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TAQMAN probe real-time fluorescent quantitative PCR method treating tree shrew IL-2 constructed plasmid as standard substance

A real-time fluorescence quantitative and IL-2 technology, applied in the field of molecular biology, can solve the problem of lack of evaluation indicators of immune cytokines in tree shrew animal models

Active Publication Date: 2013-09-04
INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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Problems solved by technology

However, there are few reports on the study of tree shrew IL-2, resulting in the lack of evaluation indicators for tree shrew animal models of immune cytokines

Method used

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  • TAQMAN probe real-time fluorescent quantitative PCR method treating tree shrew IL-2 constructed plasmid as standard substance
  • TAQMAN probe real-time fluorescent quantitative PCR method treating tree shrew IL-2 constructed plasmid as standard substance
  • TAQMAN probe real-time fluorescent quantitative PCR method treating tree shrew IL-2 constructed plasmid as standard substance

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Embodiment 1

[0052] Embodiment 1: The establishment of the TAQMAN probe fluorescent quantitative PCR method using the tree shrew IL-2 plasmid as a standard:

[0053] The experimental method of not specifying concrete condition in the embodiment, generally according to conventional conditions such as people such as Sambrook, molecular cloning: laboratory (New York: Cold Spring Harbor Laboratory Press, 1989) or people such as Peng Xiuling, genetic engineering experiment technology, (science Technical Press), or as recommended by the manufacturer.

[0054] 1PCR amplification and determination of annealing temperature

[0055] According to the characteristics of the primers and probes, annealing temperatures of 55°C, 56°C, 57°C, and 58°C were designed respectively to determine the optimal annealing temperature. Using the following reaction system and conditions, use the PMD-19T IL-2 plasmid as a template for gradient amplification using a PCR instrument: dream Mix Taq enzyme 25ul, dH 2 O20ul...

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Abstract

A TAQMAN probe real-time fluorescent quantitative PCR method treating a tree shrew IL-2 constructed plasmid as a standard substance comprises the following steps: designing a TAQMAN primer and a probe by treating a tree shrew IL-2 gene as a target, designing experiments for finding the primer annealing temperature, the primer concentration and the probe concentration under an optimal condition, purifying the tree shrew IL-2, calculating to obtain the fragment copy number of the IL-2 gene, carrying out 10-time gradient dilution by using the calculated tree shrew IL-2 plasmid to prepare positive plasmids having copy numbers of 10<5>-10<9> copies, establishing a standard curve, and detecting the sensitivity.

Description

technical field [0001] The invention belongs to the field of molecular biology, and relates to a real-time fluorescent quantitative PCR detection method based on the cloned tree shrew IL-2 plasmid as a standard product and using TAQMAN as a probe. Background technique [0002] Interleukin-2 (Interleukin-2, IL-2) is mainly a type of Th1 cytokine produced by activated lymphocytes, which can promote the differentiation and maturation of T cells, NK cells, and B cells, activate their biological activities, and induce lymphocytes. Factors activate killer cells (LAK), destroy tumor cells through lymphokines and natural killer cells, and promote the synthesis and release of many lymphokines such as interferon, tumor necrosis factor, etc., as well as antibody production, thereby greatly enhancing the immune function of the body. It plays an important role in the treatment of anti-tumor, anti-toxin, immune regulation and infectious diseases. IL-2 can also be used as an immune adjuva...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N21/64
Inventor 代解杰黄晓燕孙晓梅罕园园陆彩霞匡德宣王文广仝品芬徐娟殷安国李晓飞
Owner INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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