CN109942673A

CN109942673A - A kind of GPR1 antagonism polypeptide and its derivative and application

Info

Publication number: CN109942673A
Application number: CN201711378788.XA
Authority: CN
Inventors: 张键; 代小勇; 陈杰; 余贵媛; 蔡佳璇; 黄晨
Original assignee: Shenzhen Institute of Advanced Technology of CAS
Current assignee: Shenzhen Institute of Advanced Technology of CAS
Priority date: 2017-12-19
Filing date: 2017-12-19
Publication date: 2019-06-28
Anticipated expiration: 2037-12-19
Also published as: CN109942673B

Abstract

The invention discloses a kind of GPR1 antagonism polypeptide and its derivative and applications, more particularly to such as sequence SEQ ID No.1-8 and its derivative, the derivative of the binding peptide is on GPR1 binding peptide amino acid side groups, the aminoterminal of GPR1 antagonism polypeptide segment or c-terminus progress routinely modifys obtained product, or be to connect to be used for polypeptide or Protein Detection or the obtained product of the label of purifying on GPR1 antagonism polypeptide；The binding peptide and its derivative in vitro can be in conjunction with GPR1, by blocking the combination of chemerin and GPR1 that cAMP concentration is promoted to increase, inhibiting calcium (Ca caused by chemerin²⁺) in stream, and Cells Proliferation of Human Breast Cancer can be inhibited, block its cell cycle, promote breast cancer cell that apoptosis occurs, provided effective treatment small-molecule drug for female reproduction disease such as breast cancer etc., can be widely used in Med Biol field.

Description

A kind of GPR1 antagonism polypeptide and its derivative and application

Technical field

The present invention relates to biotechnologys and biomedicine field, specifically, the present invention is female reproduction disease target spot GPR1 receptor antagonist polypeptide LRH7-G3 and its derivative and application.

Background technique

Breast cancer (Breast cancer) is to be only second to the mortality cancer of the 2nd high mortality of the whole world of lung cancer, it continues Property destroys the life and health of the millions of women in the whole world and its family.According in the research of World Health Organization's international cancer Heart recent statistics, global women with breast cancer new cases in 2012 account for whole female malignant morbidities up to 1,670,000 22.9%；460000 women because of breast cancer deaths, 13.7%, the Zhan Suoyou women die of Zhan Suoyou female malignant death 1.7%, suffering from cancer women for about 4 just has 1 to suffer from breast cancer.Female Breast Cancer in China age-standardized incidence is 21.6/10 ten thousand, occupy the 1st of female cancer morbidity；The death rate is 5.7/10 ten thousand, occupies the 6th of female cancer death.4%~ As metastatic breast cancer when 6% breast cancer diagnosis, and the early stage patient 30%~40% for receiving adjuvant treatment can develop to turn Shifting property breast cancer, 5 years survival rates about 20% of patient.And perform the operation, the traditional treatments mode such as radiation and chemotherapy, the specificity of effect and Specificity is bad, inevitably generates lethal effect to normal cell and tissue, brings great side effect to patient. Therefore, the targeted drug for finding efficiently specificity, will be greatly improved the therapeutic effect of breast cancer！

Clinical research discovery, early-stage breast cancer often do not have typical sings and symptoms, are not easy to cause attention, often pass through Physical examination or breast cancer screening could be found.Although breast cancer early diagnosis can for breast cancer treatment win preciousness when Between, the therapeutic effect of breast cancer can be greatly improved, but the personnel ratios that China carries out early-stage breast cancer diagnosis at present are seldom.

Mammary cancer is transferred to lymph, is a kind of common metastasis symptom of breast cancer.In general, lesion on the outside of breast 2/3 is accounted for the chance of axillary lymphatic metastasis, the chance shifted by sternal is 1/3.On the inside of breast and the tumour of central portion, The chance with outside breast cancer Lymph Node Metastasis respectively accounts for 1/2 inwardly, and breast cancer is easy to happen Lung metastases and Lymph Node Metastasis.Tumour is micro- Environment is most important to the formation of tumour, determines the occurrence and development of tumour.It is that normal mammary stem cells cause at first Cancer mutation is converted into breast carcinoma stem cell, generates the tumour of prognosis mala, and then these are swollen with metastatic poor prognosis Tumor, under the influence of matrix fiber mother cell, a part of breast carcinoma stem cell is shifted, be transferred to brain, lung, liver with And marrow.

Some researches show that overexpression is presented in Chemokine receptor CXCR4 in breast cancer tissue, and ligand matrices are thin Born of the same parents' derivative factor -1 (CXCL12) is also high to be expressed in Metastasis in Breast Cancer position, in marrow, lymph node, lung and liver, CXCR4/CXCL12 signal system plays an important role in distally other organ metastasis of going back to the nest of breast cancer, therefore Become Bone of Breast Cancer and shift potential therapeutic targets, wherein CXCR4 antagonism polypeptide-CTCE-9908, comes into preclinical Experimental stage can be good at the Bone tumour for inhibiting breast cancer in breast cancer animal model.Therefore, chemotactic factor (CF) is in breast cancer It plays an important role, has using chemotactic factor (CF) as the targeted drug of target treatment breast cancer wide in occurrence and development and transfer Market prospects and application value.

Breast cancer originates from each rank conduit of mammary gland and acinar epithelia, is gradually sent out by adenomatosis to atypical hyperplasia Exhibition is carcinoma in situ, early invasive carcinoma to infiltrating cancer.The canceration that the conduit of different stage occurs, organization type are usually different. 95% or more is human malignant epithelial tumors in breast cancer, and sarcoma of breast is very rare.It is invaded for most common two kinds in histopathology Lubricant nature breast cancer is respectively as follows: infiltrating duct carcinoma (also referred to as " plain edition, NOS ") and lobular carcinoma.Both breast cancer be by What preceding wettability conduit and small leaf nodule were formed.The difference of infiltrating duct carcinoma and lobular carcinoma and other types of breast cancer Mainly as caused by the morphological differences between cell.

Molecular Targeted Therapy for Breast Cancer refers to related for mammary gland carcinogenesis, the related signal path of development and its oncogene Expression product is treated.Being remained high by Cancer Mortality is influenced, and lasting increase is presented in global anti-tumor drug market The gesture of length, the sales volume of antitumor class drug in 2010 account for the 5.8% of international drugs sales volume, occupy the world up to 459.7 hundred million dollars First, increased by 8% than 2009.Wherein, molecular targeted anti-tumor drug sales volume is 311.9 hundred million dollars, and growth rate reaches 23%.The anti-human epidermis of food and drug administration (Food and Drug Administration, FDA) approval in 1997 Molecular targeted therapy-Trastuzumab application of growth factor acceptor 2 (HER-2), has started breast cancer The new era of molecular targeted therapy.According to global best-selling drugs data statistics, global breast cancer medicines market scale has been more than within 2015 10000000000 dollars, it is Herceptin (Trastuzumab) that wherein sales volume is highest, and sales volume is 67.9 hundred million dollars, compared with the same period Increase by 4.2%.Many is the study found that EGF-R ELISA (EGFR) is the key that regulation breast cancer occurrence and development point Son, by activating downstream Ras/Raf/MEK/ERK and PI3K/AKT signal path, EGFR promotes the proliferation of breast cancer, migration And invasion；In addition, epidermal growth factor acceptor 2 (HER-2) height is expressed in breast cancer cell, by forming homodimer or different Source dimer, activation downstream PI3K/Akt and Ras/MAPK signal path promote breast cancer proliferation, angiogenesis, migrate and invade It attacks.Currently, the molecular targeted agents for breast cancer treatment mainly have: for the toltrazuril of Epidermal growth factor-recepor-2 treatment Monoclonal antibody；For the inhibitor everolimus in PI3K/AKT/mTOR access；For the Avastin of anti-angiogenic medicaments； For the iniparib of BRCA1/2 mutation；And it is directed to CDK4/6 inhibitor palbociclib.The appearance of these targeted drugs And application is that patient with breast cancer brings new dawn, so that the treatment of breast cancer is become more accurate, dramatically reduces tradition It treats bring and damages side effect！

