奶牛α-干扰素克隆表达和生物学活性分析  

Cloning and expression of interferon-alpha(IFN-α) from bovine 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]华中农业大学农业微生物学国家重点实验室,湖北武汉430070 [2]华中农业大学动物医学院预防兽医学省重点实验室,湖北武汉430070 [3]华中农业大学动物科技学院,湖北武汉430070

出　　处：《中国兽医学报》2010年第11期1495-1499,共5页Chinese Journal of Veterinary Science

基　　金：国家"973"计划资助项目(2006CB504401);湖北省自然科学基金重大项目(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我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α-干扰素(BoIFN-α)成熟蛋白基因并将其克隆到pMD18-T载体上,测序结果表明,扩增片段为牛α-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。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α-干扰素对VSV的抗病毒活性为4.26×105U/mL,对IBRV的抗病毒活性为8.91×104U/mL。结果表明,重组奶牛α-干扰素特异性好,而且抗病毒活性比较稳定。Total RNA was isolated from bovine peripheral blood lymphocytes,which were stimulated with PHA.Then the BoIFN-α 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 clone was a mature BoIFN-α gene,which had the identities of 100 % with the BoIFN-α gene published in the GenBank.It was 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.By nickel affinity chromatography purification of proteins,and using VSVMDBK/IBRV cell system,its biological activity was analysed.Recombinant BoIFN-α antiviral activity for VSV were about 4.26×105 U/mL,8.91×104 U/mL.The result indicated that the reorganization BoIFN-α specificity is good,moreover anti-viral activeness is quite stable.

关 键 词：奶牛 Α-干扰素 融合蛋白 抗病毒活性 

分 类 号：S852.4[农业科学—基础兽医学]