CN109942677B - CMKLR1 antagonistic polypeptide and derivative and application thereof - Google Patents
CMKLR1 antagonistic polypeptide and derivative and application thereof Download PDFInfo
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Abstract
The invention discloses a CMKLR1 antagonistic polypeptide and a derivative and application thereof, and particularly relates to a sequence SEQ ID No.1-15 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, change cAMP concentration by blocking the conjugation of chemerin/RvE1 and CMKLR1, inhibit calcium influx caused by chemerin, inhibit cell chemotaxis caused by chemerin, inhibit the proliferation of ovarian cancer cells, promote the apoptosis of the ovarian cancer cells and provide an effective small molecule treatment medicine for female reproductive diseases.
Description
Technical Field
The invention relates to the field of biotechnology and biomedicine, in particular to female reproductive disease target CMKLR1 receptor antagonist polypeptide LRH7-C3 and derivatives and application thereof.
Background
Some of the cancers of female reproduction are related to endocrine. Such as Breast Cancer (Breast Cancer), Endometrial Cancer (Endometrial Cancer), 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.
A review of the relationship of adipose tissue, and particularly the fat of the greater omentum, to ovarian cancer is provided by professor Ernst Lengyel in journal Nature Medicine, 10, 30.2011. 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 known as Tazarotene-Induced Gene 2(Tazarotene Induced Gene2, 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.
In 2011, 10 months, the research of natural media, which is published by the Ernst longyel doctor, finds that FABP4 plays a crucial role in the process of transferring ovarian cancer to the omentum majus, and the chemotactic factors of the omentum majus participate in the process of transferring the ovarian cancer to the omentum majus fat, and detects the chemotactic factors adiponectin, cytokines IL-6, IL-8 and the like in the omentum majus fat cells. 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 can block intracellular signaling of CMKLR1, which may be associated with family G i 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) to find novel trihydroxy-containing compounds (5, 12, 18-tri HEPE) and monohydroxy fatty acids derived from EPA, 18-hydroxy-EPA (18-HEPE) and 5-HEPE, showed 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 (immatures DCs), monocytes, neutrophils and natural killer cells, but the expression in mature DCs is reduced; 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 natural media, which is published by Ernst longyel doctor, finds that FABP4 plays a crucial role in the process of transferring ovarian cancer to the omentum majus, and the chemotactic factors of the omentum majus participate in the process of transferring ovarian cancer to omentum majus fat, and detects the chemotactic factors adiponectin and cytokines IL-6, IL-8 and the like in omentum majus fat cells, and once again indicates that: 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.
The Phage Display Technology (Phage Display Technology) is a screening Technology of specific polypeptide or protein, which can Display the polypeptide encoded by the target gene on the Phage surface in the form of fusion protein, the displayed polypeptide or protein can maintain relatively independent spatial structure and biological activity, and direct connection is established between a large number of random polypeptides and the DNA coding sequence thereof, so that the polypeptide ligands of various target molecules (such as antibody, enzyme, cell surface receptor and the like) can be rapidly identified by 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 invention provides a CMKLR1 antagonistic polypeptide, which is characterized in that the amino acid sequence is Tyr- (D) Asp-Gly-Gln-Trp-Arg-Leu-NH2(SEQ ID No.1), namely LRH 7-C3.
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 LRH7-C3 is hydrophilic polypeptide;
the purity of the LRH7-C3 synthesis reaches 99.85% by High Performance Liquid Chromatography (HPLC) and Mass Spectrum (MS).
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. 2-15:
YX1GX2WRX3SEQ ID No.2, wherein:
⑴X1is glutamic acid (E);
⑵X2is glutamic acid (E);
⑶X3is methionine (M).
YX4GX5WR SEQ ID No.3, wherein:
⑴X4is asparagine or glutamic acid (N, E);
⑵X5is glutamic acid (E).
YX6GX7W SEQ ID No.4, wherein:
⑴X6is glutamic acid (E);
⑵X7is glutamic acid (E).
GX8WRL SEQ ID No.5, wherein:
⑴X8is histidine (H).
