Abstract:Nonstructural gene 1 (NS1) of H5N2 subtype avian influenza virus QS strain was amplified by RT-PCR, whoes nucleotide sequences is 678bp in length. The fragment was cloned into the pET-28(a) vector to construct a recombinant expression plasmid. The recombi-nant expression plasmid was induced by lmmol/L IPTG for 5 hours and analyzed by 15% SDS-PAGE. The result showed that NS1 protein was highly expressed by inclusion body. Its molecular weight was 28KD as expected. The expressed NS, protein was denatured by SKL, then the denatured protein refolded by slow diluting and dialyzing method. The result indicated that the high pure and active protein was obtained. Based on the purified recombinant protein, an indirect ELISA assay for detection of anti-NSl protein antibody was developed. The best concentration of coating antigen was 2.5 g/mL, and the best dilution of serum and HRP labeled goat anti-chicken was 1:200 and 1:5000 respectively. The positive serum of ND, IB, IBD and EDS76 were all ne...更多gative to NS 1 protein by ELISA assay . The sera of virus-infected chicken and vaccinated chicken were detected. The average OD490 value of the former were 0.592, 0.623 and 0.619, positive. The average OD490 value of the latter were 0.225, 0.210 and 0.205, negative; The result demonstrated that the NS1-ELISA assay can differentiate infected from vaccinated poultry on the basis of antibody to NS 1 protein. So the approach not only afforded a kind of quick, sensitive, specific differentiating diagnostic approach, making a solid foundation of special diagnosis kit, but also can provide an effective method for early diagnosis, real-time detection, controlling and clearing of AIV.