CN111848780A

CN111848780A - Soluble receptor sIL-36R of IL-36 and application thereof

Info

Publication number: CN111848780A
Application number: CN201910333393.0A
Authority: CN
Inventors: 赖玉平; 王旺; 高晓广; 吴雅伟
Original assignee: East China Normal University
Current assignee: East China Normal University
Priority date: 2019-04-24
Filing date: 2019-04-24
Publication date: 2020-10-30

Abstract

The invention discloses a soluble receptor (sIL-36R) of interleukin-36 (IL-36), which only contains a ligand binding region of the IL-36 receptor (IL-36R), lacks a transmembrane region and an intracellular region, and has an amino acid sequence shown as SEQ ID NO. 1. The soluble receptor can inhibit NF-KB and p38MAPK signal pathways activated by IL-36, inhibit inflammatory reaction induced by IL-36, inhibit expression of antimicrobial protein REG3A induced by IL-36, relieve pathological symptoms of psoriasis, and treat psoriasis. The invention also discloses a preparation method of the soluble receptor (sIL-36R).

Description

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

Technical Field

The invention relates to the fields of biochemistry, molecular biology, immunology and dermatology, relates to a soluble receptor sIL-36R of IL-36 and application thereof, and particularly relates to a soluble receptor and an effect thereof in inhibiting psoriasis inflammatory response and treating psoriasis.

Background

Psoriasis (psoriasis), commonly known as psoriasis, is an immune-mediated chronic inflammatory skin disease typically characterized by the presence of papules, erythemas, epidermal hyperplasia of varying size and covered by silvery white scales on the skin ^1-4. Psoriasis not only causes plaques and pustules on the skin to cause red swelling and pruritus of the skin, but also can cause nail damage and arthrosis and even joint deformity, thus not only causing the patients to suffer from physical pain, but also bringing serious mental injury to the patients⁵. At present, the pathogenesis of psoriasis is still poorly understood, so that no method and medicament for radically treating psoriasis exist.

It has been found that IL-36 in IL-1 family cytokines has a significantly increased expression in skin lesions of psoriatic patients and is positively correlated with the pathological state of psoriasis⁶. The group of subjects of the invention finds that RNA released by damaged cells induces KC to express IL-36 (including IL-36 alpha, IL-36 beta and IL-36 gamma) by directly activating TLR3 on Keratinocytes (KC) in the process of researching skin damage induced psoriasis (Koebnerpenhenomenon), and the IL-36 in turn acts on KC induced KC to express REG3A to promote KC to rapidly proliferate, thereby triggering psoriasis epidermal hyperplasia^7,8. Manfred Kopf et al demonstrated that IL-36 not only acts on KC, but also induces Dendritic Cells (DC) to express IL-23 and act on Th17, gamma T and other cells to produce a large amount of IL-17. IL-17 subsequently acts on KC to induce the KC to express a large amount of chemotactic factors and IL-36 to form an inflammatory response circulation system of psoriasis ⁹. In addition, they also found that mice were not induced with psoriatic pathological symptoms by Imiquimod (IMQ) after IL-36R knockout in mice. These findings demonstrate that IL-36 is one of the key factors in the induction and maintenance of the pathological state of psoriasis, and that IL-36 and its receptor IL-36R will also serve as potential targets for the treatment of psoriasis.

The activity of IL-1 family cytokines and their induced inflammatory responses are tightly regulated by inhibitors, some of which are IL-1 family antagonistsAgents, also in part, are membrane-bound receptors lacking the intracellular segment or soluble decoy receptors in the IL-1R family. In the IL-1R family, the presence of a variety of decoy receptors has been found. IL-1R2 was the first receptor cloned in the last 80 th century. Compared with 80kDa IL-1R1, IL-1R2 has only 68kDa due to lack of intracellular domain, resulting in failure of IL-1R signaling pathway to pass down¹⁰. In addition to the membrane-bound form, the extracellular domain released from IL-1R2 cleaved by a protease on the cell membrane at a position adjacent to the extracellular domain of IL-1R2 can be used as soluble IL-1R2, and the cleaved soluble IL-1R2 can compete with IL-1R2 on the cell membrane to bind its ligand to exert inhibitory effect ^11-15. In addition, IL-33 in the IL-1 family usually binds to the receptor ST2 to play a role, while its soluble receptor sST2 can be used as a decoy receptor to compete with ST2 for binding to IL-33 to inhibit IL-33/ST2 signal channel¹⁶. However, there is no report on whether the IL-36 family contains natural soluble receptors to compete with IL-36R to bind IL-36, so as to inhibit IL-36/IL-36R signal pathway, and further inhibit, treat, alleviate and alleviate psoriasis.

Disclosure of Invention

The invention provides a soluble receptor (sIL-36R) of interleukin-36 (IL-36), and a preparation method and application thereof. The soluble receptor is obtained by utilizing affinity chromatography and ion exchange purification after being expressed by an escherichia coli system. The soluble receptor contains only the ligand binding region of the IL-36 receptor (IL-36R), with the transmembrane and intracellular regions deleted. The soluble receptor inhibits NF-KB and p38MAPK signal pathways activated by IL-36/IL-36R by competitively binding IL-36 with IL-36R, inhibits the expression of IL-36-induced antimicrobial protein REG3A, can relieve inflammatory reaction induced by IL-36 cytokine, inhibits the disease of psoriasis, relieves the pathological symptoms of psoriasis, and has therapeutic effect on psoriasis.

