CN109942682B

CN109942682B - CMKLR1 antagonistic polypeptide and derivative and application thereof

Info

Publication number: CN109942682B
Application number: CN201711387975.4A
Authority: CN
Inventors: 张键; 代小勇; 陈杰; 黄晨; 肖天霞
Original assignee: Shenzhen Institute of Advanced Technology of CAS
Current assignee: Shenzhen Institute of Advanced Technology of CAS
Priority date: 2017-12-20
Filing date: 2017-12-20
Publication date: 2021-06-18
Anticipated expiration: 2037-12-20
Also published as: CN109942682A

Abstract

The invention discloses a CMKLR1 antagonistic polypeptide and a derivative and application thereof, and particularly relates to a sequence SEQ ID No.1-17 and a derivative thereof, wherein the derivative of a binding peptide is a product obtained by carrying out conventional modification on an amino acid side chain group of the CMKLR1 binding peptide and an amino end or a carboxyl end of a CMKLR1 antagonistic polypeptide fragment, or a product obtained by connecting a label for polypeptide or protein detection or purification on the CMKLR1 antagonistic polypeptide; the conjugated peptide and the derivative thereof can be conjugated with CMKLR1 in vitro, promote the increase of cAMP concentration by blocking the conjugation of chemerin and CMKLR1, inhibit calcium influx caused by chemerin, inhibit cell chemotaxis caused by chemerin, inhibit the proliferation of ovarian cancer cells, block the cell cycle of the ovarian cancer cells, promote the apoptosis of the ovarian cancer cells, and provide effective small molecule drugs for treating female reproductive diseases such as ovarian cancer and the like.

Description

CMKLR1 antagonistic polypeptide and derivative and application thereof

Technical Field

The invention relates to the field of biotechnology and biomedicine, in particular to female reproductive disease target CMKLR1 receptor antagonistic polypeptide and derivatives and application thereof.

Background

Some of the cancers of female reproduction are related to endocrine. Such as breast cancer (Breastcancer), endometrial cancer (Endometrialcencer), and ovarian cancer (OAC). Wherein, the ovarian cancer is a malignant tumor which occurs in the ovary, 90 to 95 percent of the ovarian cancer is primary cancer, and the other 5 to 10 percent of the ovarian cancer is primary cancer metastasis of other parts to the ovary. Although the incidence of ovarian cancer in our country is not as high as that in the European and American countries, eradication-based surgical treatment, as well as cytotoxic chemotherapy, lack the effectiveness of effectively reducing the mortality of ovarian cancer. Because ovarian cancer lacks symptoms in the early stage, even if the ovarian cancer has symptoms, the ovarian cancer is not specific, and the screening effect is limited, the early diagnosis is difficult, 60 to 70 percent of cases are in the late stage, and the late stage cases have poor curative effect. Therefore, although the incidence rate of ovarian cancer is second to the cervical cancer and the endometrial cancer and is the third place of gynecological malignant tumors, the mortality rate exceeds the sum of the cervical cancer and the endometrial cancer, and the gynecological cancer is the first place of gynecological cancer, which is the biggest disease seriously threatening the health of women.

In previous clinical studies, ovarian cancer was considered to have no obvious symptomatic manifestation in early stages. However, some studies show that ovarian cancer may show clinical symptoms in early stage, such as abnormal abdominal distension, fullness, abdominal pain or back pain, lassitude, etc., but these symptoms are often ignored by patients, and metastasis often occurs when diagnosis is carried out, so that ovarian cancer is often described as "silent killer", and the prognosis of ovarian cancer is poor.

Numerous studies have shown that ovarian cancer is often transplanted to distant organs by direct spread and disseminated to the pelvic and abdominal cavities. The main pathway of ovarian cancer metastasis is intraperitoneal vegetative metastasis, which is clinically observed to be the omentum majus, with omentum metastasis being associated in 80% of women with ovarian cancer. At this time, cancer cells on the greater omentum grow at a much faster rate than at the primary lesion. The greater omentum is the peritoneum connecting the greater curvature of the stomach to the transverse colon, contains phagocytes, has an important defense function, is adipose tissue containing a large number of lipid droplets, is an important endocrine organ in the body, and participates in homeostasis. Despite the apparent fact that ovarian cancer metastasizes to the omentum majus, the mechanism has remained unclear. Because the greater omentum part is mainly adipose tissue which is the largest endocrine organ in the body and can secrete fat factors, cytokines and the like, the relationship between adipose cells in the adipose tissue and ovarian cancer cells and the mechanism thereof still need more evidence for elucidation.

With the increasing depth of research into tumors, there is increasing evidence that obesity increases the risk of mortality in cancer patients. For example, Leptin (Leptin) secreted by white adipose tissue can promote the development of breast cancer: in one aspect, leptin is able to promote breast cancer growth by activating the JAK/STAT3, MAPK-ERK1/2, or PI3K pathway; in addition, leptin can promote the generation of tumor-related blood vessels by inducing the expression of angiogenin, and leptin can also induce the transcription of human epidermal growth factor receptor 2(ErbB-2), participate in the response of insulin-like growth receptor 1(IGF-1) in triple-negative breast cancer cells, activate Epidermal Growth Factor Receptor (EGFR) and promote the invasion and metastasis of the cells. The current research also shows that leptin can promote the process of various cancers such as prostate cancer, thyroid cancer and the like, the expression level of the leptin is positively correlated with tumorigenesis, but the level of the leptin in pancreatic cancer is lower, and the relationship between the leptin and the tumorigenesis is not clear. It is also found that adiponectin has an inhibitory effect on the occurrence and development of tumors.

Obesity has also been reported to increase the risk of ovarian cancer in women. Adipose tissue affected ovarian cancer in obese women, and the investigator studied 216 women, 35 obese women and 108 women of normal weight, and found that obese women had lower survival and shorter survival times in patients with ovarian cancer than women of normal weight. Scientists found that in addition to their differences in cancer lethality and cancer recurrence rates, their tumor cells also behave differently, suggesting that hormones or proteins secreted by adipose tissue may cause ovarian cancer cells to proliferate rapidly. It has also been shown that IL-6 and IL-8 secreted by the adipocytes of the omentum majus in the peritoneal cavity can promote the metastasis of ovarian cancer cells thereto.

Detailed evidence is provided about the relationship of adipose tissue, particularly omental fat, to ovarian cancer, published by professor ernstlongyel in journal nature-medicine (NatureMedicine) at 30.10.2011. chemerin is a recently discovered adipocyte factor, also called chemerin, playing a role in immune response, inflammatory reaction, adipocyte differentiation and maturation, lipid metabolism and the like, and is related to obesity and metabolic syndrome. The chemerin gene, also called tazarotendin-inducible gene2 (tazaroteinelnducgene 2, TIG2), was first cloned in 1997 and subsequently, in 2003, Wittamer et al isolated its active protein by reverse phase high pressure liquid chromatography in ascites secondary to ovarian cancer.

In 2012, the study by Reverchon et al demonstrated that chemerin and its receptor CMKLR1 (chemokine-like receptor-1) is expressed in major human ovarian granule cells (hGCs) and human ovarian granule-like tumor cells (KGN), again mentioning the relationship of chemerin to ovarian tumors.

A study published by Dr. ErnstLengyel in naturemedine in 2011 at 10 months shows that FABP4 plays a crucial role in the process of transferring ovarian cancer to the omentum majus, chemokines of the omentum majus participate in the process of transferring the ovarian cancer to omentum majus fat, and chemokines adiponectin, cytokines IL-6, IL-8 and the like in omentum majus fat cells are detected. However, there is no mention of detection of chemerin, which is also a chemokine.

