CN103923174A - Cyr61/CCN1 protein antigenic epitope polypeptide, inhibitor and application thereof as well as monoclonal antibody - Google Patents
Cyr61/CCN1 protein antigenic epitope polypeptide, inhibitor and application thereof as well as monoclonal antibody Download PDFInfo
- Publication number
- CN103923174A CN103923174A CN201410152740.7A CN201410152740A CN103923174A CN 103923174 A CN103923174 A CN 103923174A CN 201410152740 A CN201410152740 A CN 201410152740A CN 103923174 A CN103923174 A CN 103923174A
- Authority
- CN
- China
- Prior art keywords
- cyr61
- ccn1
- protein epitopes
- polypeptide
- ccn1 protein
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/08—Peptides having 5 to 11 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Chemical & Material Sciences (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses a Cyr61/CCN1 protein antigenic epitope polypeptide and an application thereof in the preparation of medicines for detecting or treating rheumatoid arthritis synovial cell proliferation, psoriasis epidermic cell proliferation and inflammation diseases. The amino acid sequence of the Cyr61/CCN1 protein antigenic epitope polypeptide is as shown in SEQ ID No. 1. and SEQ No.2. The invention also discloses a specific anti-Cyr61/CCN1 monoclonal antibody combined with the Cyr61/CCN1 protein antigenic epitope polypeptide and a Cyr61/CCN1 protein antigenic epitope polypeptide inhibitor. The Cyr61/CCN1 protein antigenic epitope polypeptide and novel medicines related to the Cyr61/CCN1 protein antigenic epitope polypeptide have wide application prospect.
Description
Technical field
The invention belongs to field of medicaments, relate to Cyr61/CCN1 Protein Epitopes polypeptide with and preparation detect or the medicine for the treatment of quasi-wind gateway (RA) synovial cell (FLS) hyperplasia, psoriasis epidermis hyperplasia and inflammatory disease in purposes.
Background technology
Cyr61 (cysteine-rich61), be called again CCN1, that a kind of relative molecular mass by 381 amino-acid residues is the secretory protein of 42kDa, the Cyr61/CCN1 being cloned is the earliest that Lau in 1985 etc. find in the time stimulating mouse BALB/c3T3 inoblast with serum or Thr6 PDGF BB (PDGF), is named as Cyr61/CCN1 because being rich in halfcystine (containing 10% cysteine residues).Cyr61/CCN1 has obvious mitogenic activity and chemotaxis, can induce inoblast and epidermal cell proliferation and extracellular matrix secretion, participates in regulating hyperplasia, differentiation, fetal development to form, and is the basic albumen of vital movement.
In the association reaction of antigen-antibody, the position of antibody participation combination claims the contraposition (paratope) of antibody, and antigen (Cyr61/CCN1 albumen) participates in the epi-position (epitope) that the position of combination is called antigen.Epi-position is the basis of proteantigen.Determining of epitope (small-molecular peptides section) is that small-molecular peptides section and then design adopt micromolecular compound that this small-molecular peptides section and simulated albumin effect or design can simulate this peptide section for replacing effect or the design micromolecular compound of albumen or peptide molecule significant to block this albumen effect for the action site of clear and definite albumen, and this is also one of the development of current popular newtype drug in the world and preparation method.
Summary of the invention
The invention provides Cyr61/CCN1 Protein Epitopes polypeptide, its aminoacid sequence, as shown in SEQ ID NO.1, is GLECNFG.
The present invention also provides Cyr61/CCN1 Protein Epitopes polypeptide, it is the cyclic peptide section as shown in SEQ ID NO.2, add one and half Guang amino acid at the N of the peptide section of SEQ ID NO.1 end, thereby form the cyclic peptide section " C-G LECNF G " as shown in SEQ ID NO.2.
Cyr61/CCN1 Protein Epitopes polypeptide of the present invention is the 75-81 amino acids that is positioned at Cyr61/CCN1 albumen, can be with specificity resisting Cyr 61/CCN1 monoclonal antibody 093G9 in conjunction with (KD is 10-7) and in vivo and in vitro and Cyr61/CCN1 function, in the time that N end adds a C, can be cyclic peptide section " C-G LECNF G " through synthetic.
In the present invention, Cyr61/CCN1 Protein Epitopes polypeptide can prepare by protease hydrolysis, Cyr61/CCN1 Protein Epitopes polypeptide can also be by conventional solid-phase peptide synthesis synthetic or genetic engineering technique prepare.
In the present invention, according to the people Cyr61/CCN1 albumen full length sequence (GenBank:M_001554) of gene pool retrieval gained, as shown in SEQ ID NO.3, for:
Wherein, in black surround, sequence is the signal peptide of Cyr61/CCN1 albumen.
The present invention also provides specificity resisting Cyr 61/CCN1 monoclonal antibody, and described specificity resisting Cyr 61/CCN1 monoclonal antibody can be combined with Cyr61/CCN1 Protein Epitopes polypeptide of the present invention.
The present invention also provides the inhibitor of Cyr61/CCN1 Protein Epitopes polypeptide, and it can be combined with Cyr61/CCN1 Protein Epitopes polypeptide of the present invention, can be combined with GLECNFG ring texture, also can block the biological function of mediation thus.