YDGQWRL SEQ ID No.6
YDGQWRL-NH2 SEQ ID No.7
YDGQWR SEQ ID No.8
YDGQWR-NH2 SEQ ID No.9
DGQWR SEQ ID No.10
DGQWR-NH2 SEQ ID No.11
DGQW SEQ ID No.12
DGQW-NH2 SEQ ID No.13
GQWR SEQ ID No.14
GQWR-NH2 SEQ ID No.15
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;
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 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 4 amino acids; ② the amino acid composition comprises 5 amino acids; ③ 6 amino acids; and the four amino acids consist of 7 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, biotinylationFluorescent 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 Profinitye Xact, etc.;
the CMKLR1 antagonist polypeptide or derivative thereof may be of bacterial, fungal or viral origin, as well as mammalian or avian origin, such as primates (humans); rodents, including mice, rats, hamsters, rabbits, horses, cattle, canines, cats, snakes, and the like.
The derivative of the CMKLR1 antagonist polypeptide is preferably: the second amino acid residue of the CMKLR1 antagonistic polypeptide is D-configuration aspartic acid, and the end is amidated and modified to obtain Tyr- (D) Asp-Gly-Gln-Trp-Arg-Leu-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, the CMKLR1 antagonistic polypeptide LRH7-C3 and derivatives thereof have the following sequence:
1.YX1GX2WRX3(SEQ ID No.2), wherein:
⑴X1is glutamic acid (E);
⑵X2is glutamic acid (E);
⑶X3is methionine (M).
2.YX4GX5WR (SEQ ID No.3), wherein:
⑴X4asparagine, glutamic acid (N, E);
⑵X5is glutamic acid (E).
3.YX6GX7W (SEQ ID No.4), wherein:
⑴X6is glutamic acid (E);
⑵X7is glutamic acid (E).
4.GX8WRL (SEQ ID No.5), wherein:
⑴X8is histidine (H).
In addition, CMKLR1 antagonist polypeptide LRH7-C3 and derivatives thereof also include the following naturally occurring polypeptides in an organism:
SEQ ID No.6 YDGQWRL (bacteria, snake or mouse)
SEQ ID No.7 YDGQWRL-NH2(bacteria, snake or mouse)
SEQ ID No.8 YDGQWR (bacteria, plants, insects or fish)
SEQ ID No.9 YDGQWR-NH2(bacterium, plant, insect or fish)
SEQ ID No.10 DGQWR (insect)
SEQ ID No.11 DGQWR-NH2(insects)
SEQ ID No.12 DGQW (plant or insect)
SEQ ID No.13 DGQW-NH2(plant or insect)
SEQ ID No.14 GQWR (insect)
SEQ ID No.15 GQWR-NH2(insects)
In a further aspect, the present invention provides a polynucleotide encoding a polypeptide as defined in any one of SEQ ID Nos. 1 to 15.
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 medicine for preventing and/or treating female reproductive diseases comprises at least one of the above CMKLR1 antagonist polypeptide and a derivative of CMKLR1 antagonist polypeptide (SEQ ID Nos. 1-15);
in a specific experiment, the CMKLR1 antagonistic polypeptide LRH7-C3 obtained by the invention can effectively change the inhibitory effect of chemerin/RvE1 on cAMP signal pathway. Wherein, the CMKLR1 antagonistic polypeptide LRH7-C3 derivatives (SEQ ID No. 1-15) have the same effect.
In another specific experiment of the invention, the CMKLR1 antagonistic polypeptide LRH7-C3 can effectively inhibit calcium (Ca) caused by chemerin2+) And (4) internal flow effect. Wherein, the CMKLR1 antagonistic polypeptide LRH7-C3 derivatives (SEQ ID No. 1-15) 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 LRH7-C3 has a significant effect of inhibiting cell migration caused by chemerin. Wherein, the CMKLR1 antagonistic polypeptide LRH7-C3 derivatives (SEQ ID No. 1-15) have the same effect.
In further experiments, the function of CMKLR1 antagonistic polypeptide LRH7-C3 is verified by using ovarian cancer cell SKOV-3 highly expressing CMKLR1 as a cell model, and LRH7-C3 can obviously inhibit the proliferation of ovarian cancer cell SKOV-3 compared with a control group. Wherein, the CMKLR1 antagonistic polypeptide LRH7-C3 derivatives (SEQ ID Nos. 1-15) have the same effect.
In another specific experiment of the invention, the inventor adopts a flow cytometry technology to detect that CMKLR1 antagonistic polypeptide LRH7-C3 can block the SKOV-3 cycle of ovarian cancer cells and promote SKOV-3 apoptosis. Wherein, the CMKLR1 antagonistic polypeptide LRH7-C3 derivatives (SEQ ID No. 1-15) 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 LRH7-C3 and a derivative (SEQ ID No. 1-15) 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 channel.
