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
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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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