【摘要】 天花粉蛋白核糖体灭活活性部位的定位方法：羟胺特异裂解天花粉蛋白唯一ＡｓｎＧｌｙ肽键．制备性凝胶电泳获ＨＡＴｆ１和ＨＡＴｆ２二片段．免疫印迹确定天花粉蛋白上不同表位并筛选抗体．兔网织红无细胞系统测定天花粉蛋白及片段对蛋白合成的抑制活性．结果：ＨＡＴｆ１和ＨＡＴｆ２纯度各达９６６％和８０５％．ＨＡＴｆ１保留完整天花粉蛋白的抑制活性．第１４号和第１６号抗天花粉蛋白单抗与二片段显示不同免疫反应性，并用于封闭试验．第１４号单抗能封闭天花粉蛋白及ＨＡＴｆ１活性，而第１６号单抗则否．结论：天花粉蛋白抑制蛋白质生物合成的活性部位位于ＨＡＴｆ１侧，近二部分交界．更多还原

【Abstract】 AIM: To localize the active site of ribosome inactivation of trichosanthin (Tri), a Chinese herb protein. METHODS: Hydroxylamine was used to specifically cleave the unique Asn Gly peptide bond of Tri. Preparative SDS polyacrylamide gel electrophoresis was applied to get 2 cleaved fragments, HATf1 and HATf2. Western blotting was used to determine the different epitopes of Tri and screen the antibodies. A cell free system, rabbit reticulocyte lysate, was introduced to quantitate the inhibitory activity of Tri and its fragments on protein biosynthesis. RESULTS: HATf1 and HATf2 were separated with the purity of 96 9 % and 80 5 % respectively. HATf1, like intact Tri, retained the inhibitory activity on protein biosynthesis. The mAb № 14 and № 16 against Tri showed different immunoreactivities with 2 fragments and were selected as representatives in further blocking tests. The mAb № 14 hindered the activities of Tri and HATf1, whereas the mAb № 16 did not. CONCLUSION: The active site of Tri responsible for inhibitory activity on protein biosynthesis was on the HATf1 side near the junction of two portions.更多还原