Study On Cloning And Prokaryotic Expression Of H5N1 Subtype Avian Influenza Virus NA1 Gene

Highly pat hogenic avian influenza virus (HPAIV) 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 is known that a common measure against AI is to vaccinate chicken, but the poultry infected and vaccinated AIV both can product high antibody. So at present how to differentiate AI vaccinated and infected chicken is a significant open problem.One pair of primers was designed and synthesized according to the sequence of nonstructural gene one (NA1) of Avian Influenza Virus (AIV) published in GenBank. NA1 gene was amplified from AIV type H5N1 using RT-PCR. The NA1 gene was cloned into pMD-18T Vector and then transformed into E.coli DH5a.The recombinant plasmid was indentified by PCR and restriction endonuclease analysis. The result indicated that the construction contained the NA1 gene at the right location of the vector. The nucleotide and deduced amino acid sequences of these avian influenza viruses reported in GenBank were compared. It was confirmed that the nucleotide sequence amplified in the experiment shared 91% homology and the deduced amino acids homology were 86%. with the corresponding sequences published in GenBank.The cloned genomic DNA was subcloned into pET 28a(+) prokaryotic expression vector.The recombinant expression plasmid pET/NA1 was proved to be true by PCR andrestriction endonuclease analysis. The recombinant expression plasmid was transformed into E.coli BL21 and induced by 0.5mmol/L IPTG at 16℃for 20 hours, then analyzed by SDS-PAGE. The result showed that NA1 protein was highly expressed by dissolubility proteinum. Its molecular weight was 51kD. Judging from the result of Western-blotting, NA1 fusion expression protein can react with H5N1 AIV infected serum and can not react with vaccinated serum. So the expression production was specific antisera against AIV.Dissolubility proteinum NA1 protein were highly expressed by E.coli BL21 In this experiment, and we can available purity higher NA1 protein by single step purification,that is reliability safeguard to make a solid foundation of special diagnosis kit...