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Cloning, expression and purification of C-terminal of Helicobacter pylori urease enzyme C subunit for production IgY in chicken egg yolk
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Hossein Basiri   1, Seyed Latif Mousavi Gargari2 , Iraj Rasuli2 , Kazem Parivar3 , Taher Nejad Satari3 |
1- Islamic Azad University, Science and Research Campus, Tehran, Iran , hosseinbasiri@yahoo.com
2- Shahed University, Tehran, Iran
3- Islamic Azad University, Science and Research Campus, Tehran, Iran |
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Abstract: (25481 Views) |
Background: H. pylori is a spiral, microaerophilic gram negative bacterium, that multiplies and causes infection in human gastric mucosal layer. New approaches have focused on using specific treatments, such as immunotherapy, to eradicate this infection. Urease, as one of the most important virulent and antigenic factors of the bacterium, is a suitable target for this purpose.
Material and methods: In this experimental study, after purification of bacterial genomic, 3’ segment of ureC gene was amplified by polymerase chain reaction (PCR). The PCR product was ligated to pET28a. The recombinant protein was expressed followed by transformation of recombinant construct into E. coli BL21DE3. SDS-PAGE analysis was carried out and the recombinant protein was purified by Ni-NTA affinity chromatography. The purified recombinant protein was injected to hens. IgY recovered from egg yolk, was purified by aceton/chloroform precipitation. The purified IgY was analyzed by ELISA and SDS-PAGE.
Results: Sequencing of recombinant construct confirmed accuracy of cloning. SDS-PAGE analysis revealed a good expression and purification of the recombinant protein rUreCc. ELISA observation demonstrated high immunogenicity of the recombinant protein.
Conclusion: Production of rUreCc recombinant protein of H. pylori within E.coli, as host cell, provides an easy availability to antigen. In spite of recombinant antigen small size, its immunization potency is similar to total subunit of urease. Also, small size of recombinant protein has an important role in its stability, expression and purification. |
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| Keywords: Helicobacter pylori, Urease, UreC, IgY technology |
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Full-Text [PDF 592 kb]
(3096 Downloads)
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Semi-pilot: Experimental |
Subject:
Gastrology
Received: 2012/04/3 | Published: 2012/04/15
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