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Gene expression vector for adjusting and controlling NF-kB activity in cells as well as regulation and control method and application of gene expression vector

A gene expression and carrier technology, which is applied in the field of gene expression vectors that regulate the activity of NF-κB in cells, can solve problems such as side effects, and achieve the effect of simple regulation methods.

Active Publication Date: 2017-11-21
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The present invention solves the problem of excessive inhibition of NF-κB activity by small molecule NF-κB inhibitors in the prior art, resulting in serious side effects

Method used

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  • Gene expression vector for adjusting and controlling NF-kB activity in cells as well as regulation and control method and application of gene expression vector
  • Gene expression vector for adjusting and controlling NF-kB activity in cells as well as regulation and control method and application of gene expression vector
  • Gene expression vector for adjusting and controlling NF-kB activity in cells as well as regulation and control method and application of gene expression vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Evaluation of miRNA effects targeting NF-κB

[0040] method:

[0041] Materials and reagents: vector pIRES2-EGFP was purchased from Clontech, pcDNA TM 6.2-GW / EmGFP-miR-Neg was purchased from Invitrogen, pEGFP-C1 was purchased from Clontech, and pGL4.10-MP was purchased from Promega. Plasmids p-mCherry and p-RelA were constructed and provided by the laboratory of Wang Jinke's research group at the School of Biological Science and Medical Engineering, Southeast University. Plasmids p-mCherry and p-RelA are only used to provide mCherry and RelA protein coding sequences, and their sequences are the same as public genes Sequences in the database; restriction enzymes NheI, EcoRI, NotI, BamHI, KpnI, HindIII, NcoI, XbaI were purchased from Takara; HS (Premix), rTaq, T4Polynucleotide Kinase, T-Vector pMD19 (Simple), and DNA Markers were purchased from Takara; restriction enzymes AseI and NheI, type II endonuclease BsmBI, and T4DNA Ligase were purchased from NEB; LipofectamineT...

Embodiment 2

[0053] Effect Evaluation of NF-κB Specific Promoter

[0054] method:

[0055] Vector construction: a 52-bp fragment containing the classical binding site of NF-κB on the cloning vector pGL4.32 and named as decoy, its sequence is SEQ ID NO.10: 5'-GGG AAT TTC CGG GGA CTT TCC GGG AAT TTC CGGGGA CTT TCC GGG AAT TTC C-3' as NF-κB-specific regulatory elements. According to this sequence, two single-stranded oligonucleotides were designed and synthesized, and their sequences are shown in Table 2 as SEQ ID NO.11-12. The two single-stranded oligonucleotides are denatured and annealed to form a double-stranded DNA; the upstream and downstream of the double-stranded DNA are respectively introduced sequences that can anneal to the KpnI and HindIII enzyme-cut cohesive end sequences. Then the double-stranded DNA was ligated with linear pGL4.10-MP (MP, minimal promoter) cut by KpnI and HindIII double enzymes, and the recombinant vector was named pGL4.10-DMP. Thus, pGL4.10-DMP contains the...

Embodiment 3

[0065] Effect evaluation of NF-κB self-regulated miRNA expression vector

[0066] method:

[0067] Vector construction: The DMP fragment was cloned through the vector pDMP-EGFP, and AseI and NheI restriction sites were inserted upstream and downstream, respectively. The DMP fragment was connected to the recombinant vector pCMV-mCherry-amiR533 that removed the CMV promoter, and the NF-κB self-regulated miRNA expression vector was constructed, named pDMP-mCherry-amiR533 ( figure 2 ). Carrier verification was performed by PCR amplification and gene sequencing. At the same time, pCMV-mCherry-amiR533 was also constructed as a control ( figure 2 ), the vector was verified by DNA sequencing.

[0068] Cell culture: 293T cells were treated with DMEM containing 10% (v / v) FBS (HyClone Company), 100units / mL penicillin and 100g / mL streptomycin in 5% (v / v) CO 2 cultured at 37°C in a humidified incubator. cells at 1 x 10 5 cells / cm 2 seeded in a 24-well plate and incubated for 24 h...

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Abstract

The invention discloses a gene expression vector for adjusting and controlling NF-kB activity in cells as well as a regulation and control method and application of the gene expression vector. The vector comprises two sequence elements, a promoter sequence and an miRNA coding sequence, wherein the promoter sequence is used for adjusting and controlling gene expression, and the miRNA coding sequence is located at the downstream of a promoter; the promoter sequence is formed by a section of NF-kB trapper sequence and a minimal promoter sequence; the miRNA coding sequence is the miRNA coding sequence capable of encoding and targeting NF-kB miRNA. By transfecting the gene expression vector, the NF-kB activity in the overactive cells can be mildly and effectively reduced, and the activity of normal cells is not obviously influenced, so that the side effect is avoided. The regulation and control method of the gene expression vector is simple, rapid and effective; and the gene expression vector is used for preparing an NF-kB activity regulation and control reagent or a drug molecule for treating diseases closely related to NF-kB overactivity and is taken as a novel gene treatment reagent.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a gene expression carrier for regulating intracellular NF-κB (kappaB) activity, a regulating method and application thereof. Background technique [0002] Nuclear factor kappaB (NF-κB) is an important regulatory transcription factor that plays a key role in many physiological and pathological processes, such as immunity, cell proliferation, apoptosis, inflammation, and especially tumorigenesis. Some studies have shown that many diseases are related to abnormal activation of NF-κB and its signaling pathways. For example, NF-κB is aberrantly activated in many human cancers, promoting survival and malignancy by upregulating anti-apoptotic genes. It has been found that nuclear accumulation of NF-κB and a high NF-κB target gene signature lead to enrichment of the NF-κB pathway in most multiple myeloma cell lines and sensitivity to apoptosis. Therefore, many pharmaceutical co...

Claims

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Application Information

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IPC IPC(8): C12N15/63A61K48/00A61P29/00A61P35/00A61P37/02
CPCA61K48/005C12N15/63
Inventor 王进科汤缓缓
Owner SOUTHEAST UNIV
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