奶牛结核病IFN-γ基因的克隆及其在E.coli BL21(DE3)中的高效表达  

Cloning and Effective Expression of Cow Tuberculosis IFN-γGene in Escherichia coli BL21(DE3)

作　　者：豆玲[1] 豆思远 周峰[1] 王佳[1] 尚立宏[1] 曹丽萍[1] 鄂承钧 

机构地区：[1]甘肃省动物疫病预防控制中心,甘肃兰州730046

出　　处：《畜牧兽医杂志》2015年第1期18-21,共4页Journal of Animal Science and Veterinary Medicine

基　　金：甘肃省农业科技创新项目(GNCX-2011-43)

摘　　要：动物机体内γ-干扰素(IFN-γ)是可被多种物质诱导产生的生物活性蛋白,克隆和诱导表达奶牛结核病IFN-γ蛋白,对于采用ELISA诊断牛结核病有着重要意义。本研究利用RT-PCR方法克隆了奶牛结核病IFN-γ基因,并成功构建了IFN-γ基因的原核表达载体重组质粒pET28a-IFN-γ,转入E.coli BL21(DE3)中进行诱导表达,并对条件进行了优化。SDS-PAGE与Western Blot结果显示,在37℃条件下,0.75mmol/L IPTG诱导4h可见重组蛋白BovIFN-γ大量表达。本研究成功建立了利用原核表达系统获得牛IFN-γ蛋白的方法,对后续利用该蛋白作为免疫原,从而建立ELISA诊断方法奠定了基础。Abstract：Gamma--interferon （IFN-γ） is a bioactive proteins may be induced by a variety of substances in animal organism,cloning and induced expression of IFN-γ protein has important significance for ELISA diagnosis of bovine tuberculo sis. In this study, we cloned cow tuberculosis IFN-γ gene by RT-PCR, and successfully constructed the prokaryotic expression vector pET28a-IFN-γ for IFN-γ gene expression. The recombinant plasmid pET28a-IFN-γ was transformed into Escherichia coli BL21 （DE3）for expression, induced by IPTG, and the conditions of the expression was optimized. SDS-PAGE and Western Blot revealed that the best expression of recombinant proteion was induced by 0.75mmol/L IPTG at 37℃. In conclusion, this experiment has successfully established method of using prokaryotic expression system to obtain the IFN-γ protein of bovine, and those data provides the foundation for studying the ELISA diagnostic method for subsequent research of using the recombi- nant protein as immunogen.

关 键 词：奶牛 结核病 IFN-Γ基因 原核表达 

分 类 号：S852.618[农业科学—基础兽医学]