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

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S917

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    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.

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孙嘉阳,张子平,朱友芳,邹志华,韩坤煌,葛辉,王艺磊. 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[J]. Jounal of Fishery Sciences of China, 2018,[volume_no](6):1172-1182

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  • Online: December 06,2018
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