四种新型合成的CpG寡脱氧核苷酸对淡水长脚大虾的免疫调节特性  

Immunoregulatory Characteristics of Four Novel Synthetic CpG Oligodeoxynucleotides for Freshwater Giant Prawn (Macrobrachium rosenbergii)

作　　者：宋宏红 杨茵涵 赖伟萱 

机构地区：[1]东吴大学微生物学系,台湾台北

出　　处：《水产研究》2017年第4期166-179,共14页Open Journal of Fisheries Research

摘　　要：不同序列的CpG-ODNs对淡水长脚大虾的原酚氧化酵素基因propo的表现具有不同的影响。为评估不同免疫调节效应之CpG-ODNs的应用可行性,本研究使用已根据促进或抑制人类和鱼类免疫力的不同CpG-ODN序列设计之四段新的CpG-ODNs (ODN2050,ODN2051,ODN3006和ODN4006),分析其对淡水长脚大虾(Macrobrachium rosenbergii)免疫力的影响。首先,利用流式细胞仪进行ODN对血球结合率分析,四种ODNs中,ODN2050结合50%血球所需浓度(BC50)最低(1.88 μg/mL),且浓度5 μg/mL即可结合90%以上的血球细胞。以不同浓度ODN于体外直接处理虾血球后,四种ODNs都能促进释出胞外之总PO活性(POT),其中ODN2050于0.67 μg/mL不但能明显地增强POT且活性最高(p T。虾子注射ODN后1小时,测定血浆中POT发现,除ODN3006不影响POT外,其他三种ODNs都明显地增加POT,其中ODN2050促进POT表现最强,约无ODN处理之对照组的3.8倍。注射后3小时测定血球的4种免疫相关功能基因的表现显示,相较于对照组,ODN2050及ODN2051明显地增加3种与原酚氧化酵素活化系统(PAS)相关之基因(propo、pon及α2-m)的mRNA表现量;反之,ODN3006降低propo和pon的表现;至于ODN4006则对4种基因均无明显影响。最后,以脂多醣分子(LPS)注射刺激虾子1天后,进行致病菌攻击并,同时以ODN3006处理虾子,测得ODN3006组虾子的死亡率为65%,低于未经ODN处理组的85%。综合上述结果推测,四段新型且合成的CpG-ODNs可能具有调节虾子免疫反应的作用,其中的ODN2050及ODN2051为促进型,ODN3006和ODN4006可能分别为抑制型和中和型。Different sequences of CpG oligodeoxynucleotides (CpG-ODNs) have been demonstrated to result in different expressions of prophenoloxidase gene (propo) in freshwater giant prawn (Macrobrachium rosenbergii). In this study, in order to assess the possible applications of CpG-ODNs with different immunomodulatory effects that may be used in aquaculture, four novel CpG-ODNs (ODN3006, ODN4006, ODN2050 and ODN2051), which have been designed according to the different sequences of CpG-ODNs that promote or inhibit human and fish immunity, are used to determine their effect on prawn immunity. First, the binding rate of ODN to hemocytes analyzed by flow cytometry showed that the lowest concentration of ODN2005 required to binding 50% hemocytes (BC50) was at 1.88 μg/mL and that the concentration was at 5 μg/mL of ODN2050;it could bind more than 90% hemocytes. After hemocytes treated with different concentrations of ODN in vitro, all four ODNs shown increase the extracellular total PO activity (POT) secreted from hemocytes, OND2050 at 0.67 μg/mL which not only significantly enhanced POT (p T. At 1 hour after prawn was injected with ODN, the results showed that, except ODN3006 which did not affect plasma POT, the other three ODNs all significantly increase POT in plasma, and the highest POT was enhanced by ODN2050, about 3.8 times more than the control group. At 3 hours after injection, the expression of four immune-related genes showed that, in comparison with the control group, both ODN2050 and ODN2051 increased the mRNA level of three PAS-related genes (propo, pon and α2-m);in contrast, ODN3006 decreased the mRNA level of propo and pon. As for ODN4006, there was no significant effect on the four genes. After treatment with lipopolysaccharide (LPS) for 1 day, prawns were simultaneously treated with ODN3006 and challenged by Lactococcus garvieae. The mortality rate of ODN-treated group was at 60% and lower than that of the untreated control group (80%). These results suggest that all four novel CpG-ODNs may play a role in

关 键 词：新型CpG寡脱氧核苷酸 免疫调节 水产养殖虾类 淡水长脚大虾 

分 类 号：R73[医药卫生—肿瘤]