Analysis Of Helicobacter Pylori Outer Membrane Protein And Preparation Of Urease Vaccine Based On Proteomics

Helicobacter pylori（H.pylori）is a gram-negative bacterium,which was listed as a Class I carcinogen in 1994.In the world,about 50%of the people are infected with H pylori.H.pylori can induce gastritis,intestinal metaplasia and even gastric cancer when it infects normal gastric mucosa cells.H.pylori secretes a variety of toxic genes adapted to the harsh environment of the human stomach and causes chronic inflammation and tissue damage that may eventually lead to the development of peptic ulcers and stomach cancer.At present,two antibiotics combined with a proton pump inhibitor and bismuth agent are used to treat H.pylori.However,antibiotic therapy always has some side effects and bacterial resistance is gradually increasing.Therefore,we need to develop new methods to treat and prevent H.pylori infection.Our research group utilized liquid chromatography-mass spectrometry,LC-MS)and generation sequencing technology to identify and analyze the out membrane protein（OMP）and Urease B subunit（UreB）expressed by H.pylori in Shijiazhuang area.Monoclonal antibodies and vaccines against OMP and UreB were prepared according to OMP and UreB expressed in H.pylori.Monoclonal antibodies against OMP and UreB can inhibit H.pylori activity in vitro.The UreB protein vaccine can induce T cell immunity and B cell immunity in mice,providing a new idea for the treatment and prevention of H.pylori infection.Part One The changes of outer membrane protein and urease sequences of Helicobacter pylori strains were analyzed based on proteomics and generation sequencing technologyObjective:In order to analyze the differences in protein expression of OMP components in different strains,liquid chromatography-mass spectrometry（LC-MS）was used to detect the OMP components of Helicobacter pylori（H.pylori）isolated from 59 patients in Shijiazhuang area.A strain expressing Blood-group antigen-binding protein A（Bab A）was selected for culture.Then the expression changes of OMP and pathogenicity were analyzed after subculture.The diversity of Urease B subunit（UreB）sequences of different strains in Shijiazhuang was analyzed by generation sequencing.Methods:1.Fifty-nine strains of H.pylori from Shijiazhuang area were cultured by streak separation method.Urease assay and gram dyeing were used to verify the single colony,and then LC-MS assay was used to analyze the expression of OMP in 59 strains.2.H.pylori strain expressing Bab A was cultured by streak separation method.48 h of culture was one generation,and then passed through to 30generations.3.The first and 30th generation of H.pylori infected gastric cancer cell line AGS and normal gastric epithelium GES-1 cells were used to verify that the H.pylori stimulated the production of Interleukin 8（IL-8）in these cells by q RT-PCR.Bacterial adhesion experiment was used to verify the changes of adhesion ability of H.pylori to AGS and GES-1 cells after culture.4.UreB gene was amplified by PCR.The length of PCR product was determined by agarose gel electrophoresis.UreB sequences of 21 H.pylori isolates were analyzed and compared by generation sequencing.Finally,DNAMAN software was used to analyze the similarities and differences of UreB sequences.Results:1.A single colony was obtained from 59 H.pylori frozen isolates cultured by zonal streak separation method.Urease test was positive.The gram staining results showed that the single colony was red and rod-shaped,indicating that the single colony was H.pylori.2.Fifty-nine strains of OMP were identified by LC-MS technology.Bab A could be detected in 71%of the strains;47%of isolates expressed Sialic acid-binding adhesion（Sab A）protein;37%of strains expressed Bab A and Sab A protein simultaneously.3.Compared with the first generation of H.pylori,24 kinds of OMPs were detected in the 30th generation of H.pylori.The expression of 22 kinds of OMPs decreased in the 30th generation of H.pylori and 2 kinds of OMPs increased in the 30th generation of H.pylori.4.The results of q RT-PCR showed