炭疽毒素受体与人IgG1的Fc段融合蛋白ATR-Fc在CHO细胞中的表达

Expression of ATR-Fc Fusion Protein in CHO Cells

ATR_Fc是人炭疽毒素受体(ATR)的胞外区与人免疫球蛋白IgG1的铰链区、CH2区和CH3区组成的融合蛋白。表达该蛋白是为了获得结合PA的抗体样分子,通过阻断PA与细胞受体的结合,而阻止炭疽致死毒素和水肿因子进入细胞内,可作为预防和治疗炭疽感染的生物制品。将编码炭疽毒素受体N端1_227氨基酸的基因和编码Fc段的基因连接,插入到pcDNA3.1的HindⅢ和NotⅠ位点得到表达ATR_Fc融合蛋白的真核表达载体pcDNA3.1ATR_Fc,并用脂质体方法将该载体转染至CHO_K1细胞中,用G418筛选并获得ATR_Fc表达水平为10～15μg(106cells·d)的基因工程CHO细胞系ATR_Fc_1D5。采用蛋白A纯化重组蛋白,并用ELISA法鉴定ATR_Fc与PA的亲和性,表明ATR_Fc可与PA特异性结合。

ATR-Fc is a fusion protein consisting of extracellular domain of human anthrax toxin receptor （ATR） and a fragment （hinge, CH2, and CH3 domains） of the Fc of human IgG1. The aim of ATR-Fc expression is to get an antibody-like molecule binding to protective antigen （ PA）, a component of anthrax toxins, this fusion protein may compete with cell surface receptor for PA binding, and block the transport of lethal factor （LF） and edema factor （EF） into cells, thereby act as an antitoxin to prevent and treat anthrax infection. A DNA fragment encoding N-terminal amino acids 1-227 of ATR and human IgG1 Fc was inserted into the HindⅢ and Not Ⅰ sites of pcDNA3.1 to generate the eukaryotic vector pcDNA3.1/ATR-Fc for expression of ATR-Fc fusion protein. Using lipofectine-mediated gene transfer technique, pcDNA3. 1/ATR-Fc was transfected into CHO-K1 cells. After selected with G418, a recombinant CHO cell line, ATR-Fe-ID5, whose expression level was about 10- 15μg/（ 10^6eells·d）, was established. The recombinant protein expressed by the ATR-Fc-1 D5 cells was purified with protein A chromatography. The experimental results demonstrated a direct and specific interaction between ATR-Fe and PA assessed by ELISA.