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为深入研究血管紧张素原 (angiotensinogen ,AGT)的表达和组织分布特征 ,我们以原核表达的AGT蛋白C末端片段为免疫原免疫家兔 ,制备了针对AGT的多克隆抗血清 ,并对其进行了ELISA、Western印迹分析及免疫组化鉴定。结果发现 ,经多次免疫后获得的多克隆抗血清不仅效价高 (1∶2 5 60 0 ) ,而且能特异性地针对组织内源性及原核表达的AGT蛋白 ,同时在人和大鼠组织AGT之间会发生交叉反应。以此抗血清进行免疫组化染色 ,观察到人胰腺的胰岛细胞及肝内胆管上皮细胞胞浆均有AGT蛋白表达。以上结果提示 ,利用大肠杆菌融合表达的AGT蛋白可有效刺激家兔产生AGT抗体。本工作为进一步研究AGT乃至局部RAS的生理、病理意义提供了重要的实验工具
In order to further study the expression and distribution of angiotensinogen (AGT), we used the C-terminal fragment of prokaryotic expressed AGT protein as immunogen to immunize rabbits and prepared polyclonal antiserum against AGT. ELISA, Western blot analysis and immunohistochemistry identification. The results showed that the polyclonal antiserum obtained after multiple immunizations not only had high titer (1: 2.560), but also could specifically target endogenous and prokaryotic AGT proteins in tissues, while in human and rat Cross-reactivity occurs between the tissue AGTs. Using this antiserum for immunohistochemical staining, the expression of AGT protein was observed in the islet cells of human pancreas and in the cytoplasm of intrahepatic biliary epithelial cells. The above results suggest that the use of AGT protein expressed in E. coli fusion can effectively stimulate rabbits to produce AGT antibodies. This work provides an important experimental tool for further studying the physiological and pathological significance of AGT and even local RAS