CPB-ST融合基因的构建及表达研究

用限制性核酸内切酶ＥｃｏＲＩ和ＳａｌⅠ双酶切含有大肠杆菌耐热性肠毒素ＳＴ１前体蛋白基因的质粒ｐＸＳＴ１，回收３２５ｂｐ的ＳＴ基因片段，然后，通过Ｔ４ＤＮＡ连接酶将其定向连接于事先经同样的双酶切处理的带有产气荚膜梭菌β－毒素基因（ＣＰＢ）的重组质粒ｐＥＣＢ２中ＣＰＢ基因的下降，转化受体菌ＢＬ２１（ＤＥ３）中，经ＢａｍＨＩ和ＥｃｏＲＩ酶切反应鉴定重组质粒，得到了理想重组质粒ｐＥＣＢ－ＳＴ１。重组菌株Ｂｌ２１（ＤＥ３）（ｐＥＣＢ－ＳＴ１）经ＩＰＴＧ诱导后，其表达产物经ＳＤＳ－ＰＡＧＥ、Ｗｅｓｔｅｒｎ－ｂｌｏｔｉｎｇ及ＥＬＩＳＡ检测，结果表明重组菌株可以表达ＣＰＢ－ＳＴ融合蛋白，而且该融合蛋白无天然β－毒素和ＳＴ１的生物毒性。

Studies on the Construction and Expression of CPB ST Fusion Gene

おe used restriction endonucleases EcoR I and Sal I to digest plasmid pXST1 containing pre ST1 gene that encodes for heat stable enterotoxin of Escherichia coli and revovered the 325bp ST1 gene fragment. The 5′ terminus of ST1 gene was fused to 3′ terminus of CPB gene(encoding betatoxin of Clostridium perfringens)in the plasmid pECB2 that had been cleaved with EcoR I and Sal I.The recombinant pECB ST1 was studied in detail by restriction endonuclease analysis.The result showed that the recombinant plasmid carried CPB ST1 fusion gene.By transformation of E.coli BL21(DE3),we got E.coli BL21(DE3)(pECB ST1).The recombinant strain can produce CPB ST fusion protein by SDS PAGE,Western bloting and ELISA,and the fusion protein was nontoxic.