猪STAT-1α基因的原核表达及其多克隆抗体制备  

Prokaryotic expression of porcine STAT-1α gene and preparation of polyclonal antibody against STAT-1α

作　　者：李延生[1,2] 刘诚[1,2] 张珂[1,2] 李晓宁[1,2] 李晓泉[1,2] 尹珊[1,2] 谢琳娟[1,2] 何晓霞[1,2] 罗扬[1,2] 钟桃珍 罗廷荣 

机构地区：[1]广西大学动物科学技术学院 [2]亚热带农业生物资源保护利用国家重点实验室,南宁530005

出　　处：《南方农业学报》2014年第1期118-122,共5页Journal of Southern Agriculture

基　　金：国家自然科学基金项目(31060336);广西自然科学基金项目(2012GXNSFDA053009)

摘　　要：【目的】通过制备STAT-1α多克隆抗体,为进一步研究STAT-1α蛋白的功能特性及探索STAT-1α与猪瘟病毒间的相互作用机制奠定基础。【方法】利用RT-PCR从猪肾PK-15细胞中克隆出STAT-1α基因保守片段,与pET-32a(+)构建原核表达重组质粒,转化原核宿主菌(Rosetta),进行IPTG诱导表达,表达的重组蛋白经纯化和复性浓缩后,免疫小鼠制备STAT-1α多克隆抗体。【结果】STAT-1α基因能与原核表达载体pET-32a(+)构建原核表达重组质粒pET-STAT-1α,转化大肠杆菌Rosetta后的最佳体外诱导条件是IPTG 0.5 mmol/L、诱导时间6 h,其重组蛋白主要以包涵体的形式表达。STAT-1α多克隆抗体具有高效特异性,与真核细胞PK-15细胞作用后,在PK-15细胞内能检测到STAT-1α蛋白表达,可观察到大部分细胞胞浆内有绿色荧光,但胞核内几乎无荧光,即STAT-1α主要定位在正常PK-15细胞的细胞浆内表达。【结论】制备获得的猪STAT-1α多克隆抗体特异性好,既能与原核细胞大肠杆菌(Rosetta)表达的STAT-1α反应,又能与真核细胞PK-15的STAT-1α发生反应。[Objective]The polyclonal antibody STAT-1α was prepared to provide references for further research on functional characteristics of STAT-1α and interactive mechanism between hog cholera virus and STAT-1α. [Method]Using RT-PCR, STAT-1α gene conservative fragment was cloned from PK-15 cell in swine kidney, and together with pET-32a （＋）, prokaryotic expression recombinant plasmid was constructed. The STAT-1α polyclonal antibody was prepared after IPTG inducible expression of Rosetta, and purification and concentration of inclusion body protein. [Result]Prokaryotic ex- pression recombinant plasmid pET-STAT-1α was produced by STAT-1α gene and prokaryotic expression vector pET-32a （＋）. The optimal induction conditions for transforming E. coli prokaryotic cells Rosetta were IPTG concentration of 0.5 mmol/L and induction duration of 6 h. The recombinant protein must be expressed in the form of inclusion body. STAT-Iot polyclonal antibody showed the traits of high reactivity and specificity. Following cytosis with eukaryocyte PK-15, STAT-1α protein ex- pression could be detected in normal PK-15 cells. As green fluorescence was observed in the cytoplasm of most cells, it was deduced that STAT-1α was mainly localized in the cytoplasm of normal PK-15 cells. [Conclusion]With high-specificity, the prepared anti-porcine STAT-1α polyclonal antibody could react with both E. coli prokaryotic cells Rosetta DE3 and eu- karvocvte PK-15.

关 键 词：猪 STAT-1α基因 原核表达 多克隆抗体 

分 类 号：S858.28[农业科学—临床兽医学]