Effect Of HPV 16 E5 On E6 And E7 Gene Induced Human Oral Immortalized Epithelial Cell

Objective Human papillomavirus(HPV)is a sort of DNA closed virus, which presents a high affinity for humanbeing ,the only host. HPV not only infects the mucosa of women's cervix , but also infects the skin on body and mucosa inside the body.Generally,E5 has been proved to be specially related with carcinogenesis of HPV.Much attention has been taken to E6 and E7 gene,little to E5. In this study, the HPV16 E5 and the E5 mutants constructed were transfected into the E6 and E7 gene induced human immortalization oral epithelial cells ,and the host cell-related biological characteristics analysed to investigate the effect of HPV 16 E5 on E6 and E7 gene induced human oral immortalized epithelial cell.Method (1) Construction of E5 gene mutants: after comparison of HPV16 E5 complete sequences, four primers were designed for PCR to obtain E5 fragments 1-69 bp,1-171 bp,70-249 bp,172-249 bp, and construct cloning plasmids named pLEGFP-ΔE51,ΔE52,ΔE53,ΔE54. (2) Retroviruses were packaged with cell lines PA317. (3)HPV16 E5 and the mutants were transferred into human Immortalized oral cells, and RT-PCR/Real-time PCR were used to detect the expression of E5 and the mutants ,and the influence on expression of E6 and E7 mRNA lever. (4)MTT method was used to detect the effect of HPV16 E5 gene on the host cell proliferation .Result (1)Expression vector containing HPV16 E5 mutant genes , named pLEGFP-ΔE51,ΔE52,ΔE53,ΔE54 were successfully constructed . Rstriction enzyme digestion and sequencing showed that HPV16 E5 mutants were correctly inserted into the multiple cloning sites. (2)The recombinant plasmid pLEGFP-E5 and pLEGFP-ΔE51,ΔE52,ΔE53,ΔE54 were successfully transfected into PA317 cell and the retroviral vectors harvested were transfected into eukaryotic cells successfully. (3)HPV16 E5 and pLEGFP-ΔE51,ΔE52,ΔE53,ΔE54 expression in human immortalized oral epithelial cells were verificated by RT-PCR and Real-time PCR, indicating that HPV16 E5 gene and the mutants were transfected into human immortalized oral epithelial cells successfully. The testing of cell proliferation demostrated that HPV16 E5 gene could promote cell proliferation of host cell (P <0.05), and enhaced E6 and E7gene mRNA expression . However,the mutants had no effect on the cell proliferation and the expression of E6,E7 mRNA.Conclusion HPV16 E5 and the mutants were transfected into hieco successfully.E5 may promote cell proliferation and upregulte the expression of E6 and E7 gene mRNA.