hdll1^(ext)-Fc融合蛋白真核表达载体的构建及表达  

Construction of eukaryotic expression vector of hdll1^(ext)-Fc and its expression in COS-7 cells

作　　者：张萍[1] 贾洪霞[2] 周鹏[1] 韩骅[1] 

机构地区：[1]第四军医大学基础部医学遗传学与发育生物学教研室 [2]第四军医大学唐都医院血液科,陕西西安710032

出　　处：《细胞与分子免疫学杂志》2004年第1期11-14,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家重点基础研究发展规划 (973)资助 (No.0 0 1CB50 990 6);国家自然科学基金资助项目 (No .30 2 0 0 1 4 8)

摘　　要：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 。AIM: To construct an eukaryotic expression vector pEF-BOSneo-hdll1 ext-Fc, and to express the fusion protein consisting of the extracellular region of delta-like1 and Fc fragment of human IgG1 in COS-7 cells. METHODS: The extracellular region of human delta-like1 was amplified from a human brain cDNA library by PCR. The expression vector was constructed by DNA recombination. The recombinant plasmid was transfected into COS-7 cells via liposome mediation. The expression of the fusion protein was detected by RT-PCR, immunofluorescence assay and sandwich ELISA. RESULTS: DNA sequencing and restriction endonuclease digestion analysis indicated that the eukaryotic expression vector pEF-BOSneo-hdll1 ext-Fc had been constructed successfully. After recombinant plamid had been transfected into COS-7 cells, RT-PCR and DNA sequencing verified that the dll1 ext gene and IgG1Fc gene were fused correctly. The results of immunofluorescence assay were positive and the fusion protein could be detected by sandwich ELISA in culture supernatant of transfected COS-7 cells. CONCLUSION: hdll1 ext was successfully cloned and expressed in the form of Fc fusion protein, which is helpful for further study of the function of Notch pathway.

关 键 词：delta-likel 融合蛋白 基因克隆 真核表达 

分 类 号：Q786[生物学—分子生物学]