The inhibitor of Cyr61/CCN1 Protein Epitopes polypeptide of the present invention, comprises the micromolecular compound that can block Cyr61/CCN1 Protein Epitopes polypeptide biological function.Preferably, micromolecular compound SC1-SC15 as shown in the embodiment of the invention.Particularly, can design the micromolecular compound that there is simulation and suppress this peptide section biological function according to the peptide section conformational epitope of Cyr61/CCN1 Protein Epitopes polypeptide.The micromolecular compound that can block this polypeptide peptide section has the biological effect of the Cyr61/CCN1 molecular pathology of inhibition, and this becomes lead compound and all have important use at the medicine of the anti-synovial hyperplasia of preparation and osteoclasia, resisting Cyr 61/CCN1 high expression level Cells Proliferation of Human Breast Cancer, anti-epidermic cell hyperplasia and anti-fibrosis (as hepatic fibrosis) for further developing.Inhibitor of the present invention can combine with GLECNFG ring texture, also can block the pathological effect (comprise and cause inflammation, cause proliferation of fibroblast, cause Cyr61/CCN1 high expression level breast cancer cell hyperplasia and transfer, cause epidermal cell proliferation and activation etc.) of mediation thus.
Research before the present invention shows, IL-17 can raise the expression of Cyr61/CCN1, and up-regulated expression Cyr61/CCN1 can be by showing that with synovial cell integrin receptor AvB5 is combined the rear synovial cell's of promotion hyperplasia, and can promote the synthetic of metalloprotease 3 and MMP-9 and participate in cartilage and osteoclasia.The present invention studies and shows, Cyr61/CCN1 albumen can directly promote synovial cell to produce IL-6, develops thereby participate in inflammation.Further, the present invention's research shows that Cyr61/CCN1 can also promote human epidermal hyperplasia, participates in psoriatic and occurs.In experimentation on animals of the present invention, also find that Cyr61/CCN1 can mediate sjogren syndrome, Accretive Type inflammatory bowel occurs.The present invention's experiment has confirmed that neutralizing this protein expression with resisting Cyr 61/CCN1 monoclonal antibody can suppress synovial cell proliferation and IL-6 generation, alleviate arthritic clinical symptom and disorganization and inflammation in CIA model; Suppress epidermal cell proliferation, the pachyderma and the scales of skin that peel off that alleviate psoriasiform mouse produce, alleviate inflammation and the clinical symptom of sjogren syndrome and Accretive Type enteropathy animal model.
The present invention prepares can be in conjunction with the monoclonal antibody of Cyr61/CCN1 albumen.These antibody show the difference of function, and its reason is the epitope of these different monoclonal antibodies institute combinations, and namely the different sites of Cyr61/CCN1 albumen causes.
The present invention also provides Cyr61/CCN1 Protein Epitopes polypeptide to detect and/or treat the application in diseases associated with inflammation medicine in preparation.Preferably, described diseases associated with inflammation comprises rheumatism, rheumatoid arthritis, psoriatic.Preferably, described diseases associated with inflammation comprises sjogren syndrome.Preferably, described diseases associated with inflammation comprises Accretive Type inflammatory bowel.
The present invention also provides the application of Cyr61/CCN1 Protein Epitopes polypeptide in the medicine of the anti-quasi-wind gateway synovial cell proliferation of preparation.Embodiments of the invention show, the monoclonal antibody 093G9 that can specificity neutralizes this epitope suppresses inflammation and the symptom of the epidermal cell proliferation of psoriatic and animal model thereof, the symptom that suppresses sjogren syndrome animal model and inflammation, inhibition Accretive Type inflammatory bowel animal model in vivo with the external synovial cell proliferation that can suppress quasi-wind gateway and animal model thereof.
The present invention also provides the application of Cyr61/CCN1 Protein Epitopes polypeptide in the medicine of preparation inhibition breast cancer cell high expression level.The embodiment of the present invention shows, in can specificity and the monoclonal antibody 093G9 of Cyr61/CCN1 Protein Epitopes in vivo with external Cells Proliferation of Human Breast Cancer, the migration and invasion that can suppress high expression level Cyr61/CCN1 albumen, in animal model (in body), can suppress tumor growth and the lymphatic metastasis of the type.
The present invention also provides Cyr61/CCN1 Protein Epitopes polypeptide in the application of preparing in anti-fibrosis medicine.
The present invention also provides the application of Cyr61/CCN1 Protein Epitopes polypeptide in the antipsoriatic medicine of preparation.The embodiment of the present invention shows, the monoclonal antibody 093G9 that can specificity neutralizes this epitope produces with external pachyderma and the scales of skin that peel off that can suppress epidermal cell proliferation, psoriasiform mouse in vivo.
The present invention also provides the inhibitor of Cyr61/CCN1 Protein Epitopes polypeptide to treat the application in rheumatism medicine in preparation.
Being positioned at epitope on Cyr61/CCN1 albumen for the present invention carries out functional analysis and shows, the cyclic peptide of the identification polypeptide section of antibody 093G9 has the effect that stimulates quasi-wind gateway synovial cell in-vitro multiplication, and the identification epi-position of control antibodies 096B7 does not have such characteristic, as shown in Figure 1.
