Cloning And Expression Of H.pylori Neutrophil-activating Protein Gene And The Immunity Evaluation Of Its Encoding Product

Helicobacter pylori is a gram negative, spiral, microaerophylic bacterium that infects the stomach of more than 50% of the human population worldwide. It is mostly acquired during childhood and, if not treated, persists chronically, causing chronic gastritis, peptic ulcer disease, and in some individuals, gastric adenocarcinoma and gastric mucosal-associated lymphoid tissue (MALT) lymphoma. The most efficacious and economic approach to prevent infection diseases of mass population is vaccine.H. pylori infection is accompanied by a large infiltration of inflammatory cells, consisting mainly of neutrophils and monocytes, in human gastric mucosa. Several studies have provided evidence for the presence of components in H. pylori water extract that attract and activate neutrophils and other inflammatory cells. It was termed H. pylori neutrophil-activating protein (HP-NAP). HP-NAP was localized in the bacterial cytosol and is released upon autolysis. HP-NAP could bind to the external surface of the outer membrane, HP-NAP had a specific affinity to high molecular weight carbohydrates. In such a location HP-NAP might be one of the adhesion factors of H. pylori. The specific antibody against HP-NAP was detected in majority of the infected patients. HP-NAP is suggested a major virulence factor of H. pylori and also a protective antigen.To screen protective antigens for vaccine development against H. pyloriinfection, HP-napA was amplified from genomic DNA of a clinical isolated H. pylori strain MEL-HP27 by PCR technique in the present study. After sequencing , the feature of HP-napA encoding product HP-NAP was predicated by bioinformatics. A recombinant expressing vector pMAL-c2X-napA was constructed using biotechnology. The immunogenicity and immunoreactivity of rHP-NAP was studied. The protective efficacy of HP-NAP was evaluated in animal models. Methods1. Molecular cloning and sequence analysis of HP-napA from H.pylori strain MEL-HP27Genomic and proteomic databases of H pylori strain 26695 and J99 published online were analyzed by data mining tools NCBI BLAST and software Omiga2.0. HP-napA was primaryly selected as the target gene for vaccine development. Design PCR cloning primers according to H. pylori strain J99 genomic sequences using software Primer5.0. HP-napA gene was amplified from H. pylori strain MEL-HP27 with pyrobest DNA polymerase. PCR fragment was inserted into a cloning vector pNEB193. The recombinant plasmid pNEB-napA was identified using restriction enzyme digestion assay and specific PCR identification. HP-napA DNA sequencing was performed by TakaRa biotechnology Company. The nucleotide acids alignment among H. pylori strains was analysised by NCBI BLAST. The chemical feature of HP-NAP including molecular weight, solubility, pi, conserved domain and the immunal activity were predicted using bioinformatics technique before performing laboratory experiments.2. Construction of the prokaryotic expressing plasmid pMAL-c2X-napA and purification of the recombinant protein rHP-NAPHP-napA was subcloned from vector pNEB-napA into prokaryotic expressing vector pMAL-c2X. The recombinant plasmid pMAL-c2X-napA was transformed into competent E.coli TB1, then the positive transformants were selected by ampicillin resistant and blue-white screen methods. pMAL-c2X-nap^4 was identified by restriction enzyme digestion analysis and specific PCR assay. The overabundant expression of fusion protein rMBP-NAP was induced by IPTG.. The inducedrMBP-NAP was detected by SDS-PAGE and Western Blot. After bacterial lysis by sonication in ice-water bath, rMBP-NAP was purified on Amersham FPLC P-920 system by using an Amersham Superdex-200 size -exclusion chromatography column and an amylose affinity chromatography column. Factor Xa was used to cleave the protein tag MBP from fusion protein rMBP-NAP. rHP-NAP was separated from the mixture by a amersham DEAE ion-exchange column chromatography. The purity was evaluated by gel image analysis system (SynGene, USA) and the quantity was tested by Bradford assay.3. Evaluation of Immunoreactivity and immunogenicity of rHP-NAPTo investigate the immunogenicity of rHP-NAP, laboratory rabbits were immunized with purified rMBP-NAP as immunogen combinding with Freund's adjuvant. The working dilutions of antisera were determined by agarose gel immune double diffusion assay and ELISA assay. To evaluate the immunoreactivity of rHP-NAP and its role in host immune response, rHP-NAP was used as antigen in western blot assay and ELISA assay. Purified rHP-NAP and the polyclonal antibody of rHP-NAP were used in study on the inhibition interaction of H, pylori adherence to human gastric cancer cell line BGC823 in vitro.4. The protective effecacy evaluation of rHP-NAP in animal modelsrHP-NAP was used as antigen and rCTB as mucosa adjuvent to evaluated the protectiv effecacy against H. pylori chellenge after orally immunized according to different immunisation formulation. All animals were sacrificed by cervical dislocation, their stomaches were collected for H. pylori colonization assessment with urease assay, histological examination and colony forming