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目的 :构建人dll1ext(humandelta like1extracellularregion) Fc融合蛋白的真核表达载体pEF BOSneo hdll1ext Fc,并在COS 7细胞中进行表达。方法 :从人脑cDNA文库中PCR扩增人delta like1胞外段 ,通过DNA重组构建真核表达载体 pEF BOSneo hdll1ext Fc。瞬时转染COS 7细胞 ,应用RT PCR、细胞免疫荧光技术和双抗体夹心ELISA ,检测融合蛋白的表达。结果 :成功地构建了真核表达载体 pEF BOSneo hdll1ext Fc。以重组载体转染COS 7细胞后 ,RT PCR结果显示delta like1胞外段与IgG1Fc在mRNA水平正确拼接 ;细胞免疫荧光染色呈阳性反应 ;夹心ELISA法检测到细胞培养上清中有融合蛋白的表达。结论 :成功地扩增了人delta like1胞外段 ,构建了 pEF BOSneo hdll1ext Fc真核表达载体 ,并在COS 7细胞中获得表达 ,为下一步研究奠定了基础
AIM: To construct the eukaryotic expression vector pEF BOSneo hdll1ext Fc of human d11ext Fc fusion protein and express it in COS 7 cells. Methods: The human extracellular domain of human delta like1 was amplified by PCR from human brain cDNA library. The eukaryotic expression vector pEF BOSneo hdll1ext Fc was constructed by DNA recombination. Transient transfection of COS 7 cells, RT PCR, cell immunofluorescence and double antibody sandwich ELISA, the expression of the fusion protein was detected. Results: The eukaryotic expression vector pEF BOSneo hdll1ext Fc was successfully constructed. RT-PCR results showed that the extracellular domain of delta like1 was correctly spliced with IgG1 Fc at the mRNA level in COS 7 cells. The expression of fusion protein was detected by sandwich ELISA in the cell culture supernatant . CONCLUSION: The eukaryotic expression vector pEF BOSneo hdll1ext Fc was successfully amplified in vitro and expressed in COS 7 cells, which laid the foundation for the further study