人IL17-RD胞外区真核表达及其单克隆抗体的制备

Eukaryotic expression of human IL17-RD-ECD and generation of its monoclonal antibody

摘要:

为了进一步研究白介素17受体D (IL-17RD) 在IL-17信号的调节作用，探索是否可以通过单克隆抗体阻断IL-17RD介导的IL-17信号通路而缓解自身免疫疾病，利用昆虫表达载体从Sf9细胞中表达纯化人IL-17RD-ECD蛋白，免疫Balb/C小鼠30 d，取小鼠脾脏细胞并与小鼠骨髓瘤细胞SP2/0进行融合，应用有限稀释法进行筛选，经过克隆化后筛选到一株能稳定分泌抗IL-17RD-ECD的杂交瘤细胞株1F8。经过初步鉴定，该细胞株分泌的抗体类型为IgG1+kappa类，经过Western blot

Abstract:

IL-17 Receptor D (IL-17 RD) is a cytokine receptor that mediates IL-17 signaling and plays an important role in responding to the invasion of extracellular pathogens and many inflammatory and autoimmune diseases such as rheumatoid arthritis. In this study we report the generation of a mouse monoclonal antibody against human IL-17 RD. The recombinant human IL-17RD extracellular domain (hIL-17RD-ECD) was produced in the baculovirus expression system and purified from culture medium of sf9 insect cells. The purified protein was used as a T-dependent antigen to immune Balb/C mice. B cells from the spleen of immunized mice were fused with murine cell SP2/0. Hybridoma cell lines were screened for the production of the monoclonal antibody against hIL-17-RD-ECD using ELISA. A hybridoma cell line 1F8 was found to have a high production of the antibody, which was further confirmed for the specificity by both western blot and ELISA analyses. The monoclonal antibody obtained from hybridoma 1F8 was characterized to be IgG1+Kappa subclass. This study provided a base for the further therapeutic application of the antibody on the autoimmune disease including rheumatoid arthritis.