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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 an eukaryotic expression plasmid containing human papillomavirus type 58 (HPV58) E7 gene and to obtain the basic information of the gene structure of human papilloma virus (HPV58) infected human. Methods: HPV58 E7 DNA was amplified by HPV-specific primers and HPV58 E7 DNA was ligated with PBS-T vector to obtain recombinant HPV58 E7-PBS-T. After double enzyme digestion, E7 gene was compared with real The expression vector pcDNA3.1 was ligated and then identified by double enzyme digestion and sequencing. The homology of HPV58 E7 gene was analyzed by GenBank database. Results: Double enzyme digestion of recombinant DNA results showed that HPV58 E7 gene fragment and vector DNA size were consistent with expected calculation. BLAST analysis showed high homology with HPV58 E7 gene sequence in GenBank database Compared with the earliest report of HPV58 E7 gene by Japanese scholars in 1991, the difference of only one base was 125, ie, the mutation of CT. The corresponding amino acid was changed from pro to leu. CONCLUSION: The eukaryotic expression plasmid containing E58 gene of HPV58 has been constructed. Its gene sequence is highly homologous with the reported gene sequence. Whether its slight difference has any effect on function needs further study. The construction of the recombinant plasmids laid the foundation for the study of the function of HPV58 E7 gene and expressed protein.