鸡源志贺菌IpaC基因的克隆及原核表达  

Clone and prokaryotic expression of IpaC gene from chicken Shigella

作　　者：苗银萍[1] 蒋大伟[1] 许兰菊[1] 王川庆[1] 尤晓楠[1] 李克宇[1] 

机构地区：[1]河南农业大学牧医工程学院,河南郑州450002

出　　处：《中国兽医学报》2013年第9期1352-1357,共6页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(31272568)

摘　　要：应用PCR方法从ZMD-01株鸡源志贺菌中扩增IpaC目的基因,并将IpaC基因克隆至载体pET32α（＋）中,构建原核表达载体pET32α（＋）-IpaC,然后转化至大肠杆菌BL21（DE3）中诱导表达并优化表达条件,最后进行目的蛋白的可溶性鉴定及Western-blot鉴定。结果显示,扩增出大小为1 092bp的IpaC基因,与10株人源志贺菌参考株的核苷酸序列同源性为97.3%~99.8%、氨基酸序列同源性为96.4%~99.7%;进化树结果显示,与参考株AL391753相似性较大,它们在同一个分支上;SDS-PAGE结果显示,重组表达菌在约63 000处出现条带,最佳表达条件确定为诱导前培养3h、IPTG终浓度为1.0mmol/L、加入IPTG诱导4h;蛋白既存在上清中也存在包涵体中,上清蛋白纯化后纯度可达90%以上;Western-blot分析显示,融合蛋白有反应原性。以上结果表明,鸡源志贺菌IpaC基因与人源志贺菌有较高的同源性;成功表达了IpaC蛋白,为今后研究鸡源志贺菌IpaC蛋白参与的致病机制奠定基础。The purpose was to analyze the homology of IpaC gene from chicken Shigella and provide IpaC protein for researching pathopoiesis process.First,the IpaC gene was amplified by PCR technique from chicken Shigella called ZMD-01 strain,and then the combinant plasmid pET32α（＋）-IpaC was transferred into competent E.coli DH5α after being constructed.Subsequently,recombination strain was induced,and expression condition was optimized.At last,dissolubility identification and Western-blot identification of interest protein were done.The size of amplified IpaC gene was 1092 bp,the nucleotide sequence homology of IpaC genes between chicken in this paper and human（10 strains in all）was from 97.3% to 99.8%,the amino acid sequence homology was from 96.4% to 99.7%;the cladogram result showed that this gene had the highest similarity to reference strain AL391753,they were on the same branch;SDS-PAGE result showed that the fusion protein had a molecular weight of about 63000,the optimization expression condition was that cultivation time before inducing was 3h,IPTG final concentration was 1.0 mmol/L,induction time was 4h;protein was not only in supernatant but also in inclusion,the purity of protein in the supernatant was above 90% after purification;Western-blot showed that fusion protein had reactionogenicity.All results mentioned above indicated that IpaC gene from chicken had very high homology with human;IpaC protein was expressed successfully which supplied a condition for researching IpaC protein pathopviesis.

关 键 词：鸡源志贺菌 IpaC基因 克隆 

分 类 号：S852.61[农业科学—基础兽医学]