Study On Molecular Detective Analysis Of HPV16E6 In Cervical Carcinoma Specimens In Xinjiang Uygur

Objective: Cervical cancer is associated with infections by "high-risk" human papillomavirus(HPVs), HPV16 is the most frequent type. In order to detect HPV16 DNA in cervical carcinomas specimens in Xinjiang uygur. Established a method for detection of HPV16 E6 DNA in paraffin-embedded cervical carcinomas specimens. HPV16E6 has some variants, each with a different geographic distribution. The southern of Xinjiang is one region of the high incidence of cervical cancer, we have reported the variant of HPV16E6 from cervical carcinoma biopsies in Xinjiang Uygur women. This study was designed to investigate distribution of the variant in the cervical cancer of Xinjiang Uygur women, and the relationship between the variant and the high incidence of cervical cancer in Xinjiang. To establish a method of FQ-PCR (fluorescence quantitative PCR ) for detection of HPV16E6 and HPV16 E7 gene in cervical carcinomas and other cervical specimens. To study the relationship between the quantities of HPV16E6 and HPV16 E7 in cervical tissues and the course of cervical disease in Xinjiang. Utilizing HPV16E7 and HPV16E6 gene of a Chinese Uygur patient of cervical carcinoma in Xinjiang, we induced HPV16E7 and HPV16E6 protein expressed in a prokaryotic system and have obtained thispurified protein by affinity chromatography and quantification. The worldwide results have clearly shown that virtually all cervical cancer is caused by human papillomavirus infection. In order to establish fast, sensitive and cost effective method to detect HPV 16 sequences in cervical cancer, we used a visualization of aggregating gold-labeled nanoparticle probe enhanced with silver staining after sequence specific hybridizations.Methods: DNA extraction from archival formalin-fixed and paraffin-embedded specimens was performed by using a standard protocal. HPV16E6 gene was detected by PCR in tissues of 59 cervical cancer, 65 cervical intraepithelial neoplasia (CIN), 33chronic cervicitis in paraffin-embedded specimens and 96 cervical smear samples of vaginitis and cervicitis. DNA was extracted from 19 cervical carcinoma biopsies in Xinjiang Uygur women. HPV16E6 genes were amplified by PCR from the cervical carcinoma tissues. Nucleotide sequences of the HPV16E6 genes were determined by direct or cloning sequencing methods. Analysis the mutations of HPV 16 type E6 gene. The number of copies of HPV16E6 gene and β -actin was detected in parallel by FQ-PCR in tissues of 69 cervical cancer, 65 cervical intraepithelial neoplasia (CIN), 33chronic cervicitis and 96 cervical smear samples of vaginitis and cervicitis. The variation in HPV copies per genomic DNA equivalent can be estimated by dividing the HPV copy number by the β -actin copy number. The number of copies of HPV 16 E7 gene and β -actin was detected in parallel by FQ-PCR in the same tissues. In this experiment, we used the pGEX vetor of 2T-HPV16E7 to transform into E.Coli BL21(DE3), and induced by 0.1 mM IPTG to express GST-HPV16E7 fusion protein. And then the extract bacterial supernatants were purified by affinity chromatography. A450bp HPV16E6 DNA fragment was amplified by using primers designed from the coding sequence of HPV16E6 gene by PCR. The fragment containing the coding sequence of HPV16E6 proteinwas cloned into vector pMD-18T (pMD-T-HPV16E6) and transformed into E.coli JM109. Positive clone was identified successfully by enzyme digestion. Detection of HPV 16 sequences in cervical cancer with visualizing nanoparticle probes technology.Gold nanoparticle conjugated at 3'-end of SH-derivative oligonucleotide served as a detection probe, and a 5'-end NH2-derivative oligonucleotide immobili zed on glass surface as a capturing probe. Target DNA was hybridized with both of the capturing probe and the detection probe visualized based on highly sensitive "nano-amplification" and silver staining.Results: The detecting efficiency depended on DNA quality. Of 59 cervical carcinoma cases, all showed positive result of β -actin .The HPV16E6 positive rate was 83.1% (The 49 in 59 cases can be detected possitivel...