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PcDNA3.1(+)-Fbw7 (F-box and WD repeat domain-containing 7) recombinant plasmid as well as construction method and application of PcDNA3.1(+)-Fbw7 recombinant plasmid

A technology for recombinant plasmids and construction methods, which can be used in recombinant DNA technology, drug combinations, and pharmaceutical formulations to solve problems such as signal enhancement and enhancement

Inactive Publication Date: 2013-10-16
厦门大学附属成功医院 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, C / EBPδ can inhibit the expression of FBW7 in breast cancer, thereby enhancing the activity of mTOR / AKT / HIF-1 signaling pathway, which is beneficial to the occurrence and development of breast cancer; FBW7 and NOTCH1 signaling pathways also play an important role in breast cancer. Relationship, loss of FBW7 strengthens NOTCH1 signaling, further leads to failure of NOTCH1-dependent MYC regulation, and high expression of AKT

Method used

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  • PcDNA3.1(+)-Fbw7 (F-box and WD repeat domain-containing 7) recombinant plasmid as well as construction method and application of PcDNA3.1(+)-Fbw7 recombinant plasmid
  • PcDNA3.1(+)-Fbw7 (F-box and WD repeat domain-containing 7) recombinant plasmid as well as construction method and application of PcDNA3.1(+)-Fbw7 recombinant plasmid
  • PcDNA3.1(+)-Fbw7 (F-box and WD repeat domain-containing 7) recombinant plasmid as well as construction method and application of PcDNA3.1(+)-Fbw7 recombinant plasmid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] The effect of recombinant plasmid PcDNA3.1(+)-Fbw7 on the proliferation ability of cholangiocarcinoma cell line, the specific operation steps are as follows:

[0055] The cholangiocarcinoma in the logarithmic growth phase was transfected, the cells were digested and counted after 24 hours of transfection, and 5000 cells per well were seeded in a 96-well plate, and 8 replicate wells were set up for each group, with a final volume of 200 μL / Wells, and set a column without cells and only 200 μL of culture medium as blank control wells, fill the edge wells with PBS, put in 5% CO 2 Adhere to the wall overnight at 37°C in an incubator. According to the requirements of the experimental design, MTT was added in the dark after culturing for different periods of time, and the medium was removed in each space before addition, and 60 μL of medium and 10 μL of MTT solution with a concentration of 5 mg / ml were added to each well, and MTT was carefully sucked out after incubation at ...

Embodiment 2

[0058] The effect of the recombinant plasmid PcDNA3.1(+)-Fbw7 on the migration ability of cholangiocarcinoma cell line, the specific operation steps are as follows:

[0059] Serum-starved cells were treated for 24 hours, and the Migration / Invasion chamber was hydrated with DMEM serum-free culture solution: 500 μl culture solution was added to the upper layer of the chamber, and 700 μl culture solution was added to the lower layer, and placed in a 37°C cell incubator for 2 hours. Cells were digested and counted, and serum-free DMEM was used. Resuspend and dilute to 1×10 5 / mL, remove the DMEM culture medium in the small chamber, add 700 μl 10% FBS DMEM culture medium in the lower chamber, add 500 μl cell suspension in the upper layer, incubate in the incubator for 48 hours, remove the culture medium, wipe off the cells in the upper chamber with a cotton swab, and fix with methanol for 20 minutes , washed 2 times with PBS, stained with crystal violet staining solution for 30 min...

Embodiment 3

[0062] The effect of the recombinant plasmid PcDNA3.1(+)-Fbw7 on the drug sensitivity of cholangiocarcinoma cells, the specific operation steps are as follows:

[0063] After the recombinant plasmid PcDNA3.1(+)-Fbw7 was used to act on the cholangiocarcinoma cell line QBC939, the semi-lethal IC50 value of the chemotherapy drug cisplatin was used to culture for 48 hours, and the survival rate of the cells was detected by the MTT method (see Example 1 for the steps) , the result is as image 3 As shown, it shows that the recombinant plasmid PcDNA3.1(+)-Fbw7 can increase the sensitivity of cholangiocarcinoma cells to the chemotherapy drug cisplatin, and synergistically promote the chemotherapy drugs to inhibit the proliferation of cholangiocarcinoma cells QBC939.

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Abstract

The invention provides a PcDNA3.1(+)-Fbw7 recombinant plasmid as well as a construction method and an application of the PcDNA3.1(+)-Fbw7 recombinant plasmid and relates to a recombinant plasmid. The construction method comprises the following steps: (1) comparing the spectrogram information of a PSG5-Myc-Fbw7 plasmid with the spectrogram information of a PcDNA3.1(+) to determine the double-enzyme cleavage sites of EcoR I and Not I; (2) cleaving the PSG5-Myc-Fbw7 plasmid with two enzymes; (3) cleaving the carrier of the PcDNA3.1(+) plasmid with two enzymes; (4) identifying the efficiency of the double-enzyme cleavage through agarose gel electrophoresis, and connecting the carrier of the PcDNA3.1(+) plasmid with the whole length of the target gene of Fbw7; (5) transforming the recombinant plasmid into competent bacteria, screening out positive clones, performing identification through sequencing, and extracting the PcDNA3.1(+)-Fbw7 recombinant plasmid. The PcDNA3.1(+)-Fbw7 recombinant plasmid can be used for preparing medicine for curing the tumor of bile duct cancer of people.

Description

technical field [0001] The invention relates to a recombinant plasmid, in particular to the PcDNA3.1(+)-Fbw7 recombinant plasmid, its construction method and its application in the preparation of medicines for treating human cholangiocarcinoma. Background technique [0002] FBW7 (F-box and WD repeat domain-containing7) was first discovered in Escherichia coli, originally named Cdc4, also known as FBXW7, SEL-10, hAgo, hCDC4, is a complex type of SCF (SKP1 / CUL1 / F-box) Ubiquitin ligase, often involved in the ubiquitination of substrates. [0003] The so-called ubiquitination refers to a chemical reaction that converts a substrate protein into ubiquitin, resulting in the loss of substrate function and degradation. Ubiquitination is of great significance to life. In recent years, studying tumor occurrence and development from the perspective of ubiquitination has become an important part of tumor research (1.Philip Cohen and Marianna Tcherpakov.Will the Ubiquitin System Furnish ...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/66C12N5/10A61K48/00A61P35/00
Inventor 李文岗谷乐郭跃
Owner 厦门大学附属成功医院
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