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Preparing human source monoclone antibody by mouse capable of producing human IgGl weight chain-k light chain and application thereof

A monoclonal antibody and humanized technology, applied in the field of bioengineering, can solve problems such as technical difficulty and structural changes, and achieve the effects of low technical difficulty, simple operation and high recombination accuracy.

Inactive Publication Date: 2005-01-05
DALIAN D N BIO ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0015] The purpose of the present invention is to overcome the existence of the prior art: 1. It takes more genetic engineering operations to express the antibody protein with practical value; 2. The technology is difficult; 3. The whole gene of the DNA molecule up to 1.5Mb Operations are likely to bring about structural changes; 4. It is difficult to determine whether there is recombination after entering mouse cells, the impact of integration sites on expression, and whether the entire gene rearrangement pattern is normal after recombination; and provide a method for producing human cells. The mouse of IgG1 heavy chain-kappa light chain is as preparation monoclonal antibody and application, specially proposes technical solution of the present invention

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Dien Biotechnology Engineering Company uses genetic engineering technology and transgenic technology to establish a mouse capable of producing human IgG1 heavy chain-κ light chain as a platform for the development of monoclonal antibodies. The steps are as follows:

[0037] In the first step, clone the mouse Igκ chain and IgG1 chain genomic genes from the genomic DNA library of 129sv mice.

[0038] The second step is the construction of gene knock-in vector

[0039] ① Light chain vector-using the mouse k-chain genome gene fragment as the homologous region, insert the complete human Igk gene fragment into the mouse Igk gene locus, so that a small segment of the coding region at the 5'end of the mouse k gene is deleted. To ensure the inactivation of mouse k gene while inserting human k gene, insert positive selection gene neo (neomycin phosphotransferase) expression unit downstream of human k gene, and insert negative selection gene diphtheria toxin A unit (DTA) expression do...

Embodiment 2

[0056] Dien Biotechnology Engineering Company uses mice that can produce human IgG1 heavy chain-κ light chain as an application for preparing anti-tumor monoclonal antibodies.

[0057] When a cell undergoes transformation, it is often accompanied by changes in glycolipids or glycoprotein sugar chains on the cell membrane surface. sTn (NeuAcα2→6GalNAcα1→O-Ser / Thr) is a disaccharide structure that can be expressed in various adenocarcinoma tissues, but is rarely or not expressed in normal tissues. Immune preparation of monoclonal antibody mAb against the glycoantigen sTn, the purpose is to test the feasibility of the mouse strain producing human IgG1 heavy chain-κ light chain in the preparation of anti-tumor monoclonal antibodies, and to establish a method for detecting sTn Targeted antibodies provide reagents for targeted therapy, and the specific steps are as follows:

[0058] The first step is to use the antigen to immunize mice and related materials

[0059] The antigen-immunize...

Embodiment 3

[0071] Dien Biotechnology Engineering Company uses the mouse strain that produces human IgG1 heavy chain-κ light chain in the preparation of anti-hepatitis B virus monoclonal antibodies.

[0072] Hepatitis B virus is currently a very serious infectious disease. Anti-hepatitis B surface antigen antibodies have a protective effect on the human body against hepatitis B virus infection. The development of the human anti-hepatitis B surface antigen monoclonal antibody of the present invention will provide a basis for the preparation of anti-viral humanized monoclonal antibodies with mouse strains that produce human IgG1 heavy chain-κ light chain for application in human treatment. The method steps are as follows:

[0073] The first step is to immunize mice with a human IgG1 heavy chain-κ light chain antigen

[0074] The hepatitis B surface antigen (HBSAg) protein was used to immunize chimeric antibody mice, and the titers and types of anti-HBSAg antibodies in the serum were observed wi...

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Abstract

The invention relates to using a mouse able to generate human IgGl heavy chain- k light chain to prepare humanized monoclonal antibody and its application. The characteristic: respectively knocking in huma k-chain and gl-chain constant gene at the sites of gene in mouse k- light chain constant region and heavy chain gl constant region; the humanized monoclonal antibody: the constant region is fully the same as that of human antibody and the variable region is mouse sourece, and the preparing steps: obtaining mouse k-chain and gl-chain gene groups; constructing a knoncking-in carrier; making homologous recombination in ES cell; producing the mouse with light-heave conversion; further mating to obtain a mouse with complete chimeric gene; the application: taking spleen and marrow cells of an immunized chemeric mouse to make fusion and culture; screening hybridomas of humanized monoclonal antibody generating antigenic specificity; preparing the humanized monoclonal antibody with antigenic specificity. The advantages is easy to obtain high-affinity human-mouse chimeric antibody.

Description

Technical field [0001] The invention relates to the preparation and application of a humanized mouse monoclonal antibody, and belongs to the field of bioengineering. Background technique [0002] Monoclonal antibodies have broad application prospects in the diagnosis, treatment and prevention of tumors, viral infections, rheumatoid arthritis, cardiovascular diseases, and organ transplantation. Therefore, they have become the most compelling research hotspot in the field of biotechnology drugs. . [0003] Antibodies are proteins produced by the animal's immune system under the stimulation of foreign antigens, and their specificity depends on the determinants of the antigen molecule. Since various antigen molecules have many determinants, the antibodies produced by immunized animals are actually a mixture of multiple antibodies. It is not feasible to prepare antibodies by this method, because the content of antibodies is small, the separation is also difficult, and the injection of...

Claims

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Application Information

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IPC IPC(8): C07K16/18C12N5/18C12P21/08
Inventor 韩华李元李别虎薛小平黄庆生王延竹韩文君
Owner DALIAN D N BIO ENG
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