猪链球菌2型中国强毒株CovR重组蛋白及其抗体的制备

目的通过构建pET32a-covR原核表达质粒,表达并提纯CovR重组蛋白,制备其多克隆抗体。方法采用PCR法,从四川资阳病人分离株05ZYH33基因组扩增CovR的编码基因covR,构建重组表达质粒pET32a-covR,转化E.coliBL21(DE3),筛选阳性转化子进行IPTG诱导表达,产物经SDS-PAGE鉴定后,His亲和层析柱纯化CovR重组蛋白;免疫新西兰兔制备特异性抗体,Western blot检测兔多抗血清的特异性,间接ELISA检测其滴度。结果covR基因在原核细胞中得到高效表达,重组蛋白免疫新西兰兔后血清效价高达1∶204800,且重组蛋白能够与之发生特异性的反应。结论成功构建了表达载体pET32a-covR,可在E.coliBL21中高效表达,制备了特异性抗体,为进一步研究CovR的调控机理奠定了基础。

Prokaryotic expression and polyclonal antibody preparation of response regulator CovR of Streptococcus suis serotype 2

To construct prokaryotic expression vector pET32a-covR, express and purify the recombinant protein for the preparation of rabbit anti-CovR polyclonal antibody. The encoding CovR gene of 05ZYH33 strain, which was isolated from patients in Ziyang county of Siehuan province, was amplified by PCR with the designed primers, and then cloned into prokaryotie expression plasmid pET32a. The recombinant plasmid pET32a-covR was transformed into E. coli BL21. After indueed expression by IPTG, the CovR protein was analyzed with SDS-PAGE . The CovR recombinant protein was purified on a Ni2＋-NTA affinity column by its His tag, then the polyclonal antibody was prepared against it. The resuets showed the specificity of serum antibody was analyzed by Western blot, and its titer was analyzed by indirect-ELISA. The recombinant protein could be expressed in prokaryotic cells in high level. Polyclonal antibody titer of rabbit serum immunized with recombinant protein reached more than 1 ： 204 800. The recombinant protein could specifically react with it. It domonstrates that the expression vector pET32a-covR is successfully constructed. The fused gene was highly-expressed in E. coli BL21. A specific and high titer antibody has been prepared. The CovR recombinant protein and its polyclonal antibody may be useful for further functional analysis of CovR.