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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 infection of HPV58 in cervical cancer in Shaanxi Province of China and clone the major transformation genes of E6 and E7. Methods 58 samples of cervical cancer tissues were amplified by PCR using HPV GP5 + / GP6 + universal primers. Then the fluorescence polarization technique was combined with the template directed end extension reaction (TDI-FP), and HPV58 was detected by probe hybridization extension reaction. HPV58E6 and E7 genes were amplified by PCR from HPV58 positive samples randomly using specific primers. The recombinant plasmids were respectively digested with pGEM-T Easy vector and identified by sequencing. Results HPV58 was positive in 10 of 58 cervical cancer samples (17.24%). The HPV58E6 and E7 recombinant plasmids obtained by cloning were confirmed by BLAST analysis to contain the complete E6 and E7 genes of HPV58 respectively. Compared with the standard strains of GenBank, E6 and E7 Mutations of 5 and 2 bases in a gene, respectively, result in 4 and 1 amino acid substitutions in the corresponding protein amino acid sequence, respectively. Conclusion HPV58 infection is more common in human cervical cancer tissues in Shaanxi Province of China, and the nucleotide sequences of HPV58E6 and E7 transformed genes are different from the standard strains. This study lays the foundation for the further development of HPV58 related tumor diagnostic reagents and vaccines.