绵羊肺炎支原体热休克蛋白Hsp70 C末端基因原核表达  

Cloning and Partial Prokaryotic Expression of Gene Heat Shock Protein Hsp70 of Mycoplasma ovipneumoniae

作　　者：王慧[1] 周碧君[1,2] 罗成波 李益 孔令萍 文明[1,2] 覃岚[1] 程振涛[1,2] 王开功[1,2] 

机构地区：[1]贵州大学动物科学学院,贵阳550025 [2]贵州省动物疫病研究室,贵阳550025 [3]黔西南州疫病预防控制中心,贵州兴义562400

出　　处：《西北农业学报》2014年第4期130-134,共5页Acta Agriculturae Boreali-occidentalis Sinica

基　　金：黔西南州种草养羊产业发展省州科技合作专项(黔西南科合[2012]5号);贵州省自然科学技术基金(黔科合J字[2011]2332号);贵州省农业科技攻关项目(黔科合NY字[2011-3105]号);贵州省动物疫病与兽医公共卫生重点实验室培育(黔科合计Z字[2011]4008号)

摘　　要：为研究绵羊肺炎支原体(Mycoplasma ovipneumoniae,Mo)热休克蛋白Hsp70抗原特性,以Mo贵州分离株为模板扩增得到Hsp70蛋白C末端基因,将目的基因连接至pET-28a原核表达载体,经PCR、双酶切和序列测定正确的阳性重组质粒转化E.coli Rosseta表达菌,IPTG诱导表达,表达产物进行SDS-PAGE及Western blotting分析。结果显示,经PCR从Mo贵州分离株扩增获得567bp的C末端片段,并成功构建含Mo Hsp70蛋白C末端基因的原核表达质粒,经IPTG诱导表达获得分子质量约为27ku的目的蛋白,且目的蛋白能与Mo阳性血清印迹反应。说明,成功构建可有效表达Mo贵州分离株Hsp70蛋白C末端基因的原核表达载体。To explore the antigen features of heat shock protein Hsp70 of Mycoplasma ovipneumoniae （Mo） , a Mo isolate from Guizhou was served as template to amplify the fragment at C terminal of Hsp70 protein, and connecting to pET-28 a prokaryotic expression vector, transforming the right positive recombinant plasmid by PCR, conducting enzyme digestion and sequence determination into E. coli Rosseta express bacterium, and inducible express by IPTG, expression product analysis by SDS- PAGE and Western blotting. Results showed that PCR amplification gained C terminal fragment of 567 bp from Mo isolate in Guizhou, and successfully built Mycoplasma ovipneumoniae Hsp70 protein C terminal prokaryotic expression plasmid, obtained about 27 ku protein by IPTG, protein purification can react with Mo positive serum. The research results showed that the prokaryotic expression vector of the C terminal gene of Hsp70 protein in Mo isolate from Guizhou was constructedsuccessfully.

关 键 词：绵羊肺炎支原体 HSP70 克隆 原核表达 

分 类 号：S858.26[农业科学—临床兽医学]