The soluble receptor of the present invention can be produced by the alternative splicing of IL-36R pre-mRNA in the normal physiological state of an organism, such as a human.

Guanghui Yi, Structural and functional attributes of the extracellular domain of Interleukin receptor 36 was mentioned in the JBC publication of 2016 (see FIG. 16) that 335, 258 and 126-211 of the extracellular domain of Interleukin receptor 36 are also capable of inhibiting IL 36-induced activation of the IL36 signaling pathway. However, the soluble receptor sIL36R provided by the invention is different from the sequence length in the aspect of amino acid sequence comparison, the sequence of the soluble receptor sIL36R provided by the invention is shorter, the application transformation can be better, and the invention is an antagonist of a natural IL36 signal channel found in cells.

NF-KB, the nuclear factor activated B cell kappa light chain enhancer (abbreviated as NF-kappa B), is a protein complex that controls DNA transcription and consists of two subunits, p50 and p 65. NF-. kappa.B is present in almost all types of animal cells and is involved in the response of cells to many stimuli. These stimuli include stress, cytokines, free radicals, ultraviolet radiation, oxidized LDL, and bacterial or viral antigens. NF-. kappa.B plays an important regulatory role in the immune response against infection (the kappa light chain is an important component of immunoglobulins). Dysregulation of NF-. kappa.B is associated with cancer, inflammatory and autoimmune diseases, septic shock, viral infections, and immune dysplasia.

p38MAPK, mitogen-activated protein kinase (MAPK), is a group of serine-threonine protein kinases that are activated by various extracellular stimuli, such as cytokines, neurotransmitters, hormones, cellular stress and cell adhesion. MAPK is so called because it is identified by the activation of cultured cells when stimulated by mitogens such as growth factors. All eukaryotic cells express MAPK. The basic composition of the MAPK pathway is a three-level kinase mode conserved from yeast to human, and comprises MAPK kinase (MAP kinase, MKKK), MAPK kinase (MKK) and MAPK, wherein the three kinases can be sequentially activated to jointly regulate multiple important cell physiological/pathological processes such as cell growth, cell differentiation, stress adaptation to environment, cell inflammation reaction and the like. Mitogen-activated protein kinase (MAP kinase, MAPK) chains are one of the important pathways in the eukaryotic signaling network, playing a key role in gene expression regulation and cytoplasmic functional activities. MAPK chains consist of the 3-class protein kinase MAP3K-MAP2K-MAPK, which in turn phosphorylates the upstream signal to downstream responding molecules.

IL-36 refers to interleukin-36, a cytokine belonging to the interleukin-1 family, is involved in acute and chronic inflammation, and plays an important role in the innate immune response. Comprises three members of IL36 alpha, IL36 beta and IL36 gamma.

Wherein the amino acid sequence of the soluble receptor (sIL-36R) of the interleukin-36 (IL-36) is shown in SEQ ID NO. 1: MGVTSLLFCGVFFLLLLFVAADTCEDIFMHNVIISEGQPFPFNCTYPPETNGAVNLTWYKTPSKSPVSNNRHLRVHQDQTWILFLPLTLEDSGIYQCVIRNAHNCYQIAVNLTVLKNHWCDSSMEGSPVNSPDVYQQILPIGKSGSLNCHLYFPESCALDSIKWYKGCEEIKAGKKYSPSGAKLLVNNVAVEDGGSYACSARLTHLGRHFTIRNYIAVNTKEVEYGRRIPNITYPKNNSIEVPLEPMCP are provided.

Wherein the amino acid sequence shown in SEQ ID NO.1 only contains a ligand binding region of a full-length receptor (IL-36R), and does not contain a transmembrane region and an intracellular region of the full-length receptor (IL-36R). Compared with IL-36R, the soluble receptor shown in SEQ ID NO.1 of the invention has different length and different function from IL-36R.

The soluble receptor of interleukin-36 (IL-36) is obtained by transcribing and translating IL-36R mRNA which lacks the 3 rd exon, and transcription is terminated early due to the deletion of the 3 rd exon in the IL-36R mRNA, so that translated protein only contains a ligand binding region and is secreted to the outside of cells, as shown in figure 1.

The invention also provides application of the soluble receptor of the interleukin-36 (IL-36) in preparing medicines for inhibiting the production of cytokines, chemokines and antibacterial proteins induced by IL-36 gamma. In vitro experiments show that IL-36 gamma can induce the expression of a large number of chemokines CXCL1, CCL20, CXCL2 and antimicrobial protein REG3A and cytokines, namely, the soluble receptor can inhibit the expression of chemokines CXCL1, CCL20, CXCL2 and antimicrobial protein REG3A and cytokines induced by IL-36 gamma.

In the present invention, the cytokine includes Il23 and Il17 a.

In the present invention, the antimicrobial protein REG3A refers to a protein expressed in intestinal and skin epithelial cells that inhibits bacterial growth.

The invention also provides application of the soluble receptor of interleukin-36 (IL-36) in preparing a medicament for inhibiting a p38MAPK signal pathway induced by IL-36 gamma. IL-36 γ induces the expression of antimicrobial proteins REG3A and the like by activating the p38MAPK signaling pathway, i.e., the soluble receptor of the present invention can inhibit IL-36 γ -induced p38MAPK phosphorylation.

