Protection Effect Of Sucralfate On VacA-IgY In Vitro And Vivo

PARTâ… PREPARATION OF EGG YOLK IMMUNOGLOBULIN (IGY) AGAINST RECOMBINANT VACUOLATING CYTOTOXIN A PROTEIN(VACA)IN HELICOBACTER PYLORI BY GENE-ENGINEERINGObjective To prepare high titer of VacA-IgY from eggs yolk of hens immunized with recombinant protein VacA in Helicobacter pylori by gene-engineering ,which would lay a foundation for the development of therapic antibody against H.pylori infection.Methods 1,A mass of recombinant protein VacA were aquired by gene-engineering.The recombinant protein was analyzed by SDS-PAGE and purified by Ni²⁺-NAT chromatography. Protein concentratrion was detected by Bradford method. The antigenicity of VacA was identified by western blot.Rabbits were immuned with the VacA protein and then titer of the multi-clone antisera was determined by ELISA to identity the immunogenicity of VacA protein . 2. VacA-IgY was prepared by immunizing egg-laying yi-sha hens with the purified recombinant protein VacA and was isolated using the water dilution method(WD) combined with chloroform precipitation method. ELISA was used for the detection on IgY titer. Lots of high-titer VacA-IgY were mixed together and purified by salt precipitation (saturated ammonium sulphate). IgY purity was analyzed by SDS-PAGE. Protein concentration were measured by Bradford. IgY titer was determined by ELISA.Results 1. It was showed by SDS-PAGE that the recombinant protein VacA was expressed mainly as inclusion bodies with a relative molecular weight at 27000. After purified by Ni2+-NAT chromatography, the purity of target protein was over 90% and its concentration was 0.376mg/ml. After identification , VacA protein possesses both fine antigenicity and good immunogenicity. 2. Titer, purity and protein concentration of the purified IgY was about 1:2560 ,72.0%, and 6.36mg/ml,respectively.Conclusion Recombinant protein VacA at high concentration and purity was acquired. VacA-IgY of high titer, high purity and high protein concentration has been successfully prepared .PARTII PROTECTION EFFECT OF SUCRALFATE ON ACTIVITY OF VACA-IGY IN VITRO Objective To observe the protection effect of sucralfate on activity of VacA-IgY and establish base for the preparation of IgY with tolerance against gastric acid and protease.Methods Sucralfate and pepsin of various concentration was added into VacA-IgY under different pH; Protection effect of sucralfate on activity of VacA-IgY against acid and pepsin invasion was detected by ELISA. VacA-IgY containing 30%⁵0% sucralfate were enforced with freeze thawing intervention seven times; VacA-IgY (contailning 30% sucralfate) was layed in ambient temperature for 1d～4w .Relative antibody activity of VacA-IgY(AT/AC,%)was determined by ELISA.Results Under pH 1.5,2.0,3.0, activity of VacA-IgY containing 30%⁵0% sucralfate was higher than that without sucralfate; Under pH 1.5, 60% sucralfate could keep VacA-IgY activity at 68.7%. Under pH2.0,3.0, 50% and 40% sucralfate could completely protect its activity. Under pH1.5～3.0 and normal pepsin concentration, sucralfate above 30% could effectively protect its activity, improve its tolerance against freeze thawing and keep its activity above 80% after layed in ambient temperature for one month.Conclusion 30% or above sucralfate can improve the tolerance of VacA-IgY against low pH , pepsin and freeze thawing intervention which promising sucralfate a satisfactory protectant for IgY. PART III PROTECION EFFECT OF SUCRALFATE ON ACTIVITY OF VACA-IGY AGAINST HELICOBACTER PYLORI INFECTION IN VIVO OF BALB/C MOUSEObjective To observe the protection effect of sucralfate on activity of VacA-IgY against Helicobacter pylori infection in vivo of BALB/c mice and build base for the preparation of IgY with tolerance against gastric atmosphere.Methods The experimental animal model of BALB/c mice was constructed by gavage administration with H.pylori international standard strain NCTC11637.Specific immunoglobulin G against H.pylori was determined with westernblot kit for H.pylori infection. Mice with antibody-positive serum was grouped into VacA-IgY at high,middle,low dose group; sucralfate-VacA-IgY at high,middle,low dose group;sucralfate group and the infection control group. Inplantation of H.pylori and the chronic inflammation in gastric mucosa was scored according to the visual analogue scale graph of Updated Sydney System.Data analysis was launched with SPSS10.0 software.Results No therapeutic group could lighten the implantation density of H.pylori. VacA-IgY at high dose,sucralfate-VacA-IgY at high,middle,low dose relieved the infiltration of the neutrophils and chronic inflammatory cells.Both VacA-IgY at high dose(4mg/d ,7d) and sucralfate-VacA-IgY at high(4mg/d ,7d),middle(2mg/d ,7d),low dose(0.5mg/d ,7d) had a therapeutic effect on the chronic gastritis.Conclusion In vivo of BALB/c mice, sucralfate can protect the activity of VacA-IgY against H.pylori infection. While the therapeutic dose lowering from 4mg to 0.5mg, ,VacA-IgY still plays a protective effect on the gastric mucosa.