title: Cloning, expression and purification flagellar sheath adhesion of Helicobacter pylori in Escherichia coli host as a vaccination target.
abstract: OBJECTIVE
  Helicobacter pylori is a widely distributed gram-negative bacterium that infects the human stomach and duodenum. HpaA is a H. pylori-specific lipoprotein that has been shown to be an effective protective antigen against H. pylori infection. HpaA of H. pylori as a vaccine antigen is fully competent for stimulation of immune responses. The aim of this project is cloning, expression, and purification flagellar sheath adhesion of H. pylori in Escherichia coli host by fast protein liquid chromatography (FPLC) as a vaccination target.
  
  
  METHODS
  The hpaA gene was inserted into pET28a (+) as cloning and expression vectors respectively. The recombinant plasmid (pET-hpaA) was subjected to sequencing other than polymerase chain reaction (PCR) and digestion analysis. Protein expression was induced by adding 1 mM isopropyl-β-D-thiogalactoside to cultures of E. coli strain BL21 transformed with pET-hpaA. Protein expression assessed with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Protein purification of flagellar sheath adhesion was by FPLC.
  
  
  RESULTS
  The restriction endonuclease digestion, PCR amplification analysis showed that the hpaA gene of 730 bp was amplified from H. pylori DNA and sequencing analysis of the pET-hpaA confirmed the cloning accuracy and in frame insertion of hpaA fragment. SDS-PAGE analysis showed the expression of an approximately 29,000 Da protein.
  
  
  CONCLUSIONS
  Sequencing results along with SDS-PAGE analysis confirms the expression of recombinant hpaA in the heterologous E. coli BL21. Conclusion A prokaryotic expression system for hpaA gene was successfully constructed. These results indicate that production of a specific recombinant protein is an alternative and potentially more expeditious strategy for development of H. pylori vaccine.
authors: Department of Microbiology, Faculty of Biological Sciences, Shahid Beheshti University, Tehran, Iran.
authors: Neda
authors: 0000-0003-0803-9818
authors: N
authors: Soleimani
authors: Department of Bacteriology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
authors: Ashraf
authors: 0000-0002-1224-6134
authors: A
authors: Mohabati Mobarez
authors: Molecular Genetics, Cancer Research Center, Tehran University of Medical Sciences, Tehran, Iran.
authors: Baharak
authors: 0000-0002-7735-2851
authors: B
authors: Farhangi
keywords: Fast protein liquid chromatography; Helicobacter pylori; Recombinant flagellar sheath adhesion