兔出血症病毒YL株衣壳蛋白VP60基因在原核系统中的高效表达  

High expression of capsid protein (VP60) gene from rabbit hemorrhagic disease virus YL strain in E.coli

作　　者：李传山[1] 任力刚[1] 李维英[2] 杨增岐[3] 郭蔼光[1] 

机构地区：[1]西北农林科技大学生命科学学院,陕西杨凌712100 [2]西北农林科技大学林学院,陕西杨凌712100 [3]西北农林科技大学动物科技学院,陕西杨凌712100

出　　处：《西北农林科技大学学报（自然科学版）》2007年第11期24-28,共5页Journal of Northwest A&F University(Natural Science Edition)

摘　　要：为了在原核系统中高效表达兔出血症病毒(RHDV)衣壳蛋白VP60,应用RT-PCR方法从兔出血症病毒(RHDV)西北分离株YL中克隆出了衣壳蛋白VP60基因并测序,结果显示,YL株VP60基因全长1740 bp,GC含量为55.3%,已在GenBank中注册(登录号为DQ530363),VP60基因与中国RHDV最早期分离株WX84的VP60基因核苷酸序列同源性为93.0%,氨基酸序列同源性为95.8%。构建成功YL株VP60基因原核表达载体VP60-pET32a,转化到大肠杆菌表达菌株BL21(DE3)中。1 mmol/L IPTG诱导表达,SDS-PAGE检测结果显示,VP60融合蛋白分子质量约为75 ku,其表达量占总菌体蛋白的39.8%。Western blot检测结果表明,VP60融合蛋白可以和RHDV抗血清发生特异反应。本研究结果显示,兔出血症病毒在干旱半干旱条件下的遗传变异和免疫学特征无较大变化。The aim of this paper was to study the high expression of rabbit hemorrhagic disease virus（RHDV）capsid protein VP60 in E.coli.The capsid protein VP60 gene of RHDV YL Strain was cloned by employing the method RT-PCR and then sequenced.YL Strain was the first RHDV strain being sequenced in the northwest of China.The result showed that the nucleotide sequence of VP60 gene was 1 740 bp.The sequence had been submitted to GenBank（the accession number is DQ530363）.Compared with the earliest Chinese RHDV isolated WX84,the homology of VP60 gene was 93.0% in nucleotide sequence and 95.8% in amino acid sequence and the proportion of GC was 55.3%.The prokaryote expression plasmid VP60-pET32a of VP60 gene was successfully constructed,and then inserted into the expression E.coli strain BL21（DE3）.The target protein was highly expressed under the induction with 1 mmol/L IPTG.SDS-PAGE analysis showed that the molecular weight of the recombinant VP60 protein was about 75 ku.The proportion of the recombinant VP60 protein reached 39.8% of the total bacterial proteins.Western blot analysis demonstrated that the recombinant VP60 had a specific reaction with antiserum of RHDV.

关 键 词：兔出血症病毒(RHDV) 衣壳蛋白(VP60)基因 原核表达 WESTERN BLOT 

分 类 号：S852.659.6[农业科学—基础兽医学] Q786[农业科学—兽医学]