罗非鱼湖病毒核蛋白的克隆表达、抗体制备及其组织分布

近年来罗非鱼湖病毒(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,鉴于吉富罗非鱼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。本实验采用TiLV对吉富罗非鱼进行人工感染,随后在肝脏组织中克隆和测定了TiLV第6片段基因。罗非鱼湖病毒第6片段基因cDNA全长1044 bp,开放读码框(ORF)为954 bp,编码317个氨基酸,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(NP)。随后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。对吉富罗非鱼不同组织进行苏木精—伊红(H.E)染色观察,发现肝脏组织坏死并形成合胞体,脾脏部分细胞出现空泡、坏死,含铁血黄素增多,头肾细胞坏死,鳃丝上皮细胞明显解离脱落,鳃小片黏连,脑组织细胞肿大。通过蛋白印迹法(WB)和免疫组化(IHC)对人工感染TiLV的吉富罗非鱼不同组织进行检测,结果显示,NP蛋白在肝脏、脑、体肾和头肾等组织中均有表达,以肝脏组织中表达量最高。为了解吉富罗非鱼对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组织中的表达。研究表明,在感染早期(感染后12~24 h),病毒可显著抑制TNF-α和TGF-β在脾脏和头肾中的表达,可能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理论基础。

Cloning, expression, antibody preparation and tissue distribution of tilapia lake virus nucleoprotein

Recently,tilapia lake virus(TiLV)has been epidemic in many countries and posed a serious threat to Oreochromis spp.aquaculture industry.China has contributed the most amount of cultured Oreochromis spp.in the world.Up to date,there is no report of the TiLV epidemic in Oreochromis spp.in the mainland of China.However,since GIFT O.niloticus is one of the most cultured Oreochromis spp.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 which was kindly given as a gift 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 O.niloticus were determined.The length of the cDNA of the sixth genomic 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 rabbit(Albus lepus)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 O.niloticus infected with TiLV.Hematoxylin-eosin staining(H.E)was performed on different tissues of O.niloticus.The results showed that there were apparent pathological changes in the observed tissues,including hepatic necrosis and syncytium;vacuolization,necrosis and increased amount of hemosiderin in the spleen;necrosis and inclusion body in the head 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 blot and immunohistochemistry(IHC)were used to detect the expression of the NP protein in different tissues of O.niloticus infected with TiLV.The results showed that the highest amount of NP protein was expressed in the liver,followed by the brain,trunk kidney and head kidney.In order to elucidate the immune responses of O.niloticus to the TiLV infection,real-time quantitative PCR(qRT-PCR)was used to measure the mRNA expressions of TNF-αand TGF-βin 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 infection),the expression of both TNF-αand TGF-βwas 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 new light on the pathogenesis of TiLV infection and will pave a new way for the development of effective prevention and control strategy against the epidemic of TiLV in O.niloticus.