assay. Anti-rHP-NAP IgG was test by ELISA to evaluate the humoral immunity response and the T lymphocyte subgroup test was performed with flow cytometry to show the cell-mediated immunity response. Statistical analysisThe Statistical Package for the Social Sciences (SPSS 11.0) computer software was used for all analyses. The variations were tested with independent one-wayANOVA, Association between the variables were test by t test and x 2 test , significance were set at PO.05.Results1. Molecular cloning and sequence analysis of HP-nap A from H.pylori strain MEL-HP27Restriction enzyme digestion analysis and specific PCR tests confirmed that a recombinant cloning vector pNEB-napA was constructed. Sequencing result was published on GenBank (Accession No: AY366361). The homology between amplified PCR fragment and H.pylori strain J99 napA was 96.5%. The 435bp DNA fragment encodes a 144aa polypeptide chain and the homology between amplified gene encoded protein and neutrophil-activating protein of H.pylori strain J99 is 99%. The output data of BLAST search showed that the nucleotide acids homology of napA among H.pylori strains was 93% to 97%. These results indicated a complete neutrophil-activating protein encoding sequence (including the start code ATG and the end code TAA) was successfully cloned from a clinical isolated H.pylori strain MEL-HP27 in the present study.AY366361 was the first neutrophil-activating protien encoding sequence that was submitted to Genbank from China, and also one of the three neutrophil-activating protein encoding gene of H.pylori clinical strains isolated from China.Bioinformatics evaluation indicates that HP-napA was a highly conserved prokaryotic gene. The alignment between H. pylori MEL-HP 2 7 HP-napA and human genomic DNA as well as in other mammals was much lower which meet the criteria as a vaccine candidate.2. Construction of the prokaryotic expressing plasmid pMAL-c2X-napA and purification of the recombinant protein rHP-NAPRestriction enzyme digestion analysis showed that HP-napA had been inserted into vector pMAL-c2X between the restriction sites EcoR I and Sal I in the same reading fram with maltose binding protein. After screening, a high efficient expression bioengineering E.coli TB1 strain earring pMAL-c2X-?apyl had been selected. At the growth condition of 37°C 230rpm, inducing by 0.3mM IPTG for 3 hours , the total protein of E.coli TB1 contained 43.31% rMBP-NAP most of which were soluble . The asoluble rMBP-NAP contained 88% in the total induced fusion protein, and was about37.55% in the total soluble E.coli TB1 protein.SDS-PAGE assay indicated that the fusion protein started to elute from an Amersham Superdex-200 size -exclusion chromatography column within the second UV absorbance peak at 280nm. The concentration of rMBP-NAP in the elute fraction was 121mg/L, and the purity was about 91% . After the step of affinity chromatography, the purity of rMBP-NAP was about 97% and the protein tag MBP was completely cleaved in the reaction buffer containing 0.03% SDS incubated at 4°C for 16 hours by factor Xa. rHP-NAP eluted from the DEAE-Sepharose ion exchange chromatography column within the first UV absorbance peak at 280nm., and the purity of rHP-NAP was about 97%.3. Evaluation of immunogenicity and immunoreactivity of rHP-NAPSpecific antibody against rHP-NAP was detected by agarose gel immune double diffusion assay and ELISA assay. The results showed that the recombinant antigen rHP-NAP could be recognized by the host immune system and induced a specific immune response. This indicated rHP-NAP was a good immunogen.Western blot assay showed that both rMBP-NAP and rHP-NAP were recognized by human antiserum against H. pylori and rabbit antiserum against rHP-NAP. The result indicated that immunoreactivity of the recombinant neutrophil-activating protein produced by this study was significant. rHP-NAP was used as antigen in ELISA assay, it was found that the positive rate of human anti-NAP IgG in this investigation was 54%(108/200), which was close to that of Urease (57%, P>0.05). The human anti-HP-NAP IgG existed in 85% (17/20) of//, pylori positive patients. These data indicated that HP-NAP induced specific immune responses in human after infected with H.pylori.After incubated with rHP-NAP at the concentration of 20 u g/ml the mean number of//, pylori binding to the surface of a BGC823 cell was about 122.83 + 8.69 which was lower than that of PBS control 157+11.6 (PO.01). And the mean number of H. pylori binding to the surface of a BGC823 cell after treated with 10 u g/ml purified anti-HP-NAP IgG was about 38.9± 13.9 which was significantly lower than that of PBS control (P<0.01). The result showed that both rHP-NAP and anti-HP-NAP IgG produced by this study could partly inhibite the adherence of H.pylori to host cell surface. This indicated that HP-NAP was an adherence factor of H. pylori.4. The inhibition of rHP-NAP on H. pylori colonization to host stomach mucosa4.1 Establishment of animal models for H. pylori colonizationThe positive rates of SPF BALB/c mice and CV guinea pigs colonized by H.pylori were 100%. The histological examination showed that inflammatory response could be observed after 2 weeks of H.pylori colonization in guinea pigs. No typical