Chemotactic factor (CF) and its receptor in the development process of breast cancer, not only with the foundation of tumor microenvironment and metastases It is closely related, can be with the deterioration of mediate tumor cell, and promote the growth and proliferation of tumour cell.Research points out, The chemotactic factor (CF)s such as CXCL8, CXCL12, CCL2, CCL5, CCL18, which have, promotees breast cancer effect, and other chemotactic factor (CF) is such as XCL1, CXCL9, CXCL10, CXCL14, CCL16, CCL19 etc. may then have anti-breast cancer effect.Tumour associated fibroblast is thin Born of the same parents (CAFs) and cancer cell can activate and secrete a large amount of CCL2, promote the self-renewing of tumor stem cell (CSC), and then strengthen Tumour cell to change, the repellence of radiotherapy and the transfer for promoting lesion.Positive rate of the CXCR4 in breast cancer is up to 60%, CXCL12/CXCR4 effect network plays a significant role in the growth, transfer of a variety of solid tumors including breast cancer, hinders Break this biological axis can inhibit angiogenesis and tumour diffusion, increase tumour cell to chemotherapy, the sensibility of radiotherapy.In 4T1 Mouse breast cancer model can inhibit angiogenesis and metastases with the oncolytic virus treatment for carrying CXCR4 antagonist.In TNBC The expression (71%) of CXCR4 is higher than HER-2 positive breast cancer (44%) and Luminal type breast cancer (37%), CXCR4+TNBC Patient tumors are big, Yi Fasheng liver, lung, brain metastes, poor prognosis.Chemotactic factor (CF) and its receptor antagonist or inhibitor have wide Market prospects.Have statistics show about 30% small-molecule drug be with g protein coupled receptor for targeting.CXCR4 polypeptide Antagonist CTCE-9908 has been found to can inhibit the Lung metastases of Mouse Bone sarcoma and melanoma.CXCR1/CXCR2 antagonist- Repertaxin can inhibit the growth and transfer of tumour in animal model, have been used for Advanced breast cancer patient clinical trial. Therefore, effect of the research chemotactic factor (CF) in tumor development finds the novel targets of chemotactic factor (CF) treatment breast cancer, not only to explaination Metastases have great significance, and have wide pharmaceutical market prospect.

The fat correlation research with breast cancer is seen earliest in the 1960s, case-control study proposes mammary gland earliest The women of post menopausal obesity and high blood pressure is tended in the morbidity of cancer., it was also found that compared with the control group in animal experiment study, Fat mice tumors grew speed obviously increases.World Cancer Research Foundation and american cancer research institute are demonstrate,proved with epidemiology According to retrospective analysis is carried out, the result of study of animal model proves overweight and fat dramatically increases the risk to suffer from breast cancer. Meta analysis shows that, the risk to suffer from breast cancer with BMI increase and increase, BMI > 30kg/m²The wind that suffers from breast cancer of patient Danger is 1.3~2 times of normal person, and obesity can dramatically increase the risk that women suffers from breast cancer.

Obesity promotes mammary gland carcinogenesis, the mechanism of development includes the following aspects: (1) estrogen.Obesity can increase Add the synthesis of estrogen, reduces serum estradiol and sex hormone binding globulin (SHBG) is horizontal, serum estradiol is excessive, competition Estrogen receptor on target organ causes Hypothalamus-Pituitary-Ovaries gonad index disorder, breaks mammoplasia and answers Old balance promotes abnormality proliferation and the conversion of breast cancer cell；(2) chronic inflammation.It is activated in obese patient's adipose tissue Macrophage can secrete a large amount of pro-inflammatory mediators, cause chronic inflammatory reaction, a large amount of inflammatory factors and internal various cells are tumour Tumor microenvironment is formd, to promote the generation of breast cancer cell, development.When TG excessive concentration in blood, mammary gland blood vessel Lipidosis leads to the generation of chronic inflammation, the inflammatory factor of inflammatory cell secretion and Stromal fibroblasts, lymphocyte, huge Phagocyte, blood vessel and lymphatic network and extracellular matrix form tumor microenvironment, interfere the duplication and transcription of DNA, cause to dislike Property tumour occur；(3) insulin and insulin-like growth factor-i (IGF-1).Easily there is insulin resistance in obesity, makes insulin And the horizontal of IGF-1 increases, and facilitates the proliferation and revascularization of cancer cell.One research from swiss data library has collected Receive within 2005 the data of 115 000 patients of insulin therapy, sweet smart pancreas is applied alone in discovery in next 2 year Patient's pathogenesis of breast carcinoma risk of island extract for treating is obviously relatively high (RR=1.99) using other insulin analogs.(4) fat is thin Intracellular cytokine.Obesity will affect the generation of Adipocyte Factor, reduce adiponectin, increase the level of leptin, PAI-1 etc., promote thin Born of the same parents' proliferation, anti-apoptotic and acceleration angiogenesis, to promote the proliferation and growth of cancer cell.Adiponectin can not only inhibit pancreas islet Element cooperates with the Mitosis promoted with estrogen, can also be reduced by promoting Bax and p53 to promote the expression of apoptogene The transfer of cancer cell and wetting capacity.Leptin can by activate JAK/STAT3, MAPK-ERK1/2 PI3K access, from And promote the growth of breast cancer；In addition, the expression that leptin can also generate element by induction of vascular promotes the life of tumor-associated vessels At, and leptin also can induce human epidermal growth factor receptor 2 ErbB-2) transcription, and participate in triple negative breast cancer cell The reaction of middle insulin-like growth receptor 1 (IGF-1), activation EGF-R ELISA (EGFR) is to promote the invasion of cell With transfer.Obesity is the risk factor of Postmenopausal Breast Cancer, it is fat with mammary gland carcinogenesis, development it is in close relations and complicated, greatly Quantity research shows that obesity can produce certain negative regulation to the generation of breast cancer, development, diagnosis, tumour feature and prognosis. It therefore, can by the further exploration and research of Adipocyte Factor and Breast Cancer to fat, adipose tissue and its secretion Very important reference frame is provided to formulate reduction breast cancer incidence from now on and improving the corresponding measure of disease prognosis.

Adipocyte Factor Chemerin

Adipocyte Factor Chemerin is also referred to as Tazarotene-induced gene 2 (TIG2) or retinol receptor response albumen 2.? By after the clones such as Nagpal discovery, in 2003, Wittamer etc. passed through reverse phase height in the secondary ascites of oophoroma within 1997 Hydraulic fluid phase chromatography obtains its activated protein.163 amino acid of Chemerin amyloid protein precursor overall length have 6 cysteines Residue forms 3 disulfide bond, removes several amino acid of N-terminal signal peptide and C-terminal, just has bioactivity, in blood Chemerin activated form contains 134 amino acid (about 16kDa), and cecropin/cysteine proteinase suppression is belonged in structure Preparation family, when be inflamed react when, chemerin can rapidly transform into activated form under several albumen enzyme effects. Chemerin is mainly expressed in white adipose tissue, placenta and liver in the Various Tissues wide expression of human body, with 3 receptors It combines: 1. g protein coupled receptor CMKLR1；2. g protein coupled receptor GPR1；③CCRL-2.After Chemerin combination GPR1, On the one hand the calcium ion in GPR1 positive cell can be made to discharge, inhibits the aggregation of cAMP, makes map kinase phosphorylation；Other one Aspect has facilitation to stream in intracellular calcium.On the one hand Chemerin is used as chemotactic factor (CF), chemotactic Dendritic Cells And macrophage, it serves as a connection between adaptive immunity immune, chemotactic natural killer cell reaches inflammation part and participates in Inflammatory reaction；On the other hand it as new Adipocyte Factor, is secreted and is generated by adipose tissue, adjust differentiation, the steatolysis of fat cell, It can promote the biological effects such as insulin signaling pathway in fat cell.Chemerin is fat and metabolic syndrome Consequence is accounted in pathophysiological mechanism.

