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 <sup>*</sup>

doi:10.13523/j.cb.20180801

中国生物工程杂志

2018, Vol. 38

Issue (8): 1-9 DOI: 10.13523/j.cb.20180801

研究报告

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 ^*

李明英,王仁军,张帆,迟彦(

)

大连大学生命科学与技术学院辽宁省糖脂代谢重点实验室大连 116622

The Prokaryotic Expression and Activity Analysis of the Fifth Domain of β2GPⅠ and Its Mutants or Short Peptide Fragments

Ming-ying LI,Ren-jun WANG,Fun ZHANG,Yan CHI(

)

School of Life Science and Technology, Dalian University,Key Laboratory of Glycolipid Metabolism, Dalian 116622, China

全文: PDF(834 KB) HTML

摘要：

β2糖蛋白Ⅰ (beta 2- glycoprotein Ⅰ,β2GPⅠ) 是抗磷脂综合征(antiphospholipid syndrome,APS) 血清中抗磷脂抗体(antiphospholipid antibody,aPL)的主要抗原。β2GPⅠ 通过第五结构域与阴性磷脂oxLDL结合,进而被aPL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rDⅤ结合oxLDL以及APS血清中oxLDL的活性,表明rDⅤ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oxLDL结合的方法体系的建立,为APS早期实验室诊断奠定基础。

关键词： 抗磷脂综合征; β2糖蛋白Ⅰ; 心磷脂; 氧化低密度脂蛋白; 原核表达

Abstract:

Beta 2-glycoprotein Ⅰ (β2GPⅠ)is the main antigen of antiphospholipid antibody (aPL) in serum of antiphospholipid syndrome (APS). β2GPⅠ binding to oxLDL via its the fifth domian and then subsequently recognized by aPL is a key event in the development of APS arterial thrombosis. In this study, a prokaryotic expression vector encoding β2GPⅠ fifth domain (β2GPⅠ-DⅤ), β2GPⅠ-DⅤ mutant and the Phe280-Ala320 fragment of β2GPⅠ-DⅤ were constructed, their induced expression and purification were performed, and the molecular mechanism of the binding of β2GPⅠ-DⅤ to negative phospholipids was analyzed. Results showed that the spatial configuration of the Cys281-Cys288 and Ser311-Lys317 segments in β2GPⅠ-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 rDⅤ to oxLDL and oxLDL in the serum of APS were further examined, indicating that rDⅤ has biological activity consistent with that of natural β2GPⅠ. The obtainment of rβ2GPⅠ-DⅤ and the establishment of the method in rβ2GPⅠ-DⅤ binding to oxLDL in this study lay a foundation for the early laboratory diagnosis of APS.

Key words: Antiphospholipid syndrome Beta 2-glycoprotein Ⅰ Cardiolipin Oxidized low density protein Prokaryotic expression

收稿日期: 2018-03-23 出版日期: 2018-09-11

ZTFLH:

Q513

基金资助: 国家自然科学基金(81202536);国家自然科学基金(81270361);国家自然科学基金(30971232)

通讯作者: 迟彦 E-mail: chi_yan@126.com

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引用本文:

李明英,王仁军,张帆,迟彦. 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 ^*[J]. 中国生物工程杂志, 2018, 38(8): 1-9.

Ming-ying LI,Ren-jun WANG,Fun ZHANG,Yan CHI. The Prokaryotic Expression and Activity Analysis of the Fifth Domain of β2GPⅠ and Its Mutants or Short Peptide Fragments. China Biotechnology, 2018, 38(8): 1-9.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20180801 或 https://manu60.magtech.com.cn/biotech/CN/Y2018/V38/I8/1

图1 DⅤ、mDⅤ以及cDⅤ PCR 基因扩增

图2 表达载体酶切鉴定

图3 rDⅤ 、rmDⅤ和 rcDⅤ的诱导表达

图4 rDⅤ、rmDⅤ和 rcDⅤ的纯化

图5 rDⅤ、rmDⅤ和 rcDⅤ的Western blot分析

图6 rDⅤ、rmDⅤ和 rcDⅤ与CL结合的HPTLC和ELISA分析

表1 ELISA方法检测 rDⅤ、rmDⅤ和rcDⅤ与CL的结合活性

图7 ELISA方法检测 rβ2GPⅠ-DⅤ蛋白与oxLDL及APS血清oxLDL的结合活性

表2 ELISA方法检测 rβ2GPⅠ-DⅤ蛋白与oxLDL的结合活性

表3 ELISA方法检测 rβ2GPⅠ-DⅤ蛋白与APS血清oxLDL的结合活性

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