文章摘要
罗湖病毒核蛋白抗体的制备及其应用
Preparation and application of Tilapia Lake Virus Nucleoprotein antibody
投稿时间:2019-02-08  修订日期:2019-03-23
DOI:
中文关键词: 罗湖病毒  尼罗罗非鱼吉富品系  NP蛋白  多克隆抗体  组织表达  免疫因子
英文关键词: Tilapia lake virus  Nile Tilapia GIFT strain  Nucleoprotein  Polyclonal antibody  Tissue expression  Cytokine
基金项目:国家自然科学基金
作者单位E-mail
苏国茂 广东省水环境与水产品安全工程技术研究中心 1091109070@qq.com 
秦真东 广东省水环境与水产品安全工程技术研究中心  
李嘉波 广东省水环境与水产品安全工程技术研究中心  
周萌 广东省水环境与水产品安全工程技术研究中心  
莫金凤 广东省水环境与水产品安全工程技术研究中心  
张凯 广东省水环境与水产品安全工程技术研究中心  
梁日深 广东省水环境与水产品安全工程技术研究中心  
吴灶和 广东省水环境与水产品安全工程技术研究中心  
赵丽娟 广东省水环境与水产品安全工程技术研究中心  
林蠡 广东省水环境与水产品安全工程技术研究中心 linli@zhku.edu.cn 
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中文摘要:
      罗非鱼是世界重要养殖鱼类。近年来罗湖病毒(Tilapia Lake Virus,TiLV)在多个国家流行,对世界罗非鱼养殖业造成严重威胁。中国是罗非鱼第一养殖大国,尽管我国大陆还没有TiLV的正式报道,鉴于尼罗罗非鱼吉富品系((Oreochromis niloticus,GIFT strain) 是我国重要的罗非鱼养殖品种,其对TiLV的感染特性研究具有重要意义。本研究采用德国弗里德里希洛弗勒研究所Sven Bergmann 博士赠送的TiLV对尼罗罗非鱼吉富品系进行人工感染,随后在肝脏组织中克隆和测定了TiLV第六片段基因组。罗湖病毒第六片段基因组cDNA全长1044 bp,开放读码框(ORF)为954 bp,编码一个317个氨基酸蛋白,预测分子量为36.38 ku;5’非编码区(NCR)为19 bp,3’非编码区(NCR)为972 bp。系统进化树分析表明该蛋白属于TiLV核蛋白(Nucleoprotein, NP)。随后我们在大肠杆菌中表达和提纯了GST融合NP蛋白,在新西兰大白兔上制备了多克隆抗体。通过酶联免疫吸附实验 (ELISA) 检测抗体效价为1:51200,且抗体可特异性识别感染组织中的病毒NP蛋白。对罗非鱼不同组织进行苏木精-伊红 (HE) 染色观察,发现肝脏组织坏死并形成合胞体,脾脏部分细胞空泡化坏死,含铁血黄素增多;头肾细胞坏死并形成包体;体肾出现蛋白质沉淀;鰓丝上皮细胞明显解离脱落,鰓小片黏连;脑组织细胞增生和空泡化。通过蛋白印迹法(WB)和免疫组化(IHC)对人工感染TiLV的罗非鱼不同组织进行检测,结果表明NP蛋白在肝脏、脑、体肾和头肾中均有表达,以肝脏组织中表达最高。为了解罗非鱼对Tilv的免疫反应,通过实时荧光定量PCR (qRT-PCR) 测定免疫因子TNF-α和TGF-β在主要免疫器官脾脏和头肾中的表达。结果表明在感染早期(感染后12-24h),病毒可显著抑制TNF-α和TGF-β在脾脏和头肾中的表达,也许通过抑制宿主这些免疫因子来促进病毒自身早期的复制。本研究将为进一步解读TiLV的致病机理及其高效防控提供基础。
英文摘要:
      Tilapia is one of the most important cultured fish species worldwide. Recently, Tilapia Lake Virus (TiLV) has been epidemic in many countries and posed a serious threat to tilapia aquaculture industry. China has contributed the most amount of cultured tilapia in the world. Up to date, there is no report of the TiLV epidemic in tilapia in the mainland of China. However, since Nile Tilapia (Oreochromis niloticus,GIFT strain) is one of the most cultured tilapia species in the mainland, therefore it is necessary to characterize the features of the GIFT strain infected with TiLV. Taking the advantage of the TiLV was kindly gifted by Dr. Sven Bergmann from Institute of Infectology, Friedrich Loffler Institute, we performed the infection of TiLV in the GIFT strain. The whole nucleotide sequences of the sixth genomic segment of TiLV from the experimental infected tilapia were determined. The length of the cDNA of the sixth genome segment was1044 bp containing an open reading frame of 954 bp encoding a protein with 317 amino acids with predicted molecular weight of 36.38 ku. There is 5’ end non-coding region of 19 bp and 3’end non-coding region of 972 bp. The sequences and phylogenetic tree analysis showed that the sixth genomic segment encoded TiLV nucleoprotein (NP). Subsequently, GST fusion NP was expressed in Escherichia coli and purified, and it was used to immunize New Zealand white rabbits according to the conventional method to prepare rabbit anti-NP polyclonal antibody. The results showed that the antibody titer obtained by ELISA was higher than 1:51200, and the antibody could specifically recognize the NP protein from the tissues of TiLV infected tilapia. Hematoxylin-eosin staining (HE) was performed on different tissues of tilapia. The results showed that there were apparent pathological changes in the observed tissues, including hepatic necrosis and syncytium; vacuolization, necrosis and increasing hemosiderin in the spleen; necrosis and inclusion body in the head kidney; apparent protein precipitation in the trunk kidney; dissociation and shedding of the epithelial cells of the gill filament, small pieces adhered to each other; vacuoles of nerve cells in the brain tissue. Western blotting (WB) and immunohistochemistry (IHC) were used to detect the expression of the NP protein in different tissues of tilapia infected with TiLV. The results showed that the highest amount of NP protein was expressed in the liver, followed by in the brain, trunk kidney and head kidney. In order to elucidate the immune responses of tilapia to the TiLV infection, real-time quantitative PCR (qRT-PCR) was used to measure the mRNA expressions of TNF-α and TGF-β in the spleen and head kidney which are the two major immune tissues of fish. The results showed that during the early period of the infection (12-24 h post of the infection), the expression of both TNF-α and TGF-β was significantly inhibited by the viral infection, indicating that TiLV might inhibit these cytokines so as to facilitate its early replication in the host. The current study will shed a new light on the pathogenesis of TiLV infection and will pave a new way on the development of effective prevention and control strategy against the epidemic of TiLV in tilapia.
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