Yin Li-Tian, Zhu Jian-Jiang, Li Run-Hua, Wang Hai-Long, Li Ya-Qing, Yin Guo-Rong
Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. 2014 Feb;32(1):24-8.
To predict the physicochemical properties and antigenic epitopes of Toxoplasma gondii uridine phosphorylase (TgUPase), clone, and express TgUPase gene, and analyze its immunoreactivity.
The physical and chemical characters and specific epitopes of TgUPase protein were predicted by bioinformatics software tools. Total RNA was extracted from RH strain T. gondii tachyzoites. A pair of specific primers was designed according to the open reading frame of TgUPase gene (GenBank Accession No. DQ385446.1). RT-PCR product was digested with restriction enzyme and ligated into a pET-30a(+) vector. The recombinant plasmid pET-30a(+)-TgUPase was transformed into E. coli DH5alpha and the positive clones were selected by colony PCR and confirmed by double restriction enzyme digestion and sequencing. The constructed pET-30a(+)-TgUPase was then transformed into E. coli BL21(DE3) and induced with IPTG for expression. The expression product was analyzed through SDS-PAGE followed by Coomassie blue staining. Western blotting assay with His primary antibody and human anti-T. gondii serum was used to confirm the expression of rTgU-Pase and detect its immunoreactivity.
Bioinformatics prediction results showed that rTgUPase protein was 303 amino acids in length with a predicted molecular mass of M, 33 042.9, and this soluble protein had three potential T/B cell epitopes. The product of RT-PCR was 921 bp. Colony PCR, double restriction enzyme digestion and DNA sequencing confirmed that the recombinant plasmid pET-30a(+)-TgUPase was constructed. SDS-PAGE showed that bacteria containing recombinant plasmid pET-30a(+)-TgUPase expressed a soluble protein of His-TgUPase (about Mr 38,000) after being induced with IPTG. The recombinant protein reacted positively with His primary antibody and human anti-T. gondii serum by Western blotting analysis.
The recombinant plasmid pET-30a (+)-TgUPase is constructed and the soluble rTgUPase shows immunoreactivity.
预测刚地弓形虫尿苷磷酸化酶（TgUPase）的理化性质和抗原表位，克隆并表达TgUPase基因，分析其免疫反应性。
采用生物信息学软件工具预测TgUPase蛋白的理化特性和特异性表位。从刚地弓形虫RH株速殖子中提取总RNA。根据TgUPase基因（GenBank登录号：DQ385446.1）的开放阅读框设计一对特异性引物。RT-PCR产物经限制性内切酶酶切后连接到pET-30a(+)载体。将重组质粒pET-30a(+)-TgUPase转化至大肠杆菌DH5α，通过菌落PCR筛选阳性克隆，并经双酶切和测序鉴定。将构建好的pET-30a(+)-TgUPase转化至大肠杆菌BL21(DE3)，用异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达。表达产物经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE），考马斯亮蓝染色分析。用His一抗和人抗刚地弓形虫血清进行蛋白质印迹分析，以确认重组TgU-Pase的表达并检测其免疫反应性。
生物信息学预测结果显示，重组TgUPase蛋白长度为303个氨基酸，预测分子量为33 042.9，该可溶性蛋白有3个潜在的T/B细胞表位。RT-PCR产物为921 bp。菌落PCR、双酶切和DNA测序证实构建了重组质粒pET-30a(+)-TgUPase。SDS-PAGE显示，含重组质粒pET-30a(+)-TgUPase的细菌经IPTG诱导后表达出可溶性的His-TgUPase蛋白（约38 000）。蛋白质印迹分析显示，重组蛋白与His一抗和人抗刚地弓形虫血清呈阳性反应。
构建了重组质粒pET-30a(+)-TgUPase，可溶性重组TgUPase具有免疫反应性。
