Development Of Monoclonal Antibody Against The Duck Tembusu Virus And Establishment Of An Indirect ELISA

In this experiment, The E gene of duck Tembusu virus (DTMUV) was amplified by RT-PCR and inserted into the multiple clone sites of the pET32a(+) vector. The recombinant plasmid was transformed into BL21(DE3) competent cells and the cells were. induced with IPTG to express the E protein. SDS-PAGE and Western Blot assays showed that he E protein was successfully expressed and kept the activity for binding the DTMUV-specific antibody.In this experiment, An indirect ELISA was established using purified DTV (FX2010isolate) as coating antigen. The reaction conditions were optimized including1.675μg/well coating antigen of purified the virus,1:200dilution of testing serum and1:2000dilution of HRP conjugated anti-duck IgG with cut off-value of.0.432(OD450nm).The results revealed the indirect ELISA has good stability and specificity, sensitivity.In this experiment, BALB/c mice were immunized with the purified duck Tembusu virus (DTMUV) Fengxian strain (FX2010).Three monoclonal antibodies (McAb) designated J1-E5-E4,7B5and1E5-B6were obtained by lymphocyte hybridoma technique. The ascites titers of J1-E5-E4,7B5and1E5-B6were1:12800,1:6400and1:25600, respectively. All three McAbs were able to react with DTMUV specifically by indirect ELISA and indirect immunofluorescence assay (IFA) Importantly, J1-E5-E4was found to bind to protein E of DTMUV specifically in cells and neutralize DTMUV in chicken embryonated eggs. The McAb would be useful in clinical diagnosis and antigen analysis of DTMUV.