绵羊肺腺瘤病毒受体透明质酸酶-2的原核表达及纯化

为建立绵羊肺腺瘤病毒(jaagsiekte sheep retrovirus,JSRV)受体透明质酸酶-2(Hyal-2)的原核高效表达体系,本试验设计了扩增JSRV受体Hyal-2基因的特异性引物,应用PCR技术扩增出Hyal-2全长基因,将该基因定向重组于原核表达载体pGEX-4T-1中,构建pGEX-4T-1-Hyal-2重组质粒,并转化到大肠杆菌BL21(DE3)中,经IPTG诱导表达,逐步优化条件至稳定表达,经Western blotting检测融合蛋白成功表达后,通过亲和层析法对融合蛋白进行纯化。结果表明,Hyal-2基因正确地插入到原核表达载体pGEX-4T-1中;经诱导含重组质粒pGEX-4T-1-Hyal-2的表达菌高效表达了带GST标签的目的蛋白;SDS-PAGE电泳结果显示目的蛋白分子质量为80 ku,与预期大小一致,经Western blotting验证为带GST标签的融合蛋白;通过谷胱甘肽亲和层析法获得纯化的目的蛋白。本试验结果为进一步制备Hyal-2蛋白的多克隆抗体及深入研究其功能奠定基础。

Prokaryotic Expression and Purification of the Jaagsiekte Sheep Retrovirus Receptor Hyaluronidase-2

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.