【摘要】 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。更多还原

【Abstract】 Objective:To amplify the cDNA encoding complete nulcleoprotein(NP)gene from a strain of influenza virus land 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.更多还原