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。
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 47700,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∶64000,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.
作者
施玉怡
甘盛琦
刘澈
程子文
程廓
杨宝玲
王大为
SHI Yuyi;GAN Shengqi;LIU Che;CHENG Ziwen;CHENG Kuo;YANG Baoling;WANG Dawei(Department of Basic Veterinary Medicine,School of Animal Husbandry and Veterinary Medicine,Jinzhou Medical University,Jinzhou 121000,China;Department of Pathogen Biology,School of Basic Medical Sciences,Jinzhou Medical University,Jinzhou 121000,China)
出处
《吉林大学学报(医学版)》
北大核心
2025年第6期1595-1606,共12页
Journal of Jilin University:Medicine Edition
基金
辽宁省教育厅青年基金项目(JYTQN2023433,JYTQN2023435)。
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
Toxoplasma gondii
Thioredoxin 20
Subcellular localization
Protein purification
Polyclonal antibody
