Clin Exp Vaccine Res.  2016 Jan;5(1):19-25. 10.7774/cevr.2016.5.1.19.

Cloning, expression and purification flagellar sheath adhesion of Helicobacter pylori in Escherichia coli host as a vaccination target

• Soleimani
• Mohabati Mobarez A
• Farhangi B

• KMID: 2152693
• DOI: http://doi.org/10.7774/cevr.2016.5.1.19

Abstract

PURPOSE
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.
MATERIALS AND 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-beta-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.
CONCLUSION
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.

Keyword

Recombinant flagellar sheath adhesion; Helicobacter pylori; Fast protein liquid chromatography

MeSH Terms

Chromatography, Liquid
Clone Cells*
Cloning, Organism*
Digestion
DNA
DNA Restriction Enzymes
Duodenum
Electrophoresis, Polyacrylamide Gel
Escherichia coli*
Escherichia*
Helicobacter pylori*
Helicobacter*
Humans
Lipoproteins
Plasmids
Polymerase Chain Reaction
Sodium Dodecyl Sulfate
Stomach
Vaccination*
DNA
DNA Restriction Enzymes
Lipoproteins
Sodium Dodecyl Sulfate

Figure

• 

  Fig. 1 Agarose gel electrophoresis of polymerase chain reaction products stained with ethidium bromide. Lane 1, DNA marker; lanes 2, 3, 4, 5, and 6, amplified hpaA gene using pfu DNA polymerase.

• 

  Fig. 2 Evaluation of the recombinant vector enzymatic digestion. Lane 1, the hpaA gene and pET28a vector enzymatic digestion; lane 2, pET28a vector carrying the hpaA gene; lane M, DNA marker with 10 thousand base pairs length.

• 

  Fig. 3 Assessment of HpaA protein expression on 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel. Lane L, low molecular weight marker; lane 1, non-induced HpaA; lane 2, induced HpaA by 0.1 mM isopropyl-beta-thiogalactopyranoside.

• 

  Fig. 4 (A) Purification HpaA protein by fast protein liquid chromatography. (B) Purification HpaA protein in sodium dodecyl sulfate polyacrylamide gel electrophoresis gel. Lane L, low molecular weight marker; lane 1, purified HpaA protein.

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