文档介绍：Clusterin抗原的表达、抗体制备及其初步鉴定
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作者:张迎春, 刘莹, 杨金菊, 柳晓兰, 迟俊, 高建恩, 唐晓波, 朱大岭, 孙启鸿
【摘要】目的: 制备Clusterin(CLU)多克隆和单克隆抗体(mAb), 并进行特性鉴定。方法: 以成人肝cDNA 表达文库为模板, 构建重组表达质粒pGEX4T1CLU和PET32aCLU。GSTCLU 融合蛋白在大肠杆菌中表达, 被作为免疫原制备兔多抗和鼠mAb。采用ELISA法和 Western blot鉴定抗CLU抗血清在重组蛋白和天然蛋白中的特异性。采用Western blot, 间接免疫荧光, 免疫组化鉴定mAb的特异性。结果: GSTCLU 融合蛋白在相对分子质量(Mr)约54000处呈现明显表达条带。Western blot鉴定表明, 制备的抗CLU兔多克隆抗体可特异地识别成人肝总蛋白中Mr约52000和58000的CLU蛋白。获得9株可稳定分泌抗CLU的杂交瘤细胞株可识别重组人CLU 蛋白, 其中有2株可特异性结合HepG2细胞质中的蛋白, 4株可特异性结合成人肝脏组织肝细胞质中的蛋白。结论: 成功地制备出兔抗人CLU抗血清和9株抗CLU的mAb, 为进一步研究CLU在肿瘤中的功能奠定了实验基础。
【关键词】 Clusterin 原核表达多克隆抗体单克隆抗体
[Abstract] AIM: To prepare rabbit polyclonal antibodies (pAb) and mouse monoclonal antibodies (mAbs) against human cl
usterin(CLU) and characterize these antibodies’ properties. METHODS: CLU fragment was amplified from human liver cDNA library, and binant expression vectors pGEX4T1CLU and PET32aCLU were constructed. GSTCLU fusion protein was expressed in and then used as the immunogen. Properties of antiserum against human CLU were identified by ELISA, Western blot, and the mAbs against human CLU was characterized by Western blot, indirect immunofluorescent staining and immunohistochemistry staining. RESULTS: The GSTCLU fusion protein was highly expressed with a molecular weight of 54000. Western blot analysis proved that the rabbit pAb could specifically recognize 52000 and 58000 proteins in human liver total protein. All of the nine established mAbs recognized binant human CLU protein, two of which specifically bound to proteins in the cytoplasm of HepG2 cells and four of which specifically bound to proteins in the cytoplasm of adult liver tissue. CONCLUSION: pAb and mAbs against human CLU were essfully prepared, which will provide efficient tools for functional studies of CLU expressed in human tumors.
[Keywords]clusterin; prokaryotic expression; polyclonal antibody; monoclonal antibody
Clusterin(CLU)是一种普遍存在的异源二聚体硫酸化糖蛋白, 其基因