Disclosure of Invention
In order to solve the defects of the prior art, the invention aims to provide an anti-angiogenesis peptide, a screening method and application thereof, and the anti-angiogenesis peptide provided by the invention has good targeting and anti-angiogenesis activity, so that the anti-angiogenesis peptide has good practical application value.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
in a first aspect, an anti-angiogenic peptide is polypeptide 1 or polypeptide 2;
the amino acid sequence of the polypeptide 1 is shown as SEQ ID NO. 1;
the polypeptide 2 is a polypeptide derived from the polypeptide 1 by substitution and/or deletion and/or addition of one or two amino acid residues and has the same glycosyltransferase function.
In a second aspect, the method for screening the anti-angiogenic peptide comprises the steps of screening specific targeting polypeptides by using VEGF as a target protein through a phage random dodecapeptide library, performing affinity screening in an adsorption-elution-amplification process at least once through phage to obtain monoclonal phage capable of being specifically combined with the specific targeting polypeptides, obtaining an amino acid sequence of the specific targeting polypeptides through the monoclonal phage, and further screening peptides with affinity activity sequentially through ELISA detection and Surface Plasmon Resonance (SPR).
In a third aspect, a method of preparing the above anti-angiogenic peptide, the method comprising:
The anti-angiogenic peptide is prepared by microbial fermentation, or
The anti-angiogenic peptide is prepared by genetic engineering method, or
The anti-angiogenic peptide is synthesized by chemical means.
In some embodiments, the chemical method is a solid phase synthesis method.
In a fourth aspect, the use of an anti-angiogenic peptide as described above in the manufacture of a medicament, said medicament being an anti-angiogenic or anti-tumour medicament.
In some embodiments, the medicament is a composition.
In particular, the composition comprises one or more of the anti-angiogenic peptides.
Specifically, the composition comprises pharmaceutical excipients. More specifically, the pharmaceutical excipients may be pharmaceutical carriers, such as natural polymer materials (lipids, saccharides, proteins, etc.), synthetic polymer materials (polyalkylcyanoacrylates, polylactic acids, etc.), small-molecule organic materials (e.g., stearic acid, etc.), and excipients, such as binders (syrup, acacia, sodium carboxymethyl cellulose, etc.), fillers (lactose, starch, etc.), disintegrants (sodium carboxymethyl starch, microcrystalline cellulose, crospovidone, etc.), solubilizing agents (polysorbate, poloxamer, etc.), preservatives (benzyl alcohol, benzoic acid, salicylic acid, etc.), pH adjusting agents, stabilizers, etc.
In some embodiments, the dosage form of the medicament is a tablet, a granule, a freeze-dried powder injection, an injection, and the like.
In some embodiments, the drug is administered orally, subcutaneously, intravenously, intramuscularly, etc.
In some embodiments, the anti-angiogenic drug is used to treat diseases including, but not limited to, diabetic retinopathy, coronary heart disease, degenerative arthritis, and the like.
In some embodiments, the tumor includes, but is not limited to, skin cancer, head and neck cancer, lung cancer, esophageal cancer, cervical cancer, uterine cancer, pancreatic cancer, breast cancer, kidney cancer, ureter cancer, bladder cancer, squamous cell carcinoma, basal cell carcinoma, melanoma, tongue cancer, pharyngeal squamous cell carcinoma or malignant lymphoma, laryngeal squamous cell carcinoma, lung squamous cell or small cell carcinoma, esophageal squamous cell carcinoma, cervical cancer, digestive tract tumor, reproductive system tumor, lymphoid tumor, bone tumor, head and neck tumor, and the like.
The beneficial effects of the invention are as follows:
the anti-angiogenesis peptide provided by the invention has good affinity to VEGF, and a lumen formation experiment shows that the anti-angiogenesis peptide provided by the invention has good anti-angiogenesis activity, and is beneficial to realizing tumor treatment by blocking angiogenesis. In addition, cell proliferation experiments show that the anti-angiogenesis peptide can also directly inhibit proliferation of mouse breast cancer cells, so that the anti-angiogenesis peptide has good anti-tumor activity.
