【摘要】 为了研究含有分子内佐剂的CTB-IsdBid-Clfais(CIC)蛋白表达及其免疫活性,试验采用重叠PCR方法将分子内佐剂CTB与IsdBid-Clfais基因串联,并将CTB-IsdBid-Clfais(CIC)插入到表达载体pET-32a(+)中,构建pET-32a(+)-CTB-IsdBid-Clfais重组质粒,将鉴定正确的重组质粒转化到大肠杆菌BL21感受态细胞中表达目的蛋白CIC,利用Western blotting方法对其进行鉴定,并以ELISA方法检测CIC蛋白的免疫活性。结果发现,试验成功扩增了2 072bp的目的基因CIC,并将CIC正确连接到pET-32a(+)载体上,构建了pET-32a(+)-CTB-IsdBid-Clfais重组质粒;Western blotting结果证实,该重组质粒能够在大肠杆菌BL21感受态细胞中正确表达CIC蛋白,分子质量大小为95.9ku;ELISA结果显示,CIC试验组与IsdBid、Clfais蛋白组间均无显著差异(P>0.05),与BSA组间差异极显著(P<0.01)。综上所述,本研究成功构建了pET-32a(+)-CTB-IsdBid-Clfais重组质粒,该质粒在大肠杆菌BL21中成功表达了CIC蛋白,且CIC能与IsdBid、Clfais免疫小鼠血清发生反应,具有较强的免疫活性。更多还原

【Abstract】 To express CTB-IsdBid-Clfais(CIC)protein and evaluate its immunogenicity,CTB(as a molecular adjuvant)could be tandem linked with IsdBid-Clfais gene by the overlapping PCR method,then CTB-IsdBid-Clfais(CIC)was inserted into pET-32 a(+)vector to construct recombinant plasmids pET-32 a(+)-CTB-IsdBid-Clfais.The recombinant plasmids pET-32 a(+)-CTBIsdBid-Clfais were transformed into Escherichia coli(E.coli)BL21 to express the CTB-IsdBidClfa(CIC)protein,the CIC expression protein and its immune activity were detected by Western blotting and ELISA,respectively.The results showed that the length of CICgene were 2 072 bp,and CIC was correctly inserted into the pET-32 a(+)plasmids,the pET-32 a(+)-CTB-IsdBid-Clfais recombinant plasmids were successfully constructed.Western blotting result confirmed that the molecular weight of CIC proteins was 95.9 ku,which were correctly expressed by E.coliBL21 with pET-32 a(+)-CTB-IsdBid-Clfais plasmids.ELISA results showed that there was no significant difference among the CIC,IsdBid and Clfais protein groups(P>0.05),and there was extremely significant difference between CIC and BSA protein groups(P<0.01).In conclusion,the pET-32 a(+)-CTB-IsdBid-Clfais recombinant plasmids were successfully constructed,CIC proteins were correctly expressed,and were able to react with serum from mice immunized with IsdBid and Clfais,respectively,therefore,CIC proteins had strong immune activity.更多还原