| Bluetongue(BT)is a non-contact infectious disease of ruminants caused by bluetongue virus(BTV).The disease is transmitted by insects(Culicoides).It is an important animal disease designated by OIE as Class A and our country as a Class I.The susceptible animals are ruminants such as sheep,goats,cattle and deer.BTV is a worldwide infectious disease,mainly distributed in most tropical regions of the world.The fatality rate of sheep is 20-30%,sometimes as high as 80%.Most ruminants such as goats and cattle are infected by BTV recessively,and the average positive rate of BTV detection is 4.33-38.14%.There are potential risks in social and economic environment.BTV infects goats and cattle without obvious clinical symptoms,but it can continue to carry the virus to infect other susceptible animals.BTV replicates in the infected host cell to form five non-structural proteins,NS1～NS4.NS1 protein can promote the synthesis of viral protein and affect the assembly process of virion ds RNA.NS2 protein is involved in the composition of viral inclusion bodies(VIBs)and the assembly of virus core particles.NS3/NS3 a is involved in the release of progeny virus particles.NS4 protein is the smallest non-structural protein in BTV and plays an important role in virus replication.NS4 expression is low in IFN-I receptor-deficient(IFNAR-/-)mice,but cells pretreated with IFN-I confer BTV replication advantages,highlighting its ability to counteract the innate immune response after IFN activation.Studies have shown that NS4 inhibits the expression of interferon(IFN)genes and interferon stimulating genes(ISGs)in infected cells by inhibiting the activity of various promoters,thereby antagonizing the natural immune response mediated by interferon effect.As an important virulence factor in BTV,NS4 is highly conserved and highly expressed in BTV infected cells.Therefore,it is important to study the mechanism of NS4 regulating antiviral innate immunity and analyze the function of fusion protein for the prevention and control of BTV.In this study,a fusion protein of NS4 was constructed based on the acquired NS4 gene,and prokaryotic expression was induced to prepare polyclonal antibodies.By analyzing the effect of NS4 gene expression on the expression of interferon related regulatory genes,it provides a theoretical basis for further exploring the natural immune mechanism of BTV.The Viral protein NS4 can antagonize the immune response of host cells to interferon and promote BTV replication in host body.By constructing the NS4 recombinant plasmid,the fusion protein is induced and expressed,and the purified protein is used as the coating antigen to establish an ELISA detection method,which provides a new idea for the epidemiological detection and early diagnosis of BTV.The main research contents are as follows:(1)Cloning and bioinformatics analysis of BTV NS4 geneThe total viral RNA was extracted from the BTV-1 infected cell,and the NS4 gene was amplified by RT-PCR and ligated with the plasmid p GEM-T Easy to construct the cloned plasmid p GEM-T-NS4.The BTV NS4 gene sequence,the primary structure,secondary structure,transmembrane region and functional sites of NS4 protein were analyzed by various online softwares.The results showed that the NS4 gene was 234 bp in length and encoded a total of 77 amino acids.There are 5 flexible regions and epitope sites,which are 5-9,11-19,35-46,50-59,and 72-74 amino acids,respectively.Nuclear localization signal and subcellular localization prediction analysis revealed that the NS4 protein amino acid sequence has a single nuclear localization signal at positions 12-24 and multiple nuclear localization signals at positions 5-46,and the NS4 protein exists in the Golgi apparatus and the nucleus.(2)Prokaryotic expression of BTV NS4 gene and preparation of polyclonal antibodyThe NS4 gene fragment was ligated with the plasmid pCzn1 to construct a recombinant plasmid pCzn1-NS4,which was induced and expressed by IPTG to obtain the inclusion body protein.The purified fusion protein pCzn1-NS4 was immunized with New Zealand white rabbits to prepare polyclonal antibodies.The results showed that the titer of the antibody was over 1:512000,the purity was over 85%,and the yield of purified antibody was over 6.0 mg.The rabbit antibody with good immunogenicity can be obtained by inoculating New Zealand white rabbits with NS4 fusion protein,which provides experimental basis for later detection of NS4 specific expression in cells.(3)Analysis of the effect of eukaryotic expression of BTV NS4 gene on m RNA expression of IFN-β signaling pathway genesThe recombinant plasmid pc DNA3.1-NS4-e GFP and the empty vector pc DNA3.1-e GFP were transfected into HEK-293 T cells respectively,and the total cell protein was collected after the fluorescence intensity was observed by a fluorescence microscope.Use Sendai virus(Se V)to treat cells transfected with recombinant plasmids and empty vectors,and extract the total RNA from cells at 24 and 48 hours.Use q RT-PCR to analyze analyze JAK-STAT signaling pathway related genes,including the upstream recognition genes RIG-Ⅰ,MDA5,VISA,TBKI,IKK?,IRF3,TRAF3,TRAF6,IRF9,interferon genes IFN-α,IFN-β,and interferon stimulation genes ISG15,USP18.The results showed that the fusion protein pc DNA3.1-NS4-e GFP is present in the cytoplasm,nucleus and distribution along the nuclear membrane.The empty carrier protein pc DNA3.1-e GFP was about 28 k D,and the fusion protein pc DNA3.1-NS4-e GFP was about 40 k D.After Se V was induced for 24 h,the expression of RIG-I,MDA5,IFN-α,IFN-β gene m RNA were all extremely down-regulated;after 48 h of induction,the expression of TRAF6,IRF9,ISG15 was extremely down regulated.Therefore,it can be speculated that BTV NS4 antagonizes the interferon signaling pathway by regulating the expression of interferon related genes,thereby inhibiting the host’s natural immunity.(4)Establishment of indirect ELISA method for detection of BTV NS4 proteinThe NS4 gene fragment was connected with the plasmid p MAL-c5 x to construct the recombinant plasmid p MAL-c5x-NS4.Use the purified Soluble fusion protein p MAL-c5xNS4 as the coating antigen to screen the optimal coating amount,serum dilution,blocking solution,enzyme labeled antibody working concentration and the critical value of reaction to establish an ELISA detection method for BTV.And conduct antibody detection and analysis on 76 bovine clinical serum samples.The results showed that the positive coincidence rate of the tested samples was 98%,and the indirect ELISA detection method based on NS4 had good sensitivity and repeatability. |
