Cloning, Eukaryotic Construction And Expression Of N^pro Gene Of Classical Swine Fever Virus Of Wuzhou Strain In Guang Xi

Classical swine fever(CSF)is a highly-infectious disease making significant economic losses worldwide in the pig industry.N^prois the first protein of CSFV genome and is conserved in pestiviruses,with proteolytic enzyme activity.N^pro can inhibit interferon regulatory factor 3(IRF3)which involving in transcription of IFN-a/β,and then restrict the IFN-a/βinductive pathway.In order to select an optimum eukaryotic expression vector for N^proprotein, and to study the relationship between N^proand MHCI at different time point,the N^progene from CSFV typical example of Wu Zhou in Guang Xi Province was cloned,then inserted into the expression vector pIRESneo and pcDNA3.0, eucaryotic recombination expression plasmids pIRESneo-N^proand pcDNA3.0-N^prowas generated.The results showed,it was successfully to construct the eukaryotic expression vector by PCR and restriction enzyme analysis.Additionly,the target band of N^progene of pcDNA3.0-N^prowas clearer than pIRESneo-N^pro,and the value OD260/280 of pcDNA3.0-N^prowas significantly higher than that of pIRESneo-N^pro.The two recombination eukaryotic expression vector was use to transfect PK15 cells in vitro by Liposome infection protocol.The results showed,30 hours posttransfection,N^progene fragment was amplified by RT-PCR and the expression of N^proprotein was detected by IFA analysis with anti His monoclonal antibody,and found the N^proprotein was located in cytoplasm. Moreover,the protein levels of pIRESneo-N^propost transtection were higher than those of pcDNA3.0-N^pro,indicating that eukaryotic expression vector pIRESneo is more suitable for expressing N^proprotein than pcDNA3.0 in PK15 cells.