羊口疮病毒陕西分离株B2L基因的克隆、序列分析及原核表达  

Cloning,Sequence Analysis and Prokaryotic Expression of B2L Gene of Orf Virus Shaanxi Strain

作　　者：何亚鹏[1] 张琪[1] 庞文静[1] 徐丽美[1] 付明哲[1] 许信刚[1] 

机构地区：[1]西北农林科技大学动物医学院,陕西杨凌712100

出　　处：《动物医学进展》2016年第12期19-23,共5页Progress In Veterinary Medicine

基　　金：陕西省重点产业创新链项目(2016KTZDNY02-06);陕西省农业科技创新与攻关项目(2016NY-092);西北农林科技大学试验示范站(基地)科技成果推广项目(TGZX2015-32)

摘　　要：为了对羊口疮病毒(ORFV)陕西分离株B2L基因进行克隆、序列分析及原核表达,根据GenBank已登录的ORFV(JN565694.1)B2L基因序列,设计合成一对特异性引物,应用PCR技术扩增ORFV B2L基因,并将目的基因连接到原核表达载体pET-28a中,成功构建重组质粒PET28a-B2L,转化大肠埃希菌BL21感受态细胞进行诱导表达,并进行SDS-PAGE和Western blot分析。结果表明,成功克隆了ORFV陕西分离株B2L全基因序列,核苷酸序列分析表明,陕西分离株B2L基因与国内外已报道的ORFV毒株核苷酸同源性超过97.3%,氨基酸同源性超过95.0%。重组菌经IPTG诱导后成功表达分子质量约为42ku的重组蛋白,该蛋白能与羊口疮阳性血清特异性结合,具有良好的反应原性。研究结果为ORFV的分子生物学特性研究提供资料,为进一步研制ORFV单克隆抗体及抗体检测ELISA试剂盒奠定了基础。According to the published sequence of B2L gene of ORFV（JN565694.1）,a pair of specific primers were designed and synthesized for cloning, sequence analysis and prokaryotic expression of B2L gene of Orf virus Shannxi strain. The B2L gene was amplified by PCR from Orf virus of Shaanxi strain. B2L gene sequence was analyzed and cloned into pET-28a vector. The prokaryotic expression plasmid pET-B2L containing B2L gene was successfully constructed. The expression of recombinant plasmid pET-B2L in E. coli BL21competent cells was induced and detected by SDS-PAGE and Western blot. B2L gene sequence was successfully obtained, Nucleotide sequence analysis showed that B2L gene of Shaanxi strain is similar to some domestic and foreign strains. Nucleotide sequence similarity is more than 97.3% ,amino acid sequence similarity is more than 95.0%. SDS-PAGE analysis showed that 42 ku recombinanti protein was expressed by IPTG induction. Western blot analysis showed that the recombinant fusion protein had good reactiongenicity. The result provided the reference for studying molecular biology characteristics of ORFV. Furthermore,the B2L protein can be used to prepare monoclonal antibodv and ELISA detection kit.

关 键 词：羊口疮病毒 B2L基因 克隆 序列分析 原核表达 

分 类 号：S852.659.1[农业科学—基础兽医学]