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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pGEX-4T-1-CLU和PET-32a-CLU。GST-CLU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West-ern blot,间接免疫荧光,免疫组化鉴定mAb的特异性。结果:GST-CLU融合蛋白在相对分子质量(Mr)约54 000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52 000和58 000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。
OBJECTIVE: To prepare Clusterin (CLU) polyclonal and monoclonal antibodies (mAbs) and characterize them. Methods: The recombinant expression plasmids pGEX-4T-1-CLU and PET-32a-CLU were constructed with adult liver cDNA expression library as a template. GST-CLU fusion protein was expressed in E. coli and used as immunogen to prepare rabbit polyclonal and murine mAbs. The specificity of anti-CLU antiserum in recombinant and natural proteins was identified by ELISA and Western blot. West-ern blot, indirect immunofluorescence and immunohistochemistry were used to identify the specificity of mAb. Results: The GST-CLU fusion protein showed a significant expression band at about 54 000 relative molecular mass (Mr). The results of Western blot showed that the polyclonal antibody against CLU rabbit could specifically identify CLU protein of Mr about 52 000 and 58 000 in total human liver protein. Nine hybridoma cell lines secreting anti-CLU were obtained, which could recognize recombinant human CLU protein. Two of them could specifically bind to the protein in the cytoplasm of HepG2 and four of them could specifically bind to proteins in the liver cytoplasm of adult liver. CONCLUSION: The rabbit anti-human CLU antiserum and 9 anti-CLU mAbs were successfully prepared, which laid the experimental foundation for further study on the function of CLU in tumors.