CD80/IgG融合基因重组表达载体的构建及在CHO细胞中功能性表达的研究

目的 构建CD80/IgG真核表达载体,使其在中国地仓鼠卵巢细胞（Chinese hamster ovurm,CHO）中功能性表达,寻找消除白血病细胞免疫逃逸的有效方法.方法 采用定向分子克隆技术将小鼠CD80胞外段和IgG1 Fc段的cDNA串联至真核表达载体pcDNA3.0中,运用脂质体将所得的重组子转染CHO细胞,经免疫印迹、斑点ELISA检测其表达,免疫亲和层析法纯化其表达产物,用流式细胞术、MTT比色法及ELISA在体外检测该产物的生物学活性.结果 两段目的基因按预期设想克隆入空载体中并得到了表达,亲和层析纯化获得了高纯度目的蛋白,该蛋白能够将白血病细胞表面CD80分子密度提高5倍,并可显著增强同种异体小鼠淋巴细胞的增殖、对白血病细胞的杀伤以及Ⅱ-2的分泌.结论 成功构建了融合基因真核表达载体,其表达产物在体外具有相应的生物学功能,有希望成为消除白血病细胞免疫逃逸的有力工具.

Construction of CD80/IgG recombinant vector and investigation on its functionally expressing in CHO cells

Objective To construct CD80/IgG recombinant expressing vector and express it in Chinese hamster ovum cells in order to seek effective methods to eliminate immune escape of leukemic cells. Methods Two cDNAs were amplified by PCR respectively from plasmid pcDNA/B7 containing the full-length cDNA of murine CD80 and murine spleen cells and cloned to the eukaryotic expression vector pcDNA3.0 by directionally cloning and the resultant recombinant plasmid pcDNA/CD80-IgG was transfected into CHO cells with liposome transfection reagent. The constitutively expressing cells were obtained by G418 screening. Western blot and Dot ELISA were used to detect the expression of the fusion protein. Expressed fusion protein was purified with affinity chromatography and its biological activity was examined by flow cytometry, MTT colorimetry and ELISA. Results Two inserts were successfully cloned into the plasmid pcDNA3.0. Highly purified fusion protein was obtained, which was proved that it could significantly upregulate CD80 density on leukemic cells and deliberately promote the proliferation of mouse allogeneic lymphocytes and their killing rate against WEHI-3 cells from 49.7% to 84.6%(P< 0.05). ELISA assay showed that the fusion protein could also enhance IL-2 excretion of allogeneic lymphocytes activated by tumor antigen. Conclusion The recombinant vector was successfully constructed and functionally expressed in animal cells. This laid the foundation for the consequent investigations on how to eliminate the immune escape of AML cells.