TAP CHi SINH HQC, 2013,35(1): 105-109
TACH D 6 N G VA THIET K^ VECTOR TRUNG GIAN MANG GEN IL-6 CUA G A V I $ T NAM Vii Thj Thu Huyen*, Nguyen Thj Kim Cue, Le Thj Hong Minh, T r i n Thj Kim Dung, Phgni Vift Cudng Vifn Hda sinh biln, Vifn Hdn Idm KH & CN Vift Nam, *[email protected] TOM T A T : Cytokines Id mdt hp da protein c6 vai trd quylt djnh trong kilm soat hf mien djch. Chiing xac djnh ca kieu vd qui mo ciia dap img miln djch sau khi bj nhilm nguin bfnh ho$c chung ngira. Trong khudn kho bai bao ndy, gen lL-6 ciia gd da dugc tach dong va gin vdo mf t vector con thoi, la trung gian de lam ca sd cho vifc thilt kl adenovirus vector, dua gen cytokines vao gia d m dl l5ng cudng ddp irng mien djch ciia chung tdi ngudn bfnh. BSng phuong phap PCR, vdi c$p mli d?c hifu cho gen IL-6 cua ga vdi cdc Uinh ty nhdn bilt ciia enzymes gidi h ^ Kpn I, Not I vd trinh ty Kozak, da tich dong gen IL-6 cua ga (ChlL-6) vdo vector pCR2.1. Trinh ty gen ChlL-6 trong vector tach dong pCR2.1 da dupe giai, co 726 nucleotides vd sau khi BLASTn Ihly trinh ty nay tuang dong 99% so vdi mpt so trinh tu khac trong Ngan hang gen quoc te. Dl thilt ke vector trung gian pShuttIc mang gen IL-6 ciia ga, plasmid tach dong pCR2.ltChlL-6 va plasmid trung gian pShuttle dugc xir ly blng Kpn I va Not I, sau do, tien hanh phan img nli ghep gen IL-6 vao pShuUle da dugc md vong vdi su giup d5 ciia T4 ligase va chpn cac dong E.coli DH5a biln nap tren mdi tmdng co ampicilin. Kiem tra sy c6 mSt ciia gen ChlL-6 trong pShuttle blng enzymes gidi han Kpn I va Not 1 cQng nhu phuang phap PCR. Ket qua nhSn dugc cho thIy gen ChlL-6 da dugc gin vao plasmid trung gian pShuttle va tao vector tai tl hgp pShuttIe/C/(yL-5.
Tir khoa: Cytokine, ga Vift Nam, interieukin-6, tach dong, vector con thoi.
MODAU
Cytokines Id mdt hg da protein cd vai trd quylt djnh frong kilm sodt hf miln djch. Chiing xac djnh cd kieu vd qui md cua dap ling mien dich sau khi bj nhiem ngudn bfnh hodc chimg ngira. Phy thugc vdo td hgp cdc cytokiries dugc tao ra, ddp ling mien dich bdo vf cd the hodc la dap ihig trung gian qua khdng the (Th2) hodc ddp img hung gian tl bdo (Thi). Vi vdy, cytokines la cdc ling vien rIt tdt cho phep ch&a bfnh ty nhien ciing nhu td dugc cho vaccine [1,3,5].
Interieukin-6 (/i-6) la mdt interleukin boat dgng nhu mgt cytokine tien viem vd khdng viem. Cdc t l bdo T vd macrophages tilt ra IL-6 dl kich thich dap inig mien djch khi bj chin thuang, ddc bift khi bj bdng hodc cdc tdn thuang md khac ddn din viem nhilm. Trong frudng hgp ddp img mien djch cua vat chu vdi ngudn bfnh lg, thi dy d chugt, IL-6 d n dl khdng lai Streptococcus pneumoniae. IL-6 ciing la mOt "myokine", mgt logi cytokine dugc tao ra d CO, vd ham lugng tdng khi co co. Trong qud frinh luyfn tdp, ngudi ta nghl ring IL-6 boat ddng theo kiSu gilng nhu m$t hocmon, buy dOng CO chat va/hodc tdng cudng sy vdn chuyen
CO chdt. IL-6 cd the dugc macrophages tiet ra de phdn img lgi cdc phdn tu vi sinh vdt dac hifu, hay cdn ggi Id PAMPs. IL-6 cd vai trd chinh trong dilu chinh ddp ung mien dich, cdc phdn ling pha d p vd tgo mdu, boat hda lymphocytes B va T. Vai trd quan frgng cua lL-6 nhu mgt dilu biln miln djch Id khd ndng chuyen sy bift hda monocyte tu cdc tl bdo dendric thdnh macrophages [2, 6].
lL-6 da dugc nghien cim sir dyng nhu nhthig chit bd frg sinh hgc cho cdc logi vaccine gia d m cung nhu trong dilu frj khdi u. Vi vay, vifc dua ngudn cytokines nay tu ngodi vdo nhdm tdng cudng chirc ndng miln djch ciia co thi vdt chu Id vdn d l thu hut sy chii y ciia cdc nhd khoa hgc, tiln tdi dp dyng trong thyc tl khdng nhOng tren doi tugng dgng vdt ma cd thi tren cd ngudi [7].
