| Avian Influenza (AI) is an infection and/or disease syndrome caused by type Aavian influenza virus (AIV). The syndromes of AI include sub-clinical, mild upperrespiratory disease, loss of egg production and acute generalized fatal disease. Thehighly pathogenic avian influenza (HPAI), caused by the H5N1 subtype has beencausing seriously loss of economy in poultry industry and becoming a threat to thehuman health worldwidely. Therefore, it is the key for effective control the disease todevelope the technique to detect AIV quickly.Using a pair of specific prime designed according to the relevant nucleotidesequence from GenBank, the HA (nucleotide 123～870) and NA (nucleotide 417～1362)genes of H5N1 subtype AIV were amplified by RT-PCR. The RT-PCR products werecloned into pMD 18-T and pET-his to get a prokaryotic recombinant plasmid pET-HAand pET-NA. The target genes were success fully expressed in the host cellBL21 (DE3) when it was induced with IPTG. The condition of test was optimized with37℃temperature and proper inducing concentration of 0.5mmol/L IPTG and 3 hoursinduction. The recombinant protein was purified by nickel-chelating Sepharose(Amersham Biosciences, USA). So, the indirect ELISA method for detection of H5N1subtype AIV antibody in chicken serum was established.The optional working circumstances for the ELISA assay (pET-HA antigen conc-entration 320ng/well,Serum dilution 1:10,pET-NA antigen concentration 118ng/well,serum dilution1:10)was tried out with chessboard titration.Regarding sensitivity, speci-ficity, reproducibility and correlation with other methods, the developped hemagglut-inin- and neuraminidase-specific ELISAs, it will be contribution of local and system-ic immunity in resistance to influenza virus infection. |
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