The invention also provides application of the soluble receptor of interleukin-36 (IL-36) in preparing a medicament for inhibiting an NF-kB signal channel activated by IL-36 gamma. The IL-36 gamma can activate NF-kappa B to induce the expression of chemotactic factors, cytokines and the like, namely, the soluble receptor can inhibit the NF-kappa B activated by the IL-36 gamma so as to inhibit the expression of the chemotactic factors and the cytokines.

Wherein the chemokine comprises CCL20, CXCL1 and CXCL 2.

The invention also provides application of the soluble receptor of interleukin-36 (IL-36) in preparing a medicament for inhibiting keratinocyte proliferation. Wherein, the keratinocyte scratch test proves that: IL-36 gamma can induce keratinocyte proliferation to promote wound healing, and the soluble receptor can inhibit cell proliferation induced by IL-36 gamma and further inhibit wound healing.

The invention also provides application of the soluble receptor of interleukin-36 (IL-36) in preparing medicaments for inhibiting, treating, relieving and relieving psoriasis.

The soluble receptor is combined with IL-36 to inhibit the activation of an IL-36R signal channel activated by the IL-36, thereby playing a role in inhibiting, treating, relieving and relieving psoriasis. Among them, IL36 activates IL36R signaling pathway including activation of downstream NF-KB and p38 MAPK.

The soluble receptor protein injected and recombined in an IMQ-induced psoriasis mouse can relieve the pathological symptoms of the psoriasis mouse, and has the effects of inhibiting, treating, relieving and relieving psoriasis.

The invention also provides an inhibitor or a pharmaceutical composition comprising the soluble receptor sIL-36R as described above.

The invention also provides a method for inhibiting, treating, reducing and relieving psoriasis, which comprises the step of administering the soluble receptor sIL-36R as described above or the inhibitor or the pharmaceutical composition as described above to a subject individual so as to inhibit, treat, reduce and relieve psoriasis.

The invention firstly provides a method for preparing the soluble receptor sIL-36R by adopting a genetic engineering method, the method utilizes an escherichia coli system for expression, and then the soluble receptor sIL-36R is obtained by ion exchange and ultrafiltration purification.

Specifically, the method comprises the following steps:

(1) vector construction

The pET series vector (pET32a) commonly used for protein purification is used as a construction vector, the gene fragment of sIL36R cloned from keratinocytes is connected into the vector, and the constructed pET32a-sIL36R plasmid is sequenced and identified for later use.

(2) Transformation of Escherichia coli

The pET32a-sIL36R plasmid constructed and sequenced correctly in (1) was transformed into E.coli BL21(DE 3).

(3) Culturing and expressing

After the BL21(DE3) strain transformed in (2) grows colonies on a plate, inoculating a single colony for overnight culture, and transferring the overnight culture into a fresh culture medium for continuous culture; when the OD600 is 0.6-0.8, adding isopropyl thiogalactoside (IPTG) for induction expression, collecting the induced thallus, and performing bacterium breaking and washing; and respectively taking samples of the supernatant before and after induction, the bacteria-breaking supernatant and the precipitate and the washed supernatant precipitate to carry out SDS-PAGE to detect the expression condition of the target protein.

(4) Purification of

After dissolving the inclusion body, renaturation and dialysis of protein are carried out, filter membrane filtration sterilization is carried out, the obtained product is loaded on a column (anion column), elution is carried out by using elution buffer solutions with different concentrations, and the elution condition of the target protein is detected by collecting the eluate and carrying out SDS-PAGE. Collecting elution samples according to the display result, dialyzing, ultrafiltering and concentrating, collecting samples before and after ultrafiltration, carrying out SDS-PAGE to detect the expression condition of the target protein, and subpackaging the rest samples and freezing at-80 ℃ for later use.

Specifically, the preparation was as described in example 9.

The invention has the beneficial effects that: the soluble receptor of interleukin-36 (IL-36) provided by the invention can inhibit an IL-36R signal channel activated by IL-36, thereby lightening the inflammatory response of psoriasis and achieving the purpose of treating psoriasis. The soluble receptor of interleukin-36 (IL-36) provided by the invention can also inhibit the generation of inflammatory diseases caused by over-activation of an IL36 signaling pathway. In the present invention, the soluble receptor contains only part of the structure of IL36R, i.e., ligand binding domain, and does not contain transmembrane and signaling structures, and can functionally inhibit the function of IL 36R.

Drawings

FIG. 1 shows a schematic representation of the mRNA and protein structure of the soluble receptor sIL-36R.

FIG. 2 shows that the soluble receptor of the present invention has the ability to bind IL-36. gamma.

FIG. 3 shows that the soluble receptor of the present invention has the ability to compete with IL-36R for binding to IL-36 γ.

FIG. 4 shows that soluble receptors of the invention block IL-36 gamma-activated IL-36R signaling pathway in keratinocytes, where A is the soluble receptor blocking IL-36 gamma-induced phosphorylation of p38MAPK in keratinocytes; b is a soluble receptor that blocks IL-36 gamma-induced phosphorylation of NF- κ B subunit p65 in keratinocytes.

FIG. 5 shows the NF-KB promoter that the soluble receptor of the invention inhibits IL-36 γ activation.

FIG. 6 shows that the soluble receptor of the present invention inhibits IL-36 γ -activated AP-1.

