2型猪链球菌中国强毒株znuA基因的原核表达及免疫学活性分析

Prokaryotic expression and immunologic activity detection of high -affinity zinc uptake system protein ZnuA of Streptococcus suis serotype 2

目的克隆2型猪链球菌高亲和力锌吸收蛋白(High-affinity zinc uptake system protein,ZnuA)编码基因并进行原核表达,研究ZnuA蛋白的免疫学活性。方法采用PCR法,从2型猪链球菌中国强毒株05ZYH33基因组扩增基因znuA,构建重组表达质粒pET32a::znuA,转化E.coli BL21(DE3),筛选阳性转化子进行IPTG诱导表达,SDS-PAGE鉴定表达产物;His亲和层析柱纯化重组蛋白;Western blot检测ZnuA蛋白的免疫原性;重组蛋白免疫新西兰兔后收集多抗血清,间接ELISA检测多抗血清效价。结果构建的重组质粒在宿主菌中可高效表达,His亲和层析柱纯化获得较高纯度的重组蛋白;Western blot结果表明ZnuA蛋白具有良好的免疫原性;重组蛋白免疫新西兰兔后获得高效价的多抗血清。结论在原核系统中成功地表达了znuA基因编码的蛋白,且ZnuA重组蛋白具有良好的免疫原性,为进一步研究znuA基因在猪链球菌致病过程中的作用以及猪链球菌疫苗的开发奠定了基础。

To detect the immunologic activity of High-affinity zinc uptake system protein ZnuA, we cloned and expressed the gene znu- A encoding ZnuA of Streptococcus suis serotype 2. The target DNA fragment was successfully amplified from the genomic DNA of 05ZYH33 using a pair of specific primers designed for znuA, and then subcloned into pMD18-T vector. Thereafter, the znuA gene was cloned into prokaryotic expression plasmid pET32a, and the recombinant plasmid pET32a：： znuA was transformed into E .coli BL21 after identification by restriction endonuclease digestion and direct DNA sequencing. After induction by IPTG, the isolated ZnuA protein was analyzed with SDS-PAGE. Then the recombinant protein was purified by Ni2- -NTA affinity chromatography and analyzed by Western blotting. The rabbit serum was harvested after four times immunization with recombinant ZnuA protein, and analyzed by indirect-ELISA. As showed by SDS-PAGE, the recombinant protein could be expressed in prokaryotic cells, and the apparent molecular weights of the recombinant protein was similar to the predicted 53 kDa. Western blot result indicated that ZnuA could react with the serum of pig infected with S. suis2 specifically. The polyclonal antibody titer of the rabbit serum immunized with recombinant protein reached more than 1：102 400. All results indicated that the target protein can be overexpressed in E. coli BL21, which would be useful for the further investigation on function of the znuA gene.