Prokaryotic Expression, Purification Of Human Single-chain Variable Fragments Antibody Against Cyclin D1(AD9) And Analysis Of AD9 Activity

Cancer is a malignant disease for human, and there is not an effective way to cure it. Cancer cell showed that proliferation is overlimited and the cell cycle regulation is out of control. A great deal of studies has indicated that there are two checkpoints in cell cycle, G1→S and G2→M phase checkpoints. But G1-S phase checkpoint is usually out of control in the developing phases of the cancer and results in cell cycle disorder. Cyclin D1 as an important cyclin, controlls the G1→S phase of the cell cycle and shows overexpressed in many cancer cells. Inhibition and degradation of the cyclin D1 can lead to G1 phase arrest in cancer cell.ScFv is a kind of engineered antibody. It is the minimum antibody fragment which has the binding activity with antigen. Due to the small size, it can be modified by gene engineering and penetrate into the solid tumor. There is smaller immunogenicity than antibody too.In order to inactivate the activity of cyclin D1/CDK4 complex and find out the relationship between the cyclin D1 and tumor genesis,the following studies were carried out. A scFv against cyclin D1 screened in the phage antibody library. In this study, the stop codon (TAG) in the wtAD9 linker region was changed to TAT by PCR to abtain AD9 gene. The AD9 fragement was cloned into pDF vector and constructed pDF-AD9 expression vector. This vector was transformed into HB2151, and then induced by IPTG. The expressed products were purified. The binding activity of AD9 to cyclin D1 was identified by ELISA,Western blot immunofluorescence and co-immunoprecipitation. The follows are results.1. Nucleotide acid sequencing showed that the stop codon (TAG) in the wtAD9 linker region had been changed to TAT by PCR.2. The pDF-AD9/HB2151 was induced by IPTG. Then the activity of the supernatant of culture medium binding to cyclin D1 was analyzed by ELISA. The products were precipitated by ammonium sulfate, and then purified with HisTrap HP Kit. The purified protein AD9 was identified by Western blot. Then via the ELISA,Western blot analysis, it was identified that the purified AD9 had the ability of binding to cyclin D1.3. The competitive inhibition of anti-cyclin D1 polyclonal antibody to AD9 for binding cyclin D1 was analyzed by competitive ELISA. The results revealed that the rabbit anti-cyclin D1 antibody can inhibit AD9 to bind to cyclin D1. The index of inhibition is 25.9%.4. The affinity of AD9 was determined by noncompetitive ELISA, and the affinity constant binding to cyclin D1 was (4.52±0.19)×10^-8mol/L. It suggested that the AD9 had affinity of binding to cyclin D1.5. To identify the interaction between AD9 and cyclin D1 in HeLa and MCF-7 cells under fluorescence microscope, the fixed cells were incubated with the AD9, and then with anti-V5 antibody, followed by the incubation with FITC-conjugation goat anti-mouse IgG antibody and Hoechst 33342 staining chromatin. The results showed that AD9 can interact with cyclin D1 in tumor cells.6. The E-Tag and endoplasmic reticulum retained signal were introduced into AD9 by PCR, and then was inserted into pcDAN3.1 (+) vector to construct recombinant expression vector pER-ADκ. MCF-7 cells were transfected with pER-ADκand analysed after 48 hours. The scFv were Immunoprecipitated from the cell extracts using anti-E tag antibody. After separating the Immunoprecipitated complex by SDS-PAGE, they were identified by western blot and probed with anti-cyclin D1 polyclonal antibody. The results showed that there was a band in the region of 34 kDa. This result revealed that the AD9 expressed in tumor cells can bind to cyclin D1.This study provided experiment support of tumor therapy by the intrabody against cyclin D1.