Adipose factor Chemerin

The adipokine Chemerin is also known as tazarotene inducible gene 2(TIG2) or retinol receptor responsive protein 2. After cloning by Nagpal et al in 1997, Wittamer et al isolated their active proteins by reverse phase high pressure liquid chromatography in ascites secondary to ovarian cancer in 2003. The Chemerin protein precursor has 163 amino acids in length, has 6 cysteine residues, forms 3 disulfide bonds, removes an N-terminal signal peptide and several C-terminal amino acids, has biological activity, has a structure in which an activated form of Chemerin in blood contains 134 amino acids (about 16kDa), belongs to the family of cecropin/cystatin, and can be rapidly converted into an activated form by several proteases when an inflammatory reaction occurs. Chemerin is widely expressed in various tissues of the human body, mainly in white adipose tissue, placenta and liver, and binds to 3 receptors: g protein coupled receptor CMKLR 1; ② G protein coupled receptor GPR 1; ③ CCRL-2. CMKLR1 is highly expressed in macrophages, immature dendritic cells and white adipose tissues, is a main receptor for Chemerin to exert biological functions, can chemotact dendritic cells and macrophages highly expressed with CMKLR1, plays a bridge role between immunity and adaptive immunity, plays a role in immune response, inflammatory reaction, differentiation and maturation of fat cells, lipid metabolism and the like, and is related to obesity and metabolic syndrome. When Chemerin binds to CMKLR1, calcium ions are released from CMKLR1 positive cells, which inhibits cAMP aggregation and phosphorylates MAP kinase. Pretreatment with pertussis toxin blocked intracellular signaling by CMKLR1, which might be associated with the Gi family of CMKLR 1. On one hand, Chemerin is used as a chemotactic factor to chemotact dendritic cells and macrophages, plays a bridge role between immunity and adaptive immunity, and chemotactic natural killer cells to reach inflammatory sites to participate in inflammatory reaction; on the other hand, as a novel adipokine, it is secreted and produced from adipose tissue, regulates differentiation and lipolysis of adipocytes, and promotes biological effects such as insulin signaling pathway in adipocytes. Chemerin plays an important role in the pathophysiological mechanisms of obesity and metabolic syndrome.

Miticide E1(resolvin E1)

In 2002, studies by SerhanCN et al, American scholars, who analyzed inflammatory exudates using liquid chromatography tandem mass spectrometry (LC-MS/MS) and discovered novel trihydroxy-containing compounds (5, 12, 18-trihePE) and monohydroxy fatty acids, 18-hydroxy-EPA (18-HEPE) and 5-HEPE, derived from EPA, demonstrated that EPA and aspirin treatment could reduce the number of neutrophils in the mouse model of acute inflammation by 25% to 60%. When administered intravenously (100 ng/mouse), this novel trihydroxy-EPA is a potent inhibitor of neutrophil infiltration. It has the property of reducing inflammation and is named RvE 1. Through complete steric structure analysis based on the physical and biological properties of the synthesized compound, RvE1 was identified as 5S, 12R, 18R-hydroxy-6Z, 8E, 10E, 14Z, 16E-eicosapentaenoic. Bradykinin compounds (Rvs) are a group produced by EPA or DHA via epoxidation in vascular endothelial cells. According to different required substrates, D, E equal types can be divided, and each type is divided into a plurality of types. RvE1 belongs to the E family Rvs and Resolvin E1 is another lipid ligand of CMKLR1 and is capable of reducing the inflammatory response, reducing dendritic cell migration and reducing interleukin 12 production by binding to CMKLR1 and the leukotriene B4 receptor BLT 1. RvE1 can be synthesized by both aspirin-dependent and aspirin-independent pathways.

RvE1 should be stimulated to produce, act locally and be rapidly further metabolically inactivated by enzymes. In mammalian tissues, there are at least four separate pathways for RvE1 metabolism. RvE1 has a strong anti-inflammatory effect, and it can alleviate leukocyte-mediated tissue damage and down-regulate inflammatory gene overexpression. RvE1 is an important regulator of neutrophils. Nanogramme doses of RvE1 significantly reduced neutrophil infiltration at the site of inflammation in models of inflammation, including yeast-induced peritonitis and 2, 4, 6-trinitrobenzene-induced colitis. RvE1 interacts with CMKLR1 in CMKLR1 transfected cells to inhibit TNF-. alpha.induced NF- κ B activation. In vivo RvE1 can block DC migration and IL-12 production. Meanwhile, RvE1 can effectively inhibit Lipopolysaccharide (LPS) from stimulating bone marrow-derived dendritic cells (BMDCs) to release TNF-alpha, IL-6 and IL-23. RvE1 can block excessive platelet aggregation in pathological conditions, but does not interfere with collagen-stimulated physiological coagulation. RvE1 induces the shedding of L-selectin and down-regulates the expression of the human granulocyte and monocyte surface molecule CD 18. In addition, in mice, the RvE1 can rapidly reduce the rolling of white blood cell testicular muscle vein epithelial cells. RvE1 selectively induces epithelial cells to express the anti-adhesion molecule CD55, thereby promoting neutrophil clearance by epithelial cell surface CD 55. It can prevent transepithelial and transendothelial migration of inflammatory cells; neutrophils that promote phagocytosis of apoptosis by macrophages; down-regulating secretion of dendritic cell interleukin-12; up-regulating expression of T cell CCR 5; modulating expression of leukocyte adhesion molecules reduces leukocyte rolling; selective blockade of adenosine and the thromboxane receptor agonist U46619 promotes platelet aggregation. Has shown good prevention and treatment effects in various disease models such as rabbit periodontitis, mouse peritonitis, mouse retinopathy, colitis and the like.

CMKLR1 receptor

CMKLR1, also known as ChemR 23. In 2003, Meder et al demonstrated that Chemerin is a natural ligand of CMKLR1 by a reverse pharmacologic approach. The human CMKLR1 gene maps to 23.3 of the upper arm of chromosome 12 and consists of 3 exons and 2 introns. High expression in the adaptive immune system, wherein high expression is detected in macrophages, immature dendritic cells (immoturedcs), monocytes, neutrophils, natural killer cells, but reduced expression in mature DCs; thrombocytes, adipocytes, endothelial cells, oral epithelial cells, osteoclasts, vascular smooth muscle cells, and Shen glioma na cells (DBTRG-05MG) also express CMKLR 1. Activation of binding of Chemerin/RvE1 to CMKLR1 results in endocytosis which promotes intracellular calcium (Ca)²⁺) Release of ions, phosphorylating extracellular signal-regulated kinase 1/2(ERK1/2), inhibiting the accumulation of cyclic adenosine monophosphate (cAMP) by binding to G protein-coupled heterotrimers; up-regulating PI3K/Akt signaling pathway, down-regulating nuclear transcription factor kappa B (NF kappa B) signaling pathway: recruitment of immature dendritic cells and macrophages to sites of inflammation allows for the regulation of various metabolic processes, inflammatory responses, and cancer.

An increasing number of researches show that Chemerin and abnormal signal of receptors thereof are closely related to the occurrence and development of cancers, and Chemerin can induce the proliferation of endothelial cells and the formation of new vascular plexus after being combined with CMKLR1 receptors on endothelial cell membranes, thereby promoting the growth and proliferation of tumor cells. Chemerin is expressed at very low levels, some even undetectable, in most tumors, but at higher levels in paracancerous tissues, as well as in distant normal tissues, such as: colorectal cancer, lung cancer (including non-small cell lung cancer), liver cancer, melanoma, gastric cancer, and ovarian cancer. Yu et al found that Chemerin up-regulates VEGF, MMP-7 and IL-6 protein levels in gastric cancer by activating p38 and ERK1/2 signaling pathways, promoting tumor invasion and migration. In 2012, a study by Reverchon et al demonstrated that Chemerin and CMKLR1 were expressed in major human ovarian granulosa cells (hGCs) and human ovarian granulosa-like tumor cells (KGN). In 2011, 10 months, the research of naturemerine published by erntstlenyel doctors discovers that FABP4 plays a crucial role in the process of transferring ovarian cancer to the omentum majus, and chemokines of the omentum majus participate in the process of transferring ovarian cancer to omentum majus fat, and chemokines adiponectin and cytokines IL-6, IL-8 and the like in omentum majus fat cells are detected, and the following is suggested again: chemerin in adipose tissue may be associated with ovarian cancer cell migration.

The G Protein Coupled Receptor Superfamily (GPCRs) play an important role in the process of transferring extracellular signals into cells, and regulate and control cell movement, growth and gene transcription, which are the vital factors in the biology of the three cancers. Over the past decade, mediated signaling pathways have been shown to be key regulators of proto-oncogene signaling and to be good drug targets, and 60% of the drugs currently on the market have been designed for this receptor. While CMKLR1 is one of the members of GPCRs, no drug is currently available that is directed against CMKLR1 for the treatment of clinical cancer. Therefore, the CMKLR1 is taken as a target, and the screening of the novel anti-cancer polypeptide drug and the humanized antibody has important social significance and wide economic market.

Phage display technology (phagedisplay technology) is a screening technology for specific polypeptides or proteins, which can display the polypeptide encoded by a target gene on the surface of a phage in the form of fusion protein, and the displayed polypeptide or protein can maintain relatively independent spatial structure and biological activity, so that a direct connection is established between a large number of random polypeptides and the DNA coding sequence thereof, and polypeptide ligands of various target molecules (such as antibodies, enzymes, cell surface receptors and the like) can be rapidly identified by an in vitro affinity panning procedure. The phage display technology has been widely used for the screening of tumor diagnosis markers and anti-tumor lead compounds, the research of tumor specific antibodies and tumor drug targeted transportation, and the like.

