β2糖蛋白Ⅰ第五结构域及其突变体、短肽片段的原核表达及活性分析

The Prokaryotic Expression and Activity Analysis of the Fifth Domain ofβ2GPⅠ and Its Mutants or Short Peptide Fragments

β2糖蛋白Ⅰ（beta 2-glycoproteinⅠ,β2GPⅠ）是抗磷脂综合征（antiphospholipid syndrome,APS）血清中抗磷脂抗体（antiphospholipid antibody,a PL）的主要抗原。β2GPⅠ通过第五结构域与阴性磷脂ox LDL结合,进而被a PL识别,是APS动脉血栓发生的关键事件。该研究构建了编码β2GPⅠ第五结构域（β2GPⅠ-DⅤ）、β2GPⅠ-DⅤ突变体及β2GPⅠ-DⅤ的Phe280-Ala320片段的原核表达载体,对其进行诱导表达和纯化,解析了β2GPⅠ-DⅤ与阴性磷脂结合的分子机制,结果表明,β2GPⅠ-DⅤ中Cys281-Cys288以及Ser311-Lys317区段在空间上维持一定构型是与CL结合所必须的前提条件,而C245-C296,C288-C326两个二硫键在维持二者空间构型方面起到一定的作用。在此基础上,进一步检测了具有结合CL生物学活性的r DⅤ结合ox LDL以及APS血清中ox LDL的活性,表明r DⅤ具有与天然β2GPⅠ相一致的生物学活性。该研究获得的rβ2GPⅠ-DⅤ,以及与ox LDL结合的方法体系的建立,为APS早期实验室诊断奠定基础。

Beta 2-glycoprotein Ⅰ（ β2 GP Ⅰ） is the main antigen of antiphospholipid antibody（ a PL） in serum of antiphospholipid syndrome（ APS）. β2 GP Ⅰ binding to ox LDL via its the fifth domian and then subsequently recognized by a PL is a key event in the development of APS arterial thrombosis. In this study,a prokaryotic expression vector encoding β2 GP Ⅰ fifth domain（ β2 GP Ⅰ-D Ⅴ）,β2 GP Ⅰ-D Ⅴ mutant and the Phe280-Ala320 fragment of β2 GP Ⅰ-D Ⅴ were constructed,their induced expression and purification were performed,and the molecular mechanism of the binding of β2 GPⅠ-DⅤ to negative phospholipids was analyzed.Results showed that the spatial configuration of the Cys281-Cys288 and Ser311-Lys317 segments in β2 GPⅠ-DⅤ,which are maintained by the two disulfide bonds in C245-C296 and C288-C326,is a necessary precursor condition for the binding to CL. On this basis,the binding activity of r DⅤ to ox LDL and ox LDL in the serum of APS were further examined,indicating that r DⅤ has biological activity consistent with that of natural β2 GPⅠ.The obtainment of rβ2 GPⅠ-DⅤ and the establishment of the method in rβ2 GPⅠ-DⅤ binding to ox LDL in this study lay a foundation for the early laboratory diagnosis of APS.