Cloning, expression and purification flagellar sheath adhesion of Helicobacter pylori in Escherichia coli host as a vaccination target

PURPOSE: Helicobacter pylori is a widely distributed gram-negative bacterium that infects the human stomach and duodenum. HpaA is a H. pylori-specific lipoprotein that has been shown to be an effective protective antigen against H. pylori infection. HpaA of H. pylori as a vaccine antigen is fully competent for stimulation of immune responses. The aim of this project is cloning, expression, and purification flagellar sheath adhesion of H. pylori in Escherichia coli host by fast protein liquid chromatography (FPLC) as a vaccination target. MATERIALS AND METHODS: The hpaA gene was inserted into pET28a (+) as cloning and expression vectors respectively. The recombinant plasmid (pET-hpaA) was subjected to sequencing other than polymerase chain reaction (PCR) and digestion analysis. Protein expression was induced by adding 1 mM isopropyl-beta-D-thiogalactoside to cultures of E. coli strain BL21 transformed with pET-hpaA. Protein expression assessed with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Protein purification of flagellar sheath adhesion was by FPLC. RESULTS: The restriction endonuclease digestion, PCR amplification analysis showed that the hpaA gene of 730 bp was amplified from H. pylori DNA and sequencing analysis of the pET-hpaA confirmed the cloning accuracy and in frame insertion of hpaA fragment. SDS-PAGE analysis showed the expression of an approximately 29,000 Da protein. CONCLUSION: Sequencing results along with SDS-PAGE analysis confirms the expression of recombinant hpaA in the heterologous E. coli BL21. Conclusion A prokaryotic expression system for hpaA gene was successfully constructed. These results indicate that production of a specific recombinant protein is an alternative and potentially more expeditious strategy for development of H. pylori vaccine.

In silico analysis for identifying potential vaccine candidates against Staphylococcus aureus

PURPOSE: Staphylococcus aureus is one of the most important causes of nosocomial and community-acquired infections. The increasing incidence of multiple antibiotic-resistant S. aureus strains and the emergence of vancomycin resistant S. aureus strains have placed renewed interest on alternative means of prevention and control of infection. S. aureus produces a variety of virulence factors, so a multi-subunit vaccine will be more successful for preventing S. aureus infections than a mono-subunit vaccine. MATERIALS AND METHODS: We selected three important virulence factors of S. aureus, clumping factor A (ClfA), iron-regulated surface determinant (IsdB), and gamma hemolysin (Hlg) that are potential candidates for vaccine development. We designed synthetic genes encoding the clfA, isdB, and hlg and used bioinformatics tools to predict structure of the synthetic construct and its stabilities. VaxiJen analysis of the protein showed a high antigenicity. Linear and conformational B-cell epitopes were identified. RESULTS: The proteins encoded by these genes were useful as vaccine candidates against S. aureus infections. CONCLUSION: In silico tools are highly suited to study, design, and evaluate vaccine strategies.

Production of specific IgY Helicobacter pylori recombinant OipA protein and assessment of its inhibitory effects towards attachment of H. pylori to AGS cell line

PURPOSE: The common triple therapy for Helicobacter pylori is challenged by the increasing cases of antibiotic resistant infections, raising the need to explore alternative therapies. Oral administration of egg yolk immunoglobulin Y (IgY) has been previously reported as a means of passive immunization therapy for H. pylori infections. In this work, we investigated the inhibitory effect of IgY on the attachment of H. pylori to AGS cell line. MATERIALS AND METHODS: Recombinant OipA was prepared. Hens were immunized with recombinant protein three times. IgY was purified from egg yolks of immunized hens using polyethylene glycol precipitation method. The inhibitory effect of the specific immunoglobulin was evaluated in AGS cell line infected with H. pylori. RESULTS: The presence of recombinant OipA (30 kD) was confirmed via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Immunization of hens was confirmed using enzyme-linked immunosorbent assay. The purified IgY from egg yolks were assessed using SDS-PAGE and confirmed by western blot. CONCLUSION: The results showed that IgY-OipA had inhibitory effect on attachment of H. pylori to AGS cell line and may be utilized as a therapeutic or prophylaxis material.

