Preparation And Activity Study Of Novel Monoclonal Antibodies To Human Interleukin-17RA

There are five members(IL-17RA,IL-17RB,IL-17RC,IL-17RD,IL-17RD)in the IL-17 receptor family,in which IL-17RA was the first identified and well studieded.It belongs to I type single transmembrane receptor proteins with ubiquitously expressed throughout the body,especially in hematopoietic tissues.It can form heteromeric complex with IL17RB or IL-17 RC to promote the secretion of proinflammatory factor and Immune response mediated by IL17-A,IL17-F and IL-17E.The intracellular SEFIR domain of IL-17RA can recruit adoptor protein ACT1 to induce the expression and secretion of cytokines through IL-17RA-Actl-TRAF6 in downstream pathways.Subsequently,Neutrophils were mobilized to the location and result in various of diseases.Preparation and development of neutralizing antibody to block IL-17RA signal pathway will play a critical role on treatment of related disorders.In this article,we developed a novel neutralizing antibody to human Interleukin-17 RA(hIL-17RA)by recombinant technology and investigated biological activity of the antibody.This paper is divided into three parts.Firstly,the construction and expression of hIL-17RA.The recombined plasmid of IL-17RA--pSTEP2-IL17RAh,was constructed and transfected into HEK293E cells.Then hIL-17RA-his protein was expressed and purified by Nickel ion affinity chromatography,and identified by SDS-PAGE.Secondly,the preparation and identification of neutralizing antibodies to hIL-17RA.The antibody gene library was established by phage display technology,then we constructed the entire antibody plasmids and transfected them into HEK293E cells.Antibodies were purified by protein A affinity chromatography and studied by SDS-PAGE,HPLC and mass spectrometry.Their binding affinity to hIL-17RA was measured by Octet RED system.Thirdly,to investigate the biological activity of the best one choosen by binding to natural protein from four obtained antibodies.The antibody’s blocking activity to hIL-17RA and cross-reaction between species were analyzed by enzyme-linked immunosorbent assay(ELISA),Western blot,FACS,and its neutralization activity was measured by inhibiting the secretion of cytokine in HFF-1 and HT-29 cells.In summary,high purity hIL-17RA-his protein was produced and four hIL-17RA antibodies were obtained.We chose the best one to do further research on its biological activity.Secretion of IL6,IL8 and CXCL1 elicited by IL-17A,IL-17A/F,IL-17F and IL-17E was neutralized by the antibody.It possesses excellent neutralizing activity with certain cross-reaction among species.In a word,a novel neutralizing antibody to hIL-17RA was obtained,which provides research tools for studying hIL-17RA signal pathways and lays a foundation for drug development targeting hIL-17RA.