Detailed Description
In order to enable those skilled in the art to more clearly understand the technical scheme of the present invention, the technical scheme of the present invention will be described in detail with reference to specific embodiments.
Example 1:
1. four rounds of panning, DNA extraction and sequencing
(1) VEGF was formulated as a 10. Mu.g/mL solution using 0.1M sodium bicarbonate solution at pH8.6, 100. Mu.L was added to a 96 well ELISA plate and shaken overnight at 75rpm at 4 ℃.
(2) The coating was discarded, and the BSA buffer block was topped up and left at 4 ℃ for 90 minutes.
(3) The buffer was discarded, and TBST was washed 6 times with 0.1% Tween 20 for 1 minute each time, and the mixture was slapped vigorously.
(4) Phage virions 1X 1011 (90. Mu.L TBS and 10. Mu.L phage peptide library stock) were added to the coated wells and mixed well and shaken for 60 minutes at room temperature.
(5) The phage solution was discarded, and the phage was slapped with force, and in order to wash unbound phage, washed 6 times with TBST0.1% Tween 20 for 1 minute each time, and dried with force.
(6) To elute phage that specifically bound to monoclonal antibody, 100 μl of glycine buffer eluent was added and the mixture was slowly shaken at room temperature for 60 minutes.
(7) The eluate was transferred into a 1mL sterile microcentrifuge tube, and 15. Mu.L of Tris-HCl buffer neutralization solution was added to obtain a first round of phage eluate. Phage titer determination and amplification were performed on the first round of eluate.
(8) The above procedure was repeated, and rounds 2-4 of phage peptide library screening were performed. Wherein, the tween concentration in the washing step needs to be increased to 0.5% (v/v) in the second two rounds of screening, and the eluent is changed into a free target molecule solution.
(9) ER 2738 cultured overnight was diluted 1:100 and inoculated into LB medium, and 10mL was dispensed into sterile flasks.
(10) Blue plaques were picked from the fourth phage plate (total amount of plaques no more than 100) into the above culture tube and incubated on a 37℃shaker for 4.5h.
(11) The culture was transferred to a sterilizing centrifuge tube, centrifuged at 10000rpm for 30s to obtain a supernatant, and centrifuged again for 30s.
(12) 80% Of the supernatant was transferred to a new EP tube, which was the phage stock after amplification.
(13) After amplification of the picked phage monoclonal, 500. Mu.L of phage-containing supernatant was centrifuged. 200. Mu.L of PEG/NaCl was added thereto, and after mixing was reversed, the mixture was allowed to stand at room temperature for 10 minutes. Centrifuge at 10000rpm for 10 min at 4 ℃, discard supernatant.
(14) 100. Mu.L of iodide buffer was added for resuspension, 250. Mu.L of absolute ethanol was added after brief centrifugation, and the mixture was allowed to stand at room temperature for 10 minutes. Centrifuge at 10000rpm for 10 min at 4 ℃, discard supernatant.
(15) The precipitate was washed with 70% ethanol and centrifuged at 12000rpm for 5 minutes, and the supernatant was discarded.
(16) The monoclonal phage were sequenced by adding 30. Mu.L TE buffer.
(17) And analyzing the sequencing result of the monoclonal phage DNA, and finding an insertion sequence between GGTACC KPN I enzyme cutting sites and CGGCCGEAG I enzyme cutting sites, so that the amino acid sequence of the corresponding insertion polypeptide can be deduced by using a genetic code table.
2. Molecular docking
The ZDOCK module in Discovery Studio Visualizer client software is adopted to dock the target protein and the screening polypeptide, and ZDock Score in the result is used as a scoring function, and the larger absolute value of ZDock Score is, the stronger binding capacity of the receptor and the ligand is shown. The method comprises the following steps:
(1) Protein structures that downloaded VEGF were retrieved from Protein Data Bank (PDB) database as receptor proteins in molecular docking experiments. And carrying out structural prediction on the screened polypeptide from a PEP-FOLD website, and storing a prediction result to serve as a ligand protein in a molecular docking experiment.
