Preparation Of Specific Anti-Helicobacter Pylori Immunoglobulin In Egg Yolk And Evaluation Of Its Properties

Helicobacter pylori (H. pylori) is the key point of gastritis, peptic ulcer disease and World Health Organization listed it as Category I carcinogen. Currently triple or quadruple therapy based on the use of a proton-pump inhibitor and antibiotics is the primary means of treating H. Pylori-related diseases in clinical. But antibiotic resistance reduces the cure rate of this method. Therefore the development of efficient, safe and economical drugs avoiding antibiotic resistance is particularly important.Specific egg yolk immunoglobulin (IgY), the main serum immunoglobulin of poultry can be gathered in the yolk specifically. Domestic and international researches showed that specific anti-Helicobacter pylori IgY could achieve the therapeutic effect of H. pylori infection.H2O and CO2 were hydrolyzed from ammonia which was spread from blood under the action of urease when ph of stomach was less than 2. It formed "ammonia cloud1’around the bacteria and neutralize stomach acid, accelerating the metabolic rate of epithelial cells, destroying the gastric mucosal barrier and epithelial cells. Urease B subunit is the main functional units, including urease activity center. Urel was involved in controlling the passage of urease but not the biosynthesis of urease. Urel regulated the intracellular urease metabolic of H. pylori in humans and animals and played a key role to survival and multiplication.In these studies, to provide the basis for preparation of specific anti-Helicobacter pylori immunoglobulin in Egg Yolk, on the basis of previous gene research of UreB UreI biological effects anti-SS1-IgY and anti-IB-IgY were prepared successfully for antibacterial effect test in vivo and vitro. The experimental research methods, results and conclusions are as follows:1 Preparation of SS1-IgY and IB-IgYPrepared recombinant IB protein and H. pylori Sydney Strain 1 (SS1) cell with ultrasonic treatment were used as immunogens to immune hens; SS1-IgY and IB-IgY were derived from egg yolk with method of the water dilution (WD) combined ammonium sulfate precipitation and tested by SDS-PAGE, ELISA and K56000 for purity, titer and concentration. The purity of IB-IgY obtained was 85.2% with the concentration of 30.4 mg/ml, and the purity of SS1-IgY obtained was 83.2% with the concentration of 29.8 mg/ml, and the titer all of them were 1:12800.2 Evaluation of two specific IgY in growth inhibition testThe two specific IgY were mixture coated on the plates with fresh H. pylori bacteria suspension and incubated for 5 days, then the colonies were counted for statistical analysis. SS1-IgY and IB-IgY both can inhibit the growth of H. pylori in vitro.3 The performance of two IgY in rapid urease testThe solution contains large amounts of urease which was obtained from fresh H. pylori bacteria suspension through ultrasonic treatment was mixed and incubated with two IgY respectively in rapid urease test. Results show that two IgY reduced H. pylori urease activity in vitro markedly, and SS1-IgY is more effective.4 Establishment of of H. pylori infection in BALB/c mouse modelBefore bacterial infection 6-week-old mice was water fasting for 12 hours and gavaged with 0.2M sodium bicarbonate. After 10min mice were fed with 0.2ml concentration of bacterial suspension for 3 times, each time interval 1D. According to the results of gram staining, PCR, rapid urease test, H. pylori infection BALB/c mouse model was established successfully by this method.5 The inhibition of SS1-IgY and IB-IgY in vivoThe 6-week-old mice were divided into 7 groups randomly as IB-IgY 3mg group, 6mg group, SS1-IgY 3mg group,6mg group, omeprazole and clarithromycin combined group, positive infection group and the saline group. Experimental groups were deal with the same method in the H. pylori infection model and treated after 1 wk. The treatment was last for7 days at intervals of 1D. Stomach tissues were tested through gram staining, PCR, rapid urease test after 1 wk of the treatment. Data showed that when the therapeutic doses up to 6mg/mouse SS1-IgY and IB-igY did have the same therapeutic effects with omeprazole and clarithromycin combined group. These results indicate that SS1-IgY and IB-IgY could be a potential candidate overcoming tolerance of antibiotics for the treatment of H. pylori-mediated diseases as they were inhibited the growth of H. pylori and reduced H. pylori urease activity in vitro and vivo.