Cloning Of Helicobacter Pylori Omp22, HpaA And Omp22-hpaA Fusion Gene And Evaluation Of Immune Protective Efficacy Of Their Expression Products

Helicobacter pylori (H. pylori, Hp) is a common gram negative, spiral shaped,microaerophilic bacterium, which lives in the stomach and duodenum. The infection ofHelicobacter pylori is one of the most popular infections in the whole world, especiallyin the developing country, where the prevalence among middle-aged adults is 80％, andthe more high prevalence in children and infant. Helicobacter pylori infection is theprincipal cause of chronic active gastritis, peptic ulcer and a significant factor for gastricadenocarcinoma and gastric mucosa-associated lymphoid tissue (MALT) lymphoma.Recent epidemiological surveys have indicated that Helicobacter pylori infection maybe associated with coronary heart disease, diabetes, bronchitis and so on. Currenttherapies based on a proton-pump inhibitor and antibiotics have several drawbacks,including patient compliance, antibiotic resistance and recurrence of infection. Manystudies have shown that eradication of Helicobacter pylori infection improves healingand reduces the risk of recurrence or rebleeding in patients with duodenal or gastriculcer, and prevents Helicobacter pylori-related gastric carcinogenesis. Therefore, therewould be tremendous benefit to the society if safe, effective, and cost-effective vaccineswere available to prevent or cure chronic Helicobacter pylori infection.Helicobacter pylori adhesion (HpaA) is a flagellar sheath protein of Helicobacterpylori, and also is one of this bacterium main adherence factors. HpaA can bind to themany kinds of stomachs epithelial cell surface acceptor, playing an important role inadhesion stomach epithelial cell, and then further damage gastric mucosa. Some research show all Helicobacter pylori strains include hpaA gene, and considerablyconservative for its nucleotide and amino acid sequences. Furthermore, antibody againstHpaA almost could be found in all Helicobacter pylori infected patients sera, HpaA willbe an ideal antigen candidate for Helicobacter pylori vaccine.The outer membrane is a continuous structure on the surface of gram-negativebacteria, which have bilateral particular significance as a potential target for protectiveimmunity and bacterial pathogens. In other study, outer membrane protein vaccineshave been used with considerable success to induce protection against a number oforganisms. Helicobacter pylori genome include 34 outer membrane proteins whichfunctions have not been very sure, some studies indicated many outer membraneproteins from Helicobacter pylori showed strong immunogenicity. Omp22 is a majorstructural outer membrane protein of Helicobacter pylori, exposed on the surface ofHelicobacter pylori, its nucleotide and amino acid sequences were considerablyconservative. In some study, omp22 gene was predicated to have highly antigen andmaybe induce protective antibody. Omp22 protein may have the potential as a targetantigen for the development of Helicobacter pylori vaccine.In present study, we aimed to evaluate the immunogenicity of Omp22, HpaA andOmp22-HpaA fusion proteins of Helicobacter pylori (MEL-HP27) by cloning andexpressing these genes, and to evaluate their protective efficacy in mice model ofHelicobacter pylori infection.Methods1. Cloning and sequence analysis of omp22,hpaA and omp22-hpaA fusion genefrom Helicobacter pyloriomp22 gene and hpaA gene were amplified by PCR from Helicobacter pyloristrain MEL-HP27 chromosome DNA. The fragments were inserted into cloning vectorpBlueScriptⅡSK(-), and then were transformed in the E. coli strain. The recombinantplasmids were identified using restriction enzyme digestion and specific PCR. Fusiongene omp22-hpaA with an adapter was amplified from the plasmid pBlueScript-omp22 and pBlueScript-hpaA by overlap extension PCR. The sequence determination ofrecombinant plasmids was carried out by Shanghai DNA Biotechnologies Company. Inthe meantime, the sequence of omp22 and hpaA gene and amino acid were analyzed bysoftware Omiga.2.0 and DNAstar. The secondary structure and antigenicity andhydrophilicity of Omp22-HpaA fusion protein were analyzed by biological software.2. Construction of the prokaryotic expression systems expressing the omp22, hpaAand omp22-hpaA fusion gene, respectively, and optimization of expressionconditions for target genes from Helicobacter pylori in E. coliomp22, hpaA and omp22-hpaA fusion gene were subcloned from recombinantvectors into prokaryotic expressing vector pMAL-c2X. The recombinant expressingplasmids were transformed into competent E. coli TB1. Then the positive transformantswere selected by ampicillin resistant