Ⅰ型流感病毒核蛋白(NP)基因在大肠杆菌中的表达与鉴定

Expressing and identification of Nucleoprotein(NP)Gene of Influenza Virus in E.coli

目的:以Ⅰ型流感病毒RNA为模板扩增NP全基因cDNA,并在大肠杆菌中进行表达与鉴定。方法:提取流感病毒RNA,以RT-PCR法扩增编码NP基因cDNA。将扩增所得NP基因克隆于pMD18-T载体,然后定向亚克隆NP基因于原核表达载体pET-28a的多克隆位点中,经酶切鉴定后,将含有NP基因的重组质粒命名为pET-NP。用pET- NP转化受体菌BL21,在大肠杆菌BL21中表达,用诱导剂IPTG以不同浓度进行诱导,并在不同诱导时间收集样品,用SDS-PAGE和Western blot检测表达产物。结果:经SDS-PAGE电泳分析证实NP基因获得了表达,并在终浓度为1 mM的IPTG诱导下,6h其表达量达到高峰,经Western-blot分析证实表达的重组核蛋白具有免疫反应活性,大小约为60kd。结论:流感病毒核蛋白基因编码区基因获得成功克隆和构建,为流感病毒诊断试剂及单抗的研制工作提供了基础材料。

Objective：To amplify the cDNA encoding complete nulcleoprotein（NP） gene from a strain of influenza virus Iand express and identify the NP gene in E.coli.Method：Extract RNA and amplify the cDNA encoding complete nulcleoprotein（NP） gene by RT-PCR. The amplified NP gene was cloned into PMD18-T plasmid. Then the NP gene was sub-cloned into prokaryotic expression vector PET- 28a and identified by restriction endonuclease digestion, The recombinant plasmid carrying NP gene was designed as PET-NP. After transformation of BL21 with PET-NP an expressed fusion protein was identified by SDS-PAGE in E.coli.BL21,after induced by IPTG with different concentration and collected samples in different time. The expressed protein was detected by western-blotting. Result： The cloned cDNA fragment covered the whole opening frame of NP gene and reached the extremity after induced by IPTG ,the recombinant NP protein is about 60 kd in size, showed a good immunogenicity. Conclusion： The NP gene of influenza virus was successfully cloned and expressed .The study laid a foundation of development of a novel immunological diagnostic kit for influenza virus.