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[Preliminary study of HPV16 L1/E6-E7 chimeric recombinant DNA vaccine plasmid construction and expression in CHO
Jin Zheng1, Fu-ping Zhang, Lu-sheng Si
1Institute for Cancer Research, School of Life Science and Technology, Xi'an Jiaotong University, Xi'an 710061,China.
Background:
To develop HPV16 L1/E6 and L1/E7 prophylactic and therapeutic DNA vaccines.
Methods:
The nucleotides within HPV 16 E6 and E7 genes, which are responsible for viral transforming activity, were mutated by mega primer site-directed mutagenesis method. The correctly mutated E6 and E7 fragments were separately cloned into an eukaryotic expression vector pVAX1, together with HPV 16 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 HPV 16 L1 and E6 specific monoclonal antibodies.
Results:
ELISA showed the P/N ratios in the cell extracts transfected with L1E6 and L1E7 plasmids were greater than 2.1. Immunohistochemistry revealed brownish precipitant signal in cytoplasm and nuclei of the transfected cells.
Conclusions:
Successful constructions of prophylactic and therapeutic DNA vaccine plasmids may be useful for future animal experiment and clinical trial.