(2) The CMKLR1 antagonistic polypeptide and the derivative thereof (SEQ ID No. 1-15) 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 7-C3.
FIG. 2: LRH7-C3 Mass Spectrometry (MS) detection.
FIG. 3: comparison of the hydrophobic profiles of CMKLR1, chemerin, RvE1 and LRH7-C3 polypeptides. Wherein: a: CMKLR1 hydrophobic profile; b: chemerin hydrophobic profile; RvE1 hydrophobic profile; d: LRH7-C3 polypeptide hydrophobic profile; e: comparison of hydrophobic profiles for CMKLR1, chemerin, RvE1 and LRH7-C3 polypeptides. CMKLR1 contour blue, chemerin contour green, RvE1 contour red, LRH7-C3 contour purple;
FIG. 4: LRH7-C3 altered the inhibitory effect of chemerin on the cAMP signaling pathway.
FIG. 5: LRH7-C3 inhibited chemerin-induced calcium (Ca)2+) And (4) internal flow effect.
FIG. 6: LRH7-C3 inhibited chemotaxis of cells by chemerin.
FIG. 7: LRH7-C3 inhibits the proliferation of ovarian cancer cells SKOV-3.
FIG. 8: LRH7-C3 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: LRH7-C3 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: and (3) carrying out panning, amplification, purification, sequencing and synthesis on the CMKLR1 antagonistic polypeptide LRH 7-C3.
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 7-C3.
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 mug of RNA for reverse transcription (a reverse transcription kit, purchased from Promega corporation), 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.16；
Rv5’-TGATATGGATTGGGAGGAAGAC-3’SEQ ID No.17；
(viii) comparing with the transfected pSM2c-Hu-scramble RNA, 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 mul of LB-Tet liquid culture medium in a 1.5ml sterilized centrifugal tube, taking 0.2 mul 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 for 3min at room temperature, and then placing the plate in a constant temperature incubator at 37 ℃ for inversion 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 10ml of a sterilized centrifuge tube, adding 3ml of LB-Tet liquid culture medium in a sterile operation, inoculating 30 mu l of 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 fog shape (OD) by visual observation600～0.5)。
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 taken for each round of 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 taken for the next round of elutriation, and the rest amplification product is stored at-20 ℃ by 50% of glycerol.
And thirdly, measuring the titer of the phage, namely 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 later use. For each dilution of phage, 1 LB/IPTG/Xgal plate was prepared and pre-warmed in a 37 ℃ incubator for use. Will OD600Coli ER2738 E.coli 0.5 was aliquoted at 200. mu.l phage dilution per tube and stored at 4 ℃ until use. Taking 4 sterilized 1.5ml centrifuge tubes, respectively containing 100. mu.l, 90. mu.l LB-Tet culture medium, sucking 1. mu.l of bacteriophage to be tested into 100. mu.l LB-Tet culture medium, diluting according to 10-fold 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 of each dilution of phage to be titrated with 200 μ l of e.coli er2738, mix by gentle shaking, centrifuge instantaneously, incubate for 5min at room temperature. Quickly adding the mixed bacterial liquid into top agar, quickly shaking and uniformly mixing, immediately pouring into a preheated LB/IPTG/Xgal plate, uniformly flattening, cooling at room temperature for 5min, and inversely culturing the plate in a constant-temperature incubator at 37 ℃ overnight.
Amplification and purification of eluted phage: taking a 250ml conical flask, adding the overnight cultured ER2738 host bacterial liquid into 20ml of LB 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. The amplification of the monoclonal phage comprises the steps of adding overnight cultured ER2738 host bacterial liquid into 2mL of LB liquid culture medium according to the proportion 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 Loading buffer, 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 point 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. According to the analysis result, the sequence of the sample is Tyr- (D) Asp-Gly-Gln-Trp-Arg-Leu-NH2Wherein the second amino acid is in D configuration and represented by LRH7-C3, and finally the short peptide is synthesized by Shanghai Qiaozhizao Biopsis.
The purity of the LRH7-C3 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 LRH7-C3 polypeptide has a certain similarity with CMKLR1, and the similarity reaches 0.002681(PAM 250).
Example 2CMKLR1 antagonistic polypeptide LRH7-C3 was shown to be effective in promoting inhibition of the cAMP signaling pathway by chemerin.