GPR1 receptor

GPR1 is the immediate homologue of CMKLR1, with CMKLR1 it is shared be more than 40% sequence identity.It is a kind of The g protein coupled receptor to play a crucial role in eukaryocyte usually stimulates glucose, lipid accumulation and by trophic signals Pass to cAMP approach.Many signal transductions are mediated by G-protein → coupled receptor (GPCR) in eucaryote.G-protein β is even Connection receptor is present in some eucaryotes, including yeast, double flagellates and animal.GPR1 is initially the G found in the mankind G-protein linked receptor (GPCR), the receptor of chemerin is accredited as by experiment in vitro.Although not reporting Chemerin Physiological function in conjunction with GPR1, but it is reported that GPR1 in mouse animal brown adipose tissue, white adipose tissue and skeletal muscle Height is expressed, and GPR1 is mainly expressed in the vascular cell of white adipose tissue.It is struck in the GPR1 of feeding high fat diet Except in mouse, it is found that its glucose intolerance ratio WT mouse is more serious.In addition, GPR1 knocks out small in pyruvic acid resistance test The insulin level that mouse is able to suppress glucose α stimulation increases, and causes blood glucose rise.These results indicate that GPR1 is The active acceptor of Chemerin, it is adjustable it is fat during the intracorporal balance of glucose.

Using Tango bioassay, measurement after increasing the treatment of Chemerin dosage CMKLR1 and GPR1 in HTLA cell In activity.It was found that Chemerin activates CMKLR1 and GPR1 with similar effect, reach effect be 265.3 ± 182.2- and 185.6 ± 26.5- times of maximum the reaction (Emax) changed.In addition, Chemerin activation GPR1 ratio activation CMKLR1 (EC50, 54.6 ± 30.7nM) there is significant higher effect (EC50,18.2 ± 2.9nM), this proves that Chemerin has activated GPR1, together When GPR1 be also highly sensitive Chemerin receptor.

GPR1 expression cell system passes through weak Ca²⁺Mobilize and ERK1/2 activation reacts to chemerin, but its due to swash Dynamic agent in conjunction with and be effectively internalized by.Therefore, GPR1 may be as the Decoy receptors of chemerin, and so far, main thin It is intracellular or there has been no the active descriptions mediated to GPR1 in vivo.And GPR1 is in central nervous system, skeletal muscle, skin and rouge It is expressed in fat tissue, the activity of chemerin can be adjustable.

G protein coupled receptor superfamily (GPCRs) is played an important role in extracellular signal into transductive process intracellular, is adjusted Control vital factor in these three carcinobiologies of cell movement, growth and genetic transcription-.In the past more than ten years, The signal path of mediation has proven to the key regulatory person of protooncogenic signal, and is good drug target, at present The drug 60% sold on the market is designed both for this receptor.And GPR1 does not still have at present as one of GPCRs member Any drug is to treat clinical cancer directly against GPR1.Therefore, using GPR1 as target, the novel polypeptide medicine of anticancer is screened Object and humanized antibody have important social effect and wide economic market.

Display technique of bacteriophage (Phage Display Technology) is the screening of a specific polypeptide or albumen The polypeptide that target gene encodes can be showed in phage surface by technology, this technology in the form of fusion protein, and what is be demonstrated is more Peptide or protein can keep relatively independent space structure and bioactivity, make a large amount of rondom polypeptides and its DNA encoding sequence it Between establish direct connection so that the polypeptide ligand of various target molecules (such as antibody, enzyme and cell surface receptor) pass through it is external Affine biopanning procedure is able to Rapid identification.Since asking display technique of bacteriophage generation, because of its high storage capacity, efficient and convenient and clever The advantages such as screening living, phage antibody library technique are in progress very fast in recent decades, obtain in many fields of life science To extensive use, the attention that is especially just increasingly being subject in the fields such as diagnosing tumor and tumour antibody medicine preparation.Polypeptide at present The main function target spot of drug is g protein coupled receptor (GPCRs), there is the targeting of 39% polypeptide drugs in clinical studies GPCRs.Polypeptide drugs have many advantages: (1) druggability is higher than general chemicals, and bioactivity is high, high specificity；⑵ Toxic reaction is relatively weak, is not likely to produce accumulation in vivo；(3) fewer with the interaction of other drugs, with internal receptor Compatibility is relatively good.Humanized monoclonal antibodies have high sensitivity, high specificity, efficiently, serum cross reaction is few or nothing, system The advantages that standby at low cost.Polypeptide drugs and humanized antibody are screened using display technique of bacteriophage, genotype and table may be implemented The unification of type, be it is a kind of prepare the technical method that antibody is efficient, practical, the technology using expression albumen and ligand affinity, Required albumen or polypeptide are screened, the screening and system of the identification screening and antibody drug of disease marker are widely used in Among standby.

Summary of the invention

For overcome the deficiencies in the prior art and disadvantage, the primary purpose of the present invention is that providing one kind by phage display The GPR1 antagonism polypeptide that library is screened, the antagonism polypeptide and Chemerin receptor GPR1 have specific high-affinity, can The signal path of Chemerin/GPR1 is blocked by inhibiting the combination of Chemerin and GPR1, it was demonstrated that the polypeptide presses down in targeting Cells Proliferation of Human Breast Cancer processed promotes Apoptosis of Breast Cancer etc. and plays an important role, in breast cancer targeted therapy side Face has huge application value.

Another object of the present invention is to provide the derivative of above-mentioned GPR1 receptor antagonist polypeptide, which also can be with GPR1 receptor has specific high-affinity, and the binding site of Specific competition Chemerin and GPR1, is able to suppress Chemerin combination GPR1.

A further object of the present invention is to provide the applications of above-mentioned GPR1 antagonism polypeptide and its derivative.

In order to realize that above-mentioned task, the present invention take following technical solution:

GPR1 antagonism polypeptide, which is characterized in that its amino acid sequence are as follows: Gly- (D) Pro-Leu-Ile-Trp-Phe- Ser-NH₂(SEQ ID No.1), i.e. LRH7-G3.

The screening technique of above-mentioned GPR1 antagonism polypeptide, this method utilize phage random peptide library, use GPR1 plasmid first 293T cell is transfected, the stable cell lines of permanent high expression GPR1 is obtained, is control adherent cell with wild type 293T cell, The full Cell depletion screening of 5 wheels is carried out, clone's single stranded DNA sequencing is extracted in the 50 positive bacteriophage amplifications of random picking.Analyze polypeptide Amino acid sequence essential characteristic, peptides homologous compares, the high polypeptide motif of the retrieval frequency of occurrences.BLAST retrieves albumen Matter database detects the higher protein of polypeptide motif homology, biological species of the discovery containing a large amount of polypeptides, and may tie The cell surface receptor and ligand of conjunction, are conducive to subsequent a large amount of extractions and purifying obtains polypeptide.

Hydrophilicity analysis shows that LRH7-G3 is hydrophobic peptides；

High performance liquid chromatography (HPLC) and the purity of mass spectrum (MS) detection LRH7-G3 synthesis have reached 99.85%.

The derivative that another aspect of the invention provides GPR1 receptor antagonist polypeptide of the present invention is GPR1 receptor On antagonism polypeptide amino acid side groups, routinely modification obtains the progress of the aminoterminal of GPR1 receptor antagonist polypeptide fragment or c-terminus Product, or be on GPR1 receptor antagonist polypeptide connection for polypeptide or Protein Detection or the obtained production of the label of purifying Object；

Preferably, the routine is modified to amination, amidation, hydroxylating, carboxylated, carbonylation, alkylation, acetyl Change, phosphorylation, sulphation, esterification, glycosylation, cyclisation, biotinylation, fluorophor modification, polyethylene glycol PEG modification or fixed Change modification etc.；

Preferably, the label be His6, GST, EGFP, MBP, Nus, HA, IgG, FLAG, c-Myc or ProfinityeXact。

Another aspect of the present invention provides the bioactive fragment or class of GPR1 receptor antagonist polypeptide of the present invention Like object, as shown in SEQ ID No.2-8:

PLX₁WF SEQ ID No.2, in which:

⑴X₁For leucine (L).

