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

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 blotting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40000和66000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、皮质、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地实验工具。

Preparation of Polyclonal Antiserum against Human PINK1 and its Expression in Rat Brain

Objective To investigate expression of Parkinson disease(PD) related protein PINK1 in rat brain. Methods To obtain the antiserum against human PINK1, two New Zealand white rabbits(2.5 kg) were immunized with recombinant His tag fusion human PINK1 protein; the antiserum was purified by salt fractionation, the specificity and sensitivity of the antiserum were verified by Western blotting. The location and distribution of the protein 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 blotting analysis demonstrated that the antiserum detected a 40 000 band in recombinant human PINK1 protein, a 40 000 band and a 66 000 band in rat brain homogenates which is anticipated. Immunohistochemical staining on normal adult rat brain sections showed that the PINK1 immunoreactive substance presents in rat brain bulbus olfactorius, cortex, hippocampus, striatum, cerebellum, substantia nigra and red nucleus. Conclusion A polyclonal rabbit antiserum against human PINK1 was prepared and the immunoreactive PINK1 substance was widely distributed in rat CNS. The antiserum provided a useful experimental tool for profound studies on the tissue expression profile, intercellular location and biological function of PINK1.