多囊蛋白-1 LRR-WSC区单克隆抗体的制备及其在免疫组织化学诊断中的应用

Preparation of monoclonal antibody against LRR-WSC domain of polycystin-1 and distribution of polycystin-1 in kidney tissues and kidney cell lines

目的 用杂交瘤技术制备抗多囊蛋白-1 LRR-WSC区单克隆抗体,检测多囊蛋白-1在肾组织和肾细胞株中的分布和定位。方法 用多囊蛋白-1 LRR-WSC区融合蛋白PCI-e免疫BALB/c小鼠,将其脾细胞与骨髓瘤细胞株SP2/0进行细胞融合,间接酶联免疫吸附试验(ELISA)筛选出阳性克隆,有限稀释法将杂交瘤细胞株单克隆化,间接ELISA法和免疫印迹法(WB)鉴定抗体的特异性。用制备的抗多囊蛋白-1 LRR-WSC区单克隆抗体,免疫组织化学和免疫细胞化学法检测多囊蛋白-1在不同肾组织和肾细胞株中的分布。结果 细胞融合后经筛选和克隆得到的杂交瘤细胞株经WB分析表明,该细胞株分泌的单克隆抗体能特异地与多囊蛋白-1 LRR-WSC区结合。免疫组织化学显示,多囊蛋白-1主要分布于正常肾组织的远端肾小管和集合管,在胎肾囊肿组织中表达于近端肾小管,在人常染色体显性多囊肾病(ADPKD)肾囊肿组织中,表达于囊肿衬里上皮细胞,同时在ADPKD肾囊肿衬里上皮细胞系和猪近端肾小管细胞株(LLC-PK1)中也发现了多囊蛋白-1的表达。结论 本实验成功制备了抗多囊蛋白-1 LRR-WSC区的单克隆抗体,该抗体对深入研究ADPKD的发病机制具有重要意义。多囊蛋白-1在肾组织中的表达模式对肾小管的形态发生、维持肾小管结构的完整性非常重要。

Objective To prepare and identify monoclonal antibody against LRR-WSC domain of polycystin-1 and to investigate the distribution of polycystin-1 in kidney tissues and kidney cell lines. Methods BALB/c mice were immunized with fusion protein PC1-e of polycystin-1 LRR-WSC domain. The splenocytes were fused with myeloma cells by PEG 4000 and the hybridomas were selected in HAT medium. The hybridoma clones secreting antibodies against polycystin-1 LRR-WSC domain were detected by enzyme-linked immunosorbent assay ( ELISA) and cloned by limiting dilution. The specificity of anti-polycystin-1 LRR-WSC domain monoclonal antibody from hybridoma was verified by ELISA and Western blot. The distribution of polycystin-1 in tissues and cells was detected by immunohistochemical method. Results One cell line of hybridoma secreting monoclonal antibody against polycystin-1 was established. Western blot analysis showed that the monoclonal antibody reacted strongly and specifically to polycystin-1 LRR-WSC domain. Distribution of polycystin-1 in fetal kidney was localized in tubular epithelium. In normal adult kidney tissues, our study showed that polycystin-1 was mainly expressed in the medullary collecting ducts and distal convoluted tubules. Positive staining was also found in the majority of cyst-lining epithelial ceEs of cystic tissue from autosomal dominant polycystic kidney disease ( ADPKD) patients. Expressions of polycystin-1 were found in either ADPKD cyst-lining epithelia cell line and LLC-PK1, clearly plasma membrane and intracytoplasmic staining of polycystin-1 were observed. Conclusion Specific monoclonal antibody against polycystin-1 LRR-WSC domain were obtained. The antibody is important to researching the mechanism of ADPKD. The distribution of polycystin-1 in kidney tissues and cells show that polycystin-1 was important in tubular elongation and the maintenance of tubular architecture.