The Prokaryotic Expression Of AIV NS1 Gene And The Establishment Of Indirect ELISA For NS1 Protein

Avian influenza is seriously threatening the bird cultivation of the world and the human health. Along with gradual penetrating to avian influenza virus research, the people have changed the research aspect from structural protein to the nonstructural protein , which is the NS1 protein research. It has discovered that there is certain connection between the NS1 protein of avian influenza virus and apoptosis which is caused by the avian influenza virus .The NS1 protein's adjusting the effect to apoptosis is directly related to whether the cell infected by avian influenza virus produces interferon or not, and to the cells it infects. The NS1 protein can resistant the production of interferon produced by the viral infected cell, and has played a vital role .At present,many countries apply various vaccines to control and eliminate the avian influenza. Along with the widespread applying of the vaccine, it is difficult to distinguish infection group from the vaccine immune group. It has disturbed the diagnosis of avian influenza, concealed the popularity of the avian influenza, added to the difficulty of the avian influenza prevention. As the avian influenza virus nonstructural protein, the NS1 protein has the high conservative nature, and has the broad prospect in the clinical practice as the inspecting antigen in distinguishing and diagnosing immune birds and the naturally infected birds.Nonstructural gene (NS1 gene)of H9N2 subtype avian influenza virus is amplified with PCR method, on the basis a pair of specific primers designed according to the relevant nucleotide sequence from GeneBank. The fragment is successively cloned into pET-32a(+)to construct a recombinant expressed vector pET-NS1. The recombinant expressed plasmid is transformed into the host cell E.coli BL21(DE3)and induced by IPTG and then analyzed by 12% SDS-PAGE. The result shows that NS1 protein was highly expressed in inclusion body. Its molecular weight is 33 KDa as expected. The expression is optimized with proper inducing conditions of 0.7 mmol/L IPTG and 5 hours induction. Western-blot analysis proves that the recombinant protein had good immunoreactivity against H9N2 subtype AIV antibody. To purify the recombinant protein, a"His Bind Kit'is adopted. The result indicates that high purity protein is obtained. Based on the purified recombinant protein, an indirect ELISA assay for detection of anti-NS1 protein antibody is developed. The best concentration of coating antigen is 1.5μg/mL and the best dilution of serum and HRP labeled goat anti-chicken is 1:40 and 1:2000 respectively. The result demonstrats that the NS1-ELISA assay can differentiate infected poultry from vaccinated ones on the basis of antibody to NS1 protein.In conclusion, the approach can not only affords a kind of quick, sensitive, specific differentiating diagnostic approach, making a solid foundation for special diagnosis kit, but also provide an effective method for the early diagnosis, timely detection, control and purification of AIV.