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A recombinant plasmid containing wsb1 gene promoter and reporter gene and its construction method and application

A recombinant plasmid and reporter gene technology, applied in biochemical equipment and methods, recombinant DNA technology, compound screening, etc., can solve the problems of high specificity, high efficiency and rapidity that have not been seen, and achieve fast detection speed, low cost, and high efficiency. The effect of sensitivity

Active Publication Date: 2022-06-24
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, there is currently no highly specific, efficient, fast, and sensitive drug screening model for WSB1 targets

Method used

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  • A recombinant plasmid containing wsb1 gene promoter and reporter gene and its construction method and application
  • A recombinant plasmid containing wsb1 gene promoter and reporter gene and its construction method and application
  • A recombinant plasmid containing wsb1 gene promoter and reporter gene and its construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] 1 Extraction of human genomic DNA

[0039] The genomic DNA containing WSB1-P is extracted from normal human peripheral blood leukocytes or other human cells, and is extracted by the operation method of the genomic DNA purification kit or tissue and cell genomic DNA purification kit. The specific steps are as follows:

[0040] (1) For adherent cultured cells, aspirate the medium, wash the cells once with PBS, and add trypsin solution (0.1-0.25%) for digestion. Centrifuge at 800 rpm for 10 min at room temperature, collect 10 6 -10 7 cells, remove the supernatant as much as possible.

[0041] (2) Resuspend the cells with 200 μl of solution PL, add 20 μl of proteinase K and 200 μl of solution BL, and mix well.

[0042] (3) 70°C water bath for 10min, invert and mix 2-3 times during this period.

[0043] (4) Add 200 μl of absolute ethanol, invert and mix thoroughly. The addition of absolute ethanol may produce a white fibrous suspension that does not affect DNA extractio...

Embodiment 2

[0134] During the research process of the present inventors, the WSB1 high expression plasmid and other recombinant plasmids containing different WSB1 promoter sequences were constructed at the same time for the screening of antitumor drugs. include:

[0135] (1) WSB1 high expression plasmid: insert the WSB1 coding sequence shown in SEQ ID NO: 4 into the Kpn I of the pGL3-promoter vector plasmid (purchased from Promega (Beijing) Biotechnology Co., Ltd., item number: E1761) Between the site of BglII and BglII, a recombinant plasmid highly expressing WSB1 protein was constructed.

[0136] (2) According to the method described in Example 1, construct pGL3-Basic-WSB1-P(1) (WSB1-P(1) sequence is shown in SEQ ID NO: 5) and pGL3-Basic-WSB1-P (2) (WSB1-P (1) The sequence is shown in SEQ ID NO: 6) recombinant plasmid.

[0137] After the above recombinant plasmid was constructed, it was co-transfected with the pRL-TK internal reference plasmid into human colon cancer HCT-116 cells ac...

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Abstract

The invention provides a recombinant plasmid containing WSB1 gene promoter and reporter gene, its construction method and application. Specifically, the WSB1 gene promoter-specific sequence was cloned, recombined into the vector sequence containing the firefly luciferase reporter gene, and a recombinant plasmid containing the WSB1 gene promoter and reporter gene was constructed, and then the constructed recombinant plasmid was combined with Renilla luciferase The reporter gene plasmid pRL-TK was co-transfected into tumor cells to make it stably expressed. Finally, the activity of WSB1 gene promoter to initiate transcription was reflected by detecting the activity of dual luciferase, so as to be applied to targeted screening of anti-tumor drugs. The recombinant plasmid constructed by the present invention adopts the WSB1 gene promoter, and selects a specific promoter sequence. The drug screening model constructed has high specificity, high sensitivity and high accuracy, and realizes the antibacterial drug targeting WSB1. High-throughput screening of cancer drugs.

Description

technical field [0001] The invention belongs to the technical field of gene recombination and molecular cloning, relates to a recombinant plasmid of tumor angiogenesis, invasion and metastasis-related gene targets, and in particular relates to a recombinant plasmid containing a WSB1 gene promoter and a firefly luciferase reporter gene and its construction methods and applications. Background technique [0002] WD repeat and SOCS box-containing protein 1 (WSB1) is a substrate of the ECs ubiquitin ligase complex, which contains 7 WD protein complexes and a SOCS box, and is involved in osteosarcoma, neural It is overexpressed in many tumor cells including blastoma, pancreatic cancer, liver cancer and salivary gland tumors. WD-repeat protein (WD repeatProtein) is a protein with highly conserved WD motifs and diverse functions, which is involved in the regulation of various tumor cells, such as cell signal transduction, RNA splicing, transcription and vesicle transport (Cell Mol...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63C12N15/65C12Q1/66C12Q1/02
CPCG01N33/5011C12N15/63C12N15/65C12Q1/66G01N2500/10
Inventor 周利红李琦冯煜伏杰隋华王炎季青冯媛媛刘宁宁呼雪庆
Owner SHUGUANG HOSPITAL AFFILIATED WITH SHANGHAI UNIV OF T C M
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