Helicobacter pylori (Hp) is one of most common pathogens causing gastrointestinal disorder including gastric ulcer, duodenal ulcer and gastric cancer, etc. It has been verified as class I carcinogen by WHO. Nowadays, combination antibiotics and proton pump inhibitor are mainly used to erase Hp in clinical application. However, with the increased resistance of Hp, the vaccine against Hp might become the best strategy to eradicate Hp. Elements including urease, virulence factor, outer membrane protein, flagella, play an important role in Hp infection, colonization and reproduction. They have become potential candidate antigens in the development of Hp vaccine, as reported in previous studies. Presently, these antigens-centric vaccines have been tested in animal models. Therefore, this article reviews the studies on Hp vaccine with urease, virulence genes, outer membrane protein and flagella as their candidate antigens, in an attempt to provide insights for research in this regard.
Animals ; Helicobacter pylori ; Urease/genetics* ; Helicobacter Infections/prevention & control* ; Vaccines ; Membrane Proteins

BACKGROUND/AIMS: Helicobacter pylori (H. pylori) transmission route is not yet clearly understood. Isolating H. pylori from stool, saliva, and vomitus is very difficult. However, H. pylori could be cultured from feces in the setting of rapid gastrointestinal tract transit. The aim of this study was to isolate H. pylori by culture and PCR in the rectum and terminal ileum during colonoscopy. METHODS: Twenty subjects with positive UBT (urea breath test) were included. We performed polymerase chain reaction (PCR) test and culture of H. pylori with the rectal fluid and terminal ileal fluid during colonoscopy. RESULTS: H. pylori was cultured with rectal fluid from 9 (45.0%) of 20 subjects and with ileal fluid from 11 (55.0%) of 20 subjects. H. pylori was a little more frequently cultured from the terminal ileal fluid than the rectal fluid without statistical significance (p>0.05). PCR test detected flaA (16/20, 80.0% and 17/20, 85.0%), 16S rRNA gene (16/20, 80.0% and 17/20, 85.0%), cagA (10/20, 50.0% and 12/20, 60.0%), and ureC (9/20, 45% and 11/20, 54.5%) from the rectal fluid and the terminal ileal fluid, respectively. The specificity and sensitivity of ureC were 100%. CONCLUSIONS: H. pylori could be cultured from the rectal fluid and terminal ileal fluid in the setting of rapid gastrointestinal tract transit. These results suggest of fecal-oral transmission of H. pylori.
Adult ; Antigens, Bacterial/genetics ; Bacterial Proteins/genetics ; Breath Tests ; Electrolytes/administration & dosage ; Feces/microbiology ; Female ; Helicobacter Infections/*diagnosis/transmission ; Helicobacter pylori/genetics/*isolation & purification ; Humans ; Ileum/*microbiology ; Male ; Middle Aged ; Polyethylene Glycols/administration & dosage ; Polymerase Chain Reaction ; RNA, Ribosomal, 16S/genetics ; Rectum/*microbiology ; Sensitivity and Specificity ; Urea/analysis ; Urease/genetics

PURPOSE: The role of the Ferric Uptake Regulator (FUR) in the acid resistance of Helicobacter pylori (H. pylori) has been thought to be independent of urease. However, we demonstrated in this study that Fur influences urease activity. MATERIALS AND METHODS: A fur knockout mutant of H. pylori was constructed by replacing the Fur gene with a kanamycin resistant marker gene. The wild-type H. pylori and fur mutant were compared for survival. The integrity of the inner membrane of the bacteria was evaluated by confocal microscopy using membrane-permeant and -impermeant fluorescent DNA probes. Urease activity of intact H. pylori was measured between pH 3 and 8. Real time PCR of both strains was performed for urease genes including ureI, ureE, ureF, ureG, and ureH. RESULTS: The fur deletion affected the survival of H. pylori at pH 4. The urease activity curve of the intact fur mutant showed the same shape as the wild-type but was 3-fold lower than the wild-type at a pH of less than 5. Real time PCR revealed that the expression of all genes was consistently down-regulated in the fur mutant. CONCLUSION: The results of this study showed that fur appears to be involved in acid resistant H. pylori urease activity.
Bacterial Proteins/genetics/*physiology ; Helicobacter pylori/*enzymology/genetics ; Hydrogen-Ion Concentration ; Microscopy, Confocal ; Models, Biological ; Mutation ; Repressor Proteins/genetics/*physiology ; Urease/*metabolism

OBJECTIVETo clone UreB gene of Helicobacter pylori (H. pylori) isolated from children to pGEX-4T-1 expression plasmid, and do sequence analysis.

METHODSA pair of specific primer was designed according to H. pylori UreB gene in the GenBank. Using H. pylori strains isolated from children as a template, a UreB gene was obtained by PCR. After EcoR I and Not I digestion, the PCR production was linked with pGEX-4T-1 which was digested with the same enzymes. The recombinant plasmid was transformed into E.coli BL21 and identified by double enzyme digestion and sequence analysis. The sequence results were compared with the gene sequence in the GenBank.

RESULTSA UreB gene was successfully amplified from children's H. pylori strain GZCH1. It was 1710 bp in size. The objective band was identified by double enzyme digestion. DNA sequence showed that UreB was in the correct open reading frame. The sequence comparison analysis showed that DNA and amino acid sequence identities of UreB gene with other strains were 98%. The sequence of UreB of H. pylori strain GZCH1 was submitted to GenBank (accession number:FJ455126).

CONCLUSIONSUreB of H. pylori strain GZCH1 is successfully cloned to pGEX-4T-1, which provides a basis for research of oral H. pylori vaccine.

Amino Acid Sequence ; Bacterial Vaccines ; immunology ; Child ; Cloning, Molecular ; Helicobacter pylori ; enzymology ; immunology ; Humans ; Male ; Molecular Sequence Data ; Urease ; chemistry ; genetics ; immunology

OBJECTIVETo establish an effective method for purification of Helicobacter pylori UreB fragment and conduct functional analysis of the purified protein.

METHODSThe protein fragment expression was induced by IPTG and the expressed protein was purified through affinity chromatography and ion-exchange chromatography. The purity of the fragment was determined by high-performance liquid chromatography (HPLC), and the specific biological activity of the purified fragment was assayed by urease activity inhibition test.

RESULTSThe protein fragment was highly expressed in E. coli with a purity over 91%. The protein fragment showed highly specific biological activity and the specific antibody induced by this fragment in rabbits could inhibit the activity of urease in a dose-dependent manner.

CONCLUSIONThe UreB fragment with high purity and biological activity can be applied for further studies.

Amino Acid Sequence ; Animals ; Antibody Specificity ; Bacterial Proteins ; chemistry ; Bacterial Vaccines ; biosynthesis ; chemistry ; immunology ; isolation & purification ; Chromatography, High Pressure Liquid ; Electrophoresis ; Escherichia coli ; genetics ; Helicobacter pylori ; genetics ; immunology ; Molecular Sequence Data ; Peptide Fragments ; biosynthesis ; chemistry ; immunology ; isolation & purification ; Rabbits ; Urease ; antagonists & inhibitors

OBJECTIVETo prepare the live recombinant vaccine of attenuated Salmonella typhimurium SL3261 expressing Helicobacter pylori (H. pylori) B subunit (UreB) and to determine whether it could be used as an oral vaccine against H. pylori infection.

METHODSUsing genomic DNA of H. pylori Sydney strain (SSI) as template, the H. pylori UreB gene fragment was amplified by PCR and subcloned into the expression vector pTC01. The recombinant plasmid pTC01-UreB was then transferred into LB5000 to obtain modified forms, and further conversed into the attenuated Salmonella typhimurium SL3261 to obtain recombinant SL3261/pCT01-UreB as an oral immunization reagent, which was then used to orally immunize Balb/c mice twice at a three-week interval. Twelve weeks later, anti-UreB IgA antibodies in intestinal fluid and IgG antibodies in sera were determined by ELISA. The relating data in control groups (including body weight, gastric inflammation, etc.) were also collected.

RESULTSThe sequencing analysis showed that the UreB gene fragment amplified by PCR was consistent with the sequence of the H. pylori UreB gene. The restriction enzyme digestion revealed that the correct pTC01-UreB was obtained. SDS-PAGE and Western blot showed that a 61KD protein was expressed in SL3261/pTC01-UreB, which could be recognized by anti-H. pylori UreB antiserum and was absent in the control containing only Salmonella typhimurium SL3261 strain. The multiple oral immunization with SL3261/pTC01-UreB could significantly induce H. pylori specific mucosal IgA response as well as serum IgG responses. IFN-gamma and IL-10 levels were significantly increased in SL3261/pTC01-UreB group, and no obvious side effect and change in gastric inflammation were observed.

CONCLUSIONThe attenuated vaccine of Salmonella typhimurium expressing H. pylori UreB can be used as an oral vaccine against H. pylori infection.

Animals ; Antibodies, Bacterial ; blood ; Bacterial Vaccines ; administration & dosage ; immunology ; Female ; Gene Expression Regulation, Bacterial ; Gene Expression Regulation, Enzymologic ; Helicobacter Infections ; immunology ; prevention & control ; Helicobacter pylori ; enzymology ; genetics ; immunology ; Immunoglobulin G ; blood ; Interferon-gamma ; metabolism ; Interleukin-10 ; metabolism ; Mice ; Mice, Inbred BALB C ; Salmonella typhimurium ; genetics ; immunology ; metabolism ; Urease ; genetics ; immunology ; metabolism ; Vaccines, Attenuated ; genetics ; immunology ; Weight Loss

OBJECTIVETo determine the effects of lactose inducing on the expression of recombinant Helicobacter pylori rUreB and rhpaA, and Escherichia coli rLTB and rLTKA63.

METHODSBIO-RAD gel image analysis system was applied to detect the outputs of the recombinant proteins. SDS-PAGE was performed to measure the target protein expression of recombinant genes at various periods of growth, different lactose concentrations, various inducing temperatures and times. The results of the target protein expression induced by lactose were compared to those by isopropyl-beta-D-thiogalactoside (IPTG).

RESULTSLactose showed higher efficiency to induce the expression of rHpaA, rUreB, rLTB and rLTKA63 than IPTG. The expression outputs of target recombinant proteins induced at 37 degrees C were remarkably higher than those at 28 degrees C. The optimal expression parameters were 0.8 of OD600 value, 50 g/L of lactose, 4 hours of inducing time for rHpaA, and 1.2 of OD600 value, 100 g/L of lactose, 5 hours of inducing time for both the rUreB and rLtB,and 0.8 of OD600 value, 100 g/L of lactose, 4 hours for rLTKA63.

CONCLUSIONLactose, a sugar with non-toxicity and low cost, is able to induce the recombinant genes to express the target proteins with higher efficiency than IPTG.

Adhesins, Bacterial ; biosynthesis ; genetics ; Bacterial Toxins ; biosynthesis ; genetics ; Bacterial Vaccines ; biosynthesis ; genetics ; Enterotoxins ; biosynthesis ; genetics ; Escherichia coli ; genetics ; metabolism ; Escherichia coli Proteins ; biosynthesis ; genetics ; Genetic Engineering ; Helicobacter Infections ; prevention & control ; Helicobacter pylori ; genetics ; metabolism ; Humans ; Lactose ; pharmacology ; Recombinant Proteins ; biosynthesis ; genetics ; Urease ; genetics ; Vaccines, Synthetic ; biosynthesis ; genetics

The gene encoding urease subunit A (ureA) of Helicobacter pylori (H. pylori) was cloned from H. pylori isolate by polymerase chain reaction (PCR). Sterile distilled water instead of DNA served as negative control. The nucleotide sequence of the amplified product was determined. Homologous analysis of the ureA against that reported by Clayton CL and the GenBank and SwissProt databases were performed with the BLAST program at the Genome Net through the Internet. 0.8 kb PCR product was amplified from all H. pylori clinical isolators. The nucleotide sequence of the ureA was determined. The nucleotide sequence of the ureA began with ATG as the initiation codon and terminated in TAA as stop codon. The coding regions had a 44% G + C content. The DNA sequence was 98% homologous to that reported by Clayton CL (688 out of 702 residues were identical). The derived amino-acid sequences of the ureA were 99% homologous to that reported by Clayton CL (232 out of 234 residues were identical). The nucleotide sequence and the predicted protein showed significant homology to ureA of H. pylori in the NCBI Entrez database.
Base Sequence ; Cloning, Molecular ; DNA, Bacterial ; chemistry ; genetics ; Genes, Bacterial ; Genetic Code ; Helicobacter Infections ; microbiology ; Helicobacter pylori ; enzymology ; genetics ; isolation & purification ; Humans ; Molecular Sequence Data ; Polymerase Chain Reaction ; Sequence Analysis, DNA ; Transcription, Genetic ; Urease ; genetics ; metabolism

OBJECTIVESTo investigate the characteristics of virulence gene in Vibrio parahaemolyticus strains isolated from clinical patients and environment in Hangzhou, China.

METHODSThermostable direct hemolysin gene (tdh) and thermostable direct hemolysin-related hemolysin gene (trh) were determined in a total of 174 strains of V. parahaemolyticus isolated from patients and environment (seafood) in Hangzhou area by PCR.

RESULTSThe tdh was found in 92 out of 94 V. parahaemolyticus strains from food poisoning patients and in 33 out of 34 strains from sporadic diarrhea patients, and trh was not detected in all above clinical strains. Meanwhile the tdh was negative in all V. parahaemolyticus strains from environment, and the trh was also negative except one strain with urease activity. All strains with trh negative had no the activity of urease.

CONCLUSIONSThe V. parahaemolyticus strains from food poisoning patients and sporadic diarrhea patients are tdh positive and trh negative. The V. parahaemolyticus strains with tdh negative and almost trh positive in environment might be a potential pathogen in Hangzhou.

Bacterial Proteins ; genetics ; Bacterial Toxins ; genetics ; China ; Environmental Microbiology ; Foodborne Diseases ; microbiology ; Hemolysin Proteins ; genetics ; Humans ; Shellfish ; microbiology ; Urease ; genetics ; Vibrio Infections ; microbiology ; Vibrio parahaemolyticus ; classification ; genetics ; isolation & purification

The gene encoding urease subunit A (ureA) of Helicobacter pylori (H. pylori) was cloned from H. pylori isolate by polymerase chain reaction (PCR). Sterile distilled water instead of DNA served as negative control. The nucleotide sequence of the amplified product was determined. Homologous analysis of the ureA against that reported by Clayton CL and the GenBank and SwissProt databases were performed with the BLAST program at the Genome Net through the Internet. 0.8 kb PCR product was amplified from all H. pylori clinical isolators. The nucleotide sequence of the ureA was determined. The nucleotide sequence of the ureA began with ATG as the initiation codon and terminated in TAA as stop codon. The coding regions had a 44% G + C content. The DNA sequence was 98% homologous to that reported by Clayton CL (688 out of 702 residues were identical). The derived amino-acid sequences of the ureA were 99% homologous to that reported by Clayton CL (232 out of 234 residues were identical). The nucleotide sequence and the predicted protein showed significant homology to ureA of H. pylori in the NCBI Entrez database.
Base Sequence ; Cloning, Molecular ; DNA, Bacterial/chemistry ; DNA, Bacterial/genetics ; *Genes, Bacterial ; Genetic Code ; Helicobacter Infections/microbiology ; Helicobacter pylori/enzymology ; Helicobacter pylori/*genetics ; Helicobacter pylori/isolation & purification ; Molecular Sequence Data ; Polymerase Chain Reaction ; Sequence Analysis, DNA ; Transcription, Genetic ; Urease/*genetics ; Urease/metabolism