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The Food-grade Expression Of Helicobacter Pylori UreB Gene In Lactococcus Lactis And Its Immunoreactivity

Posted on:2011-01-03Degree:MasterType:Thesis
Country:ChinaCandidate:S Y ChenFull Text:PDF
GTID:2120330332958609Subject:Epidemiology and Health Statistics
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Helicobacter pylori (H. pylori) is the principal cause of chronic gastritis, pepti ulcers and stomach cancer. Current therapies are mainly based on antibiotics together with antiacid. Although the antibiotic-based triple treatments have recently acquired good therapeutic effect, it is not practical for global control for the high cost of antibiotic, problems with patient's compliance and the increasing drug resistant strains. So, under these circumstances vaccination against H. pylori infection has been considered the best way to control H. pylori infection all over the word.Urease is acid stable, appears to be essential for stomach colonization by H. pylori. The subunit B(UreB) which has been cloned in E. coli.UreB combination with immunogenic adjuvant can stimulate the mouse to producing immunoresponse against challenge of H. pylori. In all the oral immunization experiments, without immunogenic adjuvant, UreB can not protect the animals gainst challenge of H. pylori. The Cholera toxin (CT) and E. coli heat-labile toxin (LT) used to mucosal adjuvant. Because of the toxicity in volunteers, CT or LT can not be given to people.By contrast, using Lactococcus lactis (L.lactis) to deliver protective antigens to the mucosal surface may be a way to solve these problems. L. lactis is nonpathogenic food-grade bacterium that is generally recognized as safe and widely used in food industry. But these traditional genetically modified organism usually used antibiotics resistance gene screening method. So it has potential risk of the antibiotic-resistant. A number of reaserch groups have reported that L. lactis can be widespread used for the expression of heterogeneous genes.Method1.ureB gene was obtained from the recombinant vector of pMD19-T-ureB by NcoⅠand XbaⅠenzyme digestion. The ureB gene was inserted into pNZ8149 food-grade expression vector which lacF is the food-grade selection.Then the recombinant plasmid pNZ8149-ureB was electrotransformed into L. lactis NZ3900.Due to the lacF deletion, strain NZ3900 is unable to grow on lactose.2. L25 (53) orthogonal design was used to optimization the expression conditions of UreB.Different dosage of inducer, the OD6oo of culture, incubating time after iuduced were chosen. Five levels had set for each factor. Then Brandford method was used to work out the expression quantity of the target protein. ANOVA method was used to analyze the results.3.This study acquired fusion protein MBP-UreB from E. coli TB1/pMAL-C2X-UreB. The MBP-UreB fusion protein immunized mice to produce anti-UreB immune serum. Western-blot was performed to check the immunological activity of UreB expressed by food-gread gene expression system.Results1.The food-grade expression vector was successfully constructed through PCR and enzyme digestion.The recombinant plasmid was electrotransformed into L. lactis NZ3900.2.Nisin 25ng/mL was added to the medium when the recombinant grew to OD600≈0.4 then incubate 5h.As a result, the maximum yield of UreB was estimated to be 7% of total soluble cellular proteins.3.The titre of the antiserum was determined to be 1:800 by enzyme lined immunosorbent assay (ELISA).Western blot analysis showed that the UreB protein expressed by L. lactis transformant had favorable immunoreactivity.Conclusions1.Because all the fragments used to construct the expression vector system L. lactis NZ3900/pNZ8149-ureB were obtained from food-grade bacteriat, the expression vector system can be regarded as a food-grade expression vector system.2.The optimal expression conditions for targeted protein expressed by NZ3900/pNZ8149-ureB have been obtained by orthogonal design. 3.The recombinant protein UreB expressed by L. lactis was detected by Western-blot. All the results make an appealing case for construction of the food-grade vaccine for H. pylori.
Keywords/Search Tags:Helicobacter pylori, urease subunit B gene, Lactococcus lactis, food-grade expression vector
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