Abstract
Klebsiella oxytoca is a gram-negative bacterium. It is opportunistic in nature and causes hospital acquired infections. Subtractive proteomics and reverse vaccinology approaches were employed to screen out the best proteins for vaccine designing. Whole proteome of K. oxytoca strain ATCC 8724, consisting of 5483 proteins, was used for designing the vaccine. Total 1670 cytotoxic T lymphocyte (CTL) epitope were predicted through NetCTL while 1270 helper T lymphocyte (HTL) epitopes were predicted through IEDB server. The epitopes were screened for non-toxicity, allergenicity, antigenicity and water solubility. After epitope screening 300 CTL and 250 HTL epitopes were submitted to IFN-γ epitope server to predict their Interferon-γ induction response. The selected IFN-γ positive epitopes were tested for their binding affinity with MHCI-DRB1 by MHCPred. The 15 CTL and 13 HTL epitopes were joined by linkers AAY and GPGPG respectively in vaccine construct. Chain C of Pam3CSK4 (PDB ID; 2Z7X) was linked to the vaccine construct as an adjuvant. A 450aa long vaccine construct was submitted to I-TASSER server for 3D structure prediction. Thirteen Linear B cells were predicted by ABCPred server and 10 sets of discontinues epitopes for 3D vaccine structure were predicted by DiscoTope server. The modeled 3D vaccine construct was docked with human Toll-like receptor 2 (PDB ID: 6NIG) by PatchDock. The docked complexes were refined by FireDock. The selected docked complex showed five hydrogen bonds and one salt bridge. The vaccine sequence was reverse transcribed to get nucleotide sequence for In silico cloning. The reverse transcribed sequence strand was cloned in pET28a(+) expression vector. A clone containing 6586 bp was constructed including the 450 bp of query gene sequence.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
More From: International journal of peptide research and therapeutics
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.