Preliminary Development Of ELISA For Detecting Epizootic Hematopoietic Necrosis Virus

Epizootic hematopoietic necrosis (EHN) is a systemic iridoviral disease of fish. The epizootic hematopoietic necrosis virus (EHNV) causes EHN in redfin perch and rainbow trout. Along with the rapid development of aquaculture, aquatic animal disease increased, particularly by the EHN virus of epidemic diseases such as more serious harm. In this study, Culture, identification of this virus and some of its biological characteristics were done. On this basis, EHNV-MCP gene was cloned and expressd in E.coli, initially established an ELISA for detecting EHNV based on the MCP protein. The major work and results are as follows:1. Proliferation and identification of the epizootic hematopoietic necrosis virusThe EHNV was proliferated in Chinook salmon embryo cell line, and purified with sucrose density gradient centrifugation. The virus was identified by SDS-PAGE and Western blotting.2. Cloning and expression of the MCP gene of EHNVAccording to the published sequence of EHNV MCP gene, a pair of oligonucleotide primers was designed, the MCP gene was obtained from the genomic DNA of EHNV by PCR. This gene was cloned into pMD18-T vector, and the recombinant plasmid was identified by restriction enzyme and sequencing. The results showed that the nucleotide sequence of amplified MCP gene has homology of 99% with the reported sequence of MCP. The recombinant expression plasmid pET-32a-MCP was constructed, and was successfully expressed in E.coli. The expressed protein was analyzed by SDS-PAGE electrophoresis, the size was consistent with the expectancy, western blotting analysis showed that the expressed protein had a good immunogenicity.3. Preliminary development of ELISA for detecting EHNVHigh titer serum was prepared from rabbit and Balb/c mice that were immunized with purified EHNV. Rabbit anti-EHNV IgG purified with affinity was used as coating antibody, mouse anti-EHNV IgG was used as the second antibodies, the detecting EHNV antigen capture ELISA was established. The results showed that the best coating concentration of rabbit anti-EHNV IgG was about 0.5μg/mL, the optimum serum concentration of mouse anti-EHNV was about 1:12800; and it showed strong positive reaction with the standard strain of EHNV, and it was no cross reaction with the known negative samples and other fish viruses. The establishment of this method provided technical means for routine testing of aquatic EHN virus.