that compared with the first generation of H.pylori,the 30th generation of H.pylori reduced the secretion of IL-8 in AGS cells（P<0.01）.The 30th generation of H.pylori reduced the secretion of IL-8 from GES-1,but the difference was not statistically significant,which shows that gastric cancer cells may be more sensitive to H.pylori stimulation.5.Bacterial adhesion experiment results showed that the 30th generation of H.pylori had significantly increased ability to adhere to AGS and GES-1cells compared with the first generation of H.pylori（P<0.01）.6.The agarose gel electrophoresis results showed that UreB was amplified with correct location and single band.The homology of UreB DNA from 21 H.pylori strains sequence was 95%.The DNA sequence of UreB was translated into protein using DNAMAN software,and the amino acid homology was 98%.Summary:In this part,59 H.pylori strains were cultured,and different H.pylori strains expressed different OMP spectra.The type and expression level of OMP protein were changed in the process of H.pylori culture,and the ability to stimulate AGS and GES-1 cells to secrete IL-8 was decreased,while the ability to adhere to AGS and GES-1 cells was significantly increased.The homology of UreB gene DNA sequence and amino acid sequence of 21 H.pylori strains was greater than 95%,indicating that UreB gene was relatively conserved among pylori strains,which provides suggestion for the next part of the experiment.Pare two Monoclonal antibodies H.pylori Bab A_385-447（OMP28）and UreB_321-339were prepared by hybridoma technique and the neutralization capacity of the antibodies was verifiedObjective:Polypeptides were prepared with Bab A_385-447（OMP28）and UreB_321-339as targets,and monoclonal antibodies were prepared by hybridoma technique.The effects of the antibodies on the adhesion ability and urease activity of H.pylori were investigated.Methods:1.The Bab A_385-447and UreB_321-339peptides were synthesized by solid phase N-fluorene methoxycarbonyl method.Then,the purity of the synthesized peptides was determined by high performance liquid chromatography（HPLC）.2.We obtained monoclonal antibodies against two polypeptides through myeloma cell fusion,positive screening,subcloning,ascites preparation,and antibody purification.2.The titer of monoclonal antibody was detected by ELISA.3.Urease assay was used to detect the changes of urease activity inhibited by antibodies against UreB_321-339polypeptide;Bacterial adhesion assay was used to detect the changes of H.pylori’s adhesion to AGS cells inhibited by antibodies against Bab A_385-447polypeptide.4.ELISA was used to detect whether the H.pylori positive patients serum contained antibodies against Bab A_385-447（OMP28）and UreB_321-339peptides.Results:1.Bab A_385-447and UreB_321-339peptides were synthesized by solid phase N-fluorene methoxycarbonyl method.HPLC results showed that the purity of peptideswas greater than 90%.2.Monoclonal antibodies against two peptides were prepared by hybridoma technique.Four Ig G monoclonal antibodies were obtained against Bab A_385-447polypeptide.A total of 6 Ig G monoclonal antibodies were obtained against UreB_321-339polypeptide.3.ELISA results showed that the efficacy of antibodies against UreB_321-339and Bab A_385-447peptides was greater than 1:100000.Urease test results showed that the activity of H.pylori urease was inhibited by co-incubation with UreB_321-339polypeptide antibodies for 4h（P<0.01）.The adhesion test results showed that the number of H.pylori adhesion to AGS cells was inhibited by H.pylori co-incubated with Bab A_385-447polypeptide antibody for 4h（P<0.001）.4.ELISA results showed that the antibody against Bab A_385-447polypeptide and UreB_321-339polypeptides was not contained in the H.pylori positive serum.Summary:In this part,Bab A_385-447and UreB_321-339polypeptides were prepared,and then four Ig G monoclonal antibodies against Bab A_385-447polypeptide and six Ig G monoclonal antibodies against UreB_321-339polypeptide were obtained by hybridoma technique.The titer of the two kinds of antibodies was greater than1:100000.Urease