Cloning And Expression Of Truncated Human Papillomavirus18E6(HPV18E6~*) And Its Antitumor Effect On Cancer Cells

High-risk human papillomavirus (HPV) are closely related to the whole process of the boot, occurrence, development and malignant phenotype of cancer. E6and E7are the HPV proteins associated with cancer. E6from the high risk HPVs can inactivate p53, block apoptosis, activate telomerase, disrupt cell adhesion, polarity and epithelial differentiation, alter transcription and G-protein signaling, and reduce immune recognition of HPV infected cells, while truncated Human Papillomavirus E6protein (E6*) can reverse the effect of E6, restore the level of intracellular P53and caspase-8, as well as promote cell apoptosis. So E6*may be a potential anti-cancer drug caused by HPV infection. In this thesis, the cloning and expression of HPV-18E6*in E. coli and the study of its tumor suppressor effect cellular level were reported.Firstly, HPV-18E6*gene was amplified by PCR with HeLa cell cDNA as template. After cloning it into expression vector pET-28a (+), HPV18E6*protein was expressed by induction with IPTG in E.coli BL21(DE3). SDS-PAGE showed that recombinant HPV18E6*was about20%of the total bacterial proteins, and mainly expressed as inclusion bodies at37℃. So the pure protein was obtainedfrom inclusion bodies.Secondly, with the synthesized primers including oligonucleotides encoding HIV-TAT PTD11amino acids and9arginine (R9), E6*TAT and E6*R9were amplified by PCR and cloned into pET-28a(+) resulting in HPV18E6*TAT-pET28a and HPV18E6*R9-pET28a. After induction by IPTG at25℃, fusion protein HPV-18E6TAT and HPV-18E6*R9were about30%of the total E. coli proteins and mainly expressed as soluble form. Ni+affinity chromatography can not get large amout pure recombinant proteins maybe because the high positively charged TAT and R9residuces. Pure recombinant HPV18E6*-TAT and HPV18E6*-R9were obtained by ammonium sulphate salting-out at15%and20%saturation, respectively.Then, transmembrane effect by FITC labeled proteins showed that E6*TAT and E6*R9can traverse the membrane of HeLa and A549, but E6*can’t.At last, the proliferation and apoptosis experiments showed that1) all three E6*related recombinant proteins had no effect on HeLa cells;2) HPV18E6*R9and HPV18E6*TAT significantly inhibited the proliferation of A549cells with IC50of60.5ug/ml and59.Oug/ml, respectively, but HPV18E6*protein had no effect;3) AO/EB double staining visually showed the cell apoptosis-promoting effect;4) Flow cytometry indicated A549cells treated with HPV18E6*-TAT and HPV18E6*-R9protein were arrested at G2/M phase.