HPV58在宫颈癌组织中的感染分析及其转化基因的克隆(英文)

Prevalence analysis of human papilloma virus 58 and cloning of its transforming genes from biopsy samples of cervical cancer

目的分析中国陕西宫颈癌组织中HPV58的感染情况并克隆其E6、E7主要转化基因。方法采用HPVGP5+/GP6+通用引物PCR扩增58例宫颈癌组织DNA粗提物,继之将荧光偏振技术与模板指导的末端延伸反应(TDI-FP)结合,应用探针杂交延伸反应检测HPV58。用特异性引物随机从1例HPV58阳性标本中PCR扩增HPV58E6、E7基因,将其分别与pGEM-TEasy载体连接,所获得的重组质粒经内切酶消化和测序鉴定。结果58例宫颈癌标本中10例(17.24%)为HPV58阳性,克隆获得的HPV58E6、E7重组质粒经BLAST分析证实分别含有HPV58完整的E6、E7基因,与GenBank收录的标准株比较,E6、E7基因分别有5个和2个硷基发生变异,可导致相应的蛋白质氨基酸序列中分别有4个和1个氨基酸替换。结论中国陕西人宫颈癌组织中HPV58感染较为多见,且HPV58E6和E7转化基因的硷基序列与标准株有所差异。本研究为下一步HPV58相关肿瘤诊断试剂及疫苗的研制奠定了基础。

【Objective】To analyze the prevalence of human papilloma virus （HPV） 58 and clone its E6 and E7 transforming genes from biopsy samples of cervical cancer patients living in Shanxi, China. 【Methods】 HPV 58 was detected from specimens of cervical cancer by the novel method based on template-directed dyeterminator incorporation assay with fluorescence polarization detection （TDI-FP） following GP5＋/GP6＋ PCR, and HPV 58 E6 and E7 genes were randomly amplified from one HPV 58-positive sample by PCR with specific primers. The HPV 58 E6 and E7 PCR products were respectively cloned into pGEM-T Easy vector. The recombinant plasmids were identified by endonuclease restriction digestion and sequencing. 【Results】Among the 58 cervical cancer biopsy tissues, 10 （17.24%） were HPV 58-positive. E6 and E7 genes of HPV 58 were amplified from one HPV 58-positive sample and ligated with pGEM-T Easy vector. A BLAST search confirmed that the recombinant plasmids contained the complete E6 and E7 genes of HPV 58. In comparison with the prototype accepted by GenBank, there were respectively five and two point mutations in HPV 58 E6 and E7 nucleotide sequences, which might result in four and one amino acid changes in the deduced amino acid sequences of HPV 58 E6 and E7 proteins. 【Conclusion】HPV 58 is relatively prevalent in cervical cancer tissues of Chinese patients from Shanxi province. Some nucleotide sequences in HPV 58 E6 and E7 genes randomly cloned from one HPV 58-positive sample are different from the prototype. The research in this report may provide a ready source for future studies on the diagnosis and vaccine designs of HPV58-associated tumors.