Trp1707Ser突变对重组凝血因子Ⅷ轻链与血管性血友病因子结合作用的影响机制

Study on the impact of Trp1707Ser mutation on the binding mechanism of rF Ⅷ light chain with VWF

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110×10^3）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。

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× 10^3 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.