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.
Subject(s)
Antibodies, Bacterial/isolation & purification , Antigens, Bacterial/immunology , Bacterial Proteins/immunology , Helicobacter pylori/immunology , Virulence Factors/immunology , Amino Acid Sequence , Antibodies, Bacterial/genetics , Antigens, Bacterial/genetics , Antigens, Bacterial/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Biopsy , Blotting, Western , Cloning, Molecular , DNA, Bacterial/isolation & purification , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Gastric Mucosa/microbiology , Gene Expression Regulation, Bacterial , Helicobacter pylori/genetics , Helicobacter pylori/pathogenicity , Humans , Molecular Sequence Data , Polymerase Chain Reaction , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Virulence Factors/genetics , Virulence Factors/metabolismABSTRACT
BACKGROUND/AIMS: Hepatitis A virus is a global public health problem, especially in developing countries, and the most common cause of hepatitis in childhood. Hepatitis A virus is a single- stranded positive RNA virus subdivided to 6 genotypes (3 human,3 simian). The aim of this study was to determine the prevalent genotype in Turkey using sera of acute hepatitis A virus-infected patients from different geographical regions of the country. MATERIALS AND METHODS: Sera of 137 patients with acute hepatitis A virus from different geographical regions were collected for phylogenetic analysis. The VP1-2A region of the hepatitis A virus genome was amplified by real-time-polymerase chain reaction in 76 patients where possible. Amplified polymerase chain reaction fragments were sequenced, and phylogenetic analysis was done together with other reference hepatitis A virus sequences obtained from GenBank database. RESULTS: Sequencing and phylogenetic analysis of the VP1-2A junction of hepatitis A virus showed that the most prevalent genotype in Turkey is IB (100%). Comparison of Turkish isolates and reference sequences of genotype IB showed a similarity of 94.9%. The same comparison was done between Turkish isolates and reference hepatitis A virus genotype IB and HM175, and it was found that similarity between them ranged from 93.0-95.9%. When Turkish isolates were compared according to Mean Percentage Nucleotide Distance analysis, similarity ranged between 95.3%-100%. CONCLUSIONS: Phylogenetic analysis pointed out that all Turkish isolates belong to genotype IB. Sequence analysis is a useful tool in revealing hepatitis A outbreaks, and allows us to detect and distinguish the presence of epidemic and small outbreaks.
Subject(s)
Hepatitis A Antibodies/blood , Hepatitis A Virus, Human/genetics , Hepatitis A Virus, Human/immunology , Hepatitis A/epidemiology , Hepatitis A/virology , Acute Disease , Adolescent , Adult , Child , Child, Preschool , DNA, Viral/genetics , Endemic Diseases/statistics & numerical data , Female , Genotype , Hepatitis A Virus, Human/isolation & purification , Humans , Immunoglobulin M/blood , Infant , Infant, Newborn , Male , Phylogeny , Seroepidemiologic Studies , Turkey , Young AdultABSTRACT
Hepatitis delta virus (HDV) is a subviral agent of hepatitis B virus (HBV), and its life cycle is dependent on HBV. It is commonly accepted that HDV has eight distinct genotypes. In this study, the complete nucleotide sequences of HDV genomes isolated from nine Turkish patients were obtained by RT-PCR using two pairs of primers that cover the entire HDV genome. PCR products were sequenced directly. The results showed that these 9 isolates were approximately 1680 base pairs in length and clustered in the genotype HDV-1 branch when phylogenetic analysis was done with the sequences together with the complete sequences of HDV genomes representing each genotype retrieved from GenBank. Analysis of a portion of the large hepatitis D antigen (L-HDAg) gene showed that sequence similarity among these Turkish isolates is between 87.4 and 97.1%, and the Turkish isolates have the most sequence similarity to HDV-1 (90.5%), while they have the least sequence similarity to HDV-3 (64.1%). Full-genome analysis indicates that the sequence similarity is between 80.7 and 95.4%, and the highest sequence similarity is 84.8% (between the Turkish isolates and HDV-1). The lowest sequence similarity is 56.4% (between the Turkish isolates and HDV-3). In conclusion, phylogenetic analysis shows that the Turkish HDV isolates belong to HDV-1.
