title: Cloning and expression of a recombinant CagA -gene fragment of Helicobacter pylori and its preliminary evaluation in serodiagnosis.
abstract: BACKGROUND
  Helicobacter pylori strains expressing cytotoxic CagA protein are more commonly associated with peptic ulceration, atrophic gastritis and gastric adenocarcinoma than those lacking CagA. Determination of anti-CagA antibodies, therefore, acquires a relevant clinical significance in the serological detection of H. pylori infection and disease risk prediction. However, the CagA-serology has been questioned due to the differences found in their performance evaluations in different populations.
  
  
  OBJECTIVE
  To obtain a recombinant CagA fragment useful for serodiagnosis of H. pylori infection
  
  
  METHODS
  A fragment of the cagA gene was cloned into a prokaryotic T7 RNA polymerase expression vector. A recombinant C-terminal His 6 -tagged CagA was expressed, subsequently solubilized with urea and purified by immobilized metal affinity chromatography. The performance of the recombinant protein was evaluated using 180 human serum samples with an in-house Western blot assay compared to the Helicoblot 2.1 reference test.
  
  
  RESULTS
  The expressed His 6 -tagged CagA showed an immunoreactive 80kDa band as was revealed by SDS-PAGE and Western blot analysis using two different specific anti-CagA polyclonal antibodies. The recombinant protein was successfully purified obtaining a 93% of purity. The performance analysis of the purified recombinant antigen showed good immunoreactivity and exhibited values of sensitivity, specificity and accuracy of 88.1%, 100% and 92.7%, respectively.
  
  
  CONCLUSIONS
  The CagA fragment of the study may constitute a useful tool for serological diagnosis of CagA-positive H. pylori infection.
authors.affiliation: División de Enfermedades Infecciosas, Centro Nacional de Investigaciones Científicas, La Habana, Cuba.
authors.forename: Lidice
authors.initials: L
authors.lastname: González
authors.affiliation: División de Enfermedades Infecciosas, Centro Nacional de Investigaciones Científicas, La Habana, Cuba.
authors.forename: Karen
authors.initials: K
authors.lastname: Marrero
authors.affiliation: División de Enfermedades Infecciosas, Centro Nacional de Investigaciones Científicas, La Habana, Cuba.
authors.forename: Orlando
authors.initials: O
authors.lastname: Reyes
authors.affiliation: Centro de Estudios de Proteínas, Facultad de Biología, Universidad de La Habana, La Habana, Cuba.
authors.forename: Elaine
authors.initials: E
authors.lastname: Rodríguez
authors.affiliation: Departamento de Gastroenterología, Hospital Carlos J. Finlay, La Habana, Cuba.
authors.forename: Liudmila
authors.initials: L
authors.lastname: Martínez
authors.affiliation: Centro de Estudios de Proteínas, Facultad de Biología, Universidad de La Habana, La Habana, Cuba.
authors.forename: Boris L
authors.initials: BL
authors.lastname: Rodríguez
mesh_terms: D000293:Adolescent; D000328:Adult; D000942:Antigens, Bacterial / Q000096:biosynthesis / Q000097:blood* / Q000235:genetics; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000097:blood* / Q000235:genetics; D003001:Cloning, Molecular; D015870:Gene Expression; D016481:Helicobacter Infections / Q000097:blood* / Q000175:diagnosis*; D016480:Helicobacter pylori / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008875:Middle Aged; D011994:Recombinant Proteins; D012698:Serologic Tests; D055815:Young Adult
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D011994:Recombinant Proteins; C080866:cagA protein, Helicobacter pylori