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目的筛选出抑制血小板聚集的血小板膜糖蛋白(GP)Ⅱb/Ⅲa自身抗体,用噬菌体表面展示技术构建人源化抗血小板GPⅡb/Ⅲa单链噬菌体抗体(ScFv)库。方法用单克隆抗体特异性俘获血小板抗原(MAIPA)技术和血小板聚集试验筛选出血浆中含有抑制血小板聚集的血小板GPⅡb/Ⅲa自身抗体的特发性血小板减少性紫癜(ITP)患者。从筛选出患者的外周血淋巴细胞中提取mRNA,用RT PCR扩增出人免疫球蛋白的重链可变区(VH)和轻链可变区(VL)基因片断,用DNA linker将VH和VL连接成ScFv基因片断。用限制性内切酶SfiⅠ/NotⅠ酶切ScFv后克隆到噬菌体载体pHEN2,然后转化大肠杆菌TG1。用辅助噬菌体M13K07援救转化后的TG1,产生ScFv。结果95例慢性ITP患者中41例(43.2%)血浆中抗GPⅡb/Ⅲa自身抗体阳性,强阳性患者5例(5.3%)。2例(2.1%)明显抑制血小板聚集功能。扩增出380～400bp大小的VH和VL基因,用连接肽(Gly4Ser)3成功地连接成约780bp大小的ScFv片断。ScFv克隆到pHEN2并转化大肠杆菌TG1后,形成2.1×107个克隆。用辅助噬菌体M13K07援救TG1后产生的噬菌体抗体库滴度为1.62×1010cfu/ml。结论少数抗GPⅡb/Ⅲa自身抗体可抑制血小板聚集功能。用噬菌体表面展示技术构建了ScFv库,可用来筛选人源化抗血小板GPⅡb/ⅢaScFv。
Objective To screen platelet glycoprotein (GP Ⅱb / Ⅲa) autoantibodies that inhibit platelet aggregation and construct humanized anti-platelet GPⅡb / Ⅲa single-chain phage antibody (ScFv) library by phage display technique. Methods Patients with idiopathic thrombocytopenic purpura (ITP) that contained platelet GPIIb / IIIa autoantibodies that inhibited platelet aggregation in plasma were screened by monoclonal antibody specific capture platelet antigen (MAIPA) technique and platelet aggregation assay. MRNA was extracted from the peripheral blood lymphocytes of the selected patients, and the heavy chain variable region (VH) and light chain variable region (VL) gene fragments of human immunoglobulin were amplified by RT PCR. The VH and VL linked into ScFv gene fragments. ScFv was digested with restriction endonuclease Sfi I / Not I and cloned into the bacteriophage vector pHEN2, which was then transformed into E. coli TG1. Transformed TG1 is rescued with helper phage M13K07 to produce ScFv. Results Of the 95 patients with chronic ITP, 41 (43.2%) had anti-GPⅡb / Ⅲa autoantibodies in plasma and 5 (5.3%) were strongly positive. Two cases (2.1%) significantly inhibited platelet aggregation. The 380-400bp VH and VL genes were amplified and successfully ligated into the ScFv fragment of about 780bp using the linker peptide (Gly4Ser) 3. After ScFv was cloned into pHEN2 and transformed into E. coli TG1, 2.1 × 107 clones were formed. The titer of the phage antibody library generated after rescue of TG1 with helper phage M13K07 was 1.62 × 1010 cfu / ml. Conclusion A few anti-GPⅡb / Ⅲa autoantibodies can inhibit platelet aggregation. ScFv library was constructed by phage display technology and could be used to screen humanized anti-platelet GPⅡb / ⅢaScFv.