (69%). i 8 % des manns vietnamiens ayant des rapports sexuels avec des partenaires passageres dont seuls 41,89%
utilisaient des preservatifs pendant tous leurs rapports sexuels. 35% des manns vietnamiens ayant des
* DHY Hai Phong; ** Trung tam kiem dich y tS'qudc te Hal Phong Phan bien khoa hgc: PGS.TS. Tran Quang Phuc