activity was inhibited by antibodies against UreB_321-339polypeptide.Antibody against Bab A_385-447could inhibit the adhesion of H.pylori to AGS cells.In addition,we did not detect antibodies against these two peptides in H.pylori positive patients.These results indicated that the two monoclonal antibodies could be used as candidates for passive immunotherapy.Part three Polypeptide vaccine was prepared using H.pylori adhesion associated OMP,virulence factor and UreB_199-338as antigensObjective:Adhesion factor polypeptides were prepared using OMP（Bab A,Sab A,Oip A）and virulence factors（Nap A,Cag A,Fla A,GGT,Hpa A,Fec A,Vac A,UreB）as targets.Meanwhile,UreB_199-338protein vaccine was used as a target to prepare polypeptide vaccine,and the immunogenicity of recombinant UreB_199-338protein vaccine was preliminarily evaluated.Methods:1.pet28a（+）-adhesionfactorpolypeptideplasmidand p ET-22b（+）-UreB_199-338plasmid were prepared by PCR amplification,enzyme digestion,enzyme linkage,transformation and identification of positive clones.2.The plasmid was transformed into Escherichia coli BL21（DE3）receptive cells,and IPTG was added to induce the expression of adhesion factor peptide and UreB_199-338protein.3.Adhesion factor peptide protein and UreB_199-338protein were purified by Ni-NTA affinity chromatography.4.The purified adhesion factor peptides and UreB_199-338proteins were identified by Sodium dodecyl sulfate-polyacrylamide gel electrophoresis,（SDS-PAGE）,Western blot and LC-MS.5.BALB/C mice were immunized subcutaneously with 100μg Alum-UreB_199-338by mixing the adjuvant Alum and UreB_199-338protein at a volume ratio of 1:2 and the same volume of Alum.6.ELISA assay was used to detect the antibody in BALB/C mice at 21and 35 days after the last injection of Alum-UreB_199-338vaccine.7.The changes of adhesion of H.pylori to AGS cells were detected by antibody neutralization test.Results:1.Sequencing results showed that the plasmid pet28a（+）-adhesion factor polypeptide and p ET-22b（+）-UreB_199-338were successfully constructed.2.The molecular weight of adhesion factor polypeptide protein was27.96KD.Obvious blue bands were observed at 25KD-35KD by Coomath bright blue staining.Western blot confirmed bands between 25KD and 35KD.The results of LC-MS showed that more the protein sequences matched our target protein sequences,which proved that the purified adhesion factor polypeptide protein was our target protein.3.UreB_199-338recombinant protein has a molecular weight of 16.10KD.The UreB_199-338protein was purified by Ni-NTA affinity chromatography.Obvious blue bands were observed at 17KD by Coomath bright blue staining.Western blot showed obvious bands at the position of 17KD.The LC-MS results showed that the identified protein sequence matched our target protein sequence,which proved that the purified UreB_199-338protein was our target protein.4.ELISA results showed that Alum-UreB_199-338vaccine could induce a higher level of UreB_199-338specific antibody.5.The results of antibody neutralization experiment showed that BALB/C mouse serum containing specific antibody significantly inhibited the ability of H.pylori to adhere to AGS cells after incubation with H.pylori（P<0.001）.Summary:In this part,recombinant H.pylori adhesion factor polypeptide plasmid was prepared and effectively expressed in Escherichia coli.In addition,recombinant UreB_199-338protein was prepared to immunize BALB/C mice with the recombinant protein vaccine to produce a specific antibody against UreB_199-338,which significantly reduced the adhesion of H.pylori in vitro.Conclusion:In this study,monoclonal antibodies against UreB_321-339,Bab A_385-447,adhesion factor and UreB_199-338protein vaccine were prepared according to OMP profile and UreB amino acid sequence expressed by H.pylori in Shijiazhuang area.Two monoclonal antibodies can inhibit the urease activity and adhesion ability of H.pylori in vitro.The UreB_199-338vaccine can induce mice to produce specific antibodies against the UreB_199-338antigen,which can provide reference for further prevention and treatment of H.pylori.