(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 respectively5One cell/well was inoculated in 6-well cell culture plates with a medium volume of 1mL per well, placed in an incubator for 24h, starved overnight, and treated with different concentration gradients (3. mu.M, 0.3. mu.M, 0.03. mu.M) of LRH7-C3 polypeptide, Fosklin (25. mu.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; then 25 μ L of cAMP peroxidase tracer conjugate was added to each well;
c, adding 50 mu L of Anti-cAMP antibody into each hole, 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 in 293T cells highly expressing CMKLR1 (293T CMKLR 1)+/+) When LRH7-C3 (3. mu.M, 0.3. mu.M, 0.03. mu.M) was added to the solution at different concentrations, the decrease of cAMP concentration by chemerin was further promoted. In the single-acting group of the LRH7-C3 short peptide, the LRH7-C3 has no significant effect on the change of cAMP concentration. From the above, it was concluded that LRH7-C3 specifically promoted the reduction of cAMP concentration by chemerin by acting with CMKLR 1. P<0.05,**P<0.01,***P<0.001 compared to the Forskolin + chemerin group.
Example 3CMKLR1 antagonist polypeptide LRH7-C3 can effectively inhibit chemerin-induced calcium (Ca)2+) And (4) internal flow effect.
Plating cells: wild type 293T cells and 293T cells highly expressing CMKLR1 (293T CMKLR 1)+/+) At 5x10 respectively3Inoculating 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 LRH7-C3 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 (293T CMKLR 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 LRH7-C3 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 LRH7-C3 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 LRH7-C3 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 LRH7-C3 on the chemotaxis of cells induced by chemerin.
The wild typeL1.2 cells and L1.2 cells 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 LRH7-C3 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 LRH7-C3 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 LRH7-C3 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 LRH7-C3 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 5LRH7-C3 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 LRH7-C3 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 L DMSO (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 LRH7-C3 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 6LRH7-C3 can block human ovarian cancer SKOV-3 cell cycle progression.
Firstly, human ovarian cancer cell SKOV-3 is divided by 5x105Inoculating 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 LRH7-C3 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, LRH7-C3 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 LRH7-C3 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 LRH7-C3 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 7LRH7-C3 can promote apoptosis of human ovarian cancer SKOV-3 cells.
Firstly, human ovarian cancer cell SKOV-3 is divided by 5x105Inoculating 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 LRH7-C3 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;
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;
fifthly, 100 mu L of precooled 1x annexin V Binding Buffer is added to resuspend the cells;
sixthly, adding 5 mu L annexin V-FITC and 5 mu L PI, mixing evenly, and reacting for 15 minutes in a dark place at room temperature;
seventhly, adding 400 mu L of precooled 1x annexin V Binding Buffer, gently mixing, placing the sample on ice in a dark place, and detecting by a flow cytometer within 1 hour. And analyzing the detection result.
FIG. 9 shows that, through flow cytometry analysis, on one hand, with the increase of the acting concentration, the LRH7-C3 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 LRH7-C3 short peptide also obviously increases the apoptosis number of human ovarian cancer cell SKOV-3. In conclusion, the LRH7-C3 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> CP11701237C
<160> 17
<170> PatentIn version 3.3
<210> 1
<211> 7
<212> PRT
<213> Artificial sequence
<400> 1
Tyr Asp Gly Gln Trp Arg Leu
1 5
<210> 2
<211> 7
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> glutamic acid
<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223> glutamic acid
<220>
<221> MISC_FEATURE
<222> (7)..(7)
<223> methionine
<400> 2
Tyr Xaa Gly Xaa Trp Arg Xaa
1 5
<210> 3
<211> 6
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> asparagine or glutamic acid
<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223> glutamic acid
<400> 3
Tyr Xaa Gly Xaa Trp Arg
1 5
<210> 4
<211> 5
