CN108640972A - There is the small peptide and application thereof of affinity with VISTA - Google Patents
There is the small peptide and application thereof of affinity with VISTA Download PDFInfo
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- CN108640972A CN108640972A CN201810476306.2A CN201810476306A CN108640972A CN 108640972 A CN108640972 A CN 108640972A CN 201810476306 A CN201810476306 A CN 201810476306A CN 108640972 A CN108640972 A CN 108640972A
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
Abstract
The present invention is by building ELISA method screening model, provide the small peptide and application thereof that there is affinity with VISTA, the Percentage bound of the small peptide and the VISTA is all higher than 15%, application of the small peptide in developing VISTA target spots antitumor drug, treating the immunosupress class drug or anti-inflammatory drug of autoimmune disease, drug development for specific VISTA target spots inhibitor provides experimental basis, and the exploitation for VISTA target spots antitumor drug, the immunosupress class drug or anti-inflammatory drug for the treatment of autoimmune disease lays the foundation.
Description
Technical field
The present invention relates to chemical biology technical fields, more particularly to the small peptide and its use with VISTA with affinity
On the way.
Background technology
T cell activation mortifier immune globulin variable region structural domain (V-domain immunoglobulin
SuppressorofT-cell activation, VISTA) it is the negative sense immunologic test point that can inhibit T cell immune response.
VISTA is also known as PD-1H, C10orf54 and Dies1, is I type transmembrane protein, it is thin to be mainly expressed in hematopoietic cell, granulocyte and T
Born of the same parents, natural bind receptor be VSIG8 (V-Set and Immunoglobulin domain containing 8, VSIG8)/
VSIG3 (V-set and Ig domain-containingprotein 3, VSIG3).VISTA has pair of receptor and ligand
Weight function is expressed in tumor microenvironment and tumor-infiltrated lymph node and is increased, can inhibit response of the T cell to tumour antigen, presses down
T cell processed is divided into regulatory T cells (Treg), reduces the generation of interleukin 2 (IL-2), has immunosupress and is immunized
The function of adjusting, but VISTA can't have an impact the proliferation of B cell.
VISTA is related to the generation of metastatic melanoma, inductivity melanoma and age-dependent inflammation phenotype, and has
Have and is different from death protein 1 and ligand 1 (PD-1/PD-L1) signal path.In addition, VISTA is in autoimmune disease
With significant adjustment effect is also shown in organ transplant rejection's reaction, such as mouse system lupus erythematosus model and acute grafing
Object versus-host disease model.It is related to Cytotoxic T lymphocytes that VISTA is combined on mouse squamous cell tumor model
(cytotoxic T lymphocyte-associated antigen-4, CTLA-4) monoclonal antibody can effectively have a rest quiet
Phase T cell and the T cell transformation exhausted are divided into effector T cell, and clinical research confirmation, VISTA is in Human Gastric carcinoma, oral cavity squama
In shape cell cancer and non-small cell lung cancer express significantly increase, and using she wood treatment prostate cancer after VISTA expression
Can obviously it rise.
Therefore, there is the small peptide of affinity with VISTA albumen in the screening of albumen level, and obtaining can be with VISTA protein binding
Polypeptid acid sequence, exploitation specificity VISTA target spot inhibitor the problem of being those skilled in the art's urgent need to resolve.
Invention content
In view of this, the present invention provides the small peptide for having affinity with VISTA by building ELISA method screening model
And application thereof, the drug development for specificity VISTA target spot inhibitor provides experimental basis, be VISTA target spots antitumor drug,
The exploitation for treating the immunosupress class drug or anti-inflammatory drug of autoimmune disease lays the foundation.
To achieve the goals above, the present invention adopts the following technical scheme that：
Have the small peptide of affinity, the amino acid sequence of the small peptide as follows with VISTA：
Further, the Percentage bound of the small peptide and the VISTA is all higher than 15%.
Include the polypeptide variants of the small peptide, wherein the polypeptide variants are to carry out one or more repair to the small peptide
Decorations；The modification includes and human immunoglobulin(HIg) Fc segments or its variant fusion；With human serum albumins or its variant fusion；With
Tumour penetrating peptide merges；It is single-stranded or single domain antibody merges with tumor-targeting；Or with polyethylene glycol, Xtenylation,
PASylation or HESylation fusions.
Include the pharmaceutical composition of the small peptide, described pharmaceutical composition further include carrier compatible with the small peptide,
Treat the factor, excipient and diluent；Wherein pharmaceutical composition can be the solvent that each substance is directly mixed to get in the present invention
Compound；Can also be that each substance is dissolved in the water, by the way that obtained freeze-dried powder is lyophilized；
Wherein, excipient is glucose；Diluent is sodium chloride or sodium bicarbonate；Carrier is organic substance.
