Construction Of Eukaryotic Expression Vector Of Fusion Gene Sc-kit-igg-fc And Its Expression And Effects In K562 Cells

Objective To construct the eukaryotic expression vector sc-kit-IgG-Fc and to express the fusion protein in K562 cells. Investigate the effects of sc-kit-IgG-Fc on the in vitro growth so as to lays the foundation for research the role of stem cell factor in the tumor angiogenesis.Methods The two cDNAs was amplified by PCR respectively from HUVECs and human genome DNA, and cloned to the eukaryotic expression vector pIRES2-EGFP by directional cloning. The resultant recombinant plasmid pIRES2-EGFP-SCF-C-Kit-IgG was transfected into K562 cells with Lipofectamine transfection reagent. The stably expressing cells were obtained by G418 screening. PCR, Western blot , were used to detect the expression of the fusion protein. CCK-8 analysis was applied to detect the OD value of K562 cells in experimental group (recombinant plasmid transfected) with that in control groups (vector transfected, suspension only), then evaluated their proliferation capacity and apoptosis.Results DNA sequencing verified the correction of the construction of recombinant plasmid pIRES2-EGFP-SCF-C-Kit-IgG. The expressed fusion protein was detected in the transfected K562 cells and the molecular weight of the protein was similar to what we expected. Used CCK-8 to detect the cell proliferation ability, could get a conclusion that: recombinant plasmid transfected compared with control groups(vector transfected, suspension only) had a significantly increase(p<0.01),but there was no distinct variety between the two control group (p>0.05). Cell cycle distribution suggested that recombinant plasmid transfected compared with control groups(vector transfected, suspension only) had a significantly increase in s phase(p<0.01),but the apoptosis of control groups was significantly inhibited(P<0.01).Conclusion (1)The recombinant plasmid pIRES2-EGFP-SCF-C-Kit- IgG was successfully constructed and it expressed the fusion protein SCF-C-Kit- IgG in the K562 cells. (2)The transfected K562 cells could promote cell proliferation and inhibit cell apoptosis. (3) Lays the cell model for research the role of stem cell factor(SCF) and its receptor C-Kit.