肠毒素大肠杆菌定居因子CS6抗原基因在大肠杆菌中的高效表达

High expression of CS6 colonization factor of enterotoxigenic Escherichia coli (ETEC) in Escherichia coli

目的 :构建表达肠毒素大肠杆菌 (ETEC)定居因子CS6的重组质粒 ,并在大肠杆菌DH5α中高效表达。方法 :利用基因工程的方法 ,将含有CS6基因的质粒pMG2 33用限制性内切酶ClaⅠ酶切 ,得到长约 3.1kb的CS6片段 (含有CS6结构基因及调控基因的DNA片段 ) ;并与用限制性内切酶ClaⅠ酶切的表达载体质粒pBV32 1连接 ,得到含有CS6片段的重组质粒pMG2 35 ,转化至大肠杆菌DH5α。结果 :SDS PAGE、Western印迹及免疫荧光观察的结果表明 ,重组菌DH5α/pMG2 35可高效表达相对分子质量为 14 6 0 0的CS6抗原蛋白 ,并在重组菌表面表达 ,表达产物可被抗CS6多克隆抗体识别。结论 :重组菌可稳定表达肠毒素大肠杆菌定居因子抗原CS6。

Objective: To construct the recombinant plasmid pMG235 for expressing the Escherichia coli surface antigen 6 (CS6) in high level in E.coli DH5α. Methods: CS6 Gene obtained from plasmid pMG233 cut by restriction enzyme ClaⅠ was about 3.1 kb and was cloned into an expression plasmid pBV321 for constructing the recombinant plasmid pMG235, which was then transformed into E.coli DH5α. Results: The CS6 protein expressed by the recombinant strain was about 14 000 estimated by SDS-PAGE. The results of Western-blotting showed that the expressed CS6 protein of recombinant strain can be identified by CS6 antibody. Immunofluorescence analysis showed that the E.coli DH5α carrying the recombinant plasmid pMG235 can be excited to emit fluorescence on its surface. Conclusion: The recombinant strain E.coli DH5α/pMG235 can express CS6 protein in high level on its surface. This work is helpful to constructing recombinant vaccine for prevention of bacterial diarrhea.