鸡干扰素α受体Ⅱ胞外域的克隆及GST融合蛋白原核表达  

Cloning and Expression Gene of Extracellular Region of Chicken Interferon-Alpha Receptor II with GST

作　　者：牛金鹏[1] 王惠川[1] 吴国娟[1] 

机构地区：[1]北京农学院动物科学技术学院,北京102206

出　　处：《北京农学院学报》2010年第4期21-23,62,共4页Journal of Beijing University of Agriculture

基　　金：北京市自然科学基金项目(6092004);国家自然科学基金项目(30771625)

摘　　要：应用RT-PCR技术扩增鸡干扰素α受体Ⅱ胞外域(CHIFNAR2 EC)基因片段,将扩增产物克隆至pMD-18T,转化J M109感受态。重组质粒与表达载体pGEX-6P-1经双酶切后构建重组表达质粒pGEX-6P-CHIF-NAR2EC,转入BL21,提取质粒酶切和测序鉴定。测序结果与参考序列比较,核苷酸同源性为99%。用IPTG诱导表达,对诱导条件进行初步优化,表达蛋白主要以包涵体的形式存在。通过切胶的方法进行纯化,获取具有较高纯度的融合蛋白。表达产物经SDS-PAGE和Western Blot检测显示融合蛋白分子量大小约为50 KD,采用抗GST抗体进行Western Blot,成功检测到特异性目的条带。In this experiment the partial gene of coding region of interferon receptor II amplified by RT-PCR was cloned pMD-18T vector,and subcloned gene of ectodomain of interferon receptor II（CHIFNAR2EC）.The gene was further subcloned into expression vector pGEX-6P-1 for expressing in E.coli BL21.The results showed that the gene was 99 % identical to the published nucleotide sequence.SDS-PAGE analysis indicated the pGEX-6P-CHIFNAR2EC fusion protein was about 50 KD,mainly in form of inclusion body.The recombinant protein was verified by Western blot with antibody against GST.

关 键 词：干扰素α受体Ⅱ 胞外域 克隆 融合蛋白 原核表达 

分 类 号：S831[农业科学—畜牧学] Q786[农业科学—畜牧兽医]