GPLIWFS SEQ ID No.3

GPLIWFS-NH₂ SEQ ID No.4

GPLIWF SEQ ID No.5

GPLIWF-NH₂ SEQ ID No.6

PLIWFS SEQ ID No.7

PLIWFS-NH₂ SEQ ID No.8

The present invention also provides the derivative of the bioactive fragment of GPR1 receptor antagonist polypeptide or the like, the GPR1 On the amino acid side groups of the derivative of the bioactive fragment of receptor antagonist polypeptide or the like, aminoterminal or c-terminus into Row routinely modifies obtained product, or is the upper connection of bioactive fragment of GPR1 receptor antagonist polypeptide or the like for more The obtained product of label of peptide or protein detection or purifying；

In the inventive solutions, the GPR1 antagonism polypeptide and its derivative can be applied to preparation prevention And/or the drug in terms for the treatment of female reproduction disease, existence form have: 1. containing 5 amino acid；2. by 6 amino acid groups At；3. being made of 7 amino acid.The derivative of the GPR1 antagonism polypeptide is GPR1 antagonism polypeptide amino acid side groups Obtained product is routinely modified in the upper, aminoterminal of GPR1 antagonism polypeptide segment or c-terminus progress, or is GPR1 antagonism polypeptide Upper connection is for polypeptide or Protein Detection or the obtained product of the label of purifying；The described conventional modification be preferably amination, It is amidation, hydroxylating, carboxylated, carbonylation, alkylation, acetylation, phosphorylation, esterification, glycosylation, cyclisation, biotinylation, glimmering Light base group modification, polyethylene glycol PEG modification or immobilization modification etc.；The label is preferably His₆、 GST、EGFP、MBP、 Nus, HA, IgG, FLAG, c-Myc or ProfinityeXact etc.；

The GPR1 antagonism polypeptide and its derivative can derive from mammal or birds, such as primate moves Object (mankind)；Rodent, including mouse, rat, hamster, rabbit, horse, ox, canine, cat etc..

The derivative of the GPR1 antagonism polypeptide is preferred are as follows: second amino acid residue of above-mentioned GPR1 antagonism polypeptide is D Configuration proline, end carry out amidation modification, as Gly- (D) Pro-Leu-Ile-Trp-Phe-Ser-NH₂。

The acquisition of the GPR1 antagonism polypeptide and its derivative is carried out using known method in the prior art, both may be used To carry out chemical synthesis with polypeptide automatic synthesizer；By the way that short peptide sequence is derived nucleotide sequence, it is then cloned into carrier Middle carry out biosynthesis；Largely it can also be extracted and be purified out of existing organism.

Specifically, GPR1 antagonism polypeptide LRH7-G3 and its derivative have following sequence:

PLX₁WF SEQ ID No.2, in which:

⑴X₁For leucine (L).

In addition, GPR1 antagonism polypeptide LRH7-G3 and its derivative also include the following polypeptide being naturally present in biology:

SEQ ID No.3 GPLIWFS (bacterium, tobacco)

SEQ ID No.4 GPLIWFS-NH₂(bacterium, tobacco)

SEQ ID No.5 GPLIWF (bacterium, shellfish)

SEQ ID No.6 GPLIWF-NH₂(bacterium, shellfish)

SEQ ID No.7 PLIWFS (honeybee, balsamine, humpback, moth class, olive, fungi)

SEQ ID No.8 PLIWFS-NH₂(honeybee, balsamine, humpback, moth class, olive, fungi)

Another aspect of the present invention provides a kind of polynucleotide, and any one of coding SEQ ID No.1-8 is described more Peptide.

Another aspect of the present invention provides a kind of carrier, and it comprises nucleotide of the present invention, can pass through base Because technological means and promoter sequence link.

Another aspect of the present invention provides a kind of host cell, has transfected carrier of the present invention.

Another aspect of the present invention provides GPR1 receptor antagonist polypeptide of the present invention, GPR1 receptor antagonist polypeptide Derivative, the bioactive fragment or the like of GPR1 receptor antagonist polypeptide and its derivative treat chemotactic element-GPR1 in preparation Purposes in disease mediated drug.

Another aspect of the present invention provides GPR1 receptor antagonist polypeptide of the present invention, GPR1 receptor antagonist polypeptide Derivative, the bioactive fragment or the like of GPR1 receptor antagonist polypeptide and its derivative are mediated in treatment chemotactic element-GPR1 Purposes in disease.

In the inventive solutions, the chemotactic element-GPR1 it is disease mediated selected from breast cancer, fatty liver, diabetes, Inflammatory reaction, polycystic ovary syndrome.

Another aspect of the present invention provides GPR1 receptor antagonist polypeptide of the present invention, GPR1 receptor antagonist polypeptide Derivative, the bioactive fragment or the like of GPR1 receptor antagonist polypeptide and its derivative inhibit chemerin to cause in preparation CAMP concentration reduced drug in purposes.

Another aspect of the present invention provides GPR1 receptor antagonist polypeptide of the present invention, GPR1 receptor antagonist polypeptide Derivative, the bioactive fragment or the like of GPR1 receptor antagonist polypeptide and its derivative are inhibiting caused by chemerin Purposes in the reduction of cAMP concentration.

Another aspect of the present invention provides GPR1 receptor antagonist polypeptide of the present invention, GPR1 receptor antagonist polypeptide Derivative, the bioactive fragment or the like of GPR1 receptor antagonist polypeptide and its derivative inhibit chemerin to cause in preparation Calcium (Ca²⁺) effect of interior stream drug in purposes.

Another aspect of the present invention provides GPR1 receptor antagonist polypeptide of the present invention, GPR1 receptor antagonist polypeptide Derivative, the bioactive fragment or the like of GPR1 receptor antagonist polypeptide and its derivative are inhibiting calcium caused by chemerin (Ca²⁺) in the active purposes of stream.

Another aspect of the present invention provides GPR1 receptor antagonist polypeptide of the present invention, GPR1 receptor antagonist polypeptide Derivative, the bioactive fragment or the like of GPR1 receptor antagonist polypeptide and its derivative inhibit chemerin to cause in preparation Cell chemotaxis effect drug in purposes.

Another aspect of the present invention provides GPR1 receptor antagonist polypeptide of the present invention, GPR1 receptor antagonist polypeptide Derivative, the bioactive fragment or the like of GPR1 receptor antagonist polypeptide and its derivative are inhibiting thin caused by chemerin Purposes in born of the same parents' chemotaxis.

Another aspect of the present invention provides GPR1 receptor antagonist polypeptide of the present invention, GPR1 receptor antagonist polypeptide Derivative, the bioactive fragment or the like of GPR1 receptor antagonist polypeptide and its derivative treat breast cancer, fat in preparation Liver, diabetes, inflammatory reaction, polycystic ovary syndrome drug in purposes.

Another aspect of the present invention provides GPR1 receptor antagonist polypeptide of the present invention, GPR1 receptor antagonist polypeptide Derivative, the bioactive fragment or the like of GPR1 receptor antagonist polypeptide and its derivative are in treatment breast cancer, fatty liver, sugar Urinate disease, inflammatory reaction, the purposes in Stein-Leventhal syndrome.

Another aspect of the present invention provides a kind of pharmaceutical composition, wherein short of money comprising GPR1 receptor of the present invention Anti- polypeptide, the derivative of GPR1 receptor antagonist polypeptide, GPR1 receptor antagonist polypeptide bioactive fragment or the like and its spread out One of biology is a variety of as active constituent.

The pharmaceutical composition can contain a kind of or a variety of pharmaceutically acceptable carriers；

The pharmaceutically acceptable carrier is preferably diluent, excipient, filler, adhesive, wetting agent, disintegration Agent, sorbefacient, absorption carrier, surfactant or lubricant etc.；

A variety of shapes such as tablet, granula, capsule, oral solution or injection can be further made in the pharmaceutical composition The drug of formula, various dosage forms can be prepared according to the conventional method of pharmaceutical field；

The invention also discloses the drugs in terms of a kind of prevention and/or treatment female reproduction disease or tumour, include this hair The bioactivity of the bright GPR1 receptor antagonist polypeptide, the derivative of GPR1 receptor antagonist polypeptide, GPR1 receptor antagonist polypeptide Segment or the like and its at least one of derivative.

