[抗血小板IIb/IIIa抗体Fab片段在大肠杆菌中的表达及其生物学活性鉴定]

[Expression of antibody Fab against platelet IIb/IIIa in E.coli and characterization of its biological activity].

作者信息

Yang Yong-chang, Lai Chun-ning, Wang Yan, Shen Bei-fen, Li Yan

机构信息

Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing 100850, China.

出版信息

Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2004 Sep;20(5):563-7.

PMID:15367348

Abstract

AIM

To develop a Fab antibody against platelet GPIIb/IIIa.

METHODS

A mouse anti-human GPIIb/IIIa monoclonal antibody (mAb) P140 was selected by the indirect immunofluorescence assay (IFA) and platelet aggregation inhibition test. The Fd and light chain genes were cloned from the hybridoma cells secreting the mAb P140. The genes of P140 Fab were inserted into plasmid p3MH to construct recombinant expression plasmid p3MH/P140kappa-Fd. After digestion with the restriction enzyme, the recombinant plasmid was transformed into E.coli XLI-Blue. The expressed product was purified by TALON metal affinity resin. Purified P140 Fab was characterized by SDS-PAGE, ELISA, Western blot and platelet aggregation inhibition test.

RESULTS

SDS-PAGE analysis showed that relative molecular mass (M(r)) of P140 Fab was 47x10(3). The results of ELISA, Western blot and platelet aggragation inhibition test indicated that P140 Fab could specifically bind to platelet and inhibit platelet aggragation in dose-dependent manner. The mean value of IC(50) was 16.85 mg/L.

CONCLUSION

A soluble anti-platelet GPIIb/IIIa antibody P140 Fab was prepared successfully, which lays the foundation for further clinical application.

摘要

目的

制备抗血小板糖蛋白IIb/IIIa的Fab抗体。

方法

通过间接免疫荧光法（IFA）和血小板聚集抑制试验筛选出小鼠抗人GPIIb/IIIa单克隆抗体（mAb）P140。从分泌mAb P140的杂交瘤细胞中克隆Fd和轻链基因。将P140 Fab的基因插入质粒p3MH中构建重组表达质粒p3MH/P140kappa-Fd。用限制性内切酶消化后，将重组质粒转化到大肠杆菌XL1-Blue中。表达产物用TALON金属亲和树脂纯化。通过SDS-PAGE、ELISA、Western印迹和血小板聚集抑制试验对纯化的P140 Fab进行鉴定。