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
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  • 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

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
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Example Embodiment

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

[0052] Construction of human neurotrophin-3 (neurotrophin-3, NT-3) gene recombinant adenovirus expression vector.

[0053] The full-length cDNA of NT-3 gene was amplified from human brain tissue mRNA and cloned into the shuttle plasmid pShuttle to obtain an expression cassette with CMV promoter. Then connect the expression cassette with 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) Primers design and synthesis: Retrieve human NT-3 gene from GenBank, design primers at both ends for the full length of the gene: 5'-CGTCTAGAACAAGGTGTCCAT-3' and 5'-CAGGTACCAATTCATGTTCTTCCG-3'. The 5′ ends of the upstream and downstream primers contain restriction enzyme sites for XbaI and KpnI, respectively. The reaction...

Example Embodiment

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

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

[0062] (1) Immunocytochemistry: The crude lysed virus was infected with 293 cells. After about 12 hours of culture, the 293 cells were fixed with 4% paraformaldehyde. The rabbit anti-human NT-3 was used as the primary antibody for immunocytochemical staining, and DAB developed. The negative control 293 cells were infected with Ad-LacZ, and the blank control 293 cells were not infected by adenovirus. The results are shown in Figure 9, 293 cells infected with Ad-NT-3 showed strong positive staining, and 293 cells infected with Ad-LacZ and 293 cells themselves showed negative staining.

[0063] (2) ELISA: Inoculate 293 cells (4.5×105 cells / well) in a 6-well plate, add Ad-NT-3 virus solution with MOI of 1, and replace it with DMEM culture solution containing 10% newborn calf ser...

Example Embodiment

[0066] Example 3: The effect of Ad-NT-3 on the differentiation of neural stem cells in vitro

[0067] Divide the implementation materials into 4 groups, A group is NT-3 gene modified Schwann cell + neural stem cell group, B group is LacZ gene modified Schwann cell + neural stem cell group, group C is Schwann cell + neural stem cell group, and group D It is a simple neural stem cell group.

[0068] Divide two 24-well culture plates with a total of 48 wells into 4 groups, each with 12 wells, add a sterile cover glass to each well, and plate with polylysine. The Schwann cells after culture and purification are 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. After 24 hours of culture, groups A and B were respectively inoculated with Ad-NT-3 and Ad-LacZ according to the above-mentioned method. Group C was not inoculated with adenovirus, and group D only used more Polylysine paving. The cultured and purified neural stem cell...

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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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