牛分枝杆菌mpb51-mpb70融合基因的原核表达与抗原活性初步鉴定  

Prokaryotic expression of the Mycobacterium bovis fusion gene mpb51-mpb70 and preliminary identification of antigenic activity

作　　者：姜秀云[1,2] 周步丹 董阳 包艳红 陈敬蕊 刘磊 王春芳[1] 徐博文[2] 马红霞 JIANG Xiu-yun;ZHOU Bu-dan;DONG Yang;BAO Yan-hong;CHEN Jing-rui;LIU Lei;WANG Chun-fang;XU Bo-wen;MA Hong-xia(Jilin Agricultural University,Changchun 130118,China;Changchun University of Sci-Techology,Changchun 130600,China)

机构地区：[1]吉林农业大学,长春130118 [2]长春科技学院,长春130600

出　　处：《中国人兽共患病学报》2018年第9期805-810,共6页Chinese Journal of Zoonoses

基　　金：国家自然科学基金(No.31572555);吉林省科技发展计划项目(No.20170101025JC);吉林省教育厅科学技术研究项目(No.201441)资助~~

摘　　要：目的通过获得牛分枝杆菌保护性抗原MPB51、MPB70的融合蛋白MPB51-MPB70,以提高单一蛋白的抗原性。方法采用重叠拼接PCR技术将牛分枝杆菌mpb51与mpb70基因融合,获得融合基因mpb51-mpb70,克隆至pMD19中,构建重组质粒pMD-51-70。酶切、纯化后连接至pET28a(+)中,构建重组表达质粒pET-51-70。通过SDS-PAGE和Western blotting分析、鉴定融合蛋白的表达及抗原性。以间接ELISA比较MPB51、MPB70、MPB51-MPB70的抗原性。结果表达、纯化了45ku的融合蛋白MPB51-MPB70,并与牛结核血清发生特异性反应,且比单一蛋白反应性强。结论成功地获得了具有良好抗原性的牛分枝杆菌融合蛋白MPB51-MPB70。To improve antigenicity of the simple protein,Mycobacterium bovis MPB51 and MPB70 were fused using(Gly4Ser)3 to obtain a fusion protein MPB51-MPB70.The mpb51 and mpb70 genes were fused by splicing overlapping extension(SOE)polymerase chain reaction(PCR)to acquire the fusion gene mpb51-mpb70 which was then cloned into pMD19 vector to construct recombinant plasmid pMD-51-70.After enzyme digestion by Bam H I and Eco R I,the purified fusion gene was subcloned into the expression vector pET28a(+).The expression of mpb51-mpb70 was analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis(SDS-PAGE)and the antigenic activity was determined by Western-blotting.Indirect-ELISA was used to compare the antigenicity among MPB51,MPB70 and MPB51-MPB70.A 1359bp fusion gene was amplified and the recombinant plasmid pMD-51-70 was constructed successfully.The expressed fusion protein,approximately 45 ku,exhibited a reaction activity with bovine tuberculosis serum.Besides,the reaction activity was better than that of solo protein.These results indicated that the MPB51-MPB70 fusion protein was obtained successfully.

关 键 词：牛分枝杆菌 mpb51基因 mpb70基因 融合蛋白 

分 类 号：Q786[生物学—分子生物学] S855.2[农业科学—临床兽医学]