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Gene recombined virus of human neurenergen 3

A neurotrophin and recombinant virus technology, applied in the field of genetic engineering, to achieve a wide range of hosts, overcome protein instability, and good safety

Active Publication Date: 2007-05-09
GUANGZHOU DOUBLLE BIOPRODUCT CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The exogenous human NT-3 factor used for treatment is difficult to pass through the blood-brain barrier and cannot enter the central nervous system, so the route of administration of neurotrophic factor drugs has become a bottleneck limiting its clinical application

Method used

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  • Gene recombined virus of human neurenergen 3
  • Gene recombined virus of human neurenergen 3
  • Gene recombined virus of human neurenergen 3

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1: As shown in Figure 1, Figure 2 and Figure 3, construct recombinant adenovirus

[0052] The recombinant adenovirus expression vector of human neurotrophin-3 (NT-3) gene was constructed.

[0053] The full-length cDNA of NT-3 gene was amplified from human brain tissue mRNA, and cloned in the shuttle plasmid pShuttle to obtain an expression cassette with a CMV promoter. Then the expression cassette was ligated with adenovirus backbone DNA (Adeno-X viral DNA) in vitro to form a recombinant adenovirus plasmid (pAd-NT-3). Human embryonic kidney 293 cells were transfected with pAd-NT-3 and packaged into infectious recombinant adenovirus particles (Adeno-NT-3).

[0054] (1) Primer design and synthesis: The human NT-3 gene was retrieved from GenBank, and primers at both ends were designed for the full length of the gene: 5'-CGTCTAGAACAAGGTGTCCAT-3' and 5'-CAGGTACCAATTCATGTTCTTCCG-3'. The 5′ ends of the upstream and downstream primers contain XbaI and KpnI restriction...

Embodiment 2

[0060] Example 2: Functional identification of recombinant NT-3 adenovirus

[0061] 293 cells were infected with Ad-NT-3, immunocytochemistry, ELISA and Western blot were performed to detect the expression of Ad-NT-3 in vitro.

[0062] (1) Immunocytochemistry: 293 cells were infected with crudely lysed virus liquid, and after about 12 hours of culture, 293 cells were fixed with 4% paraformaldehyde, and rabbit anti-human NT-3 was used as the primary antibody for immunocytochemical staining, and DAB was used for color development. The negative control 293 cells were infected with Ad-LacZ, and the blank control 293 cells were not infected with adenovirus. The results are shown in Figure 9, the 293 cells infected with Ad-NT-3 were strongly positively stained, the 293 cells infected with Ad-LacZ and the 293 cells themselves were negatively stained.

[0063] (2) ELISA: Inoculate 293 cells (4.5×105 cells / well) in a 6-well plate, add Ad-NT-3 virus solution with an MOI of 1, and repla...

Embodiment 3

[0066] Example 3: Effect of Ad-NT-3 on neural stem cell differentiation in vitro

[0067] The implementation materials were divided into 4 groups, group A was NT-3 gene modified Schwann cells + neural stem cells, group B was LacZ gene modified Schwann cells + neural stem cells, group C was Schwann cells + neural stem cells, group D For the pure neural stem cell group.

[0068] A total of 48 wells of two 24-well culture plates were divided into 4 groups with 12 wells in each group, and a sterilized cover glass was added to each well, and plated with poly-lysine. Cultured and purified Schwann cells were trypsinized and made into 2.5×10 4 cells / mL. Add 200 μL of cell suspension to each well of groups A, B, and C, and after culturing for 24 hours, groups A and B were inoculated with Ad-NT-3 and Ad-LacZ according to the above method, group C was not inoculated with adenovirus, and group D was only inoculated with adenovirus. Polylysine decking. The cultured and purified neural ...

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Abstract

This invention discloses a recombinant adenovirus, which is constructed by inserting exogenous human NT-3 gene into E1 / E3-deleted replication-defective adenovirus to replace E1 region. The recombinant adenovirus can be directly expressed in eukaryotic cells. Expression of NT-3 gene is controlled by CMV promoter. Therefore, NT-3 gene can be specifically transferred into damaged central nerve cells through the recombinant adenovirus to express NT-3. The recombinant adenovirus can be used for repairing human central nerve damage and treating nervous system diseases.

Description

Technical field: [0001] The invention belongs to the field of genetic engineering, in particular to the preparation and application of recombinant adenovirus containing human neurotrophin-3 (NT-3) gene obtained through gene recombination. Specifically, the present invention relates to an adenovirus vector, neurotrophin-3 (NT-3), a genetic engineering product combining the two through recombination, a preparation method of the product and an application in treating nervous system diseases. Background technique: [0002] Existing scientific studies have shown that numerous human nervous system diseases are caused by damage to the central nervous system. The key to treating such diseases is to nourish and repair the damaged central nervous system, and one of the main reasons why neuron axons cannot regenerate after central nervous system injury is the lack of a microenvironment that induces the regeneration of damaged axons. [0003] Neurotrophic factors (neurotrophic factors,...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N15/12C12N15/86C12N7/08A61K48/00A61P25/00
Inventor 黄文林王俊梅
Owner GUANGZHOU DOUBLLE BIOPRODUCT CO LTD
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