Nuclear orphan receptor alpha adeno-associated virus vector related to retinoic acid in mice

A technology for orphan nuclear receptors and viral vectors, which is applied in the field of orphan nuclear receptor alpha (RORα) adeno-associated virus vectors and its construction, which can solve the problems of short time and unsuitability for the treatment of chronic immune disorders, and achieve easy preparation , Conducive to efficient transfer and long-term expression, highly stable effect

Inactive Publication Date: 2014-12-24
JIANGSU UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Adenovirus vectors express foreign genes for a short time and have strong immunogenicity, which can trigger strong

Method used

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  • Nuclear orphan receptor alpha adeno-associated virus vector related to retinoic acid in mice
  • Nuclear orphan receptor alpha adeno-associated virus vector related to retinoic acid in mice
  • Nuclear orphan receptor alpha adeno-associated virus vector related to retinoic acid in mice

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Construction of expression vector PZAC2.1-RORα

[0030]1. Cloning of mouse RORα gene and connection with pMD-18T cloning vector.

[0031] (1) Using the Oligo6.0 primer design software, according to the cDNA sequence of mouse RORα in NCBI (NM_013646.2) and the restriction site of the expression vector pZAC2.1 to be connected, the primers for amplifying the full-length RORα gene were designed as follows :

[0032] P1 (upstream primer): 5'-GAATTCACATGGAGTCAGCTCCGGCAG -3', introducing an EcoRI restriction site at its 5' end;

[0033] P2 (downstream primer): 5'-TCTAGAGCGCGACATTTACCCATCGATT-3', an XbaI restriction site was introduced at its 5' end.

[0034] (2) Using RT-RCP (reverse transcription PCR) technology to amplify the full-length coding gene of mouse RORα from mouse brain tissue.

[0035] (3) The PCR product was electrophoresed on a 1% agarose gel, and the results are shown in figure 1 , 2 and 3 in the figure are the amplified RORα...

Embodiment 2

[0044] Example 2: Packaging of rAAV2-RORα

[0045] (1) Rotate the cryopreservation tube of HEK293 cells stored in our laboratory at 37°C until thawed, inoculate 20 10cm culture dishes into DMEM complete culture medium according to 3x106, wait until the growth density reaches 70%-80%, pack For the first 12 hours, fresh culture medium was changed.

[0046] (2) Transfect 293 cells by calcium phosphate co-precipitation method, and package the virus. Among them, pZAC2.1-RORα, pAd-deltaF6, and p5E18 3 plasmids are transfected into each plate according to the content and ratio of 12.5:25:12.5 (μg). For the cells, pZAC2.1-EGFP (gifted by the University of Penesia, preserved in our laboratory) with EGFP fluorescent group was used as the control instead of pZAC2.1-RORα, and the control virus was packaged under the same conditions.

[0047] (3) Configure the transfection mixture:

[0048] Solution A: 59mL sterilized pure water, 6.5 mL 2.5mol / L CaCL2, 1300μg pAd-deltaF6 plasmi...

Embodiment 3

[0055] Example 3: r Purification of AAV2-RORα

[0056] (1) Add 4 mL of heparin agarose to the separation column used for purification, and equilibrate with PBS twice. Then add the packaged filtrate and adjust the flow rate to 1 drop / second until all the filtrate passes through the agarose column.

[0057] (2) Wash the column with the eluent (0.1M NACL PBS), the flow rate is controlled at 1 drop / second, wait until all the eluent flows through the column, and finally add the elution buffer (0.4M MACL PBS), and collect the eluate , reserved for concentration.

[0058] (3) Put the Milipore Amicon Utra-15 100k centrifugal filter device into a centrifuge tube, and rinse the filter with sterilized PBS. Add the collected purified eluate to the filter, centrifuge for 5 minutes, and then centrifuge for another 5 minutes according to the flow rate to concentrate the sample. Add PBS for washing, centrifuge according to the above steps, mix and collect in Eppendorf tubes for storage a...

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Abstract

The invention relates to a nuclear orphan receptor alpha (ROR alpha) adeno-associated virus vector related to retinoic acid in mice and a construction method of the vector and belongs to the field of gene therapy of diseases. The vector genome disclosed by the invention is small and contains a linear single-stranded DNA genome of which the size is between 4.7kb and 6kb. The rAAV-ROR alpha is packaged by virtue of a three-plasmid transfection method capable of reducing adenovirus pollution, a heparin agarose purification method without destroying AAV and AmiconUtra-15 efficient concentration effect and applied in transfection of tumor cells. The vector can infect cells in division and static stages, can be integrated into a specific domain of a host cell genome, is non-pathogenic and has weak immunogenicity, can be used in in-vivo tests and in-vitro experiments so as to give a full play to the effect of AAV vector in gene therapy as well as regulation effect of ROR alpha in a plurality of important physiological processes.

Description

technical field [0001] The invention relates to a mouse retinoic acid-related orphan receptor α (RORα) adeno-associated virus vector and a construction method thereof, belonging to the field of gene therapy for diseases. Background technique [0002] Retinoid acid receptor related orphan receptor α (retinoid acid receptor related orphan receptor, RORα, NRIFI), a member of the steroid hormone receptor superfamily transcription regulator, can directly enter the nucleus to regulate the transcription of target genes, thus participating in many important physiological The regulation of the process plays an important role in morphological development, cell proliferation and differentiation, maintenance of body homeostasis, regulation of biological metabolism, control of advanced nerve functions, and immune regulation. Studies have found that RORα gene-deficient model mice have extensive and severe functional disorders in vivo, especially prone to autoimmune diseases and allergic ...

Claims

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Application Information

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IPC IPC(8): C12N15/861C12N15/66C12N7/02C12R1/93
Inventor 吴玉敏许化溪苏兆亮王胜军应欣宇杨慧建别庆丽
Owner JIANGSU UNIV
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