【摘要】 利用前期筛选的具有良好免疫原性的S.aureus表面Clfa蛋白免疫优势片段Clfais和TraP蛋白的融合重组蛋白,加入CTB分子内佐剂,以表达CTB-Clfais-TraP(CCT)蛋白和研究CCT免疫活性。实验采用重叠PCR方法将分子内佐剂CTB与Clfais-TraP基因串联,并将CTB-Clfais-TraP插入到表达载体p ET32-a(+)上,构建p ET32-a(+)-CTB-Clfais-TraP重组质粒,经鉴定后,重组质粒转化到大肠埃希菌BL21中表达目的蛋白,利用Western blot方法对其鉴定,以ELISA方法检测CCT蛋白的免疫活性。实验结果表明表达的CCT蛋白大小为87.6 ku,CCT能与Clfais、TraP免疫小鼠血清发生反应,表明CCT具有免疫活性,不受CTB分子影响,这为进一步研究CTB提高Clfais-TraP蛋白的免疫活性奠定了良好基础。更多还原

【Abstract】 According to the recombinant proteins of Clfais as immune superiority fragment of Clfa and TraP protein of Staphylococcus aureus(S.aureus)surface that exhibited good immunogenicity,the CTB intramolecular adjuvant gene was spliced with Clfais-TraP,then CTB-Clfais-TraP(CCT)protein was expressed and its immune activity was evaluated.Initially,CTB was tandem linked with Clfais-TraP by the overlapping PCR method,then,CTB-Clfais-TraP gene was inserted into p ET32-a(+)vector to construct recombinant plasmids,p ET32-a(+)-CTB-Clfais-TraP. After the p ET32-a(+)-CTB-Clfais-TraP recombinant plasmids were identified,they were transformed into Escherichia coli(E.coli)BL21 to express the CCT,and CCT was detected by Western blot,then,immune activity of CCT was estimated by ELISA. These results showed the molecular size of CCT protein was 87.6 ku,and the CCT recombinant protein as ELISA coating antigen,could react with serum from mice immunized with Clfais,TraP,respectively. In conclusion,the p ET32-a(+)-CTB-Clfais-TraP recombinant plasmids were successfully constructed,and CCT was correctly expressed in E.coli BL21 with the p ET32-a(+)-CTB-Clfais-TraP plasmids,more importantly,CCT had strong immune activity,which would provide the foundation to further explore its immunogenicity.更多还原