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Objective To establish an indirect ELISA method with Helicobacter pylori (HP)recombinated antigene OipA6 as coating antigen after prokaryotic expression and purification.Methods The ELISA plate was coated with Hp OipA6 recom-binant protein as antigen.Serum specimens and enzyme labeled anti human IgG were added.The reaction conditions was op-timized.The results of indirect ELISA were compared with the detection of oipA gene.Results When Hp OipA6 recombi-nant protein was 1.0 mg/ml,the condition of coating was 2~8℃ one night,the dilution of serum specimens was 1∶100 and enzyme labeled anti human IgG was 1∶4 000,the incubation time of enzyme labeled anti human IgG was 40 min,the chro-mogenic time was 15 min,the result of ELISA was the best.The sensitivity and specificity of the indirect ELISA were both higher than the detection of oipA gene.Conclusion It suggested that the indirect ELISA,in which Hp OipA6 recombinant protein were coated,may be an appropriate assisted method in detection of high virulent Hp infection.
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Objective To evaluate the relationship between functional oipA gene of Helicobacterpylori and digestive disease. Methods Biopsy gastric mucosa were obtained from 360 patients who would get gastroscopy.Hp were isolated from urease positive samples and partly urease negative samples,and improved by microscope,urease experiment and 16SrRNA PCR. OipA and cagA of the isolates were obtained by PCR and the statas of oipA signal region were analysised after sequencing. Analyze the relevance between functional oipA gene of Helicobacter pylori and digestive disease.Results 106 isolated Hp were obtained,in which 72 strains with cagA gene positive,87 with oipA signal region gene positive (80 the statas of signal region was on and 1 was off;6 could not be decided).The frequency of functional oipA were both significantly higher than cagA in ulcer and atrophic gastritis.Conclusion The relationship between functional oipA gene and digestive disease was more closer than that of cagA.
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PURPOSE: The common triple therapy for Helicobacter pylori is challenged by the increasing cases of antibiotic resistant infections, raising the need to explore alternative therapies. Oral administration of egg yolk immunoglobulin Y (IgY) has been previously reported as a means of passive immunization therapy for H. pylori infections. In this work, we investigated the inhibitory effect of IgY on the attachment of H. pylori to AGS cell line. MATERIALS AND METHODS: Recombinant OipA was prepared. Hens were immunized with recombinant protein three times. IgY was purified from egg yolks of immunized hens using polyethylene glycol precipitation method. The inhibitory effect of the specific immunoglobulin was evaluated in AGS cell line infected with H. pylori. RESULTS: The presence of recombinant OipA (30 kD) was confirmed via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Immunization of hens was confirmed using enzyme-linked immunosorbent assay. The purified IgY from egg yolks were assessed using SDS-PAGE and confirmed by western blot. CONCLUSION: The results showed that IgY-OipA had inhibitory effect on attachment of H. pylori to AGS cell line and may be utilized as a therapeutic or prophylaxis material.
Subject(s)
Administration, Oral , Blotting, Western , Cell Line , Complementary Therapies , Egg Yolk , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Helicobacter pylori , Helicobacter , Immunization , Immunization, Passive , Immunoglobulins , Polyethylene Glycols , Sodium Dodecyl SulfateABSTRACT
BACKGROUND: Outer inflammatory protein A (OipA) is an outer membrane protein of Helicobacter pylori that is involved in inducing IL-8 and intracellular signaling. In this study, we have predicted exposure amino acid sequences of OipA for insertion in permissive sites of CstH subunit of Eschierchia coli CS3 pilli for bacterial surface display. MATERIALS AND METHODS: Databases: National Center for Biotechnology Institute and Protein Data Bank. Servers: PHD, SABLE, GOR 4, SignalP3.0, TBBpred, PRODIV-TMHMM, TMRPres2D, CPH Models, PHYRE, GETAREA, VADAR, Pep state and pep window. Software: Swiss PDB viewer and Discovery studio. RESULTS: In silico prediction of exposure amino acid sequences of OipA led to detection of six sequences of amino acid, 76-87, 106-112, 170-182, 222-230, 242-258, and 278-290. These sequences inserted between amino acid sequences 66-67, 100-101, and 109-110 of CstH that were predicted by Eskandari et al. as permissive sites of CstH. CONCLUSION: OipA has the ability to induce IL-8 from gastric epithelial cells and some papers are mentioned that this outer membrane protein involve to attachment and intracellular signaling. Receptor of OipA and adhesion motifs on this protein is unknown. Detection of exposure motifs aids to recognition of adhesion motifs and receptor of OipA on gastric epithelial cells. In this study, we have predicted exposure amino acid sequences for insert to subunit CstH of CS3 pilli E. coli for surface display.
