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A kind of agbl5 nucleotide sequence encoding cytoplasmic carboxypeptidase-like protein 5 and its application

A technology of nucleotide sequence and carboxypeptidase, applied in the direction of peptide/protein composition, peptidase, application, etc., can solve the problem of transducing retinal tissue cells, and achieve the effect of reducing the degree of glutamic acid

Active Publication Date: 2022-07-15
WUHAN NEUROPHTH BIOTECHNOLOGY LTD CO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Naturally occurring AAV serotypes are generally unable to transduce retinal tissue cells on the side of the vitreous cavity due to the presence of barriers such as the inner limiting membrane, glial cells, etc. that prevent the spread of AAV virions

Method used

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  • A kind of agbl5 nucleotide sequence encoding cytoplasmic carboxypeptidase-like protein 5 and its application
  • A kind of agbl5 nucleotide sequence encoding cytoplasmic carboxypeptidase-like protein 5 and its application
  • A kind of agbl5 nucleotide sequence encoding cytoplasmic carboxypeptidase-like protein 5 and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0042] Example 1: Codon-optimized AGBL5 vector construction and expression verification

[0043] (1) Construction of plasmid vector

[0044] 1. The AAV-CMV plasmid backbone, the coAGBL5 fragment and the wtAGBL5 fragment were digested simultaneously with HindIII and XhoI, respectively, and then the digested fragments were ligated to the backbone respectively.

[0045] 2. The ligation product was transformed into Escherichia coli, and a single colony was picked for digestion verification and sequencing verification.

[0046] (2) Cell transfection

[0047] 1. One day before transfection, cells were trypsinized and counted, and the cells were plated so that the density was 90% on the day of transfection.

[0048] 2. For each well of cells, dilute 0.8 μg-1.0 μg DNA in 50 μl serum-free DMEM medium.

[0049] 3. For each well of cells, dilute 1 μl-3 μl of LIPOFECTAMINE 2000 reagent with 50 μl DMEM medium. After dilution with LIPOFECTAMINE 2000, mix with the diluted DNA within 5 mi...

Embodiment 2

[0072] Example 2: In vitro cell expression and functional verification of AAV-mediated AGBL5

[0073] (1) Cell transfection

[0074] 1. One day before transfection, cells were trypsinized and counted, and the cells were plated so that the density was 90% on the day of transfection.

[0075] 2. For each well of cells, dilute 0.8 μg-1.0 μg DNA in 50 μl serum-free DMEM medium.

[0076] 3. For each well of cells, dilute 1 μl-3 μl of LIPOFECTAMINE 2000 reagent with 50 μl DMEM medium. After dilution with LIPOFECTAMINE 2000, mix with the diluted DNA within 5 minutes.

[0077] 4. Mix diluted DNA and diluted LIPOFECTAMINE 2000 and incubate at room temperature for 20 minutes.

[0078] 5. Add the complex directly to each well, shake the plate, and mix gently.

[0079] 6. At 37°C, 5% CO 2 Incubate for 24-48 hours.

[0080] 7. Lyse cells 24-72 hours after complex addition to cells.

[0081] (2) Tubulin glutamate detection

[0082] 1. Centrifuge the cell lysate at 40,000g for 20 min...

Embodiment 3

[0102] Example 3: AAV2 / 2.7M8-coAGBL5 gene therapy drug ameliorated abnormal retinal tubulin glutamination in mice

[0103] (1) Virus packaging, virus drug injection into mice

[0104] 1. HEK293T cells with a degree of polymerization of more than 90% were plated at a ratio of 1:3.

[0105] 2. About 1-2 hours before transfection, change to serum-free medium, and use transfection reagent to transfer the target gene plasmid and helper plasmid (containing AAV2.7M8 serotype element) into HEK293T.

[0106] 3. After 24h of plasmid transformation, change to a new serum-free medium

[0107] 4. 72h of transfection to collect poison. With medium, cells are blown off and centrifuged; the medium supernatant and cell pellet are then harvested separately. The virus in the supernatant of the medium was precipitated with PEG8000, and the virus pellet was collected after overnight precipitation.

[0108] 5. The virus mixture was purified by iodixanol density gradient centrifugation, and then...

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Abstract

The invention relates to the technical field of biomedical gene therapy, and discloses an AGBL5 nucleotide sequence encoding a cytoplasmic carboxypeptidase-like protein 5 and an application thereof. The nucleotide sequence of the present invention is ≥95% identical to the nucleotide sequence shown in SEQ ID NO: 3. The present invention confirms that the codon-optimized AGBL5 coding sequence has a higher protein expression efficiency than the wild-type sequence, and that HEK cells transfected with the AAV-AGBL5 plasmid containing the nucleotide sequence can express the AGBL5 protein, and the protein can function correctly . At the same time, AAV2 / 2.7M8‑coAGBL5 drug treatment can express the protein in the diseased eyes of AGBL5 knockout mice, and significantly improve the hyperglutamination of tubulin caused by AGBL5 deficiency, which has the effect of preventing or treating retinitis pigmentosa.

Description

technical field [0001] The invention relates to the technical field of biomedical gene therapy, and more particularly to an AGBL5 nucleotide sequence encoding cytoplasmic carboxypeptidase-like protein 5 and its application. Background technique [0002] AGBL5 encodes cytosolic carboxypeptidase-like protein 5 (Cytosolic carboxypeptidase-like protein 5), also known as CCP5. It is a metallocarboxypeptidase that mediates the deglutamination modification of proteins. AGBL5 belongs to a family of CCP-like proteins that specifically catalyzes the deglutamination of branch-point glutamate side chains such as those produced by post-translational glutamination of tubulin. It cannot act as a long-chain deglutaminase to shorten long polyglutamate chains, a process catalyzed by AGTPBP1, AGBL1, AGBL2, AGBL3, and AGBL4. Since AGBL5 is the only enzyme in the CCP-like protein family that recognizes branched glutamate sites, its absence in vivo may not be compensated by isozymes. AGBL5 als...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/57C12N9/48C12N15/864A61K48/00A61P27/02A61P9/10
CPCC12N9/485C12N15/86A61K48/0075A61K38/4813A61K48/005A61P27/02A61P9/10C12Y304/17022C12N2800/22C12N2750/14143A61K38/00
Inventor 李斌任盛
Owner WUHAN NEUROPHTH BIOTECHNOLOGY LTD CO
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