捕获噬菌体酶联免疫吸附法筛选抗去唾液酸糖蛋白受体单链抗体的价值

Generating the human asialoglycoprotein receptor specific scFv by capture phage ELISA

目的评价捕获噬菌体酶免疫吸附试验法(ELISA)在筛选抗去唾液酸糖蛋白受体(ASGPR)单链抗体(scFv)中的价值。方法以含ASGPR基因的质粒(CRDH1/pET3b)为模板,通过聚合酶链反应扩增获得ASGPRH1亚单位糖识别区基因(CRDH1),定向克隆至原核表达载体pET-32c,并经IPTG诱导表达,其表达产物用Ni2+螯合柱亲和纯化;然后,采用捕获噬菌体ELISA对人源噬菌体抗体库进行筛选和鉴定,同时对筛选的抗CRDH1单链抗体进行DNA测序、表达、纯化和免疫印记鉴定。然后,与间接噬菌体ELISA比较。结果CRDH1/pET-32c在原核系统中经诱导表达出分子量约35000的融合蛋白,且以包涵体形式存在;通过Ni2+亲和柱纯化获得CRDH1融合蛋白后,采用捕获噬菌体ELISA进行四轮筛选,在60个噬菌体克隆中有45株与CRDH1特异性结合,其中仅1株与重组蛋白标签有交叉结合反应。DNA序列分析表明,有9株DNA序列各异,9株可分泌性表达与CRDH1特异性结合的scFv,纯化的scFv也可特异性识别rCRDH1。计算该筛选方法的假阳性率为2%,低于间接噬菌体ELISA筛选的假阳性率(58%)。结论捕获噬菌体ELISA筛选CRDH1特异性scFv可有效降低硫氧还蛋白标签(Trx·Tag)引起的假阳性,提高筛选阳性率,并成功筛选出9株具有rCRDH1特异性的scFv。

Objective To evaluate the value of capture phage enzyme linked immunosorbent assay （ELISA） used to select anti-CRDH1 single-chain Fv （scFv） antibody. Methods The amplified CRDH1 cDNA from CRDH1/pET3b was subcloned into prokaryotic vector pET-32c. Expression was induced by IPTG. The recombinant CRDH1 was purified with Ni^2＋ chelating HiTrap HP column. After four rounds of biopanning by capture phage ELISA with recombinant CRDH1 and Trx-His-s tag, respectively, the specific anti-CRDH1 phage clones were transfected into E. coli HB2151, and induced for secreted expression of scFv antibody with IPTG. The positive colonies were sequenced, expressing, purified and identified by Western Blot. Results The recombinant CRDHI protein about 35 000 was expressed in inclusion bodies in E. coli, rCRDH1 was prepared by Ni^2＋ column purification. Forty-four positive colonies were obtained after four rounds of biopanning. There were only one colony can react with Trx/His tag, 9 of the rest 44 colonies showed differences in nucleotide sequence which induced with IPTG to produce soluble scFv antibody. Purified scFvs can only react with rCRDH1. So the false positive rate of this methods was only 2%, but that of indirect phage ELISA was 58%. Conclusions Capture phage ELISA can be used to exclude false positives caused by Trx/His tag of the recombinant protein which coated as antigen to screen a phage library. Using this methods, total 9 anti-ASGPR specific scFv antibodies have successfully and quickly screened out.