Include the pharmaceutical composition of the polypeptide variants, described pharmaceutical composition further includes being adapted with the polypeptide variants
Carrier, the treatment factor, excipient and diluent；Wherein, excipient is glucose；Diluent is sodium chloride or sodium bicarbonate；
Carrier is organic substance；Wherein pharmaceutical composition can be the solvated compounds that each substance is directly mixed to get in the present invention；
Can be that each substance is dissolved in the water, by the way that obtained freeze-dried powder is lyophilized.
The application of the small peptide, the small peptide are developing VISTA target spots antitumor drug, are treating autoimmune disease
It is applied in immunosupress class drug or anti-inflammatory drug.
Wherein, the polypeptide variants are in the immune suppression developed VISTA target spots antitumor drug, treat autoimmune disease
Application in class drug or anti-inflammatory drug processed
Further, the tumour includes fibrosarcoma, mucous membrane sarcoma, embryonal-cell lipoma, chondrosarcoma, osteosarcoma, notochord
It is tumor, angiosarcoma, endotheliosarcoma, lymphangioendothelial sarcoma, lymphangioendothelial sarcoma, synovial sarcoma, celiothelioma, Ewing' s tumor, smooth
Muscle tumor, rhabdomyosarcoma, colon cancer, cancer of pancreas, syringocarcinoma, carcinoma of sebaceous glands, papillary carcinoma, papillary adenocarcinoma, bronchiolar carcinoma,
Cephaloma, clear-cell carcinoma, kidney mother cell cancer, kidney, liver cancer, cholangiocarcinoma, choriocarcinoma, spermatogonium cancer, embryonal carcinoma, uterine neck
Cancer, orchioncus, lung cancer, Small Cell Lung Cancer, carcinoma of urinary bladder, epithelioma, glioma, astrocytoma, medulloblastoma, craniopharyhgeal canal
Tumor, ependymoma, pinealoma, hemangioblastoma, acoustic neurinoma, meningioma, oligodendroglioma, melanoma, god
Through blastoma, retinoblastoma, hematologic cancers, human primary gastrointestinal cancers, cancer of the esophagus or urinary system cancer；
The autoimmune disease includes lupus erythematosus or psoriasis；
The inflammation includes asthma, arthritis or organ-graft refection.
The screening model construction method of the small peptide, includes the following steps：
(1) VISTA fusion proteins are prepared in expression；
(2) competitive ELISA method is utilized, the affinity molecule of VISTA albumen is screened；
A VISTA albumen is coated on by physisorption on ELISA Plate, overnight by being coated with buffer solution；
B discards coating protein liquid, is cleaned using washing buffer, and confining liquid is added afterwards, is discarded after room temperature Seal treatment 2h
Confining liquid is cleaned using the washing buffer, the nonspecific binding site that removal board bottom is not saturated by albumen；
C is by sample solution and VISTA specific antibodies 1:9 are hybridly prepared into reaction solution addition, are discarded after reacting at room temperature 2h
Reaction solution, and cleaned using the washing buffer；
D is added secondary antibody and is incubated at room temperature 1h, and TMB developing solutions dark place is added after cleaning and is incubated 9min, is eventually adding terminate liquid inspection
Survey absorbance value.
Wherein, step (1) expression prepares the specific methods of VISTA fusion proteins and is：
a ExpiCHO-STMThe recovery culture of cell；
B cell transfectings；
C WesternBlot Identification of Fusion Protein；
Chromatographic purifying is carried out after d protein concentrations.
E collects albumen and carries out BCA and quantifies.
The small peptide that Percentage bound is more than 15% is filtered out, wherein
Percentage bound %=(1- (OD-OD negative controls)/(OD positive control-OD negative controls)) × 100%.
Further, VISTA specific antibodies described in step (2) c are the anti-VISTA antibody of sheep, and reaction is final concentration of
0.5μg/ml-1μg/ml；The final concentration of 1 μ g/ml-10 μ g/ml of sample solution.
Further, the washing buffer is 0.01%PBST；The coating buffer solution is 50mM carbonate buffer solutions；
The confining liquid is the PBS solution of 2.5%BSA；The terminate liquid is 2N H2SO4。
The preparation method of the wherein described washing buffer is：1ml polysorbas20s are taken to be dissolved in 100ml PBS, 0.22 μm of filter membrane
Filtering, is prepared into the mother liquor of 1%PBST, room temperature preservation is diluted to 0.01% PBST with PBS before use；
Wherein it is described coating buffer solution (50mM carbonate buffer solutions) preparation method be：
Na2CO3 0.375g
NaHCO3 0.7325g
It is dissolved in ultra-pure water, is settled to 250ml, 9.6,4 DEG C of preservations of PH；
The preparation method of wherein described confining liquid/antibody diluent (PBS solution of 2.5%BSA) is：
BSA 0.1g
PBS 10ml
It is now with the current, it is used after crossing 0.22 μm of filter, 4 DEG C of preservations；
Wherein reaction terminating liquid (2N H2SO4) preparation method be：
18N concentrated sulfuric acid 4ml ultra-pure waters are taken to be diluted to 36ml, room temperature preservation.
