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Method for constructing laryngeal cancer cell line for stably expressing green fluorescent protein

A green fluorescent protein, stable expression technology, applied in the field of cell research, can solve problems such as targeted drug screening and molecular mechanism research that limit laryngeal cancer, and achieve the effect of meeting different flux requirements and short screening period

Inactive Publication Date: 2018-12-07
FIRST HOSPITAL OF SHANXI MEDICAL UNIV
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Problems solved by technology

[0004] Currently, there is no commercially available stable cell line of laryngeal cancer expressing fluorescent protein, which largely limits the screening of targeted drugs and the study of molecular mechanisms for laryngeal cancer

Method used

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  • Method for constructing laryngeal cancer cell line for stably expressing green fluorescent protein
  • Method for constructing laryngeal cancer cell line for stably expressing green fluorescent protein
  • Method for constructing laryngeal cancer cell line for stably expressing green fluorescent protein

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Embodiment Construction

[0036] The purpose of the present invention is to provide a green fluorescent protein expression vector with a lentiviral plasmid as the backbone, and a method for obtaining laryngeal cancer cell lines stably expressing green fluorescent protein through virus packaging, infection of laryngeal cancer cells Hep-2 and screening. A detailed description will be given below in conjunction with the specific experimental process and experimental results, which is an explanation rather than a limitation to the present invention.

[0037] The construction of a laryngeal cancer cell line stably expressing green fluorescent protein includes the following steps:

[0038] First provide the source or preparation method of plasmid and main reagents:

[0039] The pLenti-puro vector and pEGFP-C1 vector plasmids were preserved for this experiment.

[0040] DH5α competent cells were purchased from Beijing Quanshijin Biotechnology Company, Lipofectamine 3000 transfection reagent was purchased from Thermo ...

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Abstract

The invention belongs to the technical field of cell research, and particularly relates to a method for constructing a laryngeal cancer cell line for stably expressing the green fluorescent protein. The method comprises the following steps: (1) designing and synthesizing a primer; (2) digesting the gene sequence of pLenti-puro carrier plasmids with BamHI and XhoI according to the gene sequence andpLenti-puro sequence of the green fluorescent protein, and connecting digested products to obtain a target carrier pLenti-puro-GFP; (3) verifying the carrier pLenti-puro-GFP with endotoxin extraction, digesting the pLenti-puro-GFP plasmids with BamHI and XhoI, and detecting the gene sequence by agarose gel electrophoresis, and sequencing the carrier pLenti-puro-GFP plasmids to detect the sequencesituation of the GFP-LC3; and (4) co-transfecting a 293T cell with the carrier pLenti-puro-GFP plasmids and a lentivirus package carrier, collecting virus supernatant, and transfecting a laryngeal cancer cell Hep-2 with the virus supernatant, infecting for 24 hours, then adding a culture medium containing puromycin, screening the cell, and observing under high content until a stable strain is obtained.

Description

Technical field [0001] The invention belongs to the technical field of cell research, and specifically relates to a method for constructing a laryngeal cancer cell line stably expressing green fluorescent protein. Background technique [0002] Green fluorescent protein (GFP) was originally a protein found in jellyfish. When jellyfish luminescent protein binds to Ca 2+ Then it emits blue fluorescence, and the blue light further excites GFP to produce green fluorescence. The fluorescence emitted by GFP has a longer duration and does not require any other auxiliary factors. After the successful cloning of GFP cDNA and its successful expression in E. coli and mammalian cells, people realized its huge application prospects. [0003] Laryngeal cancer is one of the common malignant tumors of head and neck cancer. It has the characteristics of high incidence and high recurrence rate. The incidence rate varies greatly from country to country. So far, the treatment of laryngeal cancer is st...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/867
CPCC07K14/43595C12N15/86C12N2510/00C12N2740/15043
Inventor 张宇良牛敏郭慧娜薛绪亭郑希望王斌全高伟吴勇延卢岩李会政李文奇代丰升代力殷宏雨汤叶美周江韬刘红亮
Owner FIRST HOSPITAL OF SHANXI MEDICAL UNIV
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