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目的以pOptivec为载体,以DG44细胞为宿主细胞在贴壁培养条件下表达hTNFR(p75)-IgG4融合蛋白。方法将重组质粒hTNFR(p75)-IgG4-pOptivec线性化后转染DG44细胞,经筛选得到稳定表达hTNFR(p75)-IgG4融合蛋白的DG44细胞克隆。培养上清经ProteinG亲和层析柱纯化后,进行SDS-PAGE和Western blot鉴定。然后对稳定表达目的蛋白的细胞株进行不同浓度的MTX加压以扩增目的基因,检测不同MTX加压条件下目的蛋白的表达量。结果重组质粒成功转染DG44细胞,经筛选后得到稳定表达目的蛋白的细胞株。经MTX加压后最高表达量达到15μg/mL培养基左右。结论hTNFR(p75)-IgG4融合基因片段在DG44细胞中成功表达,且其表达量能够满足实验研究的需要,为下一步实验打下了良好的基础。
Objective To express hTNFR (p75) -IgG4 fusion protein using pOptivec as a vector and DG44 cells as host cells under adherent culture. Methods The recombinant plasmid hTNFR (p75) -IgG4-pOptivec was linearized and transfected into DG44 cells. The DG44 cell clone stably expressing hTNFR (p75) -IgG4 fusion protein was screened. The culture supernatant was purified by ProteinG affinity chromatography and identified by SDS-PAGE and Western blot. The cell lines stably expressing the target protein were then subjected to different concentrations of MTX to amplify the target gene to detect the expression of the target protein under different MTX pressurization conditions. Results The recombinant plasmid was successfully transfected into DG44 cells, and the cell lines stably expressing the target protein were obtained after screening. After MTX pressurized the maximum expression reached 15μg / mL medium. Conclusion The hTNFR (p75) -IgG4 fusion gene fragment was successfully expressed in DG44 cells, and its expression level can meet the needs of experimental study, laying a good foundation for the next experiment.