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Construction method and application of TNFRSF1B gene humanized animal model

A construction method and humanization technology, applied in the field of construction of humanized animal models of TNFRSF1B gene, can solve the problems of incomplete application to humans and the like

Pending Publication Date: 2021-04-06
SHANGHAI BIOMODEL ORGANISM SCI & TECH DEV +2
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The screening and preclinical tests of human TNFRSF1B target drugs need to be evaluated in animal models, but due to the difference in species, the homology between human TNFRSF1B and mouse Tnfrsf1b protein is only 62%, and the homology of human TNFRSF1B and mouse Tnfrsf1b protein is only 62%. Targeted drugs are not fully applicable to humans, and drugs targeting human TNFRSF1B protein may not necessarily be able to recognize mouse Tnfrsf1b protein

Method used

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  • Construction method and application of TNFRSF1B gene humanized animal model
  • Construction method and application of TNFRSF1B gene humanized animal model
  • Construction method and application of TNFRSF1B gene humanized animal model

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Experimental program
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Effect test

Embodiment 1

[0066] Embodiment 1 sequence design

[0067] The mouse Tnfrsf1b gene has multiple transcripts, and the human TNFRSF1B gene contains multiple transcripts. The sequence design of this example is mainly described with one of the transcripts as an example. The corresponding human transcript is the human TNFRSF1B gene. The NCBI accession number is Transcript of NM_001066.3 (corresponding TNFRSF1B gene, its Gene ID: 7133); mouse corresponding transcript is mouse Tnfrsf1b gene NCBI accession number is NM_011610.3 transcript (corresponding Tnfrsf1b gene, its NCBI Gene ID :21938).

[0068] In the humanization strategy, the partial sequence of intron 1 of the human TNFRSF1B gene (12188496-12188795 in the NCBI accession sequence NC_000001.11, sequence information such as SEQ ID NO: 1), and the partial coding region of human TNFRSF1B (corresponding to NM_001066. 3 transcript 180-888, sequence information such as SEQ ID NO: 2), the partial coding region of mouse Tnfrsf1b (corresponding to...

Embodiment 2

[0075] Example 2 Design and Construction of Recombinant Vector PBR322-Tnfrsf1b

[0076] 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 expression cassette of human TNFRSF1B CDS-WPRE-polyA, and the 3' homology arm are shown. Wherein the 5' homology arm (SEQ ID NO: 11) is nucleotide 145229407-145233271 of NCBI accession number NC_000070.6, and the 3' homology arm (SEQ ID NO: 12) is NCBI accession number NC_000070. Nucleotides 145226171-145229003 of 6, the expression cassette sequence information of human TNFRSF1B CDS-WPRE-polyA (SEQ ID NO: 13).

[0077] 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 expression frame of human TNFRSF1B CDS-WPR...

Embodiment 3

[0089] Example 3 Verification of the vector PBR322-Tnfrsf1b

[0090] Randomly select 5 PBR322-Tnfrsf1b clones, use the restriction endonuclease SacII to carry out enzyme digestion verification, the digestion products should appear 9058bp, 6023bp size fragments in electrophoresis, and carry out plasmid amplification for the clones with correct digestion. The amplified plasmid was double-verified by the sequencing company for correct sequencing and enzyme digestion, and subsequent experiments were carried out. For the enzyme digestion results of the plasmids used in subsequent experiments, see Figure 4 .

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Abstract

The invention provides a construction method and application of a TNFRSF1B gene humanized animal model. The humanized TNFRSF1B gene modified animal model constructed in the invention can accelerate the research progress in the field related to the human TNFRSF1B gene or protein, for example, the humanized TNFRSF1B gene modified animal model is used for replacing human test drugs, and an effective model and a powerful tool are provided for preclinical experiments of TNFRSF1B target drugs.

Description

technical field [0001] The present application relates to the technical field of genetic engineering, in particular to a construction method and application of a TNFRSF1B gene humanized animal model. Background technique [0002] TNFRSF1B (TNF receptor superfamily member 1b) is a member of the TNF receptor superfamily, also known as TNFR2, whose expression is limited to suppressive immune cells, especially regulatory T cells (Tregs), myeloid suppressor cells (MDSCs), endothelial cells and selected neurons during growth. Its ligand TNF mainly recognizes TNFR1 for apoptosis, and relies on TNFRSF1B for functions related to T cell survival—inducing the non-canonical activation of NFκB by activating NFκB-inducing kinase (NIK), thereby promoting the proliferation and survival of Tregs cells. Many experiments have shown that TNFRSF1B antagonistic antibodies can inhibit the proliferation of tumor cells and Tregs, and can be used in the treatment of various tumors in theory; while a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/12C12N5/10A01K67/027A61K49/00C12Q1/02
CPCC12N15/8509C07K14/70578C12N5/0604A01K67/0278A61K49/0008G01N33/5011C12N2800/107C12N2830/48C12N2830/50A01K2217/072A01K2227/105A01K2267/0331C12N2510/00C12N2503/02G01N2500/10
Inventor 费俭孙瑞林王津津周宇池骏
Owner SHANGHAI BIOMODEL ORGANISM SCI & TECH DEV
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