基于iTRAQ技术的小瓜虫入侵相关蛋白差异研究及候选抗原筛选

Ichthyophthirius multifiliis is one of the most common and serious parasitic diseases of freshwater fish in aquaculture, and can result in heavy economic losses for aquaculture. In this study, isobaric tags for relative and absolute quantification (iTRAQ) combined with MALDI-TOF-TOF/ MS proteomics approach was first applied to screen differentially expressed proteins from infective and non-infective I.multifiliis.Then,the function and regulation of the differentially expressed proteins would be understood by bioinformatics analysis.To sereen candidate antigens for the development of vaccine against I.multifiliis, degenerate oligonucleotide primers were designed based on the identified amino acid sequences and prokaryotic expression of differentially expressed proteins gene was carried out.The results of our study may provided the basis to reveal the role of differentially expressed proteins in infecting hosts, then helps to provide new strategy for the control of I.multifiliis.

小瓜虫（Ichthyophthirius multifilis）是一种世界性分布的淡水鱼类专性寄生虫，给世界水产养殖业造成了巨大的损失。本研究采用同位素相对标记和绝对定量技术(iTRAQ)结合MALDI-TOF-TOF/ MS，对具有感染能力和不具有感染能力的小瓜虫进行比较蛋白组学研究，筛选出可能与小瓜虫入侵宿主相关的差异蛋白，并通过生物信息学手段对差异蛋白质进行功能注释；根据差异蛋白的氨基酸序列设计简并引物，克隆差异蛋白基因，构建载体并将质粒转化到大肠杆菌中进行表达，产物经纯化复性后通过体外免疫保护试验筛选小瓜虫候选抗原分子，为进一步阐明小瓜虫入侵宿主机制、小瓜虫基因工程疫苗的研制奠定基础，为小瓜虫病的防治提供新的技术手段。

项目利用iTRAQ技术研究了小瓜虫不同时期差异蛋白表达情况，并获得了1520个差异蛋白。其中滋养体特有的656个，掠食体特有84个。并利用多种软件对差异蛋白进行GO分类和KEGG Pathway分析，初步分析和预测其生物学功能，初步筛选鉴定出42个入侵相关蛋白；并克隆了其中抑动蛋白，丙酮酸激酶，α-微管蛋白等3个候选抗原基因，并进行表达、纯化；经二次注射免疫后进行免疫效果评价，其中抑动蛋白免疫效果最佳，确定其为最佳候选抗原，其对小瓜虫的免疫保护率为85%。同时，项目制备了小瓜虫口服型乳酸菌口服疫苗，经口服免疫4周后可显著降低小瓜虫感染率以及死亡率，取得较好的免疫效果。通过项目实施发表SCI论文4篇，核心期刊论文1篇，申请专利2项，其中获得授权发明专利1项。