羊口疮病毒B2L基因的原核表达、纯化及反应原性分析  

Prokaryotic expression,purification and reactogenicity of ORFV B2L gene

作　　者：石正旺 刘华南[2] 胡永浩[1] 郑海学[2] 

机构地区：[1]甘肃农业大学动物医学院,甘肃兰州730070 [2]中国农业科学院兰州兽医研究所,家畜疫病病原生物学国家重点实验室,国家口蹄疫参考实验室,甘肃兰州730046

出　　处：《甘肃农业大学学报》2016年第6期1-5,10,共6页Journal of Gansu Agricultural University

基　　金：国家“863”计划项目(2011AA10A211-1);国际原子能机构项目(16025/R);国家自然科学基金项目(31500617)

摘　　要：【目的】克隆和表达羊口疮病毒(ORFV)的B2L基因,进而纯化并分析其反应原性.【方法】根据GenBank已发表的ORFV(GQ328006)的B2L基因序列设计一对特异引物,以提取阳性临床样品的总DNA为模板,通过PCR方法获得ORFV的B2L基因,将该基因片段连接至原核表达载体pET-30a(+)上,获得重组质粒pET-30a(+)-B2L.将此重组质粒转化BL21(DE3)感受态细胞,挑取单克隆进行扩大培养,经IPTG诱导获得重组融合蛋白.用SDS-PAGE和Western-blotting对表达的目的蛋白进行分析.【结果】成功获得重组质粒pET-30a(+)-B2L,读码框正确.获得了43ku的表达产物,与预期目的蛋白大小相符;纯化后的目的蛋白能与ORFV阳性血清发生特异反应.【结论】成功对ORFV-B2L蛋白进行了原核表达,且纯化后蛋白具有良好的反应原性.[Objective] To clone and express the B2L gene of ORFV, and then purify and analyze the re- actogenicity. [Method] A pair of primer was designed according to ORFV B2L gene reported in Gen-Bank （GQ328006）, taking the DNA of this virus extracted from the pathologic materials as template and the tar- get gene was got by PCR. After purification, the product was cloned into pET-30a（q-） vector to ob-tain the recombinant plasmid pET-30a（q-）-B2L. After identification, the recombinant plasmid was trans-formed in- to BL21（DE3） competent cells and the single colony was picked to amplify and culture. After induction with IPTG, the recombinant fusion protein was expressed. The target protein was identified by SDS-PAGE and Western blotting after purification. [Result]The results showed that the B2L gene was successfully cloned into pET-30a（q-）, a protein was confirmed at position about 43 kDa by SDS-PAGE and could specif- ically react with ORFV positive serum identified by western-blot. [Conclusion]This ex-periment realize the prokaryotic expression of protein B2L,and the purified protein had good reactogenicity.

关 键 词：羊口疮病毒 B2L基因 原核表达 纯化 反应原性分析 

分 类 号：S852.6[农业科学—基础兽医学]