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为了探究α毒素氨基端及其生物学功能的关系,本研究利用PCR技术克隆了魏氏梭菌α毒素氨基端基因PLC1-250,构建了含PLC1-250基因重组质粒的BL21(DE3)(p N-PLC1-250)重组表达菌株。根据序列分析和酶切鉴定的结果,证实了重组质粒p N-PLC1-250含有PLC1-250基因,且其开放阅读框架和基因序列均正确。经过SDS-PAGE分析,结果表明PLC1-250蛋白表达量约占菌体总蛋白相对含量的18.76%。对其生物学活性的研究表明,PLC1-250蛋白与α毒素一样具有磷脂酶C活性。研究为进一步揭示α毒素的分子作用机制提供了有力的支持。
In order to explore the relationship between α-terminal amino-terminal and its biological function, we cloned α1-terminal amino acid sequence of PLC1-250 from Clostridium perfringens by PCR and constructed BL21 (DE3) (p N-PLC1-250) recombinantly expressing strain. According to the results of sequence analysis and restriction enzyme digestion, it was confirmed that the recombinant plasmid pN-PLC1-250 contains the PLC1-250 gene and the open reading frame and the gene sequence are correct. After SDS-PAGE analysis, the results showed that PLC1-250 protein expression accounted for about 18.76% of the total relative content of total bacterial proteins. Studies on its biological activity show that PLC1-250 protein has the same phospholipase C activity as alpha toxin. The research provided strong support for revealing the molecular mechanism of alpha toxin.