title: [Expression and immunogenicity analysis of a recombinant fusion protein of V. Cholera ctB and H. pylori ure I].
abstract: OBJECTIVE
  To express fusion protein of the cholera toxin B subunit (ctB) and the urea membrane channel gene (ure I) of H. pylori in E. coli, and analyze its immunogenicity.
  
  
  METHODS
  The prokaryotic expression vector pET32a+/ctB/ure I was constructed by inserting ctB gene amplified by PCR into the 5' terminus of ure I gene of expression vector pET32a+/ure I. The fusion gene was verified by endonuclease digestion and sequence analysis. The fusion protein ctB/ure I was expressed in E. coli BL21(DE3), purified by His-HP affinity chromatography, and analyzed by SDS-PAGE, Western blot and Pro-gel analyzer 4.0. The mice were immunized with purified ctB/ure I, and the immunoreactivity with ctB and ure I of the murine sera was analyzed by indirect ELISA.
  
  
  RESULTS
  The pET32a+/ctB/ure I expression vector was constructed successfully and confirmed by endonuclease digestion and sequence analysis. The expressed ctB/ure I protein with molecular weight about 58,000 was shown when induced with 1 mmol/L IPTG for 4 h at 22 degrees C, and the protein could react with horse anti-ctB and human anti-ure I sera when detected with Western blot, and the purity of the purified protein was about 94.3%. The sera from mice immunized with purified ctB/ure I protein could react with ctB, ure I, and ctB/ure I when detected with indirect ELISA.
  
  
  CONCLUSIONS
  The fusion protein expression vector pET32a+/ctB/ure I was constructed successfully. The fusion protein ctB/ure I was shown to have immunoreactivity with both anti-ctB and anti-ure I anti-sera, and could evoke production of anti-ctB and anti-ure I antibody in mice. Our work established a good foundation for further study on the new and effective H. pylori vaccines.
authors.affiliation: Department of Microbiology, School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China. wbn@china-maker.com
authors.forename: Bao-ning
authors.initials: BN
authors.lastname: Wang
authors.forename: Xiao-fang
authors.initials: XF
authors.lastname: Yang
authors.forename: Qiao-fa
authors.initials: QF
authors.lastname: Shi
authors.forename: Ming-yuan
authors.initials: MY
authors.lastname: Li
authors.forename: Cui-ping
authors.initials: CP
authors.lastname: Chen
authors.forename: Kang
authors.initials: K
authors.lastname: Cao
authors.forename: Hong
authors.initials: H
authors.lastname: Li
mesh_terms: D000818:Animals; D001428:Bacterial Vaccines / Q000235:genetics / Q000276:immunology*; D002478:Cells, Cultured; D002772:Cholera Toxin / Q000235:genetics* / Q000378:metabolism; D022121:Cholera Vaccines / Q000235:genetics / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D016480:Helicobacter pylori / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*
chemical_list: D001428:Bacterial Vaccines; D022121:Cholera Vaccines; D011993:Recombinant Fusion Proteins; D002772:Cholera Toxin