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Recombinant plasmid containing MDR1 gene promoter and reporter gene and uses thereof

An MDR1, reporter gene technology, applied in genetic engineering, plant genetic improvement, recombinant DNA technology, etc., to achieve the effects of low cost, fast detection speed and high sensitivity

Inactive Publication Date: 2009-07-15
SHANGHAI UNIV OF TRADITIONAL CHINESE MEDICINE PUTUO DISTRICT CENT HOSPITAL
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  • Recombinant plasmid containing MDR1 gene promoter and reporter gene and uses thereof
  • Recombinant plasmid containing MDR1 gene promoter and reporter gene and uses thereof
  • Recombinant plasmid containing MDR1 gene promoter and reporter gene and uses thereof

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Embodiment 1

[0024] 1. Test materials

[0025] 1. Strains and plasmids

[0026] 1) Bacterial strains: Escherichia coli E.coli DH5α, and the sensitive colorectal cancer cell line HCT-8 were purchased from the Cell Bank of the Chinese Academy of Sciences. IC of drug-resistant colorectal cancer cell line HCT-8 / VCR (commercially available) for vincristine 50 It was 250μg / ml, and the drug resistance multiple was 19.7.

[0027] 2) Plasmids: pGL3-basic and pRL-TK (commercially available).

[0028] 2. Biochemical reagents:

[0029] 1) ExTaq enzyme, restriction enzymes Nhe I, Bgl II and DNA Ligation Kit were purchased from TaKaRa Company. DL2000 marker and λDNA / HindIII marker were purchased from Shanghai MajorBio Company. PRMI1640 culture solution (Hyclone); Lipofect Transfection Reagent (TIANGEN, Beijing).

[0030] 2) Dual luciferase reporter gene detection kit was purchased from KenReal Company. Plasmid recovery kit and DNA fragment recovery kit were purchased from Tiangen Company.

[003...

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Abstract

The invention belongs to the biological technical field and relates to a method of screening a tumor multidrug resistance reversal agent. In the method, a promoter specific sequence of a multidrug resistance (MDR1) gene is cloned and regrouped into a firefly luciferase report gene vector promotor sequence, therefore, an MDR1 report gene vector is constructed. The constructed vector and the sea pansy luciferase report gene vector are cotransfected into a sensitive and drug resistant colon cancer cell line. The activities of fireflies and the luciferase are detected simultaneously to reflect the promotor transcription activity of a MDRI gene promoter. Therefore, the method of screening the tumor multidrug resistance reversal agent in a targeted way is constructed. The method has perfect application prospect in screening tumor multidrug resistance reversal agents, researching the mechanism of the tumor multidrug resistance reversal agents and mechanisms of chemotherapeutic drugs inducing humor cells to generate drug resistance, etc.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to a method for screening a tumor multidrug resistance reversal agent, in particular to a recombinant plasmid containing a multidrug resistance (MDR1) gene promoter and a reporter gene and its targeting at the MDR1 gene promoter. Application of drug screening methods for reversing or inducing tumor multidrug resistance. Background technique [0002] Tumor multidrug resistance (MDR) refers to the resistance of tumor cells to one antitumor drug and resistance to other antitumor drugs with the same or different structures and effects. MDR produced by tumor cells is an important reason for the failure of chemotherapy. Many cancer patients are not sensitive to chemotherapy drugs in clinical practice, resulting in poor chemotherapy effect and tumor recurrence and metastasis. In a sense, 90% of the cases of death from malignant tumors are related to primary or acquired drug resistance. ...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/12C12Q1/66C12Q1/68
Inventor 范忠泽许建华刘建文孙珏李琦李长龙邓皖利孙丽娟徐文山冬梅房一时王芳于有军王劲鲍文磊
Owner SHANGHAI UNIV OF TRADITIONAL CHINESE MEDICINE PUTUO DISTRICT CENT HOSPITAL
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