By the artificial section of synthesized peptide of obtained antibody identification meter position, these peptide sections are added and in cell cultures, observe its biological characteristics.Found that some peptide section can suppress synovial cell and Cyr61/CCN1 high expression level breast carcinoma cell strain hyperplasia in vitro, shows that it has the effect of simulation resisting Cyr 61/CCN1 monoclonal antibody.Therefore, the compound small molecules of these peptide sections and with it combination, all has the effect that suppresses Cyr61/CCN1 albumen, has the potential of pharmacy.
Beneficial effect of the present invention also comprises the application of Cyr61/CCN1 Protein Epitopes polypeptide in the novel drugs of research and development treatment rheumatoid arthritis, psoriatic, sjogren syndrome, Accretive Type inflammatory bowel.The sickness rate of rheumatoid arthritis in China up to 0.4-1%; The sickness rate of psoriatic in Chinese is up to 0.3-0.5%, and the sickness rate in white people is higher (if U.S.'s sickness rate is 1-3%; Norway's sickness rate is up to 7%), visible, Cyr61/CCN1 Protein Epitopes polypeptide of the present invention and the novel drugs according to the development of this epi-position will have broad prospect of application.
The meaning of peptide section G LECNF G of the present invention (114, cyclic peptide is 111) epi-position: according to " Trends Biochem Sci.2008October; 33 (10): 461-473 " report sequence: all contain such three conserved amino acids of G L C in the 75-81 amino acids in the IGFBP region of CCN family molecule sequence.
In above-mentioned, be coated with 7 conserved sequences that amino acid is this family molecule (CCN1-6) of color part, still, 093G9 can only with Cyr61/CCN1 combination.Although it is upper that G LECNF G is only positioned at Cyr61/CCN1, is the specific recognition epi-position of 093G9, wherein conservative sequence G-L-C has in all CCN family.The experimental result of not being combined with line peptide G LECNF G, being only combined with cyclic peptide * C G LECNF G according to 093G9, although prompting cyclic peptide C-G-L-C forms conformational epitope and may be arranged in CCN family molecule, G LECNF G is the specificity epitope with pathogenic effects.
The biological significance of epi-position of the present invention comprises:
Cyclic peptide 111 can stimulate the in-vitro multiplication of RA patient synovial cell and Cyr61/CCN1 high expression level mammary cancer MDA-MB-231 cell, therefore, and the pathogenic epi-position of specificity that this peptide section is Cyr61/CCN1 molecule.
The epi-position of monoclonal antibody 093G9 in can the peptide section (IGFBP) of specific recognition Cyr61/CCN1, the antibody 093G9 that blocks this epi-position has blocking-up Cyr61/CCN1 and causes the effect of synovial cell, high expression level mammary cancer MDA-MB-231 cell, other fibroblasts propagation.
Can design the chemical small molecules (SC) that there is simulation and suppress this peptide section biological function according to this peptide section conformational epitope.Wherein, can block this peptide section chemistry small molecules (SC) and there is the biological effect of the Cyr61/CCN1 molecular pathology of inhibition.This becomes lead compound and in the medicine of the anti-synovial hyperplasia of preparation and osteoclasia, resisting Cyr 61/CCN1 high expression level Cells Proliferation of Human Breast Cancer and anti-fibrosis (as hepatic fibrosis), has important purposes for further developing.
Owing to being rich in halfcystine in Cyr61/CCN1 albumen, easily form disulfide linkage, therefore, adopt gene engineering method to express very difficult, and this peptide section is easily carried out synthetic, therefore can replace Cyr61/CCN1 molecular immune to obtain the antiserum(antisera) of anti-this functional epitope, or prepare corresponding monoclonal antibody, for the further application of clinical detection and treatment.
The present invention is intended to protect the sequence of polypeptide 111, by the issuable three-dimensional arrangement of this sequence (comprising linear structure and ring texture), and combined with it and produce same function (produce antibody as promoted after synovial cell's propagation, immunity and can suppress collagen-induced sacroiliitis (CIA) mouse inflammatory effect etc.) or directly suppress the micromolecular compound of these peptide section functions.
Brief description of the drawings
Fig. 1 represents simulation conformation schematic diagram and the chemical structure of polypeptide of the present invention, wherein, (A) represents the polypeptide 114 of linear conformation; (B) polypeptide 111 of expression cyclic conformation; (C) chemical structure of expression polypeptide 111.
Fig. 2 represents that polypeptide of the present invention stimulates the external value-added experimental result of RA synovial cell.
Fig. 3 represents that polypeptide of the present invention stimulates the experimental result of MDA-MB-231 cel l proliferation.
Fig. 4 represents the inhibition of the synovial cell propagation of micromolecular compound of the present invention to the stimulation of peptide section, wherein, and the obvious inhibition of synovial cell's propagation that (A) 15 chemical small molecules of expression (SC1-15) stimulate peptide section 111 respectively; (B) represent 15 chemical small molecules (SC1-15) obvious inhibition of the synovial cell's propagation to the stimulation of Cyr61/CCN1 albumen respectively.
