Preparation And In Vitro Anti-helicobacter Pylori Effect Of The Recombinant Vaca Yolk Antibodies

Objective: To observe high titer of anti-vac IgY from eggs yolk ofhens immunized with recombinant vacA in Helicobacter pylori bygene-engineering ,which would set a foundation for the development oftreatmental antibody for H.pylori infection. Methods: A mass of recombinant vac proteins were aquired byover-culturing and inducing the engineered bacteria E.coli DH5a harboringPQE30-V vector .The recombinant protein was analyzed by SDS-PAGEand purified by Ni2+-NAT chromatography.Its protein concentration wasdetected by Bradford method,and its antigenicity and immunogenicity werecomfired by ELISA.Vac-IgY was prepared by immunized hens with thepurified recombinant protein (VacA) . The feasibility of its clinicalapplication was evaluated by comparing H.pylori diagnostic standard ofreseach with the experiment result. The relation of IgY titer andimmune-time was assessed roughly. Lots of high-titer IgY were mixed andpurified by the ammonium sulphate precipitation ,its purity was identifiedby SDS-PAGE , titer and protein concentration were measured by indirectELISA and Bradford method. Results: 1.SDS-PAGE result showed that the recombinant proteinmainly expressed as inclusion bodies. After purified, the purity of targetprotein was over 90% and its protein content was 0.67mg/mL; 2.Theindirect ELISA based on recombinant protein vac was successfullyestablished to assay the anti-vac in human serum and showed that therecombinant protein could be recognized by H.pylori antiserum of patients,its sensitivity and specificity were 92.3% and 92.0% respectivety; 3.Theindirect ELISA based on recombinant protein vac was successfullyestablished to assay the anti-vac in egg yolk extraction of hens immunizedthe recombinant protein and showed that the recombinant protein could berecognized by egg yolk extraction; 4. IgY antibody titer became higher asthe increase of the immune time:the titer was up to 1:800 after 25 daysfrom the first injection , 100 days after the first injection,the titer rised to1:6400 and could maintained some time; 5. The purity of the IgY was about65% after purified with the salt precipitation ;6. The titer and proteinconcentration of the purified IgY were 1:12800 and 25.66mg/mlrespectively. Conclusion: VacA-IgY in high titer and protein content has beensuccessfully prepared,which pave the way for further study of itsphysical-chemical and biologic properties . The indirect ELISA based onrecombinant protein vac was also successfully established to assay theanti-vac in human serum and showed a good sensitivity andspecificity ,which implied that the recombinant protein could be applied toidentify VacA+H.pylori infection .