人源化抗血小板膜糖蛋白Ⅵ单链抗体的研究  

Study of a humanized anti-platelet GP Ⅵ single chain Fv phage antibody

作　　者：冀学斌[1] 侯明[1] 张春青[1] 石艳[1] 彭军[1] 初晓霞[1] 马道新[1] 李丽珍[1] 

机构地区：[1]山东大学齐鲁医院肿瘤中心血液科,济南250012

出　　处：《中华血液学杂志》2009年第9期588-591,共4页Chinese Journal of Hematology

基　　金：国家自然科学基金（30600259、30600680、30770922）;卫生部临床学科重点项目（2007-2010）;国家973资助项目（2006CB503800）;教育部全国优秀博士论文专项基金（200561）;山东省优秀中青年科学家科研奖励基金（2008BS03009）

摘　　要：目的用噬菌体抗体库技术研究抑制血小板聚集功能的人源化抗血小板膜糖蛋白（GP）Ⅵ单链抗体。方法从特发性血小板减少性紫癜（ITP）患者血浆中用单克隆抗体特异性俘获血小板抗原（MAIPA）技术和血小板聚集实验筛选出抑制血小板聚集的抗血小板GPⅥ自身抗体。从抗GPⅥ自身抗体阳性患者外周血淋巴细胞中提取mRNA，用RT—PCR扩增出人免疫球蛋白重链可变区（VH）和轻链可变区（VL）基因片段，用连接DNA将VH和VL连接成单链抗体（ScFv）基因片段。用限制性内切酶SfiⅠ／NotⅠ酶切ScFv基因片段后克隆到噬菌体载体pHEN2，然后转化大肠杆菌TGI。用辅助噬菌体M13K07援救转化后的TG1，产生单链抗体。用抗原吸附法经过5轮吸附→洗脱→富集，得到纯化的抗GPⅥ单链抗体，并研究其对血小板聚集功能的影响。结果806例慢性ITP患者中11例（1．36％）血浆中抗GPⅥ自身抗体阳性，2例（0．24％）明显抑制胶原诱导的血小板聚集功能。扩增出380—400bp大小的VH和VL基因，用连接肽（Gly4Set）3成功地连接成约800bp大小的ScFv基因片段。将ScFv克隆到pHEN2并转化大肠杆菌TG1后，形成4．1×10^7个克隆。用辅助噬菌体M13K07援救TG1后产生的抗体滴度为2．62×10^10 cfu／ml。亲和层析后得到的纯化抗体可抑制胶原诱导的血小板聚集而对二磷酸腺苷（ADP）诱导的血小板聚集无明显影响。结论利用噬菌体抗体库技术表达的人源化抗血小板GPⅥ抗体ScFv片段可抑制血小板聚集功能。Objective To study the anti-platelet GPⅥ single chain Fv phage antibody which nan inhibit the aggregation function of platelet by using phage antibody library technology. Methods ITP patients with anti-platelet GPⅥ autoantibody that could inhibit the aggregation function of platelet were screened by MAIPA assay and platelet aggregation test. The gene fragments of heavy chain and light chain variable region （VH and VL） of immunoglobulin were amplified by RT-PCR from peripheral blood lymphoeytes mRNA of the screened patients. The VH and and VL fragments were linked through a DNA linker encoding the peptide （Gly4Ser）3 to construct single chain Fv（SeFv） gene. The ScFv gene was digested with Sfi Ⅰ/Not Ⅰ restriction enzyms and cloned into the pHEN2 phage display vector, then electrically transformed to E. eoli TG1. The TG1 containing SeFv-pHEN2 was rescued by helper phage M13K07 to produce ScFv phage antibody. The anti-platelet GPⅥ phage SeFv antibodiy was enriched and purified. The effeet of the phage antibody on platelet aggregation function was studied. Results Of 806 chronic ITP patients, 11 （ 1.36% ） were positive for antiplatelet GPⅥ autoantibody and 2 （0.24%）patients＇ plasma significantly inhibited the collagen induced platelet aggregation. The length of VH and VL fragmenls was about 380 to 400 bp, and were successfully formed SeFv fragments of about 800 bp by DNA linker, After cloning SeFv to phagemid vector pHEN2 and transforming SeFv-pHEN2 to TG1, 4.1 × 10^7 clones were obtained. After M13K07 rescue,2.62 × 10^10 cfu/ml SeFv phage antibodies were produced. The purified anti-platelet GP Ⅵ SeFv phage antibody inhibited the collagen indueed platelet aggregation. Conclusion Anti-platelet GPⅥ SeFv phage antibody produced by phage antibody library technology can inhibit the aggregation function of platelet.

关 键 词：紫癜 血小板减少性 特发性 自身抗体 血小板聚集 人源化单链抗体 噬菌体抗体库技术 

分 类 号：R686[医药卫生—骨科学]