| The non-structural protein (NS1) was a differential diagnostic marker for influenza virus infection. During the breeding time of AIV, numerous NS1 protein appeared, while in matured viruses, no NS1 protein was found. Then antibody level in wild virus-infected poultry was much higher than vaccinated poultry which had a very low or no antibody level.To obtain purified nonstructural protein (NS1), Viral ribonucleic acid (RNA) was extracted. A pair of primers were designed according to the sequence of the NS1. The NS1 gene was amplified by nested-PCR. PCR products were then inserted into a prokaryotic expression plasmid, pET-32a(+), the recombinant plasmid was pET-NS1 and was introduced into E.coli BL21 competent cells and sequenced to ensure a correct expression. E.coli colones of pET-NS1 was induced by IPTG in an optimal centigrade. Recombinant NS1 protein was analyzed by SDS-PAGE and Western blot. The amplified DNA fragment sequences were searched against the GenBank, and it shared 98% of sequence identity with the NS1 gene of one AIV virus. The expression product was about 46 kD by SDS-PAGE and Western Blot analysis. The purified product of recombinant NS1 protein was 1-3mg/mL.Purified recombinant NS1 protein of avain influenza virus was plated as coating antigen of indirect ELISA. The optimal concentration of coating antigen and the optimal dilution of antibody were determined by matrix titrations. The optimal dilution of HRP-IgG was also determined. Optimal reaction time for antigen and antibody, optimal reaction time for antibody and HRP-IgG was also determined. The optimal antigen concentration was 40μg/mL, the optimal antibody dilution was 1:200, the optimal HRP-IgG dilution was 1:10000. Optimal reaction time for antigen and antibody was 1h, optimal reaction time for antibody and HRP-IgG was lh, too. In repeating assay and specificity assay, CV<15%, it was good for repeating and had no cross reaction with other antibody serums.
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