Gene expression based on intracellular NF-kappa B activity activation effect gene in NF-kappa B overactivated cells and application

A gene expression and effector gene technology, applied in the field of medical biology, can solve problems such as side effects, physiological process interference, and low specificity

Inactive Publication Date: 2018-06-29
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, unfortunately, the specificity of these chemical drugs is low, and they produce serious side effects while inhibiting the activity of NF-κB
[0007] At present, the research and development of NF-κB inhibitor drugs mainly focus on the treatment of diseases related to NF-κB overactivation by inhibiting the activity of

Method used

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  • Gene expression based on intracellular NF-kappa B activity activation effect gene in NF-kappa B overactivated cells and application
  • Gene expression based on intracellular NF-kappa B activity activation effect gene in NF-kappa B overactivated cells and application
  • Gene expression based on intracellular NF-kappa B activity activation effect gene in NF-kappa B overactivated cells and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Expression of NF-κB RelA in different cells

[0043] experimental method:

[0044] Cell culture: HEK-293T (human fetal kidney cells), HepG2 (human liver cancer cells), A549 (human lung cancer cells), HT-29 (human colon cancer cells), HeLa (human cervical cancer cells), SKOV3 (human ovarian cancer) Cells), PANC-1 (pancreatic cancer cells), MDA-MB-453 (human breast cancer), Hepa1-6 (mouse liver cancer cells), mouse macrophages (RAW264.7), mouse melanoma cells ( B16F10), HL7702 (human normal liver cells) and MRC5 (human embryonic fibroblasts) cell culture. Cell culture uses DEME (Hepa1-6, HEK-293T, HepG2, HeLa, PANC-1, MDA-MB-453, RAW264.7, B16F10, MRC-5) or RPMI 1640 medium (A549, HT-29, SKOV) -3, HL7702), 10% fetal bovine serum (HyClone), 100units / mL penicillin and 100μg / mL streptomycin culture; the culture environment contains 5%(v / v)CO 2 Incubate at 37°C in a humidified incubator. After the cells are recovered, they are seeded into a 24-well microtiter plate (1...

Embodiment 2

[0048] Example 2 DMP-zsGreen (intracellular expression of effector gene)

[0049] experimental method:

[0050] Vector construction: construct an expression vector DMP-zsGreen; this vector contains the DMP sequence and the coding sequence of the green fluorescent protein zsGreen that can be expressed in cells under its control. Among them, DMP contains NF-κB response sequence (5'- GGGAAT TTC CGG GGA CTT TCC GGG AAT TTC CGG GGA CTT TCC GGG AAT TTC C-3', SEQ ID NO.1) and minimal promoter sequence (5'-TAG AGG GTATAT AAT GGA AGC TCG ACT TCC AG-3', SEQ ID NO. 3).

[0051] Cell culture: HEK-293T (human fetal kidney cells), HepG2 (human liver cancer cells), A549 (human lung cancer cells), HT-29 (human colon cancer cells), HeLa (human cervical cancer cells), SKOV3 (human ovarian cancer) Cells), PANC-1 (pancreatic cancer cells), MDA-MB-453 (human breast cancer), Hepa1-6 (mouse liver cancer cells), mouse macrophages (RAW264.7), HL7702 (human normal liver cells) ) And MRC5 (human embryonic f...

Embodiment 3

[0057] Example 3 DMP-Display-SBP (effect gene cell surface display expression)

[0058] experimental method:

[0059] Vector construction: construct an expression vector DMP-Display-SBP; this vector contains DMP sequence and the coding sequence for cell expression display streptavidin binding peptide (SBP). The DMP contains the NF-κB response sequence (5'-GGG AAT TTC CGGGGACTT TCC GGG AAT TTC CGG GGACTT TCC GGG AAT TTC C-3', SEQ ID NO. 1) and the minimal promoter sequence (5'-TAG AGG GTATAT AAT GGAAGC TCG ACT TCC AG-3', SEQ ID NO. 3). The SBP coding sequence is: ATG GAC GAG AAG ACC ACC GGG TGG CGG GGC GGC CAC GTT GTG GAG GGT CTCGCT GGC GAG CTG GAG CAG CTC AGG GCC CGC TTG GAG CAC CAT CCC CAG GGG CAACGCGAG CCT ATC GAT TAA (SEQ ID NO.4) . The expressed skeleton sequence is cloned from the vector pDisplay TM (Invitrogen); pDisplay TM The protein or polypeptide to be displayed on the cell membrane surface is fused to the N-terminus of the rat Igκ-chain leader sequence, which can ...

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Abstract

The invention discloses a NF-kappa B specifically activated gene expression vector and application thereof. A gene expression technique is implemented by the NF-kappa B specifically activated gene expression vector. The gene expression vector contains two elements, i.e., a regulatory gene-expressed promoter sequence and a promoter downstream effect gene coding sequence, wherein the promoter sequence consist of a section of NF-kappa B response sequence and a minimal promoter sequence. After the gene expression vector is introduced into NF-kappa B overactivated cells, an effect gene on the vector is expressed under the activation of a sequence-specific transcription factor NF-kappa B, and the effect gene generates influence on cell physiology, such as cell growth inhibition, apoptosis and death. The gene expression technique provided by the invention can be used for treating diseases related to NF-kappa B overactivation, such as inflammation and cancers. The gene expression vector constructed by the invention can be used in the preparation of gene therapy reagents and drugs for treating diseases related to NF-kappa B overactivation.

Description

Technical field [0001] The invention relates to a gene expression technology and application in a NF-κB over-activated cell based on NF-κB activity activation effect gene in a cell, and belongs to the field of medical biotechnology. Background technique [0002] NF-κB is a transcription factor found in B lymphocytes, named for its involvement in the transcriptional regulation of immunoglobulin κ light chain. It was later discovered that NF-κB is a eukaryotic transcription factor widely distributed in various cells, and an important regulator of innate immunity, inflammation, cell survival, proliferation, and apoptosis. The NF-κB family has five members: RelA (p65), RelB, c-Rel, p50 / p105 (NF-κB1) and p52 / p100 (NF-κB2), all of which contain a highly conserved Rel homology region at the N-terminus (RHD), responsible for binding to DNA, dimerization, nuclear localization, and binding to NF-κB inhibitory proteins (such as IκB). RelA / p50 is the most common NF-κB target gene regulator...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N5/10A61K31/7105A61K48/00A61P29/00A61P35/00A61P37/02
CPCA61K31/7105C07K14/47C12N15/85
Inventor 王进科王丹阳戴薇
Owner SOUTHEAST UNIV
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