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Construction method of humanized CD40 gene modified animal model and application thereof

A technology of genetic modification and construction method, which is applied in the field of genetic engineering, can solve problems such as differences in human and mouse targets, unsuitability for toxicity and safety evaluation, and damage to healthy tissues, so as to speed up the research and development process, reduce the risk of drug development, and save time. and cost effects

Pending Publication Date: 2021-04-30
SHANGHAI BIOMODEL ORGANISM SCI & TECH DEV +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The homologous gene of the human CD40 gene in mice is Cd40, both of which have similar gene structures, but in terms of amino acid identity, the amino acid identity of human and mouse CD40 proteins is only 61%, and the amino acid sequence of the extracellular region is The identity is only 59%. This difference in amino acid sequence identity is likely to cause the drug targeting the human CD40 target to fail to recognize the mouse Cd40 target, and wild-type mice cannot be used to evaluate the drug's efficacy in vivo
In addition, although immunotherapy has certain effects in treating cancer, if the immune response is too strong, it will cause great damage to healthy tissues and cause immune side effects. Strict screening for toxicity and safety
However, the commonly used wild-type mice, because of the differences in human and mouse targets, the drug cannot recognize the target in mice, and is not suitable for toxicity and safety evaluation

Method used

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  • Construction method of humanized CD40 gene modified animal model and application thereof
  • Construction method of humanized CD40 gene modified animal model and application thereof
  • Construction method of humanized CD40 gene modified animal model and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] Embodiment 1 sequence design

[0091] Both the mouse Cd40 gene and the human CD40 gene contain multiple transcripts, and the sequence design in this example is mainly described using one of the transcripts as an example. That is, exon 2 to exon 5 of mouse Cd40 gene (NCBI Gene ID: 21939) (based on the transcript whose NCBI accession number is NM_011611.2 → NP_035741.2, its mRNA sequence is shown in SEQ ID NO: 1 , the corresponding protein sequence is shown in SEQ ID NO: 2) with exons 2 to 5 of the human CD40 gene (Gene ID: 958) replaced (based on the transcript of NCBI accession number NM_001250.6→NP_001241.1, Its mRNA sequence is shown in SEQ ID NO: 3, and the corresponding protein sequence is shown in SEQ ID NO: 4), wherein, the comparison diagram of mouse Cd40 and human CD40 genes is shown in figure 1 , the schematic diagram of the transformed humanized mouse CD40 gene finally obtained is shown in figure 2 , the genomic DNA sequence (chimeric CD40 gene DNA) of the ...

Embodiment 2

[0097] Example 2 Design and Construction of Recombinant Vector PBR322-CD40

[0098] According to the sequence design, the inventor has further designed such as image 3 The targeting scheme and the vector containing the 5' homology arm, the human CD40 gene fragment, and the 3' homology arm are shown. Wherein the 5' homology arm (SEQ ID NO: 9) is the 164899679-164904205 nucleotide of NCBI accession number NC_000068.8, and the 3' homology arm (SEQ ID NO: 10) is NCBI accession number NC_000068. The nucleotides 164905758-164909778 of 8, and the human CD40 (SEQ ID NO: 11) gene fragment are nucleotides 46121820-46123219 of NCBI accession number NC_000020.11.

[0099] The construction process of the vector is as follows: design the upstream primers for amplifying the three homologous recombination fragments (LA, KI, RA), the matching downstream primers and related sequences. Among them, the 5' homology arm corresponds to the LA fragment, the insertion sequence such as the human CD4...

Embodiment 3

[0110] Example 3 Verification of carrier PBR322-CD40

[0111] Randomly select 5 PBR322-CD40 clones, and use the restriction endonuclease XbaI to carry out enzyme digestion verification, and the digestion products should have fragments of 6.3kb, 4.2kb, 3.3kb, and 2.3kb in electrophoresis. For enzyme digestion results, see Figure 4 .

[0112] The plasmid digestion results were all in line with expectations, indicating that the plasmid digestion verification results were correct. The plasmid was verified to be correct by the sequencing company, and subsequent experiments were carried out.

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Abstract

The invention provides a construction method of a humanized CD40 gene modified animal model and an application thereof, and relates to the field of gene engineering. The CD40 gene humanized model animal is successfully prepared, the humanized CD40 protein can be normally expressed in the model body, and the product can be used for CD40 gene function research and humanized CD40 antibody screening and evaluation. The animal model prepared by the method can be applied to drug screening, drug effect research, immune related diseases, tumor treatment and the like for human CD40 target sites, the research and development process of new drugs is accelerated, the time and cost are saved, and the drug development risk is reduced. A powerful tool is provided for researching the function of the CD40 protein and screening tumor drugs.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a construction method and application of a humanized CD40 genetically modified animal model. Background technique [0002] In recent years, with the launch of drugs targeting immune checkpoints such as PD-1 and CTLA4, tumor treatment is at the cusp of "immune revolution". Tumor immunotherapy has become a new treatment after radiotherapy, chemotherapy and targeted therapy Way. The most successful current tumor immunotherapy is the drug developed for immune checkpoints, which can be summarized into two main types: one is to use targeting molecules to release the inhibitory effect of immunosuppressive checkpoint molecules on the immune system to kill tumor cells , so as to kill tumors; drugs such as CTLA-4 antibody, PD-1 antibody, and PD-L1 antibody all belong to this category. The other is to use targeting molecules to activate immune checkpoint activating molecules that help t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/12C12N5/10A01K67/027
CPCC07K14/70578C12N15/8509A01K67/0278A01K2227/105A01K2267/0331
Inventor 费俭孙瑞林王津津周宇
Owner SHANGHAI BIOMODEL ORGANISM SCI & TECH DEV
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