CAJ | 학술논문

背景与目的:研究靶向血栓蛋白(RGD)_3-tTF.融合蛋白结合肿瘤血管标志物整合素α_vβ_3的能力,旨在探讨融合蛋白的RGD多肽数量和化学结构与其结合整合素α_vβ_3能力的关系及其意义. 材料与方法:用3个串联的RGD多肽与截短组织因子(truncated tissue factor,tTF)合成融合基因rRGD),-tTF,表达于大肠杆菌(E.coli)BL21(DE,),用镍柱纯化融合蛋白.通过凝血实验检测融合蛋白tTF组分的活性,运用间接酶联免疫吸附试验(ElJSA)分析其特异性结合整合素α_vβ_3的能力.并与RGD-tTF融合蛋白的活性进行比较.结果:(RGD)_3-tTF与RGD-tTF融合蛋白凝血活性相似(F=0.019,P>0.05),但(RGD)_3-tTF融合蛋白特异性结合整合素α_vβ_3的能力明显升高(F=140.17,P<0.01).当融合蛋白浓度为0.24 μmol/L时,(RGD)_3tTF融合蛋白的OD_(405nm)值是RGD-tTF融合蛋白的1.32倍(1.25/0.95).结论:(RGD)_3-tTF融合蛋白带有两个二硫键和3个RGD多肽,保留了组织因子凝血活性的同时,提高了与整合素α_vβ_3特异性结合的能力,为开展选择性肿瘤血管血栓靶向治疗的动物实验奠定了基础.
배경여목적:연구파향혈전단백(RGD)_3-tTF.융합단백결합종류혈관표지물정합소α_vβ_3적능력,지재탐토융합단백적RGD다태수량화화학결구여기결합정합소α_vβ_3능력적관계급기의의. 재료여방법:용3개천련적RGD다태여절단조직인자(truncated tissue factor,tTF)합성융합기인rRGD),-tTF,표체우대장간균(E.coli)BL21(DE,),용얼주순화융합단백.통과응혈실험검측융합단백tTF조분적활성,운용간접매련면역흡부시험(ElJSA)분석기특이성결합정합소α_vβ_3적능력.병여RGD-tTF융합단백적활성진행비교.결과:(RGD)_3-tTF여RGD-tTF융합단백응혈활성상사(F=0.019,P>0.05),단(RGD)_3-tTF융합단백특이성결합정합소α_vβ_3적능력명현승고(F=140.17,P<0.01).당융합단백농도위0.24 μmol/L시,(RGD)_3tTF융합단백적OD_(405nm)치시RGD-tTF융합단백적1.32배(1.25/0.95).결론:(RGD)_3-tTF융합단백대유량개이류건화3개RGD다태,보류료조직인자응혈활성적동시,제고료여정합소α_vβ_3특이성결합적능력,위개전선택성종류혈관혈전파향치료적동물실험전정료기출.
BACKGROUND AND AIM: To study the ability off RGD)_3-tTF thrombosis targeted protein binding to tumor vascular markers such as integrin α_vβ_3 and to investigate the relationship between the targeting ability to integrin α_vβ_3 and the chemical structure of RGD polypeptide of the fusion protein. MATERIALS AND METHODS: The (RGD) _3-tTF fusion gene was synthesized by the truncated tissue factor (tTF) and three RGD polypeptides in series and expressed in Escherichia coli(E.coli)BL21 (DE_3) . The fusion protein was then purified by Nickel affinity chromatography column. The activity of the tTF fusion protein was evaluated by clotting assay. The binding ability to intergin α_vβ_3 specially was analyzed by indirect enzyme linked immunosorbent assay (ELISA), and compared with the activity of RGD-tTF fusion protein. RESULTS: The coagulation activity of (RGD)_3-tTF and RGD-tTF purified fusion protein was alike (F = 0.019,P > 0.05). However, the ability of the (RGD)_3-tTF fusion protein binding to intergrin α_vβ_3 specifically was elevated significantly (F= 140.17, P< 0.01). When the concentration was 0.24 μmol/L, the OD_(405)nm of the (RGD) _3-tTF fusion protein was 1.3 times more than that of RGD-tTF fusion protein(1.25/0.95) . CONCLUSION: Because the (RGD)_3 -tTF fusion protein has two disufides and three RGD polypeptides, the coagulation activity of tissue factor was retained and the ability binding to integrin α_vβ_3 was increased significantly.It may provide a foundation for the further study of thrombosis-targeted therapy in animal tumor models.