2型猪链球菌中国强毒株溶血素样蛋白基因的原核表达及其抗血清制备

目的克隆2型猪链球菌溶血素样蛋白(Hemolysin-like protein,HLP)编码基因并进行原核表达,纯化重组蛋白并制备相应的多克隆抗体。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hlp,构建重组表达质粒pET32a::hlp,转化E.coliBL21(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;重组蛋白免疫新西兰兔制备多克隆抗体,间接ELISA和Western blot检测多抗血清。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;重组蛋白免疫新西兰兔后获得高效价的抗血清。结论在原核系统中成功地表达了hlp基因编码的蛋白,并以重组蛋白为抗原制备出高效价的多抗血清,为进一步研究hlp基因的功能奠定了基础。

Prokaryotic expression of hemolysin-like protein gene in highly virulent strains of Streptococcus suis serotype 2 and preparation of the polyclonal antibodies against its expressed products

To express the gene encoding hemolysinqike protein （HLP） in highly virulent strains of Streptococcus suis serotype 2 and to prepare the polyclonal antibodies against its expressed products, the hlp gene from the genomie DNA in the virulent strain 05ZYH33 was amplified by PCR using a pair of specific primers and subcloned into plasmid pMD18-T and pET32a with double digestion of BamH I and XhoI. Subsequently, the prokaryotic expression plasmid pET32a： ：hlp was transformed to E. coli BL21 （DE3） after identification by restriction endonuclease digestion and DNA sequencing. Upon induction with IPTG, E. coli BL21 containing the recombinant plasmid could express a distinct band with a molecular weight of 53 kDa, which was similar to the predicted band of HLP protein as demonstrated by SDS-PAGE. The recombinant HLP protein was purified by Ni2＋-NTA affinity chromatography and the recombinant protein was used as antigen to immunize New Zealand rabbits to prepare the polyclonal antibodies. Titers of the antibodies produced in the immunized rabbits were determined by indirect ELISA and Western blot assay. It was demonstrated that the constructed plasmid could be highly expressed in the host bacteria and the recombinant protein with high purity was obtained after His affinity chromatography. Sera of the immunized rabbits could react specifically with the purified recombinant HLP protein as demonstrated by ELISA and Western blot assay with a titer of polyclonal antibodies of 1 ： 409600. This recombinant obtained and the polyclonal antibodies produced would be useful in the further studies on the hip gene.