Study On The Molecular Mechanism Of Anti-CDK4 Single-chain Antibody

70 years later, the human cancers became the second leading cause of death. Cell cycle is closely related to the cell differentiation, growth and death. Studies show that deregulation of the cell cycle is the main reason for oncogenesis. CDK4 is the first activated kinase when the cell cycle gets into proliferative status. It is a key regulator of the transition through the G1 phase of the cell cycle. Now it is widely believed that CDK4 is an oncogene. Deregulation of the CDK4/cyclin D pathway has been identified in many cancers. Because aberrant regulation of the CDK4/cyclin D1 pathway plays an essential role in oncogenesis. CDK4 is a potential therapeutic target.An epitope. the immunocompetent area of antigen, is the Critical region of an antigen that is recognized by antibodies, and is the base of the antigen to induce the specific immune response. To exactly predict the antigen epitope is critical for diagnosis and prognosis of disease. vaccine development and the transformation of protein molecules. Although there are several ways to predict the protein epitope now, most of them only can be used for the linear epitopes analysis. Recent experiments suggest that most epitope are discontinuous. The absence of efficient methods restrains the development of the prediction of conformational epitope. In this study, we combined the phage displaying technology with molecular docking to improve the accuracy of epitope prediction. Phage displaying technology is a high-throughout screening (HTS) assay for studying the binding site of ligands and receptors. Compared to other methods for study the interaction between proteins, phage displaying technology has many advantages, such as small volume, high throughout, easy to operation, applicable to assay the continuous and discontinuous epitope. the technology have abroad application respects in all kinds of study. In our previous research, we have screened the phage library with CDK4 as the target protein, and got the scFv binding specially to CDK4. Then we transducted the scFv gene into human carcinoma cells, successfully reversed of the malignant phenotype of the cancer cell. However, it is not clear with the mechanism for the scFv suppressing the cell malignant multiplication, the crucial amino acid for CDK4 recognized by scFv. the CDK4 epitope, and so on. To adress the above questions, we carried out the following studies:At first, the protein CDK4 and the anti-CDK4 scFv were expressed and purified. Then, the scFv was solidified on the plastic ELISA plates for screening the T7 select phage diplay library. After four rounds of biopanning, a population of phages with high affinity to scFv was obtained. To prepare T7 Select phage for sequencing. PCR Amplification of Plaques was conducted. Subsequently, the sequence of selected phage recombinants was analysed with the molecular biology softwares. At last, the 3D structures of scFv, and the circular 7 mer-peptides were imitated, and the interaction of scFv binding to the 13 peptides were simulated.In this study, the protein CDK4 and the anti-CDK4 scFv were successfully purified, and a population of phages with high affinity to scFv were got. According to sequence analysis of the select phage recombinants, two groups of conserved amino acids among the peptides were found, which implys the conserved amino acids may be one part of the epitope that recognized by CDK4. And the simulated protein-protein interaction further confirmed the deduction. This study not only is helpful to interpret the interaction mechanism of scFv on bingding to CDK4. but also laid the foundation for treating tumor using anti-CDK4 intracellular scFv.