[Cytotoxicity effect of recombinant outer membrane inflammatory protein [oipA] of helicobacter pylori on a breast cancer cell line]

Breast cancer is one of the leading causes of death in women worldwide. Conventional treatments use cytotoxic drugs which have high numbers of side effects. Currently pharmacologists are searching for novel drugs with fewer side effects and maximum efficiency as breast cancer treatment. The aim of the current study is to clarify the cytotoxicity effect of the recombinant outer membrane inflammatory protein [oipA] of Helicobacter pylori [H. pylori] on a breast cancer cell line. We purified recombinant H. pylori oipA by Ni-NTA affinity chromatography. Breast cancer cells [4T1] were treated with different concentrations of recombinant oipA for various lengths of time. Cell viability was evaluated by the viability assay [MTT test]. SDS-PAGE analysis showed the expression of an approximately 34000 dalton protein. Statistical analysis showed oipA toxic effects on 4T1 cells at a concentration of 250 micro g/ml after 24 h. These findings suggested that oipA had a direct toxic effect on a breast cancer cell line [4T1] in vitro. The oipA protein might be a new tool for future therapeutic strategies in cancer immunotherapy

Production of Pentameric Cholera Toxin B Subunit in Escherichia coli

Cholera toxin B subunit [CTB] has been extensively studied as an immunogen, adjuvant, and inducer of oral tolerance in many investigations. Production of CTB has been carried out in the bacterial, plant, insect and yeast expression systems. In this study the expression of the CTB containing a 6XHis-tagged was performed by Escherichia coli [E.coli] M15. The yield of purified pentameric recombinant CTB was about 1 mg/l. Western blot analysis demonstrated that the recombinant CTB was antigenically active. In addition, GM1-ganglioside ELISA showed that recombinant CTB binds to GM1-gangelioside receptor, confirming disulfide bond formation and proper folding of the recombinant protein in E.coli. Overall, in regard to the vast applications of CTB in medicine, this bacterial expression system will be a fast, cost-effective and simple system for production of pentameric CTB and CTB conjugated proteins

[Incidence of van A, B, C, D, E in vancomycin resistant enterococcus isolated from fecal flora in Tehran]

Vancomycin-resistant enterococci [VRE] have emerged worldwide and have become an increasing problem in clinical settings. Acquired glycopeptide resistance in Enterococcus species is due to the acquisition of van A, van B, van D, van C and van E genes, resulting in the production of peptidoglycan precursors with reduced affinity for glycopeptide antibiotics. The origin of these van genes is still unknown, but recent studies have indicated that van B resistance in enterococci might arise from gene transfer from the human bowel flora. In this study, we investigated the presence of Enterococcus-associated van A, van B, van C, van D, van E genes in the feces of hospitalized patients. To determine the prevalence of vancomycin-resistant enterococci [VRE] fecal colonization of hospitalized patients, 422 Enterococcus spp. isolated from stool of patients in Amiralam hospital. Disk diffusion method was used to detect resistance to vancomycin. The MICs of vancomycin were determined by the agar dilution method. The presence of van A, B, C, D and E genes were assassed by PCR analysis. PCR was positive for van A for 6 out of 10 [60%] and van B for 4 out of 10 [40%] of VRE strains. Among the van positive enterococci, two [20%] specimens contained both van A and van B gene, whereas no van C, D and E positive enterococcal isolates were identified from these specimens. The MIC of VRE isolates were between 512- 1024 micro g/ml. Our results showed that most glycopeptide resistant Enterococcus isolated from stool of hospitalized patients carried van A and van B. It is also possible that frequency of infections caused by glycopeptide-resistant enterococci will increase in our geographical area

[Evaluation on antibiotic resistance of helicobacter pylori isolated from patients admitted to tooba medical center, Sari]

