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

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.