Trong bdi bdo nay, gen IL-6 cua gd nudi tai Vift Nam dd dugc tach ddng vd gdn vdo vector trung gian, 1dm co sd cho thiet kl vector adenovirus, mgt cdng cy chuyin gen IL-6 cho gd nhlm tdng cudng mien djch cua vgt nudi.
VAT L i f u VA PHU^ONG P H A P NGHIEN ClTU
Vu Thi Thu Huyen et al
V$t lifu
Vector tdch ddng pCR® 2.1 (Invitrogen), pShuttle (Clontech), TA cloning Kit (frivitrogen), kit tinh sach plasmid, tinh sgch sdn phdm PCR, tgo cDNA, enzyme gidi hgn Kpn I vd Not I, E. coli Top 10. Gd gilng Ri (Vifn Chdn nudi Quic gia). Cdc logi hda chit cIn thilt cho cdc ky thudt sinh hgc phdn tit cua cdc hdng Sigma, Merk...
Phuang phdp
Tdch RNA t6ng s6 tu md Id Idch ciia gd blng phuong phdp sir dyng Trizol.
Tdch ddng gen IL-6 cua gd: tiln hdnh PCR vdi cdp mdi ddc hifu gen lL-6 ciia gd dugc thilt kl cd cdc trinh ty nhgn bilt cua enzyme gidi hgn Kpn I vd Not I vd trinh ty Kozak.
ChIL-6KozakF: TATCGCGGCCGC (Not I) GATATGGCTAACTTCACCGAGGG.
ChIL-6KozakR: TCAGGGTACC (Kpn I) GGCACTGAAACTCCTGGTCTTTTTC.
Thdnh phIn PCR gdm: 0,5 pi template (cDNA hi RNA tdng sd), 18 pi nude, 2 pi buffer I Ox, 2 pi dNTPs, 1 pi ChIL-6KozakF, 1 pi ChIL-6KozakR, Taq: 0,5 pi. Chu trinh nhift cua PCR 94''C/2 phut, (94''C/1 phut, 68°C/45 gidy, 72"C/I phut) X 30 chu ky, 72°C/8 phiit va gi&
mdu d 4°C. Sdn phim PCR dugc gin vdo vector tdch ddng pCR2.1 vdi sy giup dd ciia enzyme T4 ligase.
Thilt kl vector trung gian pShuttle mang gen IL-6 ciia ga: cd hai plasmid pCR2.l/ChIL-6 va pShuttle dugc xir ly rieng blng Kpn I vd Not I d 37°C trong 2 gid. Thanh phIn phdn img cdt:
5 pi plasmid pCR2.\/ChlL-6 hodc pShuttle; 2 ^1 mdi logi enzyme; 2,5 pi buffer; 13,5pl nude khir ion. Difn di kilm tra sdn pham clt tren gel agarose 1%, tinh sgch pShuttle vd gen ChlL-6 tu agarose gel. Thyc hifn phan img ligation d 14°C qua dem vdi thdnh phdn gdm 4|il gen chlL-6, 1,5^1 T4-ligase, 1,5 ^1 buffer T4, 4 ^1 pShuttle va 4 \i\ H2O. Kiem tra hifu qud gin gen bdng enzyme gidi ban.
KET QU A VA T H A O LUAN Tdch RNA tong so tu mo Id Idch cQa gd
Ga 1 thdng tudi, dugc udng glutathione 100
mg/ngay, sau 3 ngdy, thu md Id Idch cua gd theo quy triiih thudng quy, tdch mRNA tir md Id Idch theo phuong phdp dimg trizol, u san phim mRNA frong DNAse trong 1 gid 0 3TC (hinh I).
Hinh 1. RNA tdng sd tir md la Idch ciia gd Bdng phuong phdp quang phd kl, dd xac djnh ndng dg tuong ddi va kilm fra do tinh sach ciia mau tdch chiet. Kit qua OD26o/OD2go = 1,87 va RNA cd ndng dg khodng 146,4 ^g/ml.
