产气荚膜梭菌β_2-β_1融合基因的构建及初步表达

Construction and Preliminary Expression of β_2-β_1 Fusion Gene of Clostridium perfrigens

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。

Objective To construct the β2-β1 fusion gene of Clostridium perfrigens and express in E. coli. Methods Amplify β2 toxin gene from plasmid CPB2-9 containing the gene by PCR and identify by digestion with Nco Ⅰ and BamH Ⅰ. Amplify β1 toxin gene from plasmid pXETB1 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 β2-β1 fusion gene was successfully expressed in E. coli BL21 （ DE3 ）, and the expressed product was recognized by the corresponding antibody. Conclusion The β2-β1 fusion gene of Clostridium perfrigens was successfully constructed and expressed in E. coli.