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Separation and purification method of adeno-associated virus

A technology for separation and purification of viruses, applied in the field of separation and purification of adeno-associated viruses, can solve problems such as tissue tropism affecting transduction efficiency, and achieve the effects of convenient purification of AAV, high purification efficiency and good specificity

Active Publication Date: 2020-10-23
OBIO TECH SHANGHAI CORP LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods require insertion or site-directed mutagenesis in the coat sequence, which can affect transduction efficiency or tissue tropism

Method used

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  • Separation and purification method of adeno-associated virus
  • Separation and purification method of adeno-associated virus
  • Separation and purification method of adeno-associated virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Preparation and detection of antibodies

[0042] 1. Antibody preparation

[0043] The antigen used in the present invention is based on the VP1 of AAV8, compares AAV1-13, removes most of the non-homologous sequences, obtains the VPx sequence, constructs it on the pET-21 vector, expresses and purifies the representative, and obtains the small Mouse monoclonal antibody.

[0044] 1. Preparation of Monoclonal Antibody

[0045] 1.1 Immunization of mice:

[0046] Immunization of BALB / c mice The immunization of BALB / c mice was carried out according to the conventional method. One week after the second immunization, 200 μL blood was collected by tail-docking vacuum method for antibody detection. After the second immunization, the mice were rested for two weeks for booster immunization. , fused after three days.

[0047] 1.2 Cell Fusion:

[0048] 50% PEG with a molecular weight of 1450 was used as a fusion agent for cell fusion, and the cell fusion was carried out...

Embodiment 2

[0069] Example 2 Separation and purification method of adeno-associated virus

[0070] 1. Antibody Conjugation Activated Media Step

[0071] Referring to the NHS-activated Sepharose 4 Fast Flow manual, the AAV antibody prepared in Example 1 was coupled to Sepharose 4 Fast Flow to prepare an affinity filler.

[0072] 2. Affinity purification step for AAV

[0073] 1) Equilibration: equilibrate the packed column with 8CV of 10 mM Tris-HCl, 150 mM NaCl, pH7.5 solution.

[0074] 2) Loading: Freeze-thaw, lyse and centrifuge HEK293 cells packaged with AAV1~13 respectively, filter through 0.22 μm, and then load the sample to a well-balanced chromatographic column. The loading capacity of the sample does not exceed 1E+14 vg / mL filler .

[0075] 3) Washing 1: After loading the sample, equilibrate with 8CV of 10 mM Tris-HCl, 150 mM NaCl, pH7.5 solution.

[0076] 4) Washing 2: Use 8CV of 10 mM Tris-HCl, 1 M NaCl, pH7.5 solution to wash out impurities.

[0077] 5) Wash 2: Equilibrate ...

Embodiment 3

[0082] Example 3 Separation and purification method of adeno-associated virus

[0083] 1. Antibody Conjugation Activated Media Step

[0084] Referring to the NHS-activated Sepharose 4 Fast Flow manual, the AAV antibody prepared in Example 1 was coupled to Sepharose 4 Fast Flow to prepare an affinity filler.

[0085] 2. Affinity purification step for AAV

[0086] 1) Equilibration: equilibrate the packed column with 5CV of 8 mM Tris-HCl, 130mM NaCl, pH=7.3 solution.

[0087] 2) Loading: Freeze-thaw, lyse and centrifuge HEK293 cells packaged with AAV1~13 respectively, filter through 0.22 μm, and then load the sample to a well-balanced chromatographic column. The loading capacity of the sample does not exceed 1E+14 vg / mL filler .

[0088] 3) Washing 1: After loading the sample, equilibrate with 5CV of 8 mM Tris-HCl, 130mM NaCl, pH=7.3 solution.

[0089] 4) Washing 2: Use 8 mM Tris-HCl, 0.8M NaCl, pH=7.3 solution to wash away impurities.

[0090] 5) Washing 2: Equilibrate the ...

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Abstract

The invention relates to the field of separation and purification of virus, in particular to a separation and purification method of adeno-associated virus. The method comprises the following steps: a) providing a chromatography medium which comprises a matrix support and an antibody fixed on the matrix support, and the sequences of a heavy chain variable region and a light chain variable region of the antibody are shown as SEQ ID NO: 1 and SEQ ID NO: 2 in sequence; b) co-incubating a liquid phase composition comprising an adeno-associated virus with the chromatography medium such that the adeno-associated virus binds to the antibody; and c) eluting the adeno-associated virus from the antibody, wherein the adeno-associated virus is selected from any one or more of AAV1-13. According to themethod, any one of AAV1-13 can be universally separated and purified, and the AAV does not need to be transformed, so that the AAV is more convenient to purify; the purification efficiency is high, the specificity is good, and the detectable nonspecific binding with other proteins in cells is hardly generated.

Description

technical field [0001] The invention relates to the field of isolation and purification of viruses, in particular to a method for isolation and purification of adeno-associated virus. Background technique [0002] Adeno-associated virus (adeno-associated virus, AAV), also known as adeno-associated virus, belongs to the genus of Parvoviridae and is the simplest single-stranded DNA-deficient virus found so far. It needs helper virus (usually adenovirus) virus) to participate in replication. It encodes cap and rep genes in two terminal inverted repeats (ITRs). ITRs play a decisive role in virus replication and packaging. The cap gene encodes the viral capsid protein, and the rep gene is involved in virus replication and integration. AAV can infect a variety of cells. In the presence of the rep gene product, viral DNA readily integrates into human chromosome 19. [0003] The traditional purification method for AAV is cesium chloride gradient centrifugation. In traditional ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/02C12R1/93
CPCC12N7/00C12N2750/14151
Inventor 杨佳丽贾国栋杨兴林詹霞夏清梅
Owner OBIO TECH SHANGHAI CORP LTD
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