Development Of Monoclonal Antibodies Against Protein E Of Tembusu Virus

Tembusu virus infection is a new emerging disease caused by Tembusu virus, which is characterized by egg drop and hemorrhagic ovarian inflammation. At the very start, scholars have named it as duck egg drop syndrome, duck hemorrhagic ovarian inflammation, duck viral encephalitis, ducks and geese encephalitis-oophoritis syndrome, duck infectious egg drop syndrome etc. according to its clinical symptoms and lesion. Currently, the prevalence of the disease has caused huge losses to poultry industry, and also there are no effective vaccines and drugs to control the occurrence of the disease. E protein, which is on the surface of the mature virion, is one of the major structure protein of Tembusu virus. It is involved in virus adsorption and penetration process, affecting virulence and cell tropism, and can stimulate the host to produce neutralizing antibodies, inducing a protective immune response. In this study, we got Vero cell-adapted SHYG strain by serial passage on Vero cells, obtained active truncated E protein through prokaryotic expression and developed monoclonal antibodies against protein E of Tembusu virus, which laid the foundation for further study of the structure and function of E protein, establishing diagnostic method of Tembusu virus and developing new vaccines.1. Adaptive culture of Tembusu virus on Vero cellsThe4th generation of sterile allantoic fluid of Tembusu virus SHYG strain was serially passaged on Vero cells, then the titers of SHYG strain which has respectively passaged5,10,15,20,25,30,35,40and45times on Vero cells were measured by TCID50assay. The results showed that the Tembusu virus in first generation caused cytopathic effect (CPE)7days after inoculation, the virus in20th generation caused CPE3days after inoculation; CPE of Vero cells caused by Tembusu virus was further confirmed by RT-PCR amplification of E protein gene. TCID50assay showed that the titer of virus in5th generation was101.67TCID50/mL, while the titer of virus in30th generation was106.33TCID50/mL, indicating a gradual adaptive culture of Tembusu virus on Vero cells.2. Prokaryotic expression of protein E of Tembusu virusA804-bp truncated E protein gene was amplified by PCR and inserted into the prokaryotic expression pET-32a (+) vector to construct recombinant plasmid pET-32a-E. SDS-PAGE showed that recombinant E protein was expressed effectively with molecular weight of48kD. Soluble analysis indicated that the major form of recombinant protein was inclusion body. We obtained a concentration of1.239mg/mL of recombinant protein after a process of dissolution, purification and refolding. Western blot analysis with a mouse anti-Tembusu virus serum as a primary antibody revealed that recombinant E protein had a good antigenicity.3. Development of monoclonal antibodies against protein E of Tembusu virus6-8weeks old BALB/c mice were immunized with purified recombinant E protein once every two weeks,100μg per mouse. The enhanced immunization was applied after third immunization, and spleen cells of immunized mouse were fused with SP2/0cells3days later. Three monoclonal antibodies named1D9B5,1E6C3and3G8F7were screened by indirect ELISA which using recombinant E protein as coated antigens and His protein as negative control. The asites titers of1D9B5,1E6C3and3G8F7were1:64000,1:32000and1:64000, respectively. The isotype of McAbs were determined by the isotyping reagents, results showed that1D9B5and3G8F7belongs to IgG1subtype,1E6C3belongs to IgG2a subtype. Indirect imminofluorescence assay (IFA) revealed that specific fluorescence was observed when Vero cells were infected with Tembusu virus, but no fluorescence was observed when Vero cells were infected with avian influenza viruses. Western blot showed that all three McAbs have a specific band with E protein, but not with His protein.In summary, we obtained a Vero cell-adapted strain by serial passage on Vero cells, and we gained an active recombinant E protein successfully, and subsequently, we developed three McAbs against protein E of Tembusu virus.