FIG. 7 shows that the soluble receptors of the invention inhibit IL-36 γ -induced chemokines in keratinocytes; wherein A is soluble receptor which inhibits the expression of CCL20 induced by IL-36 gamma in keratinocytes; b is soluble receptor inhibiting IL-36 gamma to induce CXCL1 expression in keratinocytes; c is soluble receptor inhibiting IL-36 gamma to induce CXCL2 expression in keratinocytes; d is that soluble receptors inhibit IL-36 γ -induced REG3A mRNA expression in keratinocytes.

FIG. 8 shows that the soluble receptor of the present invention inhibits IL-36. gamma. induced REG3A protein expression in keratinocytes.

FIG. 9 shows that the soluble receptors of the invention inhibit IL-36 γ -promoted cell proliferation and wound healing in keratinocytes; wherein A is a statistical chart of a cell scratch experiment; b is a scratch picture before transfection of the unloaded plasmid by IL-36 gamma stimulation; c is a scratch picture before the transfection of soluble receptor plasmid by IL-36 gamma stimulation; d is a scratch picture of transfected unloaded plasmid after being stimulated by IL-36 gamma; e is a scratch picture of transfected soluble receptor plasmids after stimulation with IL-36 gamma.

FIG. 10 shows the preparation of recombinant sIL-36R protein of example 9; wherein, A is sIL-36R protein induced and expressed in escherichia coli, and the protein exists in the form of inclusion body; b is sIL-36R protein eluted by NaCl with different concentrations, and 50mM-250mM NaCl is the optimal elution range; c is sIL-36R protein obtained by ultrafiltration, concentration and purification.

FIG. 11 shows that injection of recombinant sIL-36R protein can slow the disease in psoriasis-like mice.

FIG. 12 shows that injection of recombinant sIL-36R protein results in a reduction in ear thickness in psoriasis-like mice.

FIG. 13 shows that injection of recombinant sIL-36R protein inhibited expression of chemokine and inflammatory factor mRNA in psoriatic mouse lesions; wherein, A is the expression of Ccl20 in the psoriasis-like mouse skin lesion inhibited by injecting recombinant sIL-36R protein; b is the expression of Mip2 in the psoriasis-like mouse skin lesion inhibited by injecting recombinant sIL-36R protein; c is the expression of Il23 in the psoriasis-like mouse skin lesion inhibited by injecting recombinant sIL-36R protein; d is the expression of Il17a in the psoriasis-like mouse skin lesion inhibited by injecting the recombinant sIL-36R protein.

FIG. 14 shows that injection of recombinant sIL-36R protein reduced the recruitment of CD45 positive cells, dendritic cells, and neutrophils in psoriatic-like mouse lesions; wherein, A is the reduction of CD45 positive cells in psoriasis-like mouse skin lesions by injecting recombinant sIL-36R protein; b is the injection of recombinant sIL-36R protein to reduce dendritic cells which are positive for CD45 and CD11c in skin lesions of psoriasis-like mice; c is the reduction of CD 45-positive Ly 6G-positive neutrophils in psoriatic-like mouse lesions by injection of recombinant sIL-36R protein.

FIG. 15 shows that injection of recombinant sIL-36R protein inhibited the phosphorylation levels (activation) of p38MAPK, NF-KB and the expression of IL36 gamma protein in psoriatic-like mouse lesions.

FIG. 16 shows a schematic representation of the interleukin receptors mentioned in the document Guanghui Yi, JBC, 2016.

Detailed Description

The present invention will be described in further detail with reference to the following specific examples and drawings, and the present invention is not limited to the following examples. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept, and the scope of the appended claims is intended to be protected. The procedures, conditions, reagents, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art except for the contents specifically mentioned below, and the present invention is not particularly limited.

Example 1: soluble receptors have the ability to bind IL-36 gamma

Culturing human kidney epithelial cells (293T) cells, inoculating the cells in a 10cm dish, setting three groups for experiment, when the 293T cells grow to 70%, transfecting a corresponding no-load (EV) into the cells by the first group, transfecting IL-36R plasmids with Flag labels into the cells by the second group, transfecting soluble receptor sIL-36R overexpression plasmids with Flag labels, shown as SEQ ID NO.1, in the third group, changing the liquid for 6h, blowing off the cells after 36h after liquid changing, washing the cells twice by PBS in an EP tube respectively, carrying out cell lysis by using cell lysis liquid added with protease inhibitors to collect proteins, incubating the collected proteins and the Flag beads overnight, carrying out competitive elution by using Flag peptides, measuring the concentrations of the eluted proteins of the three groups, incubating equimolar no-load histones, IL-36R-Flag and sIL-36R-Flag with IL-36 gamma-6 his respectively at 4 ℃, after adding Flagbeads and further incubating for 2h, washing for 5 times by Washbuffer, and detecting the expression of IL36 gamma-his by using a protein immunoblotting method for the protein enriched by the Flagbeads.

The experimental results showed that, as shown in fig. 2, the transfection-unloaded group was the control, and no visible IL36 γ band demonstrated no binding to IL36 γ; the IL-36R transfected plasmid and the sIL-36R plasmid group both showed IL36 gamma bands by immunoblotting, demonstrating that sIL-36R has the same receptor characteristics as IL-36R and binds to its ligand IL-36 gamma.

