摘要
目的 :研制抗血小板膜糖蛋白IIb/IIIaFab抗体。方法 :通过间接免疫荧光试验和血小板聚集抑制试验 ,选取鼠源抗血小板糖蛋白IIb/IIIa单克隆抗体 (mAbP14 0 )。从分泌该mAb的杂交瘤细胞株 (P14 0 )中 ,克隆到抗体轻链基因和重链Fd段基因 ,构建原核表达重组质粒p3MH/P14 0к Fd ,并在XLI Blu菌株中进行表达。采用钴亲和层析法对P14 0Fab抗体进行纯化 ,用SDS PAGE、ELISA和Westernblot等方法 ,对P14 0Fab抗体进行检测 ,并通过血小板聚集抑制试验 ,观察P14 0Fab抗体的抗栓活性。结果 :SDS PAGE和Westernblot表明 ,纯化的P14 0Fab抗体的相对分子质量 (Mr)约为 4 70 0 0。ELISA的结果显示 ,P14 0Fab抗体可与人血小板特异性结合。在体外ADP诱导的血小板聚集试验中 ,P14 0Fab抗体对血小板聚集的抑制作用成剂量依赖性 ,IC50 的平均值为 16 .85mg/L。结论
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 cell secreting the mAb P140. The genes of P140 Fab were inserted into plasmid p3MH to construct recombinat expression plasmid p3MH/P140к-Fd. After digestion with the restriction enzyme, the recombinant 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 47×10 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.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2004年第5期563-567,共5页
Chinese Journal of Cellular and Molecular Immunology
基金
国家高技术研究发展计划 (863)重大专项资助 (No.2 0 0 2AA 2Z344B)
国家自然科学基金重大项目资助 (No.30 4 90 2 4 0 )
