Expression purification,antibody preparation,and subcellular localization analysis of Toxoplasma gondii thioredoxin 20
10.13481/j.1671-587X.20250615
- VernacularTitle: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
- Author:
Yuyi SHI
1
;
Shengqi GAN
;
Che LIU
;
Ziwen CHENG
;
Kuo CHENG
;
Baoling YANG
;
Dawei WANG
Author Information
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
- Keywords:
Toxoplasma gondii;
Thioredoxin 20;
Subcellular localization;
Protein purification;
Polyclonal antibody
- From:
Journal of Jilin University(Medicine Edition)
2025;51(6):1595-1606
- CountryChina
- Language:Chinese
-
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.
