抗血小板糖蛋白 Ibα 和 β3 的双特异性单链抗体片段 SZ-2/SZ-21 的构建与鉴定

[Construction and characterization of bispecific single-chain antibody fragments SZ-2/SZ-21 against platelet glycoprotein Ib alpha and beta3].

作者信息

Dai Kesheng, An Guangyu, Ruan Changgeng

机构信息

Jiangsu Institute of Hematology, First Affiliated Hospital of Suzhou University, Suzhou 215006, China.

出版信息

Zhonghua Yi Xue Za Zhi. 2002 Nov 10;82(21):1493-7.

PMID:12509914

Abstract

OBJECTIVE

To generate bispecific single-chain Fv (bis-scFv) antibody fragment SZ-2/SZ-21 against platelet glycoprotein (GP) Ib alpha and GP IIIa.

METHODS

The SZ-2/SZ-21 bispecific single-chain antibody expression vector pET22-21-L-2scFv was constructed by gene recombination technique with a interdomain linker fragment, SZ-21 single-chain antibody (anti- GP IIIa) gene and SZ-2 single-chain antibody (anti- GP Ib alpha) gene, identified by sequencing, and introduced into Escherichia coli strain BL21 (DE3) Plys. The bispecific protein accumulated in bacteria as insoluble inclusion bodies was harvested, denatured, refolded, and affinity-purified in vitro. SZ-2/SZ-21 bispecific single-chain antibody was added into platelet-rich plasma (PRP) and the binding of SZ-2/SZ-21 bispecific single-chain antibody with platelets was examined by histochemistry and cytometry, ELISA, or Western blotting. Restocetin, ADP, or thrombin was added into PRP with SZ-2/SZ-21 bispecific single-chain antibody to observe the platelet aggregation.

RESULTS

The expression vector pET22-21-L-2scFv was correctly constructed. Its recombinant protein was expressed mostly in the form of inclusion bodies, and the yield accounted for 14% of the total cell protein. The bispecific protein could bind to platelet. The platelet aggregation induced by restocetion, ADP, or thrombin in PRP with SZ-2/SZ-21 bispecific single-chain antibody was significantly reduced.

CONCLUSION

An SZ-2/SZ-21 bispecific single-chain antibody against two target antigens in platelet was developed and characterized with the ability to inhibit platelet aggregation induced by restocetin, ADP, and thrombin.

摘要

目的

制备针对血小板糖蛋白（GP）Ibα和GP IIIa的双特异性单链Fv（双特异性单链抗体片段，bis-scFv）抗体SZ-2/SZ-21。

方法

采用基因重组技术，构建含结构域间连接片段、SZ-21单链抗体（抗GP IIIa）基因和SZ-2单链抗体（抗GP Ibα）基因的双特异性单链抗体表达载体pET22-21-L-2scFv，测序鉴定后，导入大肠杆菌BL21（DE3）Plys。收集细菌中以不溶性包涵体形式积累的双特异性蛋白，进行变性、复性及体外亲和纯化。将SZ-2/SZ-21双特异性单链抗体加入富血小板血浆（PRP）中，通过组织化学、细胞计数、酶联免疫吸附测定（ELISA）或蛋白质免疫印迹法检测SZ-2/SZ-21双特异性单链抗体与血小板的结合情况。向含有SZ-2/SZ-21双特异性单链抗体的PRP中加入瑞斯托霉素、二磷酸腺苷（ADP）或凝血酶，观察血小板聚集情况。