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Cloning, expression and purification of the influenza A (H9N2) virus M2e antigen and truncated Mycobacterium tuberculosis HSP70 as a fusion protein in Pichia pastoris.

Abstract : Due to its conservation, the extracellular domain of the influenza A M2 protein (M2e) has the potential for being applied as a recombinant vaccine candidate against a wide range of strains, though its immunogenicity may need to be improved. The occurrence of several post-translational modifications within the structure of M2 protein may affect its immunopotency for the induction of humoral immune response. Herein, to construct a recombinant M2e-based vaccine candidate with the appropriate structural conformation and immunogenicity the corresponding nucleotide sequence from an H9N2 influenza strain was fused to the N-terminus of the truncated Mycobacterium tuberculosis HSP70(359-610), as a potent adjuvant, and following its cloning into the pPICZ alpha A plasmid the fusion gene was expressed in Pichia pastoris KM71H yeast. The secreted protein was then easily purified from the culture media, based on the presence of polyhistidine tag and used for the production of rabbit polyclonal antisera. This raised antisera could recognize the native M2e protein on the surface of H9N2 influenza virus-infected MDCK cells at a comparable level with the commercial H2N2-specific anti-M2 antibody, which was evidenced with immunofluorescence and cell-ELISA assays. These results not only re-emphasized on the conservancy of the M2e antigen, but also pointed towards the applicability of the M2e-HSP70(359-610) fusion protein for the induction of specific antibodies capable of binding to the native M2e antigen on the infected cells. Collectively, this study implied that purified M2e-HSP70(359-610) represents a promising vaccine candidate; however, its in vivo potency for the induction of protection remains to be evaluated.
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Submitted on : Wednesday, October 24, 2012 - 10:25:02 AM
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Seyyed Mahmoud Ebrahimi, Majid Tebianian, Hadi Toghyani, Arash Memarnejadian, Hamid Reza Attaran. Cloning, expression and purification of the influenza A (H9N2) virus M2e antigen and truncated Mycobacterium tuberculosis HSP70 as a fusion protein in Pichia pastoris.. Protein Expression and Purification, Elsevier, 2010, 70 (1), pp.7-12. ⟨10.1016/j.pep.2009.11.001⟩. ⟨pasteur-00744903⟩

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