Construction Trivalent Adenovirus Vector Vaccine Of HPV16/18/58 And Research Its Immune Effect In HPV58 Association Tumor

Objective: HPV infection is the primary factor which results in cervical carcinoma and cervical intraepithelial neoplasias(CIN).Its early genes E6 and E7 stemmed from early region take part in replicating virus by itself,transcription and forming tumor and so on,so it has been ideal target of cervical cancer vaccine.In our study,we turned the point mutations of HPV16,18,58 mE6E7 fusion gene to lose its transforming activity,then inserted it into the adenovirus expression vector to build the adenovirus vector vaccine which can express HPV16,18,58 mE6E7 fusion gene.At the same time,We transfect lentivirus vector containing HPV58E6E7 fusion gene into U14 cervical cancer cells of mice to build a U14 cell line which can stably express HPV58E6E7 fusion gene.At last,we studied the immune effect of AD-16/18/58 mE6E7 adenovirus vector vaccine in tumor-burdened mouse models which can express HPV58 E6 and E7 genes to providing a vaccine candidates for immunotherapy of cervical cancer and CIN.Methods :1)Acquired the gene of HPV16,18,58 mE6E7 after point mutation:through GENEBANK website,we acquired the gene sequences of HPV16,18,58 E6 and E7.According to the literature research,we incorporated the E6 and E7 gene sequence and removed the stop code,through turned key base point mutations of the E6 and E7 genes to lose its transforming activity as mutant mE6 and mE7,then using IRES gene to connect them making it become HPV16mE6E7-IRES-HPV18mE6E7-IRES-HPV58mE6E7(3480bp)fusion gene that named p GH-HPV16/18/58 mE6E7.Gene synthesis and sequencing of the gene segment was accomplished by company,then verify the gene sequences by PCR and enzyme digestion experiment.2)construct adenovirus vector vaccine AD-HPV16/18/58 mE6E7:connected the HPV16/18/58 mE6E7 gene fragments into the adenovirus expression vector to build the adenovirus vector vaccine which expressing HPV16,18,58 mE6E7 fusion gene,then detecting the translation and transcription of HPV16mE6E7,HPV18mE6E7,HPV58mE6E7 fusion gene in 293 A cells by RT-PCR and Western blot.4)construct the U14 cell line that stably expressing HPV58E6E7 fusion genes: transfect lentivirus vector that expressing HPV58E6E7 fusion gene method into the cervical cancer U14 cells of mice,and sorted out the stable transfect positive clones by the flow cytometry instrument,then detected HPV58E6E7 fusion gene expression in the stable transfect U14 cell lines by RT-PCR and Western blot and inoculated the stable transfect HPV58E6E7 fusion gene U14 cell in the right back of C57 BL / 6 mice,testing the HPV58E6E7 fusion gene expression in tumor tissue in tumor-burdened mice model by RT-PCR and Western blot.4)vaccine immunization effect research:immunized C57 BL / 6 mice by AD-HPV16/18/58 mE6E7 adenovirus vector vaccine by divided into prevention groups and therapeutic groups,then observed the morphological,tumor growth and lifetime of mice after immunization,and test serum antibody ELISA,ELISPOT,CTL humoral and cellular immunity to preliminary evaluating antitumor effect of the vaccine.Results:1.plasmid pGH-HPV 16/18/58 mE6E7 was synthesized after point mutation of HPV16,18,58 mE6E7,and the aim gene confirmed exists by the genetic sequencing,the PCR and enzyme digestion experiment;2.successfully construct AD-HPV16/18/58 mE6E7 adenovirus vector vaccine,by RT-PCR and Western blot can verify their transcription and translation of HPV16,18,58 mE6E7 fusion gene and protein in 293 A cell;3.successfully construct U14 cell lines(cervical cancer cells of mice)that stably expressing HPV58E6E7 fusion gene,through RT-PCR and Western blot HPV58E6E7 fusion gene transcription and translation can be verify in vitro and in vivo.4.Animal experiment results indicated that the vaccine can induce effective humoral immunity and cellular immunity in the immunized C57BL/6 mice,and the vaccine can inhibits the growth of HPV58 association tumor so that mice were protected from HPV58E6E7(+)tumor cells attack.1)mice subcutaneous tumor formation time in vaccine group was later than in control group,the vaccine group and the control group were statistically significant(P<0.05).2)Tumor in vaccine group mice gain significantly slower than in control group,the survival time of vaccine group was longer than control group,and the average tumor weigh of vaccine group mice is less than in control group,the inhibiting ratio of tumor in vaccine group mice is higher than in control group,the vaccine group and the control group were statistically significant(P<0.05).3)Serum antibody test by ELISA results confirmed that the vaccine immunized mice can induce specific serum antibody contraposing HPV type 58 E6 and E7 protein antigen,which can reach maximal l:25600,compared with the control group,the difference is significant(P<0.05).4)Cellular immune INF-? by ELISPOT can detect specific T cells in vaccine immunized mice which activated by HPV58E6/E7 resistance epitope peptide protein antigen and the number of spots in vaccine group are higher than in control group(P<0.05).5)Cytotoxic reaction through special CTL response of the mice immunized by AD-HPV16/18/58/mE6E7 vaccine was detected by LDH way and obtained the highest data of 28%,the data proved that the vaccine can protect the mice against U14/LV-HPV58E6E7 cells attack.Conclusion : In brief,we constructed and expressed AD-HPV16/18/ 58mE6E7 adenovirus vector vaccine,this vaccine can induce effective humoral and cellular immunity in the immunized mice and can protect the immunized mice from HPV58E6E7(+)tumor cells attack.So it can be used to treat HPV58(+)cervical Carcinoma and Cervical Intraepithelial Neoplasia as a candidate vaccine.