吉林大学学报(医学版) ›› 2025, Vol. 51 ›› Issue (6): 1595-1606.doi: 10.13481/j.1671-587X.20250615

• 基础研究 • 上一篇    

弓形虫硫氧还蛋白20的表达纯化、抗体制备和亚细胞定位分析

施玉怡1,甘盛琦1,刘澈1,程子文2,程廓2,杨宝玲2,王大为1()   

  1. 1.锦州医科大学畜牧兽医学院基础兽医学教研室,辽宁 锦州 121000
    2.锦州医科大学基础医学院 病原生物学教研室,辽宁 锦州 121000
  • 收稿日期:2024-10-26 接受日期:2025-03-01 出版日期:2025-11-28 发布日期:2025-12-15
  • 通讯作者: 王大为 E-mail:wangdawei0817@163.com
  • 作者简介:施玉怡(2000-),女,广东省汕尾市人,在读硕士研究生,主要从事人兽共患寄生虫病等方面的研究。
  • 基金资助:
    辽宁省教育厅青年基金项目(JYTQN2023433);辽宁省教育厅青年基金项目(JYTQN2023435)

Expression purification, antibody preparation, and subcellular localization analysis of Toxoplasma gondii thioredoxin 20

Yuyi SHI1,Shengqi GAN1,Che LIU1,Ziwen CHENG2,Kuo CHENG2,Baoling YANG2,Dawei WANG1()   

  1. 1.Department of Basic Veterinary Medicine,School of Animal Husbandry and Veterinary Medicine,Jinzhou Medical University,Jinzhou 121000,China
    2.Department of Pathogen Biology,School of Basic Medical Sciences,Jinzhou Medical University,Jinzhou 121000,China
  • Received:2024-10-26 Accepted:2025-03-01 Online:2025-11-28 Published:2025-12-15
  • Contact: Dawei WANG E-mail:wangdawei0817@163.com

摘要:

目的 对弓形虫硫氧还蛋白20(Trx20)进行表达纯化、抗体制备和亚细胞定位分析,为弓形虫疫苗的研制提供参考。 方法 采用生物信息学相关网站及软件对Trx20蛋白进行生物信息学分析,设计特异性引物进行目的片段扩增和原核表达载体构建,进行蛋白质的体外表达和纯化。免疫实验动物进行抗体制备,采用酶联免疫吸附试验(ELISA)法检测多克隆抗体效价,Western blotting法验证抗体的特异性和灵敏度以及确定该蛋白的天然表达情况,间接免疫荧光法(IFA)分析蛋白的亚细胞定位。 结果 生物信息学分析,Trx20蛋白是一种较稳定的亲水性蛋白,分子式为C2172H3412N548O616S20,含有424个氨基酸,预测相对分子质量为47 700,理论等电点为8.55;预测该蛋白存在1个信号肽,无跨膜区,含有1个名为“Thioredoxin like Superfamily”的结构域,并含35个磷酸化位点、1个N-糖基化位点和17个抗原决定簇;二级结构中α螺旋占氨基酸总数的41.51%,无规则卷曲占39.86%;成功构建重组质粒pET-28a-Trx20和pGEX-4T-1-Trx20,实现了可溶性重组蛋白的表达和纯化;成功制备多克隆抗体,效价高达1∶64 000,且对Trx20蛋白具有特异性识别能力;亚细胞定位,Trx20蛋白在虫体细胞质中广泛分布。 结论 刚地弓形虫Trx20蛋白是一种含有磷酸化/糖基化修饰位点和硫氧还蛋白结构域,且为定位于虫体胞质中的分泌型蛋白质。

关键词: 刚地弓形虫, 硫氧还蛋白20, 亚细胞定位, 蛋白纯化, 多克隆抗体

Abstract:

Objective To express, purify, prepare antibodies, and analyze the subcellular localization of Toxoplasma gondii thioredoxin 20(Trx20), and to provide the reference for the development of Toxoplasma gondii vaccine. Methods Bioinformatics-related websites and software were used to perform bioinformatics analysis of the Trx20 protein; specific primers were designed to amplify the target fragment and construct the prokaryotic expression vector; the protein was expressed in vitro and purified; experimental animals were immunized to prepare antibodies; enzyme-linked immunosorbent assay(ELISA) method was used to detect the titer of the polyclonal antibodies; Western blotting method was used to verify the specificity and sensitivity of the antibodies and to determine the natural expression of the protein; immunofluorescence assay (IFA) was used to analyze the subcellular localization of the protein. Results The bioinformatics analysis results showed that Trx20 protein was a relatively stable hydrophilic protein with a molecular formula of C2172H3412N548O616S20, containing 424 amino acids, a predicted relative molecular mass of 47 700, and a theoretical isoelectric point of 8.55; it was predicted that the protein had one signal peptide, no transmembrane region, contained one domain named “Thioredoxin like Superfamily”, and had 35 phosphorylation sites, one N-glycosylation site, and 17 antigenic determinants; in the secondary structure, alpha-helices accounted for 41.51% of the total amino acids, and random coils accounted for 39.86%; the recombinant plasmids pET-28a-Trx20 and pGEX-4T-1-Trx20 were successfully constructed, and the soluble recombinant protein was expressed and purified; polyclonal antibodies were successfully prepared with a titer as high as 1∶64 000, and they specifically recognized the endogenous Trx20 protein in Toxoplasma gondii; the subcellular localization results showed that Trx20 protein was widely distributed in the cytoplasm of the parasite. Conclusion Toxoplasma gondii Trx20 protein is a secretory protein containing phosphorylation/glycosylation modification sites and a thioredoxin domain, and it is localized in the cytoplasm of the parasite.

Key words: Toxoplasma gondii, Thioredoxin 20, Subcellular localization, Protein purification, Polyclonal antibody

中图分类号: 

  • S855.9