Disclosure of Invention

In order to overcome the defects and shortcomings of the prior art, the primary object of the invention is to provide a CMKLR1 antagonist polypeptide obtained by screening a phage display library, wherein the antagonist polypeptide has specific high affinity with Chemerin/RvE1 receptor CMKLR1, can block a signal path of Chemerin/RvE1-CMKLR1 by inhibiting the combination of Chemerin/RvE1 and CMKLR1, proves that the polypeptide plays an important role in targeted inhibition of ovarian cancer cell proliferation, promotion of ovarian cancer cell apoptosis and the like, and has great application value in targeted therapy of ovarian cancer.

It is another object of the present invention to provide derivatives of the aforementioned CMKLR1 receptor antagonist polypeptides, which derivatives also have specific high affinity for the CMKLR1 receptor, specifically compete for the binding site of Chemerin/RvE1 to CMKLR1, and inhibit binding of Chemerin/RvE1 to CMKLR 1.

Still another object of the present invention is to provide the use of the CMKLR1 antagonist polypeptide and derivatives thereof.

In order to realize the task, the invention adopts the following technical solution:

one aspect of the present invention provides a CMKLR1 antagonist polypeptide, wherein the amino acid sequence LRH12-C1 is: asp- (D) Tyr-His-Asp-Pro-Ser-Leu-Pro-Thr-Leu-Arg-Lys-NH₂(SEQ ID No. 1); wherein said Tyr is in the D configuration.

The screening method of the CMKLR1 antagonistic polypeptide utilizes a phage random peptide library, firstly adopts CMKLR1 plasmid to transfect 293T cells to obtain a stable cell line of permanent high-expression CMKLR1, takes wild 293T cells as control adsorption cells, carries out 5 rounds of whole cell subtractive screening, randomly picks 50 positive phage for amplification, extracts clone single-chain DNA and sequences. The basic characteristics of the amino acid sequence of the polypeptide are analyzed, polypeptide homology comparison is carried out, and a polypeptide motif with high occurrence frequency is searched. BLAST searches protein databases, detects proteins with high polypeptide sequence homology, finds biological species containing a large amount of the polypeptide, and possibly combined cell surface receptors and ligands, and is beneficial to subsequent extraction and purification of a large amount of polypeptide.

The hydrophilic analysis shows that LRH12-C1 is hydrophilic polypeptide;

the purity of LRH12-C1 synthesis detected by High Performance Liquid Chromatography (HPLC) and Mass Spectrometry (MS) reaches 99.85%;

in another aspect, the invention provides a derivative of the CMKLR1 antagonist polypeptide of the invention, which is a product obtained by conventionally modifying the amino acid side chain group of the CMKLR1 antagonist polypeptide and the amino terminal or the carboxyl terminal of the CMKLR1 antagonist polypeptide fragment, or a product obtained by connecting a tag for polypeptide or protein detection or purification to the CMKLR1 antagonist polypeptide;

preferably, the conventional modification is amination, amidation, hydroxylation, carboxylation, carbonylation, alkylation, acetylation, phosphorylation, sulfation, esterification, glycosylation, cyclization, biotinylation, fluorophore modification, polyethylene glycol (PEG) modification or immobilization modification and the like;

preferably, the tag is His6, GST, EGFP, MBP, Nus, HA, IgG, FLAG, c-Myc or ProfinityXact.

In yet another aspect, the invention provides a biologically active fragment or analog of a CMKLR1 antagonist polypeptide of the invention as set forth in SEQ ID nos. 2-18:

DYX₁X₂X₃HDX₄X₅X₆X₇SX₈PTLRK-NH₂SEQ ID No.2, wherein:

⑴X₁、X₂、X₃in order from tryptophan, aspartic acid, threonine (W, N, T);

⑵X₄selected from phenylalanine, leucine (F, L);

⑶X₅、X₆、X₇valine, alanine (V, V, A) in that order;

⑷X₈is glutamine (Q);

X₉YX₁₀X₁₁PX₁₂X₁₃PTLR-NH₂SEQ ID No.3, wherein:

⑴X₉and X₁₁Is glutamic acid (E);

⑵X₁₀is glutamine (Q);

⑶X₁₂and X₁₃In turn selected from asparagine, serine (N, S).

YHDPSX₁₄X₁₅X₁₆LX₁₇K-NH₂SEQ ID No.4, wherein:

⑴X₁₄selected from methionine or isoleucine (M, I);

⑵X₁₅is serine (S);

⑶X₁₆selected from serine or alanine (S, A);

⑷X₁₇is histidine (H).

HX₁₈X₁₉SLPTLRK-NH₂SEQ ID No.5, wherein:

⑴X₁₈selected from glutamic acid or aspartic acid (E, N);

⑵X₁₉is glycine (G).

X₂₀HX₂₁PX₂₂LPX₂₃X₂₄R-NH₂SEQ ID No.6, wherein:

⑴X₂₀、X₂₁phenylalanine and proline in sequence;

⑵X₂₂selected from alanine or glycine (A, G)

⑶X₂₃Selected from valine or serine (V, S)

⑷X₂₄Is glutamine (Q).

YHX₂₅X₂₆X₂₇PSLX₂₈X₂₉L-NH₂SEQ ID No.7, wherein:

⑴X₂₅selected from asparagine or alanine (N, A);

⑵X₂₆is histidine (H);

⑶X₂₇selected from glutamic acid or leucine (E, L);

⑷X₂₈arginine (R);

⑸X₂₉is alanine (a);

HDPSLPTLR SEQ ID No.8；

HDPSLPTL-NH₂ SEQ ID No.9；

DYHDPSLP SEQ ID No.10；

DYHDPSL-NH₂ SEQ ID No.11；

YHDPSLPT SEQ ID No.12；

YHDPSLP-NH₂ SEQ ID No.13；

HDPSLPTL SEQ ID No.14；

HDPSLPT-NH₂ SEQ ID No.15；

DPSLPTLR SEQ ID No.16；

DPSLPTL-NH₂ SEQ ID No.17。

the invention also provides a derivative of the bioactive fragment or analogue of the CMKLR1 antagonist polypeptide, wherein the amino acid side chain group, the amino terminal or the carboxyl terminal of the derivative of the bioactive fragment or analogue of the CMKLR1 antagonist polypeptide is a product obtained by conventional modification, or a product obtained by connecting a label for detecting or purifying the polypeptide or the protein to the bioactive fragment or analogue of the CMKLR1 antagonist polypeptide;

In the technical scheme of the invention, the CMKLR1 antagonist polypeptide and the derivative thereof can be applied to preparing medicines for preventing and/or treating female reproductive diseases, and the existing forms of the CMKLR1 antagonist polypeptide and the derivative thereof are as follows: contains 8 amino acids; ② the amino acid composition comprises 9 amino acids; ③ 10 amino acids; the fourth step is composed of 11 amino acids; is composed of 12 amino acids; sixthly, the amino acid is composed of 18 amino acids; is composed of 24 amino acids. The derivative of the CMKLR1 antagonistic polypeptide is a product obtained by carrying out conventional modification on an amino acid side chain group of the CMKLR1 antagonistic polypeptide and an amino terminal or a carboxyl terminal of a CMKLR1 antagonistic polypeptide fragment, or a product obtained by connecting a label for polypeptide or protein detection or purification on the CMKLR1 antagonistic polypeptide; the conventional modification is preferably amination, amidation, hydroxylation, carboxylation, carbonylation, alkylation, acetylation, phosphorylation, esterification, glycosylation, cyclization, biotinylation, fluorescent group modification, polyethylene glycol (PEG) modification or immobilization modification and the like; the tag is preferably His₆GST, EGFP, MBP, Nus, HA, IgG, FLAG, c-Myc, or ProfinityXact, etc.;

the CMKLR1 antagonist polypeptide or derivative thereof may be derived from a mammal, bird, bacterium, or nematode, such as a primate (human); rodents, including mice, rats, hamsters, rabbits, horses, cattle, dogs, cats, and the like.

