人源化抗血小板膜糖蛋白Ⅱb/Ⅲa抗体库的构建及临床价值

Construction and clinical applications of a humanized phage library of platelet GPⅡb/Ⅲa single chain Fv antibody

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 out the anti-platelet GPⅡb/Ⅲa autoantibodiy which can inhibit with aggregation function of platelet and construct a humanized anti-platelet GPⅡb/Ⅲa single chain Fv library by phage surface display technology.Methods Idiopathic thrombocytopenic purpura (ITP) patients whose plasma contains anti-platelet GPⅡb/Ⅲa autoantibodies were screened out. The antibodies can inhibit the aggregation function of platelet by using MAIPA assay and platelet aggregation test. The heavy chain and light chain variable region genes of human immunoglobulin were amplified by RT-PCR from peripheral blood lymphocytes mRNA of the patients screened out and randomly combined through a DNA linker encoding the peptide(Gly 4Ser) 3 to construct single chain Fv gene. ScFv gene was digested with SfiⅠ/NotⅠ restricted digest enzyme to ligate the pHEN2 cloning vector, then were electrically transformed to E.coli TG1.The TG1 containing ScFv-pHEN2 was rescued by helper phage M13K07 to produce ScFv phage antibody.Results Of 95 chronic ITP patients , 41(43.2%)were found positive for anti-platelet GPⅡb/Ⅲa autoantibody, 5(5.3%)were markedly positive. 2 (2.1%) patients plasma significantly inhibited the aggregation function of platelet. The lengths of VH and VL were about 380 to 400 bp. They were successfully linked by DNA linker to form ScFv fragment of about 780 bp. After cloning ScFv to phagemid pHEN2 and transforming ScFv-pHEN2 to TG1, 2.1×10 7 clones formed. After M13K07 rescue,1.62×10 10cfu/ml ScFv phage antibodies were produced. Conclusion Few anti-platelet GPⅡb/Ⅲa antibodies can inhibit the aggregation function of platelet. A phage antibody library has been constructed by phage surface display technology. Humanized anti-platelet GPⅡb/Ⅲa ScFv phage antibody can be screened from this library.