抗血小板膜糖蛋白Ⅰbα/Ⅲa双特异单链抗体的构建及其功能研究

Construction and characterization of bispecific single-chain antibody fragments SZ-2/SZ-21 against platelet glycoprotein Ib α and β_3

目的 构建、表达抗血小板膜糖蛋白 (GP)Ⅰbα GPⅢa双特异单链抗体 ,为其进一步研究、应用奠定基础。方法 应用PCR技术 ,扩增出接头片段和SZ 2 1单链抗体基因 ,克隆入pUCm T载体 ,测序分析。应用基因重组技术将接头片段、SZ 2 1单链抗体基因和SZ 2单链抗体基因重组拼接 ,构建SZ 2 SZ 2 1双特异单链抗体表达载体pET2 2 2 1 L 2scFv,并测序验证。导入大肠杆菌BL2 1 (DE3)Plys,诱导表达。流式细胞术、ELISA、Western印迹检测SZ 2 SZ 2 1双特异单链抗体与血小板结合能力 ,瑞斯托霉素、凝血酶和ADP诱导血小板聚集试验对表达产物功能进行研究。结果 表达载体pET2 2 2 1 L 2scFv构建拼接正确 ;表达产物以包涵体形式为主 ,表达量占菌体总蛋白的 1 4 % ;表达产物具有与血小板以及血小板GPⅠbα和Ⅲa结合的活性 ,可抑制瑞斯托霉素、凝血酶和ADP诱导的血小板聚集 ,此作用呈剂量依赖性。结论 成功表达了SZ 2 SZ 2 1双特异单链抗体 ,该抗体具有与相应 2个靶抗原结合的活性 ,并可抑制瑞斯托霉素。

Objective To generate bispecific single chain Fv (bis scFv) antibody fragment SZ 2/SZ 21 against platelet glycoprotein (GP) Ib α and GPⅢa. 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 GPⅢa) gene and SZ 2 single chain antibody (anti GP Ⅰb α) 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.