title: Construction of a prokaryotic expression system of vacA gene and detection of vacA gene, VacA protein in Helicobacter pylori isolates and ant-VacA antibody in patients' sera.
abstract: OBJECTIVE
  To construct a recombinant prokaryotic expression vector inserted with Helicobacter pylori vacA gene and identify the immunity of the expressed recombinant protein, and to determine prevalence of vacA-carrying/VacA expressing H pylori isolates and seroprevalence of specific ant-VacA antibody in H pylori infected patients.
  
  
  METHODS
  Polymerase chain reaction technique was used to amplify complete vacA gene of H pylori strain NCTC11637 and to detect vacA gene in 109 H pylori isolates. The amplification product of the complete vacA gene was sequenced after T-A cloning. A recombinant expression vector inserted with a complete vacA gene fragment, named as pET32a-vacA, was constructed. Expression of the target recombinant protein VacA (rVacA) was examined by SDS-PAGE. Western blot using commercial antibodies against whole cell of H pylori and an immunodiffusion assay using self-prepared rabbit anti-rVacA antibody were applied to determine immunoreaction and antigenicity of rVacA. Two ELISA methods were established to detect VacA expression in H pylori isolates and the specific anti-VacA antibody in sera from 125 patients infected with H pylori.
  
  
  RESULTS
  In comparison with the reported corresponding sequences, homologies of nucleotide and putative amino acid sequences of the cloned vacA gene were 99.82% and 100%, respectively. The constructed recombinant prokaryotic expression system efficiently produced rVacA. rVacA was able to combine with the commercial antibodies against whole cell of H pylori and to induce the immunized rabbit to produce specific antibody with an immunodiffusion titer of 1:4. All tested H pylori isolates carried vacA gene, but only 66.1% expressed VacA protein. Of the serum samples tested, 42.4% were positive for specific anti-VacA antibody.
  
  
  CONCLUSIONS
  A prokaryotic expression system of H pylori vacA gene was successfully constructed. The expressed rVacA can be used to detect specific anti-VacA antibody in human and to prepare antiserum in animals. The high frequency of vacA gene in H pylori isolates, but with a low frequency of VacA expression and specific anti-VacA antibody in H pylori infected patients implies that VacA is not an ideal antigen for H pylori vaccine.
authors.affiliation: Department of Medical Microbiology and Parasitology, College of Medical Science, Zhejiang University, 353 Yan An Road, Hangzhou 310031, Zhejiang Province, China. yanchen@mail.hz.zj.cn
authors.forename: Jie
authors.initials: J
authors.lastname: Yan
authors.forename: Ya-Fei
authors.initials: YF
authors.lastname: Mao
mesh_terms: D000907:Antibodies, Bacterial / Q000097:blood; D001426:Bacterial Proteins / Q000097:blood* / Q000235:genetics* / Q000276:immunology; D001428:Bacterial Vaccines / Q000276:immunology*; D016481:Helicobacter Infections / Q000097:blood / Q000821:virology; D016480:Helicobacter pylori / Q000201:enzymology / Q000276:immunology*; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000276:immunology*; D014614:Vaccines, Synthetic / Q000276:immunology*
chemical_list: D000907:Antibodies, Bacterial; D001426:Bacterial Proteins; D001428:Bacterial Vaccines; D011993:Recombinant Fusion Proteins; C074689:VacA protein, Helicobacter pylori; D014614:Vaccines, Synthetic