口疮病毒AH-GY13株的分离鉴定及B2L基因的原核表达  

Isolation and identification of orf virus AH-GY13 strain and prokaryotic expression of its B2L gene

作　　者：宫晓华[1] 殷冬冬[1] 王瑞[1] 唐井玉 周晓雅[1] 梅楠[1] 张雪梅[1] 兰梦蝶 陈鹏[1] 王勇[1] 王桂军[1] 

机构地区：[1]安徽农业大学动物科技学院,安徽合肥230036

出　　处：《中国兽医科学》2016年第2期185-191,共7页Chinese Veterinary Science

摘　　要：为确定安徽地区某山羊场发生的疑似口疮（orf）的病原,经过临床诊断、PCR检测以及HeLa细胞培养等途径,确定其为口疮病毒（ORFV）,并命名为AH-GY13株。根据GenBank中公布的口疮病毒B2L囊膜蛋白基因序列,设计引物,用PCR方法扩增口疮病毒B2L基因,并将此基因序列及其推导的氨基酸序列与其他不同来源的ORFV分离株进行比较。结果显示,AH-GY13株与选取的10株毒株的核苷酸序列同源性在97.4%~99.3%之间,氨基酸同源性在97.9%~99.7%之间;进化树分析显示,此毒株与辽宁省分离株属于同一分支。将该基因定向插入到原核表达载体pET-32a（＋）中,构建原核表达载体pET-32a（＋）-B2L。转化BL21,经IPTG诱导,B2L基因获得表达,以包涵体的形式存在,通过优化诱导时间和IPTG的浓度,确定了表达B2L基因的最佳诱导条件：IPTG终浓度为1 mmol/L,37℃诱导4 h。Western-blot分析表明,表达产物具有良好的免疫活性。上述结果为进一步研究ORFV B2L囊膜蛋白的结构与功能奠定了基础。AH-GY13,an orf virus（ORFV） strain, was successfully isolated from materials of goats with typical clinical symptom through passaging in He La cells and the virus was identified as the orf virus based on PCR.Using a pair of specific primers designed according to the relevant nucleotide sequence from Gen Bank,the B2 L gene of ORFV was cloned,and the nucleotide sequences and amino acids sequences were compared with other 10 ORFV strains. The homology of B2 L gene was from 97.4% to 99.3%,and the homology of the deduced amino acids sequence was from 97.9 % to 99.7 %.The phylogenetic trees showed that AH-GY13 strain was closed to Liaoning strain. The B2 L gene was cloned into pET-32a（＋） to get a prokaryotic recombinant plasmid pET-32a（＋）-B2 L.The target gene was successfully expressed in the host cell BL21. The recombinant fusion proteins were highly expressed in BL21 in the form of inclusion bodies with IPTG induction. The expression was optimized with proper inducing conditions of 1mmol/L IPTG and 4 hours at 37 ℃ induction. Western-blot analysis showed that the recombinant protein had a good reactive ability against ORFV positive serum. The studies provided fundamental data and materials for the further study on the structure and function of structural proteins of ORFV.

关 键 词：口疮病毒 分离鉴定 B2L基因 序列分析 原核表达 蛋白质免疫印迹 

分 类 号：S852.659.1[农业科学—基础兽医学]