| Fish farming plays a pivotal role in China’s aquaculture industry.Black carp,one of the four major fish in China,is an important freshwater farmed fish in this country.However,pathogenic microorganisms such as viruses pose a serious threat to the culture industry of black carp.Although fish have both innate immune system and acquired immune system,it primarily relies on the former to combat infection caused by pathogenic microorganisms.Thus,studying the anti-viral innate immune mechanism of black carp is of great significance for disease prevention and control as well as healthy culture of black carp.In this study,the TRAF family member-associated NF-κB activator(TANK)gene was cloned,recombined and functionally analyzed,which provided important data for clarifying the innate immune system of black carp.The specific research results are as follows:In mammals,TANK is linked to the regulation of the transcription of NF-κB;however,its role in interferon induction is unclear.There is no report on the TANK study of teleost fish.In order to elucidate the function of TANK in the innate immunity of teleost fish,the function of bcTANK in the anti-viral innate immunity of host cells was first identified.The open reading frame(ORF)of black carp TANK(bcTANK)comprises 1050 nucleotides and bcTANK protein contains 350 amino acids,which includes a DUF460 super family domain and a TBD super-family domain.The deduced bcTANK protein has a calculated molecular weight of 39.61 KDa and an isoelectric of 5.61.Real-time quantitative PCR(q-PCR)analysis showed that the transcription level of bcTANK gene in host cells increased with the stimulation of LPS,Poly(I:C),spring viremia of carp virus(SVCV)and grass carp reovirus(GCRV).Western blotting showed that bcTANK can be expressed in HEK293 T cells and EPC cells,whose electrophoretic migration band size is about 50 KDa.Immunofluorescence experiments showed that bcTANK was distributed in the cytoplasmic regions of EPC cells and HeLa,which clearly demonstrated that bcTANK is a cytoplasmic protein.Luciferase reporter assay analysis revealed that overexpression of bcTANK in EPC cells does not induce transcription of the interferon promoter;however,under conditions of LPS,Poly(I:C)or virus-treated cells,the IFN-activation ability of bcTANK was obviously enhanced.Crystal violet staining and plaque assay showed that under the condition of virus(GCRV or SVCV)infection,the cytopathic effect(CPE)of EPC cells overexpressing bcTANK and the virus titer in the medium were significantly lower than the control cells.It was directly demonstrated that overexpression of bcTANK in EPC cells enhances the antiviral ability of EPC cells against SVCV and GCRV.In conclusion,this study is the first one to explain the function of TANK gene in teleost fish;the research data of this paper prove that bcTANK plays an important role in activating the anti-viral innate immune system of black carp. |
