Cloning and prokaryotic expression of Cryptocaryon irritans α-tubulin gene and its mRNA expression levels in different life history stages

Abstract

PDF HTML阅读 XML下载 导出引用 引用提醒 刺激隐核虫α-微管蛋白基因的克隆、原核表达及在生活史不同阶段的mRNA表达 DOI: 作者: 作者单位: 1. 集美大学水产学院, 福建 厦门 361021;2. 福建农林大学动物科学学院, 福建 福州 350002;3. 莆田市水产科学研究所, 福建 莆田 351100;4. 福建省水产研究所, 福建 厦门 361013 作者简介: 孙嘉阳(1992-),女,硕士研究生,从事分子生物学研究.E-mail:344156662@qq.com 通讯作者: 中图分类号: S917 基金项目: 福建省海洋与渔业厅五新项目（201209200023）；福建省科技厅高校产学合作项目（2017N5002）. Cloning and prokaryotic expression of Cryptocaryon irritans α-tubulin gene and its mRNA expression levels in different life history stages Author: Affiliation: 1. Fisheries College, Jimei University, Xiamen 361021, China;2. Animal Science College, Fujian University of Agriculture, Fuzhou 350002, China;3. Putian Institute of Fishery Sciences, Putian, 351100, China;4. Fujian Fisheries Research Institute, Xiamen, 361013, China Fund Project: 摘要 | 图/表 | 访问统计 | 参考文献 | 相似文献 | 引证文献 | 资源附件 | 文章评论 摘要:从本实验室已有刺激隐核虫（）转录组数据中筛选获得α-微管蛋白基因片段，利用5'RACE和3'RACE技术首次克隆获得其cDNA，全长为1602 bp，包含1356 bp的开放阅读框，编码451个氨基酸，预测蛋白质分子量为49.78 kD。生物信息学分析表明α-微管蛋白为亲水性非跨膜蛋白，氨基酸序列的第142~148位有特异且保守的GTP核苷酸结合位点（GGGTGSG）。对刺激隐核虫α-微管蛋白氨基酸序列进行同源性比对及进化树分析，发现其与间日疟原虫（Euplotesoct ocarinatus）、尾刺耐格里原虫（）等的序列一致性高达94%~95%，且在系统进化树上聚为一支。采用实时荧光定量PCR技术对α-微管蛋白基因在刺激隐核虫3个生活史阶段的表达进行检测，结果显示α-微管蛋白基因的表达量在纤毛虫时期显著高于包囊和滋养体时期（<0.05）。我们进一步构建了α-微管蛋白重组表达载体，并转化至大肠杆菌表达菌株BL21（DE3）和Rosetta （DE3）中进行原核表达，SDS-PAGE分析表明，诱导表达的重组蛋白分子量约为50 kD，与预测的结果一致，即成功诱导表达α-微管蛋白。本实验结果为后续制备α-微管蛋白有效亚单位疫苗防治刺激隐核虫病奠定了基础。 Abstract:is an extremely destructive ciliate parasite of marine fishes, which results in severe economic costs in mariculture. Its life history is divided into three stages:trophont, tomont, and theront. Tubulin is a major component of the cytoskeleton, cilia, and flagella, which plays an important role in maintaining cell morphology, intracellular trafficking, cell division, ciliary, and flagella motility. At the same time, many studies showed that tubulin has immunogenicity and can be used as a target antigen vaccine. In this study, the α-tubulin gene fragment was obtained from the transcriptome data in our laboratory. Full-length cDNA of α-tubulin was cloned for the first time by 5'RACE and 3'RACE. The results showed that full-length cDNA of α-tubulin is 1602 bp containing a 1356 bp open reading frame, which encodes proteins with 451 amino acids. The predicted molecular mass of α-tubulin is 49.78 kD. Bioinformatics analysis showed that it is a hydrophilic non-transmembrane protein. The amino acid sequence at positions 142-148 has a unique conserved GTP nucleotide binding site (GGGTGSG). Multiple sequence alignment analysis and phylogenetic analysis revealed that the α-tubulin in was integrated with other protozoa in the phylogenetic tree and had 94%-95% sequence identity with Naegleria gruberi, and . The real-time quantitative PCR technique was used to detect the expression of α-tubulin gene in the life history of . The results showed that α-tubulin gene expression was significantly higher in the theront stage than in the tomont and trophont stages ( < 0.05). Furthermore, the α-tubulin recombinant expression vector was constructed and transformed into the BL21 (DE3) and Rosetta (DE3) of the expression strain for prokaryotic expression. SDS-PAGE analysis showed that the molecular weight of the recombinant protein was approximately 50 kD, which was consistent with the predicted result, indicating that α-tubulin protein induced expression successfully. The results of this study laid the foundation for the subsequent preparation of an effective subunit vaccine. 参考文献 相似文献 引证文献