羊布鲁氏菌BCSP31、OMP31、L7/L12基因的原核表达载体构建及表达  

Construction and expression of genes encoding BCSP31,OMP31 and L7/L12 proteins of Brucella melitensis in prokaryotic cell

作　　者：司瑞[1] 白文涛[1] 张芳琳[1] 刘艳丽[1] 胡刚[1] 吴兴安[1] 阎岩[1] 徐志凯[1] 

机构地区：[1]第四军医大学微生物学教研室,西安710032

出　　处：《中国人兽共患病杂志》2005年第11期961-964,共4页Chinese Journal of Zoonoses

基　　金：第四军医大学科研重点项目(05XJZ006)

摘　　要：目的构建羊布鲁氏菌外膜蛋白BCSP31、OMP31和L7/L12基因的重组表达质粒,并在原核系统中表达。方法根据羊布鲁氏菌M5株外膜蛋白BCSP31、OMP31和抗原L7/L12蛋白基因序列设计引物,分别扩增出大小约为1kb、730bp和380bp的目的基因片断,插入pGEM7Zf(+)中测序并进行分析。将3种基因片段亚克隆入pGEX-4T-1中,转化大肠杆菌,IPTG诱导表达,用SDS-PAGE及Western-blot进行分析鉴定。结果SDS-PAGE结果表明3个目的蛋白均以融合蛋白的方式成功表达,在相对分子量57kDa、49kDa、38kDa处有表达条带,经薄层扫描分析,目的蛋白分别占全菌蛋白的42%、36%、40%。Western-blot证实3种融合蛋白均能被布鲁氏菌免疫兔血清识别。结论成功构建了3种蛋白的表达载体并进行了高效表达,3种蛋白均具有良好的免疫原性。The purpose of this study is to express the BCSP31, OMP31 and L7/L12 proteins of Brucella melitensis in Escherichia coli to obtain the abundant recombinant proteins with high activity, and the genes of these proteins were amplified from Brucella melitensis with PCR method, then the PCR products were subcloned into sequencing vector of pGEM7Zf（ ＋ ）and expression vecotor of pGEX-4T-1, for DNA sequencing or expression. After the fight insertions were confirmed by sequencing, the expression vector were transformed into E. coli and after IPTG induction, the bacterial lysates were analyzed by SDS-PAGE and Western-blot. The SDS-PAGE analysis showed that all the three proteins were expressed successfully in fusion form and could be visualized on gel at molecular mass of 57kDa, 49kDa and 38kDa. The thin layer scan showed that the proteins reach 42%, 36% and 40% of the total bacterial protein respectively and could be detected by the positive serum from immuned rabbit by Western blot. In conclusion, the fusion genes enconding BCSP31, OMP31 and L7/L12 proteins were constructed successfully and the good immunogenicity of all the proteins with high efficiency of expression were demonstrated.

关 键 词：羊布鲁氏菌 BCSP31蛋白 OMP31蛋白 L7/L12蛋白 基因表达 

分 类 号：R378.5[医药卫生—病原生物学]