Method for constructing cell strain for stably expressing cytochrome C protein

A technology for stable expression and method construction, applied in the field of medical molecular biology

Inactive Publication Date: 2019-04-26
GUIZHOU UNIV
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  • Abstract
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Problems solved by technology

It can be seen that the release of cytochrome C from the mitochondria to the cytoplasm is a key step in triggering the apoptosis signaling pathway, and the disorder of apo...

Method used

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Embodiment

[0020] (1) cytochrome C gene amplification

[0021] 1. Cytochrome C gene PCR amplification According to the cytochrome C gene sequence (Gene ID: 54205) in NCBI, use the software LSPrimer (http: / / ccsipb.lnu.edu.cn / primer / ) to design cytochrome C gene-specific primers cytochrome C-F and cytochrome C-R, add the restriction site NotI and BamH I, the introduced restriction site NotI sequence (restriction endonuclease NotI digestion recognition site sequence) see sequence table SEQ ID 5 for details, the introduced restriction site Point BamH I sequence (restriction endonuclease BamH I digestion recognition site sequence) is detailed in the sequence table SEQ ID 6, the sequence of the specific primer cytochrome C-F (amplified people (Homosapiens) cytochrome C (Cytochrome C ) gene specific primer 1 sequence) see sequence listing SEQ ID 1 for details, and the sequence of cytochrome C-R (specific primer 2 sequence for amplifying human (Homosapiens) cytochrome C (Cytochrome C) gene) see ...

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Abstract

The invention discloses a method for constructing a cell strain for stably expressing a cytochrome C protein. The method comprises the following steps: inserting cytochrome C genes into Not I and BamHI multiple cloning sites of a pLenti-CMV-Gag-Mcherry-Puro carrier; using constructed pLenti-CMV-cytochrome C-Emcherry-Puro recombinant plasmid and virus packaging plasmid for co-transfecting 293T cells; culturing, detoxifying, collecting virus supernatant and filtering, thereby acquiring a recombinant chronic virus liquor; screening and identifying the packaged recombinant chronic virus infected293T cells by using puromycin, thereby acquiring the cell strain for stably expressing the cytochrome C protein. The invention utilizes a recombinant chronic virus system to prepare the cell strain fused with tag protein and capable of stably expressing the cytochrome C protein. The cell strain is capable of supplying an excellent tool for researching the biological function of the cytochrome C protein.

Description

technical field [0001] The invention belongs to the field of medical molecular biology, and in particular relates to a method for constructing a cell line stably expressing cytochrome C protein. Background technique [0002] Cytochrome C is encoded by nuclear genes and is located between the inner and outer membranes of mitochondria. It is one of the most evolutionarily conserved proteins. Under the action of external apoptosis stimuli, the permeability of mitochondrial membranes changes, and cytochrome C is released into the cytoplasm. In the presence of dATP, it can combine with apoptosis-related factor-1 (Apaf-1) to form a multimer, and promote the combination of pro-caspase-9 to form an apoptosis complex, activate Caspase-9, and then pass the cascade The response activates downstream Caspase-3, leading to apoptosis. It can be seen that the release of cytochrome C from the mitochondria to the cytoplasm is a key step in triggering the apoptosis signaling pathway, and the ...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N15/12
CPCC12N15/86C07K14/80C12N2740/15043C12N2800/107
Inventor 郭建军田莹檀军赵帅张书琪
Owner GUIZHOU UNIV
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