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Detection method of content of neutralizing antibody of adeno-associated virus and construction method of cell line

A construction method and detection method technology, applied in the field of medical detection, can solve the problems of inability to meet the real-time monitoring of stable cell lines, long construction period, false positives of cell lines, etc., so as to avoid the problem of drug-resistant bacteria and shorten the construction time.

Pending Publication Date: 2021-09-24
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] At present, the construction method of adeno-associated virus-infected sensitive cell lines has problems such as long construction period and false positives in the screened cell lines, which cannot meet the real-time monitoring of stable cell lines during subculture

Method used

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  • Detection method of content of neutralizing antibody of adeno-associated virus and construction method of cell line
  • Detection method of content of neutralizing antibody of adeno-associated virus and construction method of cell line
  • Detection method of content of neutralizing antibody of adeno-associated virus and construction method of cell line

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] This embodiment provides a method for constructing a cell line sensitive to adeno-associated virus infection, and the specific construction steps are as follows:

[0061] Introduce the lentiviral vector overexpressing the miniAAVR-mCherry gene into Hela cells; obtain Hela cells overexpressing miniAAVR-mCherry, that is, Hela cells sensitive to adeno-associated virus infection;

[0062] Adeno-associated virus-infected sensitive Hela cells were cultured to obtain a large number of adeno-associated virus-infected sensitive Hela cells.

[0063] Specifically, the construction process of the lentiviral vector of miniAAVR-mCherry gene is as follows (linear sequence refers to image 3 shown):

[0064] (1) Original plasmid sequence:

[0065] AAVR cDNA: Extract RNA from hela cells, reverse transcribe cDNA, use this as a template, and PCR produce AAVRcDNA.

[0066] Vector sequence: the vector is pLVX-Puro vector, purchased from transvector;

[0067] mCherry sequence: CDS region...

Embodiment 2

[0115] This embodiment provides a method for detection and content determination of adeno-associated virus neutralizing antibodies, and the specific operation steps are as follows:

[0116] The adeno-associated virus infection-sensitive Hela cells constructed in Example 1 were cultured in a 24-well plate, and the number of cells was 1×10 5 Cells / well density were plated;

[0117] Dilute the sample to be tested in a 10-fold concentration gradient, the highest dilution is 1×10 9 times, to obtain 10 different concentrations of samples to be tested;

[0118] And set the maximum value control group (do not contain AAV antibody, add neonatal umbilical cord blood serum to replace the antibody sample) and minimum value control group (do not add AAV during detection, use DMEM to replace AAV);

[0119] Take 10 samples of different concentrations to be tested, the maximum value control group and the minimum value control group, a total of 12 groups, take 10 μL respectively, mix and inc...

Embodiment 3

[0124] This embodiment provides a method for detection and content determination of adeno-associated virus neutralizing antibodies, and the specific operation steps are as follows:

[0125] (1) The adeno-associated virus infection-sensitive Hela cells constructed in Example 1 were cultured in a 24-well plate, and the number of cells was 1×10 5 Cells / well density were plated;

[0126] (2) Dilute the sample to be tested in a 10-fold concentration gradient, the highest dilution is 1×10 9 times, to obtain 10 different concentrations of samples to be tested;

[0127] (3) And set the maximum value control group (without AAV antibody, adding neonatal umbilical cord blood serum to replace the antibody sample) and the minimum value control group (do not add AAV during detection, use DMEM instead of AAV);

[0128] (4) Take 10 different concentrations of samples to be tested, the maximum value control group and the minimum value control group, a total of 12 groups, take 30 μL respectiv...

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Abstract

The invention discloses a detection method of the content of a neutralizing antibody of an adeno-associated virus and a construction method of a cell line, and relates to the technical field of medical detection. The detection method comprises the following steps of: infecting cells with an overexpression vector of a miniAAVR-fluorescence reporter gene fusion gene, and overexpressing a miniAAVR-fluorescence reporter gene to obtain a cell line sensitive to adeno-associated virus infection. By utilizing the construction method provided by the invention, various animal cells can be quickly transformed into the cell line sensitive to the adeno-associated virus. As the method does not need repeated screening of antibiotics, the construction time of the cell line can be greatly shortened. Meanwhile, the fluorescence reporter gene is used based on the screening process, so that no antibiotic marker is used, false positive stable cell strains caused by drug resistance of the cells can be avoided, and the required stable cell line can be quickly screened.

Description

technical field [0001] The invention relates to the field of medical detection, in particular to a method for detecting the neutralizing antibody content of adeno-associated virus and a method for constructing a cell line. Background technique [0002] At present, the construction method of adeno-associated virus-infected sensitive cell lines has problems such as long construction period and false positives in the screened cell lines, which cannot meet the real-time monitoring of stable cell lines during subculture. [0003] In view of this, the present invention is proposed. Contents of the invention [0004] The object of the present invention is to provide a method for detecting the neutralizing antibody content of adeno-associated virus and a method for constructing a cell line to solve the above-mentioned technical problems. [0005] The present invention is achieved like this: [0006] The invention provides a method for constructing a cell line sensitive to adeno-...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N15/65C12N15/62G01N33/68G01N33/569C12Q1/6897
CPCC12N15/86C12N15/65C07K14/705G01N33/6854G01N33/56983C12Q1/6897C12N2740/15043C12N2800/107C07K2319/60G01N2333/015G01N2469/20
Inventor 董飚叶静娅郑赵悦
Owner SICHUAN UNIV
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