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Cloning And Expressing Of Helicobacter Pylori UreB Gene In Plants

Posted on:2005-12-21Degree:MasterType:Thesis
Country:ChinaCandidate:J Y LiuFull Text:PDF
GTID:2120360125969690Subject:Genetics
Abstract/Summary:PDF Full Text Request
More than half of the population in the world is chronically infected by the gastric pathogen. Helicobacter pylori (H. pylori), which is the principal cause of most peptic ulcer disease, chronic active gastritis, gastric cancer and MALT lymphoma. Current therapies for eradicating H. pylori depend on the use of combined antibiotics. However the high cost, low patient compliance, and increasing of resistant strains make these therapies impractical on a large scale. Therefore, there is an urgent need for an effective vaccine against H. pylori infection. It is demonstrated that urease B subunit (UreB) could stimulate body to raise immunoresponse against challenge of H. pylori. In the paper, we cloned H. pylori UreB gene and transferred it into tobacco, rice and strawberry by Agrobacterium tumefaciens EHA105. The results are showed as follow:1. The UreB gene was amplified from Zhejiang clinical isolate of H. pylori chromosomal DNA by PCR technique, and was cloned into plasmid pGEM-7zf(+). The sequence of UreB gene was determined and analyzed by biological software Genedoc 3.2. After then, the UreB gene was inserted into plant expression vector pBI121 and pCAMBIA13011 under the control of CaMV 35S promoter.2. The tobacco leaves were transformed by Agrobacterium tumefaciens EHA105 with UreB gene. After twice selections of kanamycin cultures, 24 plants were regenerated from selective medium, and about 66% of them turned out to be transgenic. PCR and PCR-Southern also confirmed that the UreB gene has been integrated into the chromosomal DNA of these transgenic plants. The result of Western-blot showed that UreB protein was expressed in the part of the transgenic tobacco.3. The rice calli induced from mature embryos of zhonghuall cultivar were transformed by Agrobacterium tumefaciens EHA105 with UreB gene. After twice selections of hygromycin cultures, 12 GUS reaction positive plants were regenerated from selective medium, and about 75% of them turned out to be transgenic. In the same way, PCR and PCR-Southern confirmed that the UreB genehas been integrated into the chromosomal DNA of these transgenic plants. The result of Western-blot showed that UreB protein was expressed in the part of the transgenic rice. The leaves of strawberry were also used as materials to study the transgenic research, but the transgenic plants were not available.
Keywords/Search Tags:Helicobacter pylori, UreB, tobacco, rice, strawberry, vaccine, Agrobacterium-mediated transformation
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