PEI Enhance The Protective Of The Recombinant DNA Vaccine-Plasmid PVAX1/C-G250Peptide-C And The Heterologous Prime-boost Immunization Strategy

Research background and goalRenal cell carcinoma (RCC) is one of the most common malignant tumors of the urinary system, accounted for3%of all adults malignant tumors. Incidence of RCC has benn obvioused as a rising trend and the death was increased annually in recent years. Sincense RCC is not sensitive to radiotherapy, chemotherapy and hormone therapy, radical operation is the major treatment for it at present. However, RCC is one of the most immunoresponsive cancers in humans, immunotherapy exhibits a suitable basis for treatment. Tumor vaccine immune method to treat RCC had become the research focus of national scientists.G250is considered as a RCC-associated antigen and play an important role in RCC diagnosis, prognosis, and immunotherapy. G250is one of isomerases of the carbonic anhydrase family, which have the same genetic structure with Carbonic anhydrase IX (CAIX). G250was expressed in98%of RCC and expressed in almost all of the clear-cell subtype of RCC, while normal renal tissues do not express G250. Therefore, G250as a specific antigen for RCC and serves as an ideal target for RCC-specific tumor immunotherapy. As the exploration of CAIX, the G250peptide of amino acids249-268contains a cytotoxic T-lymphocyte epitope(CTL)and a helper T lymphocytes(Th) epitope, which was able to induce both CD4+and CD8+T cell responses and generate anti-RCC activity. Therefore, G250peptide/249is an ideal potential target for RCC-specific peptide vaccine immunotherapy. Based on the above background, in our prophase study, the gene fragments which encode mouse G250antigenic peptide/249wased inserted to replace the major immunodominant region (MIR)of the HBcAg and obtain the recombinant plasmid pET28a(+)/C-G250peptide-C, and the recombinant fusion protein. This time C-G250peptide-C was take from the pET28a (+)/C-G250peptide-C plasmid to pvaxl plasmid. Obtain the recombinant plasmid PVAX1/C-G250peptide-C. PEI as adjuvant could raise the immune response. As well as peptide vaccine could be associatided as what was called heterologous prime-boost protocol.Objective Discussed PEI as an adjuvant to enhance the protective of the recombinant DNA vaccine-plasmid PVAX1/C-G250peptide-C and a strategy of vaccination was attempted with the DNA vaccine followed by a boosts with the recombinant protein vaccine (DNA prime-protein boost vaccine). Methods For the replacement of the enzyme digestion sites, pET28a (+)/C-G250peptide-C was taken as a template. C-G250peptide-C was take from the pET28a (+)/C-G250peptide-C plasmid with the help of EcoR1、Sal1. The G250peptide was then inserted to the PVAX1which was enzyme digested by EcoR1、Xho1and obtain the recombinant plasmid PVAX1/C-G250peptide-C. Twenty-four Kunming mices were divided into fourgroups:(A) PVAX1/C-G250peptide-C DNA plasmid alone,(B) DNA-PEI polyplexes,(3) DNA prime-protein boost vaccine,(4) Blank. mice were injected at0,10,20,30days under quadriceps femoris muscle and the third group was given with the G250peptide which was made in our prophase test at the20and the30days. Immune responses were detected by indirect ELISA and flow cytometry. Results The recombinant plasmid PVAX1/C-G250peptide-C was confirmed to be correct by the restriction enzyme digestion and DNA sequencing. After four times of immunization, the titer of antibody can be detected in blood serum of the first three groups of kunming mices. DNA-PEI polyplexes was higher than DNA alone. Much higher G250-specific antibody were seen in the DNA prime-protein boost vaccine than DNA-PEI polyplexes and DNA alone. CD4+and CD8+were the same trend as the titer of antibody. Conclusion Recombinant DNA vaccine-plasmid PVAX1/C-G250peptide-C was successfully constructed. Higher G250-specific antibody was seen by indirect ELISA. CD4+and CD8+lymphocytes was tested by flow cytometry. These findings demonstrated that PEI as an adjuvant was useful to enhance the protective of the DNA vaccine. DNA prime-protein boost regimen can induce an enhanced antitumor effect in a heterologous prime-boost protocol and suggest a new way to a successful immunization against metastatic carcinoma.