It can be seen via above technical scheme that compared with prior art, the present invention provides have affinity with VISTA
Small peptide and application thereof has following technological merit：
The present invention provides the small peptide and application thereof for having affinity with VISTA by structure ELISA method screening model,
Drug development for specific VISTA target spots inhibitor provides experimental basis, for VISTA target spots antitumor drug, treats itself
The exploitation of the immunosupress class drug or anti-inflammatory drug of immunity disease lays the foundation.
Description of the drawings
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technology description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
The embodiment of invention for those of ordinary skill in the art without creative efforts, can also basis
The attached drawing of offer obtains other attached drawings.
Fig. 1 attached drawings are VISTA expressing quantities provided by the invention with expression time variation diagram.
Wherein, D2, D3, D4, D5 and D6 are respectively the 2nd day, the 3rd day, the 4th day, the 5th day, the 6th day after transfecting.
Fig. 2 attached drawings are that Western Blot provided by the invention identify VISTA protein profiles in elution fraction.
Fig. 3 attached drawings are that coomassie brilliant blue staining provided by the invention identifies VISTA albumen and foreign protein point in elution fraction
Butut.
Fig. 4 attached drawings are VISTA albumen peridium concentration Optimal Curve figure provided by the invention.
Fig. 5 attached drawings are mouse tumor volume change figure after plant tumor provided by the invention.
Specific implementation mode
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation describes, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other
Embodiment shall fall within the protection scope of the present invention.
Embodiment 1
The preparation of mouse VISTA fusion proteins
The VISTA albumen that the present embodiment uses is prepared by laboratory expression.Commercialization high yield is selected in the expression of VISTA albumen
The protein expression system of amount, with mammalian cell, that is, Chinese hamster ovary cell (Chinese hamster ovary cell,
CHO it is) expression vector, destination protein is concentrated from cell conditioned medium, then reach the mesh of purifying protein by albumen affinity column
's.
Wherein, protein expressing cells strain ExpiCHO-STMCells (Gibco, A29127) is purchased from Thermofisher
Scientific companies；ExpiCHOTMExpress culture medium (Gibco, A2910001)； Expi FectamineTMCHO
Transfection Kit (Gibco, A29129)；OptiPROTMSFM Complexation Medium (Gibco, 12309-
050)；Single-Use PETG Erlenmeyer Flasks with Plain Bottom (Nalgene, 4115-0125)；
Mouse VISTA-Fc albumen plasmid is given by the laboratories University of Wisconsin-Madison pungent medical college LilyWang.
TEMED (A610508-0100), 30% acrylamide/intersection acrylamide solution (B546017-0500), sweet ammonia
Sour (A502065-0005), three (methylol) aminomethanes (A501492-0005), SDS (A600485-0500), polysorbas20
(A100777-0500) it is purchased from Shanghai life work biotinylated biomolecule engineering services Co., Ltd；Ammonium persulfate (A3678-25G) is purchased
From Sigma-Aldrich companies；Methanol (67-56-1) is purchased from Nanjing chemical reagent limited liability company；0.22 μm of PVDF vinegar
Acid cellulose film is purchased from Bio-Rad companies of the U.S.；ECL luminescent solutions (180-5001), protein molecular weight standard (180-6003)
It is purchased from Shanghai Tian Neng Science and Technology Ltd.s；Skimmed milk power is purchased from Shanghai Bright Dairy ＆ Food Co., Ltd.；The anti-VISTA antibody of sheep
(AF7005) U.S. R＆D Systems are purchased from；Sheep secondary antibody (CW0240S) is century bio tech ltd purchased from health；Increase
Strong type BCA albumen concentration detection kit (P0009) is purchased from green skies bio tech ltd.
The preparation method of each reagent is as follows in the present embodiment：
10 × transferring film buffer solution：Tris, 60.6g；Glycine, 288g；Ultra-pure water is added to be settled to 2000ml, room temperature preservation,
10 × electrophoretic buffer 100ml is taken to add 700ml ultra-pure waters and 200ml methanol when use.
10 × TBS buffer solutions (pH 7.5)：Tris, 160.1g；Glycine, 48.4g；After adding ultrapure water dissolution, with dense salt
It is 7.6 that acid, which adjusts pH, then is settled to 2000ml with distilled water.
TBST buffer solutions (the TBS buffer solutions containing 0.1%Tween20)：10 × TBS, 100ml；
Ultra-pure water, 900ml；It can be used after Tween20,1ml mixing, matching while using.
