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目的 :用杂交瘤技术制备抗多囊蛋白 1胞外区氨基端的单克隆抗体 (mAb) ,并对其特异性进行鉴定。方法 :以肾组织总RNA为模板 ,用RT PCR扩增多囊蛋白 1胞外区氨基端的编码基因PKD1cDNA。将该基因克隆到融合蛋白表达载体pQE3 0中 ,转染大肠杆菌M15。以异丙基硫代半乳糖苷 (IPTG)诱导表达多囊蛋白 1胞外区氨基端的组氨酸融合蛋白 (PC1 e2 ) ,用亲和层析法纯化。以纯化的融合蛋白作为抗原免疫BALB/c小鼠 ,取小鼠脾细胞与小鼠骨髓瘤细胞株Sp2 / 0进行细胞融合 ,间接ELISA筛选阳性克隆 ,有限稀释法进行单克隆化。mAb的特异性用间接ELISA和Westernblot鉴定。结果 :克隆到两个编码多囊蛋白 1氨基端的cDNA片段 (50 2bp和 471bp)。构建的表达质粒经酶切和DNA测序证实 ,为所需要的质粒。表达出相对分子质量 (Mr)分别为 1980 0和 1890 0的融合蛋白 ,经Westernblot鉴定 ,均为多囊蛋白 1的融合蛋白。用Mr 为 1890 0的融合蛋白免疫小鼠 ,得到杂交瘤细胞株 7B1。Westernblot分析表明 ,该细胞株分泌的mAb能特异地与多囊蛋白 1氨基端结合。结论 :本实验表达了PKD1多拷贝区所编码的PC1 e2 ,成功地制备了抗多囊蛋白 1胞外区N端的mAb。
OBJECTIVE: To prepare the monoclonal antibody (mAb) against the amino terminal of extracellular domain of polycystin 1 by hybridoma technique and identify its specificity. Methods: The total RNA of kidney was used as a template to amplify the PKD1 cDNA encoding the amino terminal region of extracellular domain of polycystin 1 by RT PCR. The gene was cloned into the fusion protein expression vector pQE30 and transfected into E. coli M15. The histidine fusion protein (PC1 e2), which expresses the amino terminal of extracellular domain of polycystin 1, was induced with isopropylthiogalactoside (IPTG) and purified by affinity chromatography. The purified fusion protein was used as antigen to immunize BALB / c mice. The mouse spleen cells were fused with the mouse myeloma cell line Sp2 / 0 and the positive clones were screened by indirect ELISA. The monoclonal antibodies were cloned by limiting dilution. The specificity of the mAb was assessed by indirect ELISA and Western blot. Results: Two cDNA fragments (50 bp and 471 bp) encoding the amino terminus of polycystin 1 were cloned. The constructed expression plasmid was confirmed by restriction enzyme digestion and DNA sequencing as the required plasmid. The fusion proteins with relative molecular mass (Mr) of 1980 0 and 1890 0 were expressed and identified by Western blot. Mice were immunized with a fusion protein of Mr 1890 0 to obtain hybridoma cell line 7B1. Western blot analysis showed that the mAb secreted by this cell line specifically binds to the amino terminus of polycystin 1. CONCLUSION: The present study expresses PC1 e2 encoded by multiple copies of PKD1 and successfully prepared a mAb against N-terminus of extracellular domain of PC1.