利用重组蛋白制剂开发检测罗氏沼虾诺达病毒和超小病毒的免疫学方法。

Developing immunological methods for detecting Macrobrachium rosenbergii nodavirus and extra small virus using a recombinant protein preparation.

作者信息

Wang C-S, Chang C-Y, Wen C-M

机构信息

Department of Life Sciences, National University of Kaohsiung, Kaohsiung, Taiwan.

出版信息

J Fish Dis. 2016 Jun;39(6):715-27. doi: 10.1111/jfd.12404. Epub 2015 Aug 12.

Abstract

Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) have been identified as the causative agents for white tail disease (WTD) of M. rosenbergii. In this study, the gene sequences encoding MrNV and XSV capsid proteins were separately ligated into the pGEX-4T-3 expression vector and transformed into Escherichia coli. After induction, glutathione-S-transferase (GST)-tagged MrNV and XSV fusion proteins were obtained with molecular masses of 68 and 43 kDa, respectively. Specific polyclonal antibodies for MrNV and XSV against viral recombinant proteins and infected prawn tissues were verified using Western blotting. According to immunodot blot assay results, the detection sensitivities of antibodies were approximately 5 ng μL(-1) for both recombinant proteins GST-MrNV and GST-XSV. In additional, MrNV and XSV were detected at dilution levels of 1:2560 and 1:640 in the infected prawn tissues, respectively. No cross-reactions with white spot syndrome virus or grouper nervous necrosis virus were observed using immunodot blot assays. MrNV and XSV in infected muscle tissues were detected using immunohistochemistry. Although the detection limit of the immunodot blot assay was lower than that of nested reverse transcription polymerase chain reaction, these polyclonal antibodies can be useful for confirming MrNV and XSV infections in field tests.

摘要

罗氏沼虾诺达病毒(MrNV)和超小病毒(XSV)已被确认为罗氏沼虾白尾病(WTD)的病原体。在本研究中,将编码MrNV和XSV衣壳蛋白的基因序列分别连接到pGEX-4T-3表达载体中,并转化到大肠杆菌中。诱导后,分别获得了分子量为68 kDa和43 kDa的谷胱甘肽-S-转移酶(GST)标记的MrNV和XSV融合蛋白。使用蛋白质印迹法验证了针对MrNV和XSV的病毒重组蛋白及感染对虾组织的特异性多克隆抗体。根据免疫斑点印迹分析结果,针对重组蛋白GST-MrNV和GST-XSV的抗体检测灵敏度约为5 ng μL(-1)。此外,在感染的对虾组织中分别以1:2560和1:640的稀释度检测到了MrNV和XSV。使用免疫斑点印迹分析未观察到与白斑综合征病毒或石斑鱼神经坏死病毒的交叉反应。使用免疫组织化学方法检测了感染肌肉组织中的MrNV和XSV。尽管免疫斑点印迹分析的检测限低于巢式逆转录聚合酶链反应,但这些多克隆抗体可用于在现场检测中确认MrNV和XSV感染。

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