棕点石斑鱼免疫增强剂的筛选与免疫应答特异分子的鉴定

Epinephelus fuscoguttatus, which has high economic value and good development foreground, is a new culture species developed in Hainan province in recent years. However, diseases broke out frequently in its culture and thus seriously restricted its development. In order to control the eruption and spread of its diseases and improve the quality of its cultured products, this project firstly aims to screen Chinese herbal immunostimulants with good effects on improving the nonspecific immunity of Epinephelus fuscoguttatus by a self-developed new bi-screening method. Then, the Solex technology will be used to analyze the specific expression gene information in the head kidney and spleen of Epinephelus fuscoguttatus fed with Chinese herbal immunostimulants. Last, the key immune related genes in the head kidney and the spleen of Epinephelus fuscoguttatus stimulated by screened Chinese herbs would be confirmed. The results of this study will not only be helpful to develop safe and efficient drugs for controlling the fulminant diseases in Epinephelus fuscoguttatus, but also be helpful to establish and improve the fast screening methods for Chinese herbal immunostimulants for fish and reveal the immune mechanism of Epinephelus fuscoguttatus. Therefore, this study will play important roles in promoting the development and application of Chinese herbal immunostimulants for fish and the sustainable development of healthy culture of Epinephelus fuscoguttatus.

棕点石斑鱼是海南近年来发展起来的养殖新品种，具有重要的经济价值和广阔的发展前景，但在海南等地的棕点石斑鱼养殖中，病害爆发频繁并严重制约了该产业的发展，为了在保障养殖产品质量的前提下有效控制棕点石斑鱼疾病的发生与蔓延，本项目拟采用本课题组建立的鱼类免疫增强剂二联筛选法研究筛选能显著提高棕点石斑鱼非特异性免疫力的中草药免疫增强剂，并在此基础上采用Solexa高通量测序技术研究投喂或注射中草药免疫增强剂后棕点石斑鱼头肾和脾脏等免疫器官中特异表达的基因信息，研究筛选中草药免疫增强剂诱发棕点石斑鱼表达的免疫应答特异分子。本项目研究不仅有助于为棕点石斑鱼爆发性流行病的控制寻找安全高效的防治药物，还有助于建立和完善渔用中草药免疫增强剂的快速高效筛选方法和了解棕点石斑鱼的免疫机制，对推动渔用中草药免疫增强剂的开发应用和棕点石斑鱼健康养殖的可持续发展具有重大意义

棕点石斑鱼是我国南方沿海重要养殖鱼类，为安全高效控制棕点石斑鱼疾病，本项目利用“二联筛选法”从41种中草药中初筛了6种能够显著提高棕点石斑鱼白细胞呼吸爆发活性与吞噬活性的中草药。选择其中3种效果较好的中草药（鸡血藤、墨旱莲、黄柏）连续拌料投喂56d，结果表明，所筛选中草药对实验石斑鱼的增重率、特定生长率、肝体比和脾体比均无显著影响；但第7d鸡血藤组和第28d黄柏组的巨噬细胞呼吸爆发活性均显著升高。SOD、CAT、CH50、AKP等血清非特异性免疫指标检测表明，黄柏组的CH50值在第14d、AKP值在第56d显著升高，其他用药组的各项指标与对照组无显著差异甚至显著下降。溶藻弧菌攻毒结果表明：投喂墨旱莲7d能显著提高棕点石斑鱼的抗病力；投喂3种中草药14d均可显著提高石斑鱼的抗病力，以黄柏效果最好；投喂黄柏组28d实验石斑鱼抗病力略高于对照组；投喂56d后仅墨旱莲组的累积死亡率显著低于对照组。结果表明中草药免疫调节剂对鱼类免疫机能影响机制十分复杂，仅从非特异性免疫指标的变化难以确定免疫调节剂对鱼体抗病力的影响。. 利用Illumina Hiseq2000平台对投喂鸡血藤、墨旱莲、黄柏的棕点石斑鱼头肾进行转录组de novo测序分析，获得了中草药作用下棕点石斑鱼头肾参考基因组序列，并获取了大量与棕点石斑鱼免疫相关的候选通路及基因的序列信息。再以所获得的棕点石斑鱼头肾转录组测序信息作为参考基因组序列，利用数字基因表达谱(Digital Gene Expression, DGE) RNA-Seq(Quantification)技术对三种不同中草药作用下的棕点石斑鱼头肾进行测序，挖掘得到不同中草药作用下差异表达的基因及其涉及的代谢途径,主要包括：Fc gamma R介导的细胞吞噬通路中IgG-CD45-Src-Myosin基因通路，MAPK通路，IgG-BCR与TLR5基因，JAK-STAT通路中SOCS1、PIM1基因，COX-2基因等。这些通路与基因的发现，为进一步研究棕点石斑鱼对中草药免疫增强剂作用的分子应答机制奠定了基础。