产气荚膜梭菌β_2-β_1融合基因的构建及初步表达

目的构建产气荚膜梭菌β2-β1毒素融合基因并在重组大肠杆菌中表达。方法采用PCR方法,从含β2毒素基因质粒CPB2-9中扩增出β2毒素基因,经NcoⅠ和BamHⅠ双酶切后,回收0.73kb片段,再将含β1毒素基因质粒pXETB1经同样双酶切回收,与β2片段连接,转化BL21(DE3)中,进行诱导表达。结果经SDS-PAGE和Westernblot分析表明,重组菌株BL21(DE3)可表达β2-β1融合蛋白,且该蛋白可以被相应的抗体所识别。结论已成功构建了β2-β1融合基因,并在重组大肠杆菌中表达。

Construction and Preliminary Expression of β2-β1 Fusion Gene of Clostridium perfrigens

Objective To construct the β_2-β_1 fusion gene of Clostridium perfrigens and express in E.coli.Methods Amplify β_2 toxin gene from plasmid CPB2-9 containing the gene by PCR and identify by digestion with NcoⅠ and BamHⅠ.Amplify β_1 toxin gene from plasmid pXETB_1 by PCR and identify by the same method.Ligate the two gene fragments and transform to E.coli BL21(DE3) for expression.Identify the expressed product by SDS-PAGE and Western blot.Results SDS-PAGE and Western blot showed that the β_2-β_1 fusion gene was successfully expressed in E.coli BL21(DE3),and the expressed product was recognized by the corresponding antibody.Conclusion The β_2-β_1 fusion gene of Clostridium perfrigens was successfully constructed and expressed in E.coli.