重组荞麦rBTI的多克隆抗体制备及鉴定

Preparation and identification of the rat antibody against recombinant buckwheat trypsin inhibitor

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。

AIM：To prepare the rat antibody against the recombinant buckwheat trypsin inhibitor. METHODS：The recombinant buckwheat trypsin inhibitor was expressed in E.coli. BL21 （DE3） and used as an immunogen to immunize the rat. The titer and specificity of the anti-BTI antibody from the rat were analyzed by ELISA,Western blot and immunohistochemistry,respectively. RESULTS：ELISA detection indicated the titer of the antiserum was about 1∶128 000. Western blot analysis showed the antibody reacted specifically with rBTI. The immunohistochemistry analysis proved that rBTI was expressed in the cytoplast and nucleus of EC9706 cells. CONCLUSION：The rat antibody against rBTI has high titer and specificity,which is beneficial to further study on the molecular mechanisms in rBTI-induced apoptosis of tumor cells and also provide an important theory basis for further exploring the relationship between the structure and function of BTI.