关键词: 绵羊肺腺瘤病; 受体; 透明质酸酶-2; 原核表达; 蛋白纯化

Abstract: In order to construct an efficient prokaryotic expression system of jaagsiekte sheep retrovirus (JSRV) receptor hyaluronidase-2 (Hyal-2),we designed a pair of specific primers for the JSRV receptor Hyal-2 gene,using PCR amplification technique to amplify the full-length of Hyal-2 gene, and directional restructuring it into the prokaryotic expression vector pGEX-4T-1.We constructed the pGEX-4T-1-Hyal-2 recombinant plasmid which was transformed into E.coli BL21(DE3) using IPTG to induce expression, optimized conditions gradually until stable expression,and then detected fusion protein by Western blotting, at last, the fusion protein was purified by the affinity chromatography methods.The results showed that Hyal-2 gene was exactly insert into the prokaryotic expression vector PGEX-4T-1,after induction, the target protein containing GST tag was efficiently expressed by expression bacteria which included recombinant plasmid pGEX-4T-1-Hyal-2,SDS-PAGE electrophoresis result showed that the molecular weight of target protein was 80 ku,consistent with the expected size,and target protein that verified by Western blotting was a fusion protein with GST tag,the target protein which purified by the glutathione affinity chromatography would lay the foundation for further preparation of Hyal-2 protein's polyclonal antibody and in-depth study of its function.

Key words: ovine pulmonary adenomatosis; receptor; hyaluronidase-2; prokaryotic expression; protein purification