Confining liquid (the TBST buffer solutions for containing 5% skimmed milk power)：Skimmed milk power, 5g；4 DEG C of guarantors after TBST, 100ml dissolving
It deposits.
10 × electrophoretic buffer (pH=8.3)：Tris, 30.3g；Glycine, 144.0g；SDS, 10.0g add ultra-pure water fixed
Hold to 1000ml, room temperature preservation.Used time takes 10 × electrophoretic buffer 100ml to add 900ml ultra-pure waters.
10% ammonium persulfate (AP)：AP, 1g；Ultra-pure water, 10ml；After dissolving, 4 DEG C are kept in dark place, and the holding time is 1 week.
Equilibration buffer (pH 8.0)：NaCl, 2.19g；Na2HPO412H20,1.79g；After adding ultrapure water dissolution, use is dense
Hydrochloric acid tune pH is 8.0, then is settled to 250ml with ultra-pure water, is used after 0.22 μm of membrane filtration, room temperature preservation.
Elution buffer (pH 2.5)：Glycine, 1.88g；It is 2.5 with concentrated hydrochloric acid tune pH after adding ultrapure water dissolution, then
It is settled to 250ml with distilled water.It is used after 0.22 μm of membrane filtration, room temperature preservation.
Neutralization buffer (pH 8.5)：It is 8.5 with concentrated hydrochloric acid tune pH, then use after Tris, 30.285g add ultrapure water dissolution
Distilled water is settled to 50ml.It is used after 0.22 μm of membrane filtration, room temperature preservation.
PBS buffer solution：NaCl, 8.0g；KCl, 0.2g；Na2HPO4, 1.15g；KH2PO4, 0.2 g is dissolved in ultra-pure water, fixed
Hold to 1L, 7.4,121 DEG C of 20min autoclave sterilizations of PH, is used after 0.22 μm of membrane filtration, room temperature preservation.
ExpiCHO-STMThe recovery culture of cell
Freeze-stored cell goes out to take out from liquid nitrogen, and cell is dipped in thawing in 38 DEG C of water-baths and be kept stirred makes it quickly melt always
Change and be only left very small amount of ice cube into pipe, cell is taken out from water-bath, is opened in super-clean bench after being jetted through 75% alcohol,
Cell suspension (1ml) in cryopreservation tube is transferred to the cell shaking flask for being prepared with 29ml Expi CHO in advance and expressing culture medium
In, cell shaking flask is placed in 37 DEG C, 8%CO2Concentration, shaking speed be 130rpm incubator in cultivate.
After recovery three days, when cell density reaches 4 × 106–6×106Cell passage is carried out when a living cells/mL, by cell
With density for 0.3 × 106A living cells/mL is inoculated in Expi CHO expression culture mediums, cell culture volumes 30ml.It will be thin
Born of the same parents put back to 37 DEG C, 8%CO2Concentration, shaking table (amplitude 19mm) rotating speed be 130rpm incubator in cultivate.
Cell transfecting
Cell passage is carried out, until ExpiCHO-STMIt is 4 × 10 that cell, which reaches density,6–6×106A living cells/mL；
When the cell handled through above-mentioned passage reaches 4 × 106–6×106When a living cells/mL, sub-bottle (this is carried out to cell
When be D1 days), it is 3 × 10 to make the whole density of cell after sub-bottle6–4×106A living cells/mL, cell is put back in incubator and is grown
Overnight.
The second day density and living cell rate for checking cell.Cell should reach 7 × 106–10×106A living cells/mL,
Living cell rate is in 95%-99%.The fresh Expi-CHO expression culture mediums for being preheated to 37 DEG C of cell are diluted to 6 × 106It is a
Living cells/mL, guarantee transfection volume is 25ml, wherein the additive amount of each reagent is shown in Table 1.Gently turn Tissue Culture Flask is with mixing
Cell.Soft turning upside down mixes well Expi Fectamine CHO Reagent.Plasmid is diluted with OptiPRO SFM
DNA and transfection reagent shake centrifuge tube mixing.The Expi diluted is added into DNA dilutions while turn centrifuge tube
Fectamine CHO Reagent.It is incubated at room temperature Expi Fectamine CHO/DNA compounds 5min, it is then that compound is molten
Liquid is slowly added into Tissue Culture Flask, and side edged gently shakes cell bottle.Cell is finally put back to 37 DEG C, 8%CO2In incubator,
Shaking speed 130rpm.
After transfecting 20h, 150 μ L ExpiCHO Enhancer and 6ml ExpiCHO Feed are added into Tissue Culture Flask,
Then culture bottle incubator is put back to continue to cultivate.
