| Glycinin （11S） and β-conglycinin （7S）, antigenic glycoproteins found in soybeans, arethe major cause of allergic reaction, diarrhea and even death in livestock and poultry（especially in young animals）, resulting in huge economic losses. The aim of thisexperiment was to study the allergenicity and immunogenicity of11S and7S in mice andpiglets, exploring a new method of the early diagnosis and prevention, and its mechanismof soybean allergic diseases.Purification and injection preparation of soybean antigen protein.11S and7S werepurified with the Sepharose-6B gel and obtained by dislysis and cryodesiccation. Purified11S and7S were detected and analyzed by SDS-polyacrylamide gel electrophoresis. Theoptical density method was used to analyse the electrophoresis images, and its purity wascalculated. Different types of adjuvant（Freund’s adjuvant, white oil, Aluminum hydroxidecolloid, PBS and Propolis）and soybean antigen proteins were sufficiently emulsionized,and the sterility and safety were detected.Study on allergenicity of the soybean antigen protein in mice and the establishment ofenzyme-linked immunosorbent assay.510Kunming mice were randomly divided into thecontrol group, Freund’s adjuvant treatment group I （7S） and treatment group II （11S）, whiteoil adjuvant treatment group I （7S） and treatment group II （11S）, aluminum hydroxideadjuvant treatment group I （7S） and treatment group II （11S）, PBS adjuvant treatmentgroup I （7S） and treatment group II （11S）, propolis adjuvant treatment group I （7S） andtreatment group II （11S）. The mice of the control group were injected with physiologicalsaline and the treatment groups with different types of soybean protein antigen injection.Each treatment group was randomly divided into5groups of10mice each. The treatmentgroups were respectively injected soybean antigen protein injections with500μg·Kg-1,1000μg·Kg-1,2000μg·Kg-1,3000μg·Kg-1and4000μg·Kg-1（n=10）, matched by weight.At first immunization step, the emulsionized soybean antigen protein injections wereinjected by subcutaneous in the neck back of mice. The corresponding antigen components（Freund’s complete adjuvant antigen protein injection was replaced with Freundincomplete adjuvant antigen protein injection） were injected a second time after10d and athird time after20d. After10days of the third injection, blood samples were collected andfor determining the antibody titer with agarose gel diffusion tests. The ci-ELISA fordetecting soybean antigen protein antibody was established and the immunizing dose,times and immune effect were determined. Study on allergenicity and immunogenicity of soybean antigen protein in piglets.70piglets （18days old） were randomly divided into the control group,7S and11S sensitizedgroup,7S and11S immunization group,7S and11S treatment group with10piglets ineach group. The immunization groups and treatment groups were injected with7S or11Sproteins with the dose of1mg·Kg-1on18and28days old. The sensitized group andtreatment group were fed the basal diet added to4%7S or11S protein respectively on28-34and39-41days old. Agarose gel diffusion test was used to detect antibody titer on28and38days old. The skin sensitivity test of piglets was conducted on46days old. Theexperiment was conducted for50days. ELISA was adopted to detect the contents of serumIgG and IgG subclasses in piglets on28,38,48,58and68days old, exploring the changeof serum IgG and IgG subclass contents of piglets after sensitized and immunized withsoybean protein antigens.The results were as follows:1.11S and7S proteins were purified by agarose gel chromatography and the purity of11S and7S was90.6%and90.0%, respectively. There had no bacterium growth found inmedium after sterility test. Muscle was complete and had no hyperemia in rabbit legmuscles by muscle stimulation test. The results indicated that the soybean antigen proteinshad no irritant reactions by intramuscular administration.2. The antibody titers in the serum of immunized mice were1:16. The optimal coatingconcentration of11S protein and serum dilution times were5.0μg·mL-1and1:800,respectively. The optimal coating concentration of7S protein and serum dilution timeswere2.5μg·mL-1and1:1600, respectively. Both intra-and inter-assay repeatability ofELISA were less than10%. The optimal immune times and dose of11S and7S proteinwere2and1000μg·Kg-1, respectively. The optimal immune adjuvant of7S and11S werealuminum hydroxide and white oil, respectively.3. The serum antibody titers of piglets were decreased on28days old, and increased on38days old and rose above1:16, which showed that there was immune response in pigletsbecause of7S and11S. The symptom of mild diarrhea was observed in piglets on39-41days old after sensitized. The skin sensitivity test showed skin reactions of injection site inthe groups sensitized by7S or11S were obviously observed and showed above the "+"class, which indicated that the piglet skin erythema reaction showed different degrees ofpositive reaction. The allergic reactions were proved by soybean antigen proteins in pigletson46days old according to erythematic diameter of skin test sensitivity. Compared with thesensitized group, the injection point reaction was decreased in11S or7S trea group, indicating that advanced immunity could reduce allergic reactions.4. The levels of serum IgG1, IgG2and IgG4were significantly increased in piglets afterallergy, but there had no significant difference in total IgG of piglets by the sensitization of11S. The level of IgG4was significantly increased in the whole immune and sensitizationprocess, indicating that it was the major player in allergic reactions. The levels of IgG andIgG subclasses were significantly reduced after sensitized, which declined allergicreactions and improved immunity protection.The results indicated that soybean antigen proteins were highly purified. Mice andpiglets were sensitized and immunized by using the purified soybean antigen proteins, thenthe serum antibody titers of mice were measured by indirect ELISA. The optimal immunedose and times of7S and11S were1mg·Kg-1and2times, respectively. The optimalimmune adjuvants were aluminum hydroxide and white oil, respectively. The IgG contentwas not increased in the sensitized piglets immunized by11S, which showed to play a rolein immune protection. The level of IgG4was significantly increased after allergy,indicating that is the main participant in allergic reactions. |
