Preparation And Application Of Antiserum Against IHNV-N Protein And IPNV-VP2 Protein

Infectious hematopoietic necrosis virus?IHNV?and infectious pancreatic necrosis virus?IPNV?are pathogens of infectious hematopoietic necrosis and infectious pancreatic necrosis,respectively.These two viruses mainly infect salmon and trout,causing corresponding clinical diseases and extremely high mortality of fish,which has brought huge economic losses to salmon and trout aquaculture.Although vaccines against IHNV and IPNV have been commercialized in many countries,the prevalence of IHNV and IPNV is still widespread in modern aquaculture.In this study,IHNV-N gene was amplified by RT-PCR and the pTriEx-N expression vector was constructed.We co-transfected the expression vector pTriEx-N with baculovirus-linearized Bacmid DNA into Sf9 cells to harvest the recombinant virus,and IHNV-N protein was then expressed and purified using a baculovirus expression vector system.At the same time,IPNV-VP2 gene was synthesized de nova by overlapping PCR,pTriEx-VP2 expression vector was constructed,and IPNV-VP2 protein was expressed and purified from Escherichia coli prokaryotic expression system.The purified IHNV-N and IPNV-VP2 proteins were used to immunize rabbits respectively,to generate antisera.Serum titration results showed that at the dilution of 1/1000,the OD₄₅₀ value for IHNV-N antiserum was higher than 2.1 times of the negative serum value,which was considered as positive.For,the IPNV-VP2 protein antiserum,the OD₄₅₀ value at the dilution of 1/2000 was still greater than 2.1 times of the negative serum value,which was considered as positive.There was no cross-reaction between the two antisera,demonstrating the good specificity of the antibodies.The IHNV-N antiserum was also shown to be able to rapidly detect IHNV infected rainbow trout.Taken together,high-titer antisera were made in this study,which are useful reagents for the immunological detection of IHN and IPN virus infection in aquaculture.This study also laid the foundation for comprehensive monitoring and effective prevention and control of virus infections in trout fishery.