Cloning, Expression Of NapA Gene Of Helicobacter Pylori And Studies On Antigen Epitope Of NAP Protein

Background and objectiveHelicobacter pylori(Hp) is a gram negative, spiral, microaerophylic bacteriumthat infects the stomach mucosa of more than 50% of the human populationworldwide. It is mostly acquired during childhood and persists chronically, causingchronic gastritis, peptic ulcer disease, and in some individuals, gastricadenocarcinoma and gastric mucosal-associated lymphoid tissue (MALT) lymphoma,and it was regarded as gradeⅠcarcinogen by IARC of WHO in 1994.The current therapy, based on the use of a proton-pump inhibitor and antibiotics,is efficacious but faces problems such as patient compliance, antibiotic resistance,and possible recurrence of infection. The most efficacious and economic approach toprevent infection diseases of mass population is vaccine in the future. Neverthelessmany problems including antigen, adjuvant and immunity approach choice have notbeen solved efficiently so far. Various approachs have been adopted in thedevelopment of vaccines against Hp, most of which have been based on the use ofselected antigens known to be involved in the pathogenesis of the infection, such asurease, the vacuolating cytotoxin (VacA), the cytotoxin-associated antigen (CagA), and others. Although these vaccine candidates acquired certain immunoprotection inanimal experiments, However, the effect is so limited that no ideal vaccine isavailable now.Neutrophil-activating protein (NAP), an importance factor, which is correlativewith adhesion of the bacterial and inflammation occurrence, is a candidate antigen forvaccine development. It is encoded by napA gene and the molecular weight is a15Kda. Its amino acid sequence presents significant similarities with Escherichia coliDps, with Listeria innocua dodecameric ferritin (Flp) and with the two Dps-likeproteins (Dlp-1 and Dlp-2) from Bacillus anthracis. It exists in all Hp strains isolatedfrom clinical cases. It induced neutrophils to adhere to endothelial cells and stimulateendothelial to respond rapidly by upregulation of adhesion molecules such asVCAM-1, ICAM-1, and Eselectin as well as production of proinflammatorycytokines and chemokines. Finally it lead to mucosal damage via the production ofreactive oxygen intermediates(ROI). NAP antibodys were found more frequently inpatients infected by Hp in sera and that vaccination of mice with NAP induced. ThusNAP is the extremely important virulence factor and a candidate for vaccinedevelopment.In our study, we pay our attention to the epitomics of NAP protein, looking forthe precise antigen epitope of Hp by using Phage peptide library technique. In orderto select more protection antigen epitopes and make preparation formultiple-compound peptide subunits vaccine, at the same time we compared NAPantibody level in different age and different patients from healthy person, chronicgastritis, peptic ulcer and gastric cancer and to investigate the relationship of NAPand gastric cancer. Furthermore three of anti-NAP mAb were identified to find outtheir clinical application value.Methods 1. According to the nucleotide sequence of Hp 26695. Taking NCTC 11639genome as the template, we constructed the recombine done vector napA/pMD18-Tand identified them by PCR or enzyme digestion, then the clone was sequenced. Therecombinant plasmids of napA/pMD18-T were ligated with expression vector ofpGEX-4T-1 after they were digested with EcoRⅠ/XhoⅠ, then the connectedproduction transform to host strain Top10 and the fusion expression vector ofnapA/pGEX-4T-1 were constructed. And then the recombinant plasmid of napA/pGEX-4T-1 were analyzed by PCR and enzyme digestion before sequenced. And thesequences of napA gene were analyzed by bioinformatic softwares. We can make useof the BLAST tool searching homology; make use of the DNAMAN software toanalyze the DNA sequence of napA and the characteristic of encoded protein; makeuse of the on-line software of harvard university to predict antigen epitope of NAPand make use of the software of Signal P 3.0 server to analyze it whether or not signalpeptide were existed?2. The napA/pGEX-4T-1 were expressed and induced by IPTG, and inductionconditions were optimized to express largely the fusion proteins in the supernatant.Later on the expression fusion proteins NAP were purified with GST affinity column,then the purified protein were identified with SDS-PAGE and Western blot.3. The purified protein as antigen, the sera of patients and healthy persons wereanalyzed with indirect ELISA to detect the activity of the expressed protein. Toinvestigate the relations between Hp-NAP antibodies and gastric cancer by makingcomparisons of antibody level between the healthy people and patients.4. BALB/c mice were immunized with the supernatant