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目的获取人乳头瘤病毒16型早基因E6/E7的编码区序列并进行变异分析,为新型分子诊断方法的研发提供目标序列。方法通过导流杂交基因芯片技术获取人乳头瘤病毒16型感染宫颈脱落细胞。设计特异性引物,扩增人乳头瘤病毒16型早基因E6/E7的编码区序列,克隆后测序并对序列进行变异分析。结果通过导流杂交基因芯片技术进行宫颈脱落细胞的分型检测,获得了人乳头瘤病毒16型感染阳性标本;选择其中3份阳性标本核酸为模板,通过特异性引物均成功扩增出大小介于750 bp和1 000 bp之间的目的序列;分别克隆到T载体上并测序,得到3条人乳头瘤病毒16型早表达基因E6/E7的编码区序列,克隆并测序后向GenBank核酸数据库提交获收录,登录号分别为EU869316、EU869317和EU869318;经BLAST分析,3条序列与GenBank序列PPH16(Accession:K02718)的E6/E7编码区序列一致性均为99%,且存在8种碱基置换,其中4种为同义突变,另4种则可引起所编码的相应氨基酸残基改变。结论本研究得到了与HPV高危型16型PPH16高度相似的E6/E7编码序列,为HPV致病机制和新型分子检测方法研究奠定了基础。
Objective To obtain the coding sequence of human early-stage HPV16 E6 / E7 gene and carry out mutation analysis to provide the target sequence for the development of novel molecular diagnostic methods. Methods Human papillomavirus (HPV) 16 type cervical exfoliated cells were obtained by flow-through hybridization gene chip technique. Specific primers were designed to amplify the coding sequence of human papillomavirus type 16 early gene E6 / E7. The sequences were cloned and sequenced. Results The HPV type 16 positive samples were obtained by flow cytometry and microarray technology. Three positive samples were selected as template, and specific primers were used to amplify the size of the cells Were cloned into T vector and sequenced respectively. Three coding regions of HPV16 E6 / E7 gene were obtained, cloned and sequenced, and then sequenced to GenBank database The accession numbers were EU869316, EU869317 and EU869318, respectively. According to BLAST analysis, the sequences of the three sequences were 99% identical to the E6 / E7 coding region of GenBank accession PPH16 (accession: K02718), and there were 8 bases Four of them are synonymous mutations, and the other four can cause the corresponding amino acid residues encoded change. Conclusion The E6 / E7 coding sequence, which is highly similar to HPV type 16 PPH16, was established in this study, which laid the foundation for the study on the pathogenesis of HPV and new molecular detection methods.