Prokaryoti Expression And Identification Of Anti-cyclin D1 Human Derived Intrabody

The cell cycle is the basic process of the life activities of the cells, and it's disorder will lead to abnormal cellular proliferation, and tumor formation. The cell cycle is controlled by cyclins which express differently according to the different cell cycle each phase.Cyclins are the key factors of the network system. Cyclin D1 is the the first synthesized cycle protein when the cells are driven into cell cycle, it can bind CDK4 sepcially and form the cyclin D1/CDK4 complex, which promotes the cell cycle G1/S transition. Amplification and over-expression of Cyclin D1 has been found in many types of human cancer. Resently, Cyclin D1 is also found to be closely with the tumorgenesis, invasion and metastasis of tumor cells. Therefore. It becomes a potential target for the treatment of human diseases in which the control of cell proliferation is deregulated.In resent years,a new biotechnology,intrabody antibody technique, has emerged, which can specifically block intracellular target protein. Because of its highly effective, low toxic, extensive function, this technology has become the new intracellular immunotherapy and been widely used,especially for the single chain Fv antibody(scFv). Because the scFv overcome many defects in other engineering antibody, such as the complex molecular structure, the strong immunogenicity, short half-life and so on, it has become the one of major intracellular antibody forms. Up to now, people have transfered several kinds of scFv into hunman cancer cells, implied that the scFv can be used to inactivate the target protein specifically and to transform the phenotype of malignant proliferation of cancer cells. The previous data provide new ideas for the gene therapy of cancer.In this study,we obtained the AD fragment from the recombinant plasmid p3.1-AD by using PCR to add BssHⅡand NotⅠ. The AD was inserted directly into plasmid pDNA5 Which had been digested by BssHⅡand NotⅠ. Recombinant expression vertor pAD was transformed into E.coli HB2151. At the temperature of 37℃, the expression of AD gene was induced by IPTG. The AD product was purfied with HisTrap HP Kit. The protein expressed was measure by the Western blot. The recombinant vector pAD was successfully constructed and AD intrabody was expressed successfully. However.we did not get the purified AD intrabody, we supposed that many other protein in the supernatant interfere the binding of destined protein.In conclusion, intracellular anti-cyclin Dl scFv may become an effective approach for tumor gene therapy. In order to obtain the high-quality AD protein, we have to try to grope the optimal condition for purification.