TNFR1BP/IgGFc融合蛋白载体的构建、表达及其生物学活性研究

Cloning and Expression of Human TNFR1BP/IgGFc Fusion Protein in COS-7 Cells

目的构建肿瘤坏死因子受体1(TNFR1)封闭肽与IgGFc融合蛋白的真核表达载体,检测其表达和生物学活性。方法将合成的TNFR1封闭肽(TNFR1BP)基因片段插入质粒pIG/3C,构建真核表达载体pIG/3C-TNFR1BP;脂质体法将重组载体转染COS-7细胞;ELISA和Western blot法检测转染细胞的培养上清中TNFR1BP/IgGFc融合蛋白的表达;间接免疫荧光抑制实验、细胞毒抑制实验检测融合蛋白对TNFR1的封闭作用。结果经PCR和核苷酸序列测定证实TNFR1BP基因正确插入质粒pIG/3C。ELISA检测证实,在转染细胞培养上清中有融合蛋白的表达;间接免疫荧光抑制实验和细胞毒抑制实验证实融合蛋白能结合L929细胞表面的TNFR1,并能抑制分泌型肿瘤坏死因子α(sTNF-α)介导的细胞毒作用。结论成功构建并实现TNFR1BP/IgGFc融合蛋白真核表达;体外实验证实此融合蛋白可通过与细胞表面TNFR1结合,从而拮抗sTNF-α介导的生物学效应。

Objective To express the fusion protein of the TNFR1 blocking peptide with Fc fragment of human IgG1 in mammalian cells and identify the biological effect of its expressive product. Methods The eukaryotic cell expression vector pIG/3C-TNFR1BP was constructed by inserting the TNFR1 blocking peptide （TNFR1BP） gene into plasrnid pIG/3C and transfected into COS-7 cells with Lipofectamine reagent. The expression of fusion protein （TNFRIBP/IgGFc） was tested by ELISA and Western blot. Inhibition of the fusion protein to biological effects of TNF-α was detected by indirect immunofluorescence assay and cytotoxicity assay. Results DNA sequencing and PCR showed TNFRIBP gene fragment was inserted correctly into plasmid pIG/3C. ELISA and Western blot revealed that COS-7 cells transfeeted with vector pIG/3C-TNFRIBP could secrete fusion protein TNFR1BP/IgGFc. Indirect immunofluorescence assay and cytotoxicity assay indicated the fusion protein could bind to TNFR1 on the cell surface and competitively inhibit the activity of sTNF-α. Conclusion The recombinant vector pIG/3C-TNFRIBP was successfully constructed and the TNFR1BP/IgGFc fusion protein was expressed by eukaryotic cells. The fusion protein could block biological activities of TNF-α by competitively binding to TNFR1 in vitro.