人乳头瘤病毒58型E7基因重组真核表达质粒的构建与序列分析

Construction of Recombinant Eukaryotic Expression Plasmid Containing Human Papillomavirus 58 E7 Gene and Sequence Analysis

目的:构建含人乳头瘤病毒58型(HPV58)E7基因的真核表达质粒,获得HPV58感染人后病毒基因结构的基本信息。方法:用HPV型特异性引物扩增出HPV58型E7 DNA,将HPV58 E7 DNA与PBS-T载体连接,获得克隆重组体HPV58 E7-PBS-T,经双酶切鉴定后,将E7基因与真核表达载体PCDNA3.1连接,然后经双酶切、测序鉴定,通过GenBank的数据库分析软件对HPV58型E7基因进行同源性分析。结果:双酶切重组DNA结果显示,重组体中HPV58型E7基因片段和载体DNA大小与预期计算一致,经BLAST比对,与GenBank数据库中的HPV58型E7基因序列具有高度的同源性,其中与1991年日本学者最早报道HPV58型E7基因相比,仅125位一个碱基的差异,即C-T的突变;推导相应的氨基酸由脯氨酸(pro)变为亮氨酸(leu)。结论:构建了含HPV58型E7基因的真核表达质粒,其基因序列与报道的基因序列高度同源,其微小差异对功能有无影响有待探讨。该重组质粒的构建为HPV58型E7基因及表达蛋白的功能研究奠定基础。

Objective：To construct eukaryotic expression plasmid containing human papillomavirus 58 E7 gene,and to obtain basic information of HPV58 genetic structure after infecting human.Methods：HPV58 E7 gene was amplified by specific primer,and then cloned into PBS-T vector.The recombinant plasmid,HPV58 E7-PBS-T,was digested by double enzymes.Subsequently,E7 gene was cloned into eukaryotic expression vector PCDNA3.1.The recombinant plasmid was identified by double enzyme digestion and sequencing.HPV58 E7 sequence was analyzed with the GenBank data analysis software.Results：The enzyme digestion and sequencing results showed that the HPV58 E7 gene in the recombinat plasmid showed highly homology with the original HPV58 E7 gene sequence in GenBank.Only one point mutation was found on the 125 basic site which was C-T mutation compared with the HPV58 E7 gene early reported by Japanese scholars in 1991,resulting in change of the corresponding amino acid from proline to leucine.Conclusion：The recombinant eukaryotic expression plasmid containing HPV58 E7 gene was successfully constructed for the future study of HPV58 E7 gene and protein function,and the gene sequence is highly homologous with the reported gene sequence,except that its minor impact of mutation on functional differences remains for further investigation.