抗人CD3单链抗体的构建、表达及纯化

Construction, Expression and Purification of an Anti-CD3 Single-Chain Fv Antibody

采用反转录-聚合酶链反应(RT-PCR)从鼠抗人CD3杂交瘤细胞WuT3中扩增克隆出抗体重、轻链可变区基因,测序结果证实:V_H属鼠抗体重链可变区亚组Ⅱ(β)、V_L属鼠抗体Kappa轻链可变区亚组Ⅵ.将V_H、V_L基因片段克隆到单链抗体表达载体POPE51中,使V_H、V_L基因片段分别直接与一多肽连接子的两端相连,构建成单链抗体(ScFv)基因.克隆筛选证明,阳性克隆近100%.细菌用20μmol/L IPTG诱导培养,经固定金属离子亲和层析和分子筛凝胶过滤,从细胞间质中提取纯化ScFv,每升培养液可获得5mg纯度大于90%的ScFv.FACS活性检测结果表明,纯化的WuT3 ScFv与亲代结果一致.

The heavy and light chain variable domain genes were amplified by RT-PCR from murine anti-human CD3 hybridoma and cloned. Sequencing results demonstrated that the Vh belongs to murine antibody Vh domain subgroupⅡ B,and VL belongs to subgroupⅣ . The Vh and VL gene were cloned into scFv expression vector pOPE5l and bridged by a flexible peptide linker. Colonies screening indicated that nearly all the colonies were positive. WuT3 scFv was purified from periplasmic inclusion bodies of 2 liters of cultural bacteria with 20 uM/L IPTG by immobilized metal affinity chromatography (IMAC) and size-exclusion chromatography. 5 mg of monomeric scFv with a purity greater than 90% can be obtained from 1 liter of culture.FACS analysis results showed that the purified WuT3 scFv has the same specificity with the parental WuT3 McAb, but can not bind to the B cell line CD3-JOK-1 cells.