Expression Of HPV18-E6/7 Fusion Protein In Lactococcus Lactis And Its Mucosal Immunization In Mice

Cervical cancer is the second most common malignancies of women in worldwide.HPV16 and HPV18 are the two most important high-risk HPV,and their infections are associated with more than 70% of cervical cancer.E6 and E7 of the high-risk HPV16 and HPV18 play an important role in malignant transformation.It has been confirmed that HPV18 E6 and E7,as tumor rejection antigens,could induce CTL responses as a vaccine with a therapeutic effect on the tumor.In our research,we mutated E6 and E7 zinc finger sequences to reduce its carcinogenicity,without changing its immunogenicity and stability.In order to enabled E6/7 fusion protein to be highly expressed in Lactococcus lactis,the sequence of E6/7 should be optimized by Jcat software.HPV18-E6/7 mutant fusion protein was a secretory and hydrophobic protein which was expressed with inclusion bodies in Lactococcus lactis by bioinformatics analysis.The mutant fusion genes of HPV18-E6/7 were inserted into the expression vector pET30a(+),and induced its expression by IPTG.The expressed protein HPV18-E6/7 in E.coli were adopted by subcutaneous immunization to female mice.The serum of mice was collected after immunization for a month.The serum titer and specificity were analyzed by indirect ELISA and Western blot,and the mice serum titer was 1: 5000,which was used to detect HPV18-E6/7 mutant fusion protein expressed in Lactococcus lactis.The recombinant plasmids pMG36e-HPV18-E6/7 were constructed and the HPV18-E6/7fusion proteins were highly expressed in Lactococcus lactis MG1363.The HPV18-E6/7 fusion protein expressed in Lactococcus lactis were evaluated by Western blot.The results showed that Lactococcus lactis MG1363 containing pMG36e-HPV18-E6/7 expressed HPV18-E6/7 fusion protein successfully.Live Lactococcus lactis vaccines can stimulated the body to produce strong mucosal immune responses,so we adopted oral,intranasal and vaginal administration to detect the mucosal immune responses.Three kinds of mucosal immune responses were confirmed by Western blot,ELISA and IFN-? of spleen IHC.The results suggested three kind of mucosal immunization all induced immune responses,and oral administration was the best mucosal administration,then the effects of mucosal administration were followed by intranasal immunization and vaginal immunization.In conclusion,recombinant plasmid pMG36e-HPV18-E6/7 was constructed,and the best immunized means of Lactococcus lactis vaccine of high-efficiency expression of fusion protein were studied.We have studied on the prevention and treatment of Lactococcus lactis expressed HPV18-E6/7,and ultimately it provided a potential vaccine strategy for the clinical therapy of HPV18.