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

doi:10.13481/j.1671-587X.20250615

Abstract

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

CLC Number:

S855.9

Yuyi SHI,Shengqi GAN,Che LIU,Ziwen CHENG,Kuo CHENG,Baoling YANG,Dawei WANG. Expression purification, antibody preparation, and subcellular localization analysis of Toxoplasma gondii thioredoxin 20[J].Journal of Jilin University(Medicine Edition), 2025, 51(6): 1595-1606.

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References 24

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Primer	Sequence（5'-3'）	Product length（bp）
Trx20（BamH Ⅰ）-F	AAGGATCCATGGCGCCTCTGCGTGTGT	1 200
Trx20（Not Ⅰ）-R	TTGCGGCCGCCAGTTCGTCCTTCTTGTC	1 200

Amino acid	Content
Alanine	7.1
Arginine	2.8
Asparagine	2.4
Aspartic acid	6.6
Cysteine	1.9
Glutamine	2.8
Glutamic acid	6.1
Glycine	4.2
Histidine	1.9
Tryptophan	3.1
Leucine	9.9
Isoleucine	11.1
Lysine	2.8
Methionine	7.1
Phenylalanine	6.4
Proline	6.8
Threonine	5.2
Tyrosine	0.7
Serine	2.4
Valine	8.7

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

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