人乳头瘤病毒16型E6和E7基因特征分析

目的 对北京15例宫颈癌病变组织中的人乳头瘤病毒(HPV)16型E6和E7基因进行扩增及序列测定,分析E6和E7基因突变特征,并探讨宫颈癌病变中HPV16的感染情况.方法 自行设计HPVl6型E6和E7基因扩增引物,采用PCR法扩增15例宫颈癌组织中HPV16 E6和E7片段,将PCR产物克隆到TA载体,进行序列测定.通过Sequencer、Bioedit、Mega等生物学软件对E6和E7基因进行核苷酸和氨基酸序列分析.结果 15例宫颈癌中8例鳞癌检出E6和E7基因,检出率为8/15.2例腺癌、1例腺鳞癌和其他4例鳞癌中均未检出HPV16 E6E7 DNA.8例鳞癌中的4例检出的HPVl6为亚洲类似株,在E6基因178位(T→G,D25E)和E7基因647位(A→G,N29S)发生突变;另外4例为欧洲类似株,其中1例(BJ16)的HPV16在E6基因335位点发生突变(C→T,H78Y).结论 HPV16是致宫颈癌的重要因素;宫颈鳞状细胞癌HPV16感染的发生频率较腺癌和腺鳞癌高;E6基因178位点可能是区分亚洲株和欧洲株的重要位点;E6基因178位点和E7基因647位点是突变频率较高的位点,可能导致HPV16致癌能力发生改变.

Gene characterization of E6 and E7 gene of human papillomavirus of 15 cervical cancer in Beijing

Objective Conducting the gene characterization of the E6 and E7 gene of human papillomaviros 16(HPV16)isolated from 15 cases of cervical cancer at Beijing. Methods Overlapping primers were designed according to the full-length genomes of E6 and E7 from the GenBank and PCR was used to amplify the E6 and E7 fragments. TA clone was used to select a purified clone in order to have better and valuable sequencing results. Nucleotide and amino acid sequence were analyzed by the Sequencer, Bioedit, Mega et al. Results 8 of 15 (8/15) cervical samples contained HPV E6 and E7 gene, and 4 had Asian type like and 4 had Europe prototype like. There were two nucleotide mutation at E6 position 178 (T→G,D25E) and at E7 position 647 (A→G, N29S) in 4 Asian type like viruses. There were one nucleotide mutation at E6 position 335 (C→T, H78Y) in 1 of 4 Europe prototype like virus. In the cervical cancer samples, 8 of 15 contained the HPV16E 6 and E7 gene. HPV16 E6 and E7 can not be detected in denosquamous carcinoma and adenocarcinoma. Conclusion HPV16 is the main etiology of the cervical carcinoma. The HPV16 infectious ratio of squamous carcinoma is more than the ratio of adenocarcinoma. 178th nucleotide in E6 gene is the very important site to distinguish the Asia and the Europe prototype strain like. 178 nueleotide in E6 and 647 nucleotide in E7 are the frequent mutation site in cervical carcinoma. Analysis based on the E6 and E7 gene sequence of HPV 16 isolates suggests that naturally occurring sequence variants of E6 and E7 gene may have identify the oncegenie properties.