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Soluble receptor sIL-36R of IL-36 and application of soluble receptor sIL-36R

An IL-36, soluble technology, applied in the receptors of cytokines/lymphokines/interferons, medical preparations containing active ingredients, receptors/cell surface antigens/cell surface determinants, etc., to inhibit inflammation. Disease, reduce inflammation, relieve psoriasis effect

Pending Publication Date: 2020-10-30
EAST CHINA NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, regarding whether the IL-36 family contains natural soluble receptors that compete with IL-36R for binding IL-36, thereby inhibiting the IL-36 / IL-36R signaling pathway, and then inhibiting, treating, alleviating, and alleviating the technical content of psoriasis , there is no relevant report

Method used

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  • Soluble receptor sIL-36R of IL-36 and application of soluble receptor sIL-36R
  • Soluble receptor sIL-36R of IL-36 and application of soluble receptor sIL-36R
  • Soluble receptor sIL-36R of IL-36 and application of soluble receptor sIL-36R

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1: Soluble receptors have the ability to bind IL-36γ

[0056] Culture human kidney epithelial cells (293T) cells, inoculate them in 10cm dishes, and set up three groups for experiments. When the 293T cells grow to 70%, the first group transfects the corresponding empty vehicle (EV) into the cells, and the second group Transfect the IL-36R plasmid with the Flag tag into the cells, and the third group transfects the soluble receptor sIL-36R overexpression plasmid with the Flag tag as shown in SEQ ID NO.1 of the present invention, change the medium for 6h, and change the medium After 36 hours, gently blow down the cells, collect them in EP tubes and wash them twice with PBS, use the cell lysate added with protease inhibitors to lyse the cells to collect proteins, and incubate the collected proteins with Flagbeads overnight Competitive elution with Flag peptides, after measuring the concentrations of the three groups of eluted proteins, incubate equimolar empty hist...

Embodiment 2

[0058] Example 2: Soluble receptors have the ability to compete with IL-36R for binding IL-36γ

[0059]Cultivate human cervical cancer epithelial (Hela) cells and inoculate them in 6-well plates. When the Hela cells grow to 70%, a group of IL-36R plasmids with GFP tags will be transfected into the Hela cells, and the other group will be transfected into the cells together. Transfect the IL-36R plasmid with GFP tag and the soluble receptor overexpression plasmid sIL-36R shown in SEQ ID NO.1 of the present invention, change the medium for 6 hours, and add IL-36γ with 6xhis tag after 36 hours protein stimulation. After 45 minutes, the supernatant was discarded, and the cells were collected by gently blowing down, washed twice with PBS in EP tubes, stained with PE-anti-his dye for 30 minutes, then washed twice with PBS, filtered cells, and analyzed by flow cytometry The percentage of GFP+PE+ double-positive cells in the two treatment groups was analyzed by cytometer, and the ordi...

Embodiment 3

[0060] Example 3: Soluble receptors block IL-36γ-activated IL-36R signaling pathway in keratinocytes

[0061] Cultivate human keratinocytes (NHEK) cells, inoculate in 6 well plates, when NHEK cells grow to 70%, in NHEK cells, transfect the soluble receptor overexpression plasmid sIL shown in SEQ ID NO.1 of the present invention -36R, change the medium at 6 hours, wait 24 hours after the medium change, stimulate the cells with 100ng / ml recombinant IL-36γ protein for 30 minutes, collect the protein, and detect the phosphorylation of p38MAPK and p65 by Western blotting. The experimental results show that, if Figure 4 As shown, IL-36γ can induce the phosphorylation of p38MAPK and p65. It can be seen by immunoblotting that when the GAPDH internal reference protein is consistent, IL36γ can significantly induce the phosphorylation of p38MAPK and NF-κB subunit p65, that is, p- Expression of p38 and p-p65. When the sIL-36R plasmid was overexpressed, the phosphorylation bands of p38M...

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Abstract

The invention discloses a soluble receptor (sIL-36R) of interleukin-36 (IL-36). The sIL-36R only contains a ligand binding region of the IL-36R and is free of a transmembrane region and an intracellular region of the IL-36R, and has an amino acid sequence as shown in SEQ ID NO. 1. The soluble receptor can inhibit IL-36 activated NF-KB and p38 MAPK signal pathways, inhibit IL-36 induced inflammatory response, inhibit IL-36 induced expression of the antibacterial protein REG3A and relieve pathological symptoms of psoriasis, and has a treatment effect on psoriasis. The invention also discloses apreparation method of the sIL-36R.

Description

technical field [0001] The present invention relates to the fields of biochemistry, molecular biology, immunology and dermatology, relates to a soluble receptor sIL-36R of IL-36 and its application, especially relates to a soluble receptor and its role in inhibiting psoriasis inflammation Response and role in treatment of psoriasis. Background technique [0002] Psoriasis, commonly known as psoriasis, is a chronic inflammatory skin disease mediated by the immune system. Its typical features are papules and erythema of various sizes on the affected skin, epidermal hyperplasia, and the surface is covered with silvery white scales. 1-4 . Psoriasis not only causes plaques and pustules on the skin, redness, swelling, and itching, but also may cause nail damage, joint lesions, and even joint deformities. harm 5 . Because the pathogenesis of psoriasis is still poorly understood, so far there is no method and drug for radically curing psoriasis. [0003] Studies have found that...

Claims

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Application Information

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IPC IPC(8): C07K14/715C07K1/36C07K1/34C07K1/18A61K38/17A61P17/06
CPCA61K38/00A61P17/06C07K14/7155
Inventor 赖玉平王旺高晓广吴雅伟
Owner EAST CHINA NORMAL UNIVERSITY
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