title: [Construction, expression and antigenicity of bivalent vaccine candidate of human Helicobacter pylori].
abstract: OBJECTIVE
  To construct a recombinant vector containing fused gene of heat shock protein A(HspA) and outer membrane protein(OMP) with M(r) 18,000, from human Helicobacter pylori(Hp) and express the fusion protein in E.coli BL21.
  
  
  METHODS
  The gene encoding HspA was amplified from Hp chromosome by PCR. After digestion with kpn I and BamH I, the HspA gene was inserted into the prokaryotic expression vector pET32a(+). After recombinant vectors pET32a(+)/HspA and pET32a(+)/Omp (18) were digested with Hind III and BamH I, the pET32a(+)/HspA and 18,000 OMP gene segments were recovered through agarose electrophoresis, and connected by T4 ligase. The recombinant vector pET32a(+)/HspA-Omp(18) was transformed into E.coli BL21(DE3). The antigenicity of recombinant fusion protein was analysed by Western blot.
  
  
  RESULTS
  Enzyme digestion analysis and sequencing showed that the fused gene had 891 base pairs. As compared with gene reported in GenBank, there were 1.15% and 1.26% differences in cloned nucleotide sequence and amino acid sequence respectively. SDS-PAGE analysis showed that recombinant vector could be expressed in E.coli BL21. The relative molecule mass (M(r)) of expressed fusion protein was 51x10(3). And soluble expression product accounted for 18.96% of total bacterial protein.After purification via Ni-NTA agarose resin, the purity of recombinant fusion protein was about 95%. The Western blot analysis showed that recombinant fusion protein could be recognized by anti-Hp positive serum and anti-18,000 OMP mAb, suggesting that this protein had good antigenicity.
  
  
  CONCLUSIONS
  The fused gene of HspA and OMP is cloned and expressed successfully, which lays the foundation for development of protein and DNA vaccines and a diagnostic kit of Hp infection.
authors.affiliation: Department of Gastroenterology, the First Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China. jzho53@mail.china.com
authors.forename: Zheng
authors.initials: Z
authors.lastname: Jiang
authors.forename: Ai-long
authors.initials: AL
authors.lastname: Huang
authors.forename: Dan
authors.initials: D
authors.lastname: Pu
authors.forename: Xiao-hong
authors.initials: XH
authors.lastname: Tao
authors.forename: Pi-long
authors.initials: PL
authors.lastname: Wang
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000235:genetics* / Q000276:immunology; D001426:Bacterial Proteins; D001428:Bacterial Vaccines / Q000276:immunology*; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D006360:Heat-Shock Proteins / Q000235:genetics* / Q000276:immunology; D016480:Helicobacter pylori / Q000276:immunology*; D006801:Humans; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000276:immunology*; D014614:Vaccines, Synthetic / Q000276:immunology*
chemical_list: D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D001428:Bacterial Vaccines; D006360:Heat-Shock Proteins; C113725:HspA protein, bacteria; D011993:Recombinant Fusion Proteins; D014614:Vaccines, Synthetic