Cloning, expression, purification and characterization of recombinant UreB229-561 from Helicobacter pylori.
Background: 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 usefu...
| Publicado en: | Iranian Journal of Clinical Infectious Diseases Vol. 5; no. 1; pp. 18 - 25 |
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| Autores principales: | , , , , |
| Formato: | pictorial research tables/charts Journal Article |
| Publicado: |
Iranian Journal of Clinical Infectious Diseases
Winter2010
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| Acceso en línea: | Ver este registro en EBSCOhost |
| fields | @attributes: recordID: 1 pdfLink: plink: https://search.ebscohost.com/login.aspx?direct=true&db=ccm&AN=105040940&site=ehost-live header: @attributes: shortDbName: ccm uiTerm: 105040940 longDbName: CINAHL Complete uiTag: AN controlInfo: bkinfo: dissinfo: jinfo: jid: 17355109 783F jtl: Iranian Journal of Clinical Infectious Diseases issn: 17355109 maglogo: N pubinfo: dt: Winter2010 vid: 5 iid: 1 pid: 60058 pub: Iranian Journal of Clinical Infectious Diseases artinfo: ui: 105040940 105040940 2010699478 105040940 ppf: 18 ppct: 7 formats: tig: atl: Cloning, expression, purification and characterization of recombinant UreB229-561 from Helicobacter pylori. aug: au: Hajikhani B NajarPeerayeh S Soleimanjahi H Hassan ZM Goudarzi G affil: Department of Bacteriology, Faculty of Medical Sciences, Tarbiat Modarres University, Tehran, Iran sug: subj: Genetic Techniques Helicobacter Pylori Recombinant Proteins Blotting, Western Human Iran Research Methodology ab: Background: 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 in 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. Results: Having transformed competent E. coli DH5alpha 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 37degreesC, 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. Conclusion: 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. pubtype: Academic Journal doctype: pictorial research tables/charts Journal Article ougenre: Article language: English refInfo: holdings: @attributes: islocal: N |
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