title: Construction and characterization of bivalent vaccine candidate expressing HspA and M(r)18,000 OMP from Helicobacter pylori.
abstract: OBJECTIVE
  To construct a recombinant vector which can express outer membrane protein (OMP) with M(r)18,000 and heat shock protein A (HspA) from Helicobacter pylori (H. pylori) in E. coli BL21, and to exploit the possibility for obtaining the vaccine conferring protection from H. pylori infection.
  
  
  METHODS
  The target gene of HspA was amplified from H. pylori chromosome by PCR, and then inserted into the prokaryotic expression vector pET32a (+) by restrictive endonuclease enzyme kpn I, BamH I simultaneously. The recombinant vector was used to sequence, and then together with pET32a (+)/Omp(18), digested by restrictive endonuclease enzyme Hind III and BamH I simultaneously. pET32a(+)/HspA and Omp(18) were recovered from 1 % agarose gel by gel kit, and ligated with T(4) ligase by BamH I digested viscidity end. The recombinant plasmid of pET32a(+)/HspA/Omp(18) was transformed and expressed in E. coli BL21 (DE3) under induction of IPTG. After purification, its antigenicity of the fusion protein was detected by Western blot.
  
  
  RESULTS
  Enzyme digestion analysis and sequencing showed that the target genes were inserted into the recombinant vector, composed of 891 base pairs, encoded objective polypeptides of 297 amino acid residues. Compared with GenBank reported by Tomb et al, there were 1.3 % and 1.4 % differences in obtained H. pylori nucleotide sequence and amino acid residues, respectively. SDS-PAGE analysis showed that relative molecule mass (M(r)) of the expressed product was M(r) 51,000, M(r) of protein expressed by pET32a (+) was about M(r) 20,000, and soluble expression product accounted for 18.96 % of total bacterial protein. After purification with Ni(+2)-NTA agarose resins, the purification of recombinant fusion protein was about 95 %. Western blot showed that recombinant fusion protein could be recognized by the patients' serum infected with H. pylori and anti-Omp(18) monoclone, suggesting that this protein had good antigenicity.
  
  
  CONCLUSIONS
  The gene coding for H. pylori M(r)18,000 OMP and HspA was cloned and expressed successfully. The results obtained lay the foundation for development of H. pylori protein vaccine and a quick diagnostic kit.
authors.affiliation: Department of Gastroenterology, the First Affiliated Hospital, Chongqing University of Medical Sciences, Chongqing 400016, China. jianggooddoctor@mail.china.com
authors.forename: Zheng
authors.initials: Z
authors.lastname: Jiang
authors.forename: Ai-Long
authors.initials: AL
authors.lastname: Huang
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*; D001426:Bacterial Proteins; D001428:Bacterial Vaccines / Q000235:genetics* / Q000627:therapeutic use; D015870:Gene Expression; D005822:Genetic Vectors; D006360:Heat-Shock Proteins / Q000235:genetics*; D016481:Helicobacter Infections / Q000517:prevention & control; D016480:Helicobacter pylori / Q000235:genetics* / Q000276:immunology*; D006801:Humans
chemical_list: D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D001428:Bacterial Vaccines; D006360:Heat-Shock Proteins; C113725:HspA protein, bacteria