Purification And Antigenicity Evaluation Of Hen Egg White Lysozyme

Hen egg lysozyme is a natural alkaline protease, which has an ability to inhibit the growth of bacteria, and is widely used in food products. However, as one of the major allergens in egg, about 35% patients are sensitized to it. Food hypersensitivity is a complex immunodeficiency syndrome, in which many kinds of antigens and antibodies react with each other, so to make the relation of them clearly can offer some important guidances for investigation on hypoallergenic or non-allergenic foods. Therefore, this project aims to purify the hen egg white lysozyme with high activity, and to evaluate its antigenicity, which can not only develop some approach to separate lysozyme in industry, but also may give some usefull parameters for future work on developing hypo-allergenic or non-allergenic egg productions.The investigation is composed of the purification of hen egg lysozyme, the characterization of lysozyme, the preparation of rabbit polyclonal antibody against lysozyme, and the evaluation of lysozyme's antigenicity.Hen egg lysozyme was purified by CM-Sepharose Fast Flow cation-exchange chromatography, and was detected by 5%-15% sodium dodecyl sulface PAGE. It was shown that the lysozyme purity was over 90 percent, and the recovery rate was about 31.1.Partial characterization of the lysozyme was investigated using Bacillus subtilis and its cell wall as research object. The result implied that hen egg lysozyme could inhibit the growth of Bacillus subtilis significantly. Moreover, lysozyme could degradate its cell wall conspicuously at 50℃, while not at 25℃. In conclusion, temperature had a great effect on the activity of lysozyme.The preparation of rabbit polyclonal antibody against lysozyme was performed by the method of multiple subcutaneous sites ingection injection, using Freund's adjuvant to enhance the immune response, then indirect ELISA was used to detect the variety of titre of the specific rabbit serum. Before the detection of titre, we optimized the operating condition of the indirect ELISA by chessboard titration firstly:the optimal concentration of coating antigen was 0.5μg/ml,and the optimal dilution of HRP-IgG was 1:10000. The result showed that the sensitivity against lysozyme for each animal was different, the rabbit B2 and C1 were most sensitive with their titres growing up to 1:150000, on the contrary, rabbit B1 and C2 were very slow.For the evaluation of lysozyme's antigenicity, indirect ELISA, indirect competitive ELISA and western blotting were used to develop the immunoreaction between the specific rabbit serum and several antigens including hen egg lysozyme, ovotransferrin, ovalbumin, ovomucoid and a-lactalbumin. As the experiment explained, there were nonspecific immunoreaction among the rabbit serum with ovotransferrin and ovomucoid, but no reaction with a-lactalbumin, it only arised specific reaction with lysozyme, which had high specific selection.