一株鼠抗人DC-SIGN单克隆抗体的研制及DC-SIGN分子的表达分析

Preparation of a mouse anti-human DC-SIGN monoclonal antibody and identification on the expression of the DC-SIGN molecules

研制鼠抗人DC-SIGN mAb,利用所获得的mAb对DC-SIGN的表达谱进行分析。以人DC-SIGN的基因转染细胞L929/DC-SIGN为免疫原免疫BALB/c小鼠;采用B淋巴细胞融合技术,将免疫小鼠脾脏细胞与SP2/0融合;经免疫荧光标记对杂交瘤进行反复筛选和多次的克隆化培养;利用Western blot检测抗体对DC-SIGN分子的特异性识别;采用快速定性试纸法及竞争抑制结合实验分析了该mAb的亚型及抗原识别位点;利用免疫荧光法对其表达谱进行了分析。结果获得了1株持续、稳定分泌鼠抗人DC-SIGN mAb的杂交瘤细胞株;该mAb能特异性识别人DC-SIGN分子;该抗体的亚型为IgG1,其与商品化抗体E021819识别不同的抗原位点;DC-SIGN分子特异性高表达于Mo-DC,是Mo-DC的标记分子。

To prepare the mouse anti human DC SIGN monoclonal antibody and analyze the expression of the DC-SIGN molecules, BALB/c mice were immunized with L929/DC-SIGN transgenic cells, and the splenocytes of the mice were fused with SP2/0. The hybridoma cells were screened by immunofluorescenee staining. The specific recognition of the mAb with DCSIGN molecules was analyzed by Western blotting and Fast-strip analysis was performed to identify the sub-class of this mAb. The epitope recognized by this mAb was detected by competitive inhibition assay,and the expression of the DC-SIGN molecules was analyzed by immunofluorescence staining. In the end, one hybridoma cell line continuously and steadily secreting specific anti-human DC-SIGN mAb was obtained. This mAb specifically recognized DC SIGN molecules. The mAb was IgG1 subclass and it could recognize specifically different antigenic epitopes just as the same as the commercial mAb E021819. DC-SIGN molecule primarilv expresses on Mo-DC and serves as the marker of Mo-DC.