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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 a recombinant plasmid expressing enterotoxigenic Escherichia coli (ETEC) colonization factor CS6 and express it efficiently in E. coli DH5α. Methods: The plasmid pMG233 containing CS6 gene was digested with restriction endonuclease Cla I to obtain a CS6 fragment (a DNA fragment containing a CS6 structural gene and a regulatory gene) of about 3.1 kb in length by genetic engineering; The restriction endonuclease Cla I digested expression vector plasmid pBV32 1 was ligated to obtain a recombinant plasmid pMG2 35 containing the CS6 fragment and transformed into E. coli DH5α. Results: The results of SDS PAGE, Western blotting and immunofluorescence showed that the recombinant protein DH5α / pMG235 could efficiently express the CS6 protein with a molecular weight of 1460 and expressed on the surface of recombinant bacteria. The expressed product could be expressed by anti-CS6 Polyclonal antibody recognition. Conclusion: Recombinant bacteria can stably express enterotoxin Escherichia coli colonizing factor antigen.