刚地弓形虫尿苷磷酸化酶的克

发布时间：2019-06-11 09:04

【摘要】：目的预测刚地弓形虫尿苷磷酸化酶(uridine phosphorylase,TgUPase)基因编码蛋白的主要特性和抗原表位,克隆、表达TgUPase基因,并检测其免疫反应性。方法采用生物信息学在线分析程序和软件预测TgUPase蛋白的理化性质和抗原表位。提取RH株弓形虫速殖子总RNA。根据TgUPase基因全长编码序列(GenBank登录号为DQ385446.1)的开放阅读框设计引物,并用RT-PCR扩增,扩增产物经双酶切后连接入pET-30a(+)载体。用重组质粒转化大肠埃希菌(E.coli)DH5α,阳性菌落经PCR、双酶切和测序鉴定。将重组质粒pET-30a(+)-TgUPase转化至E.coli BL21(DE3),并加入异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)结合考马斯亮蓝染色检测表达产物,分别以抗His标签抗体和人抗弓形虫血清为一抗,蛋白质印迹(Western blotting)分析重组蛋白及其免疫反应性。结果生物信息学预测结果显示,TgUPase蛋白由303个氨基酸组成,相对分子质量(Mr)为33 042.9,为可溶性蛋白,有3个潜在的T/B细胞联合抗原表位。RT-PCR扩增产物约为921 bp。菌落PCR、双酶切以及测序结果表明,重组质粒pET-30a(+)-TgUPase构建成功。SDS-PAGE结果表明,经IPTG诱导获得约Mr38 000的可溶性重组蛋白(带His标签)。Western blotting结果显示,重组蛋白能被His标签抗体和人抗弓形虫血清识别。结论成功构建了重组质粒pET-30a(+)-TgUPase,获得刚地弓形虫重组尿苷磷酸化酶,且重组蛋白具有免疫反应性。
[Abstract]:Objective to predict the main characteristics and antigenic epitopes of uridine phosphorase (uridine phosphorylase,TgUPase) gene encoded by Toxoplasma gondii (Toxoplasma gondii) gene, clone, express TgUPase gene and detect its immunogenicity. Methods the physical and chemical properties and epitopes of TgUPase protein were predicted by bioinformatics online analysis program and software. Extraction of Total RNA. from Toxoplasma gondii Toxozoites of RH strain According to the open reading frame of the full length coding sequence of TgUPase gene (GenBank accession number DQ385446.1), primers were designed and amplified by RT-PCR. The amplified products were ligated into pET-30a () vector after double enzyme digestion. The recombinant plasmid was transformed into Escherichia coli (E.coli) DH5 伪. The positive colonies were identified by PCR, double enzyme digestion and sequencing. The recombinant plasmid pET-30a ()-TgUPase was transformed into E.coli BL21 (DE3) and induced by isopropyl-尾-D-galactoside (IPTG). Sodium lauryl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) combined with Coomassie brilliant blue staining was used to detect the expressed product. Anti-His labeled antibody and human anti-Toxoplasma gondii serum were used as the first antibody, respectively. The recombinant protein and its immunoreactivity were analyzed by Western imprinting (Western blotting). Results the bioinformatics prediction results showed that the TgUPase protein was composed of 303 amino acids and the relative molecular weight (Mr) was 33 042.9, which was soluble protein. There are three potential T / B cell combined antigenic epitopes. RT-PCR amplification product is about 921 bp. The results of double restriction enzyme digestion and sequencing of colony PCR, showed that the recombinant plasmid pET-30a ()-TgUPase was successfully constructed. The results of SDS-PAGE showed that about Mr38 000 soluble recombinant protein was obtained by IPTG induction (). Western blotting with His tag). The recombinant protein can be recognized by His label antibody and human anti-Toxoplasma gondii serum. Conclusion the recombinant uridine phosphorase of Toxoplasma gondii was successfully constructed by recombinant plasmid pET-30a ()-TgUPase, and the recombinant protein was immunoreactive.
【作者单位】：山西医科大学生理学系细胞生理学教育部重点实验室;山西医科大学医学寄生虫学研究所;太原师范学院生物系;
【基金】：国家自然科学基金(No.81071374) 山西省自然科学基金(No.2013011059-4)~~
【分类号】：R382.5

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