Tach ddng gen IL-6 cua gd
Mdu RNA tdng sd dugc phien ma ngugc tao cDNA, sir dyng Kit cua hdng Fermentas vdi mdi ngau nhien, sau dd, sir dyng cdp mdi d|c hifu cho gen IL-6 ciia ga de thu nhdn doan gen ChlL-6 bdng PCR (hinh 2).
1 2 3 4 5 6
Hinh 2. Difn di dd san phdm khulch dgi gen IL-6 tir cDNA 1,2, 4. Sdn phim PCR;
3. Marker DNA Ikb plus (Fermentas).
106
TAP CHI SINH HQC, 2013, 35(1): 105-109 Sdn phdm khuech dai gen dugc chuyin
tryc tilp vdo vector nhdn ddng pCR2.1 vdi sy hd frg cua enzyme T4 ligase d 14°C qua dfim.
Sau dd, vector tdi to hgp dugc biln ngp vdo tl bdo E. coli khd biln chiing DH5a. Cly trdi tl bao biln nap len mdi trudng LB d$c cd bd sung khdng sinh ampicilin 100 pg/ml vd X-gal, nuoi d 37''C qua dem. Chgn ddng t l bdo tdi t l
hgp dya tren mdu sic xanh, trlng. Nudi cdc ddng rieng rS trong mdi trudng LB cd bd sung ampicilin, tdch DNA plasmid vd kilm tra tren gel agarose 1%. D l khdng djnh dogn gen chIL-6 dd chuyin dugc vdo vector nhdn ddng pCR2.1, tiln hdnh giai trinh ty gen trong plasmid tir khudn Igc trlng vdi cdp mdi ddc hifu.
1 a t g a a c t t c a 61 c g c c g g a g a g 121 c t g c c g c c c g 181 c t g g a g g a g g 241 c t g c g c g a c c 301 a a c a g c a t g g 361 g g c t g c c t g c 421 t t c g c c t t t c 481 g t c g a g t c t c 541 a a t c c c g a t g 601 a a g t c a g a t a 661 t c g t t t a t g g 721 g c c t g a
c c g a g g g c t g c g c c c c g t c c c c g c c g c c g t aggcgggggc g c g c c g t c c a a g a t g c t c g t t c g c c g g c t t a g a c c t a c c t t g t g c t a c a g a a g t g g t c a t a g g a c t g g a t a g a a g a c c g t
c g a g g c g a c g c g g c c c c g t c c c c g c t g c c c gcggcgggcg g c t g c a g g a c c c g g a a c a a c c g a c g a g g a g g g a a t t c a t t c a c a a a g c a c c c c a g a c t c g a g a g a a a a t c g a g g g c c g t t
g g a c g g c g g c g c g c t g c t g c g c c g c c g c g g c t g c t c g a c t g a g a t g t g c a c t c a a c c t g c a a a t g c c t g a c a a g a g a c t t c t g g c g g c c a g c c g c c c a g a a c c a t g c a c c c g c t a t t t g a
cggggagcgc c g c t g c t g c t a c t : c g t c c g g g c g a g c c g c t a g a a g t t c a c c c a a g g t g a c c g a a g c t c t t t c g a t a g c g a c g a t c c g g c a a a t c c c t c c t t c a t c c t c c g a a a a g a c c a g
c g g g a g c c g c g c c g c t g c t g a g a g g t t g g g g g c c c g g g t g c g t g t g c g a g ggaggaggac c a g t g g t c t i g a a a g c a g a a c g a t g g t g a t a c g c c a a t c t g a g a c t t t a c t g a g t t t c a g t
Bdng 1. So sdnh trinh ty ChIL-6 nhdn dugc tu ga Vift Nam vdi cdc frinh tu dd dang ky trong Genbank bang chuong trinh BLASTn
Ky hifu HM 179640.1 HM367074.1 NM204628.1
Mdtd Gallus gallus mterleukin-6 (IL6) mRNA, complete eds
Gallus gallus interleukin-6 precursor, mRNA, complete eds
Gallus gallus interleukin 6 (interferon, beta 2) (IL6), mRNA >emb|AJ309540
Ddphu 97%
97%
97%
Dg ddng nhdt 99%
99%
99%
Da tien hdnh dang ky trinh ty gen Interleukin-6 ddi 726 bp cua ga Vift Nam Uen Ngdn hang gen qudc t l vdi ma sd JQ897539.
