Construction and functional evaluation of a single-chain antibody fusion protein with fibrin targeting and thrombin inhibition after activation by factor Xa
Construction and functional evaluation of a single-chain antibody fusion protein with fibrin targeting and thrombin inhibition after activation by factor Xa
复制标题
DOI：
10.1161/01.cir.101.10.1158
复制
发表时间：
2000-03-14
期刊：
影响因子：
37.8
通讯作者：
Bode, C
中科院分区：
文献类型：
--
作者：
Peter, K;Graeber, J;Bode, C
背景-重组技术被用来生产一种新的抗凝剂，它优先定位在凝块位点并具有活性。方法和结果-通过聚合酶链式反应（PCR）与杂交瘤cDNA扩增纤维蛋白特异性抗体的重链和轻链的可变区。为了获得功能性单链抗体(scFv)，通过重叠PCR引入由(Gly(4)Ser)(3)组成的连接区，将scFv克隆与编码M13噬菌体pill蛋白的DNA连接后，通过对纤维蛋白的Bβ15-22肽(β-肽)进行10轮淘选来选择高亲和力克隆。水蛭素通过基因技术融合到轻链可变区的 C 末端。为了在血块部位释放水蛭素功能必需的 N 末端，在 scFv(59D8) 和水蛭素之间引入了 Xa 因子识别位点。融合蛋白通过 SDS-PAGE 上的大小 (36 kDa)、蛋白质印迹、通过其切割成 29-kDa（单独的单链）和 7-kDa 来表征。 （水蛭素）片段，通过其与 β 肽的结合，以及 Xa 裂解后的凝血酶抑制。最后，该融合蛋白在体外比天然水蛭素更有效地抑制全血凝块的同位生长。结论：构建了一种融合蛋白，该融合蛋白与纤维蛋白特异性表位结合，并在被因子Xa激活后抑制凝血酶。这种重组抗凝剂可有效抑制体外凝块的生长。其低成本、高效、快速的生产应有助于大规模评估，以确定是否可以在没有全身出血并发症的情况下实现有效的局部抗凝血酶活性。
Background-Recombinant technology was used to produce a new anticoagulant that is preferentially localized and active at the site of the clot.Methods and Results-The variable regions of the heavy and light chains of a fibrin-specific antibody were amplified by polymerase chain reaction (PCR) with hybridoma cDNA. To obtain a functional single-chain antibody (scFv), a linker region consisting of (Gly(4)Ser)(3) was introduced by overlap PCR, After the scFv clones were ligated with DNA encoding the pill protein of the M13 phage, high-affinity clones were selected by 10 rounds of panning on the B beta 15-22 peptide of fibrin (beta-peptide). Hirudin was genetically fused to the C-terminus of the variable region of the light chain. To release the functionally essential N-terminus of hirudin at the site of a blood clot, a factor Xa recognition site was introduced between scFv(59D8) and hirudin. The fusion protein was characterized by its size on SDS-PAGE (36 kDa), by Western blotting, by its cleavage into a 29-kDa (single chain alone) and 7-kDa. (hirudin) fragment, by its binding to beta-peptide, and by thrombin inhibition after Xa cleavage. Finally, the fusion protein inhibited appositional growth of whole blood clots in vitro more efficiently than native hirudin.Conclusions-A fusion protein was constructed that binds to a fibrin-specific epitope and inhibits thrombin after its activation by factor Xa. This recombinant anticoagulant effectively inhibits appositional clot growth in vitro. Its efficient and fast production at low cost should facilitate a large-scale evaluation to determine whether an effective localized antithrombin activity can be achieved without systemic bleeding complications.