In vitro studies of IgY against fusion VacA-HpaA protein of Helicobacter pylori / 中国免疫学杂志

ABSTRACT

Objective:Researching physical-chemic and biologic speciality of VacA-HpaA IgY,which provide a solid experimental basis for the preparation of vaccine against H.pylori infection.Methods:The purified antigen of fusion protein VacA-HpaA was used to immunize hens and the VacA-HpaA IgY was extracted from the yolk by water dilution methods.(1)After the VacA-HpaA IgY was purified by deposition technique with ammonium sulfate,the purity of IgY was analyzed by SDS-PAGE,and protein content of IgY was analyzed by Bradford method.(2)The indirect ELISA was used to detect its heat-stability,acid-stability and pepsin-stability.(3)The specificity of VacA-HpaA IgY to the antigens was identified by Western blot.(4)MTT was applied to assay the neutralization of VacA-HpaA IgY to The cytotoxity of H.pylori.(5)The neutralization of IgY to AGS cells was observed with scanning electron microscopy(SEM).Results:(1)The purity of VacA-HpaA IgY was about 60%,and the protein content of IgY was 22g/L;(2)The IgY showed a good heat-stability,a favorable acid-stability,and a well ability of anti-pepsin digest;(3)Western blot exhibited the protein bands with molecular weight of 27 000 and 30 000.The titer of VacA-HpaA IgY to VacA and HpaA were 1∶3 200 and 1∶6 400;(4)A dose and time dependent correlation of the IgY to counteract the cytotoxic activity of VacA;(5)VacA-HpaA IgY could interrupt the adhesive attraction of H.pylori.Conclusion:The VacA-HpaA IgY has good stabilities and well biologic speciality,which imply that the IgY could be used to prepare treatmental antibody against H.pylori infection.