Effects Of Galectin-1 Interacting With Classical Swine Fever Virus NS5B Protein On Replication Of Classical Swine Fever Virus

Classical swine fever(CSF)is one of the swine epidemic caused by classical swine fever virus(CSFV).The typical clinical symptoms of the disease are high fever,hemorrhage,obstruction and necrosis of tissue and organ,multiple small bleeding spots,and failure in propagation.CSFV is RNA virus with single-positive strand.NS5 B protein is a non-structural protein of CSFV that has RNA-dependent RNA polymerase activity(RdRp)and is involved in the replication of viral genomic RNA.Studying the interaction between NS5 B protein and the host cell proteins is important,because it can provide an effective tool for understanding the infection of CSFV and the role of NS5 B protein in its infection process.In the previous studies of the laboratory,yeast two-hybridization with a bait plasmid containing the CSFV NS5 B gene and a porcine alveolar macrophages(PAM)cDNA library was successfully screened for lectin galactoside-binding soluble 1(LGALS 1/galectin-1)that interacts with the CSFV NS5 B protein.Galectin-1 has many functions in and outside the cell,such as inducing apoptosis in the body,regulating inflammation and immune response to resist the invasion of external pathogenic microorganisms,and it can also affect tumorigenesis,cell adhesion,and signal transduction between cells.It plays different functions through the interaction of protein-protein or protein-polysaccharide.In this study,a recombinant plasmid was constructed by cloning the CSFV NS5 B gene fragment into the prokaryotic expression vector pCold-SUMO which containing the cold promoter.Then transformed into the E.coli Rosetta(DE3)expression strain,and induced by IPTG.The recombinant protein was successfully expressed in soluble form.The protein was purified by nickel column affinity chromatography(Ni-NTA).Then immunized the mice by subcutaneous injection to prepare a polyclonal antibody against NS5 B,and verified the reactivity and specificity of the antibody by Western blot.We found that polyclonal antibody prepared in this study not only reacts with the recombinant protein expressed in prokaryotic expression,but also reacts with the protein expressed in PK-15 cells transfected with the NS5 B eukaryotic expression plasmid constructed in the laboratory,the NS5 B protein expressed transiently was detected by Western blot.However,the NS5 B protein with natural conformation obtained from the CSFV infected PK-15 cells could not react specifically.It was found that galectin-1 in CSFV-infected host cells was down-regulated at mRNA and protein levels.Then constructed the galectin-1 overexpression and interference-stable cell lines.After inoculation of CSFV,the level of transcription of CSFV and the level of viral titer released into the supernatant were tested by real-time quantitative PCR(RT-qPCR)and indirect immunofluorescence assay(IFA).We found that overexpression of galectin-1 protein can regulate CSFV RNA and virus titer down and interfering with the expression of galectin-1 gene in PK-15 cells can promote CSFV replication.In other words,galectin-1 may have biological activity to inhibit CSFV replication.It can provide a new theoretical basis for the development of anti-CSFV drugs in the future.