广州东部HPV基因型分布及其优势基因型E6／E7核酸序列分析

目的研究广州东部人乳头瘤病毒基因型分布特征，并获取优势基因型早基因E6／E7的核酸序列，进行变异分析，为本课题组下一步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进行宫颈脱落细胞标本的人乳头瘤病毒基因分型检测，分析本地区基因型分布特征，确定优势基因型；根据GenBank序列设计特异性引物，用于扩增优势基因型的早基因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标本的分型检测，检测了536份宫颈脱落细胞标本，HPV阳性占27.2％（146／536），其中多基因型感染占29.5％（43／146），高危型感染中52型为优势基因型，频数达23.3％（37／159）。HPV感染与52型HPV感染的年龄分布显示低年龄组（即A组，≤25岁）妇女最高。3份52型HPV感染阳性标本的核酸模板，通过特异性引物均成功扩增出大小接近750bp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52型早表达基因E6／E7的编码区序列，克隆后测序并对序列进行变异分析；3条序列经BLAST分析，与GenBank数据库HPV52型序列（Accession：X74481）的E6／E7编码区序列一致性均为99％，三条序列（EU924143、EU924144、EU924145）存在6种碱基置换，其中2种可引起所编码的相应氨基酸残基改变。结论本研究可确认广州东部地区妇女宫颈感染HPV中A组（17-25岁）具有最高的感染率；宫颈感染HPV的优势基因型为52型；我们通过克隆策略得到了与HPV高危型52型X74481高度相似的E6／E7编码序列，为课题组后续进行的HPV致病机制和新型分子检测方法研究提供了重要基础。

Distribution of the genotypes of human papillomavirus in east Guangzhou and coding sequences of E6/E7 of the dominant genotype

The present study was performed to investigate the distribution of human papillomavirus（HPV） in cervix of women in east Guangzhou, and to obtain coding sequences of E6/E7 from the dominant genotype of HPV and to analyze the variances inside the coding region. Flow-through hybridization and gene chips were applied in HPV subtype identification of cervical epithelium samples in this study. The distribution pattern of HPV was achieved through data comparison among age groups and genotype groups. It was found that 536 cervical epithelium samples were tested with a HPV positive rate of 27.2%（146/ 536）, in which 29.5%（43/146） were due to infection with multiple genotypes. HPV type 52 was the dominant genotype which occupied 23.3%（37/159） frequencies of 14 genotypes of high-risk HPV. Young women（A group： 17 25 years old）showed highest rate of HPV infection and HPV type 52, type 58 infection as well. We designed specific primers to amplify the E6/E7 genomic DNA fragments of dominant genotype HPV through PCR. Three of HPV type 52 positive samples were selected for E6/E7 coding sequence amplification. Each reaction produced one fragment near 750 band. Coding sequences of E6/E7 of HPV type 52 were identified through cloning with vectors pMD18 T and sequencing. By BLAST, we found the three sequences all above 99% identities with relevant coding sequence of X74481 of GenBank. Among the three sequences we found six kinds of va- riances and two of them would cause coding amino acid changes. According to our results, young women between 17 and 25 years old own highest HPV infection rate in east Guangzhou. HPV type 52 was the dominant genotype other than type 16 in most reports. The E6/E7 coding sequences of HPV type 52 and their variance profiles have been obtained.