【摘要】 从本实验室已有刺激隐核虫(Cryptocaryon irritans)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(Trypanosoma vivax)、丹氏锥虫(Trypanosoma danilewskyi)、八肋游仆虫(Euplotes octocarinatus)、尾刺耐格里原虫(Naegleria gruberi)、眼虫(Euglena gracilis)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(P<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。更多还原

【Abstract】 Cryptocaryon irritans 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 C. irritans transcriptome data in our laboratory. Full-length cDNA of α-tubulin C. irritans 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 C. irritans was integrated with other protozoa in the phylogenetic tree and had 94%-95% sequence identity with Trypanosoma vivax, T. danilewskyi, Euplotes octocarinatus, Naegleria gruberi, and Euglena gracilis. The real-time quantitative PCR technique was used to detect the expression of α-tubulin gene in the life history of C. irritans. The results showed that α-tubulin gene expression was significantly higher in the theront stage than in the tomont and trophont stages(P < 0.05). Furthermore, the α-tubulin recombinant expression vector was constructed and transformed into the BL21(DE3) and Rosetta(DE3) of the Escherichia coli 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.更多还原