Fig. 5 represents the inhibition of the breast carcinoma cell strain MDA-MB-231 propagation of micromolecular compound of the present invention to the stimulation of peptide section, wherein, (A) represent the obvious inhibition of 15 chemical small molecules (SC1-15) breast carcinoma cell strain MDA-MB-231 in-vitro multiplication that peptide section 111 is stimulated; (B) represent 15 chemical small molecules (SC1-15) obvious inhibition of the breast carcinoma cell strain MDA-MB-231 in-vitro multiplication to the stimulation of Cyr61/CCN1 albumen respectively.
Embodiment
In conjunction with following specific embodiments and the drawings, the present invention is described in further detail, and protection content of the present invention is not limited to following examples.Do not deviating under the spirit and scope of inventive concept, variation and advantage that those skilled in the art can expect are all included in the present invention, and taking appending claims as protection domain.Implement process of the present invention, condition, reagent, experimental technique etc., except the content of mentioning specially below, be universal knowledege and the common practise of this area, the present invention is not particularly limited content.
The screening of embodiment 1 Cyr61/CCN1 Protein Epitopes of the present invention polypeptide
In the present invention, employing immunoblotting screening: adopted improved tradition to measure epitope method, by a series of little peptide section of genetically engineered partitioned representation Cyr61/CCN1, then carry out Western blot (Western Blot) hybridization and screening in conjunction with epi-position with this 2 strain antibody, thus obtain can with the peptide section of 2 strain Cyr61/CCN1 monoclonal antibody combinations.
Divide the little peptide section into about 25 amino acid tablet segment length according to existing result of study by Cyr61/CCN1 albumen, then designing primer enters the gene clone of these fragments in intestinal bacteria, abduction delivering after expressing order-checking correctly, after PAGE electrophoresis, go on nitrocellulose membrane, then use respectively mouse-anti people Cyr61/CCN1 monoclonal antibody specific 093G9 (CGMCC No.
3351)with mouse-anti people Cyr61/CCN1 monoclonal antibody specific 096B7 (CGMCC No.
3299)hybridize and develop the color, observing in conjunction with situation.Can be in conjunction with the positive that develops the color, otherwise negative.Judge thus the combination epi-position of these two kinds of antibody.By aforesaid method, the epitope that filters out antibody 093G9 combination is positioned at the epi-position of the 75-81 amino acids of Cyr61/CCN1: Cyr61/CCN1
75-81gLECNFG.The position (position that bold mark) of this epi-position in Cyr61/CCN1 sequence:
Synthesizing of embodiment 2 Cyr61/CCN1 Protein Epitopes polypeptide
In the present embodiment, Cyr61/CCN1 Protein Epitopes polypeptide is synthetic is synthetic by Shanghai Qiang Yao company.The full-automatic solid-phase synthesis of ultimate principle.Its primary process is as follows: based on Fmoc chemosynthesis, first by will be synthetic the carboxyl of C-terminal amino acid of target polypeptides be connected with an insoluble macromolecule resin with covalent linkage form, then using this amino acid whose amino as the synthetic starting point of polypeptide, the carboxyl effect activated with other amino acid forms peptide bond, constantly repeat this process, can obtain polypeptide.The peptide purification of synthesized adopts HPLC purifying, and purity reaches more than 95%.The combination epi-position obtaining according to screening, has synthesized respectively linearity and ring type polypeptide, and wherein, the polypeptide of synthesized is respectively: linear polypeptide (numbering 114), and its sequence is GLECNFG; Ring type polypeptide (numbering 111), its sequence is C GLECNFG.
Conformation and the chemical structure of embodiment 3 Cyr61/CCN1 Protein Epitopes polypeptide
Adopt Cassion molecular simulation computer software, Cyr61/CCN1 Protein Epitopes peptide section of the present invention is carried out to matching in pure water solution, with the conformation that obtains may forming the most in may solution environmental in vivo, obtain the molecular structure with stereoscopy effect.As Fig. 1 (a) with the three-dimensional conformation of the Cyr61/CCN1 Protein Epitopes polypeptide (b), wherein, Fig. 1 (a) represents polypeptide 114, is linear conformation; Fig. 1 (b) represents polypeptide 111, is cyclic conformation; Fig. 1 (c) represents the chemical structure of polypeptide 111.
Embodiment 4 Cyr61/CCN1 Protein Epitopes polypeptide stimulate synovial cell's in-vitro multiplication
Quasi-wind gateway synovial cell cultivates and proliferation experiment
Clinical sample: in research, all sample standard deviations come from the RA patient of the capable knee prosthesis of clinical orthopaedics or villusectomy, and the diagnosis of all cases meets international Case definition.Patient synovial tissue is for the vitro culture of cell.In this research, clinical sample used, all knows the inside story and informs patient.
