Prokaryotic expression and subcellular localization of the matrix protein of the viral hemorrhagic septicemia virus in finfish

Abstract

PDF HTML阅读 XML下载 导出引用 引用提醒 鱼类病毒性出血性败血症病毒基质蛋白的原核表达及其亚细胞定位 DOI: 作者: 作者单位: 安徽农业大学动物科技学院, 安徽 合肥 230036 作者简介: 朱若林(1987-),男,博士,研究方向为鱼类疾病防控.E-mail:jollinz@163.com 通讯作者: 中图分类号: S941 基金项目: 国家自然科学基金青年项目（31402331）；安徽省现代农业产业技术体系（2016-2020）水产产业体系（皖农科[2016]84号）. Prokaryotic expression and subcellular localization of the matrix protein of the viral hemorrhagic septicemia virus in finfish Author: Affiliation: School of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China Fund Project: 摘要 | 图/表 | 访问统计 | 参考文献 | 相似文献 | 引证文献 | 资源附件 | 文章评论 摘要:为了对鱼类病毒性出血性败血症病毒（viral hemorrhagic septicemia virus，VHSV）基质蛋白（matrix protein，M）进行功能研究，本实验通过PCR扩增了M基因全长序列，将其克隆至原核表达载体pET-32a（+），转化至大肠杆菌Rosetta（DE3）感受态细胞后进行IPTG诱导表达，将纯化后的重组蛋白免疫BALB/c小鼠制备多克隆抗体，采用间接ELISA检测抗体效价，并运用Western blot和间接免疫荧光检测抗体特异性。结果显示，M基因全长为606 bp，IPTG诱导得到的融合蛋白主要以包涵体的形式存在，大小约为36 kD，比预计略小。间接ELISA检测抗体效价大于1：102400，Western blot检测显示该抗体可以特异性识别纯化的融合蛋白和VHSV感染的鲤上皮瘤（epithelioma papulosum cyprini，EPC）细胞中的M蛋白。间接免疫荧光结果显示M蛋白多抗能识别感染VHSV的EPC细胞中的M蛋白，且M蛋白主要定位于细胞质和细胞膜。本研究中M蛋白多克隆抗体的制备将有助于开展M蛋白的功能研究及疾病的免疫学诊断。 Abstract:Viral hemorrhagic septicemia virus (VHSV) is one of the most serious pathogens of finfish that affects over 80 marine and freshwater species in North America, Europe, and Asia. The genome of VHSV is a negative-sense, single stranded RNA containing approximately 12000 base pairs that encode six proteins, which are the nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), non-virion protein (NV), and RNA polymerase protein (L) in order from 3' to 5'. The M protein functions in a variety of rhabdovirus infection processes such as assembly and budding, inhibiting host-cell directed transcription and gene expression, inducing apoptosis of the host cell, et al., which were all thoroughly investigated in other rhabdoviruses such as vesicular stomatitis virus and rabies virus, while less so in VHSV. In order to investigate the function of the M protein of VHSV, the M gene was amplified by PCR and cloned into the prokaryotic expression vector pET-32a(+), which was transformed into Rosetta (DE3) competent cells. The recombinant protein was induced by IPTG, and the polyclonal antibody was prepared by immunizing BALB/c mice with purified recombinant protein. The titer of the antibody was detected by an indirect ELISA, and the antibody specificity was tested by western blot and indirect immunofluorescence. The results showed that the full-length M gene was 606 bp. The fusion protein induced by IPTG mainly existed in the form of an inclusion body, and the size was about 36 kDa, which was slightly smaller than expected. The indirect ELISA assay showed that the titer of the antibody was greater than 1:102400. The western blot showed that the antibody could specifically identify the purified fusion protein and M protein in VHSV infected (EPC) cells. The indirect immunofluorescence showed that the M protein antibody can recognize the M protein in VHSV infected EPC cells, and the M protein was mainly localized in the cytoplasm and cell membrane. These results suggest that the prepared polyclonal antibody can be used as an effective tool to study the function of the M protein and for the diagnosis of VHSV. 参考文献 相似文献 引证文献