and blue-white screen methods, and were identifiedby restriction enzyme digestion and specific PCR. By inducing expression, we study theinfluence of inducing opportunity, IPTG concentrations and inducing time to expressionamounts. The expression products were analyzed using the SDS-PAGE method in orderto optimize the expressing conditions of gene engineering E. coli strains.3. Purification and immunological identification of the recombinant proteinsOmp22, HpaA and Omp22-HpaAThe over abundant expression of fusion protein Omp22, HpaA and Omp22-HpaAwere induced under the optimized conditions. The offspring expressed inTB1(pMAL-c2X-omp22), TB1(pMAL-c2X-hpaA), TB1(pMAL-c2X-omp22-hpaA)were broken up with ultrasonic. The fusion proteins were purified by amylosepre-packed column after being elementary purified by salting-out, the purity wasevaluated by gel image analysis system (SynGene, USA) and the quantity was tested byBradford assay. The induced products and purified proteins were identified by Westernblot. The mice were respectively immunized with the purified rOmp22, rHpaA,rOmp22-HpaA and Helicobacter pylori whole-cell soluble antigens as immunogen withFreund's adjuvant through hypodermic. Western blot was used to test the immunoreaction of rOmp22 with mouse anti-rOmp22 sera and mouse anti-Helicobacterpylori sera, respectively. The purified rHpaA and rOmp22-HpaA were studied in thesame procedure as the rOmp22.4. The protective efficacy evaluation of romp22, rHpaA and rOmp22-HpaA inanimal experimentThe mice were divided seven groups randomly, there are fifteen mice in everygroups. The mice in the seven groups were orally immunized with rOmp22+mLT63,rHpaA+mLT63, rOmp22+rHpaA+mLT63, rOmp22-HpaA+mLT63, rUreB+mLT63,mLT63 and PBS, respectively, one time pre week for four times. One week after the lastimmunization, all the mice were attacked with 200μl Brucella broth medium containingHelicobacter pylori cells of 2×10~7 CFU for two times at ten hours intervals. All the micewere sacrificed two weeks after challenged, and the stomach was collected forperformation of histological examination, semi-quantitative and qualitative analysisbasing on urease test, Gram's stain examination, Helicobacter pylori culture, andobservation of mucosa smears for assessment of Helicobacter pylori colonization in themouse model.5. Statistical analysis of dataThe data was inputted to computer and analyzed by SAS 9.13. The quantitativevariable was analyzed with independent one-way ANOVA. The different between twogroups was analyzed with LSD test. The comparison of protection rates was analyzedwith Fisher's exact test or X~2 test. The signification level was set atα=0.05.Results1. Cloning and sequence analysis of omp22,hpaA and omp22-hpaA fusion genefrom Helicobacter pyloriThe results of restriction enzyme digestion and sequencing of the recombinant.plasmids showed that the gene omp22, hpaA and fusion gene omp22-hpaA had beencloned into the vector pBlueScriptⅡSK(-) correctly; Encoding esequence(GGTGGAGGC) of three glycine residues was inserted into omp22-hpaA fusion geneas an adaptor. The omp22 gene consists of 540 base pairs and encodes the polypeptides of 179 amino acids. The sequencing results of omp22 from strain MEB-HP27 arepublished in the GenBank, the accession number is DQ499023. The homologies of thenucleotide and putative amino acid sequences compared with three published omp22gene sequences were from 93.84%～97.41% and 90.50%～97.46%, respectively. ThehpaA gene consists of 783 base pairs and encodes the polypeptides of 260 amino acids.The sequencing results of hpaA from strain MEB-HP27 are published in the GenBanktoo, the accession number is DQ353891. The homologies of the nucleotide and putativeamino acid sequences compared with eight published hpaA gene sequences were from94.76～97.19% and 95.38～98.46%, respectively. It is necessary to predict the structureof Omp22-HpaA fusion protein because it is a new kind protein. The secondarystructure of Omp22-HpaA fusion protein was analyzed before and after fusion bybiological software Omiga2.0. It was showed that before and after fusion, the antigenactivity structure of Omp22 and HpaA protein has no change. We also analyzedantigenicity and hydrophilicity of fusion protein Omp22-HpaA, which are consistentwith antigenicity of Omp22 and HpaA protein.2. Construction of the prokaryotic expression systems expressing omp22, hpaA andomp22-hpaA fusion gene respectively, and optimization of expression conditions fortarget genes in E. coliThe omp22, hpaA and omp22-hpaA fusion gene were cut from recombinant cloneplasmids and inserted into prokaryotic expression vector pMAL-c2X, and then weretransformed into E. coli TB1 strain, selecting