利用蛋白抗体芯片筛选盐胁迫下红罗非鱼鳃差异表达蛋白

摘要

为从蛋白角度探索红罗非鱼(Oreochromis mossambicus♀ × O. niloticus♂)在盐胁迫环境下的调节适应机制,本研究采用蛋白抗体芯片结合串联质谱技术初步筛选了盐胁迫下红罗非鱼鳃组织的差异表达蛋白, 并通过免疫组化和免疫印迹技术对候选差异蛋白进行了验证.结果显示, 共筛选获得181个表达量有显著差异的蛋白质(变化倍数≥1.5), 包含上调蛋白142个, 下调蛋白39个.挑选5个蛋白进行质谱鉴定得到3个蛋白质: 中间丝蛋白(intermediate filament protein, IF)、转运蛋白63(translocation protein 63, SEC63)、蛋白质二硫键异构酶A3(protein disulfide-isomerase A3, PDIA3).免疫组化结果显示, 在淡水、盐度组中, IF和PDIA3在鳃小片基部均有阳性反应, 且随盐度升高, 阳性反应呈先降低后增强的趋势, SEC63无阳性反应.Western blot结果显示, 在淡水和盐度组中, IF和 PDIA3两种蛋白均有表达, 并且随盐度升高呈现先降低、再升高的变化趋势, SEC63蛋白无明显目的条带; 在30 盐度组中, 胁迫早期, IF蛋白表达量降低, 48 h达到最低值, 72 h有明显回升, PDIA3蛋白表达量在胁迫后96 h显著升高(P<0.05).根据研究结果推测, IF和PDIA3是在盐胁迫环境下红罗非鱼鳃组织的响应蛋白, 它们分别在细胞骨架、内质网功能维持中发挥重要调节作用.%Due to its rapid growth rate and excellent adaptability to saline environments, the red tilapia (Oreo-chromis mossambicus♀×O. niloticus♂)could be a suitable subject for studies on saline tolerance.In this study, to provide a theoretical basis for saline tolerance mechanisms, we aimed to screen and identify differentially ex-pressed proteins in the gill of Red tilapia using protein chips and mass spectrometry, which were subsequently verified by using immunohistochemistry and western blot techniques. There were 181 differentially expressed protein spots detected (change multiple ≥ 1.5), which included 142 up-regulated proteins and 39 down-regulated proteins. Among these, three proteins were identified by mass spectrometry from five differentially expressed proteins: intermediate filament protein (IF), translocation protein 63 (SEC63), and disulfide-isomerase A3 (PDIA3). The immunohistochemistry results showed that IF and PDIA3 are expressed in the gill base of red tilapia under both freshwater and saline water conditions, and showed a trend of initial decrease and then increase with an increase of saline concentration. However, no positive reaction was detected for SEC63. The western blot results showed IF and PDIA3 expression level changes in different saline waters similar to those observed using im-munohistochemistry. In the 30 g/L salinity group, the expression of IF protein decreased during the early stage of stress, reached the lowest value at 48 h, and thereafter began to rise significantly at 72 h, The expression of PDIA3 protein was significantly increased after 96 h (P < 0.05). These results suggest that IF and PDIA3 are positively responsive proteins in the gill tissues of red tilapia under salinity stress, and play important roles in the regulation of cytoskeleton and endoplasmic reticulum function.