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新城疫病毒F48E9株M和NP基因的原核表达
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闻晓波1，2，吴芬芳1，闫丽辉1，曹殿军1*，刘培欣1，刘春国1，孔宪刚1
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（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，黑龙江
哈尔滨150001； 2.黑龙江八一农垦大学 动物科技学院，黑龙江 大庆163319）
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摘要：
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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.coli BL21（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.coli BL21（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。 |
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关键词：
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新城疫病毒；M基因；NP基因；原核表达 |
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中图分类号：S 852.659.5 文献标识码：
A 文章编号：1673-4696(2008)03-0181-05 |
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Expression of M and NP genes of Newcastle disease virus
F48E9 strain in Escherichia coli |
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WEN Xiao-bo1,2,WU Fen-fang1,YAN
Li-hui1,CAO Dian-jun1,LIU Pei-xin1，
LIU Chun-guo1,KONG Xian-gang1 |
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(1.National Key Laboratory of Veterinary Biotechnology/Division of Avian Infectious Diseases，Harbin Veterinary
Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150001,China;2.College of Animal Science
and Technology,Heilongjiang August First Land Reclamation University,Daqing 163319,China )
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Abstract: According to the DNA sequence of Newcastle disease virus(NDV) F48E9 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 bacte-rial 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. |
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Key words: Newcastle disease virus;M gene;NP gene;prokaryotic expression |
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