水牛NLRP5基因原核表达载体构建及多克隆抗体制备

Construction of prokaryotic expression vector and preparation of polyclonal antibody of buffalo NLRP5 gene

为了制备水牛母源基因NLRP5多克隆抗体,采用PCR扩增水牛NLRP5基因并利用原核表达技术构建NLRP5表达载体,转化感受态细胞BL21,通过IPTG诱导表达,纯化后的重组蛋白用于免疫小鼠,制备鼠抗NLRP5多克隆抗体,用Western blot分析该基因在水牛各组织的表达特性。结果表明:通过PCR扩增成功克隆了水牛NLRP5基因CDS序列3297 bp和原核表达的480 bp目的片段,成功构建了原核表达载体p ET32a-NLRP5。在诱导最佳条件(0.5 mmol/L IPTG,30℃诱导10 h)下NLRP5重组蛋白以包涵体形式表达,纯化复性后重组蛋白能和His标签特异性反应。用该重组蛋白免疫制备获取的鼠抗NLRP5多克隆抗体与NLRP5重组蛋白反应,效价达1∶64000。用制备的多克隆抗体验证NLRP5蛋白,仅在水牛卵母细胞中特异性表达,其他组织中均无表达。

In order to prepare polyclonal antibody of buffalo maternal gene NLRP5,PCR was used to amplify buffalo NLRP5 gene and NLRP5 expression vector was constructed according to prokaryotic expression technology,transformed into competent cell BL21,and induced expression by IPTG.The purified recombinant protein was used to immunize mice to prepare mouse anti-NLRP5 polyclonal antibody.Then the expression characteristics of the gene in buffalo tissues was analyzed by Western blot.The results showed that the NLRP5 CDS sequence and the prokaryotic target fragment in length of 3297 bp and 480 bp,respectively,were successfully cloned by PCR amplification.The prokaryotic expression vector p ET32 a-NLRP5 was successfully constructed.The NLRP5 recombinant protein was expressed in the form of inclusion bodies under the optimal condition induced by IPTG 0.5 mmol/L 30℃for 10 h,which could react specifically with His-tag antibody after purification and renaturation.And then was used to immunize mice to produce mouse anti-NLRP5 polyclonal antibody.This polyclonal antibody could react with NLRP5 recombinant protein in a titer of 1∶64000.It indicates that the NLRP5 protein only specifically expressed in oocytes of buffalo,but not in other tissues.