基因免疫制备抗人BLyS单克隆抗体及单抗特性分析

Preparation and characterization of anti-hBLyS monoclonal antibody by DNA immunization

目的 :基因免疫制备抗人BLyS单克隆抗体并分析其免疫学特性。方法 :将人BLyS基因克隆到真核表达载体pcDNA3,构建重组质粒pcDNA3/hBLyS ,用其免疫BALB/c鼠 ,取鼠脾细胞与骨髓瘤细胞融合 ,用ELISA法筛选阳性克隆 ,用免疫印迹和流式细胞技术进一步鉴定抗体的特异性 ,用双向免疫扩散鉴定单克隆抗体的类别。结果 :重组质粒双酶切结果和DNA序列测定结果表明 ,pcDNA3/hBLyS重组质粒含有目的基因 ;ELISA、免疫印迹、流式细胞仪和双向免疫扩散等实验结果表明 ,9c10杂交瘤细胞株产生的单克隆抗体可以特异性结合IFN -γ激活的人T淋巴细胞膜表面BLyS蛋白的膜外区 ,属于IgG1亚类。结论 :获得了能够特异性结合人T淋巴细胞上BLyS蛋白膜外区的单克隆抗体 。

AIM: To establish the monoclonal antibody against human B lymphocyte stimulator (hBLyS) by DNA immunization and analyse its characterization. METHODS: The 858 bp DNA fragment of hBLyS was cloned into pcDNA3 plasmids. The cloned insert was identified by both sequence analysis and double digestion of the recombinant plasmid with restriction enzymes Xho I and EcoR I . After the splenocytes from BALB/c mice immunized with the recombinant plasmid of pcDNA3/hBLyS were fused with myeloma cells SP2/0,the hybridoma which can produce monoclonal antibodies against hBLyS were obtained. The specificity of anti-BLyS monoclonal antibody from hybridoma was verified by ELISA, Western blot and flow cytometry. RESULTS: The recombinant mammalian cell expression vector of pcDNA3/hBLyS was constructed, the sequence of the insert gene was identified to be the sequence encoding hBLy S antigen. The culture supernatants of hybridoma 9c10 were tested to be the monoclonal antibody with specificity against hBLyS on human peripheral blood CD3 + T cell activated by hIFN-γ by ELISA, Western blot and flow cytometry. CONCLUSION: The monoclonal antibodies against hBLyS with high activity and specificity have been established successfully, and will be an useful tool in the studies of relationship between hBLyS and human autoimmunity diseases.