青海血蜱重组蛋白Hq001的结构预测与原核表达

刘岳青, 马林源, 马静, 陈开廷, 曹美娜, 王雪薇, 王鹏, 高金亮

doi:10.11853/j.issn.1003.8280.2024.02.002

中国媒介生物学及控制杂志 >

2024 , Vol. 35 >Issue 2: 138 - 144

DOI: https://doi.org/10.11853/j.issn.1003.8280.2024.02.002

实验研究

青海血蜱重组蛋白Hq001的结构预测与原核表达

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1. 内蒙古科技大学包头医学院, 内蒙古包头 014000;
2. 鄂尔多斯市中心医院分子医学实验室, 内蒙古鄂尔多斯 017000;
3. 内蒙古科技大学包头医学院第一附属医院医学检验科, 内蒙古包头 014000;
4. 内蒙古医科大学鄂尔多斯临床医学院, 内蒙古鄂尔多斯 017000;
5. 河南中医药大学第三附属医院, 河南郑州 450003

刘岳青,男,在读硕士,主要从事蜱源抗凝血蛋白质研究,E-mail:15044857509@163.com

收稿日期: 2023-11-02

网络出版日期: 2024-05-09

基金资助

国家自然科学基金(81760375,82360406);内蒙古自治区自然科学基金(2021MS08063);内蒙古科技创新引导项目

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The recombinant protein, Hq001, in Haemaphysalis qinghaiensis: Structure prediction and prokaryotic expression

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1. Baotou Medical College, Inner Mongolia University of Science & Technology, Baotou, Inner Mongolia 014000, China;
2. Laboratory of Molecular Medicine, Ordos Central Hospital, Ordos, Inner Mongolia 017000, China;
3. Department of Medical Laboratory, The First Affiliated Hospital of Baotou Medical College, Inner Mongolia University of Science & Technology, Baotou, Inner Mongolia 014000, China;
4. Ordos School of Clinical Medicine, Inner Mongolia Medical University, Ordos, Inner Mongolia 017000, China;
5. Third Affiliated Hospital of Henan University of Traditional Chinese Medicine, Zhengzhou, Henan 450003, China

Received date: 2023-11-02

Online published: 2024-05-09

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摘要

目的对从青海血蜱唾液腺cDNA文库中克隆出的1个新基因 Hq001编码的蛋白质进行三维理论模型预测,并评估其与Kunitz型蜱源抗凝血蛋白质之间的关系。方法构建重组质粒pET-30a-Hq001并转化至感受态大肠埃希菌(Escherichia coli) BL21(DE3)中,对目的蛋白进行重组表达。使用开源软件AlphaFold v2对去除信号肽的Hq001氨基酸序列进行三级结构预测,以此得到三维理论模型。并使用开源软件GROMACS v2023对三维理论模型进行分子动力学(MD)模拟优化,应用ANOLEA和MolProbity等工具对模型质量进行评价。结果由Hq001构建的重组表达质粒pET30a-Hq001在E. coil BL21中表达后以包涵体形式存在。序列比对与结构建模结果均表明Hq001蛋白具有典型双Kunitz-BPTI型结构域,并与Bikunin、Boophilin、Ornithodorin、Ixolaris等具有抗凝血活性的Kunitz-BPTI型蛋白具有序列及结构相似性。经过1 ns的分子动力学模拟优化后理论模型均方根偏差在3 Å左右达到平衡,选取其中能量态最低的结构模型(总能量=-20 716 640 kJ/mol)作为最终模型。结论蛋白质三维理论模型证实Hq001蛋白具有保守的Kunitz-BPTI型结构域,与来自微小扇头蜱的Boophilin在序列与结构上均较为相似,Hq001蛋白可能具有Kunitz型丝氨酸蛋白酶抑制剂相似的结构与功能。

关键词： 青海血蜱; Kunitz-BPTI; AlphaFold; 格罗宁根化学模拟体系; 分子模拟; 分子动力学

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刘岳青, 马林源, 马静, 陈开廷, 曹美娜, 王雪薇, 王鹏, 高金亮 . 青海血蜱重组蛋白Hq001的结构预测与原核表达[J]. 中国媒介生物学及控制杂志, 2024 , 35(2) : 138 -144 . DOI: 10.11853/j.issn.1003.8280.2024.02.002

Abstract

Objective To construct the three-dimensional theoretical model of Hq001 protein encoded by a novel gene, Hq001, cloned from a salivary gland cDNA library of Haemaphysalis qinghaiensis, and to evaluate its relationship with tick-derived Kunitz-type anticoagulant proteins.Methods The recombinant plasmid pET-30a-Hq001 was constructed and transformed into Escherichia coli BL21 (DE3) for the expression of Hq001 protein. AlphaFold v2 software was used to predict the tertiary structure of Hq001 amino acid sequence with the signal peptide removed, to obtain a three-dimensional (3D) theoretical model. The 3D theoretical model was optimized through molecular dynamics simulation using the open-source software GROMACS v2023 and evaluated using ANOLEA and MolProbity.Results The recombinant plasmid pET30a-Hq001 was expressed as inclusion bodies in E. coli BL21. Sequence alignment and structure modeling results showed that Hq001 protein had typical double Kunitz-BPTI domains, with a similar sequence and structure to four Kunitz-BPTI-type anticoagulant proteins, such as Bikunin, Boophilin, Ornithodorin, and Ixolaris. Following 1 ns molecular dynamics (MD) simulation optimization, the root mean square deviation (RMSD) of the theoretical model attained stability at approximately 3 Å, indicative of convergence to a steady-state conformational ensemble. Concurrently, the most energetically favorable structure within this ensemble, exhibiting a total energy of -207 166 400 kJ/mol, was identified and designated as the final model.Conclusion Hq001 protein has conserved Kunitz-BPTI domains that resemble Boophilin from Rhipicephalus microplus in sequence and structure, indicating that Hq001 protein may have similar structure and function to some known Kunitz-type serine protease inhibitors.

Key words： Haemaphysalis qinghaiensis; Kunitz-BPTI; AlphaFold; GROMACS; Molecular simulation; Molecular dynamics

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