the recombinant expression plasmids. Thebioengineering E. coli TB1 strains (omp22 or hpaA or omp22-hpaA) were induced byIPTG, and target fusion proteins could be found by SDS-PAGE. Studying the influenceof inducing opportunity, IPTG concentrations and inducing time to expression amounts,We found that the gene engineering E. coli TB1 strain(pMAL-c2X-omp22) andengineering E. coli TB1 strain(pMAL-c2X-hpaA) growing two hours were induced forfour hours by the condition of 0.3 mmol/L IPTG, the expression amounts of fusionprotein take account for 25% and 30% of the total bacterium protein, respectively; Thegene engineering E. coli strain (pMAL-c2X-omp22-hpaA) growing two hours wasinduced for seven hours by the condition of 0.4 mmol/L IPTG, the expression amounts of fusion protein take account for 20% of the total bacterium protein.3. Purification and immunological identification of the recombinant proteinsOmp22, HpaA and Omp22-HpaARecombinant E. coli TB1 strain (pMAL-c2X-omp22), TB1 strain (pMAL-c2X-hpaA) and TB1 strain (pMAL-c2X-omp22-hpaA) were induced under the optimizedconditions, and the offspring expressed were collected and broken up with ultrasonic.Depositions and supernatant were analyzed by SDS-PAGE, the results showed that thetarget fusion proteins were been expressed by the soluble form in E. coli. Solubleproteins were purified by amylose pre-packed column and then the purified proteinswere confirmed to have a purity of more than 90%. The results of Western blot showedthe three proteins were recognized by the mouse anti-target protein (rOmp22, rHpaA,rOmp22-HpaA) sera, the mouse anti-Helicobacter pylori sera and sera of patient whowas infected with Helicobacter pylori, and show they had a high immunoreactivity.4. The protective efficacy evaluation of rOmp22, rHpaA and rOmp22-HpaA inmice model of Helicobacter pylori infectionTwo weeks after challenged with Helicobacter pylori, the mice were sacrificed.semi-quantitative and qualitative analysis basing on urease test, Helicobacter pyloriculture and histological examination were used to assay colonization of Helicobacterpylori in the mouse gastric mucosa. Protection rates in seven groups were as follows:46.67% (7/15) in rOmp22+mLT63 group, 53.33% (8/15) in rHpaA+mLT63 group,66.67% (8/15) in rOmp22+rHpaA+mLT63 group, 71.43% (10/14) in rOmp22-HpaA+mLT63 group, 60% (9/15) in rUreB+mLT63 group, 0% (0/15) in mLT63 group, 0%(0/15) in PBS group. The protection rates were significantly different in all the groups(P＜0.05), the protection rates in the groups immunized with rOmp22+mLT63,rHpaA+mLT63, rOmp22+rHpaA+mLT63, rOmp22-HpaA+mLT63, rUreB+mLT63showed no markedly difference (P＞0.05), but the protection rates in them weresignificantly higher than in the other groups (P＜0.05). But no significantly differencewere detected between the mLT63 group and the PBS group. Conclusions1. The omp22 gene was successfully cloned from a clinical isolated Helicobacterpylori strain MEL-HP27 for the first time in this study. The sequence was published onGenBank (No. DQ499023). This was the first omp22 sequence that has been submittedto GenBank from China. Bioinformatics evaluation indicates that omp22 was a highlyconservative prokaryotic gene, which meet the criteria as a vaccine candidate. The hpaAgene was cloned and the sequence was published on GenBank (No. DQ353891).2. The omp22-hpaA fusion gene with an adaptor (GGTGGAGGC) wassuccessfully acquired by overlap extension PCR. Omp22 and HpaA proteins could keeptheir antigen activity structure in the Omp22-HpaA fusion protein.3. The recombinant prokaryotic expression systems for omp22, hpaA andomp22-hpaA fusion gene have been constructed by DNA recombinant technique, whichcould high efficiently express Omp22, HpaA and Omp22-HpaA fusion protein by thesoluble form in E. coli TB1.4. The purification protein rOmp22 was proved with good immunoreactivity andimmunogenicity. It was proved that rOmp22 was a novel candidate antigen in vaccinedevelopment against Helicobacter pylori.5. We had obtained rHpaA and rOmp22-HpaA fusion protein with high purity andimmunoreaction after expression and purification, which could be the candidate fractionin vaccine development against Helicobacter pylori and diagnostic antigen.6. By application of the animal model, the study was performed to appraise theimmunoprotection of rOmp22, rHpaA, rOmp22-HpaA and rUreB associate withadjuvant. It discovered that the above all proteins could decrease the amount ofHelicobacter pylori colonization markedly, the protection rates were not significantlydifferent in the groups immunized. This indicated that the protective efficacy were equalbetween romp22 (rHpaA, rOmp22+HpaA) groups and rUreB group.