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Cancer cell broad spectrum high-activity promoter and usage thereof

A tumor cell and promoter technology, applied in the field of molecular biology, can solve problems such as weak expression activity, difficulty in meeting the needs of gene therapy, and shutting down expression

Active Publication Date: 2014-12-17
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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  • Cancer cell broad spectrum high-activity promoter and usage thereof
  • Cancer cell broad spectrum high-activity promoter and usage thereof
  • Cancer cell broad spectrum high-activity promoter and usage thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] Embodiment 1: Carry the construction of each promoter nucleotide sequence expression vector

[0090] 1. According to the coding sequence of Renilla luciferase (Renilla luciferase, referred to as RLuc) gene in psi-CHECK2 plasmid (purchased from Promega Company), design a pair of PCR-specific amplification primers (upstream primer F1, add EcoRI restriction Restriction site and protection base; downstream primer R1, plus SalI restriction site and protection base). The sequence of F1 and R1 is as follows:

[0091] F1: GCCgaattcGCCACCATGACTTCGAAAGT (SEQ ID NO: 5), wherein the lowercase letters represent the EcoRI restriction site

[0092] R1: TGTgtcgacTTATTGTTCATTTTTGAGAACTCGCT (SEQ ID NO: 6), wherein the lowercase letters represent the SalI restriction site.

[0093] Using the psi-CHECK2 plasmid as a template, the RLuc gene coding sequence was amplified, digested with EcoRI+SalI, and loaded into the adenovirus shuttle vector pDC315 (purchased from Beijing Benyuan Zhengy...

Embodiment 2

[0100] Example 2: Adenoviral dual-luciferase reporter lines carrying the nucleotide sequences of each promoter system construction

[0101] 1. According to the coding sequence of the firefly luciferase (Firefly luciferase, FLuc for short) gene in the psi-CHECK2 plasmid (purchased from Promega Company), design a pair of PCR-specific amplification primers (upstream primer F3, add EcoRI restriction Restriction site and protection base; downstream primer R3, plus SalI restriction site and protection base). The sequence of F3 and R3 is as follows:

[0102] F3: GCCgaattcGCCACCATGGAAGACGCC (SEQ ID NO: 9), wherein lowercase letters represent the EcoRI restriction site;

[0103] R3: TGTgtcgacTTACACGGCGATCTTTCCGC (SEQ ID NO: 10), wherein the lowercase letters represent the SalI restriction site.

[0104] Using the psi-CHECK2 plasmid as a template, the FLuc gene coding sequence was amplified, digested with EcoRI+SalI, and loaded into pENTR12 that was also digested with EcoRI+SalI ...

Embodiment 3

[0108] Embodiment 3: Utilize carrying adenovirus dual luciferase reporter system to measure the expression of each promoter active

[0109] 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 compl...

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Abstract

The invention belongs to the field of the molecular biology and relates to a cancer cell broad spectrum high-activity promoter and the usage thereof. The cancer cell broad spectrum high-activity promoter can express exogenous genes in cancer cells with broad spectrums efficiently. The expressive activity of the promoter in a series of cancer cells is higher than the existing CAG promoters and is stable in the sequence (it does not have sequence loss in the transfer process of the prokaryotic cell and the eukaryotic cell). The cancer cell broad spectrum high-activity promoter can be applied to the oncogene therapeutic process to drive the exogenous genes to be expressed efficiently in the cancer cells.

Description

technical field [0001] 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 [0002] 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 promoter can guide the holoenzyme to correctly combine with the template, activate RNA polymerase, and initiate gene transcription, thereby controlling the initiation time and degree of gene expression (transcription). The promoter is one of the important factors affecting the expression efficienc...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/89C12N15/85C12N15/861A61K48/00A61P35/00
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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