牛朊蛋白理化性质分析及其多克隆抗体的制备

Physico-Chemical Property and Polyclonal Antibody Production of Bovine Prion Protein

目的:研究酸诱导牛正常细胞朊蛋白(BoPrPC)变性过程中间体的结构,同时制备其多克隆抗体。方法:表达纯化蛋白质后通过荧光光谱法和圆二色法分析酸性条件下BoPrPC二级结构变化。同时制备多克隆抗体,通过ELISA测定多克隆抗体效价,Western blot测定其特异性。结果:酸诱导BoPrPC去折叠并在pH 4.0条件下形成去折叠中间体,易于形成淀粉样纤维聚集体。制备的多克隆抗体效价可达到1∶6 400,能特异性结合朊蛋白。结论:折叠中间体二级结构类似致病BoPrPSc能形成淀粉样纤维聚集体,成功地制备了效价及特异性良好的兔抗BoPrPC多克隆抗体,为研究阻断致病朊蛋白形成淀粉纤维聚集体防治神经细胞损伤以及临床特异性检测奠定基础。

Objective： To study the intermediate structure of denatured bovine prion protein（BoPrPC） induced by acid,and to prepare and identify its polyclonal antibody.Methods： The recombinant bovine mature prion protein（BoPrPC） has been expressed and purified.Fluorescence spectroscopy and circular dichroism were used to study the structure change of BoPrPC induced by acid.BoPrPC was used to immune the rabbit for preparing polyclonal antibody.The titer and specificity of the rabbit＇s antiserum was measured by ELISA and Western blotting.Results： BoPrPC was unfolded induced by acid,and the intermediate was at about pH 4.0 and prone to form amyloid.The polyclonal antibody was also successfully prepared after immunizing the rabbit.The titer of the antiserum measured by ELISA could achieve 1∶6 400.The specificity of antibody was proved by Western blotting analysis.Conclusion： The structure of unfolding intermediate was similar to pathogenic BoPrPSc and would form amyloid aggregates.The BoPrPC L polycl...更多onal antibody with high titer and specificity can satisfy the request of Western blot and immunohistochemistry experiment,and laid a foundation to study the characters and function of BoPrPCL.