产气荚膜梭菌α毒素基因PLC-Asp56定点突变及其特性鉴定  

Site-directed Mutagenesis PLC-Asp56 of Clostridium Perfringens Alpha-toxin Gene And Its Identification of Characterization

作　　者：陈乾坤 李晓宇 梅伟恒 陈少坚 许崇波 Chen Qiankun;Li Xiaoyu;Mei Weiheng;Chen Shaojian;Xu Chongbo(Ying Dong Collage of Life Science,Shaoguan University,Shaoguan 512005;Institute of Insect Science and Technology,School of Life Sciences,South China Normal University/Guangzhou Key Laboratory of Insect Development Regulation and Applied Research/Guangdong Provincial Key Laboratory of Insect Developmental Biology and Applied Technology,Guangzhou 510631,China)

机构地区：[1]韶关学院英东生命科学学院,广东韶关512005 [2]华南师范大学生命科学学院昆虫科学与技术研究所广州市昆虫发育调控与应用重点实验室广东省昆虫发育生物与应用技术重点实验室,广东广州510631

出　　处：《广东化工》2020年第2期230-232,共3页Guangdong Chemical Industry

基　　金：广东省自然科学基金重点项目(2018B0303110009);韶关学院重点科研项目(SZ2016KJ10);韶关学院创新创业训练计划项目(Sycxcy2017-022);广东省创新创业训练计划项目(201710576027)

摘　　要：本实验通过对A型产气荚膜梭菌α毒素PLC基因第56位氨基酸进行定点突变,将天冬氨酸Asp突变为丙氨酸Ala,构建了含有PLC-Asp56Ala突变基因的表达质粒,测序结果表明,重组质粒pPLC-Asp56Ala已经含有α毒素PLC基因的PLC-Asp56Ala突变基因。经聚丙烯酰胺凝胶电泳分析,PLC-Asp56Ala突变体蛋白含量占菌体总蛋白相对含量的22.48%。二级结构预测结果表明PLC-Asp56Ala突变体蛋白的二级结构第55位和第56位突变位点相对于α毒素由原来的无规则卷曲变成β转角。生物学活性检测表明PLC-Asp56Ala突变体蛋白已经完全丧失该酶活性。本研究结果可为今后对A型产气荚膜梭菌α毒素的致病机制的探究提供基础理论依据。A site 56(Asp→Ala)mutation which might influence the bioactivity of alpha toxin of Clostridium perfringens was induced by site-directed mutagenesis technique and a recombinant strain BL21(DE3)(pPLC-Asp56Ala)containing the PLC-mutated gene expression plasmid was constructed.The correct recombinant plasmid pPLC-Asp56Ala was constructed by nucleotide sequence,and it contained PLC-Asp56Ala mutant gene.Then SDS-PAGE analysis showed that the protein expression of the mutant accounted for 22.48%of the total protein content of the recombinant strain.At the same time,SOPMA method on EXPASY server was applied to predict and simulate the high-level structure of PLC and mutant protein.The results showed that the secondary structure of mutant protein changed from random curl to beta corner at the 56th mutation site and from random curl to beta corner at the 55th mutation site,but the tertiary structure of the two proteins was very similar.The test of Phospholipase C activity showed that the mutant protein lost the biological activity.It provide a basis to research the pathogenic mechanism of Clostridium perfringens alpha-toxin.

关 键 词：产气荚膜梭菌 Α毒素 定点突变 

分 类 号：TQ[化学工程]