关键词: 刚地弓形虫, 表面抗原相关序列蛋白, SRS67, SRS20A, 生物信息学分析, 克隆

Abstract: Objective　To predict the structures and immunogenicity of surface antigen⁃related sequence protein SRS67 and SRS20A in Toxoplasma gondii using bioinformatics methods, and to generate prokaryotic expression vectors for protein expression, so as to identify the functions of recombinant SRS67 and SRS20A proteins and their potential as vaccine candidates against T. gondii. Methods　T. gondii SRS67 and SRS20A gene and amino acid sequences were downloaded from the ToxoDB database. The open reading frames (ORFs) of SRS67 and SRS20A genes were analyzed in the ORF Finder website. The relative molecular mass, isoelectric point, amino acid composition and lipophilicit index of SRS67 and SRS20A proteins were predicted using the ProtParam software. The protein hydrophilicity/hydrophobicity of was predicted using the ProtScale tool, the transmembrane regions were predicted using the TMHMM software, the signal peptides were predicted in the SignalP⁃4.1 website, the secondary and tertiary structures of the proteins were predicted in the NPS@SPOMA and SWISS⁃MODEL websites. The phosphorylation sites of the proteins were predicted using the NetPhos⁃3.1 program, the antigenic epitopes of proteins were predicted using the Immuonmedicine Group program. B⁃cell epitopes, helper T⁃cell (Th) epitopes, and cytotoxic T lymphocyte (CTL) epitopes were predicted using the IEDB and SYFPEITHI websites, and the antigenicity scores of epitopes were evaluated using the software VaxiJen 2.0 to select the dominant epitopes. Primer sequences were synthesized based on the SRS67 and SRS20A protein⁃coding gene sequences from the ToxoDB database, and SRS67 and SRS20A genes were amplified using PCR reactions with T. gondii cDNA as a template. The amplification products were subjected to double restriction⁃enzyme digestion, and the target fragments were recovered and ligated into DH5α competent cells with T4 ligase. Positive single colonies were selected and cultured, and the pET⁃32a⁃SRS67 and pET⁃32a⁃SRS20A recombinant plasmids were extracted, transformed into competent cells for induction of recombinant protein expression. The expression of recombinant proteins was determined using sodium dodecyl sulfate⁃polyacrylamide gel electrophoresis (SDS⁃PAGE) and Western blotting. Results　Bioinformatics analysis predicted that SRS67 and SRS20A genes were 633 bp and 987 bp in length, contained 7 and 15 ORFs, and encoded 210 and 328 amino acids, respectively. The SRS67 protein had a relative molecular mass of 23 135.65, a signal peptide (D = 0.590) and no transmembrane regions, contained 22 phosphorylation sites and 8 antigenic determinants, and was a hydrophilic protein. The SRS20A protein had a relative molecular mass of 34 944.91, a signal peptide (D = 0.697) and transmembrane regions, contained 39 phosphorylation sites and 15 antigenic determinants, and was a hydrophilic protein. The SRS67 and SRS20A proteins shared similar secondary structures, both containing α⁃helices, β⁃sheets, and random coils, and their tertiary structure models exhibited typical globular characteristics, with Global Model Quality Estimation scores of 0.74 and 0.77, respectively. The average antigenic propensity score was 1.046 4 for the SRS67 protein and 1.037 4 for the SRS20A protein, respectively. SRS67 and SRS20A proteins had 7 and 8 dominant B⁃cell epitopes, 10 and 20 dominant Th⁃cell epitopes, and 2 and 3 dominant CTL epitopes, respectively. As expected, the PCR amplification products of SRS67 and SRS20A genes were approximately 633 bp and 987 bp in size. The SRS67 recombinant protein exhibited the highest expression in the precipitate following induction with 0.1 mmol/L IPTG for 16 h, and the SRS20A recombinant protein showed the highest expression following induction with 0.5 mmol/L IPTG for 16 h. SDS⁃PAGE and Western blotting confirmed successful expression of the recombinant proteins. Conclusions　The SRS67 and SRS20A proteins possess multiple cellular epitopes and exhibit favorable immunogenicity. The recombinant SRS67 and SRS20A proteins have been successfully expressed, which provides the theoretical evidence for deciphering protein functions and screening effective vaccine antigens against toxoplasmosis.

Key words: Toxoplasma gondii, Surface antigen 1?releated sequence protein, SRS67, SRS20A, Bioinformatics analysis, Cloning