Establishment Of Immunological Detection Method For ORF121 Protein Of Cyprinid Herpesvirus 2 And Expression Analysis Of Interferon Regulatory Factor In Carassius Auratus During Infection

Crucian carp is an important economic fish for freshwater aquaculture in China because of its delicious taste.However,in recent years,herpesviral haematopoietic necrosis(HVHN)has brought huge economic losses to the Chinese carp culture industry.However,in recent years,herpesviral haematopoietic necrosis(HVHN)has brought huge economic losses to the Chinese carp culture industry.The disease is highly lethal to ticks,and its pathogen is mainly CyHV-2.Therefore,it is necessary to establish a rapid and stable method for detecting whether crucian carp is infected with CyHV-2 to prevent the spread of CyHV-2.our laboratory research shows that the CyHV-2-ORF121 gene is highly expressed in various tissues and peripheral blood of diseased fish,and can be used as a potential targeted detection target for the development of diagnostic technology for CyHV-2 virus.Therefore,polyclonal antibodies against CyHV-2-ORF121 were prepared for immunological detection.Transcriptome sequencing of normal crucian carp and CyHV-2-infected crucian carp showed that the crucian carp infected with CyHV-2 was significantly up-regulated relative to the normal crucian carp interferon regulatory factor(IRF)IRF1,IRF3,IRF8 and IRF2bp2 a genes.The IRF gene plays an important role in resisting viral invasion,pathogen response,cytokine signaling,cell growth regulation,and hematopoietic cell differentiation,but its most important function is to participate in the transcriptional regulation of interferon(IFN).IRF has 11 family members in fish(IRF1-11)and is divided into 4 subfamilies based on structure and function,IRF-1 subfamily,IRF-3 subfamily,IRF-4 subfamily and IRF-5 subfamily.There is a highly conserved DNA binding domain(DBD)at the N-terminus of IRF family members,including tryptophan repeats unique to IRF family members.By investing in the eukaryotic expression plasmid of pEGFP-IFN,the laboratory found that the replication efficiency of CyHV-2 virions decreased,delaying and reducing the mortality of ticks.Because of the important role of the IRF family in the IFN signaling pathway,research on IRF family members is also increasing.In this study,the expression of IRF1,IRF3,IRF8 and IRF2bp2 a in tissues and the transcription levels under the stimulation of poly(I:C),LPS and CyHV-2 were studied.In this study,CyHV-2-ORF121 polyclonal antibody was used to establish a more immunological diagnostic technique and analyze the transcription of IRF1,IRF3,IRF8 and IRF2bp2 a under viral infection and poly(I:C)and LPS stimulation.Level.The research mainly includes the following three contents:1.The objective of this study was to produce and characterize new polyclonal antibodies against CyHV-2 ORF121 that were suitable for diagnostic use and the investigation of ORF121 function.The open reading frame(ORF)of 121 was amplified from the culture supernatant of the CyHV-2.ORF121 was subcloned into the pGEX-4T vector and recombinant protein was expressed in Escherichia coli.The purified recombinant protein ORF121 was used to immunize mice and polyclonal antibodies were obtained.The recombinant protein ORF121 were purified under urea conditions then used to 6-week-old BALA/c mice to prepare its polyclonal antibody.Western Blot and IFA assay were used to validate the ORF121 polyclonal antibody.Here,we report the recombinant protein ORF121(rORF121)could be expressed in Escherichia coli.aggregated in the form of inclusion body.The specificity of the anti-ORF121 polyclonal antibody was confirmed by Western blot.An indirect fluorescence assay(IFA)with anti-ORF121 polyclonal antibody for the detection of CyHV-2 infected RyuF-2 cells was developed in this study.In conclusion,the anti-ORF121 polyclonal antibody will be valuable tools in further studies to elucidate the mechanisms of viral infection and CyHV-2 diagnosis.2.In this study,Analysis of the sequence of the CaIRF1,CaIRF3,CaIRF8 and CaIRF2bp2 a genes obtained from the transcriptome.The CaIRF1 gene is 867 bp and encodes 288 amino acids.The predicted protein molecular mass is 33.21 ku.The N-terminal DBD domain of the CaIRF1 gene contains a 6-tryptophan repeat.CaIRF3 gene is 1 278 bp in length and encodes 425 amino acids.The predicted protein molecular mass is 48.51 Ku The N-terminal DBD domain of CaIRF3 gene contains four tryptophan repeats.The amino acid alignment shows that CaIRF3?SaIRF3?LrIRF3? MaIRF3 and OmIRF3 of homology is 85.88%,69.41%,53.30%,and 50.88%.CaIRF8 gene is 1 299 bp long and encodes 432 amino acids.The predicted protein molecular mass is 48.96 Ku.The N-terminal DBD domain of CaIRF8 gene contains a 5 tryptophan repeat.The amino acid alignment shows that CaIRF8?CcIRF8?SaIRF8?AgIRF8 and OkIRF8 of homology is 89.35%,89.35%,78.22%,and 58%,respectively.CaIRF2bp2 a gene is 1476 bp long and encodes 491 amino acids.The predicted protein molecular mass is 53.31 Ku.The N-terminal DBD domain of CaIRFIRF2bp2 a gene contains two tryptophan repeats.The amino acid alignment shows that CaIRFIRF2bp2 a ?SaIRFIRF2bp2a?CcIRFIRF2bp2a ?DrIRFIRF2bp2a and SdIRFIRF2bp2 a of homology is 93.91%,93.89%,89.54%,and 80.90%,respectively.RT-PCR analysis showed that the transcription level of IRF1,IRF3,IRF8 and IRF2bp2 a was up-regulated by poly(I:C)stimulation.The transcription level of IRF1 reached the highest value at 72 h,which was 18 times higher than the control group.The transcription level of IRF3 reached the highest at 36 h,which was 6 times higher than the control group.IRF8 reached its highest level at 36 h,19 times higher than the control group.The transcription level of IRF2bp2 a reached the highest at 72 h,which was 9 times t higher than the control group.The transcription levels of IRF1,IRF3,IRF8 and IRF2bp2 a were up-regulated after LPS stimulation.The expression level of IRF3 reached the highest at 2h,then decreased rapidly and gradually increased to normal expression level.The expression levels of IRF1,IRF8 and IRF2bp2 a reached the highest at 72 h.3.The purpose was to analyze the expression of IRF1,IRF3,IRF8 and IRF2bp2 a during viral infection.RT-PCR analysis showed that IRF1,IRF3,IRF8 and IRF2bp2 a genes were constitutively expressed in the tissues,kidney,brain,tendon,muscle and spleen,each gene was in different tissues have difference expression.CaIRF1 highest expressed in the brain,followed by sputum.CaIRF3,Ca IRF8 andCa IRF2bp2 a have the highest expression in muscle.RT-PCR analysis showed that IRF1,IRF3,IRF8 and IRF2bp2 a were up-regulated during the challenge of CyHV-2 in goldfish fin cells.this study have shown that IRF1,IRF3,IRF8 and IRF2bp2 a may play important roles in antiviral innate immunity.