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A kind of htra2 fusion gene and its application

A technology that fuses genes and DNA sequences, applied in the field of biomedicine, can solve problems such as unsatisfactory effects and inability to change the root cause of mitochondrial damage, and achieve the effect of preventing accumulation and improving protein levels

Inactive Publication Date: 2017-02-15
THE WEST CHINA SECOND UNIV HOSPITAL OF SICHUAN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since PQQ does not directly inhibit the mitochondrial transport of denatured proteins or remove denatured proteins in mitochondria, it cannot change the root cause of mitochondrial damage, and the effect is often not ideal

Method used

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  • A kind of htra2 fusion gene and its application
  • A kind of htra2 fusion gene and its application
  • A kind of htra2 fusion gene and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Construction of the MOM-HtrA2 fusion gene of the present invention and its expression vector

[0035] Specific steps are as follows:

[0036] 1) Use primer 1 containing Acc65I site and primer 2 containing BamHI site to PCR amplify the 1-201 nucleotide sequence of human mitochondrial outer membrane protein USP30 cDNA, namely USP30 (1-201) fragment.

[0037] 2) Use the primer 3 containing the BglII site and the primer 4 containing the XbaI site to PCR amplify the 400-1374 nucleotides in the protease HtrA2 cDNA, that is, the HtrA2 (400-1374) fragment.

[0038] 3) Digest the USP30 (1-201) fragment with restriction enzymes Acc65I and BamHI, and digest the HtrA2 (400-1374) fragment with BglII and XbaI.

[0039] 4) Purify and recover the cleaved fragments by agarose gel electrophoresis, and connect them into mammalian cell expression vectors that have been digested with Acc65I and XbaI. After transformation and plating, pick a single colony, extract the plasmid DNA, and use ...

Embodiment 2

[0041] The implementation mode of this embodiment is basically the same as embodiment 1, on this basis:

[0042] The DNA sequence of the primer 1 is:

[0043] 5’-GATC GGTACC GCCACC ATG ACC GCC GCC GAC AGG GCC-3’

[0044] The DNA sequence of the primer 2 is:

[0045] 5’-GATC GGATCC CAC AAG CCC TTT TCT ACG CTT-3’

[0046] The DNA sequence of the primer 3 is:

[0047] 5’-GATC AGATCT GGG GGT CGG GGT CCT CCG GCC-3’

[0048] The DNA sequence of the primer 4 is:

[0049] 5'-GATC TCTAGA TTC TGT GAC CTC AGG GGT CAC-3'.

Embodiment 3

[0051] Construction of the MOM-HtrA2 fusion gene of the present invention and its expression vector

[0052] Specific steps are as follows:

[0053] 1) Use the primer 1 containing the Acc65I site and the primer 2 containing the BamHI site to PCR amplify the 1-201 nucleotide sequence in the human mitochondrial outer membrane protein USP30 cDNA, that is, the USP30 (1-201) fragment; PCR cycle The conditions were: denaturation at 94 °C for 30 seconds, annealing at 55 °C for 30 seconds, and extension at 72 °C for 1 minute, a total of 30 cycles.

[0054] 2) Use the primer 3 containing the BglII site and the primer 4 of the XbaI site to PCR amplify the 400-1374 nucleotides in the protease HtrA2 cDNA, that is, the HtrA2 (400-1374) fragment; the conditions of the PCR cycle are: 94 ° Denaturation at C for 30 seconds, annealing at 55 °C for 30 seconds, extension at 72 °C for 1 minute, a total of 30 cycles.

[0055] 3) Digest the USP30 (1-201) fragment with restriction endonucleases Acc...

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Abstract

The invention discloses an HtrA2 fusion gene which is applied to diseases caused by mitochondria damage. The gene is formed by fusing the location sequence of mitochondria outer membrane protein USP30, a protease functional domain of HtrA2 and a PDZ functional domain. After being expressed in cells, MOM-HtrA2 protein can realize targeting location on the surface of the mitochondria to degrade various denatured proteins trying to enter the mitochondria, so as to prevent the denatured proteins from being accumulated in the mitochondria, thus fundamentally eliminating denatured proteins causing mitochondria damage and solving the problem the prior art that antioxidant medicines cure symptoms instead of diseases.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to an HtrA2 fusion gene and its application. Background technique [0002] Mitochondria damage is one of the common pathological features of organ degenerative diseases. Many of the known degenerative neurological diseases are caused by denatured proteins that damage the structure and function of mitochondria. For example, Aβ protein fragments related to Alzheimer's disease, mutant α-Synuclein protein related to Parkinson's disease, mutant SOD1 protein related to amyotrophic lateral sclerosis, etc., can all accumulate on the surface or inside of mitochondria and form insoluble protein aggregates body, destroying the mitochondrial structure, leading to neuronal death. In addition to the accumulation of denatured proteins caused by mutated genes, in neurons of the elderly, due to the decline in the accuracy of protein synthesis and the activity of proteasomes, some mitochondrial...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/62C12N15/10A61K48/00A61P25/28A61P25/16A61P25/00
Inventor 姜长安张婷胡袁
Owner THE WEST CHINA SECOND UNIV HOSPITAL OF SICHUAN