Clonaje y expresión de un fragmento recombinante del gen cagA de Helicobacter pylori y su evaluación preliminar en el serodiagnóstico (original) (raw)
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Clinical Microbiology & Infection, 2000
Helicobacter pylori has been established as a major cause of chronic active gastritis and peptic ulcer disease (PUD), also associated with gastric cancer [1,2]. The clinical diagnosis of helicobacter infection may be difficult, due to the absence of either specific symptoms or specific endoscopic findings. In addition, the results of cultures, histologic tests and urease tests on biopsies may be highly variable. Immunologic tests may therefore be of importance for the reliable diagnosis of H. pylori infections. Several serologic studies using different techniques and different antigen preparations have been performed [3]. Most of the available data have been obtained by enzyme-linked immunosorbent assays and by western immunoblotting (WB) techniques using whole H. pylori cells as antigen [4,5]. More recently, recombinant CagA (rCagA) has been used as antigen for serologic diagnosis of H. pylori infection [6-9]. We now report the detection of specific serum antibodies of the IgG class against CagA of H. pylori by using a recombinant CagA enzyme-linked immunosorbent assay (Radim, Rome, Italy). We compared it with a WB technique performed with H. pylori antigen represented by acid glycine-extracted whole bacterial cells. One hundred and seventeen subjects (60 men and 57 females, mean age 45 years, range 23-74 years) were admitted to the study. Ninety-nine patients had undergone gastroduodenoscopy for dyspepsia, and there were also 18 asymptomatic subjects, partners of H. pylori-infected, symptomatic patients. Gastric specimens were cultured for H. pylori, as previously described [10]. Histologic sections of formalin-fixed biopsy specimens were stained with hematoxylin-eosin to evaluate the morphology and the presence of Helicobacter-like organisms. Sera were prepared from blood samples obtained by routine venipuncture for serologic studies. In addition, sera were also prepared from 107 blood donors to evaluate H. pylori serology. The gene sequence that codes for the entire CagA protein (3444 bp) was amplified from strain NCTC 11637 extracted DNA, using primers derived from the sequence deposited in the Gene Bank under accession number LI1714 (primer CAG1, 5?-CCCGGATCCACTAACGAAACCATTGACCAA-3?; primer CAG2, 3?-CCACCAAAGGTTTTTAGAATTGA
Jundishapur Journal of Microbiology, 2013
Background: Helicobacter pylori is a human pathogen that causes chronic gastritis, which playsrole in gastric and duodenal ulcers, is also involved in gastric carcinogenesis and may be regarded as a possible important factor in at least a subset of patients with functional dyspepsia. Objectives: This study was aimed to construct a recombinant protein containing H. pylori antigenic CagA region and determine its antigenicity as a vaccine candidate against H. pylori. Meterials and Methods: The antigenic region of CagA gene was detected by bioinformatics techniques. In this study, the H. pylori antigenic CagA region was amplified by PCR and sub-cloned to prokaryotic expression vector pET32a. Escherichia coli BL21 (DE3) pLysS was transformed with pET32a-CagA and gene expression was induced by IPTG. The expressed protein was purified by affinity chromatography using Ni-NTA resin. The integrity of the product was confirmed by western-blot analysis using Sera of infected individual. Finally antigenicity was studied by western-blot analysis using human sera infected with H. pylori. Results: Enzyme digestion analysis, PCR and sequencing showed that the target gene (1245 bp) was correctly inserted into the recombinant vector. The expressed protein was purified using affinity chromatography by Ni-NTA resin. The data also indicated that CagA protein from H. pylori detected from patients' sera. Conclusions: Results indicates that antigenic region of recombinant CagA protein were recognized as an antigen , so it might be a candidate for the development of H. pylori vaccine, ELISA kit designs and serological diagnosis of H. pylori infections.
Journal of Immunological Methods, 2021
The CagA protein one of the key virulence factors of Helicobacter pylori plays an important role in the pathogenesis of peptic ulcer diseases. Unfortunately the cagA gene status can only be determined by PCR while serology is an alternative approach to detect antigens or antibodies. Our aim is to detect the CagA antigen in sera of infected subjects by the development of an in-house capture ELISA test. Gastric antral biopsies and serum samples were collected from 63 patients. PCR was used to determine the cagA status. Our previously developed recombinant CagA protein and monoclonal antibody were used for setting up the capture ELISA test. H. pylori positive [(38 gastritis, 14 duodenal ulcers (DU), 11 gastric ulcer (GU)] patients were determined by PCR. The cagA gene was detected in 21 (55%) of gastritis, 11 (78%) of DU and 7 (60%) of GU patients. The reagents used in setting up the capture ELISA test following optimization displayed high performance. This study showed that our developed in-house capture ELISA has the potential to detect the CagA antigen at very low concentrations even though not detected in our H. pylori infected patients sera but we are also intended to use it in saliva and stool samples.
