Sun Baozhen, Wang Zhi, Wang Ziyan, Ma Xiongchao, Zhu Fei
College of Animal Science and Technology, Zhejiang Agriculture and Forestry University, Hangzhou, China.
Front Immunol. 2017 Apr 27;8:468. doi: 10.3389/fimmu.2017.00468. eCollection 2017.
In this study, we investigated the hemocytes' immune response to white spot syndrome virus (WSSV) or infection at the protein level. The differential proteomes from crab hemocytes infected with WSSV or were analyzed using the isobaric tags for relative and absolute quantitation approach immediately after infection. Using this approach, we identified 1,799 proteins by their by LC-MS/MS spectra and sequencing data. These included 157 upregulated proteins and 164 downregulated proteins after WSSV infection. Similarly, 243 proteins were determined to be differentially expressed during infection, of these, 121 were upregulated and 122 were downregulated after infection. Interestingly, among these differentially expressed proteins, 106 were up- or downregulated significantly in both WSSV and infection. Six genes, β-actin, myosin-9, anti-lipopolysaccharide factor isoform 4, anti-lipopolysaccharide factor 4, transketolase-like protein 2-like isoform 1, and sarcoplasmic calcium-binding protein 1 were chosen for further study. The expression of these genes all showed a trend of upregulation at 24 h post-WSSV or infection except for myosin-9 in response to WSSV. To confirm the protective effects of the six genes, crabs were injected with specific dsRNAs before WSSV or challenge. The results showed that the knockdown of these genes led to an increase in the morbidity and mortality ( < 0.01) rate, and a decrease in infection time in WSSV-infected crabs. During the first 84 h, knockdown of these genes also led to an increase in the morbidity rates in -infected crabs, and results of four genes showed a higher mortality rate than that of the control after they were knocked down. This is the first report of the proteome response in crab hemocytes during WSSV or infection. These findings will contribute to our understanding of the immune response to WSSV and infection in crabs.
在本研究中,我们在蛋白质水平上研究了血细胞对白斑综合征病毒(WSSV)或感染的免疫反应。感染WSSV或后,立即使用相对和绝对定量的等压标签方法分析来自蟹血细胞的差异蛋白质组。使用这种方法,我们通过LC-MS/MS光谱和测序数据鉴定了1799种蛋白质。其中包括WSSV感染后157种上调蛋白和164种下调蛋白。同样,在感染期间确定有243种蛋白质差异表达,其中121种在感染后上调,122种下调。有趣的是,在这些差异表达的蛋白质中,有106种在WSSV和感染中均显著上调或下调。选择了六个基因,β-肌动蛋白、肌球蛋白-9、抗脂多糖因子亚型4、抗脂多糖因子4、转酮醇酶样蛋白2样亚型1和肌浆钙结合蛋白1进行进一步研究。除了肌球蛋白-9对WSSV的反应外,这些基因的表达在WSSV或感染后24小时均呈现上调趋势。为了证实这六个基因的保护作用,在WSSV或攻击前给蟹注射特异性dsRNA。结果表明,这些基因的敲低导致发病率和死亡率(<0.01)增加,并且WSSV感染蟹的感染时间缩短。在前84小时内,这些基因的敲低还导致感染蟹的发病率增加,并且四个基因敲低后的死亡率高于对照组。这是关于蟹血细胞在WSSV或感染期间蛋白质组反应的首次报道。这些发现将有助于我们了解蟹对WSSV和感染的免疫反应。