(2) And opening Discovery Studio Visualizer client software, finding files of target proteins and polypeptides from files and opening the files. Selecting a preparation Protein in an expanded menu bar, clicking a Clean Protein shortcut tool in a MannualPreparation panel group, removing ligand molecules and water molecules in a Protein crystal structure for target proteins and screening polypeptides, removing Protein multi-conformation, supplementing incomplete amino acid residues, and carrying out pretreatment such as Protein hydrogenation.
(3) The screened polypeptide molecules are docked with the treated target protein with ZDOCK. Opening the receptor and ligand Proteins in the same 3D window, clicking Macramolecules in the Tool bar, expanding Dock and Analyze Protein Complexes Tool, clicking Dock Proteins (ZDOCK), setting the opening parameters into a dialog box, clicking Input Receptor Protein parameters, selecting 2ptn:2ptn, clicking Input Ligand Protein parameters, selecting 2ptn:2sta, clicking Angular Step Size parameters, selecting 15. The Clustering parameter set is expanded, the RMSD Cutoff parameter is clicked, the value is set to 6, the INTERFACE CUTOFF parameter is clicked, the value is set to 9, the Maximum Number of Clusters parameter is clicked, and the value is set to 60. Clicking to run, and waiting for the result to finish.
(4) At the end of the calculation, the DS will appear as a Job Completed dialog box. In Job Explorer, a double-click on the completed computing task DS will newly open a report. In the Output Files section, click on the ZDockResults. Dsv link. In the Data Table, click Protein Pose. Clicking Macromolecule in the Tools toolbar, selecting Dock and Analyze Protein Complexes tool, clicking Browse in Browse Poses, selecting Top Poses IN LARGEST Clusters in the drop down menu will show the top 100 scoring conformations in the 10 largest Cluster. Selecting Chart|3D Point Plot opens a Choose Plot Axes dialog, X-axis selection ZDock Score, Y-axis selection Cluster, Z-axis selection Density, click OK. The selection ZDock Score scores higher Pose in the results, while the corresponding conformation in the molecular window is selected, at which point the corresponding ZDock Score of selection Pose is seen. The larger the absolute value of ZDock Score, the stronger the binding capacity of the receptor to the ligand, which was selected ZDock Score as a scoring function in this study.
The absolute values of ZDock Score are sequenced from the big to the small, and the first 30 screening polypeptides are selected for the next experiment.
3. ELISA experiments
ELISA tests were performed on the first 30 phage clones of the molecular docking results to identify the binding force of phage display polypeptides to target proteins. The method comprises the following steps:
(1) At the time of amplifying plaques for DNA sequencing, the remaining plaque-containing supernatant was stored at 4 ℃.
(2) ER2738 cultured overnight was diluted 1:100 in 20mL LB medium. mu.L of phage supernatant was added to each tube of ER2738 culture broth and incubated at 37℃for 4.5h with aeration.
(3) The culture was transferred to a centrifuge tube and centrifuged at 10000rpm for 10min. The supernatant was transferred to a fresh centrifuge tube and centrifuged again.
(4) 80% Of the supernatant was placed in a fresh centrifuge tube and 1/6 of the PEG/NaCl was added. The pellet was left overnight at 4 ℃.
(5) The pellet was centrifuged at 10000rpm for 15min at 4℃and the supernatant was discarded and the residual supernatant was aspirated by brief centrifugation.
(6) The pellet was resuspended in 1mL TBS, and the suspension transferred to a microcentrifuge tube and centrifuged at 4℃for 5min to remove residual cells from the pellet.
(7) The supernatant was transferred to a fresh microcentrifuge tube and reprecipitated by adding 1/6 of the PEG/NaCl. The ice is applied for 15-60min. Centrifugation was performed at 4℃for 10min, the supernatant was discarded, and a brief centrifugation was performed to remove the residual supernatant.
