Expression And Application Of Nonstructural Gene Of H₅N₁ Subtype Avian Influenza Virus In E.coli

Nonstructural gene 1 (NS₁) of H₅N₁ subtype avian influenza virus QS strain was amplified by RT-PCR, which nucleotide sequences is 678bp in length, encoding 225 amino acids. The nucleotide and deduced amino acid sequences were compared with the sequence published in GenBank, the homologies were 96.9% ⁹8.5% and 95.1 %⁹8.2%, respectively.The fragment was cloned into the pET-28(a) vector to construct a recombinant expression plasmid. The plasmid was proved to be true by PCR, restriction endonuclease analysis and the nucleotide sequences obtained by sequencing technique. The recombinant expression plasmid was induced by 1mmol/L IPTG at 37℃ for 5 hours and analyzed by SDS-PAGE. The result showed that NS₁ protein was highly expressed by inclusion body. Its molecular weight was 28KD as expected. The obtained inclusion bodies were washed three times with the buffer including 1 % triton-100 and 2M urea, respectively. The expression NS₁ 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-NS[ protein antibody was developed. The best concentration of coating antigen was 2.5μg/mL, and the best dilution of serum and HRP labeled goat anti-chicken was 1:200 and 1:5000. The border of negative and positive serum in OD490 value was 0.31,when the OD₄₉₀ value was equal or over 0.31,it was positive, or else negative. The positive serum of ND,IB,IBD and EDS₇₆ all were negative to NS₁ protein by ELISA assay ,it is directed that the NS₁ protein was only reacted to the serum of virus-infected chicken. In order to differentiate the virus-infected chicken and vaccinated chicken on the basis of antibody to NS₁, the sera were detected. The average OD₄₉₀ value of the former were 0.592, 0.623 and0.619,positive The average OD₄₉₀ value of the latter were 0.225 , 0.210 and 0.205, negative;The result demonstrated that the NSi-ELISA assay can differentiate the AIV infected birds and...