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> glutamic acid
<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223> glutamic acid
<400> 4
Tyr Xaa Gly Xaa Trp
1 5
<210> 5
<211> 5
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> histidine
<400> 5
Gly Xaa Trp Arg Leu
1 5
<210> 6
<211> 7
<212> PRT
<213> bacterium, snake or mouse
<400> 6
Tyr Asp Gly Gln Trp Arg Leu
1 5
<210> 7
<211> 7
<212> PRT
<213> bacterium, snake or mouse
<400> 7
Tyr Asp Gly Gln Trp Arg Leu
1 5
<210> 8
<211> 6
<212> PRT
<213> bacterium, plant, insect or fish
<400> 8
Tyr Asp Gly Gln Trp Arg
1 5
<210> 9
<211> 6
<212> PRT
<213> bacterium, plant, insect or fish
<400> 9
Tyr Asp Gly Gln Trp Arg
1 5
<210> 10
<211> 5
<212> PRT
<213> insect
<400> 10
Asp Gly Gln Trp Arg
1 5
<210> 11
<211> 5
<212> PRT
<213> insect
<400> 11
Asp Gly Gln Trp Arg
1 5
<210> 12
<211> 4
<212> PRT
<213> plant or insect
<400> 12
Asp Gly Gln Trp
1
<210> 13
<211> 4
<212> PRT
<213> plant or insect
<400> 13
Asp Gly Gln Trp
1
<210> 14
<211> 4
<212> PRT
<213> insect
<400> 14
Gly Gln Trp Arg
1
<210> 15
<211> 4
<212> PRT
<213> insect
<400> 15
Gly Gln Trp Arg
1
<210> 16
<211> 20
<212> DNA
<213> Artificial sequence
<400> 16
<210> 17
<211> 22
<212> DNA
<213> Artificial sequence
<400> 17
tgatatggat tgggaggaag ac 22
Claims (13)
1. The CMKLR1 antagonistic polypeptide is characterized in that the amino acid sequence is Tyr- (D) Asp-Gly-Gln-Trp-Arg-Leu-NH2。
2. A derivative of the CMKLR1 antagonist polypeptide of claim 1, wherein:
the derivative is a product obtained by connecting CMKLR1 antagonistic polypeptide with a label for polypeptide or protein detection or purification.
3. A polynucleotide encoding the polypeptide of claim 1.
4. A vector comprising the polynucleotide of claim 3 linked to a promoter sequence by gene technology means.
5. A host cell transfected with the vector of claim 4.
6. Use of a CMKLR1 receptor antagonist polypeptide of claim 1 in the manufacture of a medicament for the treatment of a chemerin/RvE1 CMKLR 1-mediated disease.
7. The use of claim 6, wherein the chemerin/RvE1-CMKLR1 mediated disease is selected from the group consisting of breast cancer, fatty liver, diabetes, inflammatory response.
8. Use of a CMKLR1 receptor antagonist polypeptide of claim 1 in the preparation of a medicament for inhibiting a decrease in cAMP concentration caused by chemerin.
9. Use of a CMKLR1 receptor antagonist polypeptide of claim 1 in the preparation of a medicament for inhibiting chemerin-induced calcium influx.
10. Use of a CMKLR1 receptor antagonist polypeptide of claim 1 in the preparation of a medicament for inhibiting chemerin-induced cell chemotaxis.
11. A pharmaceutical composition comprising the CMKLR1 receptor antagonist polypeptide of claim 1 as an active ingredient.
12. The pharmaceutical composition of claim 11, wherein the pharmaceutical composition comprises one or more pharmaceutically acceptable carriers.
13. The pharmaceutical composition of claim 12, 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.
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| CN201711386874.5A CN109942677B (en) | 2017-12-20 | 2017-12-20 | CMKLR1 antagonistic polypeptide and derivative and application thereof |
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| Publication Number | Publication Date |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104434888A (en) * | 2013-09-17 | 2015-03-25 | 深圳先进技术研究院 | Application of CMKLR1 micromolecule antagonist to control nonalcoholic fatty liver and hepatitis |
| CN104434926A (en) * | 2013-09-17 | 2015-03-25 | 深圳先进技术研究院 | Application of CMKLR1 micromolecule antagonist to control obesity and obesity metabolic syndrome |
| WO2017113101A1 (en) * | 2015-12-29 | 2017-07-06 | 深圳先进技术研究院 | Target cmklr1 for female reproductive system diseases, antagonists thereof, and related application |
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| US20070286863A1 (en) * | 2006-05-17 | 2007-12-13 | Christopher Sinal | CMKLR regulation of adipogenesis and adipocyte metabolic function |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104434888A (en) * | 2013-09-17 | 2015-03-25 | 深圳先进技术研究院 | Application of CMKLR1 micromolecule antagonist to control nonalcoholic fatty liver and hepatitis |
| CN104434926A (en) * | 2013-09-17 | 2015-03-25 | 深圳先进技术研究院 | Application of CMKLR1 micromolecule antagonist to control obesity and obesity metabolic syndrome |
| WO2017113101A1 (en) * | 2015-12-29 | 2017-07-06 | 深圳先进技术研究院 | Target cmklr1 for female reproductive system diseases, antagonists thereof, and related application |
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