Construction Of LfcinB Gene Lentiviral Vector And Its Effect On SKOV3 In Vitro

Objective To construct and identify a lentiviral vector expressing LfcinB gene and the effects of proliferation inhibition and apoptotic induction in human ovarian carcinoma SKOV3 cells, by means of wild-type LfcinB transfection, were identified, in an attempt to develop a novel therapy for human ovarian cancer.Methods According to the sequence of LfcinB in Genbank, LfcinB gene was synthesized and cloned into the pLJM1 lentiviral vector , which contained CMV promoter and green fluorescent protein(GFP).The resulting lentiviral vector was detected by PCR and sequencing. 293T cells were contransfected with lentiviral vector pLJM1,Δ8.91,pVSVG. All virus stocks were produced with the help of lipfectamine2000. The titer of virus was tested according to the expression level of GFP. LfcinB was identified by Western-blot.SKOV3 were cultured in vitro.After the recombinant vector was contransfected into the cells,the inhibition of cell proliferation was determined by MTT in vitro,the influence on the cellcycle and the apoptosis was observed with flow cytometry,apoptotic cells were examined by Hoechst 33258 staining.According to the sequence of OSP-1 which have reported,OSP-1 gene was synthesized and cloned into the pLJM1-LfcinB vector to construct a recombinant vector containing OSP-1 promoter.Results PCR and DNA sequencing confirmed that the LfcinB gene was successfully inserted into the lentiviral vector.The titer of concentrated virus was 2×108TU/ML.pLJM1-LfcinB inhibited cell growth and proliferation(P<0.05), and intruced cell apoptosis.OSP-1 promoter was successfully inserted into the recombinant vector.Conclusion In vitro, pLJM1-LfcinB affected cultured human ovarian cancer cell line SKOV3 and inducted their apoptosis. The OSP-1-LfcinB gene recobinant vector was successfully constructed, which laid the foundation for gene therapy and study of anti-ovarian cancer.