牛型布鲁氏菌外膜蛋白OMP31基因的克隆与原核表达  

Cloning and prokaryotic expression of the gene encoding outer membrane protein 31(OMP31) of Brucella abortus

作　　者：左玉柱[1] 范京惠[1] 刘媛[1] 秦建华[1] 

机构地区：[1]河北农业大学动物科技学院,河北保定071001

出　　处：《中国兽医科学》2010年第8期842-845,共4页Chinese Veterinary Science

基　　金：国家"十一五"科技支撑计划项目(2006BAD04A10-4);河北省重大技术创新专项计划项目(07227146Z-4)

摘　　要：采用PCR技术从河北省某牛场流产污物中扩增得到布鲁氏菌外膜蛋白(OMP31)基因,将其连接入pMD18-T克隆质粒并进行序列测定;测序正确后,将该基因插入pET-28a(+)载体中构建原核表达载体,转化大肠杆菌Rossetta(DE3)感受态细胞,诱导表达融合蛋白,并用Western-blot分析表达蛋白的免疫反应特性。结果显示,扩增了OMP31的全基因,基因全长723bp,共编码241个氨基酸。成功构建了布鲁氏菌OMP31基因的原核表达载体pET-OMP31,并且在大肠杆菌中表达了OMP31蛋白,经Western-blot鉴定,该蛋白可以与布鲁氏菌免疫血清产生特异性结合反应。证实布鲁氏菌外膜蛋白OMP31基因已成功表达,为布鲁氏菌病血清学诊断方法的建立及亚单位疫苗的研究提供了基础资料。The full-length outer membrane protein gene OMP31 of Brucella abortus was amplified by PCR from the aborted grime collected from one cattle farm in Hebei Province and successfully cloned into pMD18-T vector.Sequence analyses showed that the complete open reading frame with an ATG initiation codon comprised 723 nucleotides and encoded a protein of 241 amino acids.The OMP31 gene was sub-cloned into expression vector pET-28a（＋） and the recombinant plasmid was then transformed into competent Rossetta（DE3） bacteria.SDS-PAGE and Western-blot analyses revealed that the recombinant protein OMP31 with a molecular weight of 28ku was highly expressed in the host cells by the IPTG induction and could be identified by specific antisera against Brucella.

关 键 词：布鲁氏菌 外膜蛋白OMP31 克隆 原核表达 

分 类 号：S852.614[农业科学—基础兽医学] Q786[农业科学—兽医学]