Cloning And Sequence Analysis Of E6 Gene In Cervical Cancer Patients With Human Pa Pillomavirus Type16 N Dalian City

Background and Objective:Human papilloma virus belong to papovaviridae of papilloma vacuolating virus genus A, is a kind of specific infection of human skin,mucosa nonenveloped small double stranded circular DNA virus, its molecular weight is about 8 000 bp. At present,has been isolated from a variety of 130, of which about 40 type can cause human skin and mucosa squamous epithelial proliferation.For the performance of common warts, genital warts(condyloma) and other symptoms.With STD in the increased incidence of genital warts and cervical cancer, rectal cancer inc reases, people are becoming more and more attention to human papilloma virus.Acc-ording to the organization the carcinogenicity of different type can be classified as low risk and high risk, high risk type of HPV16, 18 in the cause of cervical cancer is the most common, epidemiological data showed that about 93% of the can detect the HPV DNA in cervical cancer tissue, which HPV16 type accounted for 65%.HPV E6,E7 protein is the major cause cancer transformation.HPV16E6 protein is one of the earliest expression in the process of HPV infection, E6 protein is multifunctional protein,about replication and host cells transformed and virus,sustained expression in cervical cancer cell lines and tissues,play a vital role in maintaining the malignant phenotype transformation of organization in the process of.A lot of research from animal and human body of different forms of HPV E6 vaccine proved that the E6 protein have immunogenicity, which contains many virus specific CTL epitopes.The resulting specific CTL in the eradication of the virus infection in immune response cells and related tumor cells play an important role,therefore,as a target antigen for vaccine research is of great significance of using E6. This experiment is to understand the structure characteristics of dalian area of cervical cancer patients with HPV16 type E6 protein, for the clinical diagnosis,the treatment of HPV16 infection caused by the diseases of cervical cancer and provide a theoretical basis,for the study of HPV pathogenicity and antigenicity, and research and development to provide reference for the cervical cancer vaccine. 1Methods:1 Tissue DNA extracted by usual methods, isolated type HPV16 DNA fragments;2 With the primer, application of PCR amplification type HPV16 E6 gene;3 Purpose gene(HPV16 E6 protein) connected to the p GM- T carrier, convert to DH51alpha1cells;4 Purification and extraction of plasmid DNA with restriction enzyme Eco RI enzyme identification of plasmid DNA, send positive plasmid cloning sequencing;5 In Gen Bank, with the E6 sequence of Japanese HPV standard strains as reference sequence, analysis with Blast and multiple sequence alignment software Clustal X 2.1 on the nucleotide sequence and the corresponding amino acid sequence.Results:1 In cervical cancer tissue samples extracted DNA as a template, PCR amplification get gene fragment length is about 520 bp;2 Connected to the p GM- T, construct HPV16 E6 cloned plasmid p GM- T- E6;3 Restriction enzymes Eco RI enzyme identification of plasmid DNA correct;4 Plasmid cloning sequencing found six samples with Japanese standard of HPV strains E6 sequence(sequence number KJ543728.1 respectively in Gen Bank and AB889490.1)highly homologous, there are three samples E6 gene mutates, variation sites were 28(A- C), 54(G- T), 92- C(T), 138- G(T) base site.Conelusions:1 In cervical cancer tissue of HPV- 16 positive DNA as a template type successfully1cloned1HPV161E61gene;2 Successfully constructed HPVI6 E6 protein prokaryotic expression system;3 Successfully induced the prokaryotic expression of HPV16 type E6 protein;4 Patients with cervical cancer HPV type 16 E6 gene in dalian and Japan standard of HPV E6 sequence homology, and some bases locus mutation, for the clinical diagnosis,the treatment of HPV16 infection caused by the diseases of cervical cancer and provide a theoretical basis, for the study of HPV pathogenicity and antigenicity,and research and development to provide reference for the cervical cancer1 vaccine.