Synovial cell's preparation and former culture: by the aseptic synovial tissue that obtains, after PBS cleans, use the fragment of aseptic operation scissor cut into about 1mm × 1mm × 1mm; With collagenase I or the collagenase II of 2-3 times of volume 0.5mg/ml, 37 DEG C, digest 2 hours; After 200 order gauzes filter, centrifugal going after supernatant, is resuspended in DMEM nutrient solution by cell, is placed in culture dish, and 37 DEG C, 5%CO
2cultivate.Afterwards in the time that FLS grows > 80% in blocks, had digestive transfer culture.Micro-Microscopic observation, rapidly, cellular form trends towards spindle shape in the RA FLS growth in 3-5 generation, marshalling, orientation is consistent.With specificity lymphocyte differentiation antigen as CD3, CD14, CD19, after the marks such as CD11C, detect and find negative (the < 2%CD1Ib of above-mentioned developed by molecule with flow cytometer, < 2%CD14+, < 1%CD3+, and < 5%CD19+), show the FLS that institute's cultured cells is homogeneous, rarely lymphocyte pollutes.
Synovial cell's proliferation experiment: the FLS in the vegetative period of taking the logarithm adjusts cell concn 1 × 10 after 0.25% tryptic digestion
4cells/ml, is inoculated in 96 porocyte culture plates, adds the above-mentioned peptide section (peptide section final concentration is: 2.5ug/ml, 5.0ug/ml10ug/ml) of different concns.Adopt Cyr61/CCN1 intact proteins as positive control (Proptech company, the U.S., San Diego) simultaneously.Also establish nutrient solution contrast.By peptide section or pure Cyr61/CCN1 albumen and FLS co-cultivation, within 16 hours before cultivation finishes, add the 3H (every hole) of 1 μ Ci, collecting cell, β liquid scintillation instrument detects propagation.
II Collagen Type VI inducing mouse sacroiliitis (CIA) model experiment:
Peptide section immunoprophylaxis CIA mouse generation sacroiliitis experiment: peptide section and CFA are mixed completely, DBA1 mouse back subcutaneous inoculation 3 times: for the first time: peptide+CFA, for the second time and for the third time: peptide+IFA (Freund's incomplete adjuvant), immunizing dose: 100ug peptide/mouse, back immunity 2-3 point, every minor tick 1 month, immune latter 7 days for the third time, afterbody blood sampling, detects whether produce antiserum(antisera) with ELISA; Add equivalent IFA immune mouse with CII100ug simultaneously, observe incidence, onset speed and the inflammation intensity of mouse.
The treatment of peptide section or impact CIA mouse: first prepare CIA mouse model, treat that inflammation reaches 4 timesharing peptide section and carries out mucosa delivery.Method: peptide section 2500ug/ml, 10ul/mouse, final concentration is 25ug/mouse/day.Adopt the negative contrast of solvent.
The above-mentioned peptide section of Cyr61/CCN1 Protein Epitopes polypeptide of the present invention is added respectively in the synovial cell of the former culture of quasi-wind gateway, after 48 hours, detect its stimulation ability for synovial cell's propagation, experimental result shows, peptide section 111 can obvious stimulation synovial cell proliferation, and be dose-dependently, as shown in Figure 2.This experimental result shows, is positioned at the peptide section 111 of the 75-81 position of Cyr61/CCN1, and the cyclic conformation that may form in the aqueous solution can be simulated Cyr61/CCN1 albumen stimulates the effect of synovial cell's propagation.
Embodiment 5Cyr61/CCN1 Protein Epitopes polypeptide stimulates Cyr61/CCN1 high expression level breast carcinoma cell strain (MDA-MB-231) in-vitro multiplication
By 1 × 10
4individual MDA-MB-231 cell is seeded in 96 orifice plates, then adds the peptide section of different concns (2.5ug/ml, 5ug/ml, 10ug/ml), after 4 hours, adds 3H-TdR, continue to hatch 16 hours, collecting cell, Beta scintiloscope detects c.p.m. value, for judging proliferative activity.As shown in Figure 3, peptide section 111 has the effect of significantly stimulating cellular proliferation to result.
Embodiment 6 screenings obtain with cyclic peptide 111 combinations and suppress its active micromolecular compound (SC1-15)
According to peptide section conformational epitope (ring type polypeptide C GLECNFG, numbering 111) design has simulation and suppresses the chemical small molecules (SC) of this peptide section biological function, wherein, can block this peptide section chemistry small molecules (SC) and there is the biological effect of the Cyr61/CCN1 molecular pathology of inhibition.This becomes lead compound and in the medicine of the anti-synovial hyperplasia of preparation and osteoclasia, resisting Cyr 61/CCN1 high expression level Cells Proliferation of Human Breast Cancer and anti-fibrosis (as hepatic fibrosis), has important purposes for further developing.The present invention adopts virtual screening method conventional in developing new drug, filters out the chemical molecular that may have treatment effectiveness by computing method.At present widely used is the experiment that the target of medicine is utilized to X-ray diffraction or nucleus magnetic resonance (NMR), or uses the method for bioinformation structure prediction, obtains after the three-dimension high-resolution structure of target, then carries out virtual screening.
In the present embodiment, the successful important document of medicine virtual screening comprises: (1) enough large chemical spaces (chemical space), (2) score function (scoring function) accurately, (3) high efficiency search algorithm method (searching algorithm).Chemical space, i.e. chemline used, broad sense, chemical space comprises the configuration (conformation) that part (ligand) and acceptor may exist, score function, its design is mainly from protein structure database (Protein Databank, http://www.pdb.org) the middle combination (complex) of finding the good part of resolving power and acceptor, the calculating of uses energy, add the statistical method such as multiple linear regression (multivariate linear regression), set up a mathematical function accurately, and can fast and effeciently infer that whether stably unknown part to a given acceptor combination, even predict the free energy (binding free energy) of bonding strength (binding affinity) or combination.Above-mentioned score function is energy variation situation very complicated while doing translation (translation), rotation (rotation) or topographical variations (conformational changes) according to ligand molecular with respect to acceptor, finds the binding pattern with best score value.