Each reagent additive amount when 1 25ml transfection volumes of table
WesternBlot Identification of Fusion Protein
100 μ L cell suspensions 1000rpm, 4 DEG C of centrifugation 5min are taken, supernatant is drawn.According to 5 × Loading buffer and β-
Mercaptoethanol is according to 4：1 is configured to sample-loading buffer (5 × Loadingbuffer20 μ L, 5 μ L of β-mercaptoethanol), then presses loading
Buffer solution：Sample volume ratio is 1：4 (25 μ L sample-loading buffers are added in 100 μ L samples) mix, by sample as at 100 DEG C
Thermal denaturation 10min.Postcooling centrifugation is completed, is frozen in -20 DEG C.
Judge that the molecular weight of estimating of albumen is 83.25KD according to the plasmid map of VISYA-Fc, according to various concentration point
Optimum separative capacity from glue selects 8% separation gel, the optimal separation range of various concentration separation gel to be shown in Table 2.According to table 3,
It by the volume preparative separation glue of various reagents, is poured into after mixing well in the glass plate of 1.5 mm, aqueous is added to seal, wait for 20min
Glue to be separated fully condenses.Fluid-tight water is removed, filter paper is used in combination to blot, according still further to 5% concentration glue formula glue, wherein concentrating
Glue formula is inserted into comb, waits for 20min.
The film prepared is fixed in electrophoresis tank, electrophoretic buffer is poured into, is cleaned after each loading hole, by preparation
The 2nd day to the 6th day cell conditioned medium sample sequentially adds in hole (per 10 μ L of hole) after transfection.Deposition condition is constant pressure 80V,
40min；100V, 1.5h.Electrophoresis takes out film after terminating, according to the sequence of " black glue tunica albuginea " by pvdf membrane, gel, filter paper
It is placed on the centre of transferring film folder, when transferring film is put into ice chest to ensure low temperature in transferring film slot.Transferring film condition is fixed current 200mA,
Transferring film time 2.5h.
Film is placed in confining liquid after transferring film, shaking table slowly closes 1h under room temperature, after closing, goes to seal
Liquid is closed, the anti-VISTA antibody of sheep is added and (presses antibody mother liquor：Confining liquid=1：200 dilutions) 4 DEG C be incubated overnight.Second day, recycling
Antibody washes film with TBST buffer solutions, and 80rpm is cleaned five times, each 10min.It is again that film is (female by antibody with rabbit-anti sheep secondary antibody
Liquid：Confining liquid=1：10000 dilutions) it is incubated 1h；Antibody is recycled, film is washed with TBST, 80rpm washes 10min, operates five times.Finally
Film is placed in exposure instrument, luminescent solution (A liquid is uniformly coated with：Liquid=1 B：1) it, is exposed.The results are shown in Figure 1, Fig. 1 results
Show that successful expression goes out mouse VISTA albumen in cell conditioned medium, and since the 2nd day, with the extension of expression time, in supernatant
The content of VISTA albumen gradually increases.
The optimal separation range of 2 various concentration separation gel of table
3 8%SDS-PAGE gels of table are prepared
Chromatographic purifying is carried out after protein concentration
Culture medium in shaking flask is collected into 50ml centrifuge tubes, 1000rpm low-temperature centrifugation 10min take supernatant, then
7000rpm low-temperature centrifugation 40min collect supernatant and are filtered with 0.45 μm of filter, and filtrate is collected in 50ml centrifuge tubes.According to egg
White molecular weight selects 10KD super filter tube concentrating cells supernatants, 7000 rpm to centrifuge 30min, collects the concentration in super filter tube inner tube
Liquid.
Before crossing column, first with equilibration buffer by the volume displaced at buffer components of protein concentrated solution.Displacement passes through ultrafiltration
Pipe carries out, by protein concentrated solution and equilibration buffer 1:After 1 dilution, 7000rpm low-temperature centrifugation 30min, repeatedly twice.
It fully shakes up so that resin is resuspended, draws in 2ml slurries to new chromatographic column, 2ml balances are previously added in chromatographic column
Buffer solution.It allows resin natural subsidence, flows out equilibration buffer.It is added in 10ml equilibration buffers to chromatographic column and balances resin, press
The flow velocity of about 1ml/min flows out equilibration buffer.
By sample according in the flow velocity loading to chromatographic column of about 1ml/min, efflux is collected, is labeled as Flow
Through, i.e. FT.With the equilibration buffer solution resin of 30ml, flow velocity maintains about 2ml/min, and a group is collected as per 2ml
Point.With 15ml elution buffer antibody elutions, flow velocity maintains about 1ml/min, and is added immediately 1/10 elution buffer volume
Neutralization buffer adjusts pH to 7.4, and a component is collected as per 2ml.