Li et al. (2010) [4] da tach RNA tdng sd tu lymphocytes la Idch gd 1 thang fridi sau khi dugc kich thich blng ConA trong 20 gid. Sir dyng Kit sao chep ngugc Quantscript (Trung Qudc) dl tgo cDNA va blng PCR, gen IL-6 ciia gd dd dugc tdch ddng vdo pMD18-T vector vd xdc djnh dugc ORF 726 bp. Kit qud phdn tich blng phIn mlm Bioedit cho thIy, trinh ty ndy gidng 99% so vdi trinh ty ga ciia Vift Nam.
Thiet ke vector trung gian mang gen IL-6 ciiagd
De gdn gen ChlL-6 vao vector trung gian, cd hai plasmid pCR2.1/ChIL-6 va pShuttle dlu dugc clt blng cdc enzyme gidi ban Kpn I vd Not I theo phuang phdp md td vd sdn phdm clt dugc kiem tra tren gel agarose 1% (hinh 3). Hai enzyme md vdng plasmid pShuttle vd cho mgt bdng tren difn di dd khoang 4 kb, tuong img vdi kich thudc ciia pShuttle, trong khi do, plasmid pCK2.l/ChIL-6 bj d t thdnh hai bdng, bdng Idn ban kich thudc khodng 4 kb, tuong img vdi kich thudc cua pCR2.1 vd bang dudi cd kich thudc tuong duong vdi sdn phdm PCR ciia gen ChlL-6, khodng 750 bp. Cdc bdng cua pShuttle vd gen ChlL-6 dugc 1dm sgch bdng GeneJET Gel Extraction kit (Fermentas) t& agarose gel cho phdn img gin tilp theo.
Vu Thj Thu Huyen el al
Hinh 3. Difn di dd sdn pham cdt cua pShutter (3a) vd pCR2.\lChlL-6 (3b). Marker DNA Ikb plus (Fermentas) (2).
Gen ChIL-6 dugc gin vdo vector pShuttk trong phdn img tdng thi tich 15 nl vdi sy trgi giiip cua enzyme T4 ligase d H^C qua dem.
Vector tdi t l hgp dugc biln ngp vao tl bio E colt DH5a khi biln, nudi qua dem a 37°C trfn mdi trudng LB bd sung Ampicilin 100 pg/ml dl chgn Igc ddng biln nap. Khuech dai lugng plasmid cua mOt vdi khudn Igc bat ky frong mdi trudng LB Idng cd khdng sinh qua dem. Tdch plasmid tir cdc ddng E. coli chgn Igc va kilm tnf ket qud gdn gen blng enzyme gidi han Kpn I, Not 1 vd PCR, difn di tren gel agarose 1% de kilm tra kit qua gin gen (hinh 4).
Hinh 4 Kiem tra sy cd mat cua gen ChlL-6 trong plasmid pShuttle tai id hgp A. San pham PCR vdi template la pShuttle/C/;/Z.-d va cap mdi ddc hieu cho gen ChIL-6;
B. San phdm cdt ciia pShuttle/CVz/I-d.
Plasmid tu ddng E. coli bien ngp khi dugc d t blng hai enzyme gidi ban Kpn 1 va Not 1 cho 2 bdng tren difn di dd (hinh 4B, gieng 1). Bdng tren cd kich thudc tuong img vdi kich thudc ciia pShuttle (4 kb) va bdng nhd ben dudi cd kich thudc tuong duong gen IL-6 ciia gd. Kit qua nhdn dugc cho thdy, gen ChIL-6 dd gin thanh cdng vdo vector pShuttle, tgo vector tdi td hgp pShuttle/C/;/L-6. Dieu ndy dugc khIng djnh blng kit qua PCR, khi su dyng pShuttle/C/i/i-d 1dm khudn vdi cap mdi ddc hifu cho gen ChIL-6 (binh 4A). Trinh ty dogn gen ChIL-6 gdn vao vector pShuttle da dugc gidi va phdn tich blng chuong trinh Bioedit. Kit qua cho thIy, trinh ty nucleotide ciia dogn gen khdng thay ddi so vdi trinh ty nhgn dugc trong pCKl.\IChlL-6.
Ket qua nghien cim nay la co sd dl thilt kl plasmid mang gen ChIL-6 hodc cdc gen cytokine khac dua fren adenovirus vector bdng
phuong phdp d6ng nhiem. tir dd cd thi dui cytokine tir ngoai vdo gia cam nhlm tang khi ndng mien djch cua dan, tdng sire chju dung cua gia d m vdi cdc dilu kien ngogi canh bit Ifi cung nhu cdc ngudn bfnh.
