Recombinant haemagglutinin protein of highly pathogenic avian influenza A (H5N1) virus expressed in Pichia pastoris elicits a neutralizing antibody response in mice

Subathra Murugan, Santhakumar Ponsekaran, Lingaraja Kannivel, Lakshmi Narasu Mangamoori, Dev Chandran, Srinivasan Villuppanoor Alwar, Chandrani Chakravarty, Sunil K. Lal

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Recombinant avian influenza vaccines offer several advantages over the conventional vaccines. In this study, the haemagglutinin (HA) gene of highly pathogenic avian influenza H5N1 was cloned and expressed as His tagged protein in methylotropic yeast Pichia pastoris. The expression of recombinant HA (rHA) protein was confirmed by SDS-PAGE and western blot analysis. The rHA protein was purified using Ni-NTA affinity chromatography under denaturing conditions and the functions of the protein was assessed by the haemagglutinin assay after refolding. The immunogenicity of the rHA was evaluated by immunizing four groups of mice with different payloads (2.5, 5.0, 10 and 25μg) of purified rHA and the production of rHA specific antibodies were analysed by haemagglutinin inhibition assay (HI) and enzyme-linked immunosorbent assay (ELISA). An antigen specific immune response was observed against rHA indicating that the rHA antigen could be used as a vaccine candidate against avian influenza. These results suggest that this strategy would pave the way for the development of rapid and cost effective method for the production of an avian influenza vaccine.

Original languageEnglish
Pages (from-to)20-25
Number of pages6
JournalJournal of Virological Methods
Issue number1
Publication statusPublished - Jan 2013
Externally publishedYes


  • Enzyme-linked immunosorbent assay
  • Haemaglutination
  • Haemaglutination inhibition
  • Highly pathogenic avian influenza
  • Immunogenicity

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