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Yang J.,Hubei University of Medicine | Dai L.,University of Sichuan | Pan X.,Sichuan University | Wang H.,University of Sichuan | And 5 more authors.
Pathogens and disease | Year: 2015

Chronic gastric infection by the Gram-negative bacterium Helicobacter pylori (H. pylori) is strongly associated with gastritis, gastric ulcer and the development of distal gastric carcinoma and gastric mucosal lymphoma in humans. Antibiotic treatment of H. pylori is becoming less effective because of increasing antibiotic resistance; other treatment approaches such as specifically targeted methods, etc. to destroy this organism would be beneficial. An epitope vaccine is a promising option for protection against H. pylori infection. In this study, a multi-epitope vaccine was constructed by linking cholera toxin B subunit (CTB), two antigenic fragments of H. pylori urease I subunit (UreI20-29, UreI98-107) and four antigenic fragments of H. pylori urease B subunit (UreB12-23, UreB229-251, UreB327-400, UreB515-561), resulting in the recombinant CTB-UreI-UreB (BIB). Its protective effect against H. pylori infection was evaluated in BALB/c mice. Significant protection against H. pylori challenge was achieved in BALB/c mice immunized with BIB (15/18, 83.3%), rIB plus rCTB (6/18, 33.3%) and rIB (2/18, 11.1%) separately, while no protective effect was found in the mice immunized with either adjuvant rCTB alone or PBS. The induction of significant protection against H. pylori is possibly mediated by specific serum IgA and mucosal sIgA antibodies, and a mixed Th1/Th2/Th17 cells response. This multi-epitope vaccine might be a promising vaccine candidate that helps to control H. pylori infection. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com. Source

Ding N.-N.,University of Sichuan | Ding N.-N.,Sichuan Vaccine Technology Co. | Yang J.,University of Sichuan | Yang J.,Hubei University of Medicine | And 8 more authors.
Journal of Sichuan University (Medical Science Edition) | Year: 2014

Objective To construct the multi-epitope prokaryotic expression plasmid and appropriate engineering bacteria expressing the multi-epitope fusion protein of urea membrane channel protein (Urel), urease B subunit (UreB) and adhesin (HpaA) of Helicobacter pylori, then study its microbiological characteristics. Methods: The target sequence contains multi-epitope gene sequence of Helicobacter pylori were designed and synthesized, subsequently) it was subcloned into the expression vector pET28a (+), confirmed by restriction enzyme digestion and DNA sequencing. The fusion protein rIBA was expressed in E. coli Rosseta (DE3) and analyzed by Western blot. Results: The plasmid of pET28a(+)/IBA was constructed successfully, confirmed by endonuclease digestion and sequence analyze. The expressed rIBA protein with relative molecular mass about 40 × 103 and can be detected by Western blot. Conclusion: The prokaryotic engineering bacteria expression multi-epitope of the Helicobacter pylori was constructed successfully. The recombinant protein rIBA expressed by the engineering bacteria can be identified by Sydney strain 1 of Helicobacter pylori (H. pylori SSI) specific antibody IgY, which demonstrated that the rIBA has high correlation with H. pylori SSI. Source

Wang B.,University of Sichuan | Yang J.,University of Sichuan | Yang J.,Sichuan Vaccine Technology Co. | Yang J.,Hubei University of Medicine | And 9 more authors.
International Journal of Clinical and Experimental Pathology | Year: 2014

