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Construction and application of monoclonal antibody cloning and expression vector

An expression vector and vector technology, applied in the construction and application of monoclonal antibody cloning and expression vector, can solve the problems of cumbersome experimental operation and low efficiency, and achieve the effect of improving efficiency and reducing experimental cost.

Pending Publication Date: 2022-07-12
NORTHEASTERN UNIV
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Problems solved by technology

The existing technology has the disadvantages of cumbersome experimental operation and low efficiency

Method used

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  • Construction and application of monoclonal antibody cloning and expression vector
  • Construction and application of monoclonal antibody cloning and expression vector
  • Construction and application of monoclonal antibody cloning and expression vector

Examples

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Embodiment Construction

[0034] The present invention will be further described in detail below in conjunction with the examples.

[0035] The construction of a monoclonal antibody clone and expression vector, comprising the following steps:

[0036] (1) Insert the leader sequence into the HindIII restriction site and EcoRI restriction site of the pCDNA3.1-myc-His vector, and form a C fragment with the CMV promoter sequence of the pCDNA3.1-myc-His vector without synthesizing the CMV sequence , the recombinant vector is named pCDNA-C;

[0037] (2) The antibody heavy chain constant region gene sequence (C H ), light chain kappa chain constant region DNA sequence (C k ) and the constant region DNA sequence of the light chain λ chain (C λ ), inserted into the XhoI restriction site and the Agel restriction site of the pCDNA-C recombinant vector respectively, and the bovine growth hormone PolyA signal sequence of the pCDNA3.1-myc-His vector constitutes H fragment, K fragment and L fragment respectively, ...

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Abstract

The invention relates to monoclonal antibody cloning and construction and application of an expression vector, and belongs to the technical field of molecular cloning. During construction, an antibody leader sequence is inserted into HindIII and EcoRI restriction enzyme cutting sites of a pCDNA3.1-myc-His vector, and an antibody CH sequence, an antibody Ck sequence and an antibody C lambda sequence are inserted into XhoI and Agel restriction enzyme cutting sites of a recombinant vector respectively, so that a monoclonal antibody cloning and expression vector is obtained. During application, a vector linear DNA is obtained by taking monoclonal antibody cloning and an expression vector as a template through PCR amplification, the vector linear DNA is mixed with an antibody variable region gene to serve as a template, and an antibody linear expression cassette is obtained through PCR amplification; after screening the high-activity antibody, seamlessly connecting the linear DNA of the vector with the variable region gene of the antibody to construct an antibody plasmid expression vector, and expressing the antibody. Compared with the prior art, the method has the advantages that one vector construction, two PCR reactions, two double enzyme digestion and three DNA purification are omitted, and the antibody gene cloning and expression efficiency is improved.

Description

Technical field: [0001] The invention belongs to the technical field of molecular cloning, in particular to the construction and application of a monoclonal antibody clone and an expression vector. Background technique: [0002] Monoclonal antibody (monoclonal antibody) drugs are widely used in the field of anti-pathogenic infection and tumor treatment. The preparation technology of monoclonal antibody includes hybridoma technology, phage antibody library technology and antibody gene cloning technology based on single B cells. Antibody gene cloning, expression and screening technologies based on single B cells have received increasing attention and are widely used in the field of monoclonal antibody drug development. [0003] Antibody molecules are composed of heavy chain (H chain) and light chain (K chain or λ chain). The heavy chain and light chain are divided into two parts: variable region (V) and constant region (C). The variable region is antigen-specific and binds to...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/113C07K16/10C12N15/10
CPCC12N15/85C12N15/113C07K16/10C12N15/10C12N2800/107C12N2830/50C12Q2531/113
Inventor 孙金福冯泽众
Owner NORTHEASTERN UNIV
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