| Cervical carcinoma (CC) is one of the most common malignancy among women worldwide. Statistic data from World Health Organization (WHO) shown that CC is the second leading cause of cancer death in women in the world. Its mortality and morbidity is the highest among the female cancers in the developing countries. Previous numerous studies have strongly shown that infection by certain high-risk human papillomavirus (HPV) type such as HPV16, HPV18, HPV31 and HPV45 is critical to the development of over 80 percent of cervical carcinomas. Meanwhile, 50 percent of cervical cancers are associated with HPV16 infection. HPV16 is the most common type according to the study of Molecular epidemiology. However, there is not any measure to prevent HPV 16 infection and the curative effect of chemotherapy and operation on CC is not good. DNA vaccines provide an opportunity to prevent and treat HPV infections and HPV-associated lesions. Thus the study of a kind of high-efficiency, safe and low-price HPV DNA vaccine has a more exciting foreground.In order to develop DNA vaccine, the target antigen gene must be defined, which is essential to the construction of DNA vaccine. HPV16 E7 gene is an important transforming gene, which can be consistently expressed in HPV-expressing cervical cancers; because E7 gene is more conservative than E6 gene. Furthermore, we can easily get high expression of E7. So, HPV16 E7 gene is a key antigen gene to construct effective therapeutic HPV vaccines. But E7 protein, as a transforming protein has a dangerous aspect when applied in human directly. So we must remove its transform ability and save its immunogenicity to elicit humoral or cellular immunity, which is the premise to construct HPV16 E7 DNA vaccine. Zinc-finger sequence of E7 gene at C terminal is essential for E7 to stabilize its structure and function. the mutation of Zinc-finger can remarkably reduce the stability and carcinogenicity of E7.meanwhile, which consequently enhance security of DNA vaccine.Although there are many advantages of DNA vaccine, the naked DNA vaccine can not elicit immune response strong enough to protect the animal. So, the object of our research is to improve the immunogenicity of the DNA vaccine and to reduce dangers. Recently, many data of research indicated that linkage of antigen gene to HSP70 gene can enhance potency of DNA vaccine. HSP-peptides complexes derived from cancer or virus-infected cells can elicit obviously immune response against cancer and virus infection., by cross-presentation, HSPs present peptides of external protein to MHC class I molecules and induce antigen specific CTL activity.Based on above-mentioned principle. We mutated the Zinc-finger sequence of E7 gene to decrease its stability and ability of causing cancers and increase the safety of vaccine. Meanwhile, we modified the mutated E7 gene by fusing gene coding hot shock protein70(HSP70), which can induce a stronger anti-tumor immune response by cross-presenting external protein.In this study, we inserted mutated E7 gene (91siteC—?G), wild type E7 and HSP70 separately into eukaryotic expression vector pcDNA3.1~ to construct three kinds of recombinant plasmid, respectively named pcd-mE7 (mutated)> pcd-wE7 (wild typeX pcd - HSP70.then the gene fragment that was inserted into plasmid were confirmed by PCR and enzyme analysis. In order to further study the immunogenicity of chimeric pcd-HPVi6E7-HSP7oDNA vaccine. We vaccinated the C57BL/6 mice alternatively with the above recombinant plasmids and plasmid without any inserts (pcDNA3.1). The C57BL/6 mice were vaccinated once every two weeks. Two weeks after twice boost vaccinated, we sacrificed the C57BL/6 mice to prepare spleen cell suspension, cultured in vitro with E7 protein for five days, lymphocytes proliferation were detected by method of MTT. The results showed that spleen cells from the group chimeric pcd-HPVi6 mE7-HSP7(h pcd-mE7 and pcd-wE7 can be induced specific lymphocytes proliferation with E7 protein in vitro, compared with on inserted pcDNA3.1 and PBS(p<0.01). Nonspecific lymphocytes proliferation were detected in group pcd-HSP70. we also collected the culture supernatant of spleen cell to test the concentration of IFN-y and IL-4 secreted by spleen cells through ELISA and found the concentration of IFN-y from group mice vaccinated by chimeric pcd-HPVi6 mE7-HSP70 was highest (620.15pg/ml) and that of group pcd-mE7 and pcd-wE7 was higher than control group(p<0.0l). There is no statistical difference of IL-4 concentration between groups(p>0.05). CTL activity induced by DNA vaccine was measured by the method of LDH after spleen cells were incubated with E7 expressing B16-F0 cell for 24 hours. We found that the CTL effect elicited by chimeric pcd-HPV16E7-HSP70, pcd-mE7 and pcd-wE7 was stronger than that induced by HSP70, PBS and pcDNA3.1" (p<0.01).When the rate of effect-cells to target-cells was 100:1, the mortality of target cells were respectively as follows: 55.2%, 35.5% and 26.5%. The rate of the pcd-HPV 16E7-HSP70 group was excessively higher than that of the other groups.In conclusion, we had successfully constructed pcd-mE7x pcd-wE7^ pcd-HSP70recombination plasmid and compared with chimeric pcd-HPV16E7-HSP70 to vaccinated mice . the result shown that chimeric DNA vaccine can elicit the humoral and cellular immunity effectively in mice and has more significant immunogenicity than pcd-mE7,pcd-wE7, pcd-HSP70 DNA vaccine. |
PDF Full Text Request