The invention also discloses the drugs in terms of the disease of a kind of prevention and/or the high expression GPR1 receptor for the treatment of, include GPR1 receptor antagonist polypeptide, the derivative of GPR1 receptor antagonist polypeptide, GPR1 receptor antagonist polypeptide bioactive fragment or class Like at least one of object and its derivative.

In a specific experiment of the invention, the present invention can be effective using GPR1 antagonism polypeptide LRH7-G3 obtained Alleviate chemerin to the inhibiting effect of cAMP signal path.Wherein, GPR1 antagonism polypeptide LRH7-G3 derivative (SEQ ID No.1~8) there is phase same-action.

In another specific experiment of the invention, can effectively it be inhibited using GPR1 antagonism polypeptide LRH7-G3 Calcium (Ca caused by chemerin²⁺) effect of interior stream.Wherein, GPR1 antagonism polypeptide LRH7-G3 derivative (No.1~8 SEQ ID) With phase same-action.

In further test, inventor is using the breast cancer cell MDA-MB-231 of high expression GPR1 as cell model The function of GPR1 antagonism polypeptide LRH7-G3 is demonstrated, compared with the control group, it is thin that discovery LRH7-G3 can significantly inhibit breast cancer The proliferation of born of the same parents MDA-MB-231.Wherein, GPR1 antagonism polypeptide LRH7-G3 derivative (No.1~8 SEQ ID) has identical work With.

In another specific experiment of the invention, inventor uses Flow Cytometry, detection discovery GPR1 antagonism polypeptide LRH7-G3 can block the breast cancer cell MDA-MB-231 period, promote MDA-MB-231 that apoptosis occurs.Wherein, GPR1 antagonism Polypeptide LRH7-G3 derivative (No.1~8 SEQ ID) has phase same-action.

The present invention has the following advantages and effects with respect to the prior art:

(1) described the present invention provides a kind of GPR1 antagonism polypeptide LRH7-G3 and its derivative (No.1~8 SEQ ID) Antagonism polypeptide and its derivative can specificity in conjunction with GPR1, and Specific competition Chemerin and GPR1 binding site, It is able to suppress Chemerin/GPR1 signal path.

(2) the GPR1 antagonism polypeptide and its derivative (No.1~8 SEQ ID) that present invention screening obtains can pass through blocking Chemerin/GPR1 in conjunction with and inhibit Cells Proliferation of Human Breast Cancer, promote breast cancer cell occur apoptosis, can be used as The biological species polypeptide drugs of Chemerin/GPR1 binding site, in terms of can be used for preparing prevention and/or treatment female reproduction disease Drug, such as: breast cancer, fatty liver, diabetes and inflammatory reaction.It can be obtained widely in Med Biol field Using, and generate huge social and economic effects.

Detailed description of the invention

Fig. 1: LRH7-G3 efficient liquid phase (HPLC) detection figure.

Fig. 2: LRH7-G3 mass spectrum (MS) detection figure.

Fig. 3: GPR1, chemerin and the hydrophobic profile comparative analysis of LRH7-G3 polypeptide.Wherein: the hydrophobic profile of A:GPR1 Figure；The hydrophobic profile diagram of B:chemerin；The hydrophobic profile diagram of C:LRH7-G3 polypeptide；D:GPR1, chemerin and LRH7-G3 are more The hydrophobic profile of peptide compares figure.GPR1 profile is red, and chemerin profile is blue, and LRH7-G3 profile is green；

Fig. 4: LRH7-G3 alleviates chemerin to the inhibiting effect of cAMP signal path.Wherein, A, LRH7-G3 with Effect of the Forsklin and Chemerin in 293T wild-type cell；B, LRH7-G3 are individually in 293T GPR1^+/+In work With；C, LRH7-G3 and Forsklin and Chemerin are in 293T GPR1^+/+In effect；

Fig. 5: LRH7-G3 inhibits calcium (Ca caused by chemerin²⁺) effect of interior stream.

Fig. 6: LRH7-G3 inhibits breast cancer cell MDA-MB-231 proliferation function.

Fig. 7: LRH7-G3 blocks breast cancer cell MDA-MB-231 cell cycle progression.A: flow cytomery cell Period；B: cell cycle data statistics.

Fig. 8: LRH7-G3 promotes breast cancer cell MDA-MB-231 apoptotic effect.A: flow cytomery Apoptosis； B: Apoptotic data statistics.

Specific embodiment

For a clearer understanding of the present invention, the present invention is further described referring now to the following example and attached drawing.Embodiment It is only used for explaining without limiting the invention in any way.In embodiment, each Starting reagents material is commercially available, is not specified The experimental method of actual conditions is conventional method and normal condition known to fields, or according to proposed by apparatus manufacturer Condition.

Embodiment 1: elutriation, amplification, purifying, sequencing and the synthesis of GPR1 antagonism polypeptide LRH7-G3 are carried out.

The present embodiment is primarily to screening obtains the positive bacteriophage with GPR1 specific binding, then by biting the positive Phage amplification, purifying extract phage single-chain DNA (ssDNA) and are sequenced, sequence obtained is analyzed and is compared, is finally closed At the antagonism polypeptide LRH7-G3 of high-purity.

It is specific as follows:

1. establishing the 293T cell line of permanent high expression GPR1: 293T-GPR1^+/+/LRH

1. the eugonic people's 293T cell to shine is chosen, in the day before transfection with 5 × 10⁵A/hole, is inoculated in 6 In orifice plate, after culture to second day, cell fusion degree is 60%；

2. second day is transfected, as unit of a culture hole of 6 orifice plates, the opti-MEM culture medium with 200 μ L is dilute 3 μ g plasmids are released, 6 μ L liposome Lipofectamine2000 are separately diluted with the opti-MEM culture medium of 200 μ L, are gently mixed respectively After even, it is placed at room temperature for 5 minutes；

3. two pipe dilutions are gently mixed, it is stored at room temperature after twenty minutes, is gently added 600 into mixed dilution The opti-MEM culture medium of μ L；

4. cell to be transfected is gently rinsed once with PBS, culture hole is gently then added in the dilution mixed In, it is placed in carbon dioxide incubator and cultivates；

5. transfection used medium to the greatest extent is abandoned in culture after 4~6 hours, 3mL complete medium is added into hole；

6. selecting the culture medium containing 1 μ g/mL puromycin (puromycin) to be screened after 48 hours；Not to cell It obtains stablizing the 293T cell line for expressing GPR1 after occurring death again.

7. extracting total serum IgE with TRIzol, quantitative 2 μ g RNA carry out reverse transcription, and (Reverse Transcriptase kit is purchased from Promega public affairs Department), and qPCR is carried out with specific primer sequences.

Specific primer sequences used are Hu-GPR1 primer sequence:

Fw 5’-AATGCCATCGTCATTTGGTT-3’(SEQ ID No.9)

Rv 5’-CAACTGGGCAGTGAAGGAAT-3’(SEQ ID No.10)

8. detecting the high expression level of GPR1 with comparing for pSM2c-Hu-scramble RNA has been transfected, and name Are as follows: 293T-GPR1^+/+/ LRH, it can screened for positive bacteriophage.

2. carrying out elutriation, amplification, purifying, sequencing and the synthesis of GPR1 antagonism polypeptide.