Subject(s)
Amino Acid Sequence/analysis , Bacterial Outer Membrane Proteins/analysis , Computer Simulation/methods , Escherichia coli/physiology , Epithelial Cells/microbiology , Helicobacter pylori/physiology , Intracellular Signaling Peptides and Proteins/analysis , Macrophage Inflammatory Proteins/analysis , Stomach/cytology , User-Computer InterfaceABSTRACT
Objective: To optimize Helicobacter pylori(H.pylori)oipA gene and develop strain of vaccine in Attenuated Salmonella typhimurium SL7207 haxboring recombinant plasmid pVAX1-oipA and pVAX1-optioipA, then to study its protection against H.pylori in C57BL/6 mice.Methods: oipA gene was modified by codon optimization,inserting the Kozak sequence and engineering CpG motifs.The optioipA gene and oipA gene was inserted into the eukaryotic expressing vector pVAX1 respectively to develop recombinant plasmid pVAX1-optioipA and pVAX1-oipA.The AGS cells were transfected by pVAX1-oipA,pVAX1-optioipA and pVAX1 respectively.Then the OipA protein expression was confirmed by Western blot.pVAX1-oipA,and pVAX1-optioipA were converted to LB5000 for methylation and then the modified eukaryoticvector was electrotransfered to final host SL7207 respectively, SL7207/pVAX1-oipA, SL7207/pVAX1-optioipA was identified with PCR and enzyme digestion.Specific serum IgG antibody was detected by indirect ELISA after oral inoculation with vaccine strains.Mice were challenged with live Sydney strain(SS1)three times at 0,2,4 d(5 × 10~8CFU/each mouse).Fonr weeks after challenge, the mice were sacrificed.The colonization of H.pylori in gastric mucosa were detected by rapid urease test and quantitative culture of H.pylori.Results: The AGS cells transfected with pVAX1-oipA and pVAX1-optioipA had expressed corresponding production, moreover, the expression level of pVAX1-optioipA was higher than those of pVAX1-oipA,SL7207/pVAX1-oipA and SL7207/pVAX1-optioipA confirmed with PCR,enzyme digestion;Two weeks after last immunization,immunized mice.were induced to produce anti-OipA IgG antibodies, and the levels of anti-OipA IgG antibody in pVAX1-optioipA group was obviously higher than in pVAX1-oipA group(P＜0.01); After challenged with SS1, the infection rate of pVAX1-oipA and pVAX1-optioipA group was 60%(9/15)and 26.67%(4/15), which was significantly lower than 100%(10/10)in the control group(P＜0.01).Conclusion:Attenuated Salmonella typhimurium SL7207 containing pVAX1-oipA, pVAX1-optioipA is successfully constructed.It can protect mice from H pylori infection and optimizing oipA gene can enhance the protection efficiency.
