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小鼠肝炎病毒N基因的原核表达和免疫活性初步分析
引用本文:周艳,胡建华,高诚,王宗耀.小鼠肝炎病毒N基因的原核表达和免疫活性初步分析[J].中国实验动物学报,2008,16(4):265-269.
作者姓名:周艳  胡建华  高诚  王宗耀
作者单位:[1]扬州大学比较医学中心,扬州225009; [2]上海市实验动物质量监督检验站,上海201203; [3]上海实验动物研究中心上海市计划生育科学研究所,上海201203
摘    要:目的对小鼠肝炎病毒(mouse hapetitis virus)A59毒株核蛋白(N)基因进行了克隆、表达和重组蛋白的免疫活性分析。方法根据GenBank公布的MHV-A59 N基因序列(AY700211),设计了一对特异性引物,通过RT-PCR扩增出N基因的主要抗原片段NS,将目的片段纯化后与pGEM-T-easy载体连接得到重组质粒pTN,双酶切回收目的基因片段克隆到原核表达载体pGEX-6p-1中,构建了重组质粒pGEX-NS,转化大肠杆菌BL21,用IPTG进行了诱导表达。对表达裂解物进行SDS-PAGE和Western-blotting验证。结果表达产物相对分子质量约56×103与预期相符;能与MHV阳性血清发生特异性反应,出现单一条带。结论原核表达的NP(S)蛋白有良好的抗原性,为检测小鼠肝炎病毒抗体的ELISA检测方法的研究奠定了基础。

关 键 词:小鼠肝炎病毒  N基因  克隆  原核表达

Prokaryotic Expression of Nucleoprotein Gene of Mouse Hepatitis Virus and Analysis of Its Immunological Activity
ZHOU Yan,HU Jian-hua,GAO Cheng,WANG Zong-yao.Prokaryotic Expression of Nucleoprotein Gene of Mouse Hepatitis Virus and Analysis of Its Immunological Activity[J].Acta Laboratorium Animalis Scientia Sinica,2008,16(4):265-269.
Authors:ZHOU Yan  HU Jian-hua  GAO Cheng  WANG Zong-yao
Institution:ZHOU Yan ,HU Jian-hua, GAO Cheng ,WANG Zong-yao ( 1. Comparative Medicine Center of Yangzhou University, Yangzhou 225009, China; 2. Shanghai Quality Monitoring Center for Laboratory Animals, Shanghai 201203, China; 3. Shanghai Laboratory Animal Research Center, Shanghai Institute of Planned Parenthood Research, Shanghai 201203, China)
Abstract:Nucleoprotein (N) gene of mouse hepatitis virus strain A59 (MHV-A59) was cloned, expressed and the immunological activity of the recombinant protein was analyzed. According to N gene sequence of MHV-A59 published in GenBank, a pair of primers was designed to amplify a part of N gene of MHV-A59. The PCR product was purified and cloned to pGEM-T-easy. Using enzyme digestion and T4 ligase, the target gene was further subcloned to prokaryotic expressing vector pGEX-6p-1 and after transfected into transformed E. coli strain BL21, it was induced by IPTG to express recombinant protein. The recombinant protein was identified by SDS-PAGE and Western-blot analysis. The result revealed that the protein had a molecular weight of 56 × 10^3 , which could be specifically recognized by anti-MHV antibody. Because of its good immunogenicity, the recombinant protein can be used as an antigen to detect anti-MHV antibody in sera of mice in ELISA.
Keywords:Mouse hepatitis virus(MHV)  Nucleoprotein(N) gene  Clone  Prokaryotic expression
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