PProTM plasmid as well as construction method and application thereof

A technology for constructing methods and plasmids, applied in the fields of applications, botanical equipment and methods, biochemical equipment and methods, etc., which can solve the problems of unable to screen cells, unable to screen cells with Fluc, accurate quantitative tracking, etc.

Pending Publication Date: 2022-03-25
THE AFFILIATED HOSPITAL OF GUIZHOU MEDICAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But the disadvantage is that there is no drug resistance, and cells cannot be screened by flow sorting. In addition, FLuc can only produce luminescence in living cells, and the intensity of luminescence is linearly related to the number of labeled cells.
[0005] In summary, how to overcome the shortcomings of drug-resistant cells after BSD screening, mCherry requires specific equipment and cannot be accurately and quantitatively tracked in living animals, and Fluc cannot screen cells. At present, there is no construction of the three genes of BSDR, mCherry and Fluc Reports of plasmids together

Method used

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  • PProTM plasmid as well as construction method and application thereof
  • PProTM plasmid as well as construction method and application thereof
  • PProTM plasmid as well as construction method and application thereof

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Embodiment Construction

[0101] The principles and features of the present invention will be described below in conjunction with specific drawings, and the examples given are only used to explain the present invention, not to limit the scope of the present invention.

[0102] The construction method of the pProTM plasmid of the present embodiment comprises the steps:

[0103] Step 1: Construction of BSDR-luciferase vector

[0104] Step 1.1: Take BSDR and perform PCR amplification reaction. The PCR amplification reaction system is: 10xBuffer 5μl, 2mM dNTPs 5μl, 25mM MgSO 4 2μl, 10pmol / μl of the forward primer shown in SEQ ID NO.3 1.5μl, 10pmol / μl of the reverse primer shown in SEQ ID NO.4 1.5μl, template DNA 50ng, 1.0U / μl of KOD Plus enzyme 1μl and the rest are distilled water, the total system is 50μl; the reaction program is: initial denaturation at 94°C for 2min; denaturation at 94°C for 15s, extension at 68°C for 1min, repeat 25 times; extension at 68°C for 10min, 4°C, infinite cycle, to obtain BS...

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Abstract

The invention relates to a pProTM plasmid as well as a construction method and application thereof, and belongs to the technical field of plasmid construction. The construction method of the pProTM plasmid comprises the following steps: step 1, constructing a BSDR-luciferase (BSDR-luciferase) vector; step 2, constructing a pProTM plasmid; and 3, verifying whether the pProTM plasmid is successfully constructed or not. The invention also discloses a pProTM plasmid and an application of the pProTM plasmid. The constructed pProTM plasmid integrates the advantages of the three genes of BSDR, mCherry and Fluc, on one hand, positive cells can be screened through the BSDR, and the pProTM plasmid can be used for cell drug screening; 2, the cells can be screened through mCherry or positive cells can be screened by identifying drugs; and thirdly, cells transfected by the plasmid can be tracked in a living animal through Fluc.

Description

technical field [0001] The invention relates to a pProTM plasmid, its construction method and application, and belongs to the technical field of plasmid construction. Background technique [0002] Blasticidin resistance gene (BSDR) can be constructed on a variety of vectors, and blasticidin (BSD) can be used to screen and transfect BSDR-resistant gene-positive cells to select target cells. This technology is widely used in field of medical biology research. However, some drug-resistant cells will appear after blasticidin screening, which fails to achieve the purpose of accurately screening positive cells and affects the accuracy of the research results. [0003] Red fluorescent protein (mCherry) can track cells by marking cells, and has a wide range of applications in biological and medical research. However, this technique usually requires the use of flow cytometry, and many laboratories may not have the conditions to carry out this experiment. Moreover, the signal-to-noi...

Claims

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Application Information

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IPC IPC(8): C12N15/66C12N15/65C12N15/53C12N15/85C12Q1/02
CPCC12N15/66C12N15/65C12N15/52C12N15/85G01N33/5008C12N2800/107G01N2500/10
Inventor 豆晓伟田园李青张培培
Owner THE AFFILIATED HOSPITAL OF GUIZHOU MEDICAL UNIV
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