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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.
Antibody heavy and light chain variable region genes were amplified by RT-PCR from WuT3 mouse anti-human CD3 hybridoma cells. The sequencing results confirmed that the variable heavy chain variable region of V_H mouse antibody Subgroup Ⅱ (β), V_L belongs to the subgroup of Kappa light chain variable region of murine antibody Ⅵ. The V_H and V_L gene fragments were cloned into the single chain antibody expression vector POPE51, and the V_H and V_L gene fragments were directly linked with a polypeptide The two clones were linked to each other to construct single-chain antibody (ScFv). Cloning and screening showed that the positive clones were almost 100% .The bacteria were induced by 20μmol / L IPTG and fixed by metal ion affinity chromatography and molecular sieve gel filtration, Purified ScFv was extracted from the interstitial cells, and 5 mg ScFv was obtained with a purity greater than 90% per liter of culture medium.The results of FACS assay showed that the purified WuT3 ScFv was consistent with that of the parent.