大片吸虫α-微管蛋白基因的克隆表达及免疫反应性的研究

发布时间：2018-02-24 00:14

本文关键词： 大片吸虫 α-微管蛋白基因表达诊断候选抗原　出处：《中国兽医科学》2017年04期 　论文类型：期刊论文

【摘要】：为鉴定大片吸虫α-微管蛋白基因(FgAT),并分析其作为诊断抗原的可行性,根据肝片吸虫的基因组及转录组数据设计α-微管蛋白基因的扩增引物,以大片吸虫总RN A为模板,通过RT-PCR获得大片吸虫的FgAT基因;对其进行生物信息学分析及鉴定,然后构建pGEX-6P-1(+)-FgAT重组表达载体,在大肠杆菌BL21(DE3)中进行诱导表达,再对纯化的目的蛋白进行SDS-PAGE和Western-blot分析。结果表明,大片吸虫FgAT基因的大小为1 356 bp,编码452个氨基酸残基。生物信息学分析表明,FgAT二级结构预测以无规卷曲为主,抗原表位在整个序列中均有存在。优化后的α-微管蛋白为可溶性表达,Western-blot分析结果表明其具有良好的免疫反应性。本研究首次克隆并鉴定了大片吸虫α-微管蛋白基因,表达的重组蛋白具有良好的免疫反应性,该蛋白是潜在的大片吸虫诊断靶标。
[Abstract]:In order to identify the 伪 -tubulin gene of paragonimus macrocephala, and analyze its feasibility as diagnostic antigen, a primer for amplification of 伪 -tubulin gene was designed according to the genomic and transcriptional data of paragonimiasis, and the total RNA of paragonimiasis was used as a template. The FgAT gene of paragonimiasis was obtained by RT-PCR, then the recombinant expression vector pGEX-6P-1 (pGEX-6P-1) was constructed by bioinformatics analysis and identification. The purified target protein was analyzed by SDS-PAGE and Western-blot. The result showed that the size of FgAT gene was 1 356 BP, encoding 452 amino acid residues. Bioinformatics analysis showed that the secondary structure of FGAT was mainly predicted by random coils. The antigenic epitopes were present in the whole sequence. The optimized 伪 -tubulin was expressed in a soluble way and showed good immunoreactivity. In this study, the 伪 -tubulin gene was cloned and identified for the first time. The expressed recombinant protein has good immunoreactivity and is a potential diagnostic target for paragonimiasis.
【作者单位】：黑龙江八一农垦大学动物科技学院;中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室甘肃省动物寄生虫病重点实验室;
【基金】：国家重点基础研究发展计划(973)项目(2015CB150300)
【分类号】：S852.7

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