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Plasmid vector containing 3'UTR (untranslated regions) sequence of ABCB1 gene and reporter gene as well as construction method and use of plasmid vector

A plasmid vector and gene technology, which is applied to the plasmid vector containing the ABCB1 gene 3'UTR sequence and the reporter gene and the fields of its construction and use, can solve problems such as restricting application and development, and achieve the effect of improving sensitivity and specificity

Inactive Publication Date: 2013-06-26
SHUGUANG HOSPITAL AFFILIATED WITH SHANGHAI UNIV OF T C M
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since the discovery of microRNAs began at the beginning of this century, and the research on microRNAs and MDR is still in its infancy, some problems still limit its application and development.

Method used

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  • Plasmid vector containing 3'UTR (untranslated regions) sequence of ABCB1 gene and reporter gene as well as construction method and use of plasmid vector
  • Plasmid vector containing 3'UTR (untranslated regions) sequence of ABCB1 gene and reporter gene as well as construction method and use of plasmid vector
  • Plasmid vector containing 3'UTR (untranslated regions) sequence of ABCB1 gene and reporter gene as well as construction method and use of plasmid vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Construction of a recombinant T vector containing the 3'UTR sequence of the ABCB1 gene and a reporter gene

[0041] 1. Materials

[0042] 1. Plasmid

[0043] The PMD18T vector plasmid was purchased from Promega Company in the United States, and its vector map is as follows: figure 1 shown.

[0044] 2. Main reagents

[0045] Restriction enzymes scaI and xhoI, and DNA Ligation Kit (DNA ligation kit) were purchased from TaKaRa Company. DL2000 maker was purchased from Shanghai MajorBio Company. Dual luciferase reporter gene detection kit was purchased from KenReal Company. Plasmid extraction kit and DNA fragment recovery kit were purchased from Tiangen Company.

[0046] 2. Methods and Results

[0047] 1. Blood sample collection and genomic DNA extraction

[0048] 5 mL of peripheral blood was collected from healthy volunteers, placed in EDTA anticoagulated glass test tubes, and stored in a -40°C refrigerator for later use. Genomic DNA was extracted accor...

Embodiment 2

[0088] Example 2 Construction of recombinant plasmid vector pmirGLO-ABCB1-3'UTR-promoter containing ABCB1 gene 3'UTR sequence and dual fluorescent reporter gene

[0089] 1. Materials

[0090] 1. Plasmid

[0091] The pmirGLO Dual-Luciferase-promoter dual fluorescent reporter gene vector was purchased from Promega Company in the United States, and the vector map is as follows: image 3 shown.

[0092] 2. Main reagents

[0093] Restriction enzymes scaI and xhoI, and DNA Ligation Kit (DNA ligation kit) were purchased from TaKaRa Company. DL2000 maker was purchased from Shanghai MajorBio Company. Dual luciferase reporter gene detection kit was purchased from KenReal Company. Plasmid extraction kit and DNA fragment recovery kit were purchased from Tiangen Company.

[0094] 2. Methods and Results

[0095] 1. PCR amplification of the 3'UTR sequence fragment of the ABCB1 gene

[0096] Using the PMD18-ABCB1-3'UTR plasmid obtained in Example 1 as a template, the 3'UTR sequence ...

Embodiment 3

[0114] Example 3 microRNA mimics (mimetic) / plasmid DNA / liposome co-transfection of human intestinal cancer multidrug-resistant cell line HCT116 / L-OHP

[0115] 1. Materials

[0116] 1. Cell line

[0117] Cell line: human intestinal cancer multidrug-resistant cell line HCT116 / L-OHP, purchased from Shanghai University of Traditional Chinese Medicine Integrative Tumor Intervention Institute, the multiples of resistance to chemotherapy drugs are as follows:

[0118] Table 1 Effects of four chemotherapeutic drugs on the sensitivity of HCT116 and HCT116 / L-OHP

[0119]

[0120] *P <0.01, HCT116 / L-OHP vs HCT116 group.

[0121] 2. Method

[0122] 1. Cell Culture and Plating

[0123] Human intestinal cancer multidrug-resistant HCT116 / L-OHP cells were cultured in RPMI1640 complete medium containing 10% fetal bovine serum, 100 U / ml penicillin, and 100 μg / ml streptomycin (37°C, 5% CO 2 , saturated humidity), and maintain a single layer of adherent growth. Press 5×10 5 Seed cells...

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Abstract

The invention relates to a plasmid vector. According to the plasmid vector, a 3'UTR (untranslated regions) sequence of an ABCB1 gene is inserted into the 6832-7349 bp segment of pmirGLODual-Luciferase-promoter plasmid vector. The invention also relates to a host cell containing the plasmid vector, a construction method of the plasmid vector and use of the plasmid vector in selection of relative microRNA (ribonucleic acid) of multi-drug resistance. The invention provides a fast, efficient, simple and practicable method for accurately forecasting microRNA which regulates human ABCB1 gene expression so that a new way for improving the sensitivity and specificity of microRNA detection, researching, developing and detecting drugs for reversing multi-drug resistance by virtue of gene regulation is opened.

Description

technical field [0001] The invention relates to the screening of microRNA regulating target genes, in particular, a plasmid vector containing the 3'UTR sequence of ABCB1 gene and a reporter gene, its construction method and application. Background technique [0002] Drug resistance to chemotherapy drugs is a major problem in cancer treatment today. Tumor multidrug resistance (MDR) refers to the long-term exposure of tumor cells to a certain chemotherapy drug, which not only produces drug resistance to this chemotherapy drug, but also produces crossover to other anti-tumor drugs with different structures and functions. The phenomenon of drug resistance. Since chemotherapeutic drugs began to be used in the treatment of tumors, it has been accompanied by the occurrence of multidrug resistance. Therefore, how to reverse tumor multidrug resistance has always been considered to be the research direction most closely related to clinical practice. In recent years, with the develop...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/66C12N5/10C12Q1/68C12Q1/66
Inventor 朱惠蓉李琦隋华殷佩浩王炎潘树芳靳宝辉孙建王一斐范忠泽
Owner SHUGUANG HOSPITAL AFFILIATED WITH SHANGHAI UNIV OF T C M
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