Cloning, expression, purification and characterization of recombinant UreB229-561 from Helicobacter pylori
Helicobacter pylori is a widely distributed gram negative bacterium that infects the human stomach and duodenum. Some antibiotic regimens are subjected to cure the infection but the cost of drugs, poor patient compliance and emerging of antibiotic-resistant strains are limiting the usefulness of these antibiotic therapies. Therefore, interest in a H. pylori vaccine is growing up rapidly.
Materials And Methods
We selected a fragment of B subunit of H. pylori urease enzyme consist of four important epitopes, involving in elevating host immune responses. This 1070bp fragment was amplified by PCR from genomic DNA isolated from H. pylori 22596 and then cloned into the pET28a expression vector. UreB229-561 was expressed and then affinity-purified by Ni2+-Sepharose resin. The recombinant UreB229-561 was reacted with the serum of H. pylori-infected human and rabbit anti-H. pylori polyclonal antibody in western-blot analysis.
Having transformed competent E.coli DH5α with ligation product of digested ureB fragment and pET28a, plasmid extraction from single colonies appeared in LB-agar plate after 18-24 h incubation at 37°C, using plasmid extraction kit (Bioneer, Korea). Applying both infected human serum and rabbit anti-H. pylori polyclonal antibody, brown strip corresponding to the location of the recombinant protein appeared on PVDF membrane after adding DAB solution, hence confirming the antigenicity of the protein. This recombinant fragment showed urease activity.
Our findings confirmed that a prokaryotic expression system of rUreB229-561 was successfully constructed. The results of SDS-PAGE showed that our constructed prokaryotic expression system pET28a- ureB229-561 -BL21DE3 efficiently produces target recombinant protein in the form of dissoluble inclusion body. Therefore we can suggest that these epitopes can effectively be a vaccine candidate.
Archives of Clinical Infectious Diseases, Volume:5 Issue:1, 2010
18 - 24  
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