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Human MDR1 gene expression regulator screening system and method for screening gene expression regulator

A technology of gene expression and regulators, applied in genetic engineering, plant genetic improvement, botany equipment and methods, etc., can solve the problems of difficult application, high toxicity and side effects, etc., and achieve the effect of wide applicability and simple screening method

Inactive Publication Date: 2006-05-31
NORTHEAST NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But all have its fatal weakness. The drugs that have been found to block the P-gp pump are difficult to be applied clinically due to the high toxicity and side effects (Sharma V et al. Chem Rev. 1999, 99: 2545-2560.)

Method used

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  • Human MDR1 gene expression regulator screening system and method for screening gene expression regulator
  • Human MDR1 gene expression regulator screening system and method for screening gene expression regulator
  • Human MDR1 gene expression regulator screening system and method for screening gene expression regulator

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Extraction of Human Genomic DNA

[0043] 1. Take 3ml of human peripheral blood, add anticoagulant, mix well

[0044] 2. Centrifuge at 3000rpm 4°C for 20'.

[0045] 3. Suck off the upper layer of plasma, be careful not to suck off the middle white blood cell layer.

[0046] 4. Add 5 times the volume of sterile double-distilled water, mix well, and place at room temperature for 5-10 minutes.

[0047] 5. Centrifuge at 4000 rpm for 20 minutes at 4°C and discard the supernatant. Be careful not to aspirate the leukocytes.

[0048] 6. Add 5ml of normal saline to restore the white blood cells to an isotonic environment.

[0049] 7. Centrifuge at 4000rpm for 15 minutes at 4°C and discard the supernatant.

[0050] 8. Repeat steps 6 and 7 twice for a total of 3 washes

[0051] 9. Obtain the buffy coat.

[0052] 10. Add 5ml digestion buffer (TES solution) to the white blood cells

[0053] (15mM Tris-cl, 15mM EDTA, 15mM Nacl, 0.5% SDS)

[0054] 11. Add proteinase K to a fin...

Embodiment 2

[0061] Fishing of the MDR1 promoter region

[0062] 1. Primer Design

[0063] Find the base sequence of the MDR1 promoter region from GenBank according to literature reports, and design primers for PCR amplification based on this sequence. The primer sequence is as follows: the upstream primer is: 5'-GGGGTACCCCAGTCTCTACG-3'; the downstream primer is: 5 '-CAAGCTTGTCCGACCTGAAGAG-3'. The 5' end has a Kpn I restriction site, and the 3' end has a HindIII restriction site.

[0064] 2. PCR amplification

[0065] Using human genomic DNA as a template, the primers synthesized above were used for PCR amplification. The PCR reaction conditions are: 94°C, 5min; 94°C, 1min, 62°C, 45sec, 72°C, 1min, 30 cycles; 72°C, 10min.

[0066] 3. Electrophoresis and recovery of PCR products

[0067] Perform electrophoresis on the above PCR product with 1% agarose gel. After electrophoresis, cut a small opening with a blade in front of the 2Kb band in the PCR product, insert DE81 filter paper, cont...

Embodiment 3

[0073] Construction of MDR1-p-pGL3 and MDR1-p-pEGFP1 recombinant plasmids

[0074] Amplify the above-mentioned TA clone bacteria and extract the plasmid, digest with Kpn I and HindIII, and recover a 2Kb fragment; at the same time, digest pGL3 with Kpn I and HindIII, recover the digested plasmid, and combine the recovered fragment with pGL3 The plasmid was ligated with TAKARA ligase I. When constructing the MDR1-p-pEGFP1 recombinant plasmid, the positive TA clone plasmid and the pEGFP1 vector were digested with EcoR I and the fragments were recovered for ligation. The ligation method was carried out according to the instructions of the TAKARA Ligation Kit. After ligation at 16°C for 4 hours, the ligation product was treated as above and electrotransformed into DH5α, the bacteria were spread on LB medium with ampicillin, and cultured overnight in a 37°C incubator. Pick the colony and inoculate it into 2ml of LB medium, culture it with shaking at 37°C overnight, extract the plasm...

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Abstract

This invention presents a filtration system by human MDR1 gene expression regulator, and in order to filtrate high efficientíólow poisonous dreye enhance sensitive medicament and durable remedy reverse medicament, this invention provides a method to filtrate human MDR1 gene expression regulator. This filtration system includes a regroup vector has multi-nucleotide and host cell transform or infect from the recombination vector, this recombination vector includes original vector and human gene promoter, and the original vector has a report gene without promoter, the human MDR1 gene promoter connect with the upriver of the report gene, and the human MDR1 gene promoterí»s avidity correlate with the report geneí»s expression. This invention also opens the method and use to form the filtration system. The filtration system can be used to filtrate human MDR1 gene expression regulator.

Description

technical field [0001] The invention discloses a screening system for human MDR1 gene expression regulators, and also provides a method for screening human MDR1 gene expression regulators using the system, in order to screen high-efficiency, low-toxic chemotherapy sensitizers and drug resistance reversal agents, belonging to field of biopharmaceutical technology. Background technique [0002] During the course of chemotherapy, tumor cells will develop drug resistance to a large class of drugs that have no correlation in structure and function. This phenomenon is called "multidrug resistance" (MDR). The acquisition of sex is closely related to the expression of various ATP-binding cassette (ATP-Binding cassette, ABC) carrier proteins on the cell surface. In human chromosomes, there are about 50 kinds of ABC carrier genes, among which the P-sugar protein encoded by the MDR1 gene Proteins (P-glycoprotein, P-gp or P170) mediate drug resistance most widely in tumor cells (Michih...

Claims

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Application Information

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IPC IPC(8): C12N15/12C07K14/435C12N15/63C12N15/65C12Q1/02C12Q1/68
Inventor 鲍永利李玉新孟祥颖乌垠纪雪易静雯
Owner NORTHEAST NORMAL UNIVERSITY
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