人乳头瘤病毒16型早基因E6/E7编码区序列变异研究

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。

Variation of E6/E7 coding sequence derived from human papillomavirus type 16

Objective To obtain coding sequences of E6/E7 from human papillomavirus （HPV） type 16 and analysis the variation distribution to develop a novel molecular diagnosis method for cervical cancer. Methods Flow-through hybridization and gene chips were applied in identification of HPV subtype and screen HPV type 16 positive samples from cervical epithelium samples. HPV type 16 E6/E7 coding sequences were amplified by PCR with the specific primers. The PCR products were purified and the target sequences were cloned into pMD18-T vectors and fragments were determined by sequencing analysis. Variation analyses were performed using align tools including BLAST. Results HPV type 16 positive samples were screened through flow-through hybridization from 500 cervical epithelium samples. Three of them were selected for E6/E7 coding sequence amplification. Each reaction produced one fragment with size between 750 bp and 1 000 bp. Three coding sequences of E6/E7 of HPV type 16 were cloned and sequenced. The 3 sequences had been submitted to GenBank, the assigned accession numbers are： EU869316, EU869317 and EU869318. By BLAST, all above 99% identities with corresponding coding region of PPH16 in GenBank. Among three sequences eight kinds variances and four of them would cause coding amino acid change. Conelusion E6/E7 coding sequences and their variation profiles of HPV type 16 from three women＇s cervical epithelium has been obtained. The sequences and interrelated analysis result will be helpful to the further research of HPV carcinogenesis mechanism and development of novel molecular detective methods.