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《China Animal Husbandry & Veterinary Medicine》 2010-01
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Cloning and Prokaryotic Expression of Nucleoprotein Gene of Porcine Reproductive and Respiratory Syndrome Virus

LUO Sheng-jun,HUANG Zhong,ZHOU Xiu-rong,JIA Chun-ling,YUAN Jie,WEI Wen-kang(Institute of Veterinary Medicine,Guangdong Academy of Agricultural Sciences,Guangzhou 510640,China)  
N gene fragment was amplified from PRRSV of cell RNA by RT-PCR and expressed it by prokaryotic expression vector pET-28a(+).A recombinant plasmid pET-28a-N was constructed and identified by restriction endonuclease digestion,then transformed into the E.coli BL21(DE3) plysS.The PRRSV N gene was expressed as a recombinant fusion protein and detected by SDS-PAGE and Western blotting.N gene fragment of 372 bp was amplified by RT-PCR,the recombinant bacteria was induced with IPTG and the expression protein was analyzed by SDS-PAGE.The results showed that bacteria contained the positive plasmid was induced to express with 1 mmol/L IPTG and 4 hours.A unique band was detected with the molecular mass of approximately 18 ku by SDS-PAGE.By analysis of Western blotting,the expressed production was reactive with the positive swine serum of PRRSV.PRRSV N gene was amplified and recombinant prokaryotic expression plasmid was constructed successfully.This showed that the high efficient expression of N protein was obtained which system of prokaryotic and eukaryotic expression.Using target protein after purified as coating antigen,an indirect ELISA was developed for detecting the anti-N antibody in the PRRSV serum by exploring the concentration of coating antigen and dilution degree of serum.
【CateGory Index】: S852.659.6
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