【摘要】 目的构建SarA的原核表达载体,对SarA蛋白免疫原性进行初步研究.方法首先应用生物信息学方法对SarA基因进行分析;以金黄色葡萄球菌基因组为模板,通过PCR扩增、酶切、连接等一系列反应进行原核重组质粒的构建; IPTG诱导原核重组质粒表达,对SarA蛋白进行纯化、浓缩,将浓缩后的蛋白与佐剂混合免疫家兔,收集血清,通过间接ELISA法检测血清抗体效价,并通过Western blot鉴定抗体特异性.结果成功构建重组表达质粒SarA-pET32a(+),通过IPTG诱导表达,获得以可溶形式高效表达的SarA蛋白,分子质量约为32 kDa.用纯化的SarA蛋白免疫家兔,获得了SarA多克隆抗体,其效价为1∶512 000.结论成功构建了SarA原核表达载体,且在大肠杆菌细胞中表达;得到了高效价和良好特异性的多克隆血清抗体,为进一步研究抗细菌生物膜疫苗奠定了基础.更多还原

【Abstract】 Objective The prokaryotic expression vector of SarA was constructed,and the immunogenicity of SarA protein was preliminary studied. Method The bioinformatics method was used to analyze SarA gene; Use the S.aureus genome as a template to construct a prokaryotic recombinant plasmid through a series of reactions such as PCR amplification,digestion and ligation; IPTG induces the expression of the prokaryotic recombinant plasmid,and SarA protein is purified and concentrated. The concentrated protein was mixed with adjuvant to immunize rabbits,the serum was collected,the antibody titer of serum was detected by indirect ELISA,and the specificity of antibody was identified by Western blot. Results The recombinant expression plasmid SarA-pET32a(+) was successfully constructed,and expressed through IPTG induction,and the SarA protein expressed in a soluble form was obtained with a molecular weight of about 32 kDa. Rabbits were immunized with purified SarA protein to obtain SarA polyclonal antibody with a titer of 1: 512,000. Conclusion SarA prokaryotic expression vector was successfully constructed and expressed in E. coli cells,and a polyclonal serum antibody with high titer and good specificity was obtained,laying a foundation for further research on anti-bacterial biofilm.更多还原