Example 2: soluble receptors have the ability to compete with IL-36R for binding to IL-36 gamma

Culturing human cervical carcinoma epithelial (Hela) cells, inoculating the cells into a 6-well plate, when the Hela cells grow to 70%, transfecting one group of IL-36R plasmids with GFP labels into the Hela cells, transfecting the other group of IL-36R plasmids with GFP labels and soluble receptor overexpression plasmids sIL-36R shown as SEQID NO.1 into the cells together, changing the solution for 6h, and adding IL-36 gamma protein with 6xhis labels after 36h of solution change for stimulation. After 45 minutes the supernatant was discarded, gently aspirated cells collected in an EP tube washed twice with PBS and stained with PE-anti-his dye for 30 minutes, then washed twice with PBS and filtered, and the two treatment groups were analyzed by flow cytometry for the percentage of GFP + PE + double positive cells, the ordinate SSC-H indicating the cell location. The experimental results show that, as shown in FIG. 3, in the case where sIL-36R and IL-36R exist simultaneously, sIL-36R can compete with IL-36R for binding IL-36 γ, and the cells with both GFP fluorescence and PE fluorescence decrease from 35.2% to 27.6%, which indicates that IL-36 γ binding by IL36R is reduced because of competition for binding a part of IL-36 γ after over-expression of sIL-36R.

Example 3: soluble receptors block IL-36R signaling pathway for IL-36 gamma activation in keratinocytes

Culturing human keratinocyte (NHEK) cells, inoculating the cells in a 6-well plate, transfecting a soluble receptor overexpression plasmid sIL-36R shown as SEQ ID NO.1 of the invention into the NHEK cells when the NHEK cells grow to 70%, changing the solution for 6h, stimulating the cells with 100ng/ml recombinant IL-36 gamma protein for 30 minutes after the solution is changed, collecting the protein, and detecting the phosphorylation of p38MAPK and p65 by a protein immunoblotting method. The experimental results show that as shown in FIG. 4, IL-36 gamma can induce the phosphorylation of p38MAPK and p65, and it can be seen by immunoblotting that IL36 gamma can obviously induce the phosphorylation of p38MAPK and NF-kappa B subunit p65, i.e. the expression of p-p38 and p-p65, under the condition of consistent GAPDH reference protein. After overexpression of the sIL-36R plasmid, phosphorylation bands of p38MAPK and p65 induced by IL-36 gamma are weakened, which indicates that phosphorylation of p38MAPK and p65 induced by IL-36 gamma is inhibited after overexpression of a soluble receptor.

Example 4: NF-KB promoter with soluble receptor inhibiting IL-36 gamma activation

Culturing human cervical carcinoma epithelial (Hela) cells, inoculating the cells in a 24-well plate, co-transferring a soluble receptor overexpression plasmid sIL-36R, NF-kB promoter plasmid and a Renillla plasmid shown as SEQ ID No.1 of the invention into the Hela cells when the Hela cells grow to 70%, co-transferring a no-load (pCMV) of the soluble receptor overexpression plasmid shown as SEQ ID No.1 of the invention, a NF-kB promoter plasmid and a Renillla plasmid into a control group, setting a transfection concentration gradient of 6 soluble receptor overexpression plasmids: 0ng, 50ng, 100ng, 150ng, 200ng and 250ng, changing the liquid after 6h of transfection, simultaneously adding 100ng/ml IL-36 gamma, and detecting the fluorescence reading value of each hole by using a luciferase reporter gene kit after 24 h. As shown in FIG. 5, 250ng of the transfected soluble receptor plasmid group can significantly inhibit the activity of the IL-36 gamma-activated NF- κ B promoter, indicating that the soluble receptor can well inhibit the activity of the IL-36 gamma-activated NF- κ B promoter.

Example 5: soluble receptor inhibition of IL-36 gamma-promoted activation of the AP-1 promoter

Culturing human cervical carcinoma epithelial (Hela) cells, inoculating the cells in a 24-well plate, co-transforming a soluble receptor overexpression plasmid sIL-36R, AP-1 promoter plasmid shown as SEQ ID NO.1 of the invention into the cells when the cells grow to 70%, co-transforming a no-load (pCMV) and an AP-1 promoter plasmid of the soluble receptor overexpression plasmid shown as SEQ ID NO.1 of the invention into a control group, wherein each three holes are a plurality of holes, and setting the transfection concentration gradient of 6 soluble receptor overexpression plasmids: 0ng, 50ng, 100ng, 150ng, 200ng and 250ng, changing the liquid after 6h of transfection, simultaneously adding 100ng/ml IL-36 gamma, adding a reaction substrate after 24h, and detecting the light absorption reading value of each hole.

As shown in FIG. 6, IL-36. gamma. can activate the AP-1 promoter, while soluble receptors inhibit the activity of the IL-36. gamma. activated AP-1 promoter, which can reduce the amount of substrate reacted by activated AP-1 from 60U/L to 40U/L.

Example 6: soluble receptors inhibit IL-36 gamma-induced inflammatory and chemokine in keratinocytes

Culturing human keratinocyte (NHEK) cells, inoculating the cells in a 6-well plate, transfecting a soluble receptor overexpression plasmid shown as SEQ ID NO.1 of the invention into the cells when the cells grow to 70%, changing the solution for 6h, evenly dividing the cells into 24-well plates by trypsinization after the solution is changed for 24h, adding IL-36 gamma of 100ng/ml when the cells grow to 70% by adherence, and extracting the expression of RNA detection chemokines CCL20, CXCL1, CXCL2 and antimicrobial peptide REG3A after 6 h.

