甲型流感病毒核蛋白基因的真核质粒的构建及其表达  

Construction of eukaryotic expressing plasmids for NP of influenza A virus and their transient expression in Hela cells

作　　者：冯润[1] 孙坚[1] 王农荣[1] 杨斌[1] 何士勤[1] 

机构地区：[1]南昌大学第四附属医院,南昌330003

出　　处：《江西医药》2010年第1期9-11,共3页Jiangxi Medical Journal

基　　金：国家自然科学基金资助项目(编号:C190201)

摘　　要：目的构建甲型流感病毒A/PR/8/34(H1N1)核蛋白(nucleoprotein,NP)基因的真核表达载体,转染Hela细胞,并用甲型流感病毒检测试剂盒(FluARapidTestKit)检测其表达情况。方法用特异引物从甲型流感病毒cDNA中采用PCR技术克隆出NP基因,将其插入到真核表达质粒pcDNA3.1(+)。酶切、PCR、测序鉴定后,构建好的pcDNA3.1(+)/NP用脂质体转染法转染到Hela细胞,通过甲型流感病毒检测试剂盒检测其蛋白表达。结果克隆出一个核苷酸长度为1432bp的基因,和A/PR/8/34(H1N1)(GenBank/NCB,GI:8486129)有98%的同源性,转染Hela细胞后用甲型流感病毒检测试剂盒检测到24h、48h、72h细胞内外的蛋白表达。结论成功构建甲型流感病毒A/PR/8/34(H1N1)核蛋白基因的真核表达质粒,为进一步研究理化因素对该基因的影响、基因的结构和功能打下良好的基础。Objective To construct influenza A virus（A/PR/8/34（H1N1） NP eukaryotic expressing plasmids and using Flu A Rapid Test Kit study their expression in Hela cells.Methods The NP gene was cloned by PCR from cDNA of influenza A virus and then inserted into the eukaryotic expression vector pcDNA3.1 （＋）.After identification with restriction enzyme digestion,PCR assay and sequencing ,the recombinant plasmid was transfected into Hela cells with lipofectamineT 2000 induction.The transient expression of target protein was observed by Flu A Rapid Test Kit.Results The NP gene fragment at a length of 1432bp was amplified ,which was consistent with that expected.The sequence of amplified NP gene was identical to that reported in GenBank.Flu A Rapid Test Kit proved the expression of influenza A virus NP protein.Conclusion The experiment is a success in the construction of eukaryotic ex- pressing plasmids for NP gene, thus providing a basis for further probing into the mechanism of virus infection and physical and chemical factors which can affect the gene.

关 键 词：甲型流感病毒 核蛋白 真核载体 

分 类 号：R-33[医药卫生]