| Natural antibody is a kind of immunoglobulin, which is synthesized and excreted by ripe B cell after immunocytes being activated by antigen. As an important ingredient of immunologic system, antibody provides potent protection to organisms, while is wildly used in research, diagnoses and therapeutics.Over the past 20 years, researchers have used the techniques of molecular biology to clone full repertoires of human antibody genes. By inserting such genes into an expression vector, a variety of antibody libraries have been constructed and numerous antigen-specific antibodies identified from the libraries. Currently, phage display is the most developed and widely used technology for screening and selecting specific antibodies from library through the so-called panning procedure. Humanization of mouse monoclonal antibodies (mAbs) and direct selection of fully human antibodies have dramatically advanced the development of antibody therapeutics. Thus far,22 antibodies have been approved by the U.S. FDA for the treatment of tumors, arthritis, or cardiovascular diseases.Phage display is widely used, but it has some obvious disadvantages. For example, it cannot display a full-length antibody, but only a fragment (scFv or Fab). To the best of our knowledge, no study has reported the successful expression or display of full-length human antibodies on the phage surface. During the past 10 years, many scientists have exercised considerable effort in the attempt to develop mammalian cell surface display technology. It is therefore desirable to develop technology that can display full-length antibodies, especially human antibodies, on mammalian cell surfaces. Some techniques have fused a transmembrane domain (TM) at the 3’-end of the antibody heavy chain (HC) to form the HC-TM. Co-expressing this HC-TM with a light chain, the full-length antibody could be displayed on the cell surface. Coupled with fluorescence activated cell sorting (FACS), some antigen-specific antibodies were successfully screened and identified from mammalian antibody libraries displayed on the cell surfaces. However, the diversity of mammalian display full-length antibody library developed with these techniques is limited to the range of 106, which does not meet the requirements for the screening of high-affinity antibodies with biological function.To address this drawback, we used a novel universal mammalian display vector to insert with high efficiency the full-length heavy or light chains, or a variable domain of the heavy chain. Using this unique vector, we successfully constructed heavy and light chain libraries with scales of 106 and 105 respectively. Co-transfection of both heavy and light chain libraries into mammalian 293T cells resulted in a high expression level of full-length antibodies on the cell surface, which could be detected with flow cytometry. The specific antibody-expressing cells can be selected with FACS for cloning to produce functional human monoclonal antibodies (mAbs).We have constructed a large rheumatoid arthritis (RA) specific full-length antibody library, which could be displayed on mammalian cell surfaces. The procedures of construction are as follows:Peripheral blood lymphocytes (PBL) were isolated from patients with RA. The repertoires of heavy chain variable region (VH) and kappa light chain (LCκ) of antibody were amplified by RT-PCR. Then VH was inserted into the vector pDGB-HC-TM in frame with the IgG1 constant region contained in the vector to form the full-length IgGl heavy chain library. LCκwas inserted into the vector pDGB-HC-TM to replace the HC-TM in the vector to form the full-length kappa chain library. After transformation of these ligated mixtures into competent E.coli TOPO-10 cells, the libraries of RA patients-specific antibody heavy chain (IgG1) and kappa light chain (LCκ) were constructed. Then the libraries were co-transfected into 293 T cells and the expression of full-length fully human antibody on the surface of 293 T cells was confirmed by flow cytometry. The libraries have an expressible combinatory diversity of 6.13×1010, which provided a useful platform for screen of RA specific antibodies. |
PDF Full Text Request