H5N1亚型禽流感病毒聚合酶酸性蛋白真核表达载体的构建与表达

目的构建甲型禽流感病毒H5N1亚型聚合酶酸性蛋白(PA)的真核表达载体,并表达其编码蛋白PA。方法采用RT-PCR法扩增PA基因,克隆至pMD18-T载体中构pMD18-T-PA质粒。双酶切pMD18-T-PA质粒与pXJ40-HA质粒后,胶回收并连接目的片段,构建真核表达载体pXJ40-HA-PA,鉴定后转染293T细胞。采用免疫印迹法(Western blot)鉴定重组PA蛋白的表达。结果成功构建了禽流感病毒H5N1亚型PA基因的真核表达载体,并在真核细胞中成功表达出分子量为75kD的重组蛋白。结论成功构建禽流感病毒H5N1亚型PA基因的真核表达载体,为后期进一步研究PA蛋白的功能奠定了基础。

Construction of eukaryotic expression vector of avian influenza virus H5N1 PA protein and expression of PA recombinant protein in mammalian eukaryotic cells

Objective To construct an eukaryotic expression vector of avian influenza virus（AIV）H5N1PA gene and express PA recombinant protein in 293Tcells.Methods AIV PA gene was amplified by RT-PCR and cloned into pMD18-T vector to construct pMD18-T-PA plasmid by T-A cloning.Double digestion of pMD18-T-PA and pXJ40-HA plasmids,followed by ligation of gel-purified fragments constructed pXJ40-HA-PA plasmid,which was verified by PCR,double digestion and sequencing analysis.pXJ40-HA-PA was transfected into 293Tcells.Expression of recombinant PA protein was confirmed by Western Blot analysis.Results Eukaryotic expression vector pXJ40-HA-PA was successfully constructed.A 75kD recombinant protein was expressed in mammalian eukaryotic cells,which was confirmed by Western Blot analysis.Conclusion The successful construction of AIV PA gene eukaryotic expression plasmid should lay a foundation for further research on PA gene function.