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Recombinant adeno-associated virus and application thereof

A technology of adenovirus and virus, which is applied in the field of recombinant adeno-associated virus and its application, can solve the problems of toxic side effects, unclear treatment mechanism, unpredictable application prospects, etc., and achieve the effect of reducing protein level and increasing expression

Inactive Publication Date: 2017-07-21
SHENZHEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The treatment mechanism of these drugs is not clear, and it is not sure whether they will produce exact curative effects, and some of them even have serious side effects
In addition, most of these drugs can only relieve the superficial symptoms of AD and cannot prevent the rapid development of AD
However, some drugs that have been shown to prevent and treat AD in basic research cannot predict their application prospects because they are not easy to pass through the blood-brain barrier.

Method used

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  • Recombinant adeno-associated virus and application thereof
  • Recombinant adeno-associated virus and application thereof
  • Recombinant adeno-associated virus and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Preparation of recombinant adeno-associated virus

[0037] 1. Reagents used:

[0038] 1.293 cells;

[0039] 2. Recombined adenovirus plasmid;

[0040] 3.3. Pac I restriction endonuclease;

[0041] 4. Reagents related to plasmid recovery;

[0042] 5. Reagents related to cell culture and transfection;

[0043] 6. TBS: 10mM Tris, 0.9% NaCl, pH8.1;

[0044] 7.40% CsCl: 28.45g CsCl dissolved in 42.7ml of TBS, stored at 4 degrees;

[0045] 8.15% CsCl: 9.085g CsCl dissolved in 47.69ml of TBS, stored at 4 degrees;

[0046] 9.Beckman centrifuge tube: 14*89mm, SW41 rotor;

[0047] 10. Polybrene (sigma), 10mg / ml;

[0048] 11. Dialysis bag: Spectrum Co. (supplied in rolls, with a small amount of glycerin, sulfide and heavy metals), MW=8000~14400;

[0049] 12. Sterilized glycerin.

[0050] 2. Operation process:

[0051] 1.293 cells were seeded in 1 or 2 60mm culture dishes 24 hours before transfection, so that the cell confluence rate was 50-70% at the time of t...

Embodiment 2

[0070] Example 2 Stereotaxic injection of recombinant adeno-associated virus containing SelP-H gene into mouse hippocampus CA3 region

[0071] The 3-month-old model mice were anesthetized by intraperitoneal injection of 10% chloral hydrate (3.5mg / kg), and fixed on the stereotaxic apparatus, the head hair was cut off, 75% alcohol was disinfected, and longitudinally cut along the midline of the top of the head. On the scalp, the experimental group injected 2 μL of recombinant adeno-associated virus rAAV9-GFP-SelP-H with SelP-H and green fluorescent protein genes with a microinjector, (coordinates are X=±2.30mm, Y=-2.18mm, Z= -2.10mm), the injection time is 4min, and the injection speed is 0.5μL / min. The blank control group was injected with the same amount of control adenovirus rAAV9-GFP by the same method. The rats were continued to be fed after the operation, and the rats were killed 1 month and 3 months after the operation to determine the protein expression by the fluoresce...

Embodiment 3

[0073] Embodiment 3Morris water maze detects the influence of SelP-H on the spatial exploration and memory ability of AD model mice

[0074] Experimental mice: divided into four groups, one group was 7-month-old wild-type mice injected with rAAV9-GFP, one group was 7-month-old wild-type mice injected with rAAV9-SelP-H, and one group was 7-month-old injected rAAV9 -GFP AD model mice, a group of 7-month-old AD model mice injected with rAAV9-SelP-H. 20 in each group.

[0075] The Morris water maze experiment is divided into two parts: positioning navigation and space exploration. Among them, the positioning flight lasted 5 days, and the space exploration took two days. The first day of positioning navigation is the training period. The mouse is placed on the platform for 10 seconds to adapt, and then the mouse is put into the pool from the corresponding position in different quadrants facing the wall. The recording is terminated after the mouse boards the platform for 2 seconds...

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Abstract

The invention is applicable to the technical field of biological medicines, and provides a recombinant adeno-associated virus and an application thereof. The recombinant adeno-associated virus is formed by genetic recombination of SelP-H virus and adenovirus. The recombinant adeno-associated virus provided by the invention is formed by genetic recombination of the SelP-H virus and the adenovirus, and can be stably expressed in a specific area of the head of an animal. The recombinant adeno-associated virus can improve the quantity of nissl bodies in the head of the animal, reduce a protein level of GSK3[beta] and phosphorylation in the head of the animal, meanwhile improve the expression quantity of PP2A as well as the expression quantity of neurosynaptic proteins. According to the above effects, the recombinant adeno-associated virus provided by the embodiment of the invention can play a role in controlling content and distribution of various metal ions.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, in particular to a recombinant adeno-associated virus and its application. Background technique [0002] Alzheimer's disease (AD) is a progressive neurodegenerative disease, also known as primary Alzheimer's disease. The clinical symptoms of AD are mainly manifested as progressive memory impairment, cognitive dysfunction and language impairment, etc. It is a disease that seriously affects the social life and social life of patients and brings heavy burdens to families and society. The main pathological features of AD patients are a large number of senile plaques (Senile plaque, SP) and neurofibrillary tangles (Neurofibrillary tangle, NFT) observed in the brain. Neurofibrillary tangles hypothesis caused by abnormal phosphorylation of tau protein. [0003] According to the above pathogenesis hypothesis, researchers have developed many new drugs and treatment methods for AD in recent years. Th...

Claims

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Application Information

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IPC IPC(8): C12N7/01A61K35/761A61P25/28
CPCA61K35/761C12N7/00C12N2750/14121C12N2750/14143
Inventor 都秀波谭毅彬刘琼倪嘉缵
Owner SHENZHEN UNIV
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