|
Purpose of human papillomavirus (Human papillomavirus, HPV) is a small double-stranded DNA virus closed, specificity is very strong, people are its only host. Generally, E5 gene and high-risk HPV oncogenic mechanisms with specific links. Current research on HPV mostly on its main oncogenes E6 and E7, and E5 gene for a single study is relatively rare. This experiment HPV16 E5 E5 and deletion mutants constructed transfected immortalized human oral epithelial cells, and the host cell morphology, cell proliferation and E6, E7 mRNA expression levels were analyzed, and the results will be in-depth study E5 gene on human immortalized oral epithelial cells and its relationship with E6 and E7 gene interactions provide theoretical basis. Methods (1) E5 gene deletion mutant gene expression vector: Analytical high-risk HPV16 E5 whole sequence and molecular structure, designed with one pair of restriction sites (Hind III, BamH I) of HPV E5 series of deletion mutants primers were cloned 1-69 bp ,1-171 bp ,70-249bp ,172-249 bp 4 fragments, build HPV16 E5 series of deletion mutants, PCR amplification, recovery of DNA fragments obtained E5 mutant gene fragment; The E5 mutant gene fragment was ligated into plasmid pLEGFP-N1, build pLEGFP-ΔE51, ΔE52, ΔE53, ΔE54, double digestion evacuation sequencing. (2) the use of the packaging cell line PA317 packaged into retroviral each gene. (3) E5 and its deletion mutants transfected human immortalized oral epithelial cells using RT-PCR, Real-time PCR and other methods to confirm the success of expression as well as E5 E6 and E7 mRNA expression without effect. (4) MTT assay HPV16 E5 gene on the host cell proliferation. Results (a) Construction of HPV16 E5 gene deletion mutant expression vectors pLEGFP-ΔE51, ΔE52, ΔE53, ΔE54. Digestion and sequencing results showed that the deletion mutants is correctly inserted into the multiple cloning site within the vector. (2) the pLEGFP-E5, ΔE51, ΔE52, ΔE53, ΔE54 transfected PA317 packaging cell can be obtained by a host cell transfected with the recombinant plasmid. (3) The HPV16-E5 its deletion mutants were transfected into immortalized human oral epithelial cells by RT-PCR were able to verify each target gene expression, cell proliferation after transfection experiments and Real-Time PCR showed that: HPV16 E5 gene can promote host cell proliferation (P lt; 0.05), and can indirectly increase HIECO of E6 and E7 mRNA levels, and its deletion mutants of host cells and expression of E6 and E7 no significant effect. Conclusion The gene of HPV16 E5 some extent the immortalized human oral epithelial cells, and the mRNA expression of E6 and E7 genes have some indirect regulation.
|