(8) The pellet was resuspended in 50. Mu.L TBS and phage titers were determined. Stored at 4 ℃.
(9) Each well of ELISA plate was coated with 100. Mu.L of 1. Mu.g/mL of target molecule, 3 coated wells per clone to be identified. The coating was carried out overnight at 4℃in a sealed wet box.
(10) And (5) throwing out the redundant target molecule solution, inverting the flat plate, and beating and throwing out the residual liquid on paper towels. Each well was topped up with a blocking liquid, blocking for 1h at 4 ℃. In addition, a blocking solution was also added to the uncoated well of each clone to be identified to examine the binding force of the selected sequence to the BSA coated plastic plate.
(11) The blocking solution was thrown off and the plates were washed 6 times with TBST 0.5% Tween 20, each time with the plates inverted and the washings were thrown off on clean paper towels.
(12) 1X 109 phages were added to the wells coated with target molecules. Vibrating at room temperature for 1h.
(13) Plates were washed 6 times with TBST 0.5% tween 20. HRP-labeled anti-M13 antibody was diluted in blocking solution at a ratio of 1:5000. 100 μl of diluted antibody was added to each well and the mixture was allowed to shake at room temperature for 1h.
(14) Plates were washed 6 times with TBST 0.5% tween 20. 200. Mu.L of HRP substrate solution was added to each well and allowed to react at room temperature for 1h.
(15) Absorbance at 405-415nm was recorded with a plate reader.
As a result, phages having OD values higher than that of the negative control group by more than 2 times, and having lower affinity with the blocking solution and higher affinity with the target protein were used as positive phages specifically binding to the target protein.
4. SPR experiment
The first 3 phage clones from ELISA assays were subjected to polypeptide synthesis and Surface Plasmon Resonance (SPR) was used to determine the polypeptide with the highest affinity for VEGF.
(1) And (3) system detection, namely replacing the buffer solution with 1.05 XPBS, replacing the chip with a brand new chip, selecting SYSTEM CHECK AND Pump Calibration options in a Tools module of Biacore X100 plus program control software, and carrying out system detection by using BIAtest solution and ultrapure water according to prompt. After the display is completely passed, the next operation is performed.
(2) Finding the optimal pH value of the chip coupled protein, namely determining the optimal pH value of the coupled target protein by using Find Immobilization pH modules in the kinetic/Affinity modules. According to the system instructions, the proteins were diluted to 50. Mu.g/mL with 10mM sodium acetate buffer solution at pH 4.0, 4.5, 5.0, 5.5, respectively, and the samples were sequentially taken for 180s using 50mM NaOH solution as the washing liquid. And (3) popping up a result graph after the completion, and selecting proper pH as the pH condition of the final coupled protein according to the response value and curve shape of the sample under each pH condition.
(3) Selecting Immobilize options in a kinetic/Affinity module, fixing a target protein solution at the optimal pH selected in the previous step on a CM5 chip by an amino coupling method, taking a Flow Cell 2 as a sample channel, taking a blank Flow Cell 1 as a reference channel, setting the maximum coupling quantity Rmax of the protein as 100RU according to a formula, and calculating the ligand coupling level RL according to the following formula, wherein SM is a stoichiometric ratio, is generally set to be 1, and the actual coupling quantity is RL which is 1.5 times during experiments.
(4) Affinity (KD value) determination since the polypeptide drug used for SPR was dissolved in less than 0.5% DMSO, very low concentrations of DMSO have little effect on SPR results, and no calibration curve was needed for this experiment. Preparing 100 mu M of polypeptide to be detected, and measuring the affinity of polypeptide with serial concentration gradient (sesquidilution method) and target protein. RU binding saturation period was obtained and binding affinity of target protein to polypeptide was obtained using single site interaction model fitting.
