Application of oct4 in regulation of IL-31 gene expression

A gene expression and gene technology, applied in the application field of Oct4 in regulating IL-31 gene expression, can solve problems such as unclear

Active Publication Date: 2019-03-22
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, whether Oct4 regulates the expression of IL-31 gene is still unclear.

Method used

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  • Application of oct4 in regulation of IL-31 gene expression
  • Application of oct4 in regulation of IL-31 gene expression
  • Application of oct4 in regulation of IL-31 gene expression

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1 RT-PCR method was used to confirm the influence of transcription factor Oct4 on the expression of IL-31 gene.

[0019] (1) Construction of Oct4 and shOct4 lentiviral expression vectors.

[0020] 1) Design of Oct4 gene primers.

[0021] Primer 5.0 software was used to design primers for amplifying the open reading frame of Oct4 gene. The restriction sites at both ends of the primers were XhoI and NcoI respectively. The primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd. The sequences of the primers are as follows:

[0022] Oct4 forward primer: 5'-CCGCTCGAGAGGATGGCGGGACACCTGGCTT-3'

[0023] Oct4 reverse primer: 5'-CATGCCATGGAAGGGCAGGCACCTCAGTTTG-3'.

[0024] 2) Amplify the Oct4 gene.

[0025] The Oct4 gene was amplified using the cDNA of human embryonic stem cells (purchased from Shanghai Stance Biotechnology Co., Ltd.) as a template.

[0026] The reaction system of PCR amplification is as follows:

[0027]

[0028] The reaction condition...

Embodiment 2

[0073] Example 2 The CHIP (chromatin immunoprecipitation) method was used to identify the combination of Oct4 and IL-31 gene promoter in MSCs of different passages.

[0074] The experimental steps were carried out according to the instructions of the CHIP kit, with slight changes. The CHIP kit was purchased from Wuhan Aimeijie Technology Co., Ltd.

[0075] (1) UC-MSCs (P3, P6, P9) of different generations were cross-linked and fixed with 1% formaldehyde, placed at room temperature for 10 min, and the reaction was terminated with glycine solution;

[0076] (2) Then wash the cells twice with pre-cooled PBS, centrifuge to remove the supernatant, and then add 1 ml of SDS lysate containing protease inhibitors to lyse the cells;

[0077] (3) Ultrasonic fragmentation of chromatin DNA in the cell lysate, using protein G magnetic beads to remove non-specifically bound DNA fragments;

[0078] (4) Anti-Oct4 antibody was added to the remaining supernatant, and mouse IgG antibody was adde...

Embodiment 3

[0090] Example 3 The dual-luciferase reporter gene system was used to detect the regulatory effect of Oct4 on IL-31 gene expression.

[0091] (1) Construction of a plasmid containing an IL-31 promoter.

[0092] 1) Synthesize IL-31 promoter DNA containing XhoI (tcgag) and Hind III (agctt) restriction sites, the sequence of the template strand (sense strand) is as follows:

[0093] 5'-GCTCGAGAGGCGAACACATCTGGCTCCAAGCTTCG-3'.

[0094] 2) The IL-31 promoter DNA and the pGL3-Luciferase plasmid (Promega) were subjected to XhoI and HindIII double digestion respectively, and the IL-31 promoter DNA double digestion conditions were as follows:

[0095]

[0096] The pGL3-Luciferase plasmid double digestion conditions are as follows:

[0097]

[0098]3) After digestion at 37°C overnight, run electrophoresis on 1% agarose gel, and recover the digested IL-31 promoter DNA and pGL3-Luciferase plasmid respectively;

[0099] 4) The IL-31 promoter double-enzyme digestion product was conn...

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Abstract

The invention discloses application of an Oct4 (octamer-binding transcription factor 4) in regulating IL-31 gene expression. The application is based on the achievement that the inventor detects the Oct4 can combine and activate the new target gene IL-3 in mesenchymal stem cells for the first time. A method of immune coprecipitation and double-luciferase report gene system detection is utilized to prove that the Oct4 can specifically combine the promoter of the IL-31 gene in the mesenchymal stem cells and can activate the transcription of the IL-31 gene. Therefore, the research results can provide theoretical bases for researching the expression regulation mechanism of the Oct4 for the new target gene IL-31, and can lay necessary foundations for application of the Oct4 and IL-31 in treatment of dermatitis and other diseases.

Description

technical field [0001] The invention relates to the fields of molecular biology and genetic engineering, in particular to the application of Oct4 in regulating the expression of IL-31 gene. Background technique [0002] Interleukin 31 (Interleukin 31, IL-31) is a polypeptide chain cytokine with 4 helical structures discovered by Dillon et al. 4+ Th2 cells are produced. The human IL-31 gene is located at 12q24.31, containing 904bp, with an open reading frame of 495bp, encoding a polypeptide chain consisting of 164 amino acid residues, and the mature IL-31 protein contains 141 amino acids. IL-31 binds to the heterodimer complex composed of two subunits of IL-31RA and OSMRβ, thereby activating three signaling pathways of JAK-STAT, MAPK, and PI3K / AKT to exert biological functions. The receptors of IL-31 are mainly distributed in tissues such as epithelial cells, lymphocytes, bronchi and skin, so the current research on IL-31 mainly focuses on the role of IL-31 in skin inflamma...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/47A61K48/00A61K38/17A61P17/00
CPCA61K38/00C07K14/4702
Inventor 魏星梁广铨
Owner JINAN UNIVERSITY
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