Influence Of Recombinant Adenovirus Vectors Containing HPV16 E7 Oncogene On The Expression Of Cdc25A And Cyclin E In HeLa Cells

Objective The study was designed to investigate the effect of the human papilloma-virus 16 E7 gene on cdc25A gene and CyclinE by the transfection of recombinant adenovirusvector. Methods Polymerase chain reaction (PCR) was performed to obtain Human HPV 16 E7 genefragment from human cervical carcinoma tissues. Then the human papillomavirus (HPV) 16 E7oncogene is cloned into the shuttle vector pAdTrack-CMV. Furthermore, the resultantplasmid is linearized by digesting with restriction endonuclease PmeⅠ, and subsequentlycotransformed into E. coli. BJ5183 cells with the adenoviral backbone plasmid pAdEasy-1. Finally,the recombinant plasmid is transfected into adenovirus packaging cell lines HEK-293 cells. Then theviral titer was detected and calculated with the aid of GFP expression. Laser scanning confocalmicroscope (LSCM) was performed to detect the Cycline E protein before and after transfection.RT-PCR techniques were used to evaluate the expression of Cdc25A in HeLa cell. Result GFPexpression of HEK-293 and Hela cell could be observed under the fluorescence microscope. Therecombinant adenovirus had stable efficiency of infection, The titer of the forth eneration recombinantadenovirus was 3×1011pfu/ml. RTPCR measurements showed that the expression of cdc25A mRNAincreased significantly after transfection (the gray numerical value: 0.2306±0.0964vs0.8739±0.2188, P＜0.01). LSCM analysis showed that cyclinE protein increased significantlyafter HPV16E7 genes transferred Conclusion HPV 16 E7 oncogene could espressed highly inHEK-293 and Hela cell. HPV 16 E7 oncogene could increase the proliferation of cervicalcells. HPV 16 E7 oncogene could increase the transcription of cdc25A, which may be an importantmolecule event in the mechanism of cervical carcinoma. HPV16 E7 oncogene may enhance theproliferation of cervical cell by increaseing the cyclinE expression.