FIG. 1 is a sensorgram of the interaction between V19 polypeptide and VEGF at various concentrations, and FIG. 2 is a graph of the affinity fit of the interaction between V19 polypeptide and VEGF, wherein the vertical line represents fit KD values, falling within the sample concentration range indicating that the fit data is authentic. Table 1 is the data of the results of the fitting of the interactions of the V19 polypeptide with VEGF, the smaller the affinity fit KD, indicating a stronger affinity of the polypeptide with the protein. From the results, the KD value of V19 is 9.803E-5, which indicates that the V19 polypeptide has better affinity with VEGF.
Table 1 data table of fitting results of interactions of V19 polypeptides with VEGF
| KD(M) | Rmax(RU) | Chi2(RU2) |
| 9.803E-5 | 26.76 | 0.710 |
The amino acid sequence of the screened polypeptide V19 is RTSPSPRGKHGE, and is shown as SEQ ID NO. 1. The Hangzhou Bayer pharmaceutical technology Co.Ltd.
Example 2 Effect of V19 peptide on endothelial cell lumen formation
(1) The melted Matrigel gum was added to a pre-chilled 48-well plate (80 μl/well) with a pre-chilled gun head, carefully added to avoid air bubbles.
(2) EAhy 926 cells in log phase were collected, the cell suspension concentration was adjusted to 11×104 cells/well and added to matrigel-plated well plates.
(3) The method comprises the steps of taking a hole without medicine and only added with DMEM culture medium as a blank control group, taking a hole added with 10ng/mLVEGF of serum-free DEME culture medium as a negative control group, taking a serum-free DEME culture medium containing 10ng/mLVEGF as a positive control group, taking a V19 polypeptide prepared with the serum-free DEME culture medium containing 10ng/mLVEGF as an experimental group, setting the medicine concentration to be 500 mug/mL and 1mg/mL, and setting 3 compound holes in each group.
(4) After the dosing is completed, the pore plate is continuously cultured for 6 hours, the generation condition of the cell vascular network is observed by an inverted fluorescence microscope, the cell vascular network is photographed, and the photographs are subjected to data processing by Image J software.
As shown in fig. 3, both V19 and ES2 significantly inhibited the formation of endothelial cell lumen compared to the negative control group, and the endothelial cells of the blank and negative control groups exhibited distinct cellular structures, whereas the endothelial cells of the V19 experimental group and the ES2 positive control group exhibited a single-layer adherent state with only a small amount of tubular structures. The results indicate that the V19 polypeptide has strong in vitro anti-angiogenesis capacity.
Example 3 influence of V19 peptide on proliferation of mouse breast cancer cells
The log phase growing 4T1 cells were collected, the cell suspension was adjusted to the appropriate concentration, and 5×103 cells per well were seeded into 96-well plates, placed in a carbon dioxide incubator, and the 96-well plates were cultured overnight at 37 ℃ until the cells attached. V19 and ES2 (an anti-angiogenic peptide that inhibits endothelial cell proliferation but does not act on tumor cells, as a control group herein) were then added at drug concentrations of 1 μg/mL, 5 μg/mL, 10 μg/mL, 50 μg/mL, 100 μg/mL, 500 μg/mL, 1mg/mL, respectively, with 8 multiple wells per drug. Wells containing DMEM medium alone were set as blank control groups, and wells with cells and without drug-containing medium were set as negative control groups. The 96-well plate is placed in a carbon dioxide incubator for 48 hours, the culture medium is discarded under the condition of light shielding, CCK-8 solution is added into the 96-well plate, 10 mu L of each well is placed in the incubator, the culture medium is taken out after the color of the culture medium turns orange, then the OD value of each well under the condition of 450nm wavelength is detected by an enzyme-labeling instrument, and the calculation formula of the cell proliferation rate is shown as follows, wherein the cell proliferation rate is = [ (experimental group-blank control group)/(negative control group-blank control group) ]multipliedby 100%.
As shown in fig. 4, it can be seen that the cell proliferation rate of the V19 polypeptide group significantly decreased with increasing concentration compared with the ES2 polypeptide in the control group, which indicates that the V19 polypeptide has a significant inhibitory effect on proliferation of 4T1 cells, and thus has good in vitro anti-tumor activity.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.