Objective: To study immunization procedures and preparation methods of specific IgY antibodies (IgY-Hp, IgY-IB) produced by hens immunized with Helicobacter pylori (Hp) bacterial antigen and recombinant Hp specific antigen IB, detect the inhibition effects on Hp growth and Hp urease activity, and study the effects of oral administration for treating Hp infection. Methods: By using recombinant cholera toxin subunit B (rCTB) as an adjuvant, hens received intramuscular injection immunization for continuous 7 times at an interval of 14 days. Then, the eggs were collected; IgY was purified. Results: On day 49 after hens were immunized, levels of two antibodies all reached 1:12800; after they were purified by Ammonium sulfate precipitation, their purity was over 80%. IgY-Hp could inhibit Hp growth and inhibit Hp urease activity; although in vitro, IgY-IB could not inhibit Hp growth but could inhibit Hp urease activity. The experiments in vivo found that when IgY-Hp or IgY-IB with sucralfate dual oral therapy was used to treat Hp infected mouse model, the cure rate all could reach 83.3%. Conclusion: According to immunization procedure, high titer specific IgY antibody (1:12800) can be obtained in 49 days and its titer remains stable. Oral administration of the specific IgY antibodies in Hp infected mice can reach a cure rate of 83.3%, and the antibodies are expected to become new drugs and therapeutic methods of targeted therapy against Hp infection. Source

Yang J.,University of Sichuan | Yang J.,Hubei University of Medicine | Yang J.,Sichuan Vaccine Technology Co. | Pan X.,University of Sichuan | And 8 more authors.
International Journal of Clinical and Experimental Medicine | Year: 2015

Objective: To establish high cell density cultivation process of recombinant Helicobacter pylori multi-epitope vaccine engineering bacteria BIB. Methods: Based on the results of shake flask fermentation, the process was magnified into volume of a 50 L fermenter to optimize and verify the factors affecting the yield of the target protein, such as the fermentation medium, working seed inoculation amount, inducer concentration, induction starting time, induction duration, inducer adding mode and feeding strategy. Results: After activated in modified TB medium at 37°C for 8 h, the BIB working seed was inoculated at 5% (v/v) and was induced for expression for another 11 h by the final concentration of 5 mmol/L lactose. In growth phase, glucose at rate of 80 ml/h was used as carbon source, and in induction phase, glycerol at rate of 40 ml/h was used as carbon source; ammonia water was added dropwise to control pH at about 7.0, and revolution speed is adjusted to control the dissolved oxygen at above 30%; ultimately the output of bacterial body was 70 g/L and protein expression amount was about 32%. Conclusion: After high cell density cultivation of the recombinant engineering bacteria, expression and yield of the target protein rBIB significantly increased. © 2015, E-Century Publishing Corporation. All Rights Reserved. Source

Wang B.,University of Sichuan | Pan X.,University of Sichuan | Pan X.,Sichuan Vaccine Technology Co. | Wang H.,University of Sichuan | And 5 more authors.
International Journal of Clinical and Experimental Pathology | Year: 2014

Objective: To investigate different protective effects of recombinant H. pylori multi-epitope antigen (rIB) with cholera toxin subunit B (rCTB) as the intramolecular/extramolecular adjuvant though different immunization routes in a Helicobacter pylori infected mouse model. Methods: By using rCTB as the intramolecular/extramolecular adjuvant of rIB, BALB/c mice were immunized through oral administration or intramuscular injection, on day 0, 14, 28. Every 14 days, ELISA was used to detect serum specific IgG and IgA titers after immunization. After the last immunization, H. pylori SS1 challenge was performed, and urease test, Gram staining after smearing of mouse gastric tissue, PCR, pathology and immunohistochemistry were used to evaluate preventive effect of the recombinant protein vaccine. Results: After immunization three times, intramolecular injection could induce high titers of serum specific IgG antibody, and the antibody titer in rIB group, rCTB+rIB and rBIB group was 2000, 5000 and 7500, respectively (P < 0.05). Specific IgA antibody was only detected in rBIB oral administration group. The immune protection rate in rBIB oral administration group was significantly higher than that in rBIB intramolecular injection group (33.3% vs. 83%), indicating significant difference. Conclusion: rCTB has good intramolecular/extramolecular immune adjuvant effects, and its intramolecular immune adjuvant effect is better. Both intramolecular injection and oral administration of rBIB have immune protective effect against H. pylori challenge, and oral administration of rBIB exerts better immune protective effect. Source

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