The experimental results show that IL-36 gamma can induce the expression of a large amount of chemokines CCL20, CXCL1, CXCL2 and antimicrobial peptide REG3A, and can inhibit the expression of chemokines CCL20, CXCL1, CXCL2 and antimicrobial peptide REG3A induced by IL-36 gamma after over-expressing soluble receptors, as shown in FIG. 7.

Example 7: soluble receptors inhibit IL-36 gamma-induced expression of the antimicrobial protein REG3A in keratinocytes

Human keratinocytes (NHEK) cells were cultured and seeded in 6-well plates, and when the cells grew to 70%, the experiment was divided into six groups, the first and fourth groups were not transfected, the second and fourth groups were transfected into cells with no-load control plasmid PCMV, and the third and sixth groups were transfected into cells with soluble receptor over-expression plasmid sIL-36R as shown in SEQ ID NO.1 of the present invention, and the solution was changed for 6h, 100ng/ml IL-36. gamma. was added to the fourth, fifth and sixth groups, and after 6h, the proteins were collected, and the expression of REG3A was detected by Western blotting.

As shown in FIG. 8, in the case of the consensus of the endogenous reference protein Actin, IL-36 γ induced the expression of large amounts of REG3A, while overexpression of the soluble receptor inhibited the IL-36 γ -induced expression of REG3A protein.

Example 8: soluble receptors inhibit IL-36 gamma-induced cell proliferation and wound healing in keratinocytes

Inoculating HaCaT cells into a 12-well plate, when the density of the HaCaT cells reaches 60%, dividing the experiment into four groups, wherein the first group and the third group transfect control unloaded plasmid PCMV, the second group and the fourth group transfect soluble receptor plasmids shown as SEQ ID NO.1 of the invention, changing the liquid within 6h, scratching a wound at the middle of the bottom of a culture plate by using 1ml of Tip after 24h, washing floating cells by using DPBS (digital versatile disc), adding a culture medium, adding IL-36 gamma into the second group and the fourth group of wells, and photographing to count the wound.

The experimental result shows that, as shown in fig. 9, a 40h wound picture is selected, and compared with an initial wound, the comparison result and the data statistics of the left picture clearly show that the overexpression soluble receptor plasmid can inhibit IL-36 gamma in Hacat cells to promote cell proliferation and inhibit wound healing.

Example 9: preparation of soluble sIL36R protein

The cDNA reverse transcription of total RNA of keratinocyte is taken as a template, a cloning primer is used for carrying out PCR amplification on a gene fragment of sIL36R, after a PCR product is purified, double enzyme digestion is carried out to insert a pET32a vector, and the pET32a-His-sIL36R plasmid is obtained and then is sequenced and identified. Coli BL21(DE3) was transformed with the correctly sequenced pET32a-His-sIL36R plasmid, inoculated with a single colony and cultured overnight; transferring 8mL of overnight culture into 200mL of LB (Amp +) culture medium for continuous culture; when the OD600 is 0.6-0.8, adding IPTG with the final concentration of 1mM, and inducing at 37 ℃ and 220rpm for 4 hours; centrifuging at 4 deg.C and 6000rpm for 30min, collecting thallus, resuspending with bacteria breaking buffer (Tris 50mM, NaCl 250mM, Imizol 20 mM; pH 8.0), and ultrasonic breaking thallus in ice bath; centrifuging at 12000rpm for 20min at 4 deg.C, discarding the supernatant, washing the inclusion body precipitate twice with inclusion body washing buffer (Tris 20mM, NaCl 50mM, EDTA 5mM, 1% Trition-X100; pH 8.0); and respectively taking samples of the supernatant before and after induction, the bacteria-breaking supernatant and the precipitate and the washed supernatant precipitate to carry out SDS-PAGE to detect the expression condition of the target protein. As shown in FIG. 10, the recombinant protein pET32a-sIL36R (250aa,27.927kDa, pI of 6.74) was expressed by IPTG induction, but the expressed target protein was mainly insoluble inclusion bodies. Adding 5mL of a dissolution buffer (50mM Tris, 6M Gua-HCl, 10mM DTT; pH 8.0) into the washed inclusion bodies to dissolve the inclusion bodies, blowing off the inclusion bodies by using a pipette, and placing the inclusion bodies in a shaking table at 4 ℃ for overnight; centrifuging at 12000rpm for 20min at 4 deg.C, collecting supernatant, placing in 20mL syringe, adding renaturation buffer (50mM PB, 250mM NaCl, 10mM KCl, 0.5M Arg, 1mM EDTA, 0.05% PEG4000, 0.05% Triton X-100, 1mM GSH, 0.1mM GSSG; pH 8.0) dropwise with 1mL needle, and standing at 4 deg.C for 24 hr; centrifuging at 12000rpm for 20min at 4 deg.C, collecting supernatant, dialyzing with dialysis bag for 24 hr, and replacing dialysis buffer (20mM PB) every 4 hr; centrifuging at 12000rpm for 20min at 4 deg.C, filtering with sterilized 0.22 μm filter membrane, loading onto column (anion column), eluting with 20ml of elution buffer solution with different concentrations (NaCl with different concentrations is dissolved in 20mM PB for elution, adding 40 μ l of each eluate into 10 μ l of 5 × protein loading buffer solution, decocting, performing SDS-PAGE, and detecting the elution of target protein, as shown in FIG. 10, target protein is mainly eluted in PB elution buffer solution (# 4-8) of 50-250 mM NaCl, eluting 50mM NaCl, and the combined elution samples in the range of 100-250mM NaCl are dialyzed by a dialysis bag; then, the protein eluted by 50mM and 100 mM and 250mM is concentrated by ultrafiltration through an ultrafiltration tube, and after the protein is sterilized by filtration through a sterilized 0.22 μm filter, sampling to determine protein concentration, performing SDS-PAGE detection, subpackaging the rest samples, and freezing and storing at-80 ℃ for later use.