The derivative of the CMKLR1 antagonist polypeptide is preferably: the CMKLR1 antagonistic polypeptide has a second amino acid residue of D-tyrosine, and the end of the second amino acid residue is amidated to obtain Asp- (D) Tyr-His-Asp-Pro-Ser-Leu-Pro-Thr-Leu-Arg-Lys-NH₂。

The CMKLR1 antagonistic polypeptide and the derivative thereof are obtained by adopting a known method in the prior art, and can be chemically synthesized by using an automatic polypeptide synthesizer; deducing a nucleotide sequence from the short peptide sequence, and cloning the nucleotide sequence into a vector for biosynthesis; it can also be extracted and purified in large quantities from existing organisms.

Specifically, CMKLR1 antagonist polypeptides and derivatives thereof have the following sequence:

1.DYX₁X₂X₃HDX₄X₅X₆X₇SX₈PTLRK-NH₂(SEQ ID No.2), wherein:

⑴X₁、X₂、X₃tryptophan, aspartic acid, threonine (W, N, T) in that order;

⑵X₄selected from phenylalanine, leucine (F, L);

⑶X₅、X₆、X₇valine, alanine (V, V, A) in that order;

⑷X₈is glutamine (Q).

2.X₉YX₁₀X₁₁PX₁₂X₁₃PTLR-NH₂(SEQ ID No.3), wherein:

⑴X₉and X₁₁Are all glutamic acid (E);

⑵X₁₀is glutamine (Q);

⑶X₁₂and X₁₃Asparagine, serine (N, S) in that order.

3.YHDPSX₁₄X₁₅X₁₆LX₁₇K-NH₂(SEQ ID No.4), wherein:

⑴X₁₄selected from methionine or isoleucine (M, I);

⑵X₁₅is serine (S);

⑶X₁₆selected from serine or alanine (S, A);

⑷X₁₇is histidine (H).

4.HX₁₈X₁₉SLPTLRK-NH₂(SEQ ID No.5), wherein:

⑴X₁₈selected from glutamic acid or aspartic acid (E, N);

⑵X₁₉is glycine (G).

5.X₂₀HX₂₁PX₂₂LPX₂₃X₂₄R-NH₂(SEQ ID No.6), wherein:

⑴X₂₀、X₂₁phenylalanine and proline in sequence;

⑵X₂₂selected from alanine or glycine (A, G)

⑶X₂₃Selected from valine or serine (V, S)

⑷X₂₄Is glutamine (Q).

6.YHX₂₅X₂₆X₂₇PSLX₂₈X₂₉L-NH₂(SEQ ID No.7), wherein:

⑴X₂₅selected from asparagine or alanine (N, A);

⑵X₂₆is histidine (H);

⑶X₂₇selected from glutamic acid or leucine (E, L);

⑷X₂₈arginine (R);

⑸X₂₉is alanine (A).

In addition, CMKLR1 antagonist polypeptides and derivatives thereof also include the following naturally occurring polypeptides in an organism:

SEQ ID No.8 HDPSLPTLR (bacterium)

SEQ ID No.9 HDPSLPTL-NH₂(bacterium)

SEQ ID No.10 DYHDPSLP (bacteria)

SEQ ID No.11 DYHDPSL-NH₂(bacterium)

SEQ ID No.12 YHDPSLPT (bacterium)

SEQ ID No.13 YHDPSLP-NH₂(bacterium)

SEQ ID No.14 HDPSLPTL (bacterial or nematode)

SEQ ID No.15 HDPSLPT-NH₂(bacteria or nematodes)

SEQ ID No.16 DPSLPTLR (birds)

SEQ ID No.17 DPSLPTL-NH₂(birds)

In a further aspect, the present invention provides a polynucleotide encoding a polypeptide as defined in any one of SEQ ID Nos. 1 to 17.

In a further aspect of the invention, there is provided a vector comprising a nucleotide according to the invention, which can be linked to a promoter sequence by gene technology means.

In a further aspect, the invention provides a host cell transfected with a vector according to the invention.

In still another aspect, the invention provides the use of the CMKLR1 antagonist polypeptide, derivatives of CMKLR1 antagonist polypeptide, biologically active fragments or analogs of CMKLR1 antagonist polypeptide, and derivatives thereof of the invention in the preparation of a medicament for treating a chemerin/RvE1-CMKLR1 mediated disease.

In a further aspect, the invention provides the use of a CMKLR1 antagonist polypeptide, a derivative of a CMKLR1 antagonist polypeptide, a biologically active fragment or analog of a CMKLR1 antagonist polypeptide, and derivatives thereof of the invention in the treatment of a chemerin/RvE1-CMKLR1 mediated disease.

In the technical scheme of the invention, the chemerin/RvE1-CMKLR1 mediated disease is selected from ovarian cancer, polycystic ovary syndrome, fatty liver, diabetes and inflammatory reaction.

In a further aspect, the invention provides the use of a CMKLR1 antagonist polypeptide, a derivative of a CMKLR1 antagonist polypeptide, a biologically active fragment or analog of a CMKLR1 antagonist polypeptide, and derivatives thereof of the invention in the preparation of a medicament for inhibiting a decrease in cAMP concentration caused by chemerin.

In a further aspect, the invention provides the use of a CMKLR1 antagonist polypeptide, a derivative of a CMKLR1 antagonist polypeptide, a biologically active fragment or analog of a CMKLR1 antagonist polypeptide, and derivatives thereof of the invention to inhibit the decrease in cAMP concentration caused by chemerin.

In still another aspect, the invention provides a CMKLR1 antagonist polypeptide, a derivative of a CMKLR1 antagonist polypeptide, a biologically active fragment or analog of a CMKLR1 antagonist polypeptide, and derivatives thereof of the invention for inhibiting chemerin-induced calcium (Ca) production²⁺) The use in medicine for internal flow action.

In still another aspect, the invention provides a CMKLR1 antagonist polypeptide, a derivative of a CMKLR1 antagonist polypeptide, a biologically active fragment or analog of a CMKLR1 antagonist polypeptide, and derivatives thereof of the invention for inhibiting chemerin-induced calcium (Ca)²⁺) Use in an influx effect.

In still another aspect, the invention provides the use of the CMKLR1 antagonist polypeptide, derivatives of CMKLR1 antagonist polypeptide, biologically active fragments or analogs of CMKLR1 antagonist polypeptide, and derivatives thereof of the invention in the preparation of a medicament for inhibiting cell chemotaxis induced by chemerin.

In a further aspect, the invention provides the use of a CMKLR1 antagonist polypeptide, a derivative of a CMKLR1 antagonist polypeptide, a biologically active fragment or analog of a CMKLR1 antagonist polypeptide, and derivatives thereof of the invention to inhibit cell chemotaxis induced by chemerin.

In still another aspect, the invention provides the use of the CMKLR1 antagonist polypeptide, the derivative of the CMKLR1 antagonist polypeptide, the biologically active fragment or analog of the CMKLR1 antagonist polypeptide, and the derivative thereof in the preparation of medicaments for treating ovarian cancer, polycystic ovary syndrome, fatty liver, diabetes and inflammatory reaction.

In a further aspect, the invention provides the use of the CMKLR1 antagonist polypeptide, derivatives of the CMKLR1 antagonist polypeptide, biologically active fragments or analogs of the CMKLR1 antagonist polypeptide, and derivatives thereof of the invention in the treatment of ovarian cancer, polycystic ovary syndrome, fatty liver, diabetes, inflammatory response.

In a further aspect, the present invention provides a pharmaceutical composition comprising as an active ingredient one or more of the CMKLR1 antagonist polypeptide, a derivative of the CMKLR1 antagonist polypeptide, a biologically active fragment or analog of the CMKLR1 antagonist polypeptide, and derivatives thereof as described herein.

The pharmaceutical composition may contain one or more pharmaceutically acceptable carriers;

the pharmaceutically acceptable carrier is preferably a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption enhancer, an adsorption carrier, a surfactant, a lubricant, or the like.

The pharmaceutical composition can be further prepared into various forms such as tablets, granules, capsules, oral liquid or injection, and the like, and the medicines of various formulations can be prepared according to the conventional method in the pharmaceutical field.

A medicament for preventing and/or treating female reproductive diseases, which comprises at least one of the CMKLR1 antagonist polypeptide, a derivative of the CMKLR1 antagonist polypeptide, a biologically active fragment or analog of the CMKLR1 antagonist polypeptide, and a derivative thereof.

In a specific experiment, the CMKLR1 antagonist polypeptide LRH12-C1 can effectively relieve the inhibitory effect of chemerin on cAMP signal pathway. Wherein, the CMKLR1 antagonistic polypeptide LRH12-C1 derivatives (SEQ ID No. 1-17) have the same effect.