KtT LUAN
Dogn gen hogt tinh Interleukin-6 ctia gi Vift Nam dd dugc tdch ddng va trinh ty c6 726 bp. Dd tiln hdnh ddng ki trinh ty dogn gen chIL-6 dai 726 bp cua ga Vift Nam tren Ngin hdng gen Qu6c tl vdi ma sd J(5897539.
Trinh ty dogn gen ChIL-6 vdi cdc dilm n h ^ bilt cua cdc enzyme gidi han I^n I vi Not I ciing nhu trinh ty Kozak dd dugc gin vio vector trung gian pShuttle, tao vector tai td hgp pShuttle/C/j/L-d. Trmh ty nucleotide frong vector trung gian khdng thay ddi so vdi trinh tv trong vector tich ddng pCR2.1.
TAP CHi SINH HQC, 2013,35(1): 105-109 L&i cam ffn: D I tdi nay dugc ho trg v l kinh phi
ciia chuong trinh Cdng nghf sinh hgc ndng nghifp, Bg Ndng nghifp vd Phdt friln Ndng thdn, ma sd 1121/HD_BNN_KHCN.
TAI LIEU THAM KHAO
1. Giansanti F., Giardi M. F., Botti D., 2006.
Avian Cytokines - An Overview. Current Pharmaceutical Design, 12(24): 3083-3099.
2. Kishimoto T., 2006. Proceedings:
Interieukin-6: discovery of a pleiotropic cytokine. Arthritis Research & Therapy, 8(Sup. 2): S2.
3. Kogut M. H., 2000. Cytokines and prevention of infectious diseases in poutry:
A review Avian Pathology, 29(5): 395-404.
4. Li M., Wang Y., Zou N., Cao S., Wen X., Huang Y., 2010. The polyclonal antibody
against chicken interleukin-6 prepared by immunization of recombinant eukaryotic plasmid can react efficiently with it's prokaryotic expression protein. J. Animal Veterinary Adv., 9(21): 2679-2684.
5. Schneider K., Klaas R., Kaspcrs B., Staeheli P., 2001. Chicken interieukin-6: cDNA structure and biological properties. Eur. J.
Biochem., 268: 4200-4206
6. Smolen J. S., Maini R. N., 2006.
Interleukin-6- a new therapeutic target.
Arthrhis Res. Ther., 8(Sup. 2): S5.
7. Thompson A. L., Herman F. Staats, 2011.
Review article Cytokines: The fiiture of intranasal vaccine adjuvants. Clinical &
Develop Immunol., Article ID 289597, 17 pages, doi: 10.1155/2011/289597.
C L O N I N G A N D C O N S T R U C T I O N O F E N T R Y V E C T O R C A R R Y I N G IL-6 G E N F R O M V I E T N A M E S E C H I C K E N
Vu Thi Thu Huyen, Nguyen Thi Kim Cue, Le Thi Hong Minh, Tran Thi Kim Dung, Pham Viet Cuong
Institute of Marine Biochemistry, VAST
SUMMARY
Cytokines are a diverse family of proteins that play a crucial role in controlling the immune system. They determine both the type and extent of an immune response that is generated following infection with a pathogen or after vaccination. In this paper, chicken IL-6 gene (ChlL-6) was cloned and ligatcd in a shuttle vector, as basic to construction adenovirus vector for cytokine gene delivery in chicken for improving of their immune responses to pathogens. By PCR technique, using specific primers for chicken IL-6 gen (ChIL-6) possessing recognition sites of restriction enzymes Kpn I and Not I and Kozak sequence, the ChlL-6 was cloned into pCR2.1 vector. The gene in recombinant pCKl.\IChlL-6 was sequenced, the obtained sequence had 726 nucleotides and was 99% similariy to the sequences m GenBank after BLASTn The sequence has been reported to GenBank with Accession no JQ897539. For construction pShutlle/C/i/Z.-(5 vector, the cloning vector pCR2AlChIL-6 and pShuUle were digested by Kpn I and Not 1, then the ligation reaction was perfomied with T4 ligase and Uansformed E. coli DH5a clones were selected on LB medium supplemented with ampicilin. The presence of ChlL-6 gen in pShuttle vector was verified by digestion of recombinant plasmid with Kpn I and Not I, as well as by PCR. Received results demonstrated that ChIL-6 gen was ligated into pShuttle and gave pShuttle/C///Z,-<5 vector.
Keywords: Chicken, cloning, cytokine, interleukin-6, pshutter vector.
Ngdy nhdn bdi: 25-7-2012
