Study On DNA Vaccine Of Human Single Chain Variable Fragment Of Anti-idiotype Antibody Against Nasopharyngeal Carcinoma

OBJECTIVE: To construct the recombinant eukaryotic expression vector pcDNA3.1(+)-G22 encoding human nasopharyngeal carcinoma single chain variable fragment (scFv) of anti-idiotype antibody G22, identify its expression in mammal cells and examine its effects as DNA vaccine in mice model.METHODS: The G22 gene was ligated into the sites of EcoRI and NotI of eukaryotic expression vector pcDNA3.1(+). After identification and DNA sequencing, the recombinant plasmid pcDNA3.1(+)-G22 was stably transfected into CMT-93 cells and the expression of G22 was detected by Western Blotting, flow cytometry and immunofluorescence staining. Then we constructed another recombinant plasmid pcDNA3.1 (+)-gp96 and did routine protein analysis by the same methods mentioned above. After identification the expression of G22 scFv and gp96 protein in mammal cells, we built and purified DNA vaccines. Then C57BL/6 mice were immunized intramuscularly in every two weeks. After three times immunization, one part of mice were examined by indirect ELISA test, competition inhibition ELISA test, and flow cytometry for analyzing the antibody in serum and the changes of T lymphocyte phenotype of spleen. Others were intoculated CMT-93 or CMT-93/G22 cells subcutaneously on mice's back. Tumor size and life span were observed to explore its potential as human anti-idiotype vaccine against nasopharyngeal cancer. RESULTS: Restriction analysis and DNA sequencing showed that the two recombinant plasmids were reconstructed successfully. Routine protein analysis verified that G22 scFv and gp96 protein can be expressed in eukaryotic cells in right way. In animal study, anti-tumor antibody was detected in the immunized mice sera by indirect ELISA test and it could inhibit FC2 McAb reacting with Ag of HNE2. Stimulated proliferation of CD4~+T cells and CD8~+T cells in immunized mice were also examined. Tumor size in the samples immunized with G22 scFv was obviously smaller than negative control groups and life span was also prolonged compared to control groups.CONCLUSION: The eukaryotic expression vector containing the human nasopharyngeal carcinoma anti-idiotype antibody scFv gene G22 was successfully constructed and expressed. G22 scFv can induce both humoral and cellular immunity against nasopharyngeal carcinoma in vivo and showed some advantageous effects in tumor challenge, which provided experiment evidences for clinical use of G22 scFv as anti-idiotype vaccines against nasopharyngeal carcinoma.