摘要
目的构建CD80IgG融合基因表达载体,表达并纯化该融合蛋白,初步探讨其增强抗肿瘤免疫的作用方法将小鼠CD80分子胞外段和IgGFc段cDNA串联起来,定向克隆入质粒pcDNA3.0中,使其在哺乳动物细胞中表达。采用免疫亲和层析法纯化,免疫印迹和斑点ELISA鉴定表达的融合蛋白,流式细胞术、MTT法检测其增强白血病细胞CD80分子表达及促进T细胞增殖的功能。结果DNA测序证明两段基因正确插入质粒载体;亲和层析纯化得到浓度为0.1mg/ml,纯度为95%的蛋白质溶液;免疫印迹、ELISA表明该蛋白质即为CD80IgG融合蛋白流式细胞术证明其能够提高白血病细胞表面CD80分子的表达。结论成功构建融合基因表达载体,表达并纯化出融合蛋白,该蛋白可增强CD80分子的表达。
Objective To construct CD80-IgG fusion gene expressing vector, express and purify the fusion protein and explore its enhanced anticancer immunity. Methods The cDNAs encoding the extracellular domains of mouse CD80 antigen and mouse IgG Fc fragment were cloned directionally in tandem into plasmid pcDNA3.0 and the recombinant plasmid was expressed in mammal cells. The expressed fusion protein was purified by immunological affinity chromatography, and then identified by Western blotting and dot-EIASA. Flow cytometry and MTT colorimetry were used to detect the upregulation of CD80 expression on leukemic cells and the promotion of T cell proliferation by the fusion protein. Results DNA sequencing revealed the two cDNA were inserted into pcDNA3.0. The fusion protein solution, whose concentration was 0.1mg/ml and purity was 95%, was obtained after purification. Affinity chromatography and ELISA showed its identity with CD80-IgG fusion protein, and the flow cytometry proved the upregulation of CD80 molecule on leukemic cells by this protein. Conclusion The fusion gene expressing vector was successfully constructed and expressed. The recombinant fusion protein could be purified and increase the expression of CD80 antigen on leukemic cells.
出处
《华中科技大学学报（医学版）》
CAS
CSCD
北大核心
2005年第4期420-423,共4页
Acta Medicinae Universitatis Scientiae et Technologiae Huazhong
基金
国家自然科学基金资助项目(No.30240022)
