Functions of CpG-ODN 2006 on Resistance of Zebra Fish against Vibrio vulnificus
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摘要: 为创建环境友好的免疫预防技术，分析CpG-ODN对斑马鱼抗创伤弧菌感染的作用，采用PBS（对照）和人工合成的CpG-ODN 2006（试验组）序列分别注射斑马鱼，48 h后创伤弧菌FJ03-X2进行攻毒，收集斑马鱼的肠道和肾脏组织提取RNA，逆转录成cDNA，通过Real-time RT-PCR检测相关基因的表达。结果表明，24 h后CpG-ODN 2006组的免疫保护率高达70.0%；相关免疫基因表达检测结果发现CpG-ODN 2006组肠道中lysozyme、Myd88、tlrs和tnf的mRNA水平显著下调，而il1b和il10则显著上调；肾脏中的基因表达差异更为明显：lysozyme显著上调，il1b、myd88、tnf、ifng1-2、il10、il22均显著下调。结果表明CpG-ODN 2006通过调节斑马鱼肾脏及肠道中非特异性免疫相关基因的表达，维持免疫系统的平衡，增强抗创伤弧菌FJ03-X2感染的能力。Abstract: Groups of zebra fish were injected with either PBS as controlor synthetic CpG-ODN 2006 for treatment, and 48 hours later, challenged by Vibrio vulnificus FJ03-X2 infection in this study. The immune protection of the treatment group reached a rate of 70.0% in 24 hours.RNA was extracted from the intestinal and kidney tissues and then reverse transcribed into cDNA. The mRNAs of lysozyme, Myd88, tlrs and tnf in the guts of the treated fish were significantly downregulated, while those of il1b and il10, significantly upregulated, as detected by Real-time RT-PCR.The differences on gene expression in kidney were even more apparent as the expression of lysozyme significantly upregulated, and those of il1b, myd88, tnf, ifng1-2, il10 and il22 significantly downregulated. It appeared that the CpG-ODN 2006 injection afforded the zebra fish to vitalize its immune system through regulating the expressions of non-specific immunity-related genes in the kidney and intestinesto ward off the viral attack.
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Key words:
- zebra fish /
- CpG-ODNs /
- Vibrio vulnificus /
- non-specific immunity
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图 1 斑马鱼感染创伤弧菌主要症状
注：A为对照，B为创伤弧菌感染症状。
Figure 1. Morphological changes on zebra fish before and after V. vulnificus infection
图 2 Real-time RT-PCR检测创伤弧菌感染后斑马鱼肠道中的mRNA表达水平
注：A为lysozyme、il1b和myd88的mRNA水平，B为tlr9、tlr21、tlr1、tlr3和tlr4a的mRNA水平，C为tnfa2、ifng1-2、il10、il22和il26的mRNA水平。**表示与对照组相比试验组存在极显著性差异P < 0.01。图 3同。
Figure 2. RT-PCR detection of mRNA in intestinal tract of V. vulnificus-infected zebra fish
图 3 Real-time RT-PCR检测创伤弧菌感染后斑马鱼肾脏中的mRNA表达水平
Figure 3. RT-PCR detection of mRNA in kidney tract of V. vulnificus-infected zebra fish
表 1 PCR引物序列
Table 1. PCR primer sequence
目的基因 正向引物序列(5′-3′) 反向引物序列(5′-3′) actb1 CACTTCACGCCGACTCAAAC TCGGGGATGCTTATTTGCCA tlr9 CTGGGGACTTTAGAGAGCCG TCTCATTTCCAGGCCATGTT tnfa2 GCCAACCCATTTCAGCGATTG GGCATGTGATGAAGCCAAACGAA tlr21 TTGCAGGACGTTAGCTTGGT ATTGCCCCAGTATCACACGG ifng1-2 GCCGTCTCTTGCGTTCTTTA AGCGAAAGGCTTTGTTTGAG tlr1 GTGGCAGAGGCTCCAGAAGA CAGAGCGAATGGTGCCACTAT tlr3 TGATGCCCATGCCTGTAAGA TGGAGCATCACAGGGATAAAGA lysozyme CCGTAGTCCTTCCCCGTATCA GATTTGAGGGATTCTCCATTGG il10 CCTCTTGCATTTCACCATATCC TCACGTCATGAACGAGATCC il22 CACGAGCACAGCAAAGCAAT CATCGAGGAACAACGGTGTACA il26 TTTCCCCAATATCCGATGGA TGGACTTCGCAGCACAAAATG il1b CACTTCACGCTCTTGGATGA TGGACTTCGCAGCACAAAATG tlr4a TCCACAAGAACAAGCCTTTG TGTCAAGATGCCACATCAGA myd88 TCGTCATCTAAAATTTCTTTGAGC TCCGAAAGAAACTGGGTCTG 
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表 2 CpG-ODN 2006免疫保护试验
Table 2. CpG-ODN 2006 immunity protection test
组别 攻毒尾数 死亡尾数 24 h死亡率/% 免疫保护率(RPS)/% 对照组(PBS组) 67 36 53.7 0 试验组(CpG-ODN 2006组) 62 10 16.1 70.0
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