Study On The Purification And Immune Efficacy Of Fusion Protein HspA-UreB Of Helicobacter Pylori

Helicobacter pylori is a spiral, gram-negative bacterium that infects gastric mucosa with an average prevalence rate of 50%, ranging from 8% to 87%. It is considered to be the major causative agent of acute and chronic gastritis, and a significant etiologic factor of peptic ulcer disease, gastric cancer, and mucosa-associated lymphoid tissue (MALT) lymphoma. It was regarded as grade I carcinogen by IARC of WHO in 1994. About 10-20% of those infected develop duodenal ulcers and 1-2% of those infected are likely to develop gastric carcinoma.Treatment of H. pylori involving expensive combinations of various medicines (antibiotics, proton pump inhibitors) is always effective currently. However, large-scale medical eradication is impractical due to drug resistance, cost, reinfection, and poor compliance. Consequently, investigative attention has focused on the development of prophylactic and therapeutic vaccine against H.pylori.With the development of the study, several of the candidate immunogens havefulfilled the requirements of being capable of providing significant protective immunity against H. pylori infection in the stomach. However, no single antigen used in therapeutic and prophylactic immunization experiments has induced complete eradication of colony-forming units in the stomach or has been capable of completely preventing colonization following challenge with a fully virulent dose of H. pylori. It suggests that a future vaccine should consist of a mixture of antigens that can provide protection against the most important pathogenic events in H. pylori infection.It has been proved that immunization with mixture of Urease B subunit (UreB) and Heat shock protein A subunit (HspA) almost get completely protection in animal experiment, which suggests that HspA-UreB fusion protein used as candidate antigen of H. pylori vaccine have a bright future.On the base of prokaryotic expression system BL21(pET-HU27) that has been constructed in our lab before, this study will devote to optimize the induced conditions and increase the output of recombinant HspA-UreB fusion protein of H. pylori, and to purify the soluble offspring and inclusion body, then the purified fusion protein was analysed with SDS-PAGE and detected with Western-blot test-Subsequently, the competence of the purified fusion protein to induce the protective immune response was investigated. This research was performed to appraise the value of HspA-UreB fusion protein in vaccination with the aim to build a basis for the development of highly efficient H.pylori vaccine.Methods1. Exploration of the induced conditions of recombinant E.coli BL21(pET-HU27)The induced conditions of E.coli BL21(pET-HU27) was explored in different culture medium, revulsant and content of glucose at different temperature. The offspring expressed in E.coli BL21(pET-HU27) were broken up with ultrasonic, and analysing its solubility with SDS-PAGE, detecting it with Western blot test.2. Purification of fusion protein HspA-UreBThe soluble protein was collected, and the fusion protein which was elementarilypurified by salting-out was purified by AKTA Fast Protein Liquid Chromatogram (FPLC) with HiTrap Chelating HP. The inclusion bodies of HspA-UreB fusion protein expressed in recombinant E.coli BL21 (pET-HU27) were washed, denatured and renatured by urea solution, and the fusion protein was purified by AKTA Fast Protein Liquid Chromatogram with HiTrap Chelating HP, and then the purity was analysed with SDS-PAGE by genetools, and its immunocompetence was detected with Western blot test and the concentration of it was measured with Bradford method.3. Identification of protective efficacy of vaccination with fusion protein HspA-UreB in animal experimentForty mice were divided into 4 groups randomly, there were 10 mice in every groups. The mice in the four groups were immunized with Binding buffer (200μl), recombinant cholera toxin B subunit (5μg), purified fusion protein HspA-UreB(50μg), purified fusion protein HspA-UreB (50μg) plus recombinant cholera toxin B subunit (5μg) orally respectively two doses on weeks 1 and 4, i.e. 3 weeks apart. Two weeks after the last immunization, all the mice were attacked with 200μl Brucella broth medium containing H.pylori cells of 2×107CFU for two times at three days intervals. All the mice were sacrificed 4 weeks after challenge, and the stomach was collected for performation of histological examination, semi-quantitative and qualitative analysis basing on urease test, H.pylori culture and observation of mucosa smears for assessment of H. pylori colonisation in the mouse modle.4. Observation of specific sIgA to fusion