Serum antibodies anti-H. pylori and anti-CagA: A comparison between four different assays
Journal of Clinical Laboratory Analysis, 1999
The authors compare efficacy of two ELISA assays (one supplied by DIAMEDIX [Delta Biological s.r.l.], and the other by RADIM [RADIM I]) in detecting total anti-H. pylori antibodies, and of two further ELISA methods (one supplied by EUROSPITAL [Helori CTX IgG] and the other by RADIM [RADIM 2]) in identifying anti-CagA antibodies, using sera from 69 controls (20 adults and 49 children) and from 96 patients, obtained before endoscopy. Seventy-three of the patients had H. pylori infection, while the remaining 23 were H. pylori negative (histology and polymerase chain reaction [PCR]). Fifty-two of the H. pylori positive patients, had cagA-positive strain infection, identified by PCR. The DIAMEDIX assay was found to be more sensitive (92%) than RADIM 1 (79%) in identifying H. pylori positive patients, irrespective of the infecting strain. On the other hand, the DIAMEDIX assay was less specific than RADIM 1 for H. pylori-negative patients (43% vs. 83%). However, when patients already treated for H. pylori infection were excluded from the group of H. pylori-negative patients, the DIAMEDIX assay had a specificity of 89%. In identifying anti-CagA antibodies, the kit supplied by RADIM (RADIM 2) had a sensitivity of 90% and a specificity of 94%, whereas that supplied by EUROSPITAL had a sensitivity of 100% and a specificity of 76%. The performances of the two methods in the identification of anti-CagA antibodies were found to be similar. The authors conclude that, in view of its high sensitivity, the DIAMEDIX assay may be useful in screening for H. pylori infection.
European Journal of Clinical Microbiology & Infectious Diseases, 1993
The possibility of using a recombinant fragment of the CagA (128 kDa protein) for the diagnosis ofHelicobacter pylori infection was evaluated. Following cloning of the gene coding for the CagA, a recombinant fragment of it was expressed inEscherichia coli, purified and used in Western blot and an EIA to screen sera from 82 patients with gastroduodenal disease who underwent endoscopic examination. In Western blot, good correlation was found between the serological data obtained with the recombinant antigen and those obtained using non-purified extracts ofHelicobacter pylori. The EIA using the antigen showed a sensitivity of 96.2 % and a specificity of 96.6 % compared with Western blot. These data indicate that the recombinant protein is a reliable antigen for detection of infections withHelicobacter pylori strains that are associated with disease. The EIA assay described may be used in follow-up of the progression of the illness and the results of therapy.
Serologic detection of infection with cagA+ Helicobacter pylori strains
Journal of Clinical Microbiology, 1995
Approximately 60% of Helicobacter pylori isolates possess the cagA gene and express its 120- to 140-kDa product (CagA). In this study, the cagA gene was detected in H. pylori isolates from 26 (81.3%) of 32 patients with duodenal ulcers (DU), 17 (68.0%) of 25 patients with gastric ulcers, and 23 (59.0%) of 39 patients with nonulcer dyspepsia (NUD). By Western blotting (immunoblotting) with antiserum to CagA, in vitro CagA expression was demonstrated for 95.5% of cagA+ strains compared with 0% of strains lacking cagA. Sera from patients infected with cagA+ strains (n = 66) reacted with recombinant CagA in an enzyme-linked immunosorbent assay to a significantly greater extent than either sera from patients infected with strains lacking cagA (n = 30) or sera from uninfected persons (n = 25) (P < 0.001). A strain lacking cagA was isolated from eight patients who had serum immunoglobulin G antibodies to CagA, which suggests that these patients were infected with multiple strains. Serum...