1. the preparation of ER2738 host's bacterium solution: aseptic operation first takes 200 μ l LB-Tet Liquid Cultures to be based on 1.5ml It in sterile centrifugation tube, then is frozen from the glycerol of E.coli ER2738 and 0.2 μ l bacterium solution is taken to mix well in object therewith, all drawn It is coated on LB-Tet plate, marks plate, be placed at room temperature for 3min, be subsequently placed in 37 DEG C of constant temperature incubation carton upside downs and be incubated overnight. Next day observation, grow clone after sealed with sealed membrane, 4 DEG C be kept in dark place it is spare.With sterilizing pipette tips with asptic technique picking single bacterium It falls, has been put into the 10ml sterile centrifugation tube added with 3ml LB-Tet fluid nutrient medium in advance, in 37 DEG C of constant-temperature table after label, 300 rpm/min shaken cultivations are stayed overnight.Next day, bacterium amplification liquid are stored for future use in 4 DEG C.Take 10ml sterile centrifugation tube, sterile behaviour Make be added 3ml LB-Tet fluid nutrient medium, the microbionation for taking 30 μ l to be incubated overnight wherein, 37 DEG C of constant-temperature table, 300rpm/ Min 2~3h of shaken cultivation, bacterium are in exponential phase of growth, visually observe (OD fog-like₆₀₀~0.5).

2. the elutriation of GPR1 antagonistic peptide: height expression GPR1 cell is pressed 10⁵A/culture dish is inoculated in coating poly in advance and relies 60 × 15mm of propylhomoserin²In culture dish, when routine culture to cell density 80%~90%, for eluriating (while with not expressing The cell line of GPR1 is as blank control) every wheel eluent first takes 1 μ l to survey titre, and it is remaining to add it to 20ml LB culture It is expanded in liquid, the titre after amplification is finally surveyed in repurity again, and amplified matter takes equal numbers magnitude to be used for down in 4 DEG C of short-term preservations One wheel elutriation, remaining amplified matter are saved with 50% glycerol in -20 DEG C.

3. measuring the titre of bacteriophage: taking the 10ml centrifuge tube of 4 sterilizings, each bacteriophage dilution prepares 1 sterilizing Centrifuge tube, micro-wave oven melt top agar (Agarose Top), and 3ml top agar is added in every pipe, and 45 DEG C of water-baths are spare.Each Bacteriophage dilution prepares 1 piece of LB/IPTG/Xgal plate, 37 DEG C of pre- stand-by heats of constant incubator.By OD₆₀₀~0.5 E.coli ER2738 Escherichia coli dispense according to 200 μ l/ pipe of bacteriophage dilution, and 4 DEG C save backup.Take 4 sterilizings 1.5ml centrifuge tube fills 100 μ l, 90 μ l, 90 μ l, 90 μ l LB-Tet culture mediums respectively, and bacteriophage to be measured is drawn 1 μ l and is entered In 100 μ l LB-Tet culture mediums, by 10 times of gradient dilutions, it is respectively labeled as 10^-1、10^-2、10^-3、10^-4, each dilution is gently Oscillation mixes, moment centrifugation.The bacteriophage for each dilution for taking 10 μ l to be titrated mixes with 200 μ l E.coli ER2738, gently Light oscillation mixes, and moment centrifugation is incubated at room temperature 5min.Mixed bacteria liquid is rapidly added in top agar, quick oscillation mixes, and stands The LB/IPTG/Xgal plate for pouring into preheating uniformly flattens it, the cooling 5min of room temperature, in 37 DEG C of constant incubators, is inverted Plate incubated overnight.

4. the amplification and purifying of wash-out bacteriophage: the conical flask of 250ml is taken, in 1:100 ratio, by what is be incubated overnight ER2738 host's bacterium solution is added into 20ml LB liquid medium, and 37 DEG C, the violent shaken cultivation 2h of 250rpm；It then will be wait expand The phagocytosis body fluid of increasing is added in conical flask, and 37 DEG C, the violent shaken cultivation 4.5h of 250rpm；Culture is transferred to 50ml centrifuge tube In, 4 DEG C of 10,000rpm are centrifuged 10min.Supernatant is transferred in another clean centrifuge tube, and 4 DEG C of 10,000rpm are centrifuged again 10min；It takes the 80% of supernatant to be transferred in another clean centrifuge tube, the PEG/NaCl of 1/4 volume is added, in 4 DEG C after being mixed by inversion Precipitates overnight；Second day, 4 DEG C will be deposited in, 12,000rpm centrifugation 20min.With the careful Aspirate supernatant of clean pipette tips, then 4 DEG C 12,000rpm centrifugation 1min, remove residual supernatant；Then sediment is resuspended with 1ml TBS, gently blows and beats 100 times.Then Suspension is transferred in 2ml centrifuge tube, 4 DEG C of 10,000rpm centrifugation 5min remove residual cells；1/4 volume is added in supernatant After PEG/NaCl, precipitated again in being incubated for 60min on ice；Centrifuge tube is taken out, 4 DEG C of 12,000rpm are centrifuged 20min, remove supernatant； It is resuspended and is precipitated with 200 μ l TBS, 4 DEG C of 10,000rpm are centrifuged 1min.Supernatant is transferred in another centrifuge tube.4 DEG C of short-term preservations, It can be with 50% glycerol in -20 DEG C of long-term preservations.The amplification of monoclonal phage, including (1) in 1:100 ratio, by overnight training Feeding ER2738 host's bacterium solution is added into 2mL LB liquid medium, and 37 DEG C, the violent shaken cultivation 2h of 250rpm；With sterilizing tooth Label, choose the plate for being less than 100 plaques from fourth round titre plate, and picking separates good blue plaque, is added Into culture tube, the violent shake culture 4.5h of 37 DEG C of 250r/min；Then culture is transferred in fresh centrifuge tube, 4 DEG C 10,000rpm is centrifuged 30sec.Supernatant is transferred to in fresh tube, then same centrifugation is primary；Fresh centrifugation is transferred to by the 80% of supernatant Guan Zhong, 4 DEG C of storages, can also be with 50% glycerol in -20 DEG C of long-term preservations.

5. 13 bacteriophage ssDNA of agarose gel electrophoresis identification of M: gel shaped die horizontal being placed, the comb that will be chosen Son is put well, and the space 1mm is stayed between comb bottom and mold；Weigh the conical flask that DNA electrophoresis is put into 250ml with agarose 1g In, 1 × TAE of 100ml buffer is added, after mixing, flask is placed in micro-wave oven, heating is boiled, until agarose is completely molten Solution；Electromagnetic oven is closed, conical flask is taken out, is set and is cooled to room temperature (holding flask can be resistant to) at room temperature, add bromine Change 5 μ l of second ingot, after mixing, i.e., gel solution is poured into offset plate bed board.Plastic plate used in this experiment about needs glue 100ml；Room temperature Under solidified completely to gel, take about 30 minutes, extract comb teeth, offset plate is put into electrophoresis tank；1 × TAE is added in electrophoresis tank Buffer is advisable with being higher by gel surface 2mm；It is added in offset plate after sample is diluted with Loading buffer, pays attention to adding Sample device suction nozzle should be placed in just in gel loading wells, not can pierce gel, also to prevent will be outside sample overfolw hole；Power on, Voltage is adjusted to 50 volts, after electrophoresis 90min, gel slab is taken out, observes result in the UV lamp.

6. ssDNA sequencing and sequence analysis: the M13 bacteriophage ssDNA of extraction is sent to Shanghai Invitrogen biotechnology Co., Ltd carries out DNA sequencing.Sequence analysis is carried out with Bioedit software after sequencing.By analysis result it is found that sample sequence It is classified as Gly- (D) Pro-Leu-Ile-Trp-Phe-Ser-NH₂, wherein second proline is D configuration, it is indicated with LRH7-G3, Last small peptide is synthesized by Shanghai Qiang Yao biotech firm.

Fig. 1 high performance liquid chromatography (HPLC) and the purity of Fig. 2 mass spectrum (MS) detection LRH7-G3 polypeptide are 99.85%, and And molecular size range is consistent with predicted value.The hydrophobicity edge analysis of Fig. 3, it was demonstrated that LRH7-G3 polypeptide and chemerin have centainly Similitude, similarity reaches 0.0184049 (PAM250).

Embodiment 2GPR1 antagonism polypeptide LRH7-G3 can effectively alleviate inhibition of the chemerin to cAMP signal path Effect.