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Objective:To investigate the effect of DNA vaccine expressed Helicobacter pylori(Hp) oipA on protecting against Hp infection.Methods:The ORFs of Hp oipA had been inserted into the eukaryotic expressing vector pVAX1 and SGC-7901 cells had been transfected with recombinant plasmid pVAX1-oipA. The expression of oipA had been detected by RT-PCR and ELISA. After extracted and purified, pVAX1-oipA had been injected into BALB/c mice through muscles of right leg one time each week for three weeks(100 ?g each mouse). pVAX1 blank and normal saline had been used as the control groups. Titer of antibodies had been detected by ELISA two months after the last immunization. Based on the confirmation of immunological response in the pVAX1 groups, mice had been given orogastric challenged with live Hp Sydney strain three times(0.5?108/0.5 ml each mouse). Four weeks after challenge, mice had been sacrificed. Histological change and the colonization of Hp in the gastric mucosa had been detected by urease test, the culture of Hp, and electronic microscopy.Results:SGC-7901 cells transfected with pVAX1-oipA had expressed corresponding production at the level of transcription and translation. Immunized mice had been induced anti-oipA antibodies. After challenged with bacterium, as contrast to immunized mice groups injected with pVAX1-oipA, pVAX1 blank, normal saline, the positive rate of urease test of gastric mucosa was 0(0/10), 90%(9/10), 100%(5/5)respectively,the positive rate of cultures of Hp was 20%(2/10), 90%(9/10), 100%(5/5)respectively. Histological findings: the different degree of erosion had been observed in control group, but 80%(8/10)of gastric mucosa were normal in immunized mice.Conclusion:oipA DNA could induce effective immune response in protection against Hp infection.
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0.05) between the apoptosis rate of SpcDNA3.1 and that of SoipA,SureC.Conclusion:Expression product of UreC and OipA that have antigenicity had no effect on cell function, so they could be prepared for DNA vaccine.
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Objective:To express Helicobacter pylori oipA gene with different fragments and determine the protein and its peptides with finest antigenicity.Methods:oipA gene was obtained from international standard Hp NCTC 11637 and cloned into vector pGEM-T then was identified by PCR and sequencing.Six different fragments of oipA gene were amplified by PCR with the template pGEM-T/oipA.The gene fragments and expressing vector pET-42a were ligated.The recombinant vector were transformed into BL-21.The correct recombinants were identified by PCR,enzyme digestion and sequencing.Recombination protein and the peptides were detected by Western Breeze chemiluminescent after induced by IPTG.Optimization of the time and IPTG concentration for induction were conducted.The expressed products were affinity purified by Ni-NTA of His?Bind.The antigenicity of the recombination protein was determined with Western blot indicated by goat anti-Hp polyclonal antibody,and the recombination protein with finest antigenicity was selected with indirect ELISA.Results:The six oipA gene fragments could all expressed and the products were recognized by goat-anti Hp polyclonal antibody.The finest peptide with antigenicity was the smallest peptide.Conclusion:OipA gene fragments could be expressed in prokaryotic system and the smallest peptide had the finest antigenicity.It may be used as the reagent to detect corresponding antibody in serum.
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Objective:To detect the oipA gene of Helicobacter pylori(Hp)strains NCTC11637 and Hp1,Hp2 isolated from clinical biopsies,analyze their nucleotide sequences and make a homologous comparison of nucleotide with Hp 26695.Methods:The oipA gene was detected with PCR in Helicobacter pylori(Hp)strains NCTC11637 and Hp1,Hp2 isolated from clinical gastric biopsies after routine culture.Then PCR products were sent out for nucleotide sequence analysis and compared with Hp 26695.Results:The sequence of the aim gene was obtained in NCTC11637 and Hp1,Hp2 and was made a homologous comparison of nucleotide with 26695.The number of mutation of NCTC11637,Hp1 and Hp2 and was 48,48,50 respectively.The identity was 94%,94% and 94% respectively,while the strain Hp1 was most identical to 11637 as much as 100%.The homology of Hp2 and 11637 was 97%.Conclusion:Hp1,Hp2 and NCTC11637 expresse gene oipA,but the sequences of gene oipA of different strains are distinct.