重组肉毒神经毒素A轻链(BoNT/A LC)突变体的构建、表达与活性分析  

Construction,Expression and Analysis of the Activity of Mutation of Recombinant Light Chain of Botulinum Neurotoxin Serotype A(BoNT/A LC)

作　　者：宋孚洋[1] 马臣杰[1] 高姗[2] 康琳[2] 王玉炯[1] 王景林[2] 

机构地区：[1]宁夏大学生命科学学院,中国宁夏回族自治区银川750021 [2]军事医学科学院微生物流行病研究所病原微生物与生物安全国家重点实验室,中国北京100071

出　　处：《生命科学研究》2011年第3期263-267,共5页Life Science Research

基　　金：病原微生物生物安全国家重点实验室资助课题(PBS2009B-05)

摘　　要：A型肉毒神经毒素的轻链(BoNT/A LC)是一种锌依赖性的金属内肽酶.通过X射线分析其结构并结合一些文献报道表明,轻链上的Arg362和Tyr365直接参与了酶的催化作用,而Glu350则处于其活性位点的中心位置.采用定点突变技术,对编码这3个关键性的氨基酸位点的碱基进行突变(Arg362Ala、Tyr365Phe、Glu350Ala),获得了BoNT/A LC突变体.突变体蛋白与BoNT/A的底物蛋白SNAP-25进行切割反应,结果表明,未经突变的BoNT/A轻链蛋白能够特异性地识别SNAP-25蛋白上的Q197-R198位点,而突变体蛋白则完全无法识别该位点,不具有金属内肽酶活性,成功地去除了肉毒神经毒素的毒力,为下一步的全长肉毒神经毒素重组疫苗的研究打下了基础.The light chain of botulinum neurotoxin is the catalytic domain as zinc dependent metallopro-tease.By X ray analyze its structure and some literature reports suggested that,Arg362 and Tyr365 of BoNT/A light chain may be directly involved in catalysis,Glu350 apparently occupies a central position in the active site.Site directed mutagenesis have been used to change these amino acids（Arg362Ala,Tyr365Phe,Glu350Ala）,then the mutation of the BoNT/A light chain was gotten.When the SNAP-25 was mixed with BoNT/A Lc,the reaction result between the enzyme and substrate showed that BoNT/A Lc can cleave its substrate at Q197-R198,but the BoNT/A LC mutant has no enzymatic activity.It certificates that the toxicity of BoNT/A was removed successfully,and can be considerd as foundation of studying recombi-nant holotoxoid vaccine against Botulism serotype A.

关 键 词：肉毒神经毒素 轻链 突变 

分 类 号：R996.1[医药卫生—毒理学]