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24-dehydrocholesterol reductase coded recombinant adenoviruses specifically expressed in tissue

A technology of dehydrocholesterol and recombinant adenovirus, applied in virus/phage, biochemical equipment and method, application, etc., can solve problems such as difficulty

Active Publication Date: 2012-11-21
LIAONING UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no drugs that apply gene therapy to treat AD in the market, and there are very few gene therapy drugs in trials. Purpose

Method used

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  • 24-dehydrocholesterol reductase coded recombinant adenoviruses specifically expressed in tissue
  • 24-dehydrocholesterol reductase coded recombinant adenoviruses specifically expressed in tissue
  • 24-dehydrocholesterol reductase coded recombinant adenoviruses specifically expressed in tissue

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: Construction of a defective adenovirus expressing the coding gene of DHCR24 (see Genbank).

[0049] This example describes the construction of a defective recombinant adenoviral vector carrying the gene encoding DHCR24, which is linked to the SYN promoter by genetic engineering techniques.

[0050] Firstly, HEK293 genome DNA (ATCC CRL-1573) was used as a template, Kpn I and BstX I restriction sites were added to the upstream and downstream primers, and the promoter sequence of SYN1 with a length of 500 bp was obtained by two-step nested PCR amplification ( P SYN1).

[0051] hSYN1-Ex S: 5-gcctgtgtggatgtgggagactaat-3

[0052] hSYN1-Ex AS: 5-tgcaggtctgtcatgtacccatttg-3

[0053] hSYN1-In S-KpnI: 5-ggtacctgacgaccgaccccg-3

[0054] hSYN1-In AS-BstXI: 5ccagtgtggtggggctgcgacttgggg-3

[0055] The obtained PCR amplification product of PSYN1 was cloned and sequenced by TA, the obtained positive clone was amplified by Escherichia coli, and the DNA was extracted ...

Embodiment 2

[0061] Example 2: Expression of DHCR24 gene in neural cells and other cell lines infected by Ad-DHCR24 vector in vitro.

[0062] The expression of DHCR24 after infection with Ad-PSYN1-hDHCR24 vector was tested on different cell lines: neuroblastoma cell line (N2A) and islet β-cell line (MIN6).

[0063] After infecting the cells with Ad-PSYN1-DHCR24 vector at 20 moi (multiplicity of infection) for 48 hours, the cells were collected and the total cell protein was extracted. Ad-CMV-DHCR24 was used as a control (recombinant adenovirus driven by CMV ubiquitous expression promoter to express DHCR24, for details, see the literature published by Lu et al. in Endocrinology, 149: 3267, 2008). Then Western Blotting was used to detect the expression of DHCR24 protein. The antibody uses anti-myc antibody (myc is the C-terminal tag sequence of DHCR24 protein). The result is as image 3 As shown, in the Ad-CMV-DHCR24 transfection group, both N2A nerve cells and MIN6 islet β cells were o...

Embodiment 3

[0064] Example 3: Study on the protective effect of Ad-PSYN1-DHCR24 on nerve cells.

[0065] The Ad-PSYN1-DHCR24 vector was used to infect N2A nerve cells at 20 moi, and the recombinant adenovirus Ad-CMV-LacZ (LacZ, β-galactosidase, a bacterial protein) was used as a control. 48 hours after adenovirus infection, the cells were treated with 200 μM hydrogen peroxide (oxidative stress inducer), 1 μg / ml tunicamycin (endoplasmic reticulum stress inducer) and 1 μM Aβ 1-40 (Typical pathological factors of Alzheimer's disease can lead to nerve cell apoptosis) stimulation, after 48 hours, the cells were photographed and counted with a phase contrast microscope, and the statistical results were as follows: Figure 4 shown. Ad-PSYN1-DHCR24 transfected cells in the three kinds of stimuli, compared with the control group Ad-CMV-lacZ, showed a larger number of viable cells, proving that DHCR24 has a significant protective effect on nerve cells.

[0066] All these results demonstrate tha...

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Abstract

The invention relates to belongs to 24-dehydrocholesterol reductase coded recombinant adenoviruses specifically expressed in tissue, which belongs to the Alzheimer's disease gene treatment method and the treatment field. More specifically, according to the invention, by aid of a gene adenovirus carrier, the coded gene of 24-dehydrocholesterol reductase (DHCR 24) is introduced in nerve cells, the expression of 24-dehydrocholesterol protein in nerve cells is regulated, and the nerve cells protection effect can be performed. The invention also relates to a construction of defective recombinant adenoviruses for replication comprising nerve specifically expressed DHCR 24 gene and an application of the carriers in DHCR 24 gene in cells and an application of the DHCR 24 protein in nerve cells specific expression. The prepared recombinant adenoviruses are expected to obtain positive curative effect in neurodegenerative diseases such as Alzheimer's disease or other gene treatments which require a DHCR 24 supplementary therapy.

Description

technical field [0001] The invention relates to the field of Alzheimer's disease gene therapy and treatment. More specifically, the present invention relates to the use of genetic engineering technology to construct dhcr24 Gene replication-deficient recombinant adenoviruses, and these adenoviral vectors in the future dhcr24 Application of gene transfer into cells and realization of specific overexpression of DHCR24 protein in cells such as nerve cells. The prepared recombinant adenovirus is expected to obtain positive curative effect in the gene therapy of diseases such as Alzheimer's disease and diabetes. Background technique [0002] Alzheimer's disease (AD) is a neurodegenerative disease with insidious onset, clinically characterized by memory and cognitive dysfunction, and is the most common type of senile dementia. Relevant experts predict that by 2025, 22 million people in the world will suffer from AD; it is also estimated that the market value of this treatment ar...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N15/53
Inventor 芦秀丽高兵刘剑利赵晨光贾丹
Owner LIAONING UNIVERSITY
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