[Preliminary study of HPV16 L1/E6-E7 chimeric recombinant DNA vaccine plasmid construction and expression in CHO cell].
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.
Zheng
Jin
J
Institute for Cancer Research, School of Life Science and Technology, Xi'an Jiaotong University, Xi'an 710061,China.
Zhang
Fu-ping
FP
Si
Lu-sheng
LS
Dong
Xiao-ping
XP
Wang
Yi-li
YL
D000818:Animals; D016466:CHO Cells; D036022:Capsid Proteins; D006224:Cricetinae; D003412:Cricetulus; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D016297:Mutagenesis, Site-Directed; D015514:Oncogene Proteins, Fusion / Q000096:biosynthesis* / Q000276:immunology; D009856:Oncogene Proteins, Viral / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D027383:Papillomaviridae / Q000235:genetics; D050725:Papillomavirus E7 Proteins; D010957:Plasmids / Q000235:genetics; D017354:Point Mutation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D012097:Repressor Proteins; D014162:Transfection; D019444:Vaccines, DNA / Q000096:biosynthesis* / Q000235:genetics; D014765:Viral Vaccines / Q000096:biosynthesis / Q000235:genetics
D036022:Capsid Proteins; C068651:E6 protein, Human papillomavirus type 16; D015514:Oncogene Proteins, Fusion; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D019444:Vaccines, DNA; D014765:Viral Vaccines; C059731:oncogene protein E7, Human papillomavirus type 16; C063317:L1 protein, Human papillomavirus type 16