Cloning And Expression Of The Nonstructural Gene One Of The H5N1 Subtype Avian Influenza Virus And The Establishment Of ELISA

Avian Influenza (AI) is one of the fatal infectious diseases caused by influenza A virus.The epidemic of AI has culminated in tremendous economic losses in poultry industry. It isknown that a common measure against AI is to vaccinate chicken, but the poultry infectedand vaccinated AIV both can product high antibody. So at present how to differentiate AIvaccinated and infected chicken is a significant open problem.One pair of primers was designed and synthesized according to the sequence ofnonstructural gene one (NS1) of Avian Influenza Virus (AIV) published in GenBank. NS1gene was amplified from AIV type H5N1 using RT-PCR. The NS1 gene was cloned intopMD-18T Vector and then transformed into E.coli DH5α. The recombinant plasmid wasindentified by PCR and restriction endonuclease analysis. The result indicated that theconstruction contained the NS1 gene at the right location of the vector. The nucleotide anddeduced amino acid sequences of these avian influenza viruses reported in GenBank werecompared. It was confirmed that the nucleotide sequence amplified in the experimentshared 95.2%-99.7% homology and the deduced amino acids homology were92.2%-98.7% with the corresponding sequences published in GenBank.The cloned genomic DNA was subcloned into pET-28a(+) prokaryotic expression vector.The recombinant expression plasmid pET/NS1 was proved to be true by PCR andrestriction endonuclease analysis. The recombinant expression plasmid was transformedinto E.coli BL21 and induced by 1mmol/L IPTG at 37℃for 5 hours, then analyzed bySDS-PAGE. The result showed that NS1 protein was highly expressed by inclusion body.Its molecular weight was 28kD. Judging from the result of Western-blotting, NS1 fusionexpression protein can react with H5N1 AIV infected serum and can not react withvaccinated serum. So the expression production was specific antisera against AIV.After the conditions were optimized, the recombinant protein was expressed on a largerscale. In our study, the fusion protein as envelope antigen was tested if reacted withAIV-infected and vaccinated sera by indirect ELISA. The result showed that only AIVinfected sera could be detected but vaccinated sera showed negative result. So the resultdemonstrated that the NS1-ELISA assay can differentiate the AIV infected chicken andvaccinated chicken on the basis of antibody to NS1 protein. The NS1-ELISA has thespecificity of AIV type A but it can not identify the subtype of AIV. The ELISA assay based on the expressed NS1 protein not only afforded a kind of quick,specific differentiating diagnostic approach, making a solid foundation of special diagnosiskit, but also can provide an effective methodology.