The component being collected into purification process will pass through Western Blot identifications and coomassie brilliant blue staining identification, knot
Fruit is as shown in Figures 2 and 3, and as shown in Figure 2, FT components do not detect destination protein in Fig. 2, illustrate that sample is fully incorporated in column
On material, after Equilibration buffer wash and after elution buffer elution, under VISTA albumen is successfully eluted from column material
Come.From the figure 3, it may be seen that only having destination protein molecular weight to have protein band in Fig. 3 elution fractions, other foreign protein items are had no
Band illustrates that the purity of protein that purifying obtains is higher.
Collecting protein with it is quantitative
It collects through the authenticated elution fractions containing destination protein of Western Blot, 10KD super filter tubes is used in combination to concentrate, from
Heart condition is 7000rpm low-temperature centrifugations 40min.After concentration, then albumen is preserved into system and is replaced as PBS buffer solution, i.e., it will concentration
Liquid and PBS buffer solution 1:14 mixing, 7000rpm low-temperature centrifugation 40min, repeatedly twice.Albumen is finally collected, is frozen after packing
In -80 DEG C.
Illustrate configuration protein standard substance according to kit.By working solution A and B with 50:1 ratio is made into mixed liquor, to
The protein standard substance of 20 μ l is added in 96 orifice plates per hole, then the mixed liquor of 200 μ l is added into every hole, each concentration does 3 pairs
Hole, after 37 DEG C are incubated 30min, measures OD values at 562nm wavelength, and destination protein concentration is calculated according to standard curve.Successfully
VISTA fusion proteins, a concentration of 2.3mg/ml are prepared in expression.
Embodiment 2
Reagent N aCl (A501218-0001), polysorbas20 (A100777-0500) in the present embodiment is purchased from Shanghai life work life
Object biotechnology Services Co., Ltd；KCl (analysis is pure), Na2HPO4(analysis is pure), KH2PO4(analysis is pure), Na2CO3(point
Analyse pure), NaHCO3(analysis is pure), the concentrated sulfuric acid (analysis is pure) are purchased from Nanjing Chemistry Reagent Co., Ltd.；TMB (P0209) developing solution
Purchased from green skies biological reagent company；BSA (10735078001) is purchased from BioSharp companies；Nunc MaxiSorpTM flat-
Bottom ELISA Plates (Thermofisher Scientific, 439454)；The anti-VISTA antibody (AF7005) of sheep is purchased from the U.S.
R&D Systems；Sheep secondary antibody (CW0240S) is century bio tech ltd purchased from health.
The present embodiment pilot scale agent compounding method is as follows：
Washing buffer (0.01%PBST)
1ml polysorbas20s are taken to be dissolved in 100ml PBS, 0.22 μm of membrane filtration is prepared into the mother liquor of 1%PBST, and room temperature is protected
It deposits, is diluted to 0.01% PBST with PBS before use；
It is coated with buffer solution (50mM carbonate buffer solutions)
Na2CO3 0.375g
NaHCO3 0.7325g
It is dissolved in ultra-pure water, is settled to 250ml, 9.6,4 DEG C of preservations of PH；
Confining liquid/antibody diluent (PBS solution of 2.5%BSA)
BSA 0.1g
PBS 10ml
It is now with the current, it is used after crossing 0.22 μm of filter, 4 DEG C of preservations；
Reaction terminating liquid (2N H2SO4)
18N concentrated sulfuric acid 4ml ultra-pure waters are taken to be diluted to 36ml, room temperature preservation.
Experiment group design is shown in Table 4：Used albumen is prepared by laboratory expression in experiment.Small peptide is by PBS when experiment
To required concentration, small peptide mother liquor is 10mg/ml for configuration, and screening concentration is 1 μ g/ml.Negative control is the group for not being incubated primary antibody,
Board bottom VISTA protein binding sites are not occupied at this time, which is 0%；In positive controls, primary antibody is added
And secondary antibody, the binding site of board bottom VISTA should be fully saturated by primary antibody, which is 100%；It is added in experimental group
If small peptide can should decline with VISTA protein bindings, the absorbance value of the group, Percentage bound is in 0%-100%.
Table 4 tests group
Mouse extracellular fragment fusion protein after purification is diluted to 0.09818 μ g/ml using coating buffer solution, is added per hole
The protein solution of 100 μ l, 4 DEG C of coatings are overnight.Coating protein liquid is discarded within second day, PBST is washed 3 times, and 200 μ l closings are added per hole
Liquid, room temperature discard confining liquid after closing 2h, and PBST is washed 5 times.By sample solution and antibody 1:(sample after mixing is added after 9 mixing
Final concentration of 1 μ g/ml, the final concentration of 0.5 μ g/ml of primary antibody), per 100 μ l of hole, reaction solution is discarded after reacting at room temperature 2h, PBST washes 5
It is secondary.Secondary antibody anti-sheep-HRP (1 is added:10000), per 100 μ l of hole, PBST is washed 5 times after being incubated at room temperature 1h.100 μ l are added
TMB developing solutions, dark place are incubated 9min, add after 50 μ l reaction terminating liquids the read plate survey absorbance value in 450nm at.As a result such as
Fig. 4, wherein Percentage bound %=(1- (OD-OD negative controls)/(OD positive control-OD negative controls)) × 100%
The present invention screens small peptide 318, wherein small peptide amino acid sequence of the Percentage bound more than 15% and concrete activity data
It is shown in Table 5.
