Molecular Cloning And Characterization Of NAP1 From Black Carp

Fish farming plays a pivotal role in China's aquaculture industry.Pathogenic microorganisms such as viruses pose a serious threat to the black carp breeding industry.Therefore,it is of great significance to carry out related research on black carp resistance in aquatic diseases.This article focuses on the relationship between bcNAP1 and bcMDA5 in the RIG-I-like pattern recognition receptors(RLRs)innate immune antiviral signaling pathway.NAK-associated protein 1(NAP1)is involved in NF-?B activation and IFN induction in human and mammal;however,the role of teleost NAP1 in innate immunity remains unknown.NAP1 also known as AZI2 and TBKBP2,was first identified as an activator of IKK-related kinases and involved in protecting cells from TNF-?-induced apoptosis.This article has cloned and identified the NAP1 homolog of herring(Mylopharyngodon piceus),further research on the function of black carp NAP1(bcNAP1)has been done.Gene cloning bcNAP1 cDNA has 1068 nucleotides and encodes 355 amino acids.Ex PASy software predicts that the molecular weight of bcNAP1 is 40.44 KDa and the isoelectric point is 5.45.ExPASy software predicts that the molecular weight of bcNAP1 is 40.44 KDa and the theoretical isoelectric point is 5.45.bcNAP1 migrated about 47 kDa by immunoblotting and was identified as a cytoplasmic protein by immunofluorescence experiments.bcNAP1 showed little interferon(IFN)promoter-inducing ability in the reporter assay.However,when co-expressed with black carp MDA5(bcMDA5),bcNAP1 obviously enhanced bcMDA5-mediated IFN promoter induction in reporter assay.Furthermore,transfection of the same amount of Flag-bcNAP1 and empty vector,the virus titer detected bcNAP1 did not show the anti-viral ability ofcarp spring viremia of carp virus(SVCV).Infection with grass carp reovirus(GCRV),a common virus in fish,is the same result.Accordingly,the data of plaque assay demonstrated that the antiviral activity of bcMDA5 against both GCRV and SVCV was upregulated by bcNAP1.Additionally,the association between bc NAP1 and bcMDA5 has been identified through immunofluorescent staining and co-immunoprecipitation(co-IP)assay.Thus,the data generated in this study support the conclusion that bcNAP1 interacts with bcMDA5 and up-regulates bcMDA5-mediated antiviral signaling during host innate immune activation.In summary,bcNAP1 has a very important role in the innate immune defense process of black carp and our experimental results also show that bcNAP1 activates bcMDA5-mediated signaling and is positively regulated.