CD40-IgG1Fc融合蛋白真核表达载体的构建及表达

目的 构建人CD40-IgG1Fc融合蛋白的真核表达载体CD40-IgG1Fc/pcDNA3.1( ),并在中国仓鼠卵巢细胞(CHO细胞)表达.方法 应用T-A克隆技术和亚克隆技术,构建人CD40膜外段和IgG1Fc段融合蛋白的真核表达载体CD40-IgG1Fc/pcDNA3.1( ),将其转染入CHO细胞株并筛选,进行稳定表达.通过RT-PCR、Western blot法及ELISA法证实融合基因的表达.结果 成功构建真核表达载体CD40-IgG1Fc/pcDNA3.1( ),并建立CHO细胞稳定表达株.Western blot及ELISA法检测到细胞培养上清中有融合蛋白的表达.结论 通过基因工程生产的人CD40-IgG1Fc融合蛋白,能够特异性阻断CD40-CD40L的相互作用.

Construction of Eukaryotic Expression Vector of CD40-IgG1Fc Fragment Fusion Protein and its Expression in CHO Cell

Objective To construct an eukaryotic expression vector of CD40-IgG1Fc ,and to ex-press the fusion proteinin chinese hamster ovary(CHO) cells . Methods The gene encoding the CD40-IgG1Fc fusion protein was constructed and clonedinto eukaryotic expression vector pcDNA3 .1( +) usingT-Acloning and subcloning techniques . CHO cells were transected with the expression vector CD40-IgG1Fc/pcDNA3 .1( +) ,generating cell lines that expressedfusion protein stably . The expression of thefusion gene was confirmed by RT-PCR, Western blot and ELISA. Results DNAsequencing indicatedthat the eukaryotic expression vector CD40-IgG1Fc/pcDNA3 .1( +) was successfully constructed. Aftertransected with the expression vector , CHO cells can express the fusion protein stably , which was con-firmed by Western blot and ELISA. Conclusion CD40-IgG1Fc fusion protein produced by gene engineer-ing, which can specially block CD40-CD40Linteraction,provides an original means to further study theetiopathogenesis andi mmunotherapy of autoi mmune diseases .