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| Bioinformatics Analysis and Optimized Expression of the Epitope Vaccine rCtUBE |
| GUO Le1,2, LIU Kun-mei1,2, QIN Yu-hong1, LI Xiao-kang2, DUAN Xiang-guo1, YANG Hua1, XU Guang-xian1, XI Tao2 |
1. School of Laboratory Medicine, Ningxia Medical University, Yinchuan 750004, China;
2. School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China |
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Abstract Helicobacter pylori (Hp) represents a major cause of gastroduodenal pathologies, such as chronic gastritis, peptic ulcer and gastric cancer. The aim is to design an epitope vaccine named rCtUBE with Cholera toxin B subunit (CTB) and an B cell epitope from Hp urease B subunit (UreB) by using the bioinformatics software and database, and get fusion protein rCtUBE with high purity after optimized expression and purification. Methods:An epitope vaccine rCtUBE composed of CTB and an epitope peptide named F8 involved in the active site of the urease was designed by analyzing the coupling sequence and linker between CTB and F8 through bioinformatics software. The recombinant expression vector pETCUB containing the fusion gene rCtUBE and the recombinant strain BL21(DE3)/pETCUB were constructed by gene cloning technology. After protein expression and optimization, the fusion protein rCtUBE was purified by Ni2+-charged column chromatography and anion-exchange chromatography using DEAE Sepharose FF. Then, the activity of epitope vaccine rCtUBE was investigated by intraperitoneal immunization experiments in BALB/c mice. Results:The epitope vaccine rCtUBE had a scientific and reasonable structure through the analysis of bioinformatics software. The recombinant expression vector pETCUB and recombinant strain BL21(DE3)/pETCUB were constructed successfully. After protein expression optimization and purification, about 56 mg of pure target protein was obtained from 1 L of fermentation broth and the purity of rCtUBE was 95.3%. Mice immunized with rCtUBE using aluminum hydroxide adjuvant could induce high level of antibodies specific for both CTB and F8 by ELISA. Conclusion:The epitope vaccine rCtUBE with a scientific and reasonable structure was expressed at a medium level in E. coli and had good immunological specificity. This will provide much experimental evidences for the development of epitope vaccines against Hp for human use.
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Received: 28 June 2013
Published: 25 October 2013
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| Fund: This work was supported by a Grant from National Major Special Program of New Drug Research and Development (No. 2012ZX09103-301-008) |
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[1] Marshall B J, Warren J R. Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration. Lancet, 1984,1: 1311-1315.
[2] Sette A, Fikes J. Epitope-based vaccines: an update on epitope identification, vaccine design and delivery. Curr Opin Immunol, 2003,15: 461-470.
[3] Eaton K A, Brooks C L, Morgan D R, et al. Essential role of urease in pathogenesis of gastritis induced by Helicobacter pylori in gnotobiotic piglets. Infect Immun, 1991,59: 2470-2475.
[4] Marshall B J, Barrett L J, Prakash C, et al. Urea protects Helicobacter (Campylobacter) pylori from the bactericidal effect of acid. Gastroenterology, 1990,99: 697-702.
[5] Nagata K, Mizuta T, Tonokatu Y, et al. Monoclonal antibodies against the native urease of Helicobacter pylori: synergistic inhibition of urease activity by monoclonal antibody combinations. Infect Immun, 1992,60: 4826-4831.
[6] Zhao W, Wu W, Xu X. Oral vaccination with liposome-encapsulated recombinant fusion peptide of urease B epitope and cholera toxin B subunit affords prophylactic and therapeutic effects against H. pylori infection in BALB/c mice. Vaccine, 2007,25: 7664-7673.
[7] Guo L, Li X, Tang F, et al. Immunological features and the ability of inhibitory effects on enzymatic activity of an epitope vaccine composed of cholera toxin B subunit and B cell epitope from Helicobacter pylori urease A subunit. Appl Microbiol Biotechnol, 2012,93: 1937-1945.
[8] Czinn S J, Blanchard T. Vaccinating against Helicobacter pylori infection. Nat Rev Gastroenterol Hepatol, 2011,8: 133-140.
[9] Liu W, Peng Z, Liu Z, et al. High epitope density in a single recombinant protein molecule of the extracellular domain of influenza A virus M2 protein significantly enhances protective immunity. Vaccine, 2004,23: 366-371.
[10] Livingston B, Crimi C, Newman M, et al. A rational strategy to design multiepitope immunogens based on multiple Th lymphocyte epitopes. J Immunol, 2002,168: 5499-5506.
[11] Liljeqvist S, Stahl S, Andreoni C, et al. Fusions to the cholera toxin B subunit: influence on pentamerization and GM1 binding. J Immunol Methods, 1997,210: 125-135.
[12] Hirota K, Nagata K, Norose Y, et al. Identification of an antigenic epitope in Helicobacter pylori urease that induces neutralizing antibody production. Infect Immun, 2001,69: 6597-6603. |
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