Three-cistron expression vector, preparation method and application

Abstract

The invention discloses a three-cistron expression vector, a preparation method and application and belongs to the technical field of gene engineering.The three-cistron expression vector comprises a nuclear matrix combining region sequence and a three-cistron sequence which is formed in the mode that two internal ribosomes enter a site sequence and connected; the structure of the three-cistron sequence is promoter-light chain signal peptide SPL-IRESwt-heavy chain signal peptide SPH-IRESatt-selection marker-Poly A.The vector can express a light chain, a heavy chain and a selection marker gene of an antibody at the same time, the problem that expression of a heavy chain, a light chain and a selection marker gene of a traditional vector is not balanced is solved, antibody quality is improved, and the cloned screening rate of positive cells is raised; meanwhile the nuclear matrix combining region sequence contained on the vector can further overcome transgene silencing, efficient and long-term expression of transgenes in host cells is achieved, on one hand, the antibody protein expression level is improved, and on the other hand, efficiency of screening of subsequent monoclonal antibody cell lines is improved.

Description

technical field [0001] The invention relates to a tricistronic expression carrier, and also relates to a preparation method and application of the expression carrier, belonging to the technical field of genetic engineering. Background technique [0002] Antibody is a kind of immunoglobulin with special amino acid sequence induced by antigen, synthesized and secreted by lymphocytes (plasma cells). Monoclonal antibodies (Monoclonal antibodies, mAbs) are highly specific, and thus are widely used in the treatment of various diseases, especially cancer and autoimmune diseases. Since the first monoclonal antibody drug Orthoclone Okt3 was launched in 1986, 35 monoclonal antibody drugs have been approved for marketing. At present, monoclonal antibodies are the fastest-growing class of biomedical molecules, and have attracted more and more attention from scholars and pharmaceutical companies. Most monoclonal antibodies (IgG) are produced from two identical heavy chain (HC) and two ...

AI Technical Summary

Problems solved by technology

However, the defect of this type of vector is that the ratio of heavy chain to light chain expression is unbalanced, and the ratio of the two affects the quality of monoclonal antibodies, such as aggregation and glycosylation (IRES-mediatedTricistronic vectors for enhancing generation of high monoclonal antibody expressing CHOcelllines.JBiotechnol.2012)

Although some vectors can express antibody heavy chain and light chain at the same time (patent application number: 201110380100.8, a high-efficiency expression vector of antibody and its preparation method), but because the selection marker gene is not in the same coding frame as the heavy chain and light chain genes, Some positive clones screened will not express the target protein, and false positive cell clones will appear (Vectorfragmentation:characterizing vector integrity intransfected clones by Southernblotting.BiotechnolProg.2010)

[0004] In addition, in the CHO cell expression system, it is often difficult to obtain cell lines with high levels of sustained and stable expression. This is because the random integration of transgenes into cells makes some transgenes silent or the expression level is reduced. Therefore, a large number of cell clones are required. The clonal cell lines with stable and highly expressed expression were isolated from the above, which also brought great troubles to the industrial production of genetic engineering (Astudy of monoclonal antibody-producing CHO celllines: what makes stable high producer? Biotechnol.Bioeng.2009)

Images