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.

[Molecular evaluation of the 2'-aminoglycoside nucleotidyltransferase gene in Escherichia coli isolates that produce hemolysin and are sensitive to mannose type I pili and P]

Aminoglycosides are highly potent, broad-spectrum antibiotics with many desirable properties for the treatment of life-threatening infections. Escherichia coli [E. coli] is the most common cause of urinary tract infection [UTI]. Antibiotic resistance has recently become prevalent. Enzymatic inactivation of aminoglycosides by aminoglycoside-modifying enzymes is the main mechanism of resistance to these antibiotics in E. coli. The main purpose of this research is to evaluate the presence of the 2'-aminoglycoside nucleotidyltransferase [ant[2"]-Ia] gene in E. coli isolates sensitive to mannose and hemolysin production. After collecting 276 E. coli isolates from patients that referred to Tehran Heart Center, we used the disk diffusion method to determine the resistance patterns of isolates toward Gentamicin, Tobramycin, Kanamycin, Amikacin and Netilmicin antibiotics according to the CLSI principles. We evaluated hemolysin production by assessing the ability of the isolates to grow on sheep and human blood agar media. Chromosomal DNA of the isolates was extracted using DNA extraction kits and PCR method used for the detection of the ant[2"]-Ia gene. In order to study mannose sensitivity we used human RBCs. Results obtained from antibiotic resistance determination tests showed that the highest rate of resistance was observed against tobramycin [24/63%]. Of those resistant, 6% could produce hemolysin in both sheep and human blood agar media. Mannose sensitivity was observed in 14% of isolates during agglutination. There were 24.63% of E. coli isolates resistant to Tobramycin, 23.18% resistant to kanamycin, 21.01% resistant to gentamicin, 6.15% resistant to netilmicin and 3.62% resistant to amikacin. ant[2"]-Ia gene was detected in 47.88% of E. coli isolated from urine. Due to the high prevalence of urinary tract infections caused by uropathogenic E. coli [UPEC] strains and the increasing rate of antibiotic resistance, periodic evaluations should be conducted for outbreaks of resistance in order to select the most suitable treatment to prevent routinely increasing antibiotic resistance

[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

[Evaluation of antifungal activity of essential oils of Thymus vulgaris, Petroselinum Crispum, Cuminum cyminum and Bunium persicum on candida albicans in comparison with Fluconazole]

Nowadays notable increase in acquired resistance of Candida species to antifungal drugs and necessity of using agents with antifungal properties is unavoidable. In some plants due to presence of components such as polyphenols have antimicrobial properties. In this study antifungal effects of essential oils of Thymus vulgaris, Petroselinum crispum, Cuminum cyminum and Bunium persicum on standard strain of Candida albicans were evaluated. 25 grams leafs of the Thymus vulgaris and Petroselinum crispum and seeds of the Cuminum cyminum and Bunium persicum were dried and grinded after that the essential oils of each mentioned plant were prepared by Clevenger system. Serial dilutions of essential oils were made in 96 well microtiter plates. Minimum Inhibitory Concentration [MIC] and Inhibitory zone diameter were assessed by Microdilution broth and Disc diffusion agar techniques, MIC50, MIC90 and MFC were also determined. Minimum inhibitory concentration 90 [MIC90] essential oils of Thymus vulgaris, Petroselinum crispum, Cuminum cyminum and Bunium persicum were respectively 25, 72, 412, 130 micro g/ml and Minimal Fungicide Concentration [MFC] were 48, 146, 62, 280.Inhibitory zone diameters were 28, 20, 12, 15 millimetres. In this evaluation essential oils of Thymus vulgaris, Petroselinum crispum, Cuminum cyminum and Bunium persicum showed suitable antifungal effects against growth of standard strain of Candida albicans. Thus these herbal essences after supplementary studies possibly can be suitable substitute for chemical medicine on Candida infections especially on mucocutaneous Candidiasis

[ molecular study of aac[3]-IIa [aacC2] gene in aminoglycoside resistant Escherichia coli isolated from urine]

Escherichia coli is the most prevalent etiologic agent of urinary tract infections which is the cause of about 80% of cases. Enzymatic inactivation of aminoglycosides by aminoglycosidemodifying enzymes is the main mechanism of resistance to these antibiotics in Escherichia coli. The aim of this study was the detection of aac [3] -IIa gene among aminoglycoside resistant clinical isolates of E. coli using PCR method. After collection of 250 clinical isolates of E. coli, antibiotic susceptibility patterns of isolates were determined by disk diffusion method for gentamicin, amikacin, tobramycin, kanamycin and netilmicin by considering the CLSI principles. Chromosomal and plasmid DNA of the isolates were extracted using DNA extraction Kits and PCR method was used for detection of the aac [3] -IIa gene. Results show that 96% of E. coli isolates were resistant to tobramycin, 90% resistant to kanamycin, 82% resistant to gentamicin, 30% resistant to netilmicin and 8% resistant to amikacin. aac [3] -IIa gene was detected in 54.83% of E. coli isolates. Because of high prevalence of resistance toward aminoglycoside antibiotics which is due to its transfer among bacteria by transferable elements such as transposons and plasmids. Therefor, tracing transfer routs among different bacteria is very important