Method for preparing humanized camelidae single-domain antibody through transgenic rodent

A single-domain antibody, rodent technology, applied in the fields of molecular biology and immunology, which can solve the problems of the influence of the antigen-binding activity of antibody molecules

Inactive Publication Date: 2016-07-20
长春力太生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, during the rearrangement and mutation of antibody genes, the amino acid sequence of the framework region also has a certain impact on the structure of the CDR region. Therefore, if humanization is performed in vitro, it is likely to change the spatial structure of the antibody molecule and have a negative impact on the antibody molecule. The antigen-binding activity of the

Method used

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  • Method for preparing humanized camelidae single-domain antibody through transgenic rodent
  • Method for preparing humanized camelidae single-domain antibody through transgenic rodent
  • Method for preparing humanized camelidae single-domain antibody through transgenic rodent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] 1. Humanized design of alpaca VHH gene

[0039] We selected 13 core sequences of single domain antibody genomic DNA with known sequences. By comparing with the sequence of the variable region of the human antibody heavy chain, we humanized their framework region sequence, and the nucleotide coding sequence after mutation As shown in SEQ ID NO:1-13, the humanized sites are as shown in Table 1.

[0040] Table 1. List of VHH gene humanized modification sites

[0041]

[0042] Then these humanized VHH core sequences are randomly connected using conventional molecular biology techniques, or a large DNA molecule is directly synthesized by DNA synthesis to form a single domain antibody upstream variable region DNA containing only the VHH sequence. Sequence combination. The sequence of the series in this experiment is in the order 1-13 of the number in the table above, but it can be connected in any random order, and the sequence of the series does not affect the acquisition of the ...

Embodiment 2

[0078] Example 2: Detection of alpaca single domain antibody in transgenic mice:

[0079] Take 100 microliters of blood from the mouse's orbit using a capillary tube, place it at room temperature for 1-2 hours to coagulate the blood, and centrifuge at 2000 rpm at 4 degrees for 10 minutes to separate the serum. Take the upper layer of serum and place it in a clean 1.5 ml centrifuge tube, store it at 4°C for later use.

[0080] Apply 100 microliters of rabbit anti-alpaca antibody (ab173185) diluted 1:100 at 4°C to 96-well ELISA plate overnight. After washing with PBST, add 1:10 diluted transgenic mouse serum and bind for 1 hour at room temperature. PBST Wash 3-5 times, add 100 microliters of HRP-labeled goat anti-alpaca antibody (ab112786) diluted 1:5000, react at room temperature for 1 hour, wash 3-5 times with PBST, add 100 microliters of TMB substrate, react at room temperature After 15 minutes, add 50 microliters of 2N sulfuric acid, and use a microplate reader to detect the abs...

Embodiment 3

[0081] Example 3: Preparation of humanized alpaca single domain antibody hybridomas secreted by transgenic mice and cloning and identification of single domain antibody genes

[0082] 1. Extraction of B cells from transgenic mice:

[0083] 1.1 Immunization of mice: Take 6-8 weeks old female transgenic mice, mix 100ug of chicken egg white lysozyme antigen and Freund's complete adjuvant, and then subcutaneously immunize once, and mix 100ug antigen with Freund's incomplete adjuvant at 2 weeks interval After subcutaneous immunization 2 times, intravenous booster immunization 2 weeks later, 3 to 5 days later for fusion.

[0084] 1.2 Preparation of spleen cells: the immunized transgenic mice were sacrificed by the neck, the body was disinfected with alcohol, the spleen was taken out under aseptic conditions, the connective tissue and fat were shaved, and washed once with 5ml serum-free culture medium.

[0085] 1.3 Put the spleen in a sterilized 90-100 mesh stainless steel mesh or nylon gauz...

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Abstract

The invention provides a method for preparing humanized camelidae single-domain antibody through a transgenic rodent. According to the method, a transgenic mouse can be prepared by constructing humanized alpaca single-domain antibody artificial chromosomes; meanwhile, endogenous antibodies, such as IgH, IgKappa and IgLamda gene clusters, of the mouse are subjected to knockout by adopting a homologous recombination method, and the transgenic mouse which only expresses the humanized alpaca single-domain antibody is obtained and is used for preparing a humanized monoclonal alpaca single-domain antibody. The humanized single-domain antibody can be directly obtained through a hybridoma or single cell PCR (Polymerase Chain Reaction) screening method, the obtained single-domain antibody is not required to be subjected to in-vitro mutation and humanized modification, and a gene bank is not required to be established for screening, so that the method has the advantages of high simplicity, convenience, and safety.

Description

Technical field [0001] The invention discloses a technology for preparing humanized monoclonal camelid single-domain antibodies by using transgenic rodents, and belongs to the fields of molecular biology and immunology. Background technique [0002] In higher vertebrates, immunoglobulin molecules (Ig) are generally considered to be protein tetramers (H) composed of two light chains (Lightchain, L) and two heavy chains (Heavychain, H). 2 L 2 ). In 1993, it was discovered in the humoral immune system of the mammalian camelid (Camelidae) that its immunoglobulin IgG has two types of structures: one is IgG1, which is an eterotetramer of heavy and light chains, with relative molecular mass About 150kDa; The other type is about 50% of serum IgG. IgG2 with a relative molecular weight of about 92kDa and IgG3 with about 90kDa are both homo-dimericheavy-chains. This kind of antibodies that naturally lack light chains, also known as heavy-chain antibodies (HCAb), exists in the blood of arti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/00C12N15/89
CPCC07K16/00C07K2317/24C07K2317/52C07K2317/56C07K2317/569C07K2317/64
Inventor 宋海鹏李敏王庆东
Owner 长春力太生物技术有限公司
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