Study On The Clone And Expression Of Human Interleukin-18 Gene And Its Influence On The Proliferation Ability Of Human Ovarian Cancer HO-8910 Cells

Objective:Interleukin-18(IL-18)is a new kind of cytokine which can induce interferon -γ(IFN-γ) production in T cells and natural killer(NK) cells.It plays an important role in T cell proliferation,cytotoxicity T lymphocyte(CTL) activation and enhancement of NK cell activity.IL-18 has a significant role in antitumor immune responses.The purpose of this study was to clone the human interleukin-18(IL-18) gene,construct the eukaryotic expression plasmid of IL-18 with molecular biologic technique and transfer it to Human ovarian cancer HO-8910 cells,therefore detect its expression in HO-8910 cells.The proliferation ability of Human ovarian cancer HO-8910 cells transferred with IL-18 gene was discussed.It provided experimental basis for further constructing human ovarian cancer vaccine.Methods:A pair of primer were designed according to the IL-18 sequence in Genbank,The IL-18 gene was cloned from the human placenta tissue by RT-PCR and inserted it to the pGEM-T easy vector.The pGEM-T-hIL-18 plasmid was transformed to E.coli DH5a after the IL-18 gene was confirmed conformity with the sequence from the gene bank by digesting and sequencing.We selected the positive clone into LB medium,then extracted the plasmid after cultured overnight.A pair of primer with the restriction enzyme SalI and EcoRI designed,We perforemed PCR with the pGEM-T-IL-18 plasmid again.The PCR production and the pCI-neo vector were digested with the restriction enzyme SalI and EcoRI.The recombination plasmid of pCI-hIL-18 was constructed with the production of enzymolysis and detected by digesting and sequencing.Human ovarian cancer HO-8910 cells were cultured routinely,The recombination plasmid of pCI-hIL-18 were transferred into HO-8910 ceils by Lipofectamine2000 and selected by G418.Three transferred groups were performed:HO-8910 and Lipotectamine,HO-8910 and pCI-neo,HO-8910 and pCI-hIL-18.The mRNA and protein expression of IL-18 were detected by RT-PCR and ELISA assay respectively.MTT detection was used to detecte the influence on the proliferation ability of Human ovarian cancer HO-8910 cells modified with IL-18 gene.Results:1.The IL-18 gene was successfully cloned from the human placenta tissue,the length of IL-18 gene was 605bp including the complete ORF fragment.It was conformity with the sequence from Genbank by sequencing.2.The recombination plasmid pGEM-T-IL-18 and pCI-hIL-18 were completely corrected by enzymolysis and sequencing.3.The mRNA and protein of IL-18 was expressed in the HO-8910 cells transferred with pCI-hIL-18 plasmid,but not expressed in the other two groups.4.The proliferation ability of Human ovarian cancer HO-8910 cells transferred with the pCI-hIL-18 plasmid was decreased.Conclusions:1.The human interleukin-18 gene could be cloned from the human placenta tissue2.The human interleukin-18 gene could be expressed in the HO-8910 line3.The proliferation ability of Human ovarian cancer HO-8910 cells transferred with hIL-18 gene could be decreased...