剑尾鱼IgD基因的克隆及其在嗜水气单胞菌疫苗免疫后的表达变化  

Cloning of IgD Gene in Swordtail Fish and Changes of Its Responses to Aeromonas hydrophila Vaccine Challenges

作　　者：王片片 康桦华 刘振兴 王芳[2] 刘春 WANG Pianpian;KANG Huahua;LIU Zhenxing;WANG Fang;LIU Chun(Institute of Animal Health,Guangdong Academy of Agricultural Sciences/Guangdong Key Laboratory of Livestock Disease Prevention/Guangdong Scientific Observation and Experiment Station of Veterinary Drugs and Diagnostic Techniques,Ministry of Agriculture and Rural Affairs,Guangzhou 510640,China;Pearl River Fisheries Research Institute,Chinese Academy of Fishery Science,Guangzhou 510380,China;College of Animal Science,Zhongkai University of Agricultural and Engineering,Guangzhou 510225,China)

机构地区：[1]广东省农业科学院动物卫生研究所/广东省畜禽疫病防治研究重点实验室/农业农村部兽用药物与诊断技术广东科学观测实验站,广东广州510640 [2]中国水产科学研究院珠江水产研究所,广东广州510380 [3]仲恺农业工程学院动物科学学院,广东广州510225

出　　处：《广东农业科学》2022年第2期143-150,共8页Guangdong Agricultural Sciences

基　　金：广东省农业科学院创新基金(202128);广东省自然科学基金(2021A1515010498);广州市科技计划项目(201906040005)。

摘　　要：【目的】以剑尾鱼(Xiphophorus helleri)为试验对象,克隆获得其免疫球蛋白D(Immunoglobulin D,IgD)的cDNA序列,初步探讨该基因在剑尾鱼中的组织分布及在疫苗免疫后的表达规律。【方法】根据已知的EST库设计引物,利用PCR等方法获得剑尾鱼IgD基因cDNA序列并进行分析。运用实时荧光定量PCR(RT-PCR)方法检测IgD的组织分布及嗜水气单胞菌(Aeromonas hydrophila)疫苗免疫后,在头肾、脾脏和肠中的相对表达变化。【结果】剑尾鱼IgD完整开放阅读框(Open Reading Frame,ORF)为3879 bp,编码1298个氨基酸,预测其分子量大小为145.5 ku;由可变区和恒定区组成,恒定区包含CH1-CH2-CH3-CH4-CH5-CH6-CH7和1个跨膜区(Transmembrane region,TM)。系统进化分析显示,剑尾鱼与鳜(Siniperca chuatsi)IgD的基因序列一致性最高、为70%。IgD在剑尾鱼头肾、脾和肠中均有分布,经嗜水气单胞菌疫苗腹腔注射免疫后,IgD在头肾、脾和肠中发生差异性表达,肾和脾脏中IgD表达量显著下调,肠中IgD表达量显著上调。【结论】IgD可能在剑尾鱼机体免疫应答与肠道粘膜免疫中发挥重要作用。【Objective】The swordtail fish(Xiphophorus helleri) was employed to clone the gene sequence of immunoglobulin D(IgD), and the tissue distribution and expression laws after vaccine immunization of the IgD in swordtail fish were explored.【Method】With the known EST libraries, the cDNA sequences of IgD gene from swordtail fish were cloned and analyzed, and the tissue distribution and the relative expression changes in head kidney, spleen and intestine after vaccination were detected by real-time PCR.【Result】The complete open reading frame(ORF) of swordtail fish was 3 879 bp, which encoded 1 298 amino acids, predicting that the molecular weight was 145.5 ku. The IgD of swordtail fish was composed of variable region and constant region, and the constant region contained CH1-CH2-CH3-CH4-CH5-CH6-CH7and a transmembrane region(TM). The systematic evolution analysis of gene sequences showed that swordtail fish IgD shared similarities of 70% with mandarin fish(Siniperca chuatsi). IgD mRNA expression of swordtail fish was highly detected in head kidney, spleen and intestine. After being immunized with Aeromonas hydrophila vaccine, the expression of IgD in the head kidney and spleen was significantly down-regulated, and it was significantly up-regulated in the intestine.【Conclusion】IgD may play an important role in immune response and intestinal mucosal immunity of swordtail fish.

关 键 词：剑尾鱼 免疫球蛋白D 基因克隆 实时荧光定量PCR 免疫应答 

分 类 号：Q785[生物学—分子生物学] S965[农业科学—水产养殖]