Example 10: the injection of the recombinant sIL36R protein can relieve the pathological symptoms of psoriasis mice

The soluble receptor protein as shown in example 1 was purified by injecting PBS control into the left ear and 500ng sIL-36R protein into the right ear of WT mice, applying imiquimod cream (IMQ) 10mg to the ears of mice after one day, photographing the lesion site every day, and continuously applying the ointment for 7 days.

The experimental results show that, as shown in fig. 11 and 12, six days of induction (D6) from the first day of photographing (D0), the red swelling dandruff symptoms of the mice injected with the soluble receptor protein of the present invention were significantly reduced compared to the control group. After counting data, the thickness of the ear of the PBS group is 0.6mm, while the thickness of the ear of the mouse injected with the soluble receptor protein is about 0.4, and the ear thickness is obviously reduced.

As shown in fig. 13, by extracting RNA from tissues and quantitatively measuring changes in the levels of chemokines and cytokines in mRNA, it was found that chemokine Ccl20 was hardly detected in the skin lesions of the soluble receptor-injected psoriatic mice, while the mRNA levels of chemokine Mip2 and cytokines Il23, Il17a were significantly reduced compared to the control psoriatic mice.

As shown in fig. 14, the proportion of CD 45-positive cells, neutrophils (CD 45-positive Ly 6G-positive cells) and dendritic cells (CD 45-positive CD11 c-positive cells) was lower than that of the control group.

As shown in FIG. 15, five mice in the PBS injection and the soluble receptor injection groups were tested by immunoblotting, and the level of phosphorylation of p38MAPK and NF-KB and the expression of IL36 gamma protein in the skin lesions of the psoriasis injection mice in the soluble receptor injection groups were found to be decreased compared with the PBS injection group, indicating that the phosphorylation of p38MAPK and NF-KB in psoriasis was decreased after the soluble receptor injection.

The protection of the present invention is not limited to the above embodiments. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept, and the scope of the appended claims is intended to be protected.

1Rosa,P.,Symmons,D.P.M.,Griffiths,C.E.M.&Ashcroft,D.M.Globalepidemiology of psoriasis:a systematic review of incidence andprevalence.Journal of Investigative Dermatology133,377-385(2013).

2Lebwohl,M.Psoriasis.Lancet361,1197-1204(2003).

3Griffiths,C.E.&Barker,J.N.Pathogenesis and clinical features ofpsoriasis.Lancet370,263-271(2007).

4Grine,L.,Dejager,L.,Libert,C.&Vandenbroucke,R.E.An inflammatorytriangle in psoriasis:TNF,type I IFNs and IL-17.Cytokine&Growth FactorReviews26,25-33(2015).

5Rapp,S.R.,Feldman,S.R.,Exum,M.L.,Jr,F.A.&Reboussin,D.M.Psoriasiscauses as much disability as other major medical diseases.Journal of theAmerican Academy of Dermatology41,401-407(1999).

6Lorenzi,S.IRF1 and NF-kB in neuroblastoma:from immune escape toimmune recognition.(2013).

7Jiang,Z.et al.Interleukin-36γinduced by the TLR3-SLUG-VDR axispromotes wound healing via REG3A.Journal of Investigative Dermatology137,S0022202X17327379(2017).

8Lai,Y.et al.The Antimicrobial Protein REG3A Regulates KeratinocyteProliferation and Differentiation after Skin Injury.Immunity37,74-84(2012).

9Luigi,T.et al.Psoriasiform dermatitis is driven by IL-36-mediatedDC-keratinocyte crosstalk.Journal of Clinical Investigation122,3965(2012).

10Mcmahan,C.J.et al.A novel IL-1 receptor,cloned from B cells bymammalian expression,is expressed in many cell types.Embo Journal10,2821-2832(1991).

11Cui,X.,Rouhani,F.N.,Hawari,F.&Levine,S.J.Shedding of the Type IIIL-1 Decoy Receptor Requires a Multifunctional Aminopeptidase,AminopeptidaseRegulator of TNF Receptor Type 1 Shedding.Journal of immunology171,6814-6819(2003).

12Kuhn,P.H.et al.Regulated Intramembrane Proteolysis of theInterleukin-1 Receptor II byα-,β-,and γ-Secretase.Journal of BiologicalChemistry282,11982-11995(2007).

13Lorenzen,I.et al.The membrane-proximal domain of A Disintegrin andMetalloprotease 17(ADAM17)is responsible for recognition of the interleukin-6receptor and interleukin-1 receptor II.Febs Letters586,1093-1100(2012).

14Martin,P.et al.A10.02 Deficiency in IL-1 receptor type 2 aggravatesK/BXN serum transfer-induced arthritis in mice,but has no effect inendotoxemia.Cytokine76,92-92(2015).

15Reddy,P.et al.Functional analysis of the domain structure of tumornecrosis factor-alpha converting enzyme.Journal of Biological Chemistry275,14608(2000).

