tetano
Editor, Senior Moderator
Protein Expr Purif
. 2024 Jul 4:106541.
doi: 10.1016/j.pep.2024.106541. Online ahead of print. Bacterially expressed full length Hemagglutinin of Avian Influenza Virus H5N1 forms oligomers and exhibits hemagglutination
Priyanka Panwar[SUP] 1 [/SUP], Dhwani Jhala[SUP] 1 [/SUP], Anubhav Tamrakar[SUP] 1 [/SUP], Chaitanya Joshi[SUP] 1 [/SUP], Amrutlal Patel[SUP] 2 [/SUP]
Affiliations
Avian influenza poses a significant global health threat, with the potential for widespread pandemics and devastating consequences. Hemagglutinin (HA), a critical surface glycoprotein of influenza viruses, plays a pivotal role in viral entry and serves as a primary target for subunit vaccine development. In this study, we successfully cloned, expressed, and purified hemagglutinin from the circulating strain of H5N1 influenza virus using a robust molecular biology approach. The cloning process involved insertion of the synthetic HA gene into the pET21b vector, confirmed through double digestion and sequencing. SDS-PAGE analysis confirmed the presence of the expected 60 kDa protein band post-induction. Following expression, the protein was subjected to purification via Ni-NTA affinity chromatography, yielding pure protein fractions. Native PAGE analysis confirmed the protein's oligomeric forms, essential for optimal antigenicity. Western blot analysis further validated protein identity using anti-His and anti-HA antibodies. MALDI-TOF analysis confirmed the protein's sequence integrity, while hemagglutination assay demonstrated its biological activity in binding to N-acetyl neuraminic acid. These findings underscore the potential of recombinant hemagglutinin as a valuable antigen for diagnosis and biochemical assays as well as for vaccine development against avian influenza. In conclusion, this study represents a critical guide for bacterial production of H5N1 HA, which can be a cost-effective and simpler strategy compared to mammalian protein expression. Further research into optimizing vaccine candidates and production methods will be essential in combating the ongoing threat of avian influenza pandemics.
. 2024 Jul 4:106541.
doi: 10.1016/j.pep.2024.106541. Online ahead of print. Bacterially expressed full length Hemagglutinin of Avian Influenza Virus H5N1 forms oligomers and exhibits hemagglutination
Priyanka Panwar[SUP] 1 [/SUP], Dhwani Jhala[SUP] 1 [/SUP], Anubhav Tamrakar[SUP] 1 [/SUP], Chaitanya Joshi[SUP] 1 [/SUP], Amrutlal Patel[SUP] 2 [/SUP]
Affiliations
- PMID: 38971212
- DOI: 10.1016/j.pep.2024.106541
Avian influenza poses a significant global health threat, with the potential for widespread pandemics and devastating consequences. Hemagglutinin (HA), a critical surface glycoprotein of influenza viruses, plays a pivotal role in viral entry and serves as a primary target for subunit vaccine development. In this study, we successfully cloned, expressed, and purified hemagglutinin from the circulating strain of H5N1 influenza virus using a robust molecular biology approach. The cloning process involved insertion of the synthetic HA gene into the pET21b vector, confirmed through double digestion and sequencing. SDS-PAGE analysis confirmed the presence of the expected 60 kDa protein band post-induction. Following expression, the protein was subjected to purification via Ni-NTA affinity chromatography, yielding pure protein fractions. Native PAGE analysis confirmed the protein's oligomeric forms, essential for optimal antigenicity. Western blot analysis further validated protein identity using anti-His and anti-HA antibodies. MALDI-TOF analysis confirmed the protein's sequence integrity, while hemagglutination assay demonstrated its biological activity in binding to N-acetyl neuraminic acid. These findings underscore the potential of recombinant hemagglutinin as a valuable antigen for diagnosis and biochemical assays as well as for vaccine development against avian influenza. In conclusion, this study represents a critical guide for bacterial production of H5N1 HA, which can be a cost-effective and simpler strategy compared to mammalian protein expression. Further research into optimizing vaccine candidates and production methods will be essential in combating the ongoing threat of avian influenza pandemics.