Construction of human interleukin 8 gene eukaryotic expression vector and its expression in OVCAR-3 cells

Objective:To construct eukaryotic expression vector of hIL-8 and express it in OVCAR-3 cells after transient transfection in order to facilitate for further investigation of tumorigenesis and tumor growth.Methods:The IL-8 gene was amplified from human peripheral blood mononuclear cells of healthy volunteers by RT-PCR,and was cloned to eukaryotic expression vector pcDNA3.1(+).The recombinant plasmid pcDNA3.1/hIL-8 was transfected into OVCAR-3 cells with Lipofectamine 2000.The expression of IL-8 was tested in OVCAR-3 cells by RT-PCR,ELISA and Western blot assay.The proliferative effect of IL-8 on OVCAR-3 cells was observed for 6 days by MTT.Meanwhile,the cell cycle was analyzed by FCM.Results: Agarose gel electrophoretic analysis of double digested products of recombinant plasmid showed that duplicate DNA bands were seen clearly in the right location.DNA sequencing showed that the exon of the fragment matched the gene order reported in GenBank,and the reading frame remained constant.There was significant difference in the level of mRNA and protein between the recombinant plasmid transfected group and other control groups by RT-PCR,ELISA and Western blot assay(all P0.05).MTT showed that the optical density value(D_410) of the transfected group after 3 days was higher than that of other control groups.The FCM analytic result showed that IL-8 cDNA could promote the growth of OVCAR-3 cells by increasing the S-phase cell number and upgrading the proliferation index(compared with the other groups P0.05).Conclusion: The human interleukin 8 gene is successfully subcloned into eukaryotic expression vector and expressed in OVCAR-3 cells,which confirmed the promotion of OVCAR-3 cells by IL-8 through autocrine.