In another specific experiment of the invention, the CMKLR1 antagonistic polypeptide LRH12-C1 can effectively inhibit calcium (Ca) caused by chemerin²⁺) And (4) internal flow effect. Wherein, the CMKLR1 antagonistic polypeptide LRH12-C1 derivatives (SEQ ID No. 1-17) have the same effect.

Furthermore, chemerin can act by binding to the CMKLR1 receptor, chemotactic cells for migration. In another specific experiment of the invention, the inventor utilizes a Transwell test to find that the CMKLR1 antagonistic polypeptide LRH12-C1 has a significant effect of inhibiting cell migration caused by chemerin. Wherein, the CMKLR1 antagonistic polypeptide LRH12-C1 derivatives (SEQ ID No. 1-17) have the same effect.

In further experiments, the function of CMKLR1 antagonistic polypeptide LRH12-C1 is verified by using ovarian cancer cell SKOV-3 highly expressing CMKLR1 as a cell model, and LRH12-C1 can obviously inhibit the proliferation of ovarian cancer cell SKOV-3 compared with a control group. Wherein, the CMKLR1 antagonistic polypeptide LRH12-C1 derivatives (SEQ ID No. 1-17) have the same effect.

In another specific experiment of the invention, the inventor adopts a flow cytometry technology to detect that CMKLR1 antagonistic polypeptide LRH12-C1 can block the SKOV-3 cycle of ovarian cancer cells and promote SKOV-3 apoptosis. Wherein, the CMKLR1 antagonistic polypeptide LRH12-C1 derivatives (SEQ ID No. 1-17) have the same effect.

Compared with the prior art, the invention has the following advantages and effects:

(1) the invention provides a CMKLR1 antagonistic polypeptide and a derivative (SEQ ID No. 1-17) thereof, wherein the antagonistic polypeptide and the derivative thereof can be specifically combined with CMKLR1, specifically compete a binding site of Chemerin/RvE1 and CMKLR1, and can inhibit a Chemerin/RvE1-CMKLR1 signal pathway.

(2) The CMKLR1 antagonistic polypeptide and the derivative thereof (sequence ID NO: 1-17) obtained by screening can inhibit proliferation of breast cancer cells and promote apoptosis of the breast cancer cells by blocking combination of Chemerin/RvE1-CMKLR1, can be used as a biological polypeptide drug of Chemerin/RvE1-CMKLR1 binding sites, and can be used for preparing drugs for preventing and/or treating female reproductive diseases, such as: ovarian cancer, polycystic ovary syndrome, fatty liver, diabetes, and inflammatory response. Can be widely applied in the medical and biological fields and can generate huge social and economic benefits.

Drawings

FIG. 1: high Performance Liquid Chromatography (HPLC) assay format for LRH 12-C1.

FIG. 2: LRH12-C1 Mass Spectrometry (MS) detection.

FIG. 3: comparison of the hydrophobic profiles of CMKLR1, chemerin, RvE1 and LRH12-C1 polypeptides. Wherein: a: CMKLR1 hydrophobic profile; b: chemerin hydrophobic profile; RvE1 hydrophobic profile; d: LRH12-C1 polypeptide hydrophobic profile; e: comparison of hydrophobic profiles for CMKLR1, chemerin, RvE1 and LRH12-C1 polypeptides. CMKLR1 contour was blue, chemerin contour was green, RvE1 contour was red, and LRH12-C1 contour was purple.

FIG. 4: LRH12-C1 alleviated the inhibitory effect of chemerin on the cAMP signaling pathway.

FIG. 5: LRH12-C1 inhibited chemerin-induced calcium (Ca)²⁺) And (4) internal flow effect.

FIG. 6: LRH12-C1 inhibited chemotaxis of cells by chemerin.

FIG. 7: LRH12-C1 inhibits the proliferation of ovarian cancer cells SKOV-3.

FIG. 8: LRH12-C1 blocked ovarian cancer cell SKOV-3 cell cycle progression. A: detecting the cell cycle by a flow cytometer; b: and (5) counting cell cycle data.

FIG. 9: LRH12-C1 promotes apoptosis of ovarian cancer cell SKOV-3. A: detecting apoptosis by a flow cytometer; b: and (5) counting apoptosis data.

Detailed Description

In order that the invention may be more clearly understood, it will now be further described with reference to the following examples and the accompanying drawings. The examples are for illustration only and do not limit the invention in any way. In the examples, each raw reagent material is commercially available, and the experimental method not specifying the specific conditions is a conventional method and a conventional condition well known in the art, or a condition recommended by an instrument manufacturer.

Example 1: performing panning, amplification, purification, sequencing and synthesis of the CMKLR1 antagonist polypeptide.

The embodiment mainly aims to obtain the positive phage specifically combined with CMKLR1 through screening, amplifying and purifying the positive phage, extracting phage single-stranded DNA (ssDNA) for sequencing, analyzing and comparing the obtained sequences, and finally synthesizing the high-purity antagonistic polypeptide LRH 12-C1.

The method comprises the following specific steps:

1. establishment of a 293T cell line permanently highly expressing CMKLR 1: 293T-CMKLR1^+/+/LRH

Selecting luminous human 293T cell with vigorous growth, and culturing at 5X 10/day before transfection⁵One/well, inoculating in 6-well plate, culturing until the cell fusion degree is 60% after the second day;

② the second day, with 6 hole plate culture hole as a unit, using 200 u L of opti-MEM medium dilution 3 u g plasmid, another 200 u L opti-MEM medium dilution 6 u L liposome Lipofectamine2000, after gently mixing, placed at room temperature for 5 minutes;

③ mixing the two tube dilutions gently, standing for 20 minutes at room temperature, and then adding 600 μ L of opti-MEM culture medium gently into the mixed dilutions;

rinsing the cells to be transfected with PBS slightly once, then adding the mixed diluent into the culture holes slightly, and culturing in a carbon dioxide incubator;

fifthly, after culturing for 4-6 hours, abandoning the culture medium used for transfection, and adding 3mL of complete culture medium into the hole;

sixthly, selecting a culture medium containing 1 mu g/mL puromycin (puromycin) for screening after 48 hours; obtaining the 293T cell line which stably expresses CMKLR1 after the cells are not dead any more.

Seventhly, extracting total RNA by using TRIzol, quantifying 2 mu gRNA for reverse transcription (a reverse transcription kit purchased from Promega), and performing qPCR by using a specific primer sequence. The sequence of the specific primer is Hu-CMKLR1 primer sequence:

Fw5’-GAGGCGTGACATAGAATGGA-3’SEQ ID No.18；

Rv5’-TGATATGGATTGGGAGGAAGAC-3’SEQ ID No.19；

(viii) comparing with the transfected pSM2c-Hu-scrambleRNA, detecting the high expression level of CMKLR1, and naming as: 293T-CMKLR1^+/+LRH, i.e., can be used for positive phage selection.

2. Performing panning, amplification, purification, sequencing and synthesis of the CMKLR1 antagonist polypeptide.

Preparation of ER2738 host bacterial liquid: performing aseptic technique operation, namely taking 200 mu lLB-Tet liquid culture medium to be in a 1.5ml sterilized centrifugal tube, taking 0.2 mu l of bacterial liquid from the glycerol frozen product of E.coli ER2738, fully and uniformly mixing the bacterial liquid with the glycerol frozen product, completely absorbing and coating the bacterial liquid on an LB-Tet plate, marking the plate, standing the plate at room temperature for 3min, and then placing the plate in a constant temperature incubator at 37 ℃ for inverted overnight culture. Observing the next day, sealing with sealing film after clone grows out, and storing at 4 deg.C in dark for use. Single colonies were picked by aseptic technique using a sterilized tip, placed in 10ml sterilized centrifuge tubes previously supplemented with 3ml LB-Tet broth, labeled and cultured overnight on a constant temperature shaker at 37 ℃ under shaking at 300 rpm/min. The next day, the bacterial amplification solution was stored at 4 ℃ for future use. Taking a 10ml sterilized centrifuge tube, adding 3ml LB-Tet liquid culture medium in a sterile operation, inoculating 30 mul overnight cultured bacteria, carrying out shake culture at constant temperature of 37 ℃ and 300rpm/min for 2-3 h, wherein the bacteria are in an exponential growth phase, and are in a mist shape (OD 600-0.5) by visual observation.

Panning of CMKLR1 antagonistic peptides: high-expression CMKLR1 cells are expressed according to the formula 10⁵The culture dish is inoculated on 60X 15mm which is coated with polylysine in advance²In a culture dish, when the cells are cultured to 80-90% of the cell density by a conventional method, 1 mu l of eluent is firstly used for elutriation (meanwhile, a cell line which does not express CMKLR1 is used as a blank control), the rest eluent is added into 20ml of LB culture solution for amplification, then purification is carried out, finally, the titer after amplification is measured again, an amplification product is stored at 4 ℃ for a short time, an equal numerical level is used for next round of elutriation, and the rest amplification product is stored at-20 ℃ by 50% of glycerol.

