Jaagsiekte Sheep Retrovirus Receptor Hyal-2Prokaryotic Expression And Preparation Of Polyclonal Antibody

Objective:To clone Jaagsiekte sheep virus(JSRV) receptor hyal-2gene, prepare prokaryotic expression vector, and then transform it into BL21(DE3) E.coli. After inducing expression, obtain, purify Hyal-2protein and prepara polyclonal antbody of Hyal-2. It has very important academic significance for identifing the infected cells of JSRV and to further study the tissue tropism of JSRV.Methods:According to the published sheep hyal-2gene sequence, a pair of specific primer was desiged. The gene of Hyal-2was obtained by using PCR, cloned into pGEX-4T-1expression vector, and then identified by bacteria liquid PCR and sequencing. To transform the recombinant plasmids pGEX-4T-1-hyal-2into E.coil BL21(DE3) and induce by IPTG. The best induced concentration, time, temperature and other expression conditions were optimized in order to get abundant,stable and efficient GST-Hyal-2fusion protein. To detect the expression of the fusion protein by10%SDS-PAGE, and verify the immunogenicity by Western blotting. The fusion protein was purified by glutathione affinity chromatography, and then immune rabbit, prepare the polyclonal antibody to Hyal-2protein. To detect the titers of polyclonal antibodies by double agar diffusion test and the specificity by Western blotting.Results:It was amplified the hyal-2gene fragment with about1431bp by PCR. The hyal-2gene is properly inserted into expression vector and the PGEX-4T-1-hyal-2recombinant plasmid is constructed sucessfully. The molecular weight of fusion protein is80ku, it’s immunogenicity was proved by Western blotting. Via subcutaneous injection, the concentration of fusion protein being636.1μg/ml emulsified with adjuvant, a rabbit was immuned for preraring anti-Hyal-2protein polyclonal antibody. The titers of polyclonal antibody reached1:16. Western blotting showed there was strong reactivity and specificity between Hyal-2protein and the polyclonal antibodiy.Conclusions:pGEX-4T-1-hyal-2recombination plasmid has been constructed and Hyal-2protein has been induced and expressed successfully. Polyclonal antibody to Jaagsiekte sheep virus receptor Hyal-2has been prepared.