小鼠肝炎病毒N基因的原核表达和免疫活性初步分析

Prokaryotic Expression of Nucleoprotein Gene of Mouse Hepatitis Virus and Analysis of Its Immunological Activity

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。

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.