CAJ | 학술논문

目的获得高效表达 hDll1ext-Fc 融合蛋白的细胞系以及具有生物学活性的目的蛋白。 　　方法根据已知序列设计引物，并将 hDll1ext 基因片段经PCR 扩增，酶切后连接至 pIRES2-EGFP-Fc 中，挑选阳性克隆测序，将正确的重组质粒转染至 CHO-S 细胞，筛选高表达细胞系，利用 rProtein A 亲和柱纯化目的蛋白，并通过检测 Notch 下游信号分子 Hes1的表达及双荧光素酶报告基因实验检测蛋白活性。 　　结果构建了 pIRES2-EGFP-hDll1ext-Fc 真核表达载体，并筛选了高效表达 hDll1ext-Fc 的 CHO-S 细胞系，纯化得到较高纯度的融合蛋白，配合生物活性检测实验显示，可溶性的 hDll1ext-Fc 能够激活 Hes1报告基因，并且能上调Notch 下游分子 Hes1的表达，证明其能够激活 Notch 信号通路。 　　结论在 CHO-S 细胞中成功表达了 hDll1ext-Fc 融合蛋白，为进一步研究 Delta-like-1的生物学功能奠定重要基础。
목적획득고효표체 hDll1ext-Fc 융합단백적세포계이급구유생물학활성적목적단백。 　　방법근거이지서렬설계인물，병장 hDll1ext 기인편단경PCR 확증，매절후련접지 pIRES2-EGFP-Fc 중，도선양성극륭측서，장정학적중조질립전염지 CHO-S 세포，사선고표체세포계，이용 rProtein A 친화주순화목적단백，병통과검측 Notch 하유신호분자 Hes1적표체급쌍형광소매보고기인실험검측단백활성。 　　결과구건료 pIRES2-EGFP-hDll1ext-Fc 진핵표체재체，병사선료고효표체 hDll1ext-Fc 적 CHO-S 세포계，순화득도교고순도적융합단백，배합생물활성검측실험현시，가용성적 hDll1ext-Fc 능구격활 Hes1보고기인，병차능상조Notch 하유분자 Hes1적표체，증명기능구격활 Notch 신호통로。 　　결론재 CHO-S 세포중성공표체료 hDll1ext-Fc 융합단백，위진일보연구 Delta-like-1적생물학공능전정중요기출。
Objective To obtain the CHO-S cell lines expressing hDll1ext-Fc (human Delta-like-1 extracellular domain-Fc) fusion protein and the target protein with biological activity. Methods The hDll1ext gene was amplified through PCR with the primers designed according to the known sequences, then inserted into pIRES2-EGFP-Fc vector, transformed into the competent cells. The positive clones were screened for sequencing. The recombinant plasmid pIRES2-EGFP-hDll1ext-Fc was transfected into the CHO-S cells, and then the high expressed cell lines were screened and the interest protein was purified with affinity rProtein A column. The biological activity of hDll1ext-Fc was determined by Dual-Luciferase Reporter Assay and the express of Hes1 which is a downstream factor in Notch pathway. Results We obtained the eukaryotic expression vector pIRES2-EGFP-hDll1ext-Fc, high expressed cell lines and high purified hDll1ext-Fc fusion protein. The experiment of biological activity showed that soluble hDll1ext-Fc activated Hes1 reporter gene and up-regulated the expression of Hes1. Conclusion The recombinant plasmid pIRES2-EGFP-hDll1ext-Fc is constructed and the fusion protein is expressed, which lays the important foundation of further study on its biological function.