罗氏沼虾诺达病毒重组衣壳蛋白(r-MCP43)的制备,用于罗氏沼虾(de Man)的免疫诊断方法。
Production of recombinant capsid protein of Macrobrachium rosenbergii nodavirus (r-MCP43) of giant freshwater prawn, M. rosenbergii (de Man) for immunological diagnostic methods.
作者信息
Farook M A, Madan N, Taju G, Majeed S Abdul, Nambi K S N, Raj N Sundar, Vimal S, Hameed A S Sahul
机构信息
OIE Reference Laboratory for WTD, Department of Zoology, C. Abdul Hakeem College, Melvisharam, India.
出版信息
J Fish Dis. 2014 Aug;37(8):703-10. doi: 10.1111/jfd.12156. Epub 2013 Aug 17.
White tail disease (WTD) caused by Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) is a serious problem in prawn hatcheries. The gene for capsid protein of MrNV (MCP43) was cloned into pRSET B expression vector. The MCP43 protein was expressed as a protein with a 6-histidine tag in Escherichia coli GJ1158 with NaCl induction. This recombinant protein, which was used to raise the antiserum in rabbits, recognized capsid protein in different WTD-infected post-larvae and adult prawn. Various immunological methods such as Western blot, dot blot and ELISA techniques were employed to detect MrNV in infected samples using the antiserum raised against recombinant MCP43 of MrNV. The dot blot assay using anti-rMCP43 was found to be capable of detecting MrNV in WTD-infected post-larvae as early as at 24 h post-infection. The antiserum raised against r-MCP43 could detect the MrNV in the infected samples at the level of 100 pg of total protein. The capsid protein of MrNV estimated by ELISA using anti-rMCP43 and pure r-MCP43 as a standard was found to increase gradually during the course of infection from 24 h p.i. to moribund stage. The results of immunological diagnostic methods employed in this study were compared with that of RT-PCR to test the efficiency of antiserum raised against r-MCP43 for the detection of MrNV. The Western blot, dot blot and ELISA detected all MrNV-positive coded samples as detected by RT-PCR.
由罗氏沼虾诺达病毒(MrNV)和极小病毒(XSV)引起的白尾病是对虾育苗场中的一个严重问题。将MrNV的衣壳蛋白基因(MCP43)克隆到pRSET B表达载体中。MCP43蛋白在大肠杆菌GJ1158中通过NaCl诱导表达为带有6-组氨酸标签的蛋白。该重组蛋白用于在兔中制备抗血清,可识别不同白尾病感染的后期幼体和成虾中的衣壳蛋白。采用多种免疫方法,如蛋白质印迹法、斑点印迹法和酶联免疫吸附测定(ELISA)技术,使用针对MrNV重组MCP43制备的抗血清检测感染样本中的MrNV。发现使用抗rMCP43的斑点印迹测定法能够在感染后24小时最早检测到白尾病感染后期幼体中的MrNV。针对r-MCP43制备的抗血清能够在总蛋白水平为100 pg时检测感染样本中的MrNV。以抗rMCP43和纯r-MCP43作为标准,通过ELISA估计的MrNV衣壳蛋白在感染过程中从感染后24小时到濒死阶段逐渐增加。将本研究中采用的免疫诊断方法的结果与逆转录聚合酶链反应(RT-PCR)的结果进行比较,以测试针对r-MCP43制备的抗血清检测MrNV的效率。蛋白质印迹法、斑点印迹法和ELISA检测到所有RT-PCR检测为MrNV阳性的编码样本。