inflammatory response could be observed after 2 weeks of H.pylori colonization in SPF BALB/c mouse model. But H.pylori mouse model was suitable for exact quantity assemment of colony forming units (CFUs).4.2 The inhibition of rHP-NAP on H. pylori colonized to murine gastric mucosaThe result of urease semi-quantitative assay and colony forming assay showed that density of H.pylori colonized in muose gastric mucosa was significantly lower in all the immunized groups than that of PBS and rCTB control groups (P<0.05) . The density of H.pylori colonized in muose gastric mucosa of rHP-NAP group was higher than that of groups immunized with rHP-NAP+rCTB, but the significance of the variations only was observed between rHP-NAP group and 10 Pg antigen plus adjuvant group (P<0.05 ) . These indicated mouse orally vaccination with rHP-NAP could inhibit the adherence and colonization of H. pylori to gastric mucosa with or without adjuvant. The protective efficiency could be enhanced by mucosa adjuvant rCBT. Among the groups immunized with rHP-NAP+rCBT of different amount of antigen and immunization schedule, the group of 10 u g rHP-NAP plus 10 u g rCBT immunized two doses at three weeks apart was the most appropriate formulation in this study.The mean level of anti-rHP-NAP IgG of all the immunized groups was significatly higher than that of the two control groups(P<0.05). The specific antibody in group with antigen alone was significant lower than that of the group with 10 u g antigen plus adjuvant. This result confirmed that the adjuvant could enhance the humoral immunity response induced by rHP-NAP.T lymphocyte subgroup test with flow cytometry showed that the Th subgroup in the mice of rHP-NAP+rCTB group was significantly lower than that of the PBS control group, rCTB control group, rHP-NAP group and the normal grouprespectively(P<0.05).4.3 The inhibition of rHP-NAP on H. pylori colonized to gastric mucosa of guinea pigThe result of urease semi-quantitative assay showed that there was a significant variation between rHP-NAP group and the two control groups, the result indicated that guinea pig orally immunized with rHP-NAP alone could inhibit H. pylori colonized to the stomach mucosa. The urease activity of rHP-NAP group was much more higher than that of the rHP-NAP+rCTB group (PO.05) indicating adjuvant could improve the vaccine efficacy. And the result of urease activity detection also showed there was no significant variation between groups rHP-NAP+rCTB and HpSON +rCTB (P>0.05) indicating the protection efficiency of rHP-NAP was similar to that of H. pylori whole cell sonicated antigen. The histological examination showed that guinea pigs orally immunized by rHP-NAP accompanied with rCTB adjuvant could inhibit the colonization of H.pylori to gastric mucosa as well as decrease the mucosa inflammatory response. Conclusion1. A complete neutrophil-activating protein coding sequence (including the start code ATG and the end code TAA)was successfuly cloned from a clinical isolated H. pylori strain MEL-HP27 for the first time in the present study. The sequence was published on GenBank (No.AY366361). This was the first HP-napA sequence that has been submitted to Genbank from China. Bioinformatics evaluation indicates that HP-napA was a highly conserved prokaryotic gene which meet the criteria as a vaccine candidate.2. The recombinant prokaryotic expressing vector pMAL-c2X-napA which has been constructed by DNA recombinant technique in the present study leads to a overabundant fusion expression of soluble neutrophil-activating protein of H. pylori strain MEL-HP27 with maltose binding protein in E.coli TB1.3. The purification procedures of rHP-NAP seting up on Amersham P-920 fastprotein liquid chromatography system could be directly enlarged in industry.4. HP-NAP is an important immunogen in the host immune response. The recombinant antigen rHP-NAP produced in this study could be recognized by the host immune system and induced a specific immune response. rHP-NAP with good immunoreactivity and immunogenicity could be the candidate fraction in vaccine development against H. p ylori. rHP-NAP also plays an important role as an immunityreagent in the phathogenesis and the epidemiology investigation of//, pylori.5. Rabbit anti-rHP-NAP specific multiclonal antibody has been produced in this study which would supply a immunity detection reagent for the biological functional study and applicational study of HP-NAP.6. This is the first report of H. pylori adhere to the cell surface of BGC823 could be partly inhibited by both rHP-NAP and anti-HP-NAP IgG in vitro. These results indicate HP-NAP as one of the adherence factors of//, pylori.7. SPF BALB/c mouse model and CV guinea pig model for H. pylori colonization have been established successfully in this study. It is observed that the guinea pigs were much more sensitive than BALB/c mice in mimic pathogenesis of H. p ylori infection.8. SPF BALB/c mice and CV guinea pigs orally immunized with rHP-NAP alone inhibited the colonization of the subsequent challenge with H.pylori. the protective efficacy was significantly enhanced by mocusa adjuvant rCTB. The protective efficacy of rHP-NAP could be improved by a appropriate immunization formulation in amimal models.