| PRELIMINARY STUDY OF A NOVEL HUMAN PAPILLOMAVIRUS TYPE 16L1／E6-E7 CHIMERIC RECOMBINANT DNA VACCINE |
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| 引用本文： | 郑瑾,马军,张福萍,杨筱凤,董小平,司履生,王一理.PRELIMINARY STUDY OF A NOVEL HUMAN PAPILLOMAVIRUS TYPE 16L1／E6-E7 CHIMERIC RECOMBINANT DNA VACCINE[J].西安交通大学学报(英文版),2004,16(1):45-49. |
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| 作者姓名： | 郑瑾 马军 张福萍 杨筱凤 董小平 司履生 王一理 |
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| 作者单位： | InstituteofCancerResearch,SchoolofLifeandTechnology,Xi＇anJiaotongUniversity,Xi＇an710061,China |
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| 摘 要： | Objective Preparations of HPV16 L1/E6 and L1/E7 prophylactic and therapeutic DNA vaccines. Methods The nucleotides within HPV16 E6 and E7 genes, which are responsible for viral transforming activity, were mutated by mage primer site-directed mutagenesis method. The correctly mutated E6 and E7 fragments were separately cloned into an eukaryotic expression vector pVAX1, together with HPV16 L1 gene, generating chimeric recombinants plasmids 1MpVAX1-L1E6, 2MpVAX1-L1E6, 1MpVAX1-L1E7, 2MpVAX1-L1E7 and 3MpVAX1-L1E7. CHO cells were transiently transfected with the individual DNA vaccines by calcium phosphate method. Target protein expressions in the extracts of the transfected cell lines were measured by ELISA and immunohistochemistry, with HPV16 L1 and E6 specific monoclonal antibodies. Results ELISA assays showed the P/N ratios in the cell extracts transfected with L1E6 and L1E7 plasmids were more than 2.1. Immunohistochemistry revealed brownish precipitant signal in cytoplasm and nuclei of the transfected cells. Conclusion Successful constructions of prophylactic and therapeutic DNA vaccine plasmids lay solid foundation for future animal experiment and clinical trial.
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| 关 键 词： | 疫苗DNA分子 免疫分子生物学 定向转换 HPV感染 |
PRELIMINARY STUDY OF A NOVEL HUMAN PAPILLOMAVIRUS TYPE 16 L1/E6-E7 CHIMERIC RECOMBINANT DNA VACCINE |
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| Abstract: | Objective Preparations of HPV16 L1/E6 and L1/E7 prophylactic and therapeutic DNA vaccines. Methods The nucleotides within HPV16 E6 and E7 genes, which are responsible for viral transforming activity, were mutated by mage primer site-directed mutagenesis method. The correctly mutated E6 and E7 fragments were separately cloned into an eukaryotic expression vector pVAX1, together with HPV16 L1 gene, generating chimeric recombinants plasmids 1MpVAX1-L1E6, 2MpVAX1-L1E6, 1MpVAX1-L1E7, 2MpVAX1-L1E7 and 3MpVAX1-L1E7. CHO cells were transiently transfected with the individual DNA vaccines by calcium phosphate method. Target protein expressions in the extracts of the transfected cell lines were measured by ELISA and immunohistochemistry, with HPV16 L1 and E6 specific monoclonal antibodies. Results ELISA assays showed the P/N ratios in the cell extracts transfected with L1E6 and L1E7 plasmids were more than 2.1. Immunohistochemistry revealed brownish precipitant signal in cytoplasm and nuclei of the transfected cells. Conclusion Successful constructions of prophylactic and therapeutic DNA vaccine plasmids lay solid foundation for future animal experiment and clinical trial. |
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| Keywords: | human papillomavirus type 16 DNA vaccine site-direct mutation |
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