Preparation And Biological Activity Determination Of Egg Yolk Antibody Against Helicobacter Pylori HpaA-VacA Fusion Protein

Objective To open up a new path for the treatment and prevention of Hp infection,as well as to lay the foundation for the preparation of drugs against Hp infection through genetic engineering methods to prepare Helicobacter pylori Hpa A,Vac A and Hpa A-Vac A fusion protein as immunogen, immune laying hens to produce specific yolk antibody and study its biological activity.Methods and Results 1 Polymerase chain reaction(PCR) technology was used to clone Helicobacter pylori Hpa A, Vac A and Hpa A-Vac A fusion gene.The results of cloning vector enzyme digestion and sequencing showed that Hp Hpa A, Vac A and Hpa A-Vac A fusion gene were successful to be cloned to p GEM-3Z. 2 Build Hp Hpa A,Vac A and Hpa A-Vac A fusion gene prokaryotic expression system respectively by recombinant DNA technology.SDS-PAGE results showed that the above prokaryotic expression system was build successfully and the expression of Hpa A, Vac A and Hpa A-Vac A fusion protein were efficient. 3 Purify Hpa A, Vac A and Hpa A-Vac A fusion protein by affinity chromatography, analyse its purity by SDS-PAGE, identify its immunological activity by Western blot.We found that the purity of three kinds of recombinant purified protein were more than ninety percent and were able to be identified by the serum of Hp infection patients.It also suggested that the three kinds of fusion protein had good immunological activity. 4 Immune laying hens respectively with the purified Hpa A, Vac A and Hpa A-Vac A fusion protein, separate and purify yolk antibody(Ig Y), identify its biological activity.The SDS-PAGE results showed that the purity of yolk antibody were about eighty five percent.The Western blot results suggested yolk antibody had good immunological characteristics.According to indirect ELISA results, we found the titer of Hpa A-Ig Y, Vac A-Ig Y and Hpa A-Vac A-Ig Y was 1:12800, 1:12800 and 1:6400 respectively.When Hpa A-Vac A-Ig Y antibody reacted with Hpa A and Vac A antigen respectively, the titer was 1:6400 and 1:3200.Conclusion This paper produced specific egg yolk antibody against Helicobacter pylori( Hpa A-Ig Y, Vac A-Ig Y and Hpa A-Vac A-Ig Y) successfully by genetic engineering and animal immunity, study and compared its biological activity. It suggested that three kinds of Ig Y had good biological activity,to lay the foundation for the development of drugs including prevention and treatment against Hp infection.