The present embodiment can effectively simulated on the Research foundation of biological action of Cyr61/CCN1 albumen target ring type polypeptide C GLECNFG (numbering 111), screening obtains the lead compound of the target cyclic peptide 111 with structural solid, and the compound of these small molecule structures can and suppress its activity with cyclic peptide 111 combinations.According to the chemical structure of ring type polypeptide 111, adopting present method to screen can have 15 with the micromolecular compound SC of this ring type polypeptide combination, its structure and title SC-1 to SC-15 compound as shown in Table 1 below.Structure and the synthetic method of these micromolecular compounds SC are prior art.
Embodiment 7 can suppress the effect that stimulates synovial cell to breed by cyclic peptide 111 in vitro with chemical small molecules (SC) 1-15 of peptide section 111 combinations
In the present embodiment, clinical sample, synovial cell's preparation and former culture are all with identical described in embodiment 4.
Synovial cell's proliferation experiment: the FLS in the vegetative period of taking the logarithm adjusts cell concn 1 × 10 after 0.25% tryptic digestion
4cells/ml, is inoculated in 96 porocyte culture plates, adds respectively the peptide section 111 of 10ug/ml and the Cyr61/CCN1 albumen of 2.5ug/ml (Proptech company, the U.S., San Diego).Then add respectively the 10 μ M chemistry small molecules SC-1～SC-15 that obtain by embodiment 6 method screenings, refer to following table 1, co-cultivation 48 hours.Within 16 hours before cultivation finishes, add the 3H (every hole) of 1 μ Ci, collecting cell, β liquid scintillation instrument detects propagation, as shown in Figure 4,15 chemical small molecules (SC-1～SC-15) have obvious restraining effect to synovial cell's propagation of peptide section 111 (as shown in Figure 4 A) and Cyr61/CCN1 albumen (as shown in Figure 4 B) stimulation respectively to experimental result.
Embodiment 8 can suppress to be stimulated by cyclic peptide 111 and Cyr61/CCN1 with the chemical small molecules 1-15 of peptide section 111 combinations the effect of breast carcinoma cell strain MDA-MB-231 propagation in vitro
By 1 × 10
4individual MDA-MB-231 cell is seeded in 96 orifice plates, adds the peptide section 111 of 10ug/ml and the Cyr61/CCN1 albumen of 2.5ug/ml (Proptech company, the U.S., San Diego).Then add respectively the 10 μ M chemistry small molecules SC-1～SC-15 that obtain by embodiment 6 method screenings, refer to following table 1, co-cultivation adds the 3H (every hole) of 1 μ Ci after 4 hours, continue to hatch 16 hours, collecting cell, Beta scintiloscope detects c.p.m. value, for judging proliferative activity.As shown in Figure 5, described 15 chemical small molecules (SC1-15) have obvious restraining effect to the breast carcinoma cell strain MDA-MB-231 in-vitro multiplication of peptide section 111 (as shown in Figure 5A) and Cyr61/CCN1 albumen (as shown in Figure 5 B) stimulation to experimental result.
Claims (10)
1. a Cyr61/CCN1 Protein Epitopes polypeptide, is characterized in that, its aminoacid sequence is GLECNFG, as shown in SEQ ID NO.1.
2. a Cyr61/CCN1 Protein Epitopes polypeptide, it is characterized in that, its aminoacid sequence is C-G LECNF G, and as shown in SEQ ID NO.2, it is in the cyclic peptide section that the N end interpolation half Guang amino acid of Cyr61/CCN1 Protein Epitopes polypeptide forms as claimed in claim 1.
3. specificity resisting Cyr 61/CCN1 monoclonal antibody of being combined with Protein Epitopes polypeptide as described in any one of claim 1-2.
4. a Cyr61/CCN1 Protein Epitopes peptide inhibitor, is characterized in that, described inhibitor is in conjunction with the Protein Epitopes polypeptide as described in any one of claim 1-2 and suppress its activity.
5. Cyr61/CCN1 Protein Epitopes peptide inhibitor as claimed in claim 4, is characterized in that, described inhibitor is be combined with the Protein Epitopes polypeptide described in any one of claim 1-2 and suppress its active micromolecular compound.
6. the application of the Cyr61/CCN1 Protein Epitopes polypeptide as described in any one of claim 1-2 in preparation treatment diseases associated with inflammation medicine, wherein, described diseases associated with inflammation comprises rheumatism and/or psoriatic.
7. the application of the Cyr61/CCN1 Protein Epitopes polypeptide as described in any one of claim 1-2 in the medicine of the anti-quasi-wind gateway synovial cell proliferation of preparation.
8. the application of the Cyr61/CCN1 Protein Epitopes polypeptide as described in any one of claim 1-2 in the medicine of preparation inhibition Cells Proliferation of Human Breast Cancer.
