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Designing an in‐house ELISA to detect antibody against viral haemorrhagic septicaemia virus using recombinant N protein in Iranian farmed rainbow trout ( Oncorhynchus mykiss )
Author(s) -
Rahnama Roya,
Peyghan Rahim,
Reza Seyfi Abad Shapouri Masoud,
Rezaie Anahita,
Shahbazian Nastaran
Publication year - 2019
Publication title -
aquaculture research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.646
H-Index - 89
eISSN - 1365-2109
pISSN - 1355-557X
DOI - 10.1111/are.13915
Subject(s) - rainbow trout , biology , recombinant dna , virology , virus , antibody , serology , plasmid , microbiology and biotechnology , fishery , gene , fish <actinopterygii> , immunology , biochemistry
Viral haemorrhagic septicaemia (VHS) is one of the most important viral diseases in rainbow trout that has caused great losses to Iranian rainbow trout aquaculture industry in the last 3 years. Therefore, rapid and reliable diagnosis of VHS virus infections is of great importance. An enzyme linked immunosorbent assay (ELISA) method was performed to study serum antibodies against viral haemorrhagic septicaemia virus (VHSV) using recombinant fragments of their N protein. For this purpose, the virus was first isolated from an infected farm. A part of the nucleocapsid (1–505 bp) gene was amplified by RT‐PCR using specific primers. The amplified fragment was ligated to pMALc2x vector and transferred to DH5α strain of Escherichia coli . Then, recombinant plasmids were tested for protein expression in E. coli Rosetta strain. SDS‐PAGE analysis indicated the production of a recombinant protein with an expected molecular weight of 61 KDa. Analysis of trout serum samples from seven previously infected farms and two VHS free farms showed that the designed ELISA method was effective in diagnosing the infected fish. The results revealed that the developed serological assay using designed ELISA based on recombinant protein (N) has the potential to be used in monitoring studies and to determine the prevalence of VHS in rainbow trout farms. The present data allow evaluating the levels of nonneutralizing antibodies without crude virus preparations.