Preparation Of Monoclonal Antibody Against NS1 Protein Of Duck Tembusu Virus And Establishment Of Steady Expressed Cell Line

Duck Tembusu virus disease(DTMUV)is an infectious disease caused by Duck Tembusu virus(DTMUV)which mainly infects waterfowl.The first non-structural protein,NSI,has some protective epitopes,and related to cell membrane function,First,In this study,NS1 gene was obtained from DTMUV AH-F10 strain by PCR and subcloned into pET-32a for prokaryotic expression.The expressed product was identified.Tris buffer containing urea was used to wash off the impurity protein and refolding it in gradient.The recombinant protein was purified.The mouse anti-NS1 polyclonal antibody was prepared by immunizing mice.The titer of the antibody was determined by agar diffusion test.The specificity of the antibody was identified by Western blotting and indirect immunofluore scence(IFA)test.Then PEG was used to fuse sp20 cells with spleen cells of mice,and the monoclonal antibodies to NS1 protein were obtained by clone subcloning and ELISA and IFA identification.The expression time of NS1 protein in BHK-21 cells infected with DTMUV at different time was observed by IFA test.Then the recombinant eukaryotic expression plasmid pcDNA3.1(+)-NS1 was transfected into BHK-21 cells by Lipo.2000,and the subcellular localization of NS1 protein in BHK-21 cells was observed.Results The titer was 1:8 and the results of Western blotting and IFA showed that the mouse anti-NS1 polyclonal antibody had good specificity and reactivity.Three IgG1 monoclonal antibodies were obtained,which could be used for ELISA,IFA and Western blotting detection.The prepared antibodies could recognize natural NS1 protein and had good reactivity.Using monoclonal antibodies,NS1 protein began to express in BHK-21 cells 12 hours after DTMUV infection.Subcellular localization showed that NS1 protein was mainly expressed in the cytoplasm.The expression characteristics of NS1 protein in cells were preliminarily explored.Then the recombinant eukaryotic expression vector pLOV-NS1 was constructed and transfected into 293T cells to prepare lentivirus-like particles.The supernatant of 293T cells containing lentivirus-1 ike particles was infected with BHK-21 cells.Puromycin was screened to construct a cell line capable of stably expressing NS1 protein.The nucleic acid level was detected and the protein level was identified by monoclonal antibodies.In order to further study the mechanism of action of NS1 protein and the proliferation characteristics of duck Tembusu virus,a cell line was established to stably express duck Tembusu virus NS1 protein.The results showed that NS1 protein could be stably expressed in BHK-21 cells by Western blotting detection with monoclonal antibodies.To sum up,this study successfully expressed duck Tembusu virus NS1 protein by prokaryotic expression system,prepared polyclonal and monoclonal antibodies,studied the eukaryotie expression characteristies of NS1 protein,constructed a stable expression cell line of BHK-21-NS1 secreted by NS1 protein,laid a foundation for revealing the function of NS1 protein and studying the proliferation mechanism of duck Tembusu virus.