A型魏氏梭菌α毒素羧基端蛋白基因表达与结构分析

Gene expression and structure analysis of alpha toxin carboxy-terminal protein from Clostridium welchii type A

利用PCR技术将α毒素羧基端基因CPA251-370克隆到pET-32a载体上,构建了重组表达质粒pXETCPA-C,酶切鉴定和序列分析证实其含有CPA251-370基因且序列和阅读框架均正确。对重组菌株BL21(DE3)(pXETCPA-C)表达的蛋白质进行SDS-PAGE分析,结果表明CPA251-370蛋白表达量占菌体总蛋白质相对含量的16.43%。SOPMA法预测表明CPA251-370蛋白二级结构主要由β折叠和无规则卷曲组成,其3D结构与α毒素羧基端部分相似。CPA和CPA251-370蛋白的圆二色(CD)光谱分析发现两者的CD光谱仅有一些微小变化。免疫攻毒试验表明CPA251-370蛋白免疫的小鼠能抵抗最小致死剂量(MLD) 0.5 mL·只-1(活菌数约为5×109 cfu)的A型魏氏梭菌标准株C57-1毒素攻击。该研究深入阐释了A型魏氏梭菌α毒素的分子结构,为揭示其作用的分子机制奠定了坚实基础。

The carboxy-terminal CPA251-370 gene of Clostridium welchii alpha toxin was cloned into pET-32 a by PCR and the recombinant expression plasmid pXETCPA-C was constructed. The recombinant plasmid pXETCPA-C contained CPA251-370 gene which had correct sequence and ORF by identification of endonuclease-digesting and sequence analysis. The SDS-PAGE analysis showed that the expression level of the CPA251-370 proteins was about 16.43% of total cellular protein. The secondary structure of CPA251-370 proteins was composed of beta extended strands and random coils by using SOPMA method. The three-dimensional structure of CPA251-370 protein was similar with C-terminal domain of alpha toxin. Circular dichroism(CD) spectra of CPA and CPA251-370 proteins were slightly different by CD analysis. Immunization in a mouse model with inactivated recombinant strain induced protection against at least 1 MLD of the toxin from C.welchii type A. This study lays a solid foundation for further research of the relationship between the structure and function of alpha toxin and its mechanism.