中国寄生虫学与寄生虫病杂志 ›› 2020, Vol. 38 ›› Issue (2): 159-165.doi: 10.12140/j.issn.1000-7423.2020.02.005

• 论著 • 上一篇    下一篇

伯氏疟原虫静息巯基氧化酶的生物信息学分析和体外酶活检测

郑文琪1, 冯学敏2, 曹雅明3, 韩艳秋1, 王俊瑞1,*()   

  1. 1 内蒙古医科大学附属医院 检验科,呼和浩特 010050
    2 内蒙古医科大学附属医院 外科实验室,呼和浩特 010050
    3 中国医科大学基础医学院免疫学教研室,沈阳110021
  • 收稿日期:2019-08-29 出版日期:2020-04-30 发布日期:2020-05-11
  • 通讯作者: 王俊瑞
  • 作者简介:郑文琪(1988-),女,博士,助理研究员,从事疟原虫的抗感染免疫方面研究。E-mail: zhengwenqi2011@163.com
  • 基金资助:
    国家自然科学基金(81760367);内蒙古自治区卫生和计划生育委员会科研计划项目(201703106);内蒙古自治区高等学校“青年科技英才计划”(NJYT-20-B14)

Bioinformatics analysis and enzymatic activity test of quiescin sulfhydryl oxidase from Plasmodium berghei

Wen-qi ZHENG1, Xue-min FENG2, Ya-ming CAO3, Yan-qiu HAN1, Jun-rui WANG1,*()   

  1. 1 Department of Laboratory Medicine, Hohhot 010050, China
    2 Laboratory of Surgery, Affiliated Hospital of Inner Mongolia Medical University, Hohhot 010050, China
    3 Department of Immunology, College of Basic Medical Sciences, China Medical University, Shenyang 110021, China
  • Received:2019-08-29 Online:2020-04-30 Published:2020-05-11
  • Contact: Jun-rui WANG
  • Supported by:
    Supported by the National Natural Science Foundation of China(81760367);the Scientific Research Project of Health and Family Planning Commission of Inner Mongolia(201703106);the Youth Science and Technology Talents Program of Colleges and Universities of Inner Mongolia(NJYT-20-B14)

摘要:

目的 克隆、表达伯氏疟原虫静息巯基氧化酶(PbQSOX)基因,纯化重组PbQSOX蛋白(rPbQSOX),并对其生物信息学特点和酶活性进行分析。方法 分别采用BLAST、SMART、ClustalW对PbQSOX蛋白的信号肽、蛋白结构域、活性位点、同源性进行分析;通过BLAST和MAGA5构建PbQSOX的系统进化树;应用SWISS-MODEL对PbQSOX蛋白的三级结构进行预测。BALB/c雌性小鼠经腹腔感染伯氏疟原虫(1 × 10 7/鼠),于感染后第4天提取疟原虫基因组DNA。PCR扩增PbQSOX基因,经测序正确后,将目的片段连接至原核表达载体pET30a(+),构建重组质粒pET30a(+)-PbQSOX。将鉴定正确的重组质粒转化至大肠埃希菌BL21中,经异丙基-β-D-硫代半乳糖苷(IPTG)诱导后,取菌液进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质免疫印迹(Western blotting)分析;应用镍氨三乙酸琼脂糖柱纯化可溶性的rPbQSOX,并以三(2-羧乙基)膦(TCEP)或二硫苏糖醇(DTT)为酶反应底物分析rPbQSOX的酶活性。 结果 BLAST分析结果鉴定出了PbQSOX大部分其他物种的同源基因。多序列蛋白比对与蛋白结构域分析结果显示,PbQSOX蛋白含1个信号肽,存在Trx1、ψErv和Erv/ALR结构域,但完全缺失了Trx2结构域。PbQSOX蛋白含3个关键的CXXC活化基序(C为半胱氨酸Cys,X为任意氨基酸),即CPAC、CRNC和CNYC;采用MAGA5邻接法构建的系统进化树显示,PbQSOX与约氏疟原虫QSOX(PyQSOX)的亲缘关系最近;SWISS-MODEL同源建模法分析结果显示,用已知的布氏锥虫QSOX(TbQSOX)可预测PbQSOX蛋白的三级结构。PCR扩增PbQSOX基因,获得1 486 bp目的条带,经测序为PbQSOX基因序列;pET30a(+)-PbQSOX质粒经 Nde Ⅰ、Hind Ⅲ双酶切与测序鉴定正确。SDS-PAGE和Western bloting分析结果显示,rPbQSOX重组蛋白主要以可溶形式表达,相对分子质量约59 000。纯化后的rPbQSOX对TCEP的催化活性为0.84 ± 0.18,DTT的催化活性为0.78 ± 0.14(P < 0.01)。结论 PbQSOX蛋白为单Trx1结构域的伯氏疟原虫静息巯基氧化酶,构建、表达的rPbQSOX蛋白为可溶性蛋白,具有一定的静息巯基氧化酶催化活性。

