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

摘要： 目的　采用生物信息学方法预测刚地弓形虫表面抗原相关序列蛋白SRS67和SRS20A的结构和免疫原性，并构建原核表达载体进行蛋白表达，以探讨重组蛋白功能及成为弓形虫病疫苗候选分子的潜力。方法　自ToxoDB数据库下载刚地弓形虫SRS67和SRS20A基因及氨基酸序列，于ORF Finder网站对SRS67和SRS20A基因开放阅读框进行分析。采用ProtParam软件预测刚地弓形虫SRS67和SRS20A蛋白相对分子质量、等电点、氨基酸组成及脂溶性指数等理化性质，采用ProtScale工具预测蛋白亲/疏水性，采用TMHMM软件预测蛋白跨膜区域，在SignalP⁃4.1网站预测蛋白信号肽，通过NPS@SPOMA和SWISS⁃MODEL网站预测蛋白的二级结构和三级结构，采用NetPhos⁃3.1程序预测蛋白磷酸化位点，应用Immuonmedicine Group程序预测蛋白抗原决定簇，通过IEDB网站和SYFPEITHI网站分别预测蛋白B细胞表位、辅助性T细胞（helper T cell，Th）抗原表位和细胞毒性T淋巴细胞（cytotoxic T lymphocyte，CTL）抗原表位，并采用VaxiJen 2.0软件对表位进行抗原性评分以筛选优势表位。根据ToxoDB数据库中刚地弓形虫SRS67和SRS20A基因序列合成引物序列，以弓形虫cDNA为模板，通过PCR反应扩增SRS67和SRS20A基因，并将扩增产物基因进行双酶切，回收目的片段。通过T4连接酶将目的片段转化至DH5α感受态细胞中，挑取阳性单菌落培养后提取pET⁃32a⁃SRS67和pET⁃32a⁃SRS20A重组质粒，并将其转化至表达感受态中进行小量诱导表达。采用十二烷基硫酸钠⁃聚丙烯酰胺凝胶电泳（sodium dodecyl sulfate ⁃ polyacrylamide gel electrophoresis，SDS⁃PAGE）及Western blotting检测重组蛋白表达水平。结果　经生物信息学预测发现，刚地弓形虫SRS67基因与SRS20A基因长度分别为633、987 bp，分别具有7个和15个开放阅读框、编码210个和328个氨基酸。SRS67蛋白相对分子质量为23 135.65，有信号肽（D = 0.590），无跨膜区，含有22个磷酸化位点、8个抗原决定簇，为亲水性蛋白；SRS20A蛋白相对分子质量为34 944.91，有信号肽（D = 0.697）和跨膜区，含39个磷酸化位点、15个抗原决定簇，为亲水性蛋白。SRS67蛋白和SRS20A蛋白二级结构相似，均包含α⁃螺旋、β⁃折叠及无规则卷曲；三级结构模型呈典型球状特征，全球模型质量评估（Global Model Quality Estimation）分值分别为0.74和0.77。SRS67蛋白和SRS20A蛋白分别具有22个和39个磷酸化位点。SRS67蛋白平均抗原倾向指数为1.046 4，含有8个抗原决定簇；SRS20A蛋白平均抗原倾向指数为1.037 4，含有15个抗原决定簇。SRS67蛋白具有7个优势B细胞表位；SRS20A蛋白具有8个优势B细胞表位。对于Th细胞表位，SRS67与SRS20A分别筛选出10个和20个优势表位；对于CTL细胞表位，SRS67与SRS20A分别筛选出2个和3个优势表位。PCR扩增SRS67基因及SRS20A基因产物长度分别约为633 bp和987 bp，与预期相符。分别构建含目的基因的重组质粒，诱导表达发现SRS67重组蛋白在0.1 mmol/L IPTG诱导16 h后在沉淀中表达量最多、SRS20A重组蛋白在0.5 mmol/L IPTG诱导16 h后在沉淀中表达量最多，且SDS⁃PAGE及Western blotting检测结果表明重组蛋白表达成功。结论　SRS67和SRS20A蛋白具有多个细胞表位和良好免疫原性。本研究成功表达了重组SRS67和SRS20A蛋白，为后续研究蛋白功能及筛选有效的弓形虫病疫苗抗原提供了理论依据。

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