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Chimeric strong promoter, and applications thereof

A technology of promoters and uses, applied in the field of molecular biology, can solve the problems of weak expression activity, closed expression, and difficulty in meeting the needs of gene therapy.

Active Publication Date: 2017-08-15
HEPATOBILIARY SURGERY HOSPITAL SECOND MILITARY MEDICAL UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, some virus-derived constitutive promoters are prone to be shut down by epigenetic modification despite high transient expression activity (such as CMV promoter); while some human-derived natural constitutive promoters or tumor-specific promoters Although the expression is stable, the expression activity is relatively weak, which is difficult to meet the needs of gene therapy

Method used

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  • Chimeric strong promoter, and applications thereof
  • Chimeric strong promoter, and applications thereof
  • Chimeric strong promoter, and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] Embodiment 1: Carry the construction of each promoter nucleotide sequence expression vector

[0091] 1. According to the coding sequence of the Renilla luciferase (Renilla luciferase, referred to as RLuc) gene in the psi-CHECK2 plasmid (purchased from Promega Company), a pair of PCR-specific amplification primers (upstream primer F1, plus EcoRI restriction enzyme) were designed at the beginning and end. cutting site and protecting base; downstream primer R1, plus SalI restriction enzyme cutting site and protecting base). The sequence of F1 and R1 is as follows:

[0092] F1: GCCgaattcGCCACCATGACTTCGAAAGT (SEQ ID NO: 5), wherein the lowercase letters represent the EcoRI restriction site

[0093] R1: TGTgtcgacTTATTGTTCATTTTTGAGAACTCGCT (SEQ ID NO: 6), wherein the lowercase letters represent the SalI restriction site.

[0094] Using the psi-CHECK2 plasmid as a template, the RLuc gene coding sequence was amplified, digested with EcoRI+SalI, and loaded into the adenovirus...

Embodiment 2

[0101] Embodiment 2: Construction of the adenoviral dual luciferase reporter system carrying each promoter nucleotide sequence

[0102] 1. According to the coding sequence of the firefly luciferase (Fireflyluciferase, referred to as FLuc) gene in the psi-CHECK2 plasmid (purchased from Promega Company), a pair of PCR-specific amplification primers (upstream primer F3, plus EcoRI restriction enzyme) were designed at the beginning and end. cutting site and protecting base; downstream primer R3, plus SalI restriction enzyme cutting site and protecting base). The sequence of F3 and R3 is as follows:

[0103] F3: GCCgaattcGCCACCATGGAAGACGCC (SEQ ID NO: 9), wherein lowercase letters represent the EcoRI restriction site;

[0104] R3: TGTgtcgacTTACACGGCGATCTTTCCGC (SEQ ID NO: 10), wherein the lowercase letters represent the SalI restriction site.

[0105] Using the psi-CHECK2 plasmid as a template, the FLuc gene coding sequence was amplified, digested with EcoRI+SalI, and loaded in...

Embodiment 3

[0109] Example 3: Determination of the activity of each promoter using the adenovirus dual luciferase reporter system

[0110] The low-passage HEK293, Hep3B, Huh7, HepG2, PLC / PRF / 5, BEL-7404, H460, H1299 cell lines (all purchased from ATCC) in good growth state were divided into 1×10 4 Cells / well spread 96-well plate, set at 37°C, 5% CO 2 Culture in the incubator for 24 hours; according to the multiplicity of infection MOI=5, respectively infect Ad-CMV-2Luc, Ad-CAG-2Luc, Ad-CAC-2Luc, Ad-CACU-2Luc, Ad-CCAU-2Luc, Ad-4CAU- 2Luc and other 6 kinds of recombinant viruses were set up with 4 duplicate wells for each group; 2 Incubator culture; after 24 hours, the cells were lysed, and placed in a microplate reader to measure the enzymatic activity of RLuc by a dual-luciferase detection kit (purchased from Promega Company), and the enzymatic activity of FLuc was used as an internal reference to obtain RLuc / FLuc relative ratio. The specific operation steps were completed according ...

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Abstract

The invention belongs to the field of molecular biology, and relates to a chimeric strong promoter, and applications thereof. The chimeric strong promoter is capable of realizing wide spectrum high efficiency expression of exogenous genes in tumor cells, the expression activity of the chimeric strong promoter in a series of tumor cells is higher than traditional CAG promoter, and the sequence is stable (sequence missing is not caused in prokaryotic cell and eukaryotic cell migration process). The chimeric strong promoter is suitable to be used in tumor gene therapy process to drive high efficiency expression of exogenous genes in tumor cells.

Description

[0001] The present invention is a divisional application of the parent application with application number 201410495099.7. The application date of the parent application is September 25, 2014, and the title of the invention is "a broad-spectrum high-activity promoter for tumor cells and its use". technical field [0002] The invention belongs to the field of molecular biology, and relates to a tumor cell broad-spectrum high-activity promoter and its application. The promoter is an artificially synthesized chimeric promoter, which has broad-spectrum high activity in tumor cells. The present invention also relates to a recombinant vector containing the promoter, a recombinant virus, and the use of the promoter controlling a therapeutic gene for gene therapy. Background technique [0003] The promoter is a part of the gene, usually located upstream of the 5' end of the structural gene, and is a DNA sequence that RNA polymerase recognizes, binds and initiates transcription. The...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/85A61K48/00A61P35/00
CPCA61K48/0058C12N15/85C12N2800/107C12N2830/15
Inventor 钱其军金华君李振海吕赛群吴红平丁娜李林芳俞德超吴孟超
Owner HEPATOBILIARY SURGERY HOSPITAL SECOND MILITARY MEDICAL UNIV
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