Preparation Of Human Papillomavirus18E6Single-codon Mutants And Their Effects On Tumor Cell Growth

Human papilloma virus (HPV) infection is the leading cause of cervical cancer. High-risk HPVs, HPV-16and HPV-18, were related to more than90%of cervical cancer. Early protein E5/E6/E7of HPV could interact with PDZ domain from host cell, degrade the tumor-suppressor protein p53and build a kind of suitable environment for its transformation, proliferation and inducing the occurrence of tumor in vivo. It was reported that a single-codon mutation (F47R) in HPV16-E6oncoprotein converts it into a potential tumor suppressor recently. HPV18-E6was so similar to HPV16-E6. Whether the similar mutation in HPV18-E6has the similar anticancer effect? In this study, plasmids expressing HPV18-E6with F49R, F127R and F49R-F127R mutantions were constructed, then their effects on tumor cell growth and apoptosis were compared with that of nomal HPV18-E6, to understand the basis of HPV18-E6mechanism and verify the possibility of these mutants as anti-uterine cancer drugs. The results were as follows.The gene encoding HPV-18E6was amplified from the genome of Hela cells and cloned into eukaryotic expressing vectors to obtain pcDNA3.1-E6and pEGFP-C1-E6. Genes encoding single condon mutations (F49R or F127R) of HPV-18E6were amplified by overlap PCR and cloned into vectors pcDNA3.1and pEGFP-C1to get expression plasmids pcDNA3.1-E6F49R, pcDNA3.1-E6F127R and pEGFP-C1-E6F49R, pEGFP-C1-E6F127R. Plasmids expressing double condon mutation (F49R/F127R), pcDNA3.1-E6F49R-F127R and pEGFP-C1-E6F49R-F127R, were constructed by site-directed mutagenesis kit.After transfecting the eukaryotic expressing vectors into Hela cells for48hr, RT-PCR showed that there were significant differences between the HPV18-E6transcription and expression level in the HeLa cells for the first time. In vitro, MTT assay, acridine orange/ethidium bromide fluorescent staining method anddifferential analysis of growth curve were used to firstly show that the effect of HPV18-E6protein on the proliferation of cancer cells and theinhibitoryeffects of all HPV18-E6mutations on different cells. Their effects on HeLa cells expressing truncated E6were weaker than those on cells expressin full-length E6. Their effects on cell proliferation and apoptosis in stable transfected cells were much better than those on transient transfected cells. Meantime, the inhibitory effects of the single mutant HPV18-E6F49R and the double mutant HPV18-E6F49R-F127R on cells were better than HPV18-E6F127R in comparison. To economicly and efficiently prepare potiential antitumor drug HPV18-E6F49R, prokaryotic expression plasmids pET28a-HPV18-E6F49R-TAT and pET28a-HPV18-E6F49R-R9were constructed and then fusion proteins were expressed in Escherichia coli. After optimization, induction with0.06mmol/L IPTG and0.1mmol/L Zn2+at15℃for20h were most suitable for soluble expression of recombinant fusion protein HPV18E6F49R-TAT and HPV18E6F49R-R9. Although recombinant protein contains His tags, conventional Ni2+column affinity chromatography can not effectively purify target proteins.More than85%pure recombinant fusion protein can be obtained by anion exchange chromatography combined with Zn2+affinity chromatography. The IC50of HPV18E6F49R-TAT and HPV18E6F49R-R9were280ug/ml and260ug/ml, respectively.