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Recombinant adeno-associated virus packaging plasmid, recombinant adeno-associated virus and application of recombinant adeno-associated virus packaging plasmid

A virus packaging and adenovirus technology, applied in the biological field, can solve the problem that the retrograde labeling efficiency needs to be further improved, and achieve the effect of wide application value and market prospect, good tools and technical support

Pending Publication Date: 2022-03-18
SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The reported recombinant adeno-associated virus rAAV2 / retro has higher retrograde labeling efficiency than other adeno-associated viruses (Neuron,2016,92(2):372-382.), but there is brain region selectivity (NeurosciBull,2020 ,36(3):202-216.), mainly infected cortical neurons, and its retrograde labeling efficiency needs to be further improved

Method used

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  • Recombinant adeno-associated virus packaging plasmid, recombinant adeno-associated virus and application of recombinant adeno-associated virus packaging plasmid
  • Recombinant adeno-associated virus packaging plasmid, recombinant adeno-associated virus and application of recombinant adeno-associated virus packaging plasmid
  • Recombinant adeno-associated virus packaging plasmid, recombinant adeno-associated virus and application of recombinant adeno-associated virus packaging plasmid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: Construction of recombinant adeno-associated virus packaging plasmid

[0042] According to the AAV11 genome sequence (GenBank: AY631966.1), the Cap11 gene sequence was synthesized, and as a template, the Cap11 fragment was amplified using Takara Primerstar Polymerase (Takara Company), and the sequence of the forward primer Cap11-F was shown in SEQ ID NO.1, The sequence of the reverse primer Cap11-R is shown in SEQ ID NO.2; the PCR reaction system is 50μl: 5×Reaction Buffer 10μl, 10mM dNTPs 1μl, 10μM forward primer 2.5μl, 10μM reverse primer 2.5μl, template DNA 0.5 μl, DNA Polymerase 0.5 μl, ddH 2 O 33 μl. The amplification conditions were: 98°C for 3 min, 98°C for 20 s, 60°C for 20 s, 72°C for 2 min, 72°C for 10 min, 16°C for 30 min, 30 cycles; the amplified DNA fragment was recovered with a gel recovery kit (Omega).

[0043] SwaI and AgeI (New England Biolabs) were used to digest the pAAV-RC2 / 1 vector (purchased from Addgene, number: 112862) and the Cap11 ...

Embodiment 2

[0044] Example 2: Preparation of recombinant adeno-associated virus

[0045]The recombinant adeno-associated virus packaging plasmid pAAV-RC2 / 11 expression vector was used to package the recombinant adeno-associated virus, and the adeno-associated virus core plasmid pAAV-EF1α-EGFP-WPRE-hGH polyA was combined with pAAV-RC2 / 11 The viral element helper plasmid pAd-Helper (purchased from Addgene, number: 112867) was co-transfected into HEK-293T cells according to the number of plasmid molecules 1:1:1. After 72 hours of transfection, the supernatant and cell pellet were collected and processed separately: containing AAV The HEK-293T cell pellet of virus particles was resuspended in the lysate (15 dishes plus 9 mL), and after repeated freezing and thawing in liquid nitrogen and 37°C water bath, added Benzonase nuclease (Sigma, E1014-25KU) to digest at 37°C for 1 hour. Then add a final concentration of 150mM NaCl and shake it at 37°C for 30min; after the cell supernatant is treated w...

Embodiment 3

[0046] Example 3: In vivo application of recombinant adeno-associated virus

[0047] (1) The prepared rAAV2 / 11-EF1α-EGFP-WPRE-hGH polyA (300nL / mouse) virus (rAAV2 / 11) was injected into 8-10 week old C57BL / 6 mice (purchased from Hunan The caudoputamen (Caudoputamen, CPu) region of Slack Jingda Experimental Animal Co., Ltd., after 3 weeks of sufficient expression, injected 0.5ml 1% pentobarbital sodium-0.9% sodium chloride solution in the mouse intraperitoneal cavity Anesthesia was performed, and the brain tissue was stripped by cardiac perfusion with phosphate buffered saline (Phosphate Buffer Saline, PBS) and 4% paraformaldehyde solution (Paraformaldehyde Solution, PFA). Mouse brain tissue was fixed with PFA solution overnight and then dehydrated with 30% sucrose-PBS solution. The dehydrated brain tissue was fully embedded with tissue embedding medium and cut into 40 μm thick brain slices with a cryostat. Olympus It was imaged with a VS120 slide scanning microscope (Olympus, ...

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Abstract

The invention discloses a recombinant adeno-associated virus packaging plasmid, a recombinant adeno-associated virus and application of the recombinant adeno-associated virus packaging plasmid. The recombinant adeno-associated virus packaging plasmid comprises a Rep gene of a type 2 adeno-associated virus and a Cap gene of a type 11 adeno-associated virus. The recombinant adeno-associated virus is obtained by co-transfecting a packaging cell line with the recombinant adeno-associated virus packaging plasmid, an adeno-associated virus core plasmid and an adenovirus element helper plasmid. The recombinant adeno-associated virus prepared by the invention has the characteristics of marking a specific brain region and an upstream connection network thereof, and the retrograde marking efficiency is higher. The problem that an existing retrograde marking method is low in efficiency is solved, better tools and technical supports are provided for neuroscience research, disease model establishment, gene therapy and the like, and the method has wide application value and market prospects.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a recombinant adeno-associated virus packaging plasmid, a recombinant adeno-associated virus and applications thereof. Background technique [0002] Viral vectors, especially those that allow efficient gene transfer from axon terminals to the central nervous system (CNS), are useful for dissecting the structure and function of neural circuits in specific brain regions, and for delivering piggybacked therapeutic genes to distant sites. It has become one of the most potential and promising therapeutic tools. In contrast to traditional retrograde tracers, viral vectors can express genes for manipulation of neuronal activity or gene therapy. Some neurotropic viruses have retrograde infection ability, such as herpes simplex virus (HSV), pseudorabies virus (PRV), lentivirus (LV), canine adenovirus (CAV), rabies virus (RABV), etc., but the toxicity is high or the preparation pro...

Claims

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Application Information

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IPC IPC(8): C12N15/864C12N7/01C12N15/65C12N15/34
CPCC12N15/86C12N7/00C12N15/65C07K14/005C12N2750/14121C12N2750/14122C12N2750/14143C12N2750/14152
Inventor 林坤章韩增鹏骆能松徐富强
Owner SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
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