构建甲型流感病毒核蛋白融合基因三种方法的比较研究

Comparison of three methods in construction fusion gene of influenza A virus Nucleoprotein

目的比较构建甲型流感病毒核蛋白NP融合基因的三种方法，以获取成功构建含绿色荧光蛋白融合基因的方法。方法PCR扩增甲型流感病毒核蛋白NP基因片段，运用三种不同的方法构建NP表达绿色荧光蛋白的融合基因：①含限制性内切酶酶切位点的NP基因片段与pEGFP-N1克隆构建融合基因；②NP基因片段与pMD19-TVector连接、TA克隆后获得限制性内切酶酶切位点，经pEGFP—N1克隆构建融合基因；③含限制性内切酶酶切位点的NP基因片段与pMD19-TSimpleVector连接、TA克隆后经pEGFP—N1克隆构建融合基因。结果含限制性内切酶酶切位点的NP基因片段TA克隆后经pEGFP—N1克隆，获得高效且稳定的绿色荧光核蛋白融合基因，第一、第二两种方法构建绿色荧光核蛋白融合基因成功率低。结论含限制性内切酶酶切位点的NP基因片段经TA克隆与pEGFP-N1连接克隆，成功构建了绿色荧光蛋白的甲型流感病毒核蛋白NP融合基因pEGFP-N1-NP，该方法高效且重复性好。该研究为进一步了解NP蛋白的生物功能及甲型流感病毒的致病机理奠定了基础。

Objective To develop a best method of constructing influenza NP fusion gene containing enhanced green fluorescent protein （EGFP）. Methods The full-length NP gene of influenza A was amplified by RT-PCR and was inserted into an eukaryotic expression vector pEGFP-N1 in order to construct a fusion gene of pEGFP-N1-NP using three different methods. Method one, NP gene containing restriction endonucleases and pEGFP-N1 were both digested using the same restrict enzymes and ligated, yielding the fusion gene of pEGFP-N1-NP. Method two, NP gene was cloned into pMD19-T Vector to construct a plasmid of pMD19-T-NP. The pMD19-T-NP cloned into pEGFP-N1 to construct the fusion gene of pEGFP-N1-NP; Method three, NP gene containing restriction endonucleases was cloned into pMD19-T Simple Vector to construct a plasmid of pMD19-T-NP. The pMD19-T-NP cloned into pEGFP-N1 to construct the fusion gene of pEGFP-NI-NP. Results The fusion genc of recombinant eukaryotic expression vector pEGFP-N1-NP was successfully constructed by using method three. Conclusions The full-length NP gene is obtained and its fusion gene of recombinant eukaryotic expression plasmid is successfully constructed. This study provides foundation for further understanding the biological function of NP protein and the mechanism of diseases induced by influenza A virus.