| Peanut allergy is considered to be a serious food safety issues,which belongs to a immediate hypersensitivity. There are a lot of people suffer form peanut allergices, and it is life threatening.So far,foreign scholars have done a number of related research in the treament of peanut allergy. However, no good treatment is available. Avoiding intake of peanut is still the best approach to fight against peanut allergy.Since chitosan has good histocompatibility, a few toxic side effects, no harm to human. and chitosan nanoparticles can be prepared as a sustained-release system, resulting in active ingredients release easily and resist to be digested. The recombinant Ara h 2-chitosan nanoparticles were prepared and its antiallergic effect was evaluated in the mice model of peanut allergy.The main contents include prokaryotic expression and purification of the major peanut allergen Ara h 2, preparation of recombinant Ara h 2 chitosan nanoparticles, establishment of a mouse model of peanut allergy, and antiallergic effect in the animal experiment of the recombinant Ara h 2 chitosan nanoparticles.The main research results and conclusions are as follows.1 The expression vector pET-32a-Ara h 2 was constructed using synthetic Ara h 2 gene as a target, and it was transformed into E.coli origami.The positive recombinant was identified by restriction enzyme and DNA sequence analysis.The results showed that the construction was successful. The SDS-PAGE analysis of expression products showed that the expression protein (35kD) was the same size as the theoretical value when the cell was induced with 1.0mmol/IPTG. Moreover, the induced cell was collected and sonicated, followed by collecting the supernatant after centrafigation. Then, the SDS-PAGE analysis of the supernatant and pellet indicated that the expression protein is partially soluble.Finally, the recombinant Ara h 2 with His tag was purified by Ni2+ affinity chromatography, and the SDS-PAGE analysis of the elution peaks showed recombinant Ara h 2 protein with high purity was obtained.2 The recombinant Ara h 2-chitosan nanoparticles were prepared by ionic gelation method.The protein concentration was determined by BCA assay and the Association Efficiency and Loading Capacity of the recombinant Ara h 2-chitosan nanoparticles were also calculated. SDS-PAGE analysis of the nanoparticles showed that the recombinant Ara h 2 was successfully wrapped in chitosan, and the nanoparticles diameter was identified to be 230nm-670nm by scanning electron microscopy (SEM).Moreover, western-blotting analysis of the nanoparticles indicated that the recombinant Ara h 2-chitosan nanoparticles with immune activity was successfully preparaed.3 SDS-PAGE analysis of the extraction showed that successfully extracted crude peanut protein. Crude peanut protein(CPE), extracted by defatting with acetone, was used to sensitized C57BL/C mice by subcutaneous injection to all groups expect the Naive group.Then the sensitized mice was treated with recombinant Ara h 2 chitosan nanoparticles by gavage, and recombinant Ara h 2 and chitosan nanoparticles were used as control respectively. The levels of the specific serum IgE,IgG2a,plasma histamine, and IL-4,IL-5,IL13 and INF-r sereted from spleen cell were determined by enzyme-linked immunosorbent assay.It was shown that the levels of specific serum IgE and plasma histamine significantly reduced after treatment with the recombinant Ara h 2 chitosan nanoparticle compared to other groups. CPE could upregulate IFN-r, down-regulate IL-4,IL5and IL-13 derived from cultured spleen cells from mice receiving ecombinant Ara h 2 chitosan nanoparticle treatment.These indicates that the treatment of recombinant Ara h 2 chitosan nanoparticle can reverse the balance of Thl/Th2, resulting in Thl based immune response. The antiallergic effects of recombinant Ara h 2 chitosan nanoparticle is obvious. |
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