sflk1-IFN-γ双功能蛋白基因重组质粒的构建和表达及生物学活性鉴定

Construction,expression and identification of recombinant plasmid encoding bifunctional protein sflk1-IFN-γ

目的:构建表达可溶性血管内皮生长因子受体2(sVEGFR2,在小鼠又称sflk1)与IFN-γ双功能蛋白的重组质粒pcDNA3.1(+)/sflk1-IFN-γ,对表达的sflk1-IFN-γ重组蛋白的生物学活性进行鉴定。方法:以RT-PCR法分别扩增出sflk1与小鼠IFN-γ基因片段,克隆入pcDNA3.1(+)载体,将该重组质粒表达于真核细胞,以ELISA和W estern blotting分别检测胞外及胞内sflk1-IFN-γ双功能重组蛋白的表达,并对该蛋白的生物学活性进行鉴定。结果:构建的pcDNA3.1(+)/sflk1-IFNγ-重组质粒能够在真核细胞中有效表达,且表达的sflk1-IFN-γ重组蛋白兼有sflk1和IFN-γ两者的生物学活性。结论:成功构建和表达了sflk1-IFN-γ双功能蛋白基因重组质粒,为进一步研究该双功能蛋白的抗肿瘤作用打下了基础。

Objective： To construct,express and identify the recombinant plasmid pcDNA3.1（＋）/sflk1-IFN-γ encoding bifunctional protein sflk1-IFN-γ（soluble fetal liver kinase 1 and interferon-γ）.Methods： sflk1 and IFN-γ gene fragments were cloned by RT-PCR,and then inserted into pcDNA3.1（＋） plasmid between BamHI-EcoRI and XhoI-XbaI restriction sites to form the recombinant plasmid pcDNA3.1（＋）/sflk1-IFN-γ.The recombinant sflk1-IFN-γ transiently expressed in COS-7 cells was detected by ELISA and Western blotting.Bioactivities of sflk1-IFN-γ fusion protein were identified by proliferation inhibition assay with H5V cells and NK activity assay.Results： pcDNA3.1（＋）/sflk1-IFN-γ can be effectively expressed in COS-7 cells.Concentrations of sflk1 and IFN-γ in culture supernatants of pcDNA3.1（＋）/sflk1-IFN-γ transfected COS-7 cells were（20.85±2.48）ng/ml and（1.08±0.09）ng/ml,respectively.Western blotting showed that the molecular weight of sflk1-IFN-γ fusion protein was about 130 kDa,while that of sflk1 was 115 kDa.The supernatants of transfected cells significantly inhibited the proliferation of H5V cells stimulated by mouse VEGF 164 and enhanced the NK activity of splenocytes,demonstrating that sflk1-IFN-γ fusion protein possessed the bioactivities of both sflk1 and IFN-γ.Conclusion： The constructed plasmid pcDNA3.1（＋）/sflk1-IFN-γ can be effectively expressed in eukaryotes.The expressed sflk1-IFN-γ fusion protein has the biological activities of both sflk1 and IFN-γ.