| Helicobacter pylori (H. pylori, Hp) is the most common cause of gastritis and gastric ulcers and plays a pivotal role in the development of gastric carcinomas. Successful treatment of Hp infections most often employs antibiotic therapy, consisting of some combination of antibiotic, and either bismuth or a proton pump inhibitor. However, antibiotic therapy fails in10%to15%of cases due to the development of antibiotic resistance. It is important to seek new therapies for a wider means of treating, suppressing, or preventing H. pylori infection without drug resistance problems. In this study, different culture media were tested to culture and preserve Hp. The mature chickens were procedurally immunized with inactivated Hp cells, agglutination test were used to determine the titer of egg yolk antibody, and the pattern of immune response, bacteriostasis in vitro, physical and chemical characteristic of immune yolk were investigated. All these studies were of great significance to the industrial production of high immune yolk antibody against Hp and correlatively biologic medication.1Effect of different methods on Hp culture and preservationThe strain was cultured on Columbia Agar Base (CAB) supplemented with mixed antibiotics containing2500IU/l Polymyxin B Sulfate,5mg/l Trimethoprim,10mg/l Vancomycin HCl and10mg/l Amphotericin B and6%defibrinated sheep blood or6%horse serum respectively in a microaerophilic atmosphere of5%O2,10%CO2and85%N2at37℃for72h. With the two methods, good effect was observed. In condition of sterilization with no supplementation with mixed antibiotics, the strain grew well without contamination. When Brucella Broth was used an culture medium with combined antibiotics and horse serum in microaerophilic bag, Hp grew rapidly but was easily to form spheroid without replacing the bag on time. The strain was revived successively with cryopreservation at-70℃to-80℃.2Preparation and bacteriostasis in vitro of egg yolk antibody against Helicobacter pyloriPreparation and bacteriostasis in vitro of egg yolk antibody against Hp were studied. Chickens were procedurally immunized with inactivated bacterial cells at150,165and180days old, successively. Agglutination test showed that the egg yolk antibody against HP was produced from immunized hens, the antibody titer reached to1:1024at the45th day after the first immunization, the high level of antibody (>1:128) lasted for2months, then went down gradually to1:8after130days of age. Bacteriostasis in vitro showed that Hp was hypersensitive to egg yolk antibody.3Physical and chemical characteristic of high immune yolk against Helicobacter pyloriThe physical and chemical characteristic of high immune yolk (HIY) against Hp was studied. The results showed that the pH value and temperature of environment had obvious influence on anti-Hp HIY, while pepsin enhaced the influence. When only with pH value, the sensitive range was pH<3, when with pH value and pepsin, the sensitive range was pH≤4. HIY had better thermal stability at the temperature of pasteurize. All these characteristics were of great significance to the industrial production of high immune yolk antibody against Hp.4Establishment of Helicobacter pylori infection model in Mongolian gerbilsTwo methods were used to establish models of Hp (NCTC11637) infection in the Mongolian gerbil stomach. In the first method, Mongolian gerbils were forbidden to eat for18hours, then feeded0.5mL of50%alcohol and inoculated Hp three times at a6h-,12h-, and24h-interval. In the second method, Mongolian gerbils was treated as same as the above method, but taken Ranitidine orally everyday, with5mg/kg body weight. On the7th,15th,35th, and the45th day after inoculation, the infection and pathological changes in gastric mucosa were detected by Hp antigen diagnostic ELISA kit, bacterial culture, and histological section. The results showed that there were much Helicobacter pylori in the stomach in35days postinfection, combining the gastric mucous membrane damaged by alcohol with Ranitidine by oral administration per day, and the pathological changes were the same with that in natural infected cases.5Inhibitory effect of egg-yolk antibody on experimental Helicobacter pylori infection in Mongolian gerbilsInhibitory role of egg-yolk antibody on experimental Hp infection in Mongolian gerbil were observed as soon as egg-yolk antibody against Hp was prepared. The experiment gerbils were divided into4groups(36mice/group) and then taken orally normal saline in group Ⅰ, combination of antibiotics in Group Ⅱ, egg-yolk antibody against Hp in GroupⅢ for13days(one time/d). The egg-yolk antibody was injected hypodermically twice with a48h interval in groupIV.48h after first time administration the mice were inoculated with2.75×108colony forming units(CFUs) of Hp (ATCC43504) bacteria grown in Brucella broth. There were much Hp in the stomach on the7th,15th,30th,45th day after inoculation. The infection rate was100%in group Ⅰ, but low than23%in Group Ⅱ, GroupⅢ or groupⅣ. The results showed that the egg-yolk antibody against Hp by oral or injected route might decrease Hp infection in the stomach of Mongolian gerbils, and there is no significantly difference between group with egg-yolk antibody and group with antibiotic (p<0.05)6Effects of egg-yolk antibody on the treatment of Helicobacter pylori infection in the stomach of Mongolian gerbilsTo observe the effects of treatment by egg-yolk antibody aganist H. pylori infection, the experiment Mongolian gerbils were orally inoculated with H. pylori grown in Brucella broth (ATCC43504,1.15×108CFU) twice at48-hour-interval, and divided into4groups (16mice/group) on the seventh day after first inoculation time. Then the gerbils took orally normal saline in group Ⅰ, association of antibiotics in Group Ⅱ, egg-yolk antibody against H. pylori in GroupⅢ for12days (one time/d). The egg-yolk antibody was injected hypodermically twice with a48-hour-interval in groupⅣ. There were much H. pylori in the stomach before medication, with infection rate of100%. On the seventh day after medication the clearance rate of H. pylori was60%in group Ⅱ, and there were few H. pylori in group Ⅲ and group Ⅳ. On the twelfth day after taking orally drugs, the clearance rate was60%in both of group Ⅱ and group Ⅲ, but40%in group Ⅳ. Taking orally or injecting the egg-yolk antibody against H. pylori might decrease H. pylori infection in the stomach of Mongolian gerbil, and there is significantly difference between group treated with egg-yolk antibody and group treated with antibiotic.7Clone and expression of UreB gene from Helicobacter pylori in prokaryotic expression vectorIn order to develop genetic engineering vaccine against Helicobacter pylori or provide immunogen for preparation of egg-yolk antibody, a prokaryotic expression vector expressed UreB gene encoded protective antigen from Hp was constructed. UreB gene was PCR amplified and cloned in pGEM-T Easy vector. After sequence analysis of verification, the UreB gene was digested with restriction endoenzyme and ligated to pET32a (+), transformed to E.coli BL21(DE3). The molecular weight of fusion protein was about80KDa when the recombinant bacterium was induced by IPTG and detected by SDS-PAGE. The fusion protein was obtained in the supernatant of recombinant bacterium and purified by Ni+affinity chromatography when it was induced at30℃. The fusion protein could be recognized by corresponding antibody of mice sera immunized by inactivated Hp in western bolt, indicating this fusion protein had good immunocompetence. |
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