| As a new type of antibody,nanobody has higher solubility and antigen binding ability,is more suitable for gene manipulation means and lower artificial manufacturing cost,so it is widely used in scientific research and clinical application.More and more diseases have been found to be associated with abnormal activation/deactivation of protein modifications after translation,which has led to the development and research of modified antibodies.Phosphorylation antibodies are one of them,as the phosphorylation state of many important proteins has a significant impact on their activity and function.This study explores the development of a method for screening phosphospecific antibodies through a fully synthesized nanoantibody library.Phosphorylation is a common protein modification method that changes the structure and function of proteins by adding phosphate groups to them.Multipoint phosphorylation adds phosphate groups to multiple sites in a protein,resulting in more complex structural changes and even affecting the stability and function of the entire protein complex.Therefore,the development of single or multipoint phosphospecific antibodies may imply different difficulties and requirements for methods,which is worth systematic exploration and provides a basis for method development.Previously,our study found that mental stress can activate phosphorylation at the S181 site of RBM24,while RBM38 S117/T132 two-point phosphorylation can be induced by hypoxia.The proline rich domain of Tau protein is located in proline directed protein kinases(such as GSK3 β)Multiple phosphorylation modifications occur at multiple sites in the Ser Pro/Trr Pro motif under the action of.The occurrence of abnormal phosphorylation of Tau protein is closely related to various neurodegenerative diseases.This study aimed to develop phosphorylated nanobody and constructed a fully synthesized nanobody display library.Specific nanobodies targeting phosphorylation at the RBM24 S181 site,RBM38 S117/T132 site,and TAU T117/T231/S235 site were screened through phage surface display technology and a combination of phage and yeast surface display screening.We screened nanobodies targeting RBM24 S181 single point phosphorylation using phage surface display technology;Nanobodies targeting RBM38 S117/T132 site phosphorylation were successfully screened through a combination of phage and yeast surface display screening technology.However,no nanobodies targeting phosphorylation at TAU T117/T231/S235 sites were found.Therefore,through this study,we demonstrate that the fully synthesized nanobody display library can be used for the development of phosphorylated nanobodies.The discovery of these nanobodies will contribute to the in-depth study of the phosphorylation modification of these proteins and their mechanisms of action in diseases. |
