Method for detecting recombinant lentivirus titer on basis of gag gene

A recombinant lentivirus and gene detection technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve problems such as time-consuming, labor-intensive and cost-effective, and achieve the effects of convenient operation, wide application range and accurate detection results.

Inactive Publication Date: 2017-12-05
安徽安龙基因科技有限公司
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Problems solved by technology

[0004] The purpose of the present invention is to solve the problem of time-consuming, labor-intensive and high-cost detection method of recombinant le...

Method used

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Embodiment 1

[0025] A method for detecting recombinant lentivirus titer based on gag gene, comprising the steps of:

[0026] (1) Primer design for the lentiviral gag gene

[0027] (2) To construct a standard product for detecting lentivirus, the steps are as follows:

[0028] S1. The corresponding fragment is amplified by ordinary PCR, and a new cloning plasmid is constructed by using T vector;

[0029] S2, transforming the new cloning plasmid into Escherichia coli, identifying, culturing, and extracting the plasmid;

[0030] S3. Calculate the corresponding copy number according to the concentration and molecular weight of the plasmid, and use this as a standard.

[0031] Among them, the copy number calculation formula is: copy number = 6.02 × 1023 × nucleic acid concentration ÷ (DNA length × 660); the unit of copy number is copies / ml, and the unit of nucleic acid concentration is g / ml;

[0032] (3) Fluorescent quantitative PCR method was used to determine the titer, and the steps were ...

Embodiment 2

[0041] A method for detecting recombinant lentivirus titer based on gag gene, comprising the steps of:

[0042] (1) Primer design for the lentiviral gag gene

[0043] According to the lentiviral gene sequence (GenBank: D86068.1), PCR primers were designed. gag upstream primer sequence: 5'GACCAGCGGCTACACTA 3', gag downstream primer sequence: 5'TATGTGCCCTTCTTTGC 3';

[0044] (2) To construct a standard product for detecting lentivirus, the steps are as follows:

[0045] S1. The corresponding fragment is amplified by ordinary PCR, and a new cloning plasmid is constructed by using T vector;

[0046] S2, transforming the new cloning plasmid into Escherichia coli, identifying, culturing, and extracting the plasmid;

[0047] S3. Calculate the corresponding copy number according to the concentration and molecular weight of the plasmid, and use this as a standard;

[0048] Among them, the copy number calculation formula is: copy number = 6.02 × 1023 × nucleic acid concentration ÷ (...

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PUM

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Abstract

The invention discloses a method for detecting recombinant lentivirus titer on the basis of gag gene. The method comprises the following three parts: designing a primer of lentivirus gag gene, constructing a standard substance for detecting lentivirus and measuring titer by a fluorescent quantitative PCR method. The method is convenient to operate, and the detection result is accurate. As lentivirus gag gene codes protein precursor of p55 and the protein precursor undergoes protease cleavage to form nucleocapsid protein (p7), inner membrane protein (p17) and capsid protein (p24) of the virus, the protein coded by gag is necessary to virion maturation. The primer is designed with the gene gag which must be carried by the lentivirus, and recombinant lentivirus titer is detected by fluorescent quantitative PCR. In comparison with the prior art, the detection primer for real-time quantitative PCR is designed in the gag area of lentiviral vector. Therefore, the detection is not dependent on an exogenous gene and is suitable for lentivirus of non-reporter gene vector, and the application range is wider.

Description

technical field [0001] The invention relates to the technical field of recombinant lentivirus titer detection, in particular to a method for detecting the titer of recombinant lentivirus based on gag gene. Background technique [0002] Lentiviral vectors are currently one of the most widely used gene delivery tools, and have shown broad application prospects in gene therapy research and the preparation of transgenic animals. Lentiviral vector is a kind of retroviral vector, among which human immunodeficiency virus (HIV-1) vector is the most intensively studied. Recombinant lentiviral vectors can infect both dividing and non-dividing cells. It can carry a large foreign gene and stably integrate and express it, and the host immune response induced by it is relatively small, so that the recombinant lentiviral vector has a broad application prospect. [0003] Regardless of the purpose of using recombinant lentivirus, it is necessary to accurately measure the virus titer. At p...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68
CPCC12Q1/702C12Q1/686C12Q2600/158C12Q2561/113C12Q2563/107C12Q2545/114
Inventor 韦玉军李航凌发忠苏军吴远航
Owner 安徽安龙基因科技有限公司
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