新城疫病毒F48E9株M和NP基因的原核表达  

Expression of M and NP genes of Newcastle disease virus F_(48)E_9 strain in Escherichia coli

作　　者：闻晓波[1] 吴芬芳[1] 闫丽辉[1] 曹殿军[1] 刘培欣[1] 刘春国[1] 孔宪刚[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室禽传染病研究室

出　　处：《中国兽医科学》2008年第3期181-185,共5页Chinese Veterinary Science

基　　金：国家自然科学基金项目(30371076);黑龙江省科学技术计划项目(GB02B505)

摘　　要：根据新城疫病毒(NDV)F48E9国内标准强毒株编码序列设计了2对特异性引物,分别用于扩增M和NP基因。经序列测定,将其克隆到原核表达载体pET-30a(+)上,转化E.coliBL21(DE3),筛选表达NDV F48E9株M蛋白和NP蛋白的菌株。IPTG诱导表达后,SDS-PAGE分析结果表明,NDV M蛋白在E.coliBL21(DE3)内以可溶形式存在,而NP蛋白以包涵体的形式表达。用纯化的M和NP蛋白免疫鸡制备超免疫血清,进行Western-blot分析;结果,它们可以与真核表达系统表达的M和NP蛋白以及NDV发生特异性反应,表明,原核表达系统表达的NDV M和NP蛋白具有良好的免疫原性和反应原性。According to the DNA sequence of Newcastle disease virus（NDV） F48 E9 strain in China, two pairs of primers were designed to amplify M and NP genes of NDV. The amplified M and NP genes were cloned into pMD18-T vector. The recombinants were sequenced and compared with the reference sequences. The M and NP genes were subcloned respectively into the prokaryotic expression vector pET-30a （＋）. After identification with PCR and digestion with restriction endonuclease,the recombinant prokaryo- tic expression vector harboring the M or NP genes was transformed into Escherichia coli competent cell BL21（DE3） to screen the recombinant bacterial strains expressing the M or NP genes. The positive bacterial strain was induced with IPTG and the expression of M or NP genes was confirmed by SDS-PAGE and Western-blotting. The results indicated that the recombinant M protein existed in the soluble proteins whereas NP protein existed in the form of inclusion bodies. The purified recombinant M and NP proteins were emulsified and injected into chicken to prepare hyperimmune sera separately. The anti-M or NP protein hyperimmune sera could react specifically with NDV or M or NP proteins expressed in mammalian cells. The results demonstrated that the recombinant M and NP proteins expressed in E. coli possessed good immunogenicity and reactivity.

关 键 词：新城疫病毒 M基因 NP基因 原核表达 

分 类 号：S852.659.5[农业科学—基础兽医学]