奶牛β-干扰素在大肠杆菌中高效表达和生物活性分析  

High Expression of Interferon-beta from Bovine in E. coli and its Antiviral Activity Analysis

作　　者：刘颖[1,2] 刘华兰[1,2] 杨利国[3] 陈焕春[1,2] 郭爱珍[1,2] 

机构地区：[1]湖北省华中农业大学农业微生物学国家重点实验室,武汉430070 [2]湖北省华中农业大学动物医学院预防兽医学省重点实验室,武汉430070 [3]湖北省华中农业大学动物科技学院,武汉430070

出　　处：《中国畜牧兽医》2010年第1期123-128,共6页China Animal Husbandry & Veterinary Medicine

基　　金：国家973项目(2006CB504401);湖北省自然基金重大项目(2008CDA073);艾滋病和病毒性肝炎等重大传染病防治科技重大专项(2009ZX10602-14;2008ZX10301)

摘　　要：提取经植物血凝素诱导培养的中国健康奶牛外周血淋巴细胞总RNA,应用RT-PCR方法扩增出奶牛β-干扰素成熟蛋白基因并将其克隆到pMD18-T载体上。测序结果表明,扩增片段为奶牛β-干扰素成熟蛋白序列,与GenBank上发表的干扰素序列同源性为100%。将其重组到原核表达载体pET32a(+)上,并在大肠杆菌BL21中实现了高效表达。表达产物以His-Tag融合蛋白的形式存在,表达量约占细菌总蛋白的40%。用镍亲和层析法对蛋白进行纯化,并利用VSV-MDBK/IBRV细胞系统分析其生物活性。重组奶牛β-干扰素抗病毒活性分别约3.16×105U/mL,7.5×104U/mL。结果表明,重组奶牛β-干扰素特异性好,而且抗病毒活性比较稳定。本研究为研制和开发重组奶牛β-干扰素类生物制品研发奠定了基础。Total RNA was isolated from bovine peripheral blood lymphocytes,which were stimulated with PHA.Then the bovine IFN-β cDNA was amplified by reverse transcription chain reaction.The amplified fragment was cloned into vector pMD18-T and then sequenced.The result indicated that the cloned gene was mature bovine IFN-β gene,which had the identities of 100% with the BoIFN-β gene published in the GenBank.Subcloned into the pET32a（＋） expression vector and expressed in E.coli host BL21 with IPTG induction.To express the product of His-Tag fusion protein existed in the form of the expression of the total bacterial proteins account for about 40%.By nickel affinity chromatography purification of proteins,and using VSV-MDBK/IBRV cell system analysis its biological activity,reorganization BoIFN-β anti-virus activeness distinction recombinant BoIFN-β antiviral activity were about 3.16×10^5,7.5×10^4 U/mL.The result indicates that the reorganization BoIFN-β specificity is good,moreover anti-viral activeness is quite stable.This research to develop and develops the reorganization BoIFN-β class biological preparations research and development to lay the foundation.

关 键 词：奶牛 Β-干扰素 融合蛋白 抗病毒活性 

分 类 号：Q78[生物学—分子生物学]