Taking 4 sterilized 10ml centrifuge tubes, preparing 1 sterilized centrifuge tube for each phage dilution, melting top agar (agar top) by a microwave oven, adding 3ml top agar into each tube, and carrying out water bath at 45 ℃ for standby. For each dilution of phage, 1 LB/IPTG/Xgal plate was prepared and pre-warmed in a 37 ℃ incubator for use. And (3) subpackaging the E.coli ER2738 escherichia coli with OD 600-0.5 according to the phage dilution of 200 mu l/tube, and storing at 4 ℃ for later use. Taking 4 sterilized 1.5ml centrifuge tubes, respectively containing 100 μ l, 90 μ lLB-Tet culture medium, sucking 1 μ l of bacteriophage to be tested into 100 μ lLB-Tet culture medium, diluting according to 10 times gradient, respectively marking as 10^-1、10^-2、10^-3、10^-4And each dilution is mixed evenly by gentle oscillation and then is centrifuged instantly. Mix 10 μ l each diluted phage to be titrated with 200 μ l e. Quickly adding the mixed bacterial liquid into top agar, quickly shaking and uniformly mixing, immediately pouring into a preheated LB/IPTG/Xgal plate, and uniformly mixingHomogenizing, cooling at room temperature for 5min, culturing at 37 deg.C in constant temperature incubator, and standing on inverted plate overnight.

Amplification and purification of eluted phage: taking a 250ml conical flask, adding the overnight cultured ER2738 host bacterium liquid into a 20mLLB liquid culture medium according to the proportion of 1:100, and carrying out vigorous shaking culture at 37 ℃ and 250rpm for 2 h; then adding the phage liquid to be amplified into an erlenmeyer flask, and carrying out vigorous shaking culture at 37 ℃ and 250rpm for 4.5 h; the culture was transferred to a 50ml centrifuge tube and centrifuged at 10,000rpm at 4 ℃ for 10 min. Transferring the supernatant into another clean centrifugal tube, and centrifuging again at 10,000rpm at 4 ℃ for 10 min; transferring 80% of the supernatant into another clean centrifuge tube, adding 1/4 volume of PEG/NaCl, reversing, mixing uniformly, and precipitating at 4 ℃ overnight; the next day, the pellet was centrifuged at 12,000rpm for 20min at 4 ℃. Carefully sucking the supernatant with a clean gun head, centrifuging at 4 deg.C and 12,000rpm for 1min, and removing the residual supernatant; the pellet was then resuspended in 1ml TBS and gently pipetted 100 times. Then transferring the suspension into a 2ml centrifuge tube, and centrifuging at 4 ℃ and 10,000rpm for 5min to remove residual cells; adding 1/4 volume of PEG/NaCl to the supernatant, and incubating on ice for 60min for reprecipitation; taking out the centrifuge tube, centrifuging at 4 deg.C and 12,000rpm for 20min, and removing supernatant; the pellet was resuspended in 200. mu.l TBS and centrifuged at 10,000rpm for 1min at 4 ℃. The supernatant was transferred to another centrifuge tube. Short-term storage at 4 deg.C, or long-term storage at-20 deg.C with 50% glycerol. Amplification of monoclonal phage, comprising adding overnight cultured ER2738 host bacterial liquid into 2mLLB liquid culture medium according to a ratio of 1:100, and carrying out vigorous shaking culture at 37 ℃ and 250rpm for 2 h; selecting a plate with less than 100 plaques from the fourth round of titer plates by using a sterilizing toothpick, picking well-separated blue plaques, adding the blue plaques into a culture tube, and carrying out violent shake culture at 37 ℃ and 250r/min for 4.5 h; the culture was then transferred to a fresh centrifuge tube and centrifuged at 10,000rpm for 30sec at 4 ℃. Transferring the supernatant into a fresh tube, and centrifuging once again; 80% of the supernatant was transferred to fresh centrifuge tubes and stored at 4 ℃ or stored with 50% glycerol for a long period at-20 ℃.

Identifying M13 bacteriophage ssDNA by agarose gel electrophoresis: horizontally placing a gel forming mold, placing the selected comb, and reserving a space of 1mm between the bottom of the comb and the mold; weighing 1g of agarose for DNA electrophoresis, putting the agarose into a 250ml triangular flask, adding 100ml of 1 XTAE buffer solution, uniformly mixing, putting the flask into a microwave oven, heating and boiling until the agarose is completely dissolved; and (3) closing the induction cooker, taking out the triangular flask, cooling the triangular flask to room temperature (which can be tolerated by holding the flask by hand), adding 5 mu l of ethidium bromide, and pouring the gel solution into a rubber plate paving plate after uniform mixing. The rubber plate used in the experiment needs about 100ml of rubber solution; after the gel is completely solidified at room temperature and takes about 30 minutes, pulling out the comb teeth, and putting the rubber plate into an electrophoresis tank; adding 1 XTAE buffer solution into the electrophoresis tank, preferably 2mm higher than the surface of the gel; diluting the sample with a Loadingbuffer, adding the diluted sample into a gel plate, and paying attention to that a suction head of a sample injector is just placed in a gel spot sample hole, the gel cannot be punctured, and the sample is prevented from overflowing out of the hole; switching on a power supply, adjusting the voltage to 50V, performing electrophoresis for 90min, taking out the gel plate, and observing the result under an ultraviolet lamp.

Sequencing and sequence analysis of ssDNA: the extracted M13 phage ssDNA was sent to Shanghai Yingji Biotechnology Ltd for DNA sequencing. Sequencing was followed by sequence analysis using Bioedit software. The analysis result shows that the sequence of the sample is Asp- (D) Tyr-His-Asp-Pro-Ser-Leu-Pro-Thr-Leu-Arg-Lys-NH₂Wherein the second tyrosine is in the D configuration, represented by LRH12-C1, and finally the short peptide is synthesized by Shanghai Qiaozhou Bio.

The purity of the LRH12-C1 polypeptide detected by High Performance Liquid Chromatography (HPLC) in FIG. 1 and Mass Spectrometry (MS) in FIG. 2 was 99.85%, and the molecular weight was consistent with the predicted value. The hydrophobic profile analysis of FIG. 3 shows that the LRH12-C1 polypeptide has a certain similarity with CMKLR1, and the similarity reaches 0.002681(PAM 250).

Example 2CMKLR1 antagonistic polypeptide LRH12-C1 was found to be effective in alleviating the inhibitory effect of chemerin on the cAMP signaling pathway.

(1) Cyclic adenosine monophosphate (cAMP) enzyme-linked immunosorbent assay:

plating cells: wild type 293T cells and 293T cells highly expressing CMKLR1 (293TCMKLR 1)^+/+) At 5x10 respectively⁵Inoculating each well into 6-well cell culture plate with culture medium volume of 1mL, culturing in incubator for 24 hr, starving overnight, and adding different concentration gradients (3)μ M, 0.3 μ M, 0.03 μ M) of LRH12-C1 polypeptide, Fosklin (25 μ M) and chemerin (30nM) for 6 h;

preparing a sample: adding 300 mu L of cell lysate into each hole, standing at 4 ℃ for 20 minutes, scraping and collecting cells by using a cell scraper, turning upside down and uniformly mixing, centrifuging at 12,000rpm for 10 minutes, and collecting supernatant;

③ measuring the concentration of the sample: the sample concentration was determined by BCA method;

enzyme-linked immunosorbent assay of cyclic adenosine monophosphate (cAMP):

a, preparing required reagents, and preparing 3 multiple wells for each sample;

b, adding 50 mu L of sample or standard substance into a 96-well plate coated with the antibody; adding 25 mu LcAMP peroxidase tracer conjugate into each hole;

c, adding 50 mu of Lanti-cAMP antibody into each hole, and slowly incubating for 30 minutes on a shaking table at room temperature;

d, washing 5 times by using eluent, adding 100 mu L of chemical light-reflecting agent into each hole, and incubating for 5 minutes at room temperature;

and e, reading the plate by the microplate reader, and recording the luminescence value.

