CN112630444A - Polypeptide screening method based on target protein - Google Patents
Polypeptide screening method based on target protein Download PDFInfo
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- CN112630444A CN112630444A CN202011447276.6A CN202011447276A CN112630444A CN 112630444 A CN112630444 A CN 112630444A CN 202011447276 A CN202011447276 A CN 202011447276A CN 112630444 A CN112630444 A CN 112630444A
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Abstract
The invention provides a polypeptide screening method based on target protein. The screening method comprises the following steps: detecting the target protein with the fluorescent marker by utilizing a polypeptide chip technology to obtain a polypeptide set with different binding signal strengths; sequencing the polypeptide sequences in the polypeptide collection according to the binding signal intensity; selecting a predetermined number of polypeptide sequences which are ranked at the top as candidate polypeptides of the target protein. By utilizing the polypeptide chip technology, a large number of target proteins can be screened for a plurality of potential candidate functional polypeptides in a short time, and the method has the characteristics of low cost, high flux, high candidate polypeptide binding rate, high accuracy and the like. Particularly for target protein S of the new coronary pneumonia, the screening method can screen out polypeptide which can competitively reduce or inhibit the combination of the S protein and ACE2 protein in high flux, thereby weakening or inhibiting the infection of the new coronary virus to human body.
Description
Technical Field
The invention relates to the field of polypeptide screening, in particular to a polypeptide screening method based on target protein.
Background
The screening method based on the target protein can screen and obtain peptide fragments with specific functions, for example, the screened peptide fragments can have specific medical applications, for example: drugs, anti-infective agents, and the like. The method can screen active small molecular polypeptide with direct effect on target. Unlike phenotype-based screening, target-based polypeptide screening is a quantitative assay for biochemical level interactions of proteins with specific physiological functions or distinct mechanisms mixed with candidate compounds, commonly used methods include enzyme-linked immunosorbent assay, fluorogenic visualization, nuclear magnetic resonance, and the like.
The traditional polypeptide screening method based on the target protein has certain limitation, generally needs to synthesize candidate polypeptide first and then carry out screening after mixing with the target protein, has low flux and high cost, and is difficult to carry out simultaneous screening on a plurality of binding sites of the same target.
Therefore, there is still a need for improvement of the existing screening method for candidate polypeptides of target proteins to improve the screening throughput of the existing screening method.
Disclosure of Invention
The invention mainly aims to provide a polypeptide screening method based on a target protein, and provides a basis for developing a new polypeptide aiming at the target protein.
In order to achieve the above objects, according to one aspect of the present invention, there is provided a method for screening a polypeptide based on a target protein, the method comprising: detecting the target protein with the fluorescent marker by utilizing a polypeptide chip technology to obtain a polypeptide set with different binding signal strengths; sequencing the polypeptide sequences in the polypeptide collection according to the binding signal intensity; selecting a predetermined number of polypeptide sequences which are ranked at the top as candidate polypeptides of the target protein.
Further, detecting the target protein with the fluorescent label by using a polypeptide chip technology to obtain a polypeptide set with different binding signal intensities comprises: diluting the fluorescence-labeled target protein at multiple concentrations to obtain multiple samples to be detected at different concentrations; and respectively incubating a plurality of samples to be detected with the polypeptide chip, thereby obtaining a polypeptide set with different binding signal intensities with the target protein under different concentrations.
Further, ordering the polypeptide sequences in the collection of polypeptides by binding signal strength comprises: calculating the signal intensity value of the corresponding characteristic of each polypeptide sequence in the polypeptide set combined with the target protein under each concentration, and sequencing according to the signal intensity value; preferably, the mean or median of the signal intensity of the corresponding features of each polypeptide sequence in the collection of polypeptides that bind to the target protein at each concentration is calculated and ranked according to the mean or median; more preferably, the signal intensity values for the corresponding features of each polypeptide sequence at each concentration are log10 transformed and sorted by mean or median of the transformed log10 values.
Further, selecting a predetermined number of top-ranked polypeptide sequences as candidate polypeptides for the target protein includes: selecting as a first candidate set a first predetermined number of polypeptide sequences, all ordered in a set ratio of concentration ranges; removing a second predetermined number of polypeptide sequences from the first candidate set that are top ranked in the blank control and the standard control to obtain a second candidate set; calculating the average value of each peptide fragment sequence in the second candidate set under different concentrations; and sorting each peptide segment in the second candidate set according to the average value, and selecting a third predetermined number of the polypeptide sequences as candidate polypeptides.
