A型魏氏梭菌α毒素氨基端PLC结构分析与生物学活性鉴定

摘要

利用PCR扩增技术克隆A型魏氏梭菌α毒素氨基端的PLC1-250基因,构建含PLC1-250基因表达质粒的BL21(DE3)(pN-PLC1-250)重组菌株,序列分析和酶切鉴定证实构建的pN-PLC1-250重组质粒含有目的基因且基因序列与阅读框架均正确.SDS-PAGE分析表明,PLC1-250蛋白表达量占菌体总蛋白相对含量的18.76%.利用SOPMA法预测PLC1-250蛋白分子的二级结构,且同源模建了其3D结构,结果表明,PLC1-250蛋白分子的二级结构主要为α螺旋和无规则卷曲,三级结构与α毒素相类似.此外,还对其生物学活性进行了鉴定,可为进一步探索α毒素作用的分子机制,以及其分子结构与生物学功能的关系奠定基础.%Amino-terminal PLC1-250 gene of Clostridium welchii α-toxin was amplified by PCR.The recombinant strain BL21 (DE3)(pN-PLC1-250) containing PLC1-250 gene was constructed.It was shown that the recombinant plasmid pN-PLC1-250 contained PLC1-250 gene with correct sequence and ORF by identification of endonuclease-digesting and sequence analysis.SDS-PAGE analysis showed that the expression level of PLC1-250 proteins were about 18.76% of total cellular proteins.The secondary structure and three-dimensional structure of PLC1-250 proteins were predicted by SOPMA method on EXPASY website.The results showed that the secondary structure of PLC1-250 protein was composed of alpha helices and random coils.The three-dimensional structure of PLC1-250 protein was similar with amino-terminal domain of α-toxin protein.The study of biological activity laid foundation for further research of the molecular mechanism of α-toxin and the relation of its molecular structure and biological function.