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Building method of PDGFB promoter activity report plasmids

A technology of reporter plasmid and construction method, which is applied in the field of construction of PDGFB promoter activity reporter plasmid to achieve the effect of promoting tumor and liver fibrosis

Pending Publication Date: 2020-02-11
NANTONG UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is currently no suitable reporter plasmid for studying the promoter activity of the PDGFB gene

Method used

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  • Building method of PDGFB promoter activity report plasmids
  • Building method of PDGFB promoter activity report plasmids

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] A kind of construction method of PDGFB promoter activity reporter plasmid, described mouse PDGFB gene promoter sequence is as shown in SEQID:NO:1, construction method comprises the following steps:

[0029] S1. Extract the peripheral blood of normal mice, use the blood DNA extraction kit (available in various biological reagent companies, this experiment was purchased from Sangon Bioengineering (Shanghai) Co., Ltd.) to extract mouse genomic DNA, and test the DNA purity and backup for completeness;

[0030] S2, database search, the promoter sequence of the transcription start site (TSS) region of the mouse PDGFB gene with a length of 600bp (-499-100bp) is shown in SEQ ID: NO: 1;

[0031] S3, PCR amplification of the PDGFB gene promoter sequence, adding the corresponding restriction endonuclease sequence and protective bases at both ends of the primer; the sequence of KpnI is shown in SEQ ID: NO: 3, and the sequence of Hind III is shown in SEQ ID: NO: 4 shown; the sequen...

Embodiment 2

[0040] S11. Plasmid identification step: the plasmid constructed in step S10 was preliminarily identified by restriction endonuclease double digestion, and further sent to Shanghai Invitrogen Company for sequencing to sequence the constructed plasmid, and named it pPDGFB-luc to construct the plasmid The sequence, that is, the sequence of pPDGFB-luc is shown in SEQ ID: NO:2.

[0041] Such as figure 1 As shown, the above-mentioned plasmid was digested with Kpn I and Hind III and then detected by agarose gel electrophoresis to obtain two bands of about 600bp and about 5000bp, which can be used for preliminary verification of the plasmid constructed in the present invention, and then confirmed by further sequencing. The plasmid sequence is correct.

Embodiment 3

[0043] S12. The function of the constructed plasmid was verified by detecting the promoter luciferase activity after transfecting mouse HSCs cells. The results further confirmed that the constructed PDGFB promoter activity reporter plasmid pPDGFB-luc was functional and used for the PDGFB promoter active research.

[0044] In the step S12, verifying the function of constructing the plasmid by detecting the promoter luciferase activity after transfecting the mouse HSCs cells includes the following steps: inoculating the mouse HSCs cells on a 12-well plate one day before the transfection, and culturing until the cell density reaches About 70%. Dilute 1.6 ug plasmid and 30 ng renilla with 300 ul Optin-MEM, according to the Lipo 2000 liposome transfection reagent instructions, respectively pPDGFB-luc (constructed in step S10) and pGL3-Basic-vector (negative control group) Transfected into cells, set up 3 duplicate wells. After 24 hours, the cells were collected using the dual flu...

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Abstract

The invention discloses a building method of PDGFB gene promoter activity report plasmids. The method comprises the following steps of extracting peripheral blood of normal mice; extracting mouse genomic DNA by a DNA extraction kit; detecting the DNA purity and integrity for use; performing database retrieval to obtain a promoter sequence of a mouse PDGFB gene; performing PCR amplification on thepromoter sequence of the mouse PDGFB gene; adding corresponding restriction endonuclease sequences and protection basic groups to the two ends of a primer; performing recovery and purification on an amplification product through a DNA gel recovery kit; performing double enzyme digestion on a PCR product subjected to recovery and purification and luciferase report plasmid vectors pGL3-Basic-vectorrespectively by Kpn I and Hind III; performing recovery and purification on an enzyme digestion product through the gel recovery kit; recovering target gene fragments and vector fragments; performingconnection over the night at 16 DEG C by using T4 ligase; converting a connection product into competent escherichia coli on a next day; and coating a solid LB culture medium plate over the night.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a method for constructing a PDGFB promoter activity reporter plasmid. Background technique [0002] Platelet-derived growth factor (PDGF) is currently the strongest known mitogen. The traditional PDGF family includes PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC, and PDGF-DD. PDGF has a strong ability to promote cell proliferation. , differentiate and lead to tumors and liver fibrosis, among which the role of PDGF-BB in the process of liver fibrosis is particularly prominent. The expression of PDGFB gene is regulated by many factors, among which the regulation of its promoter activity is the most important regulation method. However, there is currently no suitable reporter plasmid for studying the promoter activity of PDGFB gene. Contents of the invention [0003] The invention provides a method for constructing a PDGFB promoter activity reporter plasmid to solve the problems in the prior a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/70C12N15/66
CPCC12N15/70C07K14/49
Inventor 翟旭光
Owner NANTONG UNIVERSITY
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