16Palmer,G.,Lipsky,B.P.,Smithgall,M.D.&Meininger,D.The IL-1 receptoraccessory protein(AcP)is required for IL-33 signaling and soluble AcPenhances the ability of soluble ST2 to inhibit IL-33.Cytokine42,358-364(2008).

SEQUENCE LISTING

<110> university of east China

<120> soluble receptor sIL-36R of IL-36 and application thereof

<160>1

<170>PatentIn version 3.3

<210>1

<211>249

<212>PRT

<213> Artificial sequence

<400>1

Met Gly Val Thr Ser Leu Leu Phe Cys Gly Val Phe Phe Leu Leu Leu

1 5 10 15

Leu Phe Val Ala Ala Asp Thr Cys Glu Asp Ile Phe Met His Asn Val

20 25 30

Ile Ile Ser Glu Gly Gln Pro Phe Pro Phe Asn Cys Thr Tyr Pro Pro

35 40 45

Glu Thr Asn Gly Ala Val Asn Leu Thr Trp Tyr Lys Thr Pro Ser Lys

50 55 60

Ser Pro Val Ser Asn Asn Arg His Leu Arg Val His Gln Asp Gln Thr

6570 75 80

Trp Ile Leu Phe Leu Pro Leu Thr Leu Glu Asp Ser Gly Ile Tyr Gln

85 90 95

Cys Val Ile Arg Asn Ala His Asn Cys Tyr Gln Ile Ala Val Asn Leu

100 105 110

Thr Val Leu Lys Asn His Trp Cys Asp Ser Ser Met Glu Gly Ser Pro

115 120 125

Val Asn Ser Pro Asp Val Tyr Gln Gln Ile Leu Pro Ile Gly Lys Ser

130 135 140

Gly Ser Leu Asn Cys His Leu Tyr Phe Pro Glu Ser Cys Ala Leu Asp

145 150 155 160

Ser Ile Lys Trp Tyr Lys Gly Cys Glu Glu Ile Lys Ala Gly Lys Lys

165 170 175

Tyr Ser Pro Ser Gly Ala Lys Leu Leu Val Asn Asn Val Ala Val Glu

180 185 190

Asp Gly Gly Ser Tyr Ala Cys Ser Ala Arg Leu Thr His Leu Gly Arg

195 200 205

His Phe Thr Ile Arg Asn Tyr Ile Ala Val Asn Thr Lys Glu Val Glu

210 215 220

Tyr Gly Arg Arg Ile Pro Asn Ile Thr Tyr Pro Lys Asn Asn Ser Ile

225 230235 240

Glu Val Pro Leu Glu Pro Met Cys Pro

245

Claims

1. A soluble receptor for IL-36 sIL-36R, wherein said receptor comprises only the ligand binding domain of IL-36R; the amino acid sequence is shown in SEQ ID NO. 1:

MGVTSLLFCGVFFLLLLFVAADTCEDIFMHNVIISEGQPFPFNCTYPPETNGAVNLTWYKTPSKSPVSNNRHLRVHQDQTWILFLPLTLEDSGIYQCVIRNAHNCYQIAVNLTVLKNHWCDSSMEGSPVNSPDVYQQILPIGKSGSLNCHLYFPESCALDSIKWYKGCEEIKAGKKYSPSGAKLLVNNVAVEDGGSYACSARLTHLGRHFTIRNYIAVNTKEVEYGRRIPNITYPKNNSIEVPLEPMCP。

2. the soluble receptor sIL-36R according to claim 1, wherein said amino acid sequence comprises the ligand binding domain of the full-length receptor IL-36R only, and does not comprise the transmembrane and intracellular domains of the full-length receptor IL-36R.

3. Use of the soluble receptor sIL-36R according to claim 1 in the preparation of a medicament for inhibiting IL-36-induced production of cytokines, chemokines and antimicrobial proteins.

4. The use of claim 3, wherein said cytokine comprises Il23, Il17 a.

5. The use of claim 3, wherein said chemokine comprises CXCL1, CCL20, CXCL 2.

6. Use of the soluble receptor sIL-36R according to claim 3 for the preparation of a medicament for inhibiting IL-36-induced expression of the antimicrobial protein REG 3A.

7. Use of the soluble receptor sIL-36R of claim 1 in the manufacture of a medicament for inhibiting IL-36-activated p38 MAPK and/or NF-KB.

8. Use of the soluble receptor sIL-36R according to claim 1 in the preparation of a medicament for inhibiting keratinocyte proliferation.

9. The use of the soluble receptor sIL-36R according to claim 1 in the preparation of a medicament for the inhibition, treatment, alleviation or amelioration of psoriasis.

10. The use according to any one of claims 3 to 9, wherein the receptor binds to IL-36 and inhibits activation of the IL-36R signalling pathway activated by IL-36, thereby acting to inhibit, treat, alleviate or ameliorate psoriasis.

11. An inhibitor or a pharmaceutical composition comprising the soluble receptor sIL-36R according to claim 1.

12. A method of inhibiting, treating, reducing or ameliorating psoriasis, wherein a soluble receptor sIL-36R according to claim 1, or an inhibitor or pharmaceutical composition according to claim 11, is administered to a subject in order to inhibit, treat, reduce or ameliorate psoriasis.

13. The method of claim 1 or 2, wherein the soluble receptor sIL-36R is obtained by expression using an e.coli system followed by purification by ion exchange and ultrafiltration.