Cloning and Expression of HA2 gene of Avian Influenza A (H5N1 HA2) Virus in Pichia pastoris
Keywords:Avian influenza, Haemagglutinin, H5N1, HA2, Pichia pastoris
Avian influenza or bird flu is a highly infectious viral disease and is one of the most major concerns to both poultry industry and human health. The haemagglutinin (HA) protein is one of the markers for detection of influenza viral infection. There is no efficient protection and therapy for the disease, hence, rapid and accurate detection of avian influenza virus infection is an important tool to control outbreaks.
In this study, the HA2 gene was amplified and cloned into pPICZA expression vector. The recombinant plasmid was verified by PCR, restriction analysis and nucleotide sequencing. After transformed into Pichia pastoris, the integration of HA2 gene into host genome was confirmed. The expression of HA2 was performed and the protein was internally expressed as tagged fusion protein. The recombinant HA2 protein was extracted and purified from cell lysate using nickel affinity chromatography under native condition.
A single band of 35-40 kDa was observed by SDS-PAGE. Western blot analysis revealed that the protein could be reacted with His DetectorTM and anti myc antibody, indicating that this was protein of interest.
The HA2 recombinant protein could react with serum sample from patients recovered from avian influenza infection who had their hemaglutination inhibition (HI) titer over 1:40, while it did not react with the non-exposed individual serum. The recombinant HA2 expressed by P. pastoris could be useful as a potential antigen for epidemiological study of highly pathogenic avian influenza (HPAI) infection.
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