Cloning and expression of HpaA protein of Helicobacter pylori
Abstract number: P915
Najar Peerayeh S., Atoofi J., Farshchian M., Azimian A., Razeghi M., Samimi R.
Introduction:Helicobacter pylori is associated with the chronic gastritis, peptic ulcer, gastric adenocarcinoma, and gastric mucosa-associated lymphoid tissue (MALT) lymphoma. The antibiotic therapies do not protect from potential re-infection, and have a risk for development of drug resistance. Therefore, the prophylactic vaccine mediated protection against H. pylori is an attractive clinical interest. H. pylori adhesin A (HpaA) is a conserved surface lipoprotein and plays important roles in pathogenesis of infection. In this study an hpaA gene cloned in pET expression vector and the recombinant protein (rHpaA) was over expressed in E. coli.
Methods: Genomic DNA of the standard H. pylori strain 26695 was isolated as the template and hpaA gene was amplified by PCR. Prokaryote expression vector pET28a inserted with hpaA gene (pET28a-hpaA) was constructed. The recombinant plasmid was used to transform competent E. coli DH5alpha, and positive clones were selected. Then the recombinant plasmid was used to transform E. coli BL21DE3 for expression of recombinant protein HpaA. The expression of recombinant protein induced by isopropythio-beta-D-galctoside (IPTG) at different concentration was examined by SDS-PAGE. Western blot assay were used to determine immunoreactivity of rHpaA by a rabbit polyclonal antibodies against whole cell of H. pylori.
Results: In comparison with the reported corresponding sequences, the nucleotide sequence homology of hpaA gene was 98.8%. HpaA fusion protein was able to react with the rabbit polyclonal antibody against whole cell of H. pylori.
Conclusion: A prokaryotic expression system with high efficiency of H. pylori hpaA gene was successfully established and HpaA fusion protein showed satisfactory immunoreactivity. These results indicate that production of specific recombinant protein is an alternative and potentially more expeditious strategy for development of H. pylori vaccine.
|Session name:||18th European Congress of Clinical Microbiology and Infectious Diseases|
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