人BLyS基因克隆、表达和抗人BLyS单克隆抗体的制备

Molecular Cloning, Expression of hBLyS and Preparation of Anti-hBLyS Monoclonal Antibody

【目的】研制抗人BLyS单克隆抗体 ,以进一步探讨人BLyS与自身免疫性疾病的关系。【方法】将人BLyS基因克隆到融合蛋白原核表达载体 pGEX 4T 1中 ,构建重组质粒 pGEX 4T 1/hBLyS ,用此重组质粒转化大肠杆菌BL2 1,加IPTG诱导大肠杆菌表达GST hBLyS融合蛋白 ,用此融合蛋白免疫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仪进一步鉴定抗体的特异性。【结果】重组质粒双酶切结果和DNA序列测定以及表达蛋白SDS PAGE电泳结果表明 ,pGEX 4T 1/hBLyS重组质粒可以正确表达GST hBLyS融合蛋白 ;ELISA、免疫印迹和流式细胞仪检测结果表明 ,1c6杂交瘤细胞株产生的单克隆抗体可以特异性结合人T淋巴细胞膜表面BLyS的膜外区 ,属于IgG2b亚类。【结论】所获得的单克隆抗体具有能够结合人T淋巴细胞上的BLyS特异性 。

To obtain monoclonal antibodies against human BLyS(B lymphocyte stimulator) The DNA fragment of hBLyS was cloned into pGEX 4T 1 plasmid, and transformed into E coli BL21 The expression of GST hBLyS fusion protein was induced by IPTG in E coli BL21 This protein was used as antigen to immunize BALB/c mice The splenocytes of immunized mice were fused with myeloma cells SP2/0 to produce hybridoma cell line which could secrete anti hBLyS antibodies The verification of antibodies specificity were characterized by ELISA, Western blot, and flow cytometry The cloned insert was identified by both sequencing analysis of recombinant pGEX 4T 1/hBLyS plasmid and double digestion of recombinant pGEX 4T 1/hBLyS plasmid with restriction enzymes EcoRⅠand SalⅠ The expression of recombinant GST hBLyS fusion protein was confirmed by SDS PAGE The monoclonal antibody generated from hybridoma 1c6 was obtained with specificity for extracellular domain of hBLyS on human peripheral blood CD3 + T cell activated by hIFN γ The monoclonal antibody of hybridoma 1c6 belongs to IgG2b [Conclusion] The monoclonal antibody against hBLyS with high activity and specificity have been established successfully It provides us a basis for further studies of relationship between hBLyS and autoimmune diseases