FIG. 4 shows that chemerin can reduce cellular cAMP concentration at a concentration of 30nM, but in 293T cells highly expressing CMKLR1 (293TCMKLR 1)^+/+) When LRH12-C1 (3. mu.M, 0.3. mu.M, 0.03. mu.M) was added to the solution at different concentrations, the cAMP concentration was significantly increased. In wild 293T cells, the CMKLR1 receptor was not expressed, so LRH12-C1 had no significant inhibitory effect on the effect of chemerin in reducing cAMP concentration. Similarly, in the single-action group of LRH12-C1 short peptide, LRH12-C1 did not increase cAMP concentration. From the above, it was concluded that LRH12-C1 specifically inhibited the decrease of cAMP concentration by chemerin by interacting with CMKLR 1. P<0.05,**P<0.01,***P<0.001 compared to the Forskolin + chemerin group.

Example 3CMKLR1 antagonist polypeptide LRH12-C1 can effectively inhibit calcium (Ca) caused by chemerin²⁺) And (4) internal flow effect.

Plating cells: wild type 293T cells and 293T cells highly expressing CMKLR1 (293TCMKLR 1)^+/+) Respectively, respectivelyAt 5x10³Inoculating each cell/well into a 96-well cell culture plate, wherein the volume of a culture medium in each well is 200 mu L, placing the cell culture plate in an incubator for 24 hours, and then starving the cell culture plate overnight;

preparing a reagent: dissolving probenecid into 1mL of buffer solution to prepare probenecid with the concentration of 250nM, shaking up, and adding into a fluorescent reagent for later use;

③ removing the cell culture medium, adding LRH12-C1 polypeptide with different concentration gradients (30. mu.M, 3. mu.M, 0.3. mu.M, 0.03. mu.M, 0.003. mu.M) and chemerin (0.3nM) for 30 minutes, and then adding 100. mu.L of the above fluorescent reagent into each well;

fourthly, placing the mixture for 30 minutes at 37 ℃ and then placing the mixture for 30 minutes at room temperature;

measuring fluorescence absorbance at 494nm for exciting light and 516nm for emitting light.

FIG. 5 shows the expression of CMKLR1 in 293T cells (293TCMKLR 1)^+/+) In the case of chemerin, calcium (Ca) was promoted at an action concentration of 0.3nM²⁺) Flowing signal pathway, increasing calcium ion (Ca)²⁺) And (4) concentration. After different concentrations of LRH12-C1 short peptide are added, calcium ions (Ca) can be remarkably reduced²⁺) Concentration, inhibition of chemerin on calcium (Ca)²⁺) Activation of the flow signal path. However, chemerin is on calcium (Ca) in wild type 293T cells not expressing CMKLR1²⁺) The signaling pathway of the flow is inactive, and LRH12-C1 is calcium (Ca)²⁺) The flow signaling pathway also had no effect, and it was concluded from this experiment that chemerin can activate calcium (Ca) by binding to the receptor CMKLR1²⁺) The signal path flows, and the LRH12-C1 short peptide can specifically inhibit the chemerin/CMKLR1 signal path to reduce calcium ions (Ca)²⁺) And (4) concentration. P<0.05,**P<0.01,***P<0.001 compared to the chemerin group.

Example 4 the Transwell assay detects the inhibition of the CMKLR1 antagonist polypeptide LRH12-C1 on the chemotaxis of cells induced by chemerin.

Wild type L1.2 cell and L1.2 cell highly expressing CMKLR1 (L1.2CMKLR1)^+/+) At 1x10 respectively⁶Inoculating each cell in a 96-well Transwell cell culture plate with the size of 5 mu m, culturing for 6h, and starving for 1 h;

② adding LRH12-C1 polypeptide with different concentration gradients (30. mu.M, 3. mu.M, 0.3. mu.M, 0.03. mu.M, 0.003. mu.M) and chemerin (0.3nM) into the lower chamber of the culture plate for 2 h;

and counting the number of cells chemotactic from the upper chamber to the lower chamber by using a flow cytometer.

FIG. 6 shows that in L1.2 cells highly expressing CMKLR1 (L1.2CMKLR1)^+/+) In the method, chemerin can remarkably promote cells to chemotact from an upper chamber to a lower chamber, and after the same concentration of LRH12-C1 short peptide is added, the chemotactic number of the cells can be remarkably reduced, and the chemotactic effect of the chemerin on the cells is inhibited. However, in L1.2 cells which do not express CMKLR1 in the wild type, chemerin has no chemotactic effect on the cells, and the short peptide LRH12-C1 has no effect on the cells. It can be concluded from this experiment that chemerin can promote cell chemotaxis by binding to receptor CMKLR1, while LRH12-C1 short peptide can specifically block chemerin/CMKLR1 signaling pathway to inhibit cell chemotaxis. P<0.05,**P<0.01,***P<0.001 compared to the chemerin group.

Example 5LRH12-C1 can significantly inhibit the proliferation of human ovarian cancer SKOV-3 cells.

Firstly, human ovarian cancer cell SKOV-3 is expressed as 5x10³Inoculating each well into a 96-well cell culture plate, culturing for 24h with the culture medium volume of 200 mu L per well, and then starving overnight;

adding LRH12-C1 polypeptide with different concentration gradients (100 mu M, 10 mu M, 1 mu M, 0.1 mu M, 0.01 mu M and 0.001 mu M) to culture for 24 hours, 48 hours and 72 hours respectively;

③ adding 20 mu L of MTT working solution into each hole, and continuously putting into a carbon dioxide incubator to culture for 4 hours;

and fourthly, abandoning the supernatant in the culture plate, adding 150 mu LDMSO (dimethyl sulfoxide), shaking for 10 minutes, selecting 490nm wavelength on a microplate reader for detection, and drawing a growth curve of the cells.

In the previous work of the laboratory of the inventor, the SKOV-3 of any ovarian cancer cell is detected to highly express CMKLR1 through a q-PCR test. FIG. 7 shows that different concentrations of LRH12-C1 short peptide can significantly inhibit SKOV-3 cell proliferation, and the effect of the short peptide on inhibiting SKOV-3 cell proliferation is more significant with the increase of action time.

Example 6LRH12-C1 can block human ovarian cancer SKOV-3 cell cycle progression.

Firstly, human ovarian cancer cell SKOV-3 is expressed by 5 multiplied by 10⁵Inoculating each cell/well in 6-well cell culture plates, culturing for 24h with the volume of culture medium per well being 1mL, and then starving overnight;

adding LRH12-C1 polypeptide with different concentration gradients (1 mu M, 0.1 mu M and 0.01 mu M) to culture for 24 hours, 48 hours and 72 hours respectively;

③ carefully collecting the cell culture solution into a centrifugal tube for standby. Digesting the cells with pancreatin until the cells can be blown down by a pipette or a gun head, adding the previously collected cell culture solution, blowing down all adherent cells, and blowing off the cells gently. Again collected in the centrifuge tube. Centrifuging about 1000g for 3-5 min to precipitate cells;

fourthly, carefully sucking the supernatant, and about 50 microliters of culture solution can be left to avoid sucking away the cells. Approximately 1ml of ice-cooled PBS was added, the cells were resuspended, and transferred to a 1.5ml centrifuge tube. The cells were pelleted by centrifugation again and the supernatant carefully aspirated, approximately 50 microliters or so of PBS could remain to avoid aspiration of the cells. Lightly flicking the bottom of the centrifugal tube to properly disperse cells to avoid cell agglomeration;

fifthly, adding the mixture into 1ml of ice bath precooled 70% ethanol, lightly blowing and uniformly mixing, and fixing for 12 hours at 4 ℃. Centrifuging at about 1000g for 3-5 min to precipitate cells. The supernatant was carefully aspirated, and about 50. mu.l or so of 70% ethanol remained to avoid aspiration of the cells. Approximately 1ml of ice-cold PBS was added to resuspend the cells. The cells were pelleted by centrifugation again and the supernatant carefully aspirated, approximately 50 microliters or so of PBS could remain to avoid aspiration of the cells. Lightly flicking the bottom of the centrifugal tube to properly disperse cells to avoid cell agglomeration;

sixthly, 0.5 ml of propidium iodide staining solution is added into each tube of cell sample, and the cell sediment is slowly and fully resuspended, and is bathed for 30 minutes in the dark at the temperature of 37 ℃. Then storing at 4 ℃ or in ice bath in dark place;

and detecting red fluorescence and light scattering at the position of the wavelength of 488nm by using a flow cytometer. The cell results were analyzed.

