Recombinant adeno-associated virus, preparation method thereof and application thereof in antibody detection

A virus vector and virus technology, applied in the field of recombinant adeno-associated virus and its preparation, can solve the problem of incomplete detection of adeno-associated virus antibodies in serum, inability to meet multiple uses of recombinant adeno-associated virus, and affect rAAV-mediated exogenous Efficiency and other issues, to achieve reliable marking effect, wide application value, and convenient collection

Active Publication Date: 2020-12-29
SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Related studies have shown that pre-existing antibodies in vivo affect rAAV-mediated exogenous efficiency
[0004] However, most of the current recombinant adeno-associated viruses only express a foreign protein, such as luciferase or fluorescent protein, which cannot meet the multiple purposes of a recombinant adeno-associated virus, and cannot save the cost and time of production
Some recombinant adeno-associated viruses that can express two foreign proteins at the same time have low protein expression and have higher requirements for protein expression and purification, or the brightness of the fluorescent protein is not enough, and the expected effect cannot be achieved when used
At present, the methods for detecting AAV antibodies in serum are not perfect, and there is an urgent need for a method that is easy to operate and can accurately detect AAV antibodies in serum

Method used

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  • Recombinant adeno-associated virus, preparation method thereof and application thereof in antibody detection
  • Recombinant adeno-associated virus, preparation method thereof and application thereof in antibody detection
  • Recombinant adeno-associated virus, preparation method thereof and application thereof in antibody detection

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preparation example Construction

[0038] The invention provides a method for preparing recombinant adeno-associated virus, comprising the following steps:

[0039] (1) Preparation of clones capable of simultaneously expressing luciferase and green fluorescent protein

[0040] Using homologous recombination, SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 were sequentially inserted into the core skeleton of the adeno-associated virus vector. The vector was obtained from the Addgene plasmid library, and the vector number was Addgene#20298 , the obtained clone was named pAAV-EF1α-Gluc-2A-mNeonGreen; where SEQ ID NO.1 and SEQ ID NO.3 are the nucleotides of luciferase Gaussia luciferase and green fluorescent protein mNeonGreen after individual base substitutions, respectively sequence. Although the final translated amino acid sequence was the same as before the substitution, the expression of luciferase and green fluorescent protein was stronger after the base substitution. The luciferase expressed in the present invent...

Embodiment 1

[0048] Example 1 Preparation of a recombinant adeno-associated virus expressing luciferase and green fluorescent protein simultaneously

[0049] 1. Preparation of clones capable of simultaneously expressing luciferase and green fluorescent protein

[0050] First, the Gaussia Luciferase gene (SEQ ID NO.1) was synthesized by whole gene synthesis and inserted into the vector pUC57 to obtain pUC57-Gaussia Luciferase, and the fluorescent protein gene mNeonGreen (SEQ ID NO.3) was synthesized and inserted into the vector pUC57 to obtain To obtain pUC57-mNeonGreen, sequentially insert SEQ ID NO.1, SEQ ID NO.2 (2A gene) and SEQ ID NO.3 into the vector AAV core skeleton (Addgene #20298) by means of homologous recombination, and the obtained clone was named pAAV-EF1α-Gluc-2A-mNeonGreen;

[0051] The primers for amplifying the corresponding sequence are respectively as follows: the primers of the DNA fragment Gaussia Luciferase: SEQ ID NO:4 and SEQ ID NO:5, and the template is pUC57-mNeo...

Embodiment 2

[0056] Example 2 Detection of luciferase activity of recombinant adeno-associated virus expressing luciferase and green fluorescent protein simultaneously

[0057] According to the ratio of 1:100, the recombinant virus was diluted with PBS, and 80 μL of the virus was taken to infect 293T cells (96-well plate), at 37 ° C, 5% CO 2 After culturing, the supernatant and cell lysate were collected respectively after 6, 12, 24, 48 and 72 hours after infection, and the activity of luciferase was detected, and the results were as follows: figure 2 shown. The results showed that: (1) the activity of luciferase could be detected 6 hours after infection, and its activity gradually increased with the prolongation of time, reaching the peak value at 24 hours after infection, and then the activity showed a downward trend; (2) ) the luciferase activity in the supernatant was significantly higher than that in the cell lysate. This result provides effective support for the subsequent detecti...

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Abstract

The invention relates to a recombinant adeno-associated virus, a preparation method thereof and an application thereof in antibody detection. An expression vector is constructed by utilizing an EF1[alpha] promoter and nucleotide sequences of Gaussia luciferase and green fluorescent protein mNeonGreen after base substitution, and the recombinant adeno-associated virus for simultaneously expressingthe Gaussia luciferase and the green fluorescent protein is prepared. The recombinant adeno-associated virus and serum to be detected are diluted and mixed, cells are added for culture, supernate is taken after infection, and the activity of the Gaussia luciferase is detected so as to detect whether adeno-associated virus antibodies exist in the serum or not. The recombinant adeno-associated virusalso has a wide application value in the aspect of neural circuit marking.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a recombinant adeno-associated virus, its preparation method and its application in antibody detection. Background technique [0002] Adeno-associated virus (Adeno-associated Virus, AAV) is a single-stranded DNA virus belonging to the family Parvoviridae and the genus Dependovirus. The genome of AAV is a single-stranded DNA molecule with a length of about 4.7Kb, and the virus particle has no envelope, and its structure is an icosahedron with a diameter of about 20-25nm. Recombinant adeno-associated virus (rAAV) vector is a new type of gene vector modified on the basis of non-pathogenic wild-type AAV. The DNA of the rAAV vector generally replaces the AAV coding gene with foreign gene expression elements, and only the ITR sequence required for virus replication and packaging is retained. rAAV is produced by trans-compensation of AAV's non-structural protein Rep gene, stru...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/864C12N15/53C12N15/12C12Q1/66C12R1/93
CPCC12N7/00C12N15/86C12N9/0069C07K14/43595C12Q1/66C12N2750/14121C12N2750/14143G01N2333/90241
Inventor 贾凡徐富强
Owner SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
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