CAJ | 학술논문

目的探索Trp1707Ser突变对重组凝血因子Ⅷ轻链(rFⅧLC)与血管性血友病因子(VWF)结合作用的影响机制.方法以长链PCR法构建野生型和Trp1707Ser突变型rFⅧLC原核表达质粒,应用BL21原核表达系统进行蛋白表达,梯度复性法对表达蛋白进行结构复性,SDS-PAGE和Western blot对所得蛋白进行鉴定.复性后蛋白使用GST柱纯化并收集,表面等离子共振法(SPR)检测B区缺失重组FⅧ(BDD-rFⅧ)、野生型和突变型rFⅧLC与VWF的结合活性.结果 SDS-PAGE电泳和Western blot结果显示所表达蛋白(相对分子质量110×103)与目的蛋白相符.蛋白纯化峰图显示野生型蛋白表达量高于突变型.SPR检测结果显示BDD-rFⅧ、野生型rFⅧLC、突变型rFVⅧLC蛋白与VWF的结合活性均呈浓度依赖性增强.BDD-rFⅧ与VWF结合KD值(12.2)明显低于野生型rFⅧLC(48.9)和突变型rFⅧLC(46.3).结论 Trp1707Ser突变对原核表达rFⅧLC与VWF的结合作用无明显影响,重链对FⅧ与VWF结合作用的影响更为重要.
목적 탐색Trp1707Ser돌변대중조응혈인자Ⅷ경련(rFⅧLC)여혈관성혈우병인자(VWF)결합작용적영향궤제.방법 이장련PCR법구건야생형화Trp1707Ser돌변형rFⅧLC원핵표체질립,응용BL21원핵표체계통진행단백표체,제도복성법대표체단백진행결구복성,SDS-PAGE화Western blot대소득단백진행감정.복성후단백사용GST주순화병수집,표면등리자공진법(SPR)검측B구결실중조FⅧ(BDD-rFⅧ)、야생형화돌변형rFⅧLC여VWF적결합활성.결과 SDS-PAGE전영화Western blot결과현시소표체단백(상대분자질량110×103)여목적단백상부.단백순화봉도현시야생형단백표체량고우돌변형.SPR검측결과현시BDD-rFⅧ、야생형rFⅧLC、돌변형rFVⅧLC단백여VWF적결합활성균정농도의뢰성증강.BDD-rFⅧ여VWF결합KD치(12.2)명현저우야생형rFⅧLC(48.9)화돌변형rFⅧLC(46.3).결론 Trp1707Ser돌변대원핵표체rFⅧLC여VWF적결합작용무명현영향,중련대FⅧ여VWF결합작용적영향경위중요.
Objective To disclose the impact of Trp1707Ser mutation on the binding mechanism ofrFⅧ light chain (rFⅧ LC) with VWF.Methods Using long-chain PCR technique,we constructed rF Ⅷ LC plasmids of both wild type and Trp 1707Ser mutant type.BL21 competent cells were used for protein expression.Gradient renaturation was employed to refold protein.SDS-PAGE and Western blot were performed to identify the molecular weight of expressed protein.GST-Sefinose was used for protein purification and surface plasmon resonance (SPR) was employed to detect binding of B-domain-deleted rF Ⅷ (BDD-rFⅧ),wild and mutant rFⅧ LC with VWF,respectively.Results The results of SDS-PAGE and Western blot showed a molecular weight of 110× 103 of expressed proteins,which were consistent with objective proteins.The expression quantity of wild type was higher than that of mutant type.A concentration-dependent combination of the 3 testing proteins with VWF was found.The KD value of BDDrFⅧ (12.2) was lower than that of both rFⅧ LCs (wild type 48.9 and mutant type 46.3),whereas there was no discrepancy between wild rFⅧ LC and mutant rFⅧ LC.Conclusions Trp1707Ser mutation didn't impact the binding of rFⅧ LC expressed by BL21 competent cells with VWF.The heavy chain played a more important role in impacting the binding of FⅧ with VWF.