| Peanut is one of the eight kinds of sensitive foods and its allergy symptom is serious, the occurrence rate is high, It does not reduce with age causing of allergic patients and has attracted wide attentionfor peanut allergy. Ara h2 accounts for about 10% of the total protein content of peanutamong the 13 types of peanut allergy proteins that have been discovered,it is identified by the serum about 90% of peanut allergy patients, which is one of the major allergens in peanut. Therefore, it is an urgent task to obtain the allergen protein, which is of great value in research. The work of this paper is that the immunoaffinity chromatography column was prepared by preparing the corresponding antibody and the home-made column was applied to the purification of Ara h2.The research includes purification of Ara h2 from natural peanut and rabbit polyclonal antibody against Ara h2 and preparation of antibody and antigen affinity chromatography column and purification of Ara h2 in different peanut materials. The main methods, results and conclusions of the study are as follows:1.Ara h2 was purified by column chromatography from natural peanut and identified by SDS-PAGE. By the high purity Ara h2, the rabbit polyclonal antibody against Ara h2 was obtained by multi-point subcutaneous injection, and the titer and specificity were detected by ELISA and Western blotting.An Affinity chromatography column was prepared for purification ofpolyclonal antibody againstAra h2 from rabbit serum againstAra h2 with Hydrogen bromide activated sepharose-4B, and SDS-PAGE and blot werstern were used for identification.The result showed that the purity of Ara h2 was above 90%. The coupling ratio was 95.1%, and the antibody / column ratio was 17.1mg/g. The SDS-PAGE results showed that the anti peanut allergen Ara h2 specific antibody was highly purified by column separation; Western blotting showed that the antibody specificity was very high, detection of ELISA titer was 1:6400, compared with the serum sample purification factor was at least 100.2.Specific rabbit antibody was raised against Ara h2 from Japanses rabbits immunized with a routine method. The antigenicity of Ara h2 was evaluated by indirect ELISAand western blotting.Another immuno affinity chromatography was prepared by pAb against Ara h2 coupling with Hydrogen bromide activated sepharose-4B column for isolation of Ara h2.,and The coupling ratio, column capacity, recovery rate and use times were characterizedThe results showed that the titre of the antibody have high specificity.The coupling ratio is 90.1%, the medium density is 18.1mg/g, and the capacity of the self-made column is 0.55-0.60 mg, the recovery rate is between 92.7%-97.8%, and the column capacity is still 87.5% after using 11 times.3.Raw crude peanut extract were boiled 15, 30, 45, 60, 90 min,and two kinds of samples(raw and boiled crude peanut extract) were in vitro simulated gastrointestinal digestion for 15 min, 30 min, 45 min, 60 min, 90 min and 5min, 15 min, 30 min, 60 min, 90 min in vitro simulated gastrointestinal digestion. With Gly-HCl(pH2.7) as the elution solution, Ara h2 was isolated by home-made columnfrom raw peanut, boiled peanuts and its stomach digestion, intestinal digestion products, and the product was identified by SDS-PAGE and werstern blot, The ELISA was used to measuredthe extraction rate of raw peanut, boiled peanuts and its stomach digestion, intestinal digestion products.The results showed that boiled treatment resulted in a complex aggregation of peanut crude protein, the difference was not significant, especially the Ara h2 protein bands;That had different levels of digestion in vitro gastric digestion for raw and boiled peanuts crude extracts, and Ara h2 had a strong anti gastric digestion;There were very significant digestion forraw and boiled peanuts crude extracts, and Ara h2 digestion was significant in vitro gastrointestinal students.SDS-PAGE results showed that the raw peanuts crude extracts purity was 87.4%, boiled peanuts crude extract purity was 79.5%, Thepurity of raw and boiledcrude extracts were 72.7% and 70.7%. and two polymer of boiled peanuts crude extract was separated.The extraction rates were 94.4%、88.7%、73.4%、79.2% by ELISA, respectively.It was showed that the home-made IAC had good separation ability. |
