牛病毒性腹泻病病毒Changchun184株E2基因的克隆及在大肠杆中的高效表达  

Cloning of the E2 gene of bovine virol diarrhea virus strain of Changchun184 and its expression in Escherichia coli

作　　者：徐兴然[1,2] 涂长春[1] 余兴龙[1] 肖昌[1,2] 张青婵[1] 张丽颖[1] 查云峰[1] 史子学[1] 

机构地区：[1]军事医学科学院军事兽医研究所 [2]西南农业大学,重庆北碚400716

出　　处：《中国预防兽医学报》2005年第2期98-101,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家"973"计划资助课题 (N0 G19990 1190 5 )

摘　　要：根据GenBank已发表的多个BVDV_1序列的比较分析结果设计引物 ,应用RT_PCR及套式PCR克隆得到包含Changchun184 (CC_184 )株E2基因的片段F2 /R2 ,克隆、测序分析结果表明该片段大小为 1391bp ,软件分析结果表明CC_184株E2基因长度为 112 2bp(GenBankaccessionnumber:AF5 2 6 380 )。通过基因操作构建得到表达完整E2蛋白和去除E2蛋白C_端疏水区的重组质粒 pET2 8a_BE2和 pET2 8a_BE2m ,转化大肠杆菌并诱导目的蛋白表达 ,SDS_PAGE检测结果表明重组菌能表达目的蛋白 ,其表达量分别占菌体总蛋白的 6 2 5 %和 35 7%。The major antigenic coding gene E2 of the earliest Chinese isolates of bovine viral diarrhea virus,bovine isolate Changchun184(CC_184) was obtained by use of reverse transcription polymerase chain reaction (RT_PCR)and nest_PCR.Sequencing result showed that the E2 gene of CC_184 has 1 122 nucleotides in length,which encode 374 amino acid residues.Two prokaryotic expression plasmids,pET28a_BE2 and pET28a_BE2m,which contains the integrity of E2 and truncated E2 gene without the trans_membrane region,were contains the integrity of E2 and truncated E2 gene without the trans_membrane region,were obtained and transformed into E.coli.,followed by inducing expression of the target protein,respectively.The SDS_PAGE and Western_blot result showed that the recombinant host can express the E2 protein.The lamina scan results demonstrate that the E2 protein shared the 6.25 % and 35.7 % proportion of the gross protein of the host cell,respectively.

关 键 词：牛病毒性腹泻病病毒 E2基因 克隆 表达 

分 类 号：S852.65[农业科学—基础兽医学] Q786[农业科学—兽医学]