title: [Production of a recombinant CagA protein for the detection of Helicobacter pylori CagA antibodies].
abstract: At present, Helicobacter pylori infections affect approximately 50% of the world population. It is known that H.pylori is related with several gastric diseases including chronic atrophic gastritis, peptic and gastric ulcers as well as gastric carcinomas. CagA (Cytotoxin-associated gene A) protein which is one of the most important virulence factors of H.pylori, is thought to be responsible for the development of gastric cancer. CagA is a 128 kDa hydrophilic protein which binds to the epitelial stomach cells and is known to be phosphorylated on its EPIYA regions. The EPIYA regions are highly variable and carry a higher risk of developing gastric cancer than CagA negative strains. The aim of this study was to construct a prokaryotic expression system expressing a recombinant CagA protein, which can be used for the detection of anti-CagA antibodies. For the isolation of H.pylori genomic DNA, a total of 112 gastric biopsy samples obtained from patients who were previously found positive for rapid urease (CLO) test, were used. H.pylori DNAs were amplified from 57 of those samples by polymerase chain reaction (PCR) and of them 35 were found positive in terms of cagA gene. Different EPIYA motifs were detected in 25 out of 35 cagA positive samples, and one of those samples that contained the highest number of EPIYA motif, was chosen for the cloning procedure. Molecular cloning and expression of the recombinant fragment were performed with Champion Pet151/D expression vector (Invitrogen, USA), the expression of which was induced by the addition of IPTG (Isopropyl-beta-D-thiogalactopyranoside) into the E.coli culture medium. Expression was observed with anti-histidin HRP (Horse Radish Peroxidase) antibodies by SDS-PAGE and Western Blot (WB) analysis. In our study, two clones possessing different fragments from the same H.pylori strain with three different EPIYA motifs were succesfully expressed. Since CagA antigen plays a signicant role in the pathogenesis of H.pylori infections, the detection of anti-CagA antibodies done by non-invasive commercial ELISA or WB methods, is important in diagnosis. Recombinant CagA protein produced in this study could easily be used in the ELISA tests to be developed for screening anti-CagA antibodies in the patients' sera. However, since an ELISA method using this antigen has not been developed yet, its diagnostic performance could not be evaluated in this study. In conclusion, the construction of such a system for recombinant CagA antigen expression may be a pilot study for the development of our own ELISA tests in Turkey, and also will help the clinicians for the prediction of disease outcome and decision of the appropriate antimicrobial treatment by the help of anti-CagA antibody detection.
authors: Ankara University Faculty of Medicine, Institute of Hepatology, Ankara, Turkey. mryakgc@yahoo.com.
authors: Miray
authors: M
authors: Akgüç
authors: Ersin
authors: E
authors: Karatayli
authors: Esra
authors: E
authors: Çelik
authors: Duygu
authors: D
authors: Koyuncu
authors: İnci
authors: İ
authors: Çelik
authors: Senem Ceren
authors: SC
authors: Karatayli
authors: Ali
authors: A
authors: Özden
authors: A Mithat
authors: AM
authors: Bozdayi
mesh_terms: D000595:Amino Acid Sequence; D000907:Antibodies, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D000942:Antigens, Bacterial / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D001706:Biopsy; D015153:Blotting, Western; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005753:Gastric Mucosa / Q000382:microbiology; D015964:Gene Expression Regulation, Bacterial; D016480:Helicobacter pylori / Q000235:genetics / Q000276:immunology* / Q000472:pathogenicity; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D037521:Virulence Factors / Q000235:genetics / Q000276:immunology* / Q000378:metabolism
chemical_list: D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D037521:Virulence Factors; C080866:cagA protein, Helicobacter pylori