Helicobacter pylori, which infect approximately one half of the world's population, are an important risk factor in chronic gastritis, peptic ulcer disease, and gastric cancer. H. pylori eradication is now widely recommended as the most effective treatment of peptic ulcer disease. One of the most important reasons for treatment failure is H. pylori resistance to the antimicrobials usage in therapy. The aim of this study was to determine susceptibility patterns of H. pylori isolates in 6 routine anti-microbial agents in Northern Iran. 125 patients from Tooba Medical Center in Sari with endoscopic evidence of dyspepsia complaints were used for obtaining gastric biopsies specimens. Biopsies were sent to the laboratory in thioglycolate broth [transport medium]. Bacteria were primarily cultured on Columbia agar supplemented with 7% horse blood, 7% fetal calf serum. Urease, Catalase and Oxidase activities were used for H. pylori identification. Bacterial suspensions equivalent to 3 Mc. Farlands were spread on plates, along with antibiotic disks and placed in the diameter zone. Inhibition was measured after 3 days of incubation in micro-aerophilic condition. H. pylori were isolated from 116[92.8%] subjects, a total of 125 biopsy specimens. Resistance to metronidazole, amoxicillin, clarithromycin, tetracycline, furazolidone and ciprofloxacin were 71%, 35%, 25%, 9%, 24% and 25%, respectively. Multiple resistance [amoxicillin-clarithromycin-metronidazole] were found in [6]5% of the isolates. Comparison of our data with previous results showed that prevalence of H. pylori resistance to clarithromycin, furazolidone and metronidazole has increased in Iran considerably. Resistance to amoxicillin in our study was too high in comparison with foreign studies. The present study demonstrates the need for continuous monitoring of the antimicrobial susceptibility in H. pylori in order to determine the optimal drug regimens

Detection of Brucella abortus by alkB and IS711 based primers

Brucellosis is a zoonotic disease, which involves both animals and human. Although the conventional methods have been widely used for its laboratory diagnosis, the PCR techniques have proved to be useful due to specificity, sensitivity and the rapidness. Various target sequences of brucella bacterium such as OMP2, 16s RNA and IS711 have been used for the primer designing. All primer sets have shown different sensitivities and specificities. In present investigation, PCR protocol and primer designated based on IS711 and a fragment of chromosomal DNA all were optimized with standard genome and clinical samples. Numerous tissue samples [liver, kidney, lymph node, and uterus] were prepared and were cultured by the bacteriological standard methods along with the serology positive human samples. PCR protocol was optimized and the primer's sensitivity and the specificity were checked using pure genome of B. abortus. All samples were tested by the standard bacteriological methods. The samples were then subject to PCR amplification and the PCR product was confirmed using the RFLP technique. The culture results indicated a poor sensitivity as it was previously reported. The PCR product 157 bp was observed on the agarose gel indicating that significant number of clinical samples [human brucellosis cases] were positive by PCR but not by the culture method. Although B. abortus DNA was detected in all the culture positive veterinary specimens, some cross-reactions with close related bacteria were observed that might influence the interpretation of the results. The sensitivity of the present PCR protocol was significantly higher when alk B and IS711 based primers were used in compare to each of the alkB and IS711 based primers alone. More research will be needed to improve the specificity and sensitivity of the PCR protocol before recommending for routine laboratory works

Isolation of legionella from BAL samples of legionella pneumonia patients, by PCR and Culture methods

Among the members of legionellaceae, Legionella Pneumophila is involved in 95% of cases of severe pneumonia. Isolation of the causative agent from bronchoalveolar lavage [BAL] fluid specimen is a delicate process and also time-consuming. Moreover, it has been shown that some Legionella strains may be viable but cannot be cultured. The aim of this study was comparison of culture and PCR for detection of Legionella Pneumophila from bronchoalveolar lavage [BAL] fluid specimens. In this study, 70 BAL fluid specimens were collected from patients suspected to Legionnaires' disease. These samples were cultured on selective buffered charcoal-yeast extract agar [BCYE] and then tested with specific L. Pneumophila primers for mip gene. Among 70 BAL samples, three [4.2%] were positive with culture and six [8.4%] of specimens were positive by PCR. The three culture positive samples were all positive after specific DNA amplification. Among 63 culture-negative samples, 3 were positive after amplification. The clinical features of the patients were in accordance with legionellosis. The accurate diagnosis of Legionella Pneumophila has an important implication for the treatment of infection. Analysis of the results showed that PCR is faster and more sensitive for isolation and identification of L. pneumophila to apply on BAL fluid specimens than culture. Therefore, specific Legionella PCR can be a good option for isolation and identification of Legionella Pneumophila from bronchoalveolar lavage [BAL] fluid specimens in patient of severe pneumonia