阅读说明：本技术 一种IL-36的可溶性受体sIL-36R及其应用 (Soluble receptor sIL-36R of IL-36 and application thereof ) 是由 赖玉平 王旺 高晓广 吴雅伟 于 2019-04-24 设计创作，主要内容包括：本发明公开了一种白介素-36(IL-36)的可溶性受体(sIL-36R),其仅含有IL-36受体(IL-36R)的配体结合区域,缺失跨膜区和胞内区,其氨基酸序列如SEQ ID NO.1所示。该可溶性受体可以抑制IL-36激活的NF-KB和p38MAPK信号通路,抑制IL-36诱导的炎症反应,抑制IL-36诱导的抗菌蛋白REG3A的表达,以及可以减轻银屑病的病理症状,对银屑病有治疗作用。本发明还公开了该可溶性受体(sIL-36R)的制备方法。(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).)

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.

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.