9. the Cyr61/CCN1 Protein Epitopes polypeptide as described in any one of claim 1-2 is in the application of preparing in anti-fibrosis medicine.
10. the nucleotide sequence of coding Protein Epitopes polypeptide as described in any one of claim 1-2.
Priority Applications (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610897943.8A CN106492188B (en) | 2014-02-08 | 2014-04-16 | Cyr61/CCN1 protein epitope polypeptide, inhibitor and monoclonal antibody thereof, and application thereof |
| CN201911091007.8A CN111150832B (en) | 2014-02-08 | 2014-04-16 | Application of inhibitor SC-2 of Cyr61/CCN1 protein epitope polypeptide |
| CN201911090995.4A CN111068040B (en) | 2014-02-08 | 2014-04-16 | Application of inhibitor SC-4-SC-15 of Cyr61/CCN1 protein epitope polypeptide |
| CN201410152740.7A CN103923174B (en) | 2014-02-08 | 2014-04-16 | Cyr61/CCN1 protein antigenic epitope polypeptide, inhibitor and application thereof as well as monoclonal antibody |
| CN201911091016.7A CN111012895B (en) | 2014-02-08 | 2014-04-16 | Application of inhibitor SC-3 of Cyr61/CCN1 protein epitope polypeptide |
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410045817.0 | 2014-02-08 | ||
| CN201410045817 | 2014-02-08 | ||
| CN2014100458170 | 2014-02-08 | ||
| CN201410152740.7A CN103923174B (en) | 2014-02-08 | 2014-04-16 | Cyr61/CCN1 protein antigenic epitope polypeptide, inhibitor and application thereof as well as monoclonal antibody |
Related Child Applications (4)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911091016.7A Division CN111012895B (en) | 2014-02-08 | 2014-04-16 | Application of inhibitor SC-3 of Cyr61/CCN1 protein epitope polypeptide |
| CN201911091007.8A Division CN111150832B (en) | 2014-02-08 | 2014-04-16 | Application of inhibitor SC-2 of Cyr61/CCN1 protein epitope polypeptide |
| CN201610897943.8A Division CN106492188B (en) | 2014-02-08 | 2014-04-16 | Cyr61/CCN1 protein epitope polypeptide, inhibitor and monoclonal antibody thereof, and application thereof |
| CN201911090995.4A Division CN111068040B (en) | 2014-02-08 | 2014-04-16 | Application of inhibitor SC-4-SC-15 of Cyr61/CCN1 protein epitope polypeptide |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103923174A true CN103923174A (en) | 2014-07-16 |
| CN103923174B CN103923174B (en) | 2017-01-11 |
Family
ID=51141588
Family Applications (5)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911091007.8A Active CN111150832B (en) | 2014-02-08 | 2014-04-16 | Application of inhibitor SC-2 of Cyr61/CCN1 protein epitope polypeptide |
| CN201911091016.7A Active CN111012895B (en) | 2014-02-08 | 2014-04-16 | Application of inhibitor SC-3 of Cyr61/CCN1 protein epitope polypeptide |
| CN201911090995.4A Active CN111068040B (en) | 2014-02-08 | 2014-04-16 | Application of inhibitor SC-4-SC-15 of Cyr61/CCN1 protein epitope polypeptide |
| CN201610897943.8A Active CN106492188B (en) | 2014-02-08 | 2014-04-16 | Cyr61/CCN1 protein epitope polypeptide, inhibitor and monoclonal antibody thereof, and application thereof |
| CN201410152740.7A Active CN103923174B (en) | 2014-02-08 | 2014-04-16 | Cyr61/CCN1 protein antigenic epitope polypeptide, inhibitor and application thereof as well as monoclonal antibody |
Family Applications Before (4)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911091007.8A Active CN111150832B (en) | 2014-02-08 | 2014-04-16 | Application of inhibitor SC-2 of Cyr61/CCN1 protein epitope polypeptide |
| CN201911091016.7A Active CN111012895B (en) | 2014-02-08 | 2014-04-16 | Application of inhibitor SC-3 of Cyr61/CCN1 protein epitope polypeptide |
| CN201911090995.4A Active CN111068040B (en) | 2014-02-08 | 2014-04-16 | Application of inhibitor SC-4-SC-15 of Cyr61/CCN1 protein epitope polypeptide |
| CN201610897943.8A Active CN106492188B (en) | 2014-02-08 | 2014-04-16 | Cyr61/CCN1 protein epitope polypeptide, inhibitor and monoclonal antibody thereof, and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (5) | CN111150832B (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101709088A (en) * | 2009-12-04 | 2010-05-19 | 上海市免疫学研究所 | Monoclonal antibody for resisting Cyr61 protein and application thereof |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5326751A (en) * | 1992-06-26 | 1994-07-05 | Arizona Board Of Regents On Behalf Of The University Of Arizona | Enkephalin analogs |
| IL121134A0 (en) * | 1997-06-22 | 1997-11-20 | Yeda Res & Dev | Peptides and antiallergic compositions comprising them |
| CN101492495B (en) * | 2009-02-24 | 2011-08-31 | 中国农业科学院哈尔滨兽医研究所 | A group of antigen epitope polypeptide and uses thereof |
| TW201102086A (en) * | 2009-06-04 | 2011-01-16 | Hoffmann La Roche | Antibodies against human CCN1 and uses thereof |
