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Recombinant plasmid containing MRP (Multidrug Resistance-associated Protein)-2 genes 3' UTR (Untranslated Region) and reporter genes and application thereof

A MRP-2, reporter gene technology, applied in the field of biomedicine, to achieve the effect of the best application prospects

Inactive Publication Date: 2014-05-07
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at this stage, there are few research reports on multidrug resistance and microRNA, and there are even fewer reports on MRP-2 protein and microRNA. At present, there is no simple and fast method to screen for the ability to inhibit multidrug resistance. microRNA

Method used

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  • Recombinant plasmid containing MRP (Multidrug Resistance-associated Protein)-2 genes 3' UTR (Untranslated Region) and reporter genes and application thereof
  • Recombinant plasmid containing MRP (Multidrug Resistance-associated Protein)-2 genes 3' UTR (Untranslated Region) and reporter genes and application thereof
  • Recombinant plasmid containing MRP (Multidrug Resistance-associated Protein)-2 genes 3' UTR (Untranslated Region) and reporter genes and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1, construction contains the recombinant plasmid of MRP-2 gene 3 ' UTR and reporter gene

[0026] 1. Test materials

[0027] 1. Strains and plasmids

[0028] (1) Strains: Escherichia coli E.coliDH5α, drug-resistant colorectal cancer cell line HCT116 / L-OHP induced by the Pharmacology Laboratory of East China University of Science and Technology, IC for oxaliplatin 50 It was 167μg / ml, and the drug resistance multiple was 9.7;

[0029] (2) Plasmids: pGL3-promoter and pRL-SV40 are preserved by our laboratory.

[0030] 2. Biochemical reagents

[0031] (1) ExTaq enzyme, restriction endonuclease HindIII, NcoI and DNA Ligation Kit were purchased from TaKaRa Company. DL2000 marker and λDNA / HindⅢ marker were purchased from Shanghai MajorBio Company. PRMI 1640 culture medium (Hyclone); Lipofect Transfection Reagent (TIANGEN, Beijing);

[0032] (2) Dual luciferase reporter gene detection kit was purchased from KenReal Company. Plasmid Recovery Kit and DNA Fragment...

Embodiment 2

[0070] Example 2. Screening for microRNAs that have an impact on the activity of the 3' UTR of the MRP-2 gene

[0071] 1. Cell culture

[0072] Human colon cancer cell line oxaliplatin-resistant HCT116 / L-OHP was cultured in RPMI1640 complete medium containing 10% newborn bovine serum, 100 U / ml penicillin, 100 μg / ml streptomycin (37°C, 5% CO 2 , saturated humidity), and maintain a single layer of adherent growth. The medium of HCT116 / L-OHP cells was supplemented with 5 mg / mL oxaliplatin to maintain their drug resistance.

[0073] 2. Cell plating

[0074] Press 5×10 5 Seed cells in exponential growth phase in 6 empty plates at 37°C 5% CO 2 Incubate overnight in the incubator until the cells are 80% confluent.

[0075] 3. Co-transfection

[0076] Configure mimics (microRNA mimics) / DNA / liposome complexes in EP tubes as follows: Dilute plasmid DNA (4μg / well) and mimics (80nM / well) into RPMI 1640-SF (250μL / well) respectively, dilute Liposomes (10 μL / well) to RPMI 1640-SF (250...

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Abstract

The invention discloses a recombinant plasmid containing MRP (Multidrug Resistance-associated Protein)-2 genes 3' UTR (Untranslated Region) and reporter genes and application thereof. Constructed MRP-2 gene 3' UTR and reporter gene carriers and pRL-SV40 carriers and microRNA (Ribonucleic Acid) to be screened are co-transfected into a drug-resistance colon cancer cell line; inhibitory activity of microRNA to MRP-2 gene 3' UTR region is responded by simultaneously detecting activities of firefly and renilla luciferase; and thus, a method for targeted screening of reversal tumor multidrug resistance microRNA is established. The method has excellent application prospect in aspects of screening the reversal tumor multidrug resistance microRNA, researching acting mechanism between the reversal tumor multidrug resistance and microRNA and researching the mechanism of the tumor cell multidrug resistance microRNA level.

Description

technical field [0001] The invention relates to the field of biomedicine, and more specifically relates to a recombinant plasmid containing a 3' UTR of MRP-2 gene and a reporter gene, its production method and application. Background technique [0002] Tumor multidrug resistance (MDR) refers to the cross-resistance of tumor cells to other anti-tumor drugs with different structures, different targets and different modes of action while developing resistance to multiple drugs. Multidrug resistance is the most important and common phenomenon of tumor drug resistance, and it has become one of the biggest obstacles in tumor treatment. Multidrug resistance-associated protein MRP-2 is a member of the adenosine triphosphate (ATP) binding cassette transporter gene family, also known as ABCC2 (ATP-binding cassette, subfamily C, member 2), also known as tubular multispecific organic anion transporter (cMOAT), is a newly discovered gene related to tumor multidrug resistance in recent y...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63C12N15/66C12Q1/68C12Q1/66C12Q1/02
Inventor 刘建文徐可梁欣沈克江林程卓安
Owner EAST CHINA UNIV OF SCI & TECH
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