重组A型产气荚膜梭菌α毒素C端基因在大肠杆菌中的高效表达

High Expression of Recombinant Alpha-toxin's C-Terminal Gene of Clostridium perfringens Type A in E.coli

在成功克隆A型产气荚膜梭菌α毒素C端基因 (cpa40 8基因 )后 ,发现基因N端ATG后密码子在大肠杆菌中的使用频率普遍偏低 ,在不改变氨基酸的前提下 ,对N端基因进行修饰 ,构建重组表达质粒pBV2 2 0 cpa40 8,导入大肠杆菌DH5α中 ,经温度诱导 ,cpa40 8基因获得了高效表达 ,表达量达菌体总蛋白的 43 75 %,表达产物以可溶形式存在。经Western blot分析 ,表达产物能被标准抗α毒素血清识别。

After having been cloned and digested by the restriction endoenzyme EcoR Ⅰ and Pst Ⅰ,the modified alpha toxin's C terminal gene was connected with the expression vector pBV220 and then transferred into the host cell E.coli DH5α.Induced by 42℃,the cpa408 gene was highly expressed ,which expression amount was to the 43 75 percent of the totall bacterial protein.By analysis of SDS PAGE,the molecular weight of the expression protein is about 15 5×10 3Da.Analysised by Western blot,the protein could specificly react with the standard anti alpha toxin.