5 small peptide amino acid sequence of table and concrete activity data summarization
Wherein H-histidine；D-aspartic acid；R-arginine；F-phenylalanine；A-alanine；Half Guang ammonia of C-
Acid；G-glycine；Q-glutamine；E-glutamic acid；K-lysine；L-leucine；M-methionine；N-asparagine；
S-serine；Y-tyrosine；T-threonine；I-isoleucine；W-tryptophan；P-proline；V-valine.
Embodiment 3
Influence of the small peptide to mouse melanoma
B16-F10 cell culture waits for that cell is in exponential phase, grows in 1640 complete mediums (containing 10%FBS)
When degrees of fusion is 80%~90%, pancreatin had digestive transfer culture.Wherein, B16-F10 cells are provided by military science research institute.
Using PBS as blank control, using small peptide as drug, 8 groups altogether, including control group, SEQ ID NO:1- SEQ ID
NO:7 groups (20 μ g/ are only), 200 μ l of administered volume, every group of 6 mouse, experiment repeat 3 times.
1) anesthetized mice：EP pipes equipped with cell are overturned into mixing 6 times, removes syringe needle and inhales cell culture fluid extremely
0.4ml；
2) the both sides groin intracutaneous injection B16F10 cells for giving every mouse, per 100 μ l of side.It is denoted as D0 on the day of planting tumor
It, starts to be administered for D3 days, is administered every 1 day, every group of 0 μ l of every mouse peritoneal injection protein 20.
Start within D9 days, with isoflurane by mouse anesthesia, weigh, with vernier caliper measurement left and right side tumor size.It waits for pair
When being about 2000mm3 according to the gross tumor volume of group, stop administration, put to death mouse, terminates zoopery.Mouse tumor volume after plant tumor
6 and Fig. 5 are shown in Table, by table 6 and Fig. 5 it is found that SEQ ID NO:1- SEQ IDNO:7 small peptide is to there is the work for inhibiting mouse tumor
With.
Table 6 plants mouse tumor volume after tumor
The present invention provides the small peptide and application thereof for having affinity with VISTA by structure ELISA method screening model,
Drug development for specific VISTA target spots inhibitor provides experimental basis, for VISTA target spots antitumor drug, treats itself
The exploitation of the immunosupress class drug or anti-inflammatory drug of immunity disease lays the foundation.
Each embodiment is described by the way of progressive in this specification, the highlights of each of the examples are with other
The difference of embodiment, just to refer each other for identical similar portion between each embodiment.For device disclosed in embodiment
For, since it is corresponded to the methods disclosed in the examples, so description is fairly simple, related place is said referring to method part
It is bright.
The foregoing description of the disclosed embodiments enables those skilled in the art to implement or use the present invention.
Various modifications to these embodiments will be apparent to those skilled in the art, as defined herein
General Principle can be realized in other embodiments without departing from the spirit or scope of the present invention.Therefore, of the invention
It is not intended to be limited to the embodiments shown herein, and is to fit to and the principles and novel features disclosed herein phase one
The widest range caused.
Sequence table
<110>China Medicine University
<120>There is the small peptide and application thereof of affinity with VISTA
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 15
<212> PRT
<213> Artificial Sequence
<400> 1
Ile Val Asn Pro Gly Gly Ser Asn Leu Thr Tyr Thr Ile Glu Arg
1 5 10 15
<210> 2
<211> 9
<212> PRT
<213> Artificial Sequence
<400> 2
Asp Asn Leu Pro Tyr Leu Val Ala Tyr
1 5
<210> 3
<211> 17
<212> PRT
<213> Artificial Sequence
<400> 3
Val His His Gln Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn
1 5 10 15
Lys
<210> 4
<211> 7
<212> PRT
<213> Artificial Sequence
<400> 4
Cys Pro Leu Lys Gly Val Arg
1 5
<210> 5
<211> 7
<212> PRT
<213> Artificial Sequence
<400> 5
Gly Ser Asn Leu Thr Tyr Thr
1 5
<210> 6
<211> 5
<212> PRT
<213> Artificial Sequence
<400> 6
Lys Gly Gly Gly Lys
1 5
<210> 7
<211> 6
<212> PRT
<213> Artificial Sequence
<400> 7
Ser Asn Leu Thr Tyr Thr
1 5
Claims (10)
1. having the small peptide of affinity with VISTA, which is characterized in that the amino acid sequence of the small peptide is as follows：
IVNPGGSNLTYTIER SEQ ID NO:1；
DNLPYLVAY SEQ ID NO:2；
VHHQKLVFFAEDVGSNK SEQ ID NO:3；
CPLKGVR SEQ ID NO:4；
GSNLTYT SEQ ID NO:5；
KGGGK SEQ ID NO:6；
SNLTYT SEQ ID NO:7。
2. the small peptide with VISTA with affinity according to claim 1, which is characterized in that the small peptide with it is described
The Percentage bound of VISTA is all higher than 15%.
