title: [Construction, expression and purification of UreB-Omp11 fusion protein of Helicobacter pylori and its immunocompetence].
abstract: OBJECTIVE
  To construct H.pylori vaccine candidate strain expressing UreB-Omp11 recombinant fusion protein of H.pylori. To express and purify the fusion protein UreB-Omp11 and to determine its immunocompetence.
  
  
  METHODS
  The two genes were amplified by PCR, and the fusion gene ureB-omp11 was amplified by over lap extension PCR and then cloned into the fusion expression vector pET30a(+), pET28a(+) and pMAL-c2X. The appropriate expression system was selected, and the recombinant UreB-Omp11 fusion protein was expressed and indentfied by SDS-PAGE and Western blot analysis. Then the fusion protein was purified by MBP affinity chromatography and the purity was indentfied by SDS-PAGE. Then the fusion protein was immunized to mice. The immunized mice sera were analyzed by Western blot with purified fusion protein.
  
  
  RESULTS
  The ureB-omp11 fusion gene was correctly insected into pET30a(+) and confirmed by Enzyme digestion and sequencing analysis; Results in SDS-PAGE and optical density scanning demonstrated that this fusion protein MBP-UreB-Omp11 was expressed in the recombinant strain of E.coli TB1(pMAL-ureB-omp11). The fusion protein UreB-Omp11 was recognized by the mice sera immunized by H.pylori, the human sera infected with H.pylori and The purity of fusion protein was 90% after purification. The fusion protein purified could be recognized by corresponding antibody of mice sera immunized by this fusion piotein, This fusion protein has strong immunoantigenicity and immunoreactivity.
  
  
  CONCLUSIONS
  The prokaryotic expression system TB1 (pMAL-c2X-ureB-omp11) was successfully constructed and selected. The results obtained lay the foundation for research on development of protein and DNA vaccine of Hp.
authors.affiliation: Department of Clinical Medicine, Henan Province Xinyang Vocational and Technical College, Xinyang, China. zhaoyuxia888@126.com
authors.forename: Yu-Xia
authors.initials: YX
authors.lastname: Zhao
authors.forename: Jian-Hua
authors.initials: JH
authors.lastname: Qi
authors.forename: Han
authors.initials: H
authors.lastname: Zhang
authors.forename: Guang-Cai
authors.initials: GC
authors.lastname: Duan
authors.forename: Yuan-Lin
authors.initials: YL
authors.lastname: Xi
mesh_terms: D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification; D001428:Bacterial Vaccines / Q000096:biosynthesis / Q000276:immunology* / Q000302:isolation & purification; D015153:Blotting, Western; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D016480:Helicobacter pylori / Q000235:genetics / Q000276:immunology*; D007121:Immunocompetence; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification
chemical_list: D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D001428:Bacterial Vaccines; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins