Generation And Application Of The NOK/STYK1Monoclonal Antibody

Protein tyrosine kinases (PTKs)-mediated signaling transduction pathways play pivotal roles inthe regulation of key functions during embryogenesis, development, organgenesis, metabolismand immunity. PTKs consist of about90members that can be classified as two subgroups: thetransmembrane receptor PTKs (RTKs) and the nonreceptor PTKs (NRTKs). Over the past decades,amounts of evidence showed that PTKs enhance the cell proliferation, mobility and viability byactivating multiple downstream signaling transduction pathways, whereas abnormal PTKscatalytic activity often leads to oncogenesis as well as other various diseases, such as leukemia,breast cancer, cervical cancer and immunity system defect. Thus, the kinase activity of PTKs isunder strictly spatiotemporal control. Recently, more and more researchers focus on making smallmolecular inhibitors or generating monoclonal antibodies to target particular PTKs to blockspecific signaling transduction for cancer treatment.NOK/STYK1（Novel oncogene with Kinase domain, also known as serine/threonine andtyrosine receptor protein kinase）is a new member of RTKs that has been identified as anotheroncogene. Compared with the canonical RTKs, NOK/STYK1contains a transmembrane helix andan intracellular kinase domain, but lacks intact extracellular ligand binding domain. Evidenceshowed that NOK/STYK1is overexpressed in multiple cancer cells, including breast cancer,ovarian cancer and lung cancer, suggesting that the NOK/STYK1expression level could be acts asone of the hallmarks of cancer. However, the mechanism of NOK/STYK1participates in theregulation of signaling transduction and oncogenesis remains elusive. Moreover, to generate aspecific monoclonal antibody to illustrate the functions of NOK/STYK1is really in need. In thisresearch, we purified a short peptide of NOK/STYK1(AAIKTADDEA) by SF9insect cellsexpression system. Then, we used this peptide as an antigen to immunize Balb/C mice to obtainthe B cells of spleen. Subsequently, we fused the B cells to murine cells SP2/0and then screenedand identified the positive hybridoma cells line for production of the monoclonal antibody against NOK/STYK1by ELISA. Fortunately, we found that a hybridoma cell line7G9has a highproduction of monoclonal antibody, which was confirmed for the specificity and efficiency byboth western blotting and ELISA. Furthermore, this monoclonal antibody was verified to bepowerful for detecting the expression of NOK/STYK1in various cancer cell lines and tissues byboth immunefluorescence and immunohistochemical staining respectively.Overall, we successfully generated a monoclonal antibody of NOK/STYK1for the first time,and we believe that this antibody will help us to get a deep insight into the mechanism ofNOK/STYK1participating in oncogenesis. Importantly, this study provided us with promisingtherapeutic application of the monoclonal antibody on the cancer treatment by blocking theNOK/STYK1mediated-cellular signaling pathways.