Generation and characterization of recombinant single chain Fv antibody that recognizes platelet glycoprotein Ibα
Generation and characterization of recombinant single chain Fv antibody that recognizes platelet glycoprotein Ibα
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DOI：
10.1016/s0049-3848(03)00152-x
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发表时间：
2003-01-25
影响因子：
7.5
通讯作者：
Ruan, CG
中科院分区：
文献类型：
--
作者：
Dai, KS;Zhu, HP;Ruan, CG
制备并鉴定了抗血小板膜糖蛋白(GP)Ibalpha的重组单链抗体(ScFv)。单链抗体是从SZ-2杂交瘤产生的，该杂交瘤产生了对GPIbalpha有反应的抗血小板抗体。用逆转录聚合酶链式反应(RT-PCR)从SZ-2杂交瘤细胞中扩增出VH和VL基因片段。克隆入pUCm-T载体后，分别对两个不同克隆的VH和VL基因的DNA序列进行分析，得到了相同的结果。SZ-2可变区与Kabat数据库的比较表明，VH属于小鼠Ig Heavy家族XV，而VL属于小鼠Ig kappa家族XXVI。为了组装SZ-2scFv，先后将Vu和VL片段克隆到pSW1-scFv中。单链抗体以VH-VL方向排列，与15个氨基酸(Gly(4)Ser)(3)连接。扩增出单链抗体的编码序列，并将其克隆到带有PEL B前导序列的pET22b载体的框内，以指导蛋白的分泌。将重组表达载体转化大肠杆菌BL21(DE3)PlysS，用异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达单链抗体。重组抗体的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析表明，重组抗体有一个表观分子量约为31,000的蛋白质。通过考马斯亮蓝染色的SDS-PAGE比较，SZ-2单链抗体的产量约占菌体总蛋白的25%。用Ni-NTA亲和层析成功地纯化了重组SZ-2ScFv抗体，产率为120 mg/L，经酶联免疫吸附试验和流式细胞仪检测表明，SZ-2ScFv抗体能与血小板结合。经Western印迹分析，它能与血小板GPIb结合。它保留了亲本SZ-2单抗(Moab)的结合能力。在功能研究中，SZ-2scFv分别抑制瑞斯托菌素和凝血酶诱导的血小板凝集和聚集，但对ADP诱导的血小板聚集无影响。因此，SZ-2单链抗体具有作为抗血栓药物的潜力。(C)2003爱思唯尔科学有限公司。保留所有权利。
A recombinant single chain Fv (scFv) fragment with specific activity against platelet glycoprotein (GP) Ibalpha was developed and characterized. The scFv was generated from the SZ-2 hybridoma, which produced an anti-platelet antibody reactive to GPIbalpha. VH and VL gene segments were generated from the SZ-2 hybridoma by reverse transcribed-polymerase chain reaction (RT-PCR). After cloning into pUCm-T vector, the DNA sequences of both VH and VL genes were analyzed from two different clones, respectively, the same results were obtained. Comparison of SZ-2 variable region to the Kabat database showed that VH belonged to the mouse Ig heavy family XV while VL belonged to the mouse Ig kappa family XXVI. For assembly of the SZ-2 scFv, VU and VL fragments were cloned into pSW1-scFv successively. The scFv was arranged in VH-VL orientation, being joined together with a 15-amino-acid (Gly(4)Ser)(3) linker. The scFv encoding sequence was amplified and cloned into pET22b vector in-frame with a pel B leader sequence to direct secretion of the protein. Escherichia coli strain BL-21(DE3)PlysS was transformed with the recombinant plasmid, and expression of the scFv was induced using isopropyl-beta-D-thiogalactopyranoside (IPTG). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the recombinant antibody revealed a protein with apparent molecular weight of approximately 31,000. By comparing band intensity on a Coomassie brilliant blue-stained SDS-PAGE, the production yield of SZ-2 scFv was about 25% of the total cellular proteins. The recombinant SZ-2 scFv antibody was successfully purified using Ni-NTA affinity chromatography with a yield of 120 mg/l. The SZ-2 scFv antibody could bind to platelets demonstrated by enzyme-linked immunosorbent assay (ELISA) and flow cytometry. Analyzed by Western blot, it could bind to platelet GPIb. It retained the binding capacity of its parental SZ-2 monoclonal antibody (MoAb). In functional studies, SZ-2 scFv inhibited platelet agglutination and aggregation induced by ristocetin and thrombin, respectively, but had no effect on ADP-induced platelet aggregation. Therefore, SZ-2 scFv has the potential to be used as an antithrombotic agent. (C) 2003 Elsevier Science Ltd. All rights reserved.