Construction And Immunogenicity Of The Safe And Effective HPV 16E7 DNA Vaccines

Background &Objectives: Recently,the association between human papillomavirus (HPV) infection and many diseases has been firmly established by the study on molecular biology. Increased incidence of HPV-induced lesions in immunosuppressed patients and the spontaneous regression of some lesions,such as ,warts and precancerous lesions,indicate an important role of the immune system against HPV infecion and development. Immune-intervention therapy will be a potential method to treat HPV infection in the future. DNA vaccine has emerged as an attractive approach for generating antigen-specific immunotherapy. Because HPV E7 proteins are expressed persistently in most of cervical carcinoma cells,it is suggest that HPV 16E7 protein can be chosen as a model antigen for treating HPV-associated cervical malignancies and precancerous lesions. However,safety and limited potency of HPV DNA vaccine are the biggest barriers for widely clinical use. Baced on the studies above,in order to develop safe DNA vectors with low or no transforming activity ,we constructed two DNA vaccines by two different methods, mutation on the zinc-binding motifs of E7 and fusion E7 with HSV-1 VP22.Methods: (1) Vector construction:To induce the mutation on 58 and 91 amino acids of E7,we designed two sites of primers that contains the mutatant sites and amplified the fragments which contain the double mutation (58Cys → Gly,91Cys → Gly)by overlapping polymerase chain reaction for 4 times.The ORF of wild E7 and E7 mutant was inserted into the plasmid pcDNA3.1 BamHI and EcoRI cut sites to generate pcDNA-16E7 and pcDNA-16ME7.Meanwhile,pcDNA-VP22/E7 was constructed by inserting the VP22 fragment into pcDNA-E7 . For large scale preparation of plasmid DNA,transformed Escherichia coli DH5 α , bacteria were cultured in LB medium in the presence of ampicillin,and plasmids were extracted by the alkaline lysis followed by the...