| CN101708328A (en) * | 2009-11-06 | 2010-05-19 | 上海市免疫学研究所 | Pharmaceutical application of CYR61 protein |
| WO2013049830A2 (en) * | 2011-09-30 | 2013-04-04 | The Washington University | Tip-1 and grp-78 binding peptides and method of identifying peptide receptors |
-
2014
- 2014-04-16 CN CN201911091007.8A patent/CN111150832B/en active Active
- 2014-04-16 CN CN201911091016.7A patent/CN111012895B/en active Active
- 2014-04-16 CN CN201911090995.4A patent/CN111068040B/en active Active
- 2014-04-16 CN CN201610897943.8A patent/CN106492188B/en active Active
- 2014-04-16 CN CN201410152740.7A patent/CN103923174B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101709088A (en) * | 2009-12-04 | 2010-05-19 | 上海市免疫学研究所 | Monoclonal antibody for resisting Cyr61 protein and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| NCBI: "CID 10234652", 《PUBCHEM COMPOUND DATABASE》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111068040B (en) | 2023-03-07 |
| CN111068040A (en) | 2020-04-28 |
| CN111150832A (en) | 2020-05-15 |
| CN111012895B (en) | 2023-03-07 |
| CN103923174B (en) | 2017-01-11 |
| CN111012895A (en) | 2020-04-17 |
| CN106492188A (en) | 2017-03-15 |
| CN106492188B (en) | 2019-12-20 |
| CN111150832B (en) | 2023-03-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1699408A (en) | Peptide for high performance inhibition of angiogenesis and method for preparing same and use thereof | |
| CN105112383B (en) | Penetratin hPP5 and application thereof | |
| CN102099372A (en) | Anti-amyloid immunogenic compositions, methods and uses | |
| CN108676073B (en) | Anti-obesity decapeptide LLVVYPWTQR and application thereof | |
| CN108893449B (en) | Hybridoma cell, monoclonal antibody of anti-human cyclophilin A and application thereof | |
| CN111840530A (en) | Preparation method of Eimeria tenella recombinant polypeptide vaccine VNQS and application method thereof in chicken coccidiosis resistance | |
| CN116970058B (en) | Tumor neoantigen polypeptide aiming at TP53 gene R249S mutation and application thereof | |
| CN102901826A (en) | New potential human cytokine TMEM98 and uses thereof | |
| CN103923174B (en) | Cyr61/CCN1 protein antigenic epitope polypeptide, inhibitor and application thereof as well as monoclonal antibody | |
| ES2355265T3 (en) | A KIT FOR IMMUNE DETECTION OF NUROGLOBULIN LINKED TO ENZYME AND USE OF THE SAME. | |
| CN103626865A (en) | Secreted protein with chemotactic activity, as well as coding sequence and medicament usage thereof | |
| CN109652555A (en) | A kind of molecular marker of diagnosis and treatment malignant tumour | |
| CN102241776B (en) | RANKL-TNF (Receptor Activator of Nuclear Factor Kappa-B Ligand-Tumour Necrosis Factor) sample region fusion protein and preparation method and application thereof | |
| CN115850377A (en) | Tumor neoantigen polypeptide based on NRAS gene Q61K mutation and application thereof | |
| CN105646701B (en) | Recombinant human endostatin albumen of different aminoacids structure and its preparation method and application | |
| CN103728456A (en) | ELISA (enzyme linked immunosorbent assay) diagnostic kit for thymidine kinase (TK) | |
| CN102242125A (en) | BK channel blocker gene, recombinant vector thereof and cloning method thereof | |
| CN1712542B (en) | Screen and use for labelled proto-protein 18 of protein molecule related to hepatocellular carcinoma | |
| CN103204939A (en) | CD147-HAb18MAb compound crystal structure and application thereof | |
| CN116948004B (en) | Tumor new antigen polypeptide aiming at CTNNB1 gene H36P mutation and application thereof | |
| CN103374067A (en) | Method for preparing human epididymis protein 4 (HE4) recombinant protein in mammalian cell | |
| CN103642812B (en) | A kind of bolti antiapoptotic factors FasL gene and application thereof | |
| CN101200720B (en) | Mitochondrial membrane potential decrease related polynucleotide as well as coded polypeptide and uses thereof | |
| CN103613644B (en) | A kind of little peptide LTF and application thereof with antitumous effect | |
| CN110105454A (en) | A kind of antineoplastic amalgamation protein and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C41 | Transfer of patent application or patent right or utility model | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20160728 Address after: 201318 Shanghai city Pudong New Area Tianxiong road 588 Lane Shanghai International Medical Park Business Park Building No. 16 Applicant after: DiaSys Diagnostic Systems (Shanghai) Co., Ltd. Address before: 300457 No. 220, Dongting Road, Tianjin Development Zone, Binhai New Area, Tianjin, Tianjin international biological medicine joint research laboratory, 14 floor, N411 Applicant before: TIANJIN HAIJIN JIUHE BIOLOGICAL TECHNOLOGY CO., LTD. |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant |