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To investigate the effect of patchouli alcohol on inhibiting Helicobater pylori urease activity, and its effect on expression levels of related genes, and lay the foundation for further research on the effect of patchouli alcohol on H. pylori colonization and infection. H. pyloriwas cultured and identified by gram staining, rapid urease test (RUT) and PCR method. Then agar dilution method was used to detect the bacterial survival after 1 h intervention by different concentrations of patchouli alcoholin the acidic (pH 5.3) and neutral (pH 7.0) conditions; berthelot method was used to detect urease activity and RT-qPCR method was used to detect the expression changes of ureA, ureB, ureE, ureH, ureI, and nixA related urease genes. The results showed that the survival rate of H. pyloriwas not significantly changed but the urease activity was obviously decreased after intervention by different concentrations of patchouli alcohol; meanwhile, the expression levels of ureA, ureB, ureE, ureH, ureI, and nixA were decreased to different degrees. Therefore, patchouli alcohol could inhibit H. pylori urease activity in both acidic and neutral conditions, and the mechanism may be related to down-regulation of urease gene expression., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Chinese Pharmaceutical Association.)

Objective: To prepare and purify anti-Helicobacter (H.) pylori Sydney Strain 1 (SS1) immunoglobulin Y (IgY) and anti-recombinant Ure I-UreB protein (rIB) IgY and investigate their antibacterial effects in vitro., Methods: rIB protein and SS1 cell solution were treated by ultrasonic disruption and used as immunogens to immune Leghorn hens. SS1-IgY and IB-IgY were derived from egg yolk with method of the water dilution (WD) combined with ammonium sulfate precipitations and analyzed by growth inhibition test (GIT) and rapid urease test (RUT)., Results: Two specific IgY with purity of over 80% and titer over 1:12 800 were prepared successfully. Both SS1-IgY and IB-IgY inhibited the growth of H.pylori and reduced H.pylori urease activity in vitro markedly., Conclusion: SS1-IgY and IB-IgY can inhibit the growth of H.pylori and reduce the urease activity in vitro.

Objective: To prepare and characterize the monoclonal antibody (mAb) against Helicobacter pylori (Hp) and establish a competitive ELISA used for detection of Hp antibodies in the sera of Hp-infected patients., Methods: BALB/c mice were immunized with inactivated Hp to generate Hp mAb using the hybridoma technology. Hp mixed proteins including cytotoxin-associated gene A (CagA), vacuolating cytotoxin A (VacA) and urease, as well as inactivated Hp were applied to screen positive hybridoma. Selected Hp mAb was analyzed and characterized with ELISA and Western blotting, and then labeled with horseradish peroxidase (HRP) for establishing a competitive ELISA to detect Hp antibodies in the sera of Hp-infected patients., Results: One Hp mAb named as C3 was selected after screening large amount of hybridoma, and the C3 Hp mAb was special for IgG2a subtype with the affinity titer of 1×10(7). Western blotting, ELISA and mass spectrum analysis indicated that the C3 Hp mAb could recognize Hp urease subunit B specifically. Using the C3 Hp mAb, we developed a competitive ELISA which could be used to detect Hp antibodies in the sera of Hp-infected patients., Conclusion: We successfully obtained one mAb that could specifically recognize Hp urease subunit B and developed a competitive ELISA using the Hp mAb.

Objective: To clone UreB gene of Helicobacter pylori (H. pylori) isolated from children to pGEX-4T-1 expression plasmid, and do sequence analysis., Methods: A 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., Results: A 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)., Conclusions: UreB of H. pylori strain GZCH1 is successfully cloned to pGEX-4T-1, which provides a basis for research of oral H. pylori vaccine.

Objective: To investigate the effects of the two-valence vaccine consisting of Helicobacter pylori (H. pylori) catalase and urease subunit UreB in preventing H. pyloriinfection in mice., Methods: C57BL/6 mice were divided into 7 groups and immunized with intragastric administration of catalase and UreB (both 100 microg) plus cholera toxin (CT, 2 microg), catalase (100 microg) plus CT (2 microg), UreB (100 microg) plus CT (2 microg), catalase (100 microg), UreB (100 microg), CT (2 microg), or PBS, respectively, once a week for 4 consecutive weeks. Two weeks after the last immunization, all the mice were challenged by live H. pylori, and sacrificed 4 weeks after the challenge to obtain the gastric mucosa samples for detecting H. pylori using semi-quantitative bacterial culture assay., Results: The total protection rate in mice immunized with the two-valence vaccine, single-valence vaccine of catalase, and single-valence vaccine of UreB was 83.3% (20/24), 41.7% (10/24) and 54.2% (13/24), respectively, and the rate in the other 4 groups were all 0. The H. pyloricolony density in mice with vaccination was significantly lower than that of other 4 groups (P<0.05). The total protection rate and H. pylori colony density differed significantly between the two-valence vaccination group and the single-valence vaccination groups (P<0.05)., Conclusion: The two-valence vaccine consisting of catalase, UreB and adjuvant has better immunoprotective effects than the single-valence vaccines.

We analyzed the distribution of glutathione-S-transferase P1 gene (GSTP1) polymorphism in a population from northern China and the relationship between the polymorphic BsmAI site in its exon 5 and gastric cancer susceptibility and evaluated the combined effect of GSTP1 polymorphism and H. pylori infection on gastric cancer. Blood samples were taken from 1,612 subjects in areas of high and low incidence of gastric cancer. Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) were performed to analyze the genotype of a GSTP1 polymorphism in exon 5 (Ile105Val). Serum levels of anti-H. pylori IgG were measured by enzymed-linked immunosorbent assay. We found that the GSTP1 Val variant allele frequency was 22%, which was significantly different from the Western people. There was a significant difference in GSTP1 allele gene distribution between the area of high incidence of gastric cancer(23%) and low incidence(20%). The frequency of GSTP1 Val/Val genotype was statistically higher in the gastric cancer group compared to the non-gastric cancer population. Analysis showed a statistically significant 1.587-fold increase in gastric cancer risk associated with the GSTP1 Val allele. Moreover, there was a statistically significant interaction (odds ratio, 17.571; 95% confidence interval, 6.207 - 49.742) between GSTP1 Val/Val genotype and positive H. pylori IgG status. Our results indicate that the distribution of GSTP1 polymorphism has geographic differences. Individuals with the GSTP1 Val allele gene show an increased risk for gastric cancer. Association of the GSTP1 (Val/Val) genotype with H. pylori IgG positive status could significantly increase gastric cancer risk.

Aim: To construct the recombinant plasmid containing catalase (KatA) of Helicobacter pylori (Hp), analyze its nucleic acid sequence, express it in E. coli and study its antigenicity., Methods: KatA fragments were amplified from Hp chromosomal DNA by PCR. Its T-A was cloned, sequenced and compared with other HP strains on the GenBank. Then the gene cloned into pGEX-4T-1 fusion expression vector was expressed in E. coli and purified by GST-affinity chromatography. The purified product was used to identify 29 stains of mouse anti Hp monoclonal antibodies and analyze antigenicity with serum of Hp-infected patients by Western blot., Results: KatA fragments were composed of 1,515 bp (GenBank No. DQ333889) and the nucleotide homology with other Hp strains on the GenBank was 96%-97%. 85 kDa of the recombinant KatA-pGEX-4T-1 was expressed in E. coli. 4 of 29 anti-Hp mouse monoclonal antibodies were against KatA. Western blot analysis proved that KatA was specifically recognized in the serum of Hp-infected patients., Conclusion: The recombinant KatA has original antigenicity. It is of great value to clinical sero-diagnosis and vaccine study of Hp.

To investigate the effect of recombinant plasmid of Helicobacter pylori ureB gene on gastric epithelial cell. The full length sequence of ureB gene from NCTC11637 was amplified by PCR. The recombinant plasmid was constructed by cloning the open reading frame (ORF) of ureB into the eukaryotic expression vector pcDNA3.1, and was transfected SGC-7901 cells, then the clones resisting Hygromycin were screened. mRNA expression of ureB of transfected cells was detected by RT-PCR. The effect of recombinant plasmid of Hp ureB gene on cell phenotype was observed by fluorescence strain, on proliferation by MTT, on apoptosis and cell cycles by flow cytometry, respectively. The positive clones of ureB (SureB) appeared cell membrane budding and cell shrinkage. MTT assay showed there was no statistic significance between the SureB and SpcDNA3.1 which were transfected only by pcDNA3.1 (P > 0.05), suggesting that the growth of SureB were not inhibited. The apoptosis rate of SureB was higher than that of SpcDNA3.1 (P = 0.007). Analysis for cell cycle showed that in SureB cells the proportion of S phase increased, the proportion of both G2/M and G0/G1 phase decreased. Positive transfection of ureB gene into SGC-7901 can change cell phenotype and induce cell apoptosis.

Objective: To investigate the effects of recombinant Helicobacter pylori catalase (rHpCAT)on oxidative stress in rat colonic mucosal epithelial cells., Methods: Oxidative stress model was established by hydroxyl generated from Fenton reaction in cultured colonic mucosal epithelial cells isolated from normal rats, in the model of which the effects of rHpCAT were observed. The cells were divided into normal control, model, 5-aminosalicylic acid (5-ASA, 0.1 mmol/L), and rHpCAT (1 x 10(5), 1 x 10(6), and 1 x 10(7) U/kg, respectively) groups. At the end of the experiment, the content of lactic dehydrogenase (LDH), malondialdehyde (MDA), myeloperoxidase (MPO), glutathione peroxidase (GSH-Px), catalase (CAT) and, superoxide dismutase (SOD) were detected in the culture supernatant., Results: The contents of LDH, MDA and MPO were elevated while those of GSH-Px, CAT and SOD reduced in the model group. rHpCAT at different doses reduced the release of LDH, depressed the contents of MDA and MPO, and increased the contents of GSH-Px, SOD and CAT., Conclusion: rHpCAT has protective effects against rat colonic mucosal oxidative damage.

Objective: To perform genetic recombination of the urease B subunit (UreB) of Helicobacter pylori (Hp) and examine the biological properties of the recombinant protein., Methods: The gene fragment encoding Hp UreB was isolated clinically from Chinese subjects by means of PCR, and cloned subsequently into an expression vector pET-11C-UreB for the non-fusion protein expression in E.coli BL21 (DE3) strain., Results: The expression of recombinant UreB was achieved in E.coli BL21 with a relative molecular weight of approximately 62,000 at the expression ratio of 26%, and the first 15 amino acids of recombinant UreB were MKKISREYVSMYGP. The results of peptide mapping and amino acid compositional analysis were consistent with previous theoretical prediction, and enzyme-linked immunosorbent assay together with Western blotting indicated strong immunogenicity and reactivity of the recombinant protein in BalB/c mice, which were specifically recognized by polyclonal BalB/c mice anti-Hp sera or human sera infected with Hp., Conclusion: The results of this study has laid an solid immunological foundation for incorporating recombinant UreB as a subunit vaccine component against Hp.

Objective: To clone Helicobacter pylori ureB gene, to construct prokaryotic expression system of the gene and to identify immunogenicity of the fusion protein., Methods: The ureB gene from a clinical isolate Y06 of H.pylori was amplified by high fidelity PCR. The nucleotide sequence of the target DNA amplification fragment was sequenced after T-A cloning. The expression vector pET32a with inserted ureB gene was constructed. ureB fusion protein was expressed in E.coli strain BL21DE3 induced by IPTG at different dosages. Western blot using antibody against whole cell of H.pylori as well as immunodiffusion assay using antiserum of rabbit against the fusion protein was applied to determine immunogenicity of the fusion protein., Results: In comparison with the reported corresponding sequences, the homology of nucleotide sequence of the cloned ureB gene was from 96.88% approximate, equals 97.82%, while the homology of its putative amino acid sequence was as high as 99.65% approximate, equals 99.82%. The expression output of UreB protein in pET32a-ureB-BL21DE3 system was approximately 40%of the total bacterial proteins. UreB protein was able to combine with antibody against whole cell of H.pylori and induce rabbit to produce high titer antibody after the animal was immunized with the protein., Conclusion: An expression system with high efficiency of H.pylori ureB gene has been established successfully. The expressed UreB protein with satisfactory immunogenicity and immunoreactivity can be used as antigen in H.pylori vaccine.

Objective: To construct a non-resistant attenuated Salmonella typhimurium (S.typhimurium) strain capable of expressing Helicobacter pylori (Hp) catalase., Methods: After PCR amplification, the gene fragment encoding Hp catalase was inserted into the expression vector pYA248 containing asd gene, and the recombinant vector was then introduced into the host S.typhimurium strain X4072 depleted of genes encoding adenylate cyclase (delta cya), cyclic adenosine monophosphate receptor protein (delta crp) and aspartate-beta-semialdehyde dehydrogenase (delta asd). Bridged enzyme-linked immunosorbent assay (ELISA) was employed to measure the antigenicity of the catalase expressed in the sonicate and culture supernatant. According to Meacock's method and with the assistance of the growth curve, the stability of the recombinant strain was evaluated. A half lethal oral dose test was conducted to evaluate the safety of recombinant strain., Results: S.typhimurium X4072 (pYA248-CAT) with expected capacity was successfully constructed, and bridged ELISA demonstrated higher catalase levels in the culture supernatant than in the sonicate of the recombinant strain X4072 (pYA248-CAT). After the strain was passaged for 100 generations without selection pressure, all the randomly selected colony of the recombinant strain grew well with positive catalase antigenicity as identified by ELISA. The growth curve of the recombinant strain showed comparable growth status of the 2 strains X4072 (pYA248) and X4072 (pYA248-CAT). The survival rate of C57BL/6 mice was 100% 30 d after oral administration of 1.0x10(10) cfu X4072 (pYA248-CAT)., Conclusion: Non-resistant S. typhimurium vaccine X4072 (pYA248- CAT) is constructed successfully, which is stable in vitro and safe as confirmed by animal experiment. This vaccine provides a new candidate for viable oral vaccine against Hp infection.

Objective: To investigated the effect of attenuated Salmonella typhimurium vaccine strain expressing Helicobacter pylori (Hp) catalase (KatA) on protection against Hp infection., Methods: Recombinant plasmid expressing KatA was constructed. Expression of KatA was induced with IPTG, and analyzed by SDS-PAGE and Western blot. The recombinant plasmid were introduced into attenuated Salmonella typhimurium SL3261 strain to construct live oral vaccine strain. C57BL/6 mice was orally immunized with this vaccine strain and then given orogastric challenge with live Hp Sydney strain. The stomach samples were submitted to a rapid urease test and quantitative culture., Results: The SDS-PAGE showed a dominant additional protein with molecular weight of 79kDa which could specially react with antibody against GST and accounted for 19% of total bacteria proteins. Animal experiment indicated that this vaccine strain could protect mice against Hp infection., Conclusion: Attenuated Salmonella typhimurium vaccine strain expressing KatA could induce effective immune response in protection against Hp infection,which may play an important role in preventing and treating Hp infection and related diseases.

Objective: To evaluate the immune prophylaxis of recombinant urease subunit A or B (rUreA or rUreB)., Methods: The BALB/C mice were divided into seven groups: negative control group, control group with CTB, control group with rUreA, control group with rUreB, experimental group with rUreA + CTB and experimental group with rUreB + CTB, experimental group with sonicate + CTB, which were gastrically inoculated with 150 ml PBS, 5 micrograms CTB, 50 micrograms rUreA, 50 micrograms rUreB, 50 micrograms rUreA + CTB, 5 micrograms rUreB + CTB and 400 micrograms sonicate + 5 micrograms CTB, respectively. Two weeks later, the mice were gastrically infected with H. pylori (10(7)-10(8) CPU per time). 4, 8 and 12 weeks later, the mice were killed and the immune protection of PBS, CTB, rUreA, rUreB, rUreA + CTB, rUreB + CTB and sonicate + CTB were evaluated by the presence of H. pylori in gastric tissue stained with Giemsa by microscopy. The mice in the side-effecting groups of rUreA + CTB and rUreB + CTB were not infected with H. pylori. 12 weeks later, the mice in the two groups were killed, and the side effects of rUreA + CTB and rUreB + CTB were evaluated by the infiltration of inflammation cells in gastric tissue stained with HE by microscopy., Results: The mice gastrically inoculated with CTB, rUreA or rUreB had significantly less H. pylori. The immune protective effect of rUreA, rUreB or sonicate of H. pylori combined with CTB was stronger than that of rUreA, rUreB or sonicate of H. pylori alone. The immune protective effect of rUreA + CTB occurred later than that of rUreB + CTB or sonicate of H. pylori + CTB. The mice immunized with rUreA + CTB, rUreB + CTB or sonicate of H. pylori + CTB were not completely protected against H. pylori infection. The mice immunized with rUreA + CTB, rUreB + CTB or sonicate of H. pylori + CTB were less infected with H. pylori, and had mild inflammation of gastric tissue., Conclusion: Mice immunized with rUreA + CTB, rUreB + CTB or sonicate of H. pylori + CTB could not be completely protected against H. pylori infection and have alleviated inflammation of gastric tissue.

In this study serum anti-urease antibodies of Helicobacter pylori was examined in order to investigate the prevalence of H. pylori infection in 394 children between age 1 and 13. The results showed that the average infection rate was 15.7% with a tendency of increase infection rate with age, and by the age of 12 the H. pylori infection rate reached 30%, which was higher than those found in developed countries. We also noticed that there was a comparatively higher serapositivity among 1 and 7 year olds. This may possibly be due to the close contact between family members and school mates. In addition, the serum positive rate of antibody to H. pylori urease in children with recurrent abdominal pain was much higher than those asymptomatic children (P < 0.05).

The urease extracted from 6 strains of Helicobacter pylori (HP) was used as antigen to detect sera anti-urease antibodies of HP in 136 patients with gastric diseases and 13 persons with normal gastric mucosa by enzyme-linked immunosorbent assay (ELISA). The antigen proved to be pure and specific for ELISA. For a titre of 450EU or over 450EU the test had a sensitivity of 91.96% and a specificity of 86.49%. The results of the ELISA showed excellent correlation with microbiological detection of HP and histological examination of the antrum. The level of sera antibody showed a relation to the bacterial amount of HP in gastric mucosa, but no connection with the severity of gastritis. While ELISA may be effective for the serodiagnosis of HP infection and is able to distinguish patients with normal antrum mucosa from those with gastritis. The test does not help to distinguish those with antrum gastritis from those with peptic ulcer. It also provides a reliable method for epidemiological investigation of HP infection.