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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。
Objective To clone and express the high-affinity zinc uptake system protein (ZnuA) gene of Streptococcus suis type 2 and to study the immunogenicity of ZnuA protein. Methods The gene znuA was amplified from genomic DNA of Streptococcus suis serotype 2 Streptococcus suis type 2 by PCR. The recombinant plasmid pET32a :: znuA was constructed and transformed into E. coli BL21 (DE3). The positive transformants were screened for IPTG induction and SDS -PAGE. The recombinant protein was purified by His affinity chromatography. The immunogenicity of ZnuA protein was detected by Western blot. The recombinant protein was used to immunize New Zealand rabbits to collect multiple antisera. ELISA was used to detect the titer of multiple antisera. Results The constructed recombinant plasmids were highly expressed in host bacteria, and purified by His affinity chromatography to obtain higher purity of recombinant protein. The result of Western blot showed that ZnuA protein had good immunogenicity. The recombinant protein was immunized with high titer of New Zealand rabbit Multi-antiserum. Conclusions The protein encoded by znuA gene was successfully expressed in prokaryotic system and the recombinant protein ZnuA has good immunogenicity, which lay a foundation for further study on the role of znuA gene in the pathogenesis of Streptococcus suis and the development of Streptococcus suis vaccine basis.