Tudy Of Anti VacAA-HpaA Chicken Egg Yolk Antibody (VacAA-HpaA IgY) Against The Infection Of Helicobacter Pyolori In Vivo And Its Biological Avaibility

Objective: To observe high titer of HpaA-VacA IgY from eggs yolk of hens immunized with recombinant fusion protein HpaA-VacA in Helicobacter pylori by gene-engineering ,which would lay a foundation for the development of treatment antibody against H.pylori infection.Methods: 1,A mass of recombinant fusion protein HpaA-VacA protein were aquired by gene-engineering.The recombinant protein was analyzed by SDS-PAGE and purified by Ni2+-NAT chromatography. Its protein concentratrion was detected by Bradford method. Its immunogenicity was comfired by ELISA.2. HpaA-VacA IgY was prepared by immunizing Laying hens with the purified recombinant protein HpaA-VacA and was isolated by using the water dilution method(WD) combined chloroform precipitation method. Lots of high-titer HpaA-VacA IgY were mixed together and purified by salt precipitation (saturated ammonium sulphate). 3.Its purity was showed by SDS-PAGE, its specificity and titer were identified by ELISA,its protein concentration and purity were measured by indirect Bradford and SDS-PAGE respectively.Results: 1.SDS-PAGE showed that the recombinant fusion protein of HpaA-VacA mainly expressed as inclusion bodies. After purified, the purity of target protein was over 90% and its concentration was 1.2mg/ml;It had the Immunogenicity against both VacA and HpaA.2. The purity,the titer and the protein content of purified IgY was about 60%,1:12800 and 22mg/ml. respectively,it had the specificity against both VacA and HpaA.Conclusion: HpaA-VacA IgY with high titer, hith purity,high protein concentration and high titer has been successfully preparation .PARTⅡIN VIVO STUDIES OF CHICKEN EGG YOLK ANTIBODY (HpaA-VacA IGY) AGAINST HELICOBACTER PYLORIObjective: To verificate the HpaA-VacA IgY prevent and cure the infection of Helicobacter pylori in vivo,and provide a solid experimental basis for the preparation of oral vaccine with the effection of both prevention and curence against Helicobacter pylori.Methods: All the mice were disparted into such 4 groups as treatment group,preservation group,positive control(only with H. pylori) and negative group(only with bouillon). In the light of dosage magnitude,the mice of the treatment and preservation groups were respectively disparted into 6 groups:1mg/ml,2mg/ml,4mg/ml,6mg/ml,8mg/ml and control with PBS. As the treatment group, the mice were given with different dosage of HpaA-VacA IgY by oral 4 weeks after infected with H.pylori for three times within 5 days, and the mice were killed 8 weeks after disposal to observe the rate of currence.As the preservation group,the mice were given with HpaA-VacA IgY by oral,and 30 minutes later were infected with H.pylori for three times within 5 days. The mice were killed 12 weeks after disposal to observe the rate of preservation.Results:The total rate of infection of the positive control was 70.4%.As to the treatmen group, we got ideal result with the dosage of 6mg/ml of IgY. As to the preservation group,we got ideal result with the dosage of 6mg/ml of IgY.Conclusion:We have setup the model of mice infected with H.pylori successfully. The HpaA-VacA IgY have the ability of both currence and preservation against H.pylori in vito. It provide a solid experimental basis for the preparation of oral vaccine with the effection of both prevention and curence against Helicobacter pylori.PARTⅢSTUDY OF BIOLOGICAL AVAILABILITY OF HPAA-VACA IGYObjective: To research the protection of carbohydrates to the acid-stability of HpaA-VacA IgY by ELISA , to observe the biding quantity and antibody activity of HpaA-VacA IgY in rabit's gastral cavity at different time, and its antibody activity on the surface of gastral mucose , provide a solid experimental basis for the experiment of research of the IgY's biological availability and find out a way to enhance its biological availability.Methods: 1,Different carbohydrates were added into the IgY and its acid-stability was measured by indirect ELISA after 2h at acid condition of differet PH at37℃.2,Different concentratin of sorbitol and alantin were added into HpaA-VacA IgY to observe the peak concentratin of them which can enhance the IgY's acid-stability at PH2.0. 3,50ml of HpaA-VacA IgY with the concentration of 1mg/ml was infused into the rabit's gastral cavity with the way of intragastrical. The remaining HpaA-VacAIgY was expulsed some time after being infused .its PH,volum and its antibody activity were detected .The gastral mucose were reciped to detect the antibody activity of the IgY.Results: 1.The acid stability of HpaA-VacAIgYwas enhanced in the presence of all carbohydrate we used but was mostly enhanced in the presence of sorbitol and alantin after it.2. The peak concentration of sorbitol and alantin which can enhance the acid stability of IgY were 40% and 30% respectively at PH 2.0.3,78%,46% and 14% of the IgY liquid remained after 1h,4h and 7.5h respectively.4,The antibody activity of the IgY fell-off with the time moved and remain 68.8%,47.6%,29.1% afer 40min,4h,7.5h respectively.5,The PH of the IgY liquid fell but ascended again,it fell to the lowest level with 5.0 at 3h. 6,The antibody activity of the IgY. of the gastral rabit was not observed.Conclusion: The acid stability of IgY was enhanced in the presence of all carbohydrate we used but was mostly enhanced in the presence of sorbitol and alantin after it.The biding time and biding quantity of HpaA-VacAIgY in rabit's gastral cavity were detected successfully. Therefore,we had provide a solid experimental basis for the experiment of research of the IgY's biological availability and find out a way to enhance its biological availability.