PK-15细胞ANXA8膜联蛋白原核表达及多克隆抗体制备

Expression and preparation of the polyclonal antibody against the ANXA8 protein of PK-15 cells

为了制备ANXA8膜联蛋白的多克隆抗体,本研究以PK-15细胞ANXA8基因为模板,通过RT-PCR扩增得到猪源ANXA8基因,并克隆到pGEX6p1载体。加入His-tag标签,成功构建了重组质粒pGEX6p1-ANXA8,鉴定正确后将其转化至大肠杆菌BL21(DE3)感受态细胞中诱导表达,纯化后的重组蛋白利用SDS-PAGE、Western blot进行鉴定。以纯化后的重组蛋白作为抗原,免疫SPF级昆明鼠,获得针对ANXA8蛋白的多克隆抗体。经过ELISA检测,抗体效价达1∶25600,经过Western blot和间接免疫荧光(IFA)检测,抗体可以与ANXA8蛋白产生特异性结合反应。本试验成功地应用大肠杆菌表达ANXA8蛋白,表达的蛋白具有免疫原性,并获得鼠源阳性血清,为ANXA8蛋白结构及功能的研究奠定基础。

In order to prepare polychonal antibodies against ANXA8 protein,the ANXA8 gene from PK-15 cells was used as a template,and the pig-derived ANXA8 gene was amplified by RT-PCR and cloned into the pGEX6p1 vector.The His-tag was added to successfully construct the recombinant plasmid pGEX6p1-ANXA8.When the identification was confirmed to be correct,the recombinant plasmid pGEX6p1-ANXA8 was transformed into E.coli BL21(DE3)competent cells and was successfully induced and expressed.Then,the recom⁃binant protein was purified and identified by SDS-PAGE and Western blot.The purified recombinant protein was used as the antigen to im⁃munize SPF-grade Kunming mice to obtain polyclonal antibodies against the ANXA8 protein.ELISA detection showed that the antibody titer reached 1∶25600.Western blot and IFA detection showed that the antibody could specifically bind to the ANXA8 protein.In this experi⁃ment,Escherichia coli was successfully used to express ANXA8 proteins.The expressed protein was immunogenic and mouse-derived positive serum was obtained,which laid a foundation for research on the structure and function of the ANXA8 protein.