坏死梭杆菌白细胞毒素基因SH片段的克隆与表达  

Cloning and Expression of the Leukotoxin Gene SH from Fusobacterium necrophorum

作　　者：赵利芳[1,2] 陈立志[2] 张秀华[2] 冯书章[3] 王克坚[4] 刘晓颖[2] 陆伟[2] 冯凯[1,2] 王秀东[2] 姚志利[2] 

机构地区：[1]江苏科技大学生物与环境工程学院,江苏镇江212018 [2]中国农业科学院特产研究所,吉林吉林132109 [3]中国人民解放军军事医学科学院军事兽医研究所,吉林长春130062 [4]厦门大学海洋环境科学教育部重点实验室环境科学研究中心,福建厦门361005

出　　处：《特产研究》2009年第1期14-18,共5页Special Wild Economic Animal and Plant Research

基　　金："十五"国家科技攻关子课题(2002BA518A04);吉林省科技发展计划项目(20070570);吉林市科技发展计划项目(200805)

摘　　要：坏死梭杆菌白细胞毒素是坏死杆菌病的主要致病因子,白细胞毒素基因(lkt)是其编码基因。以分离到的国内牛源坏死梭杆菌FN(A)菌株F4基因组DNA为模板,应用PCR方法扩增白细胞毒素基因SH片段,克隆至pMD18-T载体上,以BamHⅠ和HindⅢ酶切的目的片段SH与相应酶切的pET32a载体连接构建pET32a-SH重组表达质粒,经转化E coli BL21(DE3)后用IPTG进行蛋白诱导,SDS-PAGE检测重组蛋白表达情况。结果表明:扩增基因序列大小为1800bp,SDS-PAGE检测重组蛋白有效表达,表达得到大小为80.2kDa的目的蛋白,采用镍柱亲和层析方法纯化SH重组蛋白,获得了纯度达95%的重组蛋白;经West-ern-blot证实,该蛋白对抗坏死杆菌阳性血清具有反应活性。The leukotoxin of Fusobacterium necrophorum （FN） is considered to be one of the main virulence factors. The lkt gene encodes for FN. In this study, the SH fragment of lkt gene was amplified by PCR using the F4 genome as the template, which was isolated from the Chinese Fusobacterium necrophorum strain. The fragment was then cloned to the pMD18-T vector for sequencing. Thereafter, the SH fragment was subcloned into the multiple cloning sites of the pET32 to construct pET32a-SH recombinant plasmid, which was then trans-formed into E.coli BL21 （DE3） with IPTG induction for expression. SDS-PAGE was used to analyze the recombinant protein. The results showed that the SH fragment of about 1800 bp was amplified and was about 80.2 kDa. The fusion protein was purified by Ni-NTA affinity chromatography under denature conditions, and their purity was above 95%. Western-blOt analysis indicated the SH fragment had antigenicity against Fusobacterium necrophorum.

关 键 词：坏死梭杆菌 SH 克隆 表达 

分 类 号：S855.12[农业科学—临床兽医学] Q786[农业科学—兽医学]