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目的:制备重组荞麦胰蛋白酶抑制剂(rBTI)的多克隆抗体。方法:通过大肠杆菌原核表达系统制备高纯度的rB-TI蛋白,免疫大白鼠,制备多克隆抗体。用Western blot、间接ELISA及免疫细胞组织化学法检测该抗体的效价和特异性。结果:间接ELISA法检测所制备抗体的效价达1∶128000以上;Western blot显示该抗体能与BTI蛋白特异结合;免疫组织化学法检测结果显示rBTI主要存在于EC9706细胞的胞质和细胞核内。结论:所制备的大鼠抗rBTI的抗血清具有较高的免疫反应性和特异性,为rBTI诱导肿瘤细胞凋亡的机制研究及进一步揭示荞麦胰蛋白酶抑制剂的结构与功能的关系提供重要的基础。
Objective: To prepare polyclonal antibody of recombinant rat buckwheat trypsin inhibitor (rBTI). Methods: High purity rB-TI protein was prepared by E. coli prokaryotic expression system and immunized to prepare polyclonal antibody. The antibody titer and specificity were detected by Western blot, indirect ELISA and immunocytochemistry. Results: The titer of the prepared antibody was over 1:128000 by indirect ELISA. The result of Western blot showed that the antibody could specifically bind with BTI protein. Immunohistochemical results showed that rBTI mainly existed in the cytoplasm and nucleus of EC9706 cells. Conclusion: The anti-rBTI antiserum has high immunoreactivity and specificity. It is important to study the mechanism of rBTI-induced tumor cell apoptosis and further reveal the relationship between structure and function of buckwheat trypsin inhibitor basis.