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Preparation method of novel PD1 FAB monoclonal antibody in DG44 expression system

An expression system and expression vector technology, applied in the field of preparation of novel PD1FAB monoclonal antibody in DG44 expression system

Inactive Publication Date: 2017-05-31
SHANGHAI MEDICILON INC
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Problems solved by technology

However, there is no report on the use of DG44 cells to express PD1 FAB antibody

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  • Preparation method of novel PD1 FAB monoclonal antibody in DG44 expression system
  • Preparation method of novel PD1 FAB monoclonal antibody in DG44 expression system
  • Preparation method of novel PD1 FAB monoclonal antibody in DG44 expression system

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[0026] The present invention utilizes means such as genetic engineering, utilizes pOptiVEC TM As an expression vector, DG44 cells are used as hosts to express PD1 FAB antibody fragments. In one example, the preparation method of the present invention includes: cloning the heavy chain and light chain of the PD1 FAB antibody respectively carrying the Ac NPVgp64 signal peptide into the vector pOptiVEC TM In the process, the recombinant expression vector was constructed, and the recombinant plasmid was transposed into the competent cell Top10 to obtain the recombinant plasmid DNA and transfected into the cells. By the limited dilution method, a high-expression monoclonal cell line was obtained, expressing the target protein of PD1 FAB, and obtained by centrifugation. The supernatant product containing the target protein; the resulting protein is purified by chromatography to obtain the PD1 FAB protein. Hereinafter, the production method of the present invention will be exemplaril...

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Abstract

The invention relates to a preparation method of a novel PD1 FAB monoclonal antibody in a DG44 expression system. The method comprises the steps of (a) cloning a gene of an amino acid sequence with a light chain and a heavy chain of an Fab fragment of signal peptide at N end separately to an expression vector with dual promoters to build a recombinant expression vector; (b) transposing the obtained recombinant expression vector into a competent cell, screening recombinant bacterial plaques, extracting recombinant plasmids, transfecting the recombinant plasmids to a DG44 cell, further amplifying the recombinant cell, expressing Fab protein by using the amplified and cultured cell, and centrifugally collecting a supernatant product containing the Fab protein; and (c) expressing the supernatant by the obtained cell, carrying out ultrafiltration concentration by using a membrane coating, replacing a buffer solution, and then carrying out chromatographic column purification to obtain a fully human monoclonal antibody Fab fragment of the monoclonal antibody PDI.

Description

technical field [0001] The invention relates to a preparation method of a novel PD1 FAB monoclonal antibody in a DG44 expression system, and belongs to the field of biotechnology. Background technique [0002] PD1 (programmed death 1) monoclonal antibody is a world-renowned immuno-oncology therapeutic antibody in recent years. It is the first monoclonal antibody drug approved by the FDA for immunotherapy. It is known as a breakthrough discovery in cancer treatment. Its typical Representatives include Nivolumab (BMS) and Keytruda (Merk). PD-1 programmed death receptor is an important immunosuppressive molecule and a member of the CD28 superfamily. PD-1 is mainly expressed in activated T cells and B cells, and its function is to inhibit the activation of cells. This is a normal homeostasis mechanism of the immune system, because excessive T / B cell activation can cause autoimmune diseases, so PD-1 is a talisman for our human body. The ability of tumor cells to evade the immu...

Claims

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Application Information

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IPC IPC(8): C07K16/28C07K1/36C07K1/34C07K1/16C12N15/85
CPCC07K16/2818C12N15/85C12N2800/107
Inventor 王涛汪俊陈春麟周南梅
Owner SHANGHAI MEDICILON INC
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