含有分子内佐剂的CIC蛋白表达及免疫活性初步研究

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2 072 bp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95.9 ku；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。

Study on Expression and Preliminary Immunocompetence of CIC Protein Containing Molecular Adjuvants

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-32a(+) vector to construct recombinant plasmids pET-32a(+)-CTB-IsdBid-Clfais. The recombinant plasmids pET-32a(+)-CTB-IsdBid-Clfais were transformed into Escherichia coli (E.coli) BL21 to express the CTB-IsdBid-Clfa(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 CIC gene were 2 072 bp, and CIC was correctly inserted into the pET-32a(+) plasmids, the pET-32a(+)-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.coli BL21 with pET-32a (+)-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-32a(+)-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.