关键词: 疟原虫, 生物信息学, 蛋白表达, 伯氏疟原虫静息巯基氧化酶

Abstract:

Objective To clone and express the gene of Plasmodium berghei quiescin sulfhydryl oxidase (PbQSOX), purify the recombinant protein rPbQSOX, and analyze its bioinformatics characteristics and enzymatic activity.Methods The signal peptides, protein domains, active sites and homology of PbQSOX protein were analyzed by BLAST, SMART and ClustalW softwares. The phylogenetic tree of PbQSOX was constructed by BLAST and MAGA5. The tertiary structure of PbQSOX protein was predicted by SWISS-MODEL. Female BALB/c mice were intraperitoneally injected with 1 × 10 7 P. berghei protozoan/mouse. On day 4 after infection, P. berghei genomic DNA was extracted. The PbQSOX gene was amplified by PCR. After confirming the product by sequencing, the target product was cloned into pET30a(+) vector to construct recombinant plasmid pET30a(+)-PbQSOX. The verified plasmid was transfected into E. coli BL-21 after induction by isopropyl β-D-thiogalactoside, the bacteria culture medium was analysed underwent by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. The soluble recombinant PbQSOX (rPbQSOX) was purified by Ni-triacetate agarose column, and the enzyme activity was analyzed using TCEP [Tris(2-carboxyethyl)phosphine] or DL-Dithiothreitol (DTT) as the enzyme reaction substrate. Results BLAST analysis identified homologues of PbQSOX from other species. Multiple-sequence blasting and domain analysis showed that PbQSOX contained a signal peptide, and Trx1, ψErv and Erv/ALR domains, but lacked the Trx2 domain. The PbQSOX protein contained three key CXXC activation motifs (C, cysteine; X, any amino acid): CPAC, CRNC and CNYC. The phylogenetic tree constructed by MAGA5 using the neighbor joining method showed that the PbQSOX was genetically most close to the P. yoelii QSOX (PyQSOX). SWISS-MODEL analysis showed that the tertiary structure of PbQSOX can be predicted using known Trypanosoma brucei QSOX (TbQSOX). PCR amplification of PbQSOX resulted in a specific band of 1 486 bp, sequence of which was confirmed by sequencing. The PET30a (+) -PbQSOX plasmid was verified by sequencing after Nde Ⅰ and Hind Ⅲ digestion. SDA-PAGE and Western blotting analysis showed that rPbQSOX was expressed in soluble form with a relative molecular weight of about 59 000. The enzymatic activities of purified rPbQSOX toward TCEP and DTT were 0.84 ± 0.18 and 0.78 ± 0.14, respectively.Conclusion The expressed PbQSOX protein contains a single Trx1 domain. The rPbQSOX is a soluble protein that has some catalytic activity of the quiescin sulfhydryl oxidase.

Key words: Plasmodium, Bioinformatics, Protein expression, Plasmodium berghei quiescin sulfhydryl oxidase

中图分类号: