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AGBL5 nucleotide sequence for encoding cytoplasm carboxypeptidase protein 5 and application of AGBL5 nucleotide sequence

A nucleotide sequence and carboxypeptidase technology, applied in the direction of peptide/protein components, peptidase, application, etc., can solve the problem of transducing retinal tissue cells, so as to prevent or treat retinitis pigmentosa and improve retinal pathological symptoms Effects of improving and restoring normal function

Active Publication Date: 2021-03-12
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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  • AGBL5 nucleotide sequence for encoding cytoplasm carboxypeptidase protein 5 and application of AGBL5 nucleotide sequence
  • AGBL5 nucleotide sequence for encoding cytoplasm carboxypeptidase protein 5 and application of AGBL5 nucleotide sequence
  • AGBL5 nucleotide sequence for encoding cytoplasm carboxypeptidase protein 5 and application of AGBL5 nucleotide sequence

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0043] (1) Plasmid vector construction

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

[0045] 2. The ligation product was transformed into E. coli, and a single colony was picked for enzyme 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 on the day of transfection was 90%.

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

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

[0...

Embodiment 2

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

[0073] (1) Cell transfection

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

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

[0076] 3. For each well of cells, use 50 μl of DMEM medium to dilute 1 μl-3 μl of LIPOFECTAMINE 2000 reagent. After dilution of 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 culture plate, and mix gently.

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

[0080] 7. Cells were lysed 24-72 hours after adding the complex to the cells.

[0081] (2) Detection of tubulin glutamate

[0082] 1. Centrifuge the...

Embodiment 3

[0102] Example 3: AAV2 / 2.7M8-coAGBL5 gene therapy drugs improve the abnormal glutamate of retinal tubulin in mice

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

[0104] 1. HEK293T cells with a degree of polymerization above 90% are transferred in a ratio of 1:3.

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

[0106] 3. After 24 hours of plasmid transformation, replace with new serum-free medium

[0107] 4. The virus was harvested after 72 hours of transfection. With the culture medium, blow down the cells and centrifuge; then harvest the culture supernatant and cell pellet separately. The virus in the medium supernatant was precipitated with PEG8000, and the virus pellet was collected after precipitation overnight.

[0108] 5. The virus mixture was purified by iodixanol density gradient ce...

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Abstract

The invention relates to the technical field of biomedical gene therapy, and discloses an AGBL5 nucleotide sequence for encoding cytoplasm carboxypeptidase protein 5 and application of the AGBL5 nucleotide sequence. The nucleotide sequence disclosed by the invention is more than or equal to 95% identical to the nucleotide sequence shown in SEQ ID NO: 3. According to the present invention, the efficiency of the codon-optimized AGBL5 coding sequence expression protein is proved to be higher than the efficiency of the wild-type sequence, the AAVAGBL5 plasmid transfected HEK cell containing the nucleotide sequence can express the AGBL5 protein, and the protein can correctly perform the function. Meanwhile, the AAV2 / 2.7 M8-coAGBL5 drug treatment can be used for knocking out lesion eye expression protein of a mouse in AGBL5 and remarkably improving tubulin high glutamic acid caused by defects of the AGBL5, and has the effect of preventing or treating retinal pigment degeneration.

Description

technical field [0001] The invention relates to the technical field of biomedical gene therapy, and more specifically relates to an AGBL5 nucleotide sequence encoding cytoplasmic carboxypeptidase protein 5 and its application. Background technique [0002] AGBL5 encodes cytoplasmic carboxypeptidase-like protein 5 (Cytosolic carboxypeptidase-like protein5), also known as CCP5. It is a metallocarboxypeptidase that mediates the deglutamation modification of proteins. AGBL5 belongs to the CCP-like protein family and can specifically catalyze the deglutamination of branch point glutamate side chains generated by post-translational glutamylation of tubulin. It does not act as a long-chain deglutaminase to shorten long polyglutamic acid chains, a process catalyzed by AGTPBP1, AGBL1, AGBL2, AGBL3, and AGBL4. Since AGBL5 is the only enzyme in the CCP-like protein family that can recognize branched glutamation sites, its absence in vivo may not be compensated by isozymes. AGBL5 als...

Claims

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Application Information

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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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