Preparation Of Anti-H.pylori Yolk Antibody By DNA Immunization

Helicobacter pylori(H.pylori,Hp.)has a high infection rate in the world.It is the only pathogen that colonizes the stomach acid environment.Colonization of H.pylori changes the acidic environment of the stomach and releases toxic substances that induces a series of chronic or even malignant stomach diseases.Yolk immunoglobulin(IgY)produces natural antibodies that are transported to the yolk via serum stimulated by specific receptors in bird B lymphocytes.By generating passive immune protection,the IgY has a therapeutic effects on human and animal diseases caused by pathogenic bacteria,viruses,etc.Objective: A recombinant eukaryotic expression plasmid containing H.pylori immunogenicity pathogenic factors(Hpa A),urease B subunit(Ure B),and I subunit(Ure I)was prepared.The recombinant plasmid was used as an immunogen to immunize laying hens to produce specific yolk antibody(IgY)and then tested for its biological activity.This study will lay a foundation for the research and development of preventive health foods or clinical treatment and diagnostic reagents for Helicobacter pylori.Method and result: The Hpa A,Ure B and Ure I genes of H.pylori were amplified by PCR and cloned into p ET28a(+)and p CMV-tag2 B plasmids by restriction enzyme digestion,respectively.The recombinant plasmid was transformed into E.coli and transfected into Hep G2 cells to express the target protein,respectively.The active target protein was obtained and purified by gel chromatography.The expression of the target protein was identified by SDS-PAGE and Western blot.The results showed that both eukaryotic and prokaryotic expression of the protein had a good specific immune response,indicating the feasibility of DNA vaccine with the in vivo immune.Preparation of three recombinant p CMV-tag2 B plasmids with endotoxin removal.The layer hens were immuned by p CMV-tag2B-Hpa A ? p CMV-tag2B-Ure B ? p CMV-tag2B-Ure I and the equal mixtures of three kinds of plasmids,respectively.The purified Hpa A,Ure B and Ure I protein antigens were used as a control group to be immunized together.The eggs were collected,diluted by water,precipitated by PEG,and purified by DEAE-Sephorose Fast Flow gel filtration chromatography.The biological activity of the specific yolk antibody(Hp.-IgY)against H.pylori was determined.Western blot results showed that all groups of Hp.-IgY could specifically bind to the corresponding antigen and had good immunoreactivity.The results of indirect ELISA showed that the specific IgY produced by DNA immunization layer chickens could reach the same level by protein antigen immunization.Agglutination test showed that Hp.-IgY and H.pylori could undergo agglutination and precipitation in vitro..Conclusion: This study demonstrated the feasibility of constructing Hpa A,Ure B and Ure I gene vaccines through the above experiments,and obtained a specific yolk antibody protein with similar potency to traditional immune methods through DNA immunization.We also proved that the specific IgY antibody had a good biological activity.A simple and economical method for the preparation IgY antibody from Helicobacter pylori was preliminarily explored.The study will provide conditions for the development of preventive health foods and clinical treatment and diagnostic reagents for Helicobacter pylori..