金黄色葡萄球菌FnbpA、ClfA、Ebps抗原表位的串联表达及多克隆抗体的制备

Tandem Expression of FnbpA,ClfA,Ebps Antigen Epitopes of Staphylococcus aureus and the Preparation of the Polyclonal Antibody

为获得金黄色葡萄球菌特异性抗原蛋白并以此制备多克隆抗体,应用DNAStar软件筛选金黄色葡萄球菌FnbpA、Clf A、Ebps的抗原表位,以柔性氨基酸序列连接后进行全基因合成,将目的基因克隆到pGEX-4T-1原核表达载体,转化至大肠杆菌感受态细胞,诱导表达,采用Sepharose 4B柱亲和层析纯化表达蛋白,制备抗原后对家兔进行皮下多点注射,于第42天采集外周血,获得血清。结果表明,本试验成功构建了金黄色葡萄球菌抗原基因表达载体,并获得了高免血清,经ELISA的方法检测血清效价可达到1∶10000以上,成功制备多克隆抗体,为以后采用免疫学方法快速检测金黄色葡萄球菌奠定了基础。

In order to achieve the special antigen protein and prepare polyclonal antibody,so as to lay the foundation of the rapid detection of Staphylococcus aureus,this experiment screened the antigen epitopes of FnbpA,ClfA,Ebps of Staphylococcus aureus using the DNAStar software,then connected the epitopes with a linker and synthesized the whole genes.The objective gene was cloned into expression vector pGEX-4T-1,followed by transformation into E.coli BL21.The expression production was purified through Sepharose 4B affinity chromatography method,and the purified protein was injected into rabbits to prepare polyclonal antibody,at the 42 days peripheral blood was collected and the serum was obtained.The results revealed that this experiment has successfully established the expression vector,and the antibody titer was more than 1∶10000 analysised by ELISA.These results were benefit to develop the rapid detection of Staphylococcus aureus using immunological methods.