and precipitation ofcultured Hp after ultrasonication, and mAbs were obtained by means of hybridomatechnique. The resultant mAbs was evaluated for subtype, titer, affinity, and furtheridentified with NAP prepared by recombinant expression. NAP mAbs and enteropathogenic bacterium were reacted to identify the antibody specificy byImmunohistochemistry. NAP mAbs and Hp and mucosa samples from tissues ofgastric cancer were reacted to identify the antibody sensitivity byImmunohistochemistry.5. To identify epitope of NAP using the mouse monoclonal antibodies againstNAP as selective molecule and immunoscreening phage display random 7-peptideslibrary, the positive clones were identified by sandwich ELISA and competitiveELISA.Result1. napA fragment was successful enlarged from Hp NCTC11639. It is composedof 435 base pairs (GenBank No. DQ341279).2. The result shows that the nucleotide homology of napA from NCTC11639with other Hp strains on the GenBank was 94%～98% by BLAST homologysearching tool. The coding protein NAP is structurally but not functionally similar tothe DNA binding protein Dps of E. coli, the protein has significant antigenicity,furthermore their homology to be low in compared with other species.3. The result shows that there are four high antigenicity peptides locating in 4～24, 55～77, 95～103, 118～140 amino acid sequence by analyzing secondarystructure and tertiary structure and hydrophilicity and high antigenic peptide. Theiraverage antigenic index is 1.0236, and the peptide from 118～140 may be a goodantigen epitope because its high antigenic propensity is 1.15 and it got a high scorefor hydrophilicity.4. napA gene in Hp clinical isolates was detected by PCR. The napA gene existsin all of Hp clinical isolates, it is in accord with basic characteristic of vaccine.5. napA-PGEX-4T-1-E. coli Top 10 solubility expressing system was constructedand confirmed that the best expressing condition of inducing concentration is 1mmol/L IPTG and temperature at 30℃and time for 4h by optimizing expressionconditions.6. The molecular weight of the recombinant napA-pGEX-4T-1 expressed inE. coli was 44kDa. The recombinant NAP protein can reacted with sera of patients andRabbit anti human whole bacterium antibody, while not reacted with sera of healthypeople, the result shows that the recombinant NAP protein has good immunoreaction.7. The prevalence of Hp IgG antibody of healthy was 64% by Hp IgG detectreagent. But the prevalence of gastric cancer was 70% and it was higher than that ofpeptic ulcer (68%) and chronic gastritis (64%), their average value is 0.46±0.27,0.55±0.13, 0.43±0.21, 0.68±0.13 respectively.8. The level of NAP antibodies of healthy was 60%. But the antibody level ofgastric cancer was 97.5% and it was higher than that of peptic ulcer (92.9%) andchronic gastritis (85.7%), their average value is 0.61±0.14; 1.01±0.13; 0.89±0.07;0.98±0.17 respectively.9. Totally 29 hybridoma cell lines were established which secreted specificmAbs, and the prepared mAbs showed specific reaction against NAP (3 strains:E006,E019,E023), Western blot analysis proved that the recombinant NAP wasspecifically recognized by the sera of Hp infected patients. All of mAbs against NAPwere identified as immunoglobulin G1 (IgG1) and their titers in the culturesupernatant and ascites was 1:16 to 1:32 and 1:32000 to 1:64000 respectively. ThemAbs showed specific reaction with recombinant NAP and Hp clinical strains.10. From the experiment and sequencing comparison results, a different linearepitope was confirmed as mimotope of NAP protein, it is FAHLATQ.Conclusion1. The research indicates that we were the first one to clone napA gene(GenBank No.DQ341279) from genome DNA of Hp NCTC 11639. 2. We succeeded in constructing a prokaryotic solubility expression system ofHp-napA-PGEX-4T-1-E. coli Top10. The recombinant NAP has originalimmunoreaction and it is of great value to clinical sero-diagnosis and vaccine study ofHp.3. NAP is a conservative gene and a good immunogen. NAP have four highantigenic peptides and there is may be a good antigen epitope in peptide of 118～140.4. We succeeded obtaining three mAbs against NAP which got high specificityand sensitivity, and there may be facilitate for further study of detection and vaccinedevelopment of Hp.5. The infection rate of Hp is quite high in Guangdong province. There are somerelations between NAP antibodies and gastric cancer and NAP is a risky factor ofgastric cancer. This study provided basis for pathogenesis mechanisms, diagnosis andvaccine research of the gastric cancer associated with Hp infection.6. The mimotope (FAHLATQ) of NAP was obtained by a 7 mer phagepeptide library, and the result was in accordance with the peptide of 118～140 frombioinformatics analysis, The results provide a new approach for diagnosis and vaccineof NAP.