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目的将噬菌体抗体库中筛选到的抗52-k Da FK506结合蛋白(FKBP52)人源单链抗体(sc Fv)转换为完整人IgG并在真核细胞中表达。方法噬菌体抗体库获得的5株抗人FKBP52-sc Fv,PCR扩增其轻重链可变区(V_H/V_κ),利用同源重组技术将5对V_H、V_κ拼接至分别包含有人κ链及IgG1恒定区的真核表达载体p CMV/R-L、p CMV/R-H上,转染HEK293F细胞进行完整抗体表达,ELISA鉴定抗体活性,蛋白A柱分离纯化活性最高的抗体,SDS-PAGE、Western blot法鉴定纯化后抗人FKBP52-IgG。结果 PCR及测序结果证实正确构建得到5对抗人FKBP52真核表达载体,转染HEK293F细胞后经ELISA鉴定均有完整抗体表达,对活性最高的5号克隆进行抗体分离纯化,经SDS-PAGE及Western blot验证获得高纯度的特异性人源抗FKBP52-IgG。结论成功将原核表达的抗人FKBP52-sc Fv转换为真核表达的完整人IgG。
OBJECTIVE: To screen human phage antibody (sc Fv) against 52-k Da FK506 binding protein (FKBP52) screened in phage antibody library for complete human IgG and to express in eukaryotic cells. Methods Five anti-human FKBP52-sc Fv obtained from the phage antibody library were used to amplify the variable region of light chain and heavy chain (V_H / V_κ) by PCR. Five pairs of V_H and V_κ were spliced to each of the human κ chain and IgG1 The recombinant plasmid pCMV / RL was transfected into HEK293F cells and expressed in E.coli. The antibody activity was detected by ELISA, and the highest activity was detected by SDS-PAGE and Western blot Anti-human FKBP52-IgG purified. Results The PCR and sequencing results confirmed that the eukaryotic expression vector of 5 pairs of human FKBP52 was constructed correctly. After transfected into HEK293F cells, the whole antibody was identified by ELISA. The highest activity of clone 5 was isolated and purified by SDS-PAGE and Western blot blot to obtain high purity of specific human anti-FKBP52-IgG. Conclusion The prokaryotic expression of anti-human FKBP52-sc Fv was successfully transformed into eukaryotic expressed human IgG.