罗非鱼罗湖病毒ORF10蛋白的亚细胞定位及组织表达分析

为了研究罗非鱼罗湖病毒(tilapia lake virus, TiLV) ORF10 蛋白的功能, 从染病莫桑比克罗非鱼(Oreochromis niloticus)脾脏和肝脏组织中克隆获得 TiLV ORF10 基因编码序列, 并成功构建了荧光定位载体 pEGFP-ORF10 和重组表达载体 pET32a-ORF10。将荧光定位载体 pEGFP-ORF10 转染鲤(Cyprinus carpio)上皮瘤细胞(Epithelioma papulosum cyprinid, EPC), 经荧光显微镜观察, 在 EPC 的细胞核中观察到绿色荧光信号, 表明该蛋白定位于细胞核中。将重组载体 pET32a-ORF10 转化大肠杆菌 BL21(DE3)进行目的融合蛋白表达, 聚丙烯酰胺凝胶电泳(SDS-PAGE)检测表明, His-TiLV ORF10 融合蛋白成功表达并主要存在于上清液中。随后, 采用 Ni-NTA 亲和层析的方法纯化融合蛋白 His-TiLV ORF10, 以其为抗原多次免疫 Balb/C 小鼠, 制备多克隆抗体。蛋白印迹法(WB)分析显示, 制备的抗体可以特异性识别 His-TiLV ORF10。利用蛋白印迹法进一步对感染 TiLV 莫桑比克罗非鱼的不同来源组织进行检测, 结果显示, TiLV ORF10 蛋白在染病莫桑比克罗非鱼肌肉、脾脏、肝脏和肾脏中的表达存在较大差异, 其中以肝脏和肾脏组织表达量为最高, 脾脏次之, 肌肉中表达量最低。本研究为深入了解 TiLV ORF10 蛋白的功能和病毒侵染与致病等机理提供了重要基础。

Subcellular localization of Tilapia Lake Virus ORF10 protein and its tissue expression analysis

To study the protein function of Tilapia Lake Virus (TiLV) ORF10, the TiLV ORF1 0.coding region was amplified from the cDNA of spleen and kidney tissues of TiLV-infected tilapia.The eukaryotic expression vector pEGFP-ORF1 0.and recombinant expression vector pET3 2.a-ORF1 0.were then constructed.pEGFP-ORF1 0.was transfected into Epithelioma papulosum cyprini (EPC) cells for fluorescence microscopy observation.Green fluorescence signals were observed in the nuclei of EPC, indicating that TiLV ORF1 0.was localized in the nucleus.The recombinant vector pET3 2.a-ORF1 0.was transformed into E.coli DE 3.for protein expression analysis, and the results showed that His-TiLV ORF1 0.was successfully expressed in the supernatant form.Furthermore, the fusion protein His-TiLV ORF1 0.was purified by Ni-NTA affinity chromatography and was subsequently used to immunize Balb/C mice to prepare polyclonal antibodies.Western blot analysis showed that mice antibodies can specifically recognize His-TiLV ORF10.To further analyze the protein content of TiLV ORF1 0.in the muscle, spleen, liver, and kidney tissues of TiLV-infected tilapia, the polyclonal antibodies were used for western blot detection.The results revealed differential protein contents of TiLV ORF1 0.in the muscle, spleen, liver, and kidney tissues.The highest expression was observed in the liver and kidney tissues, followed by the spleen.The lowest expression was found in the muscle tissues.The subcellular localization, polyclonal antibody preparation, and tissue expression analysis of TiLV ORF1 0.in this article provide an important preliminary basis for the further study of the function of the ORF1 0.protein.