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

1. plating cells: by 293T cell (the 293T GPR1 of wild type 293T cell and high expression GPR1^+/+), respectively with 5×10⁵A/hole is inoculated in 6 porocyte culture plates, and every hole culture volume is 1 mL, is placed in incubator and is cultivated for 24 hours Afterwards, overnight starvation, be added various concentration gradient (3 μM, 0.3 μM, 0.03 μM) LRH7-G3 polypeptide, Fosklin (25 μM) with And chemerin (30nM) acts on 6h；

2. sample preparation: the cell pyrolysis liquid of 300 μ L is added in every hole, and 4 DEG C are placed 20 minutes, is scraped and is collected with cell scraper Cell, after mixing of turning upside down, supernatant is collected in 12,000rpm centrifugations 10 minutes；

3. sample concentration measures: sample concentration is measured by BCA method；

4. cyclic adenosine monophosphate (cAMP) Enzyme-linked Immunosorbent Assay is tested:

A, reagent needed for preparing, 3 multiple holes of each preparation of samples；

The sample of 50 μ L or standard items addition are coated in 96 orifice plates of antibody by b；25 μ L are added in every hole again CAMP peroxidase tracer conjugate；

C, every hole are added 50 μ L Anti-cAMP antibody, room temperature, are slowly incubated for 30 minutes on shaking table；

D is cleaned 5 times using eluent, and 100 μ L chemistry reflective agents are added in every hole, is incubated at room temperature 5 minutes；

E, microplate reader read plate record luminous value.

Fig. 4 shows that chemerin can reduce cell cAMP concentration when concentration is 30nM, but express in height 293T cell (the 293T GPR1 of GPR1^+/+) in (3 μM, 0.3 μM, 0.03 μM) of LRH7-G3 of various concentration effects are added after, CAMP concentration can be made significantly to increase.And in wild type 293T cell, GPR1 receptor, therefore LRH7-G3 pairs are not expressed Chemerin reduces the effect of cAMP concentration without obvious inhibiting effect.Likewise, in LRH7-G3 small peptide independent role group, LRH7-G3 can not be such that cAMP concentration increases.In conclusion it can be concluded that LRH7-G3 can specificity by with GPR1 effect acts on to further suppress chemerin to the reduction of cAMP concentration.^*P<0.05,^*P<0.01,^{* *}P<0.001 Compared with Forskolin+chemerin group.

Embodiment 3GPR1 antagonism polypeptide LRH7-G3 can effectively inhibit calcium (Ca caused by chemerin²⁺) in stream make With.

1. plating cells: by 293T cell (the 293T GPR1 of wild type 293T cell and high expression GPR1^+/+), respectively with 5ⅹ10³A/hole is inoculated in 96 porocyte culture plates, and every hole culture volume is 200 μ L, is placed in incubator and is cultivated After for 24 hours, overnight starvation；

2. reagent configures: probenecid being incorporated in 1mL buffer solution, the probenecid that concentration is 250nM is configured to, shakes up Afterwards, it is added in fluorescent reagent and prepares for use；

3. removing cell culture medium, various concentration gradient (30 μM, 3 μM, 0.3 μM, 0.03 μM, 0.003 μM) are added LRH7-G3 polypeptide and chemerin (0.3nM) are acted on 30 minutes, and then the 100 above-mentioned fluorescent reagents of μ L are added in every hole；

4. 37 DEG C are placed 30 minutes, it is then placed at room temperature for 30 minutes；

5. measuring fluorescence light absorption value at exciting light 494nm and transmitting light 516nm.

Fig. 5 is shown, in 293T cell (the 293T GPR1 of high expression GPR1^+/+) in, chemerin is in activity When 0.3nM, calcium (Ca can be promoted²⁺) stream signal path, increase calcium ion (Ca²⁺) concentration.And various concentration is being added After LRH7-G3 small peptide, calcium ion (Ca can be significantly reduced²⁺) concentration, inhibit chemerin to calcium (Ca²⁺) stream signal path Activation.But in the 293T cell that wild type does not express GPR1, chemerin is to calcium (Ca²⁺) signal path is flowed without activation work With LRH7-G3 is to calcium (Ca²⁺) stream signal path is also without function and effect, by the experiment it can be concluded that chemerin can lead to It crosses in conjunction with receptor GPR1 and activates calcium (Ca²⁺) stream signal path, and the inhibition that LRH7-G3 small peptide can be specific Chemerin/GPR1 signal path reduces calcium ion (Ca²⁺) concentration.^*P<0.05,^*P<0.01,^{* *}P < 0.001 with Chemerin group compares.

Embodiment 4LRH7-G3 can significantly inhibit the proliferation of human breast cancer cell MDA-MB-231.

1. by human breast cancer cell MDA-MB-231 with 5 X 10³A/hole is inoculated in 96 porocyte culture plates, every Kong Pei Supporting matrix product is 200 μ L, is cultivated for 24 hours, then overnight starvation；

2. the LRH7-G3 polypeptide of various concentration gradient (100 μM, 10 μM, 1 μM, 0.1 μM, 0.01 μM, 0.001 μM) is added It cultivates 24 hours, 48 hours, 72 hours respectively；

3. the MTT working solution of 20 μ L is added in every hole, continues to be put into carbon dioxide incubator and cultivate 4 hours；

4. abandoning the supernatant in culture plate, 150 μ L DMSO (dimethyl sulfoxide) are added, shakes 10 minutes, is selected in microplate reader It selects 490nm wavelength to be detected, draws the growth curve of cell.

Inventor laboratory finds breast cancer cell MDA-MB-231 high in previous work, through q-PCR testing inspection Express GPR1.Fig. 6 shows that the LRH7-G3 small peptide of various concentration can significantly inhibit the increasing of breast cancer cell MDA-MB-231 cell It grows, and with the increase of action time, small peptide inhibits the effect of breast cancer cell MDA-MB-231 cell Proliferation also more aobvious It writes.

Embodiment 6LRH7-G3 can block human breast cancer cell MDA-MB-231 cell cycle progression.

1. by human breast cancer cell MDA-MB-231 with 5 X 10⁵A/hole is inoculated in 6 porocyte culture plates, every hole culture Matrix product is 1mL, is cultivated for 24 hours, then overnight starvation；

2. the LRH7-G3 polypeptide that various concentration gradient (1 μM, 0.1 μM, 0.01 μM) is added cultivate respectively 24 hours, it is 48 small When, 72 hours；

3. spare in careful collection cell culture fluid a to centrifuge tube.With trypsin digestion cell, until cell can be by gently When being blown and beaten with pipette or pipette tips, the cell culture fluid that addition front is collected, all attached cells under piping and druming, and gently Dispel cell.It is collected into centrifuge tube again.1000g or so is centrifuged 3-5 minutes, sedimentation cell；

4. carefully absorbing supernatant, about 50 microlitres or so of culture solution can be remained, to avoid cell is siphoned away.About 1 milli is added The PBS of ice bath pre-cooling is risen, cell is resuspended, and be transferred in 1.5 milliliters of centrifuge tubes.Centrifugation cell again, it is careful to absorb Clearly, about 50 microlitres or so of PBS can be remained, to avoid cell is siphoned away.Gently attack centrifuge tube bottom is kept away with appropriate cell dispersion It is agglomerating to exempt from cell；

5. 1 milliliter of ice bath is added to be pre-chilled in 70% ethyl alcohol, gently piping and druming is mixed, 4 DEG C of fixed 12h.1000g or so centrifugation 3-5 minutes, sedimentation cell.It is careful to absorb supernatant, about 50 microlitres or so of 70% ethyl alcohol can be remained, to avoid cell is siphoned away. The PBS of about 1 milliliter of ice bath pre-cooling is added, cell is resuspended.Centrifugation cell again carefully absorbs supernatant, can remain about 50 Microlitre or so PBS, to avoid cell is siphoned away.Gently attack centrifuge tube bottom avoids cell agglomerating with appropriate cell dispersion；

6. 0.5 milliliter of propidium iodide stain liquid is added in every solencyte sample, it slowly and is sufficiently resuspended cell precipitation, 37 DEG C It is protected from light warm bath 30 minutes.Then storage can be protected from light with 4 DEG C or ice bath；

7. detecting red fluorescence at excitation wavelength 488nm wavelength with flow cytometer, while detection light scatters situation.Point Analyse cell results.

