蚯蚓纤溶酶去糖基化和免疫反应性分析

 目的对去糖基化的蚯蚓纤溶酶进行免疫反应性分析。方法①将变性的蚯蚓纤溶酶全组分与免疫佐剂混合,免疫家兔制得抗血清,酶联免疫吸附剂测定(enzyme linked immunosorbent assay,ELISA)和Western-blotting检测抗血清的效价。②三氟甲基磺酸去除蚯蚓纤溶酶全组分及6.5#,7#单组分的糖链,SDS-PAGE和ConA-HRP亲和印迹检测糖链的去除情况。③ELISA和Western-blotting检测糖链去除前后蚯蚓纤溶酶免疫反应性是否有变化。结果去除糖链后蚯蚓纤溶酶全组分及6.5#,7#单组分的免疫反应性基本没有变化。结论糖链的存在与否对蚯蚓纤溶酶组分免疫反应性的影响不大,提示蚯蚓纤溶酶分子上的寡糖链可能不构成其抗原决定簇。

Deglycosylation of Earthworm Fibrinolytic Enzymes and Immunoreactivity Assay

OBJECTIVE To investigate the immunoreactivity of the deglycosylated EFEs.METHODS The antisera against earthworm fibrinolytic enzymes(EFEs) were developed by immunising rabbits with heat denatured EFEs.The oligosaccharides of EFEs and the component 6.5#,7# were removed by trifluoromethanesulfonate(TFMS) with the indication of SDS-PAGE and ConA-HRP affinity blotting.The methods of ELISA and Western-blotting were used to detect the differences of immunoreactivity between the samples and the deglycosylated ones.RESULTS There were no obvious changes of the immunoreactivity of EFEs and the component 6.5#,7# after being deglycosylated by TFMS.CONCLUSION The oligosaccharides of EFEs and the component 6.5#,7# show no influences on their immunoreactivities markedly.The oligosaccharides were not involved in the antigen determinants of EFEs.