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Method for assembling bacteriophage gene engineering antibody library gene

A technology of genetically engineered antibodies and bacteriophages, applied in the fields of biomedical research and clinical applications, can solve the problems of large mutation probability, low fidelity, poor stability, etc., and achieve the effect of small mutation probability, improved fidelity and good stability

Inactive Publication Date: 2008-07-02
SHAANXI CHAOYING BIOTECH
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Problems solved by technology

[0008] The present invention provides a method for gene assembly of a phage genetically engineered antibody library to overcome the poor stability, low efficiency, high mutation probability and low fidelity in the prior art, and the inability to be directly used for human-derived Issues in scFv research

Method used

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Embodiment Construction

[0026] The present invention will be described in detail below by way of examples.

[0027] The principle of the method of the present invention is to use two methods of PCR and enzyme digestion and connection at the same time. After obtaining the human antibody heavy chain and light chain variable region genes by RT-PCR, the enzyme digestion sites at both ends of the primers are pre-designed. Realize splicing of antibody heavy chain variable region, Linker and antibody light chain variable region.

[0028] Specifically, the present invention designs 6 5' heavy chain variable region primers and 7 3' light chain variable region primers, and 6 5' light chain variable region primers according to the characteristics of human antibody variable regions. and 13 primers for the variable region of the light chain at the 3' end, a total of 32 primers were designed and synthesized at the same time a pair of Linker containing two restriction sites XhoI and ApaI (that is, the light chain a...

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Abstract

The invention relates to a gene assembly method of phage genetically engineered antibody library, belonging to the field of biomedical study and clinical applications. The method aims to overcome the problems of the prior art, including poor stability, low efficiency, high mutation probability and low fidelity, and indirect application to humanized single chain antibody study. The technical proposal adopted by the invention comprises the following steps of: obtaining heavy chain variable region gene and light chain variable region gene of a humanized antibody by RT-PCR, and ligating the antibody heavy chain variable region, a linker and the antibody light chain variable region with the pre-designed restriction enzyme cutting sites on two ends of primers using PCR and restriction enzyme method at the same time.

Description

Technical field: [0001] The invention relates to the field of biomedical research and clinical application, in particular to a method for gene assembly of a phage genetic engineering antibody library. Background technique: [0002] In recent years, genetic engineering technology has developed rapidly, and a series of genetic engineering drugs have been widely used in clinical practice, and their application scope mainly focuses on genetic engineering drugs and monoclonal antibodies for tumors and AIDS. At present, there are more than 2,200 biotechnology drugs under development worldwide, of which more than 1,700 have entered the clinical trial stage. There are more than 300 biologic drugs in late-stage clinical trials in the United States. In 2001, FDA approved 8 kinds of biotechnology drugs and vaccines and 3 new indications of drugs; in 2002, FDA approved a total of 20 new biotechnology drugs and 15 new indications of biotechnology drugs. In 2003, FDA approved 35 new dru...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12N15/13
Inventor 杜孟刚何文亮赵育英步雪
Owner SHAANXI CHAOYING BIOTECH
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