抗CLCN5单克隆抗体的制备及鉴定

Preparation and identification of the monoclonal antibodies against chloride channel 5

目的:制备抗氯离子通道蛋白5(CLCN5)单克隆抗体(mAb)并对其进行鉴定。方法:用人CLCN5为抗原免疫BLAB/c小鼠,用常规方法进行细胞融合,经筛选及克隆化建立可稳定分泌抗CLCN5mAb的杂交瘤细胞株;用ELISA及Westernblot进行抗体的鉴定。结果:筛选到4株可稳定分泌抗CLCN5mAb的细胞株;Westernblot显示,在4株抗体中有1株在相对分子量为83kDa处出现1条特异性条带,说明该单克隆抗体可与HSG胞浆蛋白中的CLCN5蛋白特异性地结合。结论:本实验成功获得1株可特异性识别CLCN5的单克隆抗体细胞株,可用于肾结石、X-连锁综合征的进一步研究、临床诊断及相关试剂盒等的开发研制。

Objective： To prepare and identify monoclonal antibody against chloride channel 5 （CLCN5） . Methods：We prepared hybridoma cell lines secreting CLCN5 mAb from immunized BALB/c mice with antigen CLCN5, then by cell fusion, screened with indirect ELISA and cloned with limited dilution methods. The properties of antiserum against CLCN5 were characterized by ELISA and Western blot. Results：Four hybridoma cell lines secreting CLCN5 mAb had been developed, and the ELISA titre of the mAbs was respectively detected. According to the Western blot,we could see that the CLCN5 protein was highly expressed with a molecular weight of 83 kDa, it meaned that the monoclonal antibodies specially recognized a protein band expressed in the human submandibular gland （HSG） cell line. Conclusion：We gets a CLCN5 mAb which can be specially recognized by CLCN5. The successful generation monoclonal antibodies protein will provide efficient affinity reagents for the further functional studies of clcn5 gene expressed in human body and it＇s relation with kidney stone dent＇s disease and X-linked disorder.