3. including the polypeptide variants of small peptide described in claims 1 or 2, which is characterized in that the polypeptide variants are to the small peptide
Carry out one or more modifications；The modification includes and human immunoglobulin(HIg) Fc segments or its variant fusion；With human seralbumin egg
White or its variant fusion；It is merged with tumour penetrating peptide；It is single-stranded or single domain antibody merges with tumor-targeting；Or with polyethylene glycol,
Xtenylation, PASylation or HESylation are merged.
4. including the pharmaceutical composition of small peptide described in claims 1 or 2, which is characterized in that described pharmaceutical composition further include with
The compatible carrier of the small peptide, the treatment factor, excipient and diluent.
5. including the pharmaceutical composition of polypeptide variants described in claim 3, which is characterized in that described pharmaceutical composition further include with
The compatible carrier of the polypeptide variants, the treatment factor, excipient and diluent.
6. the application of small peptide described in claims 1 or 2, which is characterized in that the small peptide is in exploitation VISTA target spot antineoplastics
Object, the immunosupress class drug for treating autoimmune disease or the application in anti-inflammatory drug.
7. the application of small peptide according to claim 6, which is characterized in that the tumour includes fibrosarcoma, mucous membrane sarcoma, fat
Fat sarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangioendothelial sarcoma, lymphangioendothelial sarcoma, cunning
Film sarcoma, celiothelioma, Ewing' s tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, cancer of pancreas, syringocarcinoma, carcinoma of sebaceous glands, breast
Head cancer, papillary adenocarcinoma, bronchiolar carcinoma, cephaloma, clear-cell carcinoma, kidney mother cell cancer, kidney, liver cancer, cholangiocarcinoma, chorion
Cancer, spermatogonium cancer, embryonal carcinoma, cervical carcinoma, orchioncus, lung cancer, Small Cell Lung Cancer, carcinoma of urinary bladder, epithelioma, glioma, star
Shape cytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neurinoma, meningioma,
Oligodendroglioma, melanoma, neuroblastoma, retinoblastoma, hematologic cancers, human primary gastrointestinal cancers, cancer of the esophagus or
Urinary system cancer；
The autoimmune disease includes lupus erythematosus or psoriasis；
The inflammation includes asthma, arthritis or organ-graft refection.
8. the screening model construction method of small peptide according to claim 1 or claim 2, which is characterized in that include the following steps：
(1) VISTA fusion proteins are prepared in expression；
(2) competitive ELISA method is utilized, the affinity molecule of VISTA albumen is screened；
A VISTA albumen is coated on by physisorption on ELISA Plate, overnight by being coated with buffer solution；
B discards coating protein liquid, is cleaned using washing buffer, and confining liquid is added afterwards, confining liquid is discarded after room temperature Seal treatment,
It is cleaned using the washing buffer, the nonspecific binding site that removal board bottom is not saturated by albumen；
C is by sample solution and VISTA specific antibodies 1：9 are hybridly prepared into reaction solution addition, and reaction is discarded after reacting at room temperature 2h
Liquid, and cleaned using the washing buffer；
Secondary antibody incubation at room temperature is added in d, and TMB developing solutions dark place is added after cleaning and is incubated, and is eventually adding terminate liquid detection absorbance value.
9. the construction method of small peptide screening model according to claim 8, which is characterized in that described in step (2) c
VISTA specific antibodies are the anti-VISTA antibody of sheep, react final concentration of 0.5 μ g/ml-1 μ g/ml；The sample solution is dense eventually
Degree is 1 μ g/ml-10 μ g/ml.
10. the construction method of small peptide screening model according to claim 8, which is characterized in that the washing buffer is
0.01%PBST；The coating buffer solution is 50mM carbonate buffer solutions；The confining liquid is the PBS solution of 2.5%BSA；Institute
It is 2N H to state terminate liquid2SO4。
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| CN112824427A (en) * | 2019-11-18 | 2021-05-21 | 杨小骏 | Short peptide for inhibiting glioma and application thereof |
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