Method for treating adeno-associated viruses and kit

A virus and solid-phase carrier technology, applied in the biological field, can solve the problems of adeno-associated virus purification, identification and titer determination methods to be studied, etc.

Active Publication Date: 2019-01-11
CAPITAL UNIVERSITY OF MEDICAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, the purification, identification and titer determination methods of adeno-associated virus are still to be studied

Method used

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  • Method for treating adeno-associated viruses and kit
  • Method for treating adeno-associated viruses and kit
  • Method for treating adeno-associated viruses and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] 1. Obtain purified AAV virus particles from a small amount of crude virus extract by SAV-AAVR-SBP magnetic bead method

[0067] 1.1 Expression and purification of AAVR-derived AAVR protein ( figure 2 )

[0068] The present invention firstly produces and purifies the protein of the PKD1-5 domain in the AAVR protein sequence (referred to as AAVR protein), and carries a His tag and a biotin-affinity SBP tag at the C-terminal of the AAVR protein.

[0069] 1) PET-28a-AAVR and PET-28a-AAVR-SBP fusion plasmids were constructed using PET-28a as a vector.

[0070] 2) The two fusion plasmids were transformed with 100 μL of BL21 competent cells as host bacteria, and cultured at 37°C for 14 hours.

[0071] 3) Pick a monoclonal colony, inoculate it in 5 ml of kanamycin-resistant liquid LB, shake the bacteria at 37°C at 220 rpm for 8 hours, and inoculate it in 1 L of kanamycin-resistant liquid LB medium at a ratio of 1:1000, under the same conditions Cultivate until the OD value ...

Embodiment 2

[0092] 2. Purified AAV virus particles were obtained from a large number of crude virus extracts by AAVR-SAV prepacked column chromatography ( Figure 4 )

[0093] Figure 4 A shows the schematic diagram of the reaction flow, using SAV as the solid carrier to load the AAVR-SBP protein. After immobilizing the PKD protein, the crude extract containing AAV particles was passed through the prepacked column, and the AAV was bound to the AAVR-SBP protein and eluted with a high salt solution to obtain the AAV virus.

[0094] 2.1 Preparation of AAVR-SAV prepacked column

[0095] A 5 ml streptavidin agarose (SAV) prepacked column (ThermoFisher Company, USA) was used, the loading buffer was 20 mM Tris-HCl, pH=7.4, and the loading flow rate was 1 ml / min. The loading buffer was equilibrated for 5 times the column volume, and a total of 5 ml of 1 mg / ml AAVR-SBP protein solution (obtained in step 1.1 of Example 1) was loaded, and the flow-through was collected and loaded twice. Then, eq...

Embodiment 3

[0103] 3. The titer of purified AAV virus was identified by AAVR protein-coated ELISA kit ( Figure 5 )

[0104] Figure 5 A shows the schematic diagram of the kit. The AAVR protein is used as the capture antibody, and the AAV virus is added in a series of dilutions, and the AAVR-SBP protein is used as the primary antibody. SAV-HRP was used as secondary antibody, ABTS was used as HRP reaction substrate, and the degree of color development of each sample was determined by 405nm absorption light of microplate reader.

[0105] 3.1 Coating of AAVR protein

[0106] The AAVR protein was diluted to 1 mg / ml with coating diluent (0.05mol / L sodium carbonate-sodium bicarbonate buffer, pH 9.6). Add 100 μL of diluted AAVR protein to each well of the ELISA plate and incubate at 4°C overnight. Ensure the ambient humidity, complete the coating after overnight, and discard the liquid in the wells.

[0107] 3.2 ELISA plate blocking

[0108] Block with 5% calf serum at 37°C for 40min. When...

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Abstract

The invention provides a method for treating adeno-associated viruses and a kit. The method comprises the following steps: contacting the adeno-associated viruses with adeno-associated virus receptorsconnected with lag proteins so as to obtain an adeno-associated virus-adeno-associated virus receptor-label protein complex; and contacting the adeno-associated virus-adeno-associated virus receptor-label protein complex with a binding protein, thereby obtaining the adeno-associated virus-adeno-associated virus receptor-label protein-binding protein complex, wherein the binding protein is capableof being specifically bound to the lag protein. Therefore, by utilizing the method for treating adeno-associated viruses disclosed by the invention, the aims of performing separation, purification, identification, titer detection and the like on the adeno-associated viruses can be achieved, and the method disclosed by the invention has the advantages of being simple and convenient to operate, high in accuracy, excellent in repeatability, low in cost and the like and is applicable to large-scale application.

Description

technical field [0001] The present invention relates to the field of biology. Specifically, the present invention relates to methods and kits for treating adeno-associated viruses. Background technique [0002] Viral vectors are widely used in gene transfer and gene therapy. Among them, adeno-associated virus (AAV) has the characteristics of non-pathogenicity, stable physicochemical properties, weak immunogenicity, and can be integrated into eukaryotic chromosomes to mediate the long-term stable expression of foreign genes. It has attracted more and more attention and has broad application prospects in the field of gene transfer and gene therapy. [0003] However, methods for purification, identification and titer determination of adeno-associated viruses remain to be studied. SUMMARY OF THE INVENTION [0004] The present invention aims to solve at least one of the technical problems existing in the prior art at least to a certain extent. [0005] It should be noted th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/02G01N33/569G01N33/531C12R1/93
CPCG01N33/531G01N33/56983C12N7/00G01N2469/10C12N2750/14151
Inventor 丁卫崔梦甜程杉卢雅彬张晨光牛静
Owner CAPITAL UNIVERSITY OF MEDICAL SCIENCES
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