FIG. 8 shows that, through flow cytometry analysis, on one hand, as the acting concentration is increased, LRH12-C1 short peptide can obviously reduce the number of human ovarian cancer cells SKOV-3G2/M phase cells, increase the number of G0/G1 and S phase cells, and block normal progress of cell cycle; on the other hand, as the action time is prolonged, the LRH12-C1 short peptide also obviously reduces the cell number of G2/M phase, increases the cell number of G0/G1 and S phase, and blocks the normal progress of the cell cycle. From the above, it can be concluded that LRH12-C1 short peptide inhibits the proliferation of human ovarian cancer cell SKOV-3 by blocking the cell cycle. P<0.05,**P<0.01 compared to control group stage G0/G1;^#P<0.005 compared to control S phase;^&P<0.005 compared with the control group G2/M period.

Example 7LRH12-C1 can promote apoptosis of human ovarian cancer SKOV-3 cells.

thirdly, collecting the supernatant into a centrifuge tube, and then carefully digesting and collecting the cell culture solution into the centrifuge tube by using pancreatin without EDTA. Centrifuging about 500g for 5 minutes to precipitate cells;

fourthly, washing the cells twice by using precooled PBS, and centrifuging about 500g for 5 minutes to collect the cells;

adding 100 μ L of precooled 1 × annexin VBindingbuffer, and resuspending the cells;

sixthly, adding 5 mu LannexinV-FITC and 5 mu LPI, mixing evenly and reacting for 15 minutes at room temperature in a dark place;

seventhly, adding 400 mu L of precooled 1 Xannexin VBindingbuffer, gently mixing, placing the sample on ice in a dark place, and detecting by a flow cytometer within 1 h. And analyzing the detection result.

FIG. 9 shows that, through flow cytometry analysis, on one hand, with the increase of the acting concentration, the LRH12-C1 short peptide can obviously promote the apoptosis of human ovarian cancer cell SKOV-3, and on the other hand, with the increase of the acting time, the LRH12-C1 short peptide also obviously increases the apoptosis number of human ovarian cancer cell SKOV-3. In conclusion, the LRH12-C1 short peptide can remarkably promote apoptosis of human ovarian cancer cell SKOV-3. P <0.05, P <0.01 compared to control.

SEQUENCE LISTING

<110> Shenzhen advanced technology research institute

<120> CMKLR1 antagonistic polypeptide, derivative and application thereof

<130> CP11701201C

<160> 19

<170> PatentIn version 3.3

<210> 1

<211> 12

<212> PRT

<213> Artificial sequence

<400> 1

Asp Tyr His Asp Pro Ser Leu Pro Thr Leu Arg Lys

1 5 10

<210> 2

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<212> PRT

<213> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (3)..(3)

<223> Tryptophan

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> aspartic acid

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> threonine

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<221> MISC_FEATURE

<222> (8)..(8)

<223> phenylalanine or leucine

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<221> MISC_FEATURE

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<223> valine

<220>

<221> MISC_FEATURE

<222> (11)..(11)

<223> alanine

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<223> Glutamine

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Asp Tyr Xaa Xaa Xaa His Asp Xaa Xaa Xaa Xaa Ser Xaa Pro Thr Leu

1 5 10 15

Arg Lys

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<211> 11

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<221> MISC_FEATURE

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<223> glutamic acid

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<223> serine

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Xaa Tyr Xaa Xaa Pro Xaa Xaa Pro Thr Leu Arg

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<210> 4

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Tyr His Asp Pro Ser Xaa Xaa Xaa Leu Xaa Lys

1 5 10

<210> 5

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> glutamic acid or aspartic acid

<220>

<221> MISC_FEATURE

<222> (3)..(3)

<223> Glycine

<400> 5

His Xaa Xaa Ser Leu Pro Thr Leu Arg Lys

1 5 10

<210> 6

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> Phenylalanine

<220>

<221> MISC_FEATURE

<222> (3)..(3)

<223> proline

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> alanine or glycine

<220>

<221> MISC_FEATURE

<222> (8)..(8)

<223> valine or serine

<220>

<221> MISC_FEATURE

<222> (9)..(9)

<223> Glutamine

<400> 6

Xaa His Xaa Pro Xaa Leu Pro Xaa Xaa Arg

1 5 10

<210> 7

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (3)..(3)

<223> asparagine or alanine

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> histidine

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> glutamic acid or leucine

<220>

<221> MISC_FEATURE

<222> (9)..(9)

<223> arginine

<220>

<221> MISC_FEATURE

<222> (10)..(10)

<223> alanine

<400> 7

Tyr His Xaa Xaa Xaa Pro Ser Leu Xaa Xaa Leu

1 5 10

<210> 8

<211> 9

<212> PRT

<213> bacterium

<400> 8

His Asp Pro Ser Leu Pro Thr Leu Arg

1 5

<210> 9

<211> 8

<212> PRT

<213> bacterium

<400> 9

His Asp Pro Ser Leu Pro Thr Leu

1 5

<210> 10

<211> 8

<212> PRT

<213> bacterium

<400> 10

Asp Tyr His Asp Pro Ser Leu Pro

1 5

<210> 11

<211> 7

<212> PRT

<213> bacterium

<400> 11

Asp Tyr His Asp Pro Ser Leu

1 5

<210> 12

<211> 8

<212> PRT

<213> bacterium

<400> 12

Tyr His Asp Pro Ser Leu Pro Thr

1 5

<210> 13

<211> 7

<212> PRT

<213> bacterium

<400> 13

Tyr His Asp Pro Ser Leu Pro

1 5

<210> 14

<211> 8

<212> PRT

<213> bacteria or nematodes

<400> 14

His Asp Pro Ser Leu Pro Thr Leu

1 5

<210> 15

<211> 7

<212> PRT

<213> bacteria or nematodes

<400> 15

His Asp Pro Ser Leu Pro Thr

1 5

<210> 16

<211> 8

<212> PRT

<213> birds

<400> 16

Asp Pro Ser Leu Pro Thr Leu Arg

1 5

<210> 17

<211> 7

<212> PRT

<213> birds

<400> 17

Asp Pro Ser Leu Pro Thr Leu

1 5

<210> 18

<211> 20

<212> DNA

<213> Artificial sequence

<400> 18

gaggcgtgac atagaatgga 20

<210> 19

<211> 22

<212> DNA

<213> Artificial sequence

<400> 19

tgatatggat tgggaggaag ac 22

Claims

Use of a CMKLR1 antagonist polypeptide in the manufacture of a medicament for the treatment of a chemerin/RvE1 CMKLR 1-mediated disease; the amino acid sequence of the CMKLR1 antagonistic polypeptide is Asp- (D) Tyr-His-Asp-Pro-Ser-Leu-Pro-Thr-Leu-Arg-Lys-NH₂。
2. The use of claim 1, wherein the chemerin/RvE1-CMKLR1 mediated disease is selected from the group consisting of breast cancer, fatty liver, diabetes, inflammatory response.
Use of a CMKLR1 antagonist polypeptide in the preparation of a medicament for inhibiting a decrease in cAMP concentration caused by chemerin; the amino acid sequence of the CMKLR1 antagonistic polypeptide is Asp- (D) Tyr-His-Asp-Pro-Ser-Leu-Pro-Thr-Leu-Arg-Lys-NH₂。
Use of a CMKLR1 antagonist polypeptide in the preparation of a medicament for inhibiting chemerin-induced calcium influx; the amino acid sequence of the CMKLR1 antagonistic polypeptide is Asp- (D) Tyr-His-Asp-Pro-Ser-Leu-Pro-Thr-Leu-Arg-Lys-NH₂。
Use of a CMKLR1 antagonist polypeptide in the preparation of a medicament for inhibiting chemerin-induced cell chemotaxis; the amino acid sequence of the CMKLR1 antagonistic polypeptide is Asp- (D) Tyr-His-Asp-Pro-Ser-Leu-Pro-Thr-Leu-Arg-Lys-NH₂。
6. A pharmaceutical composition comprising as an active ingredient a CMKLR1 antagonist polypeptide; the amino acid sequence of the CMKLR1 antagonistic polypeptide is Asp- (D) Tyr-His-Asp-Pro-Ser-Leu-Pro-Thr-Leu-Arg-Lys-NH₂。
7. The pharmaceutical composition of claim 6, wherein the pharmaceutical composition comprises one or more pharmaceutically acceptable carriers.
8. The pharmaceutical composition of claim 7, wherein the pharmaceutically acceptable carrier is a diluent, excipient, filler, binder, wetting agent, disintegrant, absorption enhancer, adsorptive carrier, surfactant, or lubricant; the pharmaceutical composition is further prepared into tablets, granules, capsules, oral liquid or injections, and the medicines of various dosage forms are prepared according to conventional methods in the pharmaceutical field.