Build And Verify C33A Cervical Cancer Cell Line Which Is Stable Expression 58 Type Of Human Papillomavirus(HPV58) E6E7 Fusion Gene

Purpose Make human cervical carcinoma cell C33A transfected by human lentivirus,which contains targeting human HPV58E6E7 fusion gene(E6E7 fusion gene);build a stable fusion gene expression HPV58E6E7 C33A cell lines and try to verify experimentally for its stable expression in vitro and in vivo.Methods Transfect the HPV58E6E7 fusion gene to human cervical carcinoma cell C33A by Lentviral-mediated,filter the stably transfected positive clones by flow cytometry.Using realtime fluorescence quantitative PCR,Western-blot and other detection methods to verify HPV58E6E7 fusion gene’s expression in stably transfected cell lines C33A.Inoculate the stably transfected HPV58E6E7 fusion gene C33A cells in the left armpit of nude mice to be tumorigenic,using realtime fluorescence quantitative PCR,Western blot to verify the tumor tissue HPV58E6E7 fusion gene expression.Results LV-HPV58E6E7 fusion gene which has been successfully constructed transfection C33A cells,select strong fluorescence of cell lines。Fluorescence quantitative PCR results showed:The load of HPV58E6E7 fusion gene is 0.95 in the genome in transfection HPV58E6E7 fusion,in the transfection no-load group and control group are 0;Through the western-blot detection,we can visible the fusion protein expression with His-tag in the genome in transfection HPV58E6E7 fusion,the othertwo groups are not seen。The MTT assay results showed:Growth rate of the C33A cells transfection HPV58E6E7 fusion gene significantly faster than the C33A cells transfection no-load group and control group(P<0.05);The cell cycle experiment results showed:G1 phase cells of the C33A cells transfection HPV58E6E7 fusion gene is 42.16±1.63,is apparently lower than that in the transfection control group(54.09±2.75 P<0.05)and the transfection no-load group(54.09±2.83,P<0.05);G2 phase cells is 23.33±1.63,is apparently higher than that in the transfection control group(16.59±3.18,P<0.05)and the transfection no-load group(17.71±.48 P<0.05);S phase cells is 34.51 ±2.01,apparently higher than that in the transfection control group(25.89±3.07,P<0.05)and the transfection no-load group(27.18±2.22,P<0.05)。C33A cells transfection HPV58E6E7 fusion gene which has been built and validated and control cells respectively injected into left armpit of 12 nude mice skin,21 days find that both the maximum diameter of about 1 cm long around tumor tissue,living imager camera results showed:Transfection with slow virus carrier cells into a tumor in the two groups are visible green fluorescent protein,C33A group is not seen;Take each group of three tumor tissue for fluorescence quantitative PCR,the results showed:The load of HPV58E6E7 fusion gene were 0.90,0.90,0.92 in the genome in transfection HPV58E6E7 fusion group,the rest of the viral load of two group were 0;Through the western-blot detection,we can visible the fusion protein expression with His-tag in the genome in transfection HPV58E6E7 fusion group,the other two groups are not seen。Conclusion Successfully established a stable gene expression for HPV58E6E7 C33A cell lines,both in vitro and in vivo.