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Serotype 8 recombinant adeno-associated virus preparation method

A technology of recombinant adenovirus and serum, which is applied in the field of preparation of recombinant adeno-associated virus, can solve the problems of long time consumption, small flux, and reducing the immune response of the body, and achieve the goal of increasing virus titer, large specific surface area, and large virus output Effect

Active Publication Date: 2018-06-19
BRAINVTA (WUHAN) CO LTD
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AI Technical Summary

Problems solved by technology

The recombinant adeno-associated virus obtained by density gradient centrifugation has high purity and can remove empty shell virus, which can effectively reduce the immune response of the body in the field of gene therapy, but its disadvantage is that the throughput is small, and it is only suitable for small-scale purification; ion exchange purified virus obtained The recombinant virus has high purity and is suitable for large-scale purification of recombinant virus, but it cannot remove empty shell virus, and it is expensive and time-consuming
Since the recombinant adeno-associated virus of serotype 8 is mainly secreted in the culture medium during the preparation process, and currently the virus is mostly prepared by serum culture, the collected virus liquid contains a large amount of protein, and it is very difficult to use ion exchange or density gradient centrifugation. effort

Method used

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  • Serotype 8 recombinant adeno-associated virus preparation method
  • Serotype 8 recombinant adeno-associated virus preparation method
  • Serotype 8 recombinant adeno-associated virus preparation method

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preparation example Construction

[0036] The present invention provides a preparation method of recombinant adeno-associated virus of serotype 8, comprising the following steps:

[0037] (1) 293T cells were cultured in a sheet carrier using serum-free medium.

[0038] 293T cells were inoculated on the sterilized and soaked sheet carrier, and the cells were cultured at a temperature of 37°C, a pH of 6.8-7.6, a DO of 30%-60%, and a stirring speed of 60 rpm. The amount of cells on the sheet carrier reaches 1×10 8 ~3×10 8 cells / g. Animal cells are inoculated on the sheet-like carrier, under the controlled culture conditions of the bioreactor, the nutrient consumption is detected, and the liquid is replenished or changed, and the cultured cells reach 1×10 8 ~3×10 8 cells / g, the cells on the sheet carrier form a healthy and dense monolayer, which is conducive to the transfection of the plasmid. Excessively high cell density needs to consume a large amount of medium, and the DNA-transfection complex cannot be use...

Embodiment 1

[0053] Taking a 500ml basket bioreactor as an example, the culture medium adopts 293Free style serum-free medium produced by Life Company to produce and prepare serotype 8 recombinant adeno-associated virus.

[0054] The amount of the sheet carrier is about 10g, made of polyester fiber, round, 1cm in diameter, figure 1 is the appearance picture of the sheet carrier used in the present invention; figure 2 It is the surface morphology and structure shown by the high-resolution pictures of the sheet carrier used in the present invention. First, soak it with PBS, then rinse it with PBS, then soak it with culture medium overnight, then soak it with PBS and sterilize it by autoclaving, and remove it before use. All PBS.

[0055] Inoculate 5 × 10 on the sheet carrier in the bioreactor 7 A 293T cell, DO is controlled at 30-60%, pH value is maintained at 7.0-7.2, cultured and grown at 37°C for 5-7 days, and glucose digestion is detected during the stirring paddle speed of 60rpm / min,...

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Abstract

The invention belongs to the field of bioengineering, and more specifically relates to a method for preparing serotype 8 recombinant adeno-associated virus, The method comprises the following steps: 1) using a serum-free medium for culturing 293T cell in a sheet carrier; 2) performing transient transfection on the cultured 293T cell by adeno-associated viral vector system plasmid to obtain the transfected cell; and the adeno-associated viral vector system plasmid comprises AAV expression vector plasmid, auxiliary plasmid pHelper and AAV capsid plasmid pRC8; and 3) continuously culturing the transfected cell, collecting and replacing the serum-free medium on time to obtain a medium containing the recombinant adeno-associated virus. The preparation method can guarantee production stability,preparation and purifying steps are saved, downstream virus purifying difficulty is obliviously reduced, and the preparation method is especially suitable for industrial scale-production of the serotype 8 recombinant adeno-associated virus.

Description

technical field [0001] The invention belongs to the field of bioengineering, and more specifically relates to a preparation method of a serotype 8 recombinant adeno-associated virus. Background technique [0002] At present, there are mainly two methods for preparing recombinant AAV on a large scale. One is the method of producing AAV based on the baculovirus expression system. The recombinant AAV prepared by this method has high yield, low cost and is easy to operate. Cells, during the preparation of AAV, are easy to introduce insect genes into the virus, and at the same time, in the downstream purification process, it is difficult to remove the baculovirus mixed in AAV, which brings unpredictable risks to clinical drug use. Moreover, the AAV produced by the baculovirus expression system has a high empty shell rate, which has caused the body's immune response. Therefore, this method is not favored by people in the field of biopharmaceuticals. Another method is to prepare A...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/864
CPCC12N7/00C12N15/86C12N2750/14121C12N2750/14143
Inventor 罗振曹佩奚源张齐玉张胜
Owner BRAINVTA (WUHAN) CO LTD
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