CTLA4Ig融合蛋白在CHO细胞中的表达

CTLA4Ig是人CTLA4胞外区与人免疫球蛋白铰链区、CH2区、CH3区组成的融合蛋白，可以与B7结合，通过阻断B7与CD28的结合，从而阻断B7介导的T细胞活化必需的共刺激信号，可作为免疫抑制剂用于器官移植。将CTLA4Ig融合分子克隆到真核表达载体pCI-dhfr，并用脂质体方法转染到COS7和CHO-dhfr-细胞中，用氨甲喋呤筛选转染的CHO-dhfr-细胞。用RT-PCR、ELISA、细胞免疫荧光染色和Western-blot鉴定重组蛋白的表达。采用A蛋白纯化重组蛋白。

Expression of CTLA4Ig fusion protein in CHO cells

CTLA4Ig is a soluble chimeric protein consisting of extracellular domain of human CTLA4Ig and a fragment (hinge,CH2,and CH3domains)of the Fc of human IgG1.CTLA4Ig binds to B7molecules on APCs and thereby blocks the B7mediated co-stimulatory signal for T-cell activation and so may be used as immunosuppressant in organ transplan-tation.In this study,CTLA4Ig chimeric molecule was cloned into eukaryotic expression vector pCI-dhfr.Using lipofectine-mediated gene transfer technique,pCI-CTLA4Ig was transfected into CHO-dhfr-cell,and the transfectants were selected with methotrexate.Expression of the recombinant protein was assessed by PR-PCR,ELISA,cell immunofluorescense stain-ing and Western-blot,and at last the recombinant protein purification was performed by protein A chromatography.