Bioluminescent reporter gene for monitoring DNA (Deoxyribonucleic Acid) synthesis phase and application thereof

A DNA synthesis phase, bioluminescence technology, applied in recombinant DNA technology, DNA/RNA fragmentation, determination/inspection of microorganisms, etc., can solve problems such as inability to repeat experiments, and it is difficult to continuously observe tumor growth and metastasis.

Inactive Publication Date: 2012-02-22
SHANTOU UNIV MEDICAL COLLEGE +1
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

This invasive research method needs to kill a large number of animals, and it is impossible to repeat the experiment on the same group of animals. It is difficult to directly observe the growth and metas...

Method used

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  • Bioluminescent reporter gene for monitoring DNA (Deoxyribonucleic Acid) synthesis phase and application thereof
  • Bioluminescent reporter gene for monitoring DNA (Deoxyribonucleic Acid) synthesis phase and application thereof
  • Bioluminescent reporter gene for monitoring DNA (Deoxyribonucleic Acid) synthesis phase and application thereof

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Embodiment Construction

[0029] (1) Construction of eukaryotic recombinant expression vector pGL3-CYCA-Luc

[0030] 1. Primer design Use primer software to design a pair of primers. A protective base, a HindIII restriction site and a Kozak sequence were added to the 5' end of the upstream primer, and a protective base and a HindIII restriction site were added to the 5' end of the downstream primer. The sequenced primers were synthesized by Songbao Bioengineering (Dalian) Co., Ltd.

[0031] 2. PCR amplification target fragment Reaction system 50 μL, Premix ExTaq 25 μL; Template (PBS-cyclinA2) 5 μL; Primer 1 20 μm, 1.5 μL; Primer 2 20 μm, 1.5 μL; Sterilized water 17 μL. After mixing well, complete the following operations on the PCR instrument: 94°C for 5 min, 98°C for 10 s, 56°C for 30 s, 72°C for 1 min, 30 cycles.

[0032] 3. Enzyme digestion and recovery of plasmids and PCR products Total system 20 μL, HindⅢ 1 μL, 10 × Mbuffer 2 μL, PCR product or plasmid DNA <1 μg, make up to 20 μL with sterilized...

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Abstract

The invention relates to gene synthesis in molecular biology, mainly relates to a bioluminescent reporter gene for monitoring a DNA (Deoxyribonucleic Acid) synthesis phase, and also relates to application of the gene to anti-tumor medicament screening. According to the invention, a plasmid PBS (pBluescript) containing a full-length human cyclinA2 gene is taken as a template, primers are designed on two ends of the cyclinA2 gene, cDNA (Complementary DNA) full length of the cyclinA2 is amplified and cloned to an end 5' of a Luc gene of a pGL3-control vector to form an eukaryon recombinant expression vector, namly pGL3-CYCA-Luc, which is capable of expressing fusion protein. The vector can express the fusion protein CYCA-Luc after being transfected into a tumor cell. In-vitro experiments show that after being degraded through an ubiquitination dependency pathway, the fusion protein changes in a cell cycle dependency manner and can be used for monitoring the action effect of a specific anti-tumor medicament of a phase S. The invention plays an important role in the fields of screening the anti-tumor medicaments specific to the phase S, and the like.

Description

technical field [0001] The invention relates to the field of cell and molecular biology, and mainly relates to a bioluminescent reporter gene for monitoring the DNA synthesis period, and also relates to the application of the gene in antitumor drug screening. Background technique [0002] From the end of one cell division, through the accumulation of substances, until the end of the next cell division, it is called a cell cycle. A cell cycle can be artificially divided into four phases, namely the early DNA synthesis phase (G1 phase), the DNA synthesis phase (S phase), the late DNA synthesis phase (G2 phase) and the mitotic phase (M phase). The operation of the normal cell cycle is controlled by various mechanisms to ensure the orderly progress of cell division, and when the regulation of the cell cycle is out of control, it will lead to the occurrence of tumors. The biggest difference between tumor cells and normal cells is that they can proliferate indefinitely, which is ...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N15/63C12Q1/66C12Q1/02G01N21/64A61K49/00
Inventor 陈志鸿张国君赵瑞君
Owner SHANTOU UNIV MEDICAL COLLEGE
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