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