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目的研制抗rhEndoglin-scFv(single chain variable fragment)抗体。方法将具有抗原结合活性的抗rhEndoglin-scFv噬菌体感染大肠杆菌E.coliHB2151,经IPTG诱导表达,SDS-PAGE和Western Blot鉴定分析。用HiTrap Anti-ETag柱亲和层析纯化scFv抗体,HiTrap Desalting柱行缓冲液更换。用间接ELISA测scFv抗体的亲和常数,用竞争性ELISA和间接免疫荧光检测scFv抗体的抗原结合活性。结果成功诱导表达和纯化抗rhEndoglin-scFv抗体,表达产物主要位于周质腔中,相对分子质量约为2.9×104。测定scFv的亲和常数为(2.14±0.84)×107L/mol,它能与抗rhEndoglin单抗竞争性结合同一抗原表位,且竞争作用随scFv浓度增加而加强。间接免疫荧光实验证实该scFv抗体能识别HUVEC胞膜表达的Endoglin抗原。结论制备的抗rhEndoglin-scFv抗体具有与rhEndoglin和天然Endoglin结合的能力,可望将其用作肿瘤诊断和治疗的靶向载体。
Objective To develop anti-rhEndoglin-scFv (single chain variable fragment) antibody. Methods Anti-rhEndoglin-scFv phage with antigen-binding activity was used to infect E. coli HB2151, induced by IPTG, and identified by SDS-PAGE and Western Blot. The scFv antibody was purified by HiTrap Anti-ETag column affinity chromatography and replaced with HiTrap Desalting column buffer. The affinity constants of the scFv antibodies were measured by indirect ELISA and the antigen-binding activity of the scFv antibodies was tested by competitive ELISA and indirect immunofluorescence. Results The anti-rhEndoglin-scFv antibody was successfully expressed and purified. The expressed product was mainly located in the periplasmic space with a relative molecular mass of about 2.9 × 104. The scFv was determined to have an affinity constant of (2.14 ± 0.84) × 107 L / mol, which competes with the anti-rhEndoglin mAb for binding to the same epitope, and its competition is enhanced as the concentration of scFv is increased. Indirect immunofluorescence assay confirmed that the scFv antibody could recognize the Endoglin antigen expressed on the membrane of HUVEC. Conclusion The prepared anti-rhEndoglin-scFv antibody has the ability of binding with rhEndoglin and native Endoglin, and is expected to be used as a target vector for tumor diagnosis and treatment.