Development Of An Indirect ELISA Kit For Detecting Bovine Akabane Disease Using Recombination Nucleocapsid Protein

To obtain recombinant nucleocapsid (N) protein of Akabane virus (AKAV) for diagnostic method research, S fragment from OBE-1 strain was amplified by the reverse transcription-polymerase chain reaction (RT-PCR). The amplified fragment was cloned into the pMD18-T vector and N gene was subcloned into the pET-28a (+) expression vector after digestion with BamH I and Xho I. The recombinant plasmid was transformed into E. coli BL21 (DE3). The N gene of AKAV was over-expressed in E. coli as a fusion protein. The result of SDS-PAGE and Western blotting assay showed that the target protein was about 27 kD with good immunological activity. The fusion protein is about 58.5% of total soluble protein of host cell and can be purified under native conditions with purity of 99%. The rabbits were vaccinated with the recombinant N protein to produce its antiserum. Using the antiserum as first antibody, AKAV antigen was specially detected in the inoculated BHK21 cell and brain of baby mice by indirect immunofluorescence assay. Additionally, an indirect enzyme linked immunosorbant assay (ELISA) kit was developed for detection of antibodies against AKAV using the purified recombinant N protein. The optimal reaction conditions of ELISA were determined: the concentration of the recombinant N protein was 1μg/100μl/well for coating ELISA plates; the dilution folds of the rabbit anti-bovine IgG conjugated with peroxidase and the sample sera were 1:8000 and 1:100 respectively. The ELISA kit was confirmed to be specific and reproducible. Bovine serum samples from Yun Nan (89) and Inner Mongolia (100) were detected by the ELISA kit and SN test. Comparing to SN test the result revealed that the threshold of ELISA was determined as 0.411 and 0.303, then 72.7% (56/77) and 91.4% (85/93) agreement was obtained in two districts respectively. It was shown that the kit was stable in 37℃ for 3 days. These results will be primary to develop diagnostic methods for Akabane disease.