Human Papillomavirus Type 16 E6E7 Gene Vaccineexperimental Study

HPV (Human papillomavirus, HPV) which belongs to Papovaviridae Division, is a kind of the double-stranded closed circular DNA virus, which infects human skin and mucous membranes specificly causing lesions to occur in sites of infection and can not grow in the medium. More than 100 types HPV have been isolated, and they can been divided into low-risk type and high-risk type. The low risk type consists of the 6,11 ,34,44type and so on, relates to the benign tumor of the cervix, cause to genital papilloma, condyloma, low-grade uterine intraepithelial neoplasia mainly.The high-risk type mainly consists of 16,18,31,33 , 35,39 type, etc., are relates to the cervical cancer closely, cause to cervical intraepithelial high and cervical cancer mainly, HPV can be detected in 90% of the cervical cancer ,of which the highest proportion of HPV16 detection rate is more than 50%, so in vaccine research ,the development of research aimed at HPVl6 cervical cancer prevention is of great significance.HPV gene can be devided into three parts, the early genes (E region), the late genes (L region), and the non-coding long control sequence (LCR). E area consists of six open reading frames (El, E2, E4, E5, E6, E7), in which the high-risk HPV E6, E7 protein is mainly encoded to the transformation of normal cells conceration and to accelarate the viral replication, to make the infected cells continuing proliferation, and to make the failure of tumor suppressor genes, HPV E6 jnont to work with p53 tumor suppressor gene function, to lead to the loss of the error correction of DNA in the cell cycle. HPV E7 is inhibited to PRb, and then activate to promote cell cycle genes continuous loop, so that the tissue hyperplasia persisted. Other advanced cervical cancer gene loss,but E6, E7 persist in cervical cancer may play an important role in sustainable development, therefore, vaccine research aimed at E6, E7 protein which are safe, effective and economical HPV vaccine for cervical cancer prevention is of great significance.In this study, recombinant plasmid pcDNA-E6E7 is successfully constructed with HPV16E6E7, to detect its expression, to evaluate HPV16E6E7DNA vaccine induced humoral and cellular immunity in mice. Methods: The digestion of recombinant plasmid pGEM-T-E6E7, E6E7 gene was inserted into the eukaryotic expression vector pcDNA3.1 (-) to construct recombinant HPV16E6E7 plasmid pcDNA-E6E7, by restriction enzyme and sequence detection and identification, transfection COS-7 cells by IFA method validation HPV16E6E7 in vitro transient expression in eukaryotic cells successfully. Package the purified plasmid with adjuvant polyethylene amine (PEI), inject of 6 weeks BABL / c mice with intramuscular, while setting the original vector pcDNA3.1 (-) and normal saline as the control group ,inject once every 2 weeks,after three times of injection, collect the retinal venous blood of mice the two weeks after the last immunization, separate of serum, analysis the humoral immune response of genetic immunization induced in mice with IFA method. Results: Construction of recombinant plasmid HPV16E6E7 pcDNA-E6E7, can be expressed temporarily in COS-7 cells, the anti-E6E7 antibody can be detected in the serum in the immunized mice ,the original vector pcDNA3.1 (-) and saline injection, the specific E6E7 antibody can not be detceted; Detected by quantitative ELISA to analysis IL-2 and IFNγsecretion, in the experimental group IL-2 produced was significantly higher than the control group, P <0.01; IFNγproduced by the experimental group was significantly higher than the control group, P <0.05 differences between groups statistically are significant, the experimental results show that the vaccine was successfully constructed and the HPV16E6E7 DNA analysis by IFA and ELISA quantitative detection of DNA vaccines are effective absorption, mice can induce specific humoral and cellular immunity.In this paper, the DNA vaccine HPV 16E6E7 basic experimental offered research an experimental platform for the related development of vaccines which provided an effective way for the treatment of HPV ,and made an useful attempt for the development of HPV16-DNA vaccine. Therefore, the next step of this study will use DNA vaccines to prevent HPV infections laid the foundation for pre-clinical experiments, while the results of this study HPV infection in the disease will have potential application.