protein HspA-UreB in mice after immunization.Mensurating the level of specific sIgA to fusion protein HspA-UreB in gastric juice and fecal in mice after immunization with ELISA, and discovering the variational disciplinarian of specific sIgA to fusion protein HspA-UreB.5. Statistical analysis of dataThe data was inputed to computure and analyzed by SAS 8.2. The quantitative variable were analyzed with independent one-way ANOVA to examine whether there were difference in OD levels, the difference between two groups was analyzed with LSD-t test. The comparation of protection rates was analyzed with Fisher's exact test.The evaluation of histology was analyzed with Kruskal-Wallis rank sum test. The signification level was set at a=0.05.Results1. Results of the induced conditions of recombinant E.coli BL21(pET-HU27)The percentage of fusion protein in E.coli BL21(pET-HU27) (21.51%) was highest in LB culture medium in 0.3mmol/L IPTG concentration for 5h at 37 ℃, and the percentage of fusion protein in E.coli BL21(pET-HU27) (17.28%) was highest in SOC culture medium in 1.0mmol/L IPTG concentration for 8h at 25℃, and the percentage of fusion protein in E.coli BL21(pET-HU27) in LB culture medium containing 1.0g/L glucose in 0.3mmol/L IPTG concentration for 5h at 37℃(9.92%) was higher than that containing 2.0g/L, 4.0g/L glucose. The offspring induced at different condition was characterized by Western blots and has a high immunocompetence..The offspring expressed in E.coli BL21(pET-HU27) were broken up with ultrasonic, the percentage of soluble fusion protein and inclusion body was 50.02%, 18.35% respectively in SOC culture medium in 1.0mmol/L IPTG concentration at 25 ℃ for 8h, and the percentage of soluble fusion protein and inclusion body was 20.33% , 58.62% respectively in LB culture medium in 0.3mmol/L IPTG concentration at 37℃ for 4h..2. Results of purification for fusion protein HspA-UreBThe soluble fusion protein and inclusion body were purified by AKTA Fast Protein Liquid Chromatogram with HiTrap Chelating HP. The purified recombinant HspA-UreB fusion protein in soluble protein and inclusion body was confirmed to have a purity more than 90%, and content of 0.15 mg/ml, 0.25mg/ml respectively, and a high immunocompetence characterized by Western blot test.3. Identification of protective efficacy of vaccination with fusion protein HspA-UreB in animal experiment of H.pylori infectionFour weeks after challenge with H.pylori, protection rates in 4 groups were as follows: 0% (0/10) in Binding buffer group, 10% (1/10) in rCTB group, 12.5% (1/8)in fusion protein HspA-UreB group, and 60% (6/10) in fusion protein HspA-UreB + rCTB group. The protection rates were significantly different in the groups (P<0.05), and the colonization of H.pylori showed by urease semi-quantitative trial were significantly different in the groups (P<0.05), and the changes of histology in gastric tissue were also significantly different among the groups (P<0.05).4. Results of specific sIgA to fusion protein HspA-UreB in mice after immunization.Twelve days later after the second immunization, the specific sIgA to fusion protein HspA-UreB was appeared in gastric juice and fecal, and the peak was appeared at the sixteenth day. However, the specific sIgA to fusion protein HspA-UreB was not detected in gastric juice and fecal in these days before the second immunization.Conclusion1. The expression condition of E.coli BL21 (pET-HU27) is optimized in this study. The output of soluble fusion protein is increased with the change of induced condition, such as time, temperature, culture medium, concentration of revulsant etc. An effective method for purifying recombinant HspA-UreB fusion protein of H. pylori from soluble offspring and inclusion body expressed in recombinant E.coli BL21 (pET-HU27) is established by AKTA Fast Protein Liquid Chromatogram with HiTrap Chelating HP. The purified recombinant HspA-UreB fusion protein is confirmed to have a purity of more than 90%.2. A more effective immunization is achieved by immunized on the first and the 21st day instead of other complex immunisation regimes. By application of the animal model, the study is performed to appraise the value of HspA-UreB fusion protein plus rCTB in vaccine with the aim to build a prophylactic H.pylori vaccine, and the petection rate is 60%, and the study shows that fusion protein HspA-UreB used as candidate antigen of H.pylori vaccine is safe.3. On the day 12th after the second immunization, the specific sIgA to fusion protein HspA-UreB is observed in gastric juice and fecal, and the peak is appeared atthe sixteenth day, which will be helpful to select more resultful procedure and to investigate the immunological mechanism in the further study.