Fig. 7 is shown, is found by flow cytometry analysis, on the one hand with the raising of activity, LRH7-G3 small peptide It can be substantially reduced MDA-MB-231G2/M phase cell number, increase G0/G1 and S issue mesh, block cell cycle normal progression； On the other hand with the extension of action time, LRH7-G3 small peptide is significantly reduced G2/M phase cell number, increases G0/G1 the and S phase Number blocks cell cycle normal progression.In conclusion can be inferred that LRH7-G3 small peptide by blocking the cell cycle to press down The proliferation of MDA-MB-231 processed.^*P<0.05,^*P < 0.01 is compared with the control group G0/G1 phase；^#P < 0.005 and control group S phase It compares；^&P < 0.005 is compared with the control group G2/M phase.

Embodiment 7LRH7-G3 can promote human breast cancer cell MDA-MB-231 Apoptosis.

3. first supernatant is collected into centrifuge tube, then with without EDTA pancreatin carefully digest collection cell culture fluid to from Heart pipe.500g or so is centrifuged 5 minutes, sedimentation cell；

4. washing cell twice with the PBS of pre-cooling, 500g or so is centrifuged 5 minutes collection cells；

5. 1 X AnnexinV Binding Buffer of 100 μ L pre-cooling is added, cell is resuspended；

6. 5 μ L AnnexinV-FITC and 5 μ L PI are added, mix gently, room temperature is protected from light 15 minutes；

7. 1 X AnnexinV Binding Buffer of 400 μ L pre-cooling is added, mix gently, by sample as keeping away on ice Light is placed, and uses flow cytomery in 1h.Analysis detection result.

Fig. 8 is shown, is found by flow cytometry analysis, on the one hand with the raising of activity, LRH7-G3 small peptide Breast cancer cell MDA-MB-231 can be obviously promoted, apoptosis occurs, on the other hand with the extension of action time, LRH7-G3 is short Peptide also significantly increases breast cancer cell MDA-MB-231 apoptosis number.In conclusion LRH7-G3 small peptide can remarkably promote people Apoptosis occurs for breast cancer cell MDA-MB-231.^*P<0.05,^*The compared with the control group of P < 0.01.

SEQUENCE LISTING

<110>Shenzhen Xianjin Technology Academe

<120>a kind of GPR1 antagonism polypeptide and its derivative and application

<130> CP11701241C

<160> 10

<170> PatentIn version 3.3

<210> 1

<211> 7

<212> PRT

<213>artificial sequence

<400> 1

Gly Pro Leu Ile Trp Phe Ser

1 5

<210> 2

<211> 5

<212> PRT

<213>artificial sequence

<220>

<221> MISC_FEATURE

<222> (3)..(3)

It<223>is leucine

<400> 2

Pro Leu Xaa Trp Phe

1 5

<210> 3

<211> 7

<212> PRT

<213>bacterium or tobacco

<400> 3

Gly Pro Leu Ile Trp Phe Ser

1 5

<210> 4

<211> 7

<212> PRT

<213>bacterium or tobacco

<400> 4

Gly Pro Leu Ile Trp Phe Ser

1 5

<210> 5

<211> 6

<212> PRT

<213>bacterium or shellfish

<400> 5

Gly Pro Leu Ile Trp Phe

1 5

<210> 6

<211> 6

<212> PRT

<213>bacterium or shellfish

<400> 6

Gly Pro Leu Ile Trp Phe

1 5

<210> 7

<211> 6

<212> PRT

<213>honeybee, balsamine, humpback, moth class, olive or fungi

<400> 7

Pro Leu Ile Trp Phe Ser

1 5

<210> 8

<211> 6

<212> PRT

<213>honeybee, balsamine, humpback, moth class, olive or fungi

<400> 8

Pro Leu Ile Trp Phe Ser

1 5

<210> 9

<211> 20

<212> DNA

<213>artificial sequence

<400> 9

aatgccatcg tcatttggtt 20

<210> 10

<211> 20

<212> DNA

<213>artificial sequence

<400> 10

caactgggca gtgaaggaat 20

Claims

1. a kind of GPR1 antagonism polypeptide, which is characterized in that its amino acid sequence is Gly- (D) Pro-Leu-Ile-Trp-Phe- Ser-NH₂SEQ ID No.1。

2. a kind of bioactive fragment or the like of GPR1 antagonism polypeptide, as shown in SEQ ID No.2-8:

PLX₁WF SEQ ID No.2, in which:

⑴X₁For leucine (L)；

GPLIWFS SEQ ID No.3

GPLIWFS-NH₂ SEQ ID No.4

GPLIWF SEQ ID No.5

GPLIWF-NH₂ SEQ ID No.6

PLIWFS SEQ ID No.7

PLIWFS-NH₂ SEQ ID No.8。

3. the bioactive fragment of GPR1 antagonism polypeptide described in claim 1 or GPR1 antagonism polypeptide as claimed in claim 2 Or the like derivative, it is characterised in that:

The derivative is on the amino acid side groups of GPR1 antagonism polypeptide or its bioactive fragment analog living, ammonia Obtained product is routinely modified in cardinal extremity or c-terminus progress, or is connection for polypeptide or Protein Detection or the label of purifying institute Obtained product；

Preferably, the routine be modified to amination, amidation, hydroxylating, carboxylated, carbonylation, alkylation, acetylation, Phosphorylation, esterification, glycosylation, cyclisation, biotinylation, fluorophor modification, polyethylene glycol PEG modification or immobilization modification；Institute The label stated is His₆, GST, EGFP, MBP, Nus, HA, IgG, FLAG, c-Myc or ProfinityeXact.

4. a kind of polynucleotide can encode any one of the SEQ ID No.1-8 polypeptide.

5. a kind of carrier, it comprises any bar polynucleotides described in claim 4, and by gene technology means with Promoter sequence link.

6. a kind of host cell has transfected carrier described in claim 5.

7. GPR1 receptor antagonist polypeptide described in claim 1, bioactive fragment as claimed in claim 2 or the like and Purposes of the derivative as claimed in claim 3 in treatment chemerin-GPR1 is disease mediated；

Preferably, the chemerin-GPR1 is disease mediated is selected from breast cancer, fatty liver, diabetes, inflammatory reaction, more capsule ovum Nest syndrome.

8. GPR1 receptor antagonist polypeptide described in claim 1, bioactive fragment as claimed in claim 2 or the like and Purposes in the reduced drug of cAMP concentration caused by derivative as claimed in claim 3 inhibits chemerin in preparation.

9. GPR1 receptor antagonist polypeptide described in claim 1, bioactive fragment as claimed in claim 2 or the like and Calcium (Ca caused by derivative as claimed in claim 3 inhibits chemerin in preparation²⁺) effect of interior stream drug in purposes.

10. GPR1 receptor antagonist polypeptide described in claim 1, bioactive fragment as claimed in claim 2 or the like with And the purposes in the drug of derivative as claimed in claim 3 cell chemotaxis effect caused by preparation inhibition chemerin.

11. a kind of pharmaceutical composition, wherein including GPR1 receptor antagonist polypeptide described in claim 1, as claimed in claim 2 Bioactive fragment or the like and one of derivative as claimed in claim 3 or a variety of, as active constituent；

Preferably, the pharmaceutical composition can contain a kind of or a variety of pharmaceutically acceptable carriers；

The pharmaceutically acceptable carrier be preferably diluent, excipient, filler, adhesive, wetting agent, disintegrating agent, Sorbefacient, absorption carrier, surfactant or lubricant etc.；

The diversified forms such as tablet, granula, capsule, oral solution or injection can be further made in the pharmaceutical composition, respectively The drug of kind dosage form can be prepared according to the conventional method of pharmaceutical field.