• Biomolecules & Therapeutics
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• v.9 no.3
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• pp.162-166
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• 2001

Helicobacter pylori is a major cause of gastric-associated diseases. In our previous study, the Adhesin/CTXA2B was expressed as insoluble recombinant chimeric protein derived from the H. pylori adhesin genetically coupled to CTXA2B subunit in Escherichia coli. Since it is very important to optimize IPTG concentration, culture temperature and composition of medium to maximize cell growth and productivity, these conditional growth factors were determined for increasing the productivity of the expressed Adhesin/CTXA2B chimeric protein in Escherichia coli JM101 carrying pTEDhpa/ctxa2b. Our data demonstrate that optimal medium for increased production of chimeric protein was a YCP/Glu medium composed of 2% yeast extract, 1% casamino acid, phosphate solution [0.3% $KH_2P0_4$, 0.4% $Na_2HP0_4$, 0.25% ($NH_4)_2HPO_4$], and 0.5% glucose. In addition, optimal concentration of IPTG was 1 mM and culture temperature, $37^{\circ}C$.

• Journal of Microbiology and Biotechnology
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• v.10 no.1
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• pp.56-62
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• 2000

The hpa gene genetically linked to the ctxa2b gene was cloned into the pTED expression vector, and the constructed pTEDhpa/ctxa2b was transformed into Excherichia coli. The fusion protein, the adhesin fused to the cholera toxin subunit A2B (CTXA2B) subunit, was expressed to high levels as inclusion bodies in E. coli. The expressed protein was partially purified by washing the inclusion bodies with working solution containing 8M Urea and 0.1M DTT. Refolding of denatured fusion protein was carried out in the presence of glutathione redox buffer. The refolded fusion protein was purified by size exclusion chromatography. The expressed fusion protein was verified by SDS-PAGE, western blotting with antibodies to both antigenic components of adhesin and cholera toxin subunit B (CTXB), and its N-terminal amino acid sequence was analyzed. The orderly assembled fusion protein was confirmed by modified Gm1-ganglioside ELISA with Abs to adhesin. The results indicate that the purified fusion protein is an Adhesin/CTXA2B protein containing the H. pylori adhesin and $G_{m1}4-ganglioside binding activity of CTXB and the expressed fusion protein in E. coli could be easily purified by the refolding process, Its molecular weight was 168kDa as estimated by size exclusion chromatography. The Adhesin/CTXA2B protein may be used as a candidate antigen for oral immunization against H. pylori.

• Microbiology and Biotechnology Letters
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• v.44 no.2
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• pp.218-226
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• 2016

Helicobacter pylori primarily colonizes the human stomach. Infection by this bacterium is associated with various gastric diseases, including inflammation, peptic ulcer, and gastric cancer. Although there are antibiotic regimens for the eradication of H. pylori, the resistance of this species against antibiotics has been continuously increasing. The natural compound plumbagin has been reported as an antimicrobial and anticancer molecule. In this study, we analyzed the inhibitory effect of plumbagin on H. pylori strain ATCC 49503 as well as the expression of various molecules associated with H. pylori growth or virulence by immunoblotting and reverse transcription polymerase chain reaction (RT-PCR) analyses. We demonstrated the minimal inhibitory concentration of plumbagin on H. pylori through the agar dilution and broth dilution methods. Furthermore, we investigated the effect of plumbagin treatment on the expression of the RNA polymerase subunits and various virulence factors of H. pylori. Plumbagin treatment decreased the expression of RNA polymerase subunit alpha (rpoA), which is closely associated with bacterial survival. Moreover, the mRNA and protein levels of the major CagA and VacA toxins were decreased in plumbagintreated H. pylori cells. Likewise, the expression levels of urease subunit alpha (ureA) and an adhesin (alpA) were decreased by plumbagin treatment. Collectively, these results suggest that plumbagin may inhibit the growth, colonization, and pathogenesis of H. pylori by the mechanism demonstrated in this study.

• Korean Journal of Microbiology
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• v.48 no.2
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• pp.109-115
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• 2012

Helicobacter pylori is an important factor of chronic gastritis, digestive ulcer, and stomach cancer. CagL, a virulence factor of H. pylori, is well-known as a pilus protein which acts as adhesion to host cell and a component of Type 4 secretion system. In this study, we evaluated the protective response of recombinant CagL protein (rCagL) using Mongolian gerbil animal model for H. pylori infection. The cagL gene was cloned from 26695 H. pylori followed by over-expression and purification of the protein in E. coli. Mongolian gerbils were immunized with rCagL protein mixed with aluminum adjuvant via intramuscular injections once a week during 4 weeks. At a week after the last immunization, the Mongolian gerbils were administrated with H. pylori 7.13 strain into the stomach and sacrificed to measure antibody titer on rCagL by ELISA and bacterial colonization in the stomach, and to examine the histopathological changes and cytokine expression at 6 week after challenge. Antibody titers on recombinant protein were significantly increased from a week after the first immunization. There was no significant change of the number of bacterial colony between control group and immunized group. The relative stomach weight was significantly decreased in immunized group, but the significant change of histopathological assessment was not observed in the stomach. Cytokine expression such as IL-$1{\beta}$ and KC also was not significantly different between control and immunized groups. These results indicate that rCagL could effectively induce the formation of the specific IgG antibodies. However, bacterial colonization and histopathological lesions could not be inhibited by the immunization in the stomach, indicating not enough protection against H. pylori infection. We consider that along with CagL other adequate antigens could be needed stimulating immune response and inducing protective effects against gastric disease, and also a better adjuvant could be considered.

• Pediatric Infection and Vaccine
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• v.14 no.1
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• pp.97-103
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• 2007

Purpose : Lewis antigen has been known to have a role in the attachment of H. pylori to the gastric mucosa, but its expression pattern in children with H. pylori infection is still unclear. The recently described blood group antigen-binding adhesin BabA is known to mediate adherence of H. pylori to Lewis B receptors on gastric epithelium. We investigated the expression of Lewis antigen in gastric mucosa of Korean children with H. pylori infection. Methods : The expression of Lewis A ($Le^a$), B ($Le^b$), X ($Le^x$), and Y ($Le^y$) was evaluated by immunohistochemistry in H. pylori positive biopsy specimens from 35 children (antral gastritis in 30, peptic ulcer in 5) and in H. pylori negative specimens from 19 children. PCR assays for cagA and babA2 gene of H. pylori were performed. Results : We confirmed the expression of $Le^a$ in 60%, $Le^b$ in 97%, $Le^x$ in 100%, and $Le^y$ in 100% of the superficial epithelium of the 35 H. pylori positive children. In H. pylori negative patients, $Le^a$, $Le^b$, $Le^x$, and $Le^y$ expression was 52%, 100%, 89%, and 100%, respectively. The cagA gene was detected in 65% and babA2 gene in 25% of 35 patients. No differences in neutrophil activity and chronic inflammation were found according to the presence of cagA and babA2 genes in H. pylori. Conclusion : $Le^b$, $Le^x$ and $Le^y$ antigen were highly expressed in gastric mucosa of Korean children, but they were not associated with the status of H. pylori infection and the positivity of babA2 gene. Further studies for other mucosal receptors and toxins are needed to define the immune responses to H. pylori infection in gastric mucosa of Korean children.