Construction of eukaryotic expression vector of open reading frame of unknown KH gene and its effect on proliferation of K562 cell / 重庆医科大学学报

Objective:To construct the eukaryotic expression vector of open reading frame of KH gene(KH-ORF),an unknown gene obtained from K562 cells,and investigate its function.Methods:The KH-ORF cDNA was cleaved from plasmid expression vector cut with the same restriction endonucleases.pCI-KH-ORF,the pGEM-T-easy-KH-ORF with EcoRI and SalI and subcloned into the pCl-neo mammalian recombinant expression plasmid,was transfected into K562 cells with liposome and screened by G418.KH-ORF expression was tested by RT-PCR.The effect of expression product on cell proliferation was tested by growth-curve and LDH detection.Results:KH-ORF was expressed in K562 cells.The effects of increased proliferation were significant.Conclusion:The product of KH-ORF may be a functional protein that is related to the proliferation of K562 cells,and KH gene may be a functional unknown leukemia gene.

The expression of open reading frame of unknown KH gene from K562 cells in E.coli / 重庆医科大学学报

Objective:To construct the prokaryotic expression system of open reading frame of KH gene (KH-ORF),an unknown gene in K562 cells.Methods:The 225bp KH-ORF cDNA was cleaved from plasmid pGEM-T-easy-KH-ORF with EcoRI and SalI and subcloned into the pCI-neo Mammalian Expression Vector cutted with the same restriction endonucleases.The recombinant expression plasmid was transfected into BL21 (DE3) plysS by hot-shock assay.Induced by IPTG, the recombinant protein product was tested by SDS-PAGE.Results:24kD KH-ORF product was obtained as inclusion bodies in BL21 (DE3) plysS after the expression vector had been induced by IPTG for 30 minutes Conclusion :The construction of prokaryotic expression system of KH- ORF is successful.