Selection Of Anti-CDK4 Antibodies Using The Intracellular Antibody Capture Technology

Cancer is a malignant disease for human, and there is not an effective way to cure it. Cancer cells 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. Yet G1-S phase checkpoint is usually out of control in the developing phases of the cancer and results in cell cycle disorder. CDK4 as an important CDK, controlls the G1→S phase of the cell cycle and shows overexpressed in many cancer cells. Inhibition and degradation of the CDK4 can lead to G1 phase arrest in cancer cell.Intrabody is a new gene therapy method which is expressed in cell and oriented to subcellular organelle, binding with intracellular target molecule and affecting its processing, secretion and biological effects so that gene phenotype knockout is brought into effect. In the past research, isolated intrabodies in vitro are often unstable and insoluble suppressing their functions. The intracellular antibody capture technology which is based on the high-throughput selection of stable and soluble functional antibodies for target antigen. is a useful method to isolate antibodies that are suitable for eukaryotic intracellular expression. In order to prepare the efficient anti-CDK4 intrabodies to inactivate CDK4 kinase and in this study, a phage scFvs library screened by CDK4 with two panning cycles was cloned into yeast vectors by homologous recombination or ligation in vitro for obtaining anti-CDK4 human scFvs yeast library. Twenty-five positive clones were screened out by yeast two-hybrid-system. The terms of the study are that:1. Using pET28a-CDK4 plasmid as template and nucleotide acid sequence including EcoRI and SalI sites as primers, CDK4 gene was obtained by PCR, then directly cloned into pGBKT7 to construct yeast expression plasmid pGBKT7-CDK4 for the bait protein human CDK4.Agarose gel electrophoresis showed two DNA bands with molecular weight of 7300 bp and 910 bp. Nucleotide acid sequencing showed that the insertion sequence was consistent with the experimental design and the reading frame was correct. It showed that the yeast expression plasmid pGBKT7-CDK4 for the bait protein human CDK4 was successfully constructed.2. The plasmid pGBKT7-CDK4 was transformed into yeast strain Y187. PGBKT7-CDK4/Y187 was spread on SD/-Trp,SD/-His/-Trp and SD/-Ade/-Trp plate. The result showed that only yeast appeared on SD/-Trp plate. It showed the bait protein was not able to activate the report genes ADE and HIS. By X-gal filter screen, it was showed that bait protein CDK4 can not be self-activated report gene LacZ, so that this bait protein is available for yeast two hybrid test.3. After the scFv fragments library with the complete SMARTIII and CDSIII obtained by two round PCR and linear pGADT7-Rec vector were cotransformed into AH109 yeast, 71 yeast transformant were obtained. The yeast cocultivations with pGBKT7-CDK4/Y187 were selected by X-gal filter. The result showed that nine positive clones were screened out under the positive and negative controls being effective, which could interact stably with CDK4 in eucaryoti cells.4. The pGAD424-BNN plasmid was reconstructed by orthomutation to MCS of pGAD424 with BssHⅡand NheⅠsite. It makes the construction of yeast antibody library from phage antibodies libraries in future.5. The pGAD424-BNN-scFv yeast antibody library was successfully construct by ligation in vitro. Sixteen positive antibodies against CDK4 in eukaryotic cells were screened out by IACT.This study provided the technicalplatform for the preparation of of high effective intrabodiesby IACT.