抗多囊蛋白1氨基端单克隆抗体的制备和鉴定

Preparation and characterization of monoclonal antibody against N-terminal domain of polycystin 1

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。

AIM: To prepare and identify monoclonal antibody (mAb) against N-termin al domain of polycystin 1. METHODS: Total RNA was extracte d from kidney tissue of a healthy man. Gene sequence encoding polycystin 1 N-te rminal domain was amplified by one-step RT-PCR. The target gene was inserted i nto prokaryotic expression vector pQE30 and transformed into competent cells E .coli M15. The fusion protein was expressed under IPTG induction and purified by affinity chromatography. The purified fusion protein was then used to immuniz e BALB/c mice. The splenocytes from immunized mice were fused with myeloma cells Sp2/0 by PEG 4000 mediator method and the hybridomas were selected in HAT mediu m. The hybridoma clones secreting mAb against polycystin 1 amino-terminal domai n were detected by ELISA and cloned by limiting dilution. The specificity of mAb against polycystin 1 N-terminal domain was verified by ELISA and Western blot. RESULTS: cDNA encoding polycystin 1 extracellular region was o btained. Fusion protein of polycystin 1 N-terminal domain were expressed in pQE 30 expression system. The relative molecular masses (M r) of the two fusion proteins were 19 800 and 18 900, respectively. One hybridoma cell 7B1 secreting specific mAb was obtained. Western blot analysis showed that the mAb reacted st rongly and specifically to pol-ycystin 1 N-terminal domain. CONCLUSION: polycystin 1 N-terminal fusion proteins have been expressed in E.col i M15. Anti-fusion protein mAb with antigen-binding activity has been prepar ed successfully.