Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

A kind of eukaryotic promoter and its preparation method and application

A promoter and eukaryotic technology, applied in the biological field, can solve problems such as single activity, lack of highly active promoters, inability to flexibly control gene expression output, etc., and achieve a high success rate

Active Publication Date: 2020-08-11
SOUTHEAST UNIV
View PDF1 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the wild-type human cytomegalovirus promoter (wild-type CMV, wtCMV) in the prior art has a single activity and cannot flexibly control the gene expression output (output) in synthetic biology, and lacks a highly active promoter

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • A kind of eukaryotic promoter and its preparation method and application
  • A kind of eukaryotic promoter and its preparation method and application
  • A kind of eukaryotic promoter and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Construction of promoter and reporter gene expression plasmids

[0049] Promoter Construction: By using an improved SELEX-Seq method, the DNA binding specificity of NF-κB was successfully characterized (PLoS ONE, 2013, 8(10):e76109). By analyzing SELEX-Seq data, sequences with high affinity for NF-κB dimer were found. The human CMV IE promoter in the pEGFP-N1 plasmid (Clontech) was used for transformation, replacing its natural NF-κB binding site with the high-affinity sequence found in the SELEX-Seq experiment. The human wtCMV promoter sequence SEQ ID NO.15 contains four NF-κB binding sites, as shown in SEQ ID NO.16-SEQ ID NO.19 in Table 1. Two high-affinity sequences (T and P) selected by SELEX were used in the transformation, as shown in SEQ ID NO.20 and SEQ ID NO.21 in Table 2. Use the sequence named S that does not contain known TFBS, shown in SEQ ID NO.22 in table 2 as the NF-κB binding site knockout control ( figure 1 ).

[0050] In the study, the human wtCMV...

Embodiment 2

[0078] Promoter evaluation using HepG2 cells

[0079] method:

[0080] Cell culture: HepG2 cells were cultured in DMEM medium. The medium contained 10% (v / v) fetal bovine serum, 100 units / mL penicillin and 100 g / mL streptomycin. Cells at 37°C, 5% (v / v) CO 2 cultivated in. Cells were obtained from the Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

[0081] Cytoplasmic expression reporter gene system to evaluate promoter activity (dual luciferase detection): Cells were divided into 1×10 5 The density of cells / well was seeded in 24-well plates and incubated for more than 12 hours. Cells were then co-transfected with pGL4.10 plasmid (0.5 μg / well) with various promoters and internal control plasmid pGL4.75 (0.05 μg / well). Co-transfection of the plasmid pGL4.10 without inserting the promoter sequence and the internal reference plasmid pGL4.75 was used as a negative control. Cells were incubated with Lipofectamine 2000 and co-tr...

Embodiment 3

[0086] Evaluation of other promoters using HepG2 cells

[0087] method:

[0088] Cell culture: HepG2 cells were cultured in DMEM medium. The medium contains 10% fetal bovine serum, 100 units / mL penicillin and 100 g / mL streptomycin. Cells at 37°C, 5% CO 2 cultivated in. Cells were obtained from the Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

[0089] Cytoplasmic expression reporter gene system to evaluate promoter activity (dual luciferase detection): Cells were divided into 1×10 5 The density of cells / well was seeded in 24-well plates and incubated for more than 12 hours. Cells were then co-transfected with pGL4.10 plasmid (0.5 μg / well) with various promoters and internal control plasmid pGL4.75 (0.05 μg / well). Co-transfection of the plasmid pGL4.10 without inserting the promoter sequence and the internal reference plasmid pGL4.75 was used as a negative control. Cells were incubated with Lipofectamine 2000 and co-trans...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a eukaryotic promoter and a preparation method and application thereof. The eukaryotic promoter comprises any of 14 mutant CMV promoters with different transcriptional activities, and is obtained by substituting artificial base sequences with NF-kappaB binding sites in human wild-type CMV promoters through different combinations. A series of novel mammalian promoters are prepared, three promoters (T1P2, T1 and P2) thereof have stronger transcriptional activities than the human wild-type CMVs, and the three promoters with high transcription activities have potential important application value in the field of biomedicine. In addition, a series of engineered mammalian promoters with various transcriptional activities is also produced, and can be applied to flexible control of gene expression output in synthetic biology. The preparation method of the eukaryotic promoter, as a novel method, is used for producing the promoters with different transcriptional activities, and is simple and convenient and high in success rate.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a eukaryotic promoter and its preparation method and application. Background technique [0002] Expression of foreign genes in mammalian cells is essential for transgene therapy trials, DNA vaccines, and production of pharmaceutical products and basic research in cell biology. The expression level of foreign genes in mammalian expression systems is mainly related to the transcriptional strength of the promoters in the expression vectors. Therefore, for the expression of foreign genes in eukaryotic cells, it is very important to have appropriate promoters. These promoters have important application value in the production of medicine, medicine and biomedicine, gene therapy, and basic research of life science. The development of eukaryotic promoters is of particular value in the fields of gene therapy and drug production. [0003] Thirty years ago, researchers identified ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/85
CPCC12N15/113C12N15/85C12N2830/60
Inventor 王进科王丹阳
Owner SOUTHEAST UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com