Medical Microbiology and Immunology, 2019
Currently, Western-type CagA is used in most commercial Helicobacter pylori CagA ELISA kits for CagA detection rather than East Asian-type CagA. We evaluated the ability of the East Asiantype CagA ELISA developed by our group to detect anti-CagA antibody in patients infected with different cagA genotypes of H. pylori from four different countries in South Asia and Southeast Asia. The recombinant CagA protein was expressed and later purified using GST-tag affinity chromatography. The East Asian-type CagA-immobilized ELISA was used to measure the levels of anti-CagA antibody in 750 serum samples from Bhutan, Indonesia, Myanmar, and Bangladesh. The cutoff value of the serum antibody in each country was determined via Receiver Operating Characteristic (ROC) analysis. The cutoff values were different among the four countries studied (Bhutan, 18.16 U/mL; Indonesia, 6.01 U/mL; Myanmar, 10.57 U/mL; and Bangladesh, 6.19 U/ mL). Our ELISA had better sensitivity, specificity, and accuracy of anti-CagA antibody detection in subjects predominantly infected with East Asian-type CagA H. pylori (Bhutan and Indonesia) than in those infected with Western-type CagA H. pylori predominant (Myanmar and Bangladesh). We found positive correlations between the anti-CagA antibody and antral monocyte infiltration in subjects from all four countries. There was no significant association between bacterial density and the anti-CagA antibody in the antrum or the corpus. The East Asian-type CagA ELISA had improved detection of the anti-CagA antibody in subjects infected with East Asian-type CagA H. pylori. The East Asian-type CagA ELISA should therefore be used in populations predominantly infected with East Asian-type CagA.
Serological differentiation of Helicobacter pylori CagA(+) and CagA(-) infections
Archivum immunologiae et therapiae experimentalis, 2003
Many Helicobacterpylori strains causing gastroduodenal diseases have a cagA gene encoding CagA protein, a virulence factor of these bacteria. Anti-CagA antibodies produced by the majority of people infected with CagA(+) strains can indicate such an infection. In this study, the efficacy of three immunoenzymatic tests for detecting CagA(+) and CagA(-) infections were compared: immunoblot (Milenia ID Blot H. pylori IgG; MB) and ELISA conducted either with a recombinant immunodominant fragment of CagA (rCagA) or the full-length CagA molecule (flCagA). The 13C-urea breath test (13C-UBT) was used for establishing H. pylori status. The serum samples from 157 individuals were used for serodiagnosis. H. pylori CagA(+) infection was detected in H. pylori-infected individuals with similar frequencies by MB (64%) and flCagA-ELISA (60%) and a little less frequently by rCagA-ELISA (53%). There was a high coincidence between the negative results of these three tests for H. pylori-uninfected indiv...
Production of a monoclonal antibody against the 128 kDa (CagA) protein of Helicobacter pylori
Journal of Immunological Methods, 1996
Helicobacrer pylon' is recognised as an important factor in gastroduodenal pathology. The 128 kDa CagA protein has been established as a useful marker of H. pyfori strains associated with more severe forms of disease. A mouse monoclonal antibody raised against the CagA protein has been produced and characterised as belonging to the IgGl subtype. It identified the protein in all clinical isolates (lo/IO) from this laboratory and in two NCTC reference strains (NCTC 11637 and NCTC 11961). No cross-reacting proteins were detected in H. pylori L2, a well characterised strain known not to contain the cugA gene, or in four Helicobacter sp. from non-human sources (H. cunis, H. mustelidae. H. muridarum and H. a&onyx). The monoclonal antibody was used to develop an antigen capture ELISA system for detecting the presence of antibodies to the CagA protein in human serum samples.
A positive assay for identification of cagA negative strains of Helicobacter pylori
Journal of Microbiological Methods, 2003
A new PCR protocol was developed for the positive identification of cagA negative Helicobacter pylori strains. Amplification of a portion of the genome across the insertion point of the cag pathogenicity island (the ES-''empty site'') generated a 106-bp fragment, which produces a positive signal for cagA negative strains. Combined with the results of the cagA assay, the signals for ES allowed the complete characterization of the patients' cagA status. DNA sequencing analysis confirmed the identity of the ES fragment. The new protocol and cagA assay were applied to 22 DNA preparations isolated from stools from H. pylori infected adult patients and to 21 DNA preparations isolated from stools from H. pylori infected children. The same analysis was also performed on nine colonies of H. pylori derived from gastric biopsies of nine of the adult patients. The total number of cagA positive cases from adult patients was 14 or 63.6% (11 mono-and 3 mixed) and of the cagA negative cases (or ES positive) was 9 or 40.9% (6 mono-and 3 mixed). Of the 21 stool DNA samples from children, 6 (28.6%) were cagA positive, 12 (57.1%) were cagA negative and 3 (14.3%) were positive for cagA and for the ES simultaneously. The proportions of mixed cagA positive and cagA negative H. pylori infections were almost equal in adults and children (13.6% and 14.3%, respectively). No reaction products of the proper fragment sizes for cagA or the empty site (ES) were obtained from any of the stool DNA samples of 10 H. pylori uninfected subjects (100% specificity). This noninvasive assay discriminates consistently cagA negative cases from cagA positive strains and from amplification failures. It can be a useful tool for clinical and epidemiological studies of H. pylori infection.