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人PINK1抗血清的制备及其免疫活性物质在大鼠脑组织中的表达
发表时间：2010-04-15 浏览量：1565 下载量：837
| 全部作者： | 李继胜，李尧华，李昕，于顺，王年强，孙晓红，杨慧 |
| 作者单位： | 首都医科大学北京神经科学研究所，北京神经再生修复研究重点实验室，教育部神经变性病学重点实验室；首都医科大学宣武医院老年病研究所, 教育部神经变性病重点实验室 |
| 摘 要： | 目的：制备帕金森病相关蛋白PINK1抗血清并检测其在大鼠脑组织中的表达特征。方法：制备人PINK1基因重组蛋白，免疫新西兰大耳白兔获得抗PINK1抗血清；通过盐析法初步纯化、Western blot法鉴定抗血清的敏感性和特异性、免疫组化法检测PINK1在大鼠脑中的分布。结果：人PINK1基因重组蛋白以包涵体形式在大肠杆菌中高效表达；纯化蛋白制备兔抗血清经盐析纯化后，兔抗人PINK1抗血清特异性识别人PINK1基因重组蛋白及大鼠脑匀浆中40 ku和66 ku的蛋白质，与预期结果一致；免疫组化染色显示PINK1广泛分布在嗅球、皮层、海马、纹状体、小脑、黑质和红核。结论：制备了一种兔抗人PINK1多克隆抗血清；PINK1广泛分布于大鼠中枢神经系统；为以后深入研究PINK1表达的组织分布、细胞内定位及其生物学功能提供了有效的实验工具。 |
| 关 键 词： | 神经生物学；PINK1；抗血清；大鼠；重组蛋白 |
| Title： | Preparation of polyclonal antiserum against human PINK1 and its expression in rat brain |
| Author： | LI Jisheng, LI Yaohua, LI Xin, YU Shun, WANG Nianqiang, SUN Xiaohong, YANG Hui |
| Organization： | Beijing Institute for Neuroscience, Capital Medical University, Beijing Center for Neural Regeneration and Repair, Key Laboratory for Neurodegenerative Disease of Ministry of Education；Beijing Institute of Geriatrics, Key Laboratory for Neurodegenerative Diseases of Ministry of Education, Xuanwu Hospital of Capital University of Medical Sciences |
| Abstract： | Objective: To prepare Parkinson disease (PD) related protein PINK1 polyclonal antiserum and investigate its expression in rat brain. Methods: To obtain the antiserum against human PINK1, two New Zealand white rabbits were immunized with recombinant His-tag fusion human PINK1 protein. Purified by salt fractionation, the specificity and sensitivity of the antiserum were verified by Western blot method. The location and distribution were further determined by immunohistochemistry. Results: The human recombinant PINK1 gene was highly expressed as inclusions in prokaryotic expression system. After first salt fractionation, western blot analysis demonstrated that the antiserum detected a 40 ku band in recombinant human PINK1 protein and a 66 ku band in rat brain homogenates, which were anticipated. Immunohistochemical staining on normal adult rat brain sections showed that the PINK1-immunoreactive substance presented in rat brain bulbus olfactorius, cortex, hippocampus, striatum, cerebellum, substantia nigra and red nucleus. Conclusion: the experiment has prepared a polyclonal antiserum against human PINK1 and the PINK1-immunoreactive substance was widely distributed in rat central nervous system. The antiserum provided a useful experimental tool to profoundly research the tissue expression profile, intercellular location and biological function of PINK1. |
| Key words： | neurobiology; PINK1; polyclonal antiserum; rat; recombinant protein |
| 发表期数： | 2010年4月第7期 |
| 引用格式： | 李继胜，李尧华，李昕，等. 人PINK1抗血清的制备及其免疫活性物质在大鼠脑组织中的表达[J]. 中国科技论文在线精品论文，2010，3（7）：686-691. |
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