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Methods, compositions and kits for extraction and purification of adeno-associated virus and adenovirus from host cells

An adenovirus and kit technology, applied in the fields of biotechnology and pharmaceutical research, can solve problems such as the existence of gradient medium health risks, cumbersome operating procedures, and reducing infection activity.

Active Publication Date: 2018-05-29
贺道耀
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this technique has some serious limitations in application: (1) significant reduction in infectious activity, (2) health risks associated with the gradient medium, (3) possible toxicity of the gradient medium, (4) limitations in the scale of production and (5) The operating procedures are cumbersome

Method used

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  • Methods, compositions and kits for extraction and purification of adeno-associated virus and adenovirus from host cells
  • Methods, compositions and kits for extraction and purification of adeno-associated virus and adenovirus from host cells
  • Methods, compositions and kits for extraction and purification of adeno-associated virus and adenovirus from host cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0113] Example 1: Lysis, extraction and purification of adeno-associated virus and adenovirus from host cells

[0114] This example tests methods for the lysis, extraction and purification of AAV and AdV virus particles from their packaging cells (host cells). This example demonstrates that the present method achieves higher recovery and purity of virus particles compared to conventional methods. Furthermore, the method is easy to operate and can be scaled up readily.

[0115] Methods and Materials

[0116] Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS) and cell culture dishes were purchased from Fisher Scientific. 6-well and 12-well cell culture plates were purchased from Santa Cruz Biotechnology. Plasmid DNA Maxiprep kit was purchased from Qiagen. Lipofectamine 2000 Transduction Reagent (TransfectionReagent) was purchased from Life Technologies. DNase I, Maxima Sybr Green qPCR master mix (MasterMix, 2X), Pierce BCA protein assay kit, SDS-PAGE minige...

Embodiment 2

[0175] Embodiment 2: virus purification kit

[0176] This example tests a collection of reagents and materials, referred to as virus purification kits, for the purification of some virus particles. This example illustrates that the kit can greatly improve the quality and efficiency of virus purification.

[0177] Methods and Materials

[0178] Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS) and cell culture dishes were purchased from Fisher Scientific. 6-well and 12-well cell culture plates were purchased from Santa Cruz Biotechnology. Plasmid DNA Maxiprep kit was purchased from Qiagen. Lipofectamine 2000 transduction reagent was purchased from Life Technologies. DNase I, Maxima Sybr Green qPCR master mix (2X), Pierce BCA protein assay kit, SDS-PAGE mini gels, Amicon Ultra-4 centrifugal filters and all chemicals were purchased from FisherScientific. Silica-based chromatography columns were purchased from Agilent Technologies, Inc.

[0179] cell cult...

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Abstract

The present invention provides a method of tertiary purification for extraction and purification of viral particles from a sample containing host cells that package viral particles. The third-stage purification method comprises: using a first fractionation solution to precipitate and remove most of the impurity protein; using a second fractionation solution to precipitate and collect virus particles and to remove some residual impurities; and further using column chromatography on the collected virus particles to remove remaining impurities, thereby obtaining an ultrapure virus solution. The method also includes the steps of collection of viral host cells, cell suspension, and cell lysis. The purification method is simple and convenient to operate, and it is convenient to obtain a high-yield ultra-purity virus solution, which is suitable for large-scale and small-scale preparation, and the solution and the operation steps do not contain toxic substances to the cells. Therefore, the method demonstrates a highly desirable purification technique for vector viruses including adeno-associated viruses and adenoviruses. The invention also includes kits or packages designed in accordance with the above techniques.

Description

technical field [0001] The invention belongs to the field of biotechnology and medical research, and generally relates to the purification of virus particles, more specifically, to a method for extracting and purifying carrier virus particles such as adeno-associated virus and adenovirus packaged in host cells from host cells and Its components and kits. Background technique [0002] The delivery of exogenous genes and small non-coding RNAs (such as siRNA and miRNA) mediated by viral vectors is a very effective tool that is widely used in basic research and medical research and development. Viral vectors currently in use include adenovirus, retrovirus, lentivirus, adeno-associated virus (AAV), and herpes simplex virus (HSV), with retroviruses and lentiviruses being the most frequently used in recent research literature. Retroviruses and lentiviruses belong to the class of retroviruses, which are secreted in the culture medium of the host cells after packaging in the host ce...

Claims

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Application Information

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IPC IPC(8): C12N7/02
CPCC12N7/00C12N2710/10051C12N2750/14151C12N15/86C12N2710/10351
Inventor 贺道耀
Owner 贺道耀
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