延边白鹅IFN-γ基因的原核表达及其多克隆抗体的制备  

Prokaryotic Expression of Yanbian Goose IFN-γ Gene and Preparation of Its Polyclonal Antibody

作　　者：张颖[1] 刘恩平[1] 陈海迪[1] 高旭[1] 张立媛[1] 于清洋[1] 鲁承[1] 梁晚枫[1] 

机构地区：[1]延边大学动物医学系,吉林延吉133400

出　　处：《河南农业科学》2015年第12期126-129,共4页Journal of Henan Agricultural Sciences

基　　金：国家自然科学基金项目(31460661);延边大学第六届大学生科研项目(20130108)

摘　　要：为制备抗延边白鹅IFN-γ蛋白的多克隆抗体,以已制备的pMD-IFN-γ质粒为模板,通过PCR扩增到延边白鹅IFN-γ基因,经TA克隆和BamHⅠ、EcoRⅠ双酶切,将目的基因亚克隆至pGEX-4T-1原核表达载体,转化大肠杆菌BL21(DE3),进行IPTG诱导表达,将经SDS-PAGE及Western blot鉴定正确的重组蛋白用GST标签纯化试剂盒纯化,以纯化的重组蛋白作为抗原,免疫BALB/c小鼠制备多克隆抗体并进行ELISA检测。结果显示,成功构建了原核表达载体p GEXIFN-γ,表达的重组蛋白以包涵体形式存在,分子质量约为43 ku,制备的小鼠抗IFN-γ多克隆抗体效价为1∶16 000。In order to prokaryotic express Yanbian white goose IFN-γ gene,and prepare polyclonal antibody against goose IFN-γ protein. The goose IFN-γ gene was amplified from p MD-IFN-γ plasmid by PCR,the target gene was cloned into pGEX-4T-1 vector to construct the prokaryotic express plasmid. The recombinant plasmid was transformed into E. coli BL-21（ DE3） strain,and induced by IPTG,which can express fused protein GST-IFN-γ,the product was identified by SDS-PAGE and Western blot. Then the antiserum against GST-IFN-γ was prepared in BALB/c mice and the antibody titer was determined by ELISA. The results showed that fusion protein GST-IFN-γ was about 43 ku,the polyclonal antibody against GST-IFN-γ was prepared,and the antibody titer was 1∶ 16 000 by ELISA.

关 键 词：延边白鹅 IFN-Γ基因 原核表达 多克隆抗体 

分 类 号：S852.4[农业科学—基础兽医学]