Further, the target protein is a protein derived from a virus or a target protein of a tumor; preferably, the virus is selected from any one of respiratory tract infection viruses; more preferably, the respiratory infection virus includes any one of influenza virus, parainfluenza virus, syncytial virus, coronavirus, metapneumovirus and adenovirus; preferably, the coronavirus includes any one of SARS-CoV, SARS-CoV-2 and MERS-CoV; preferably, the tumor is selected from any one of: lung cancer, gastric cancer, colorectal cancer, liver cancer, breast cancer, esophageal cancer, thyroid cancer, cervical cancer, brain cancer, and pancreatic cancer; more preferably, the target protein for lung cancer is a voltage-gated calcium channel; the target protein of the gastric cancer is an estrogen receptor; the target protein of the colorectal cancer is an epidermal growth factor receptor erbB 1; the target protein of the liver cancer is cyclooxygenase or Beta adrenergic receptors (Beta adrenergic receptors); the target protein of the breast cancer is a glucocorticoid receptor; the target protein of the esophageal cancer is Gamma-aminobutyric acid A receptor (Gamma-aminobutyric acid A receptor); the target protein of the thyroid cancer is a stem cell growth factor receptor or a vascular endothelial growth factor receptor 2; the target protein of the cervical cancer is an interferon alpha/beta receptor; the target protein of brain cancer is glucocorticoid receptor; the target protein for pancreatic cancer is cyclooxygenase.
Further, the target protein is any one of the following proteins: spike protein of SARS-CoV-2, spike protein of SARS-CoV, AKT protein kinase, bromodomain-containing protein, glucagon receptor, Syk tyrosine kinase, TrkA receptor, TLR-4, CD40 ligand receptor, mTOR complex 2, G protein-coupled receptor 44, beta amyloid protein, phosphate receptor 1, Hsp 90, PDE 10, oxalate VR1, cyclin-dependent enzyme 9, nucleoprotein alpha, MAP kinase, t-cell surface glycoprotein CD8, tau-polymerization and apoptosis protein inhibitors; preferably, the target protein is a recombinant protein of the receptor binding region of the spike protein of SARS-CoV-2; or the target protein is a target protein of a rare disease; preferably, the rare disease is selected from any one of: hemophilia, pituitary adenoma, Duchenne muscular dystrophy, spinocerebellar ataxia, autosomal dominant polycystic kidney disease, primary dystonia, myasthenia gravis, cushing's syndrome, congenital adrenal cortical hyperplasia, hyperphenylalaninemia, multiple sclerosis, early onset muscular dystrophy, and idiopathic hypogonadotropic hypogonadism; more preferably, the target protein for hemophilia is any one of coagulation factor X, prothrombin and fibrinogen; the target protein of pituitary adenoma is glucocorticoid receptor or dopamine D2 receptor; the target protein of Duchenne muscular dystrophy is glucocorticoid receptor or voltage-gated calcium channel; the target protein of spinocerebellar ataxia is any one of erythropoietin receptor, potassium ion channel and peroxisome proliferation activating receptor; the target protein of autosomal dominant polycystic kidney disease is any one of epidermal growth factor receptor erbB1, receptor protein tyrosine kinase erbB-2 and nuclear factor kappa B; the target protein of the primary dystonia is cyclooxygenase or glucagon-like peptide 1 receptor; the target protein of myasthenia gravis is acetylcholinesterase or a 5-hydroxytryptamine receptor; the target protein of the cushing syndrome is any one of glucocorticoid receptor, HMG-CoA reductase and lanosterol 14 alpha demethylase; the target protein of the congenital adrenal cortical hyperplasia is any one of glucocorticoid receptor, mineralocorticoid receptor and adrenocortical hormone releasing factor receptor 1; the target protein of hyperphenylalaninemia is phenylalanine hydroxylase; the target protein of multiple sclerosis is glucocorticoid receptor; the target protein of the early muscular dystrophy is any one of glucocorticoid receptor, cyclooxygenase-2 and cyclooxygenase-1; the target protein of the idiopathic hypogonadotropic hypogonadism is any one of estrogen receptor, androgen receptor and gonadotropin-releasing hormone receptor.
Further, the target protein is a recombinant protein of a receptor binding region of a spike protein of SARS-CoV-2, and the obtaining of a plurality of samples to be detected with different concentrations by performing a plurality of concentration gradient dilutions on the fluorescence labeled target protein comprises: the recombinant protein with the fluorescent label is respectively expressed according to the weight ratio of 1:500, 1:1000, 1:5000, 1:10000, 1:50000 and 1: 500000 and 1: diluting at 5000000 concentration to obtain multiple samples to be detected with different concentrations.
Further, after obtaining the candidate polypeptide, the screening method further comprises: candidate polypeptides are synthesized according to the sequence and further validated to determine the peptide fragment of interest.
Further, further validation of the candidate polypeptide to determine the peptide fragment of interest includes: carrying out in-vitro binding experiment verification on the candidate polypeptide and the target protein and determining a target peptide segment according to a verification result; and/or incubating the candidate polypeptide with SARS-CoV-2 and host cells to carry out cell experiment verification and determining the target peptide segment according to the verification result.
Further, before the polypeptide chip technology is used for detecting the target protein with the fluorescent label, the screening method further comprises the following steps: selecting a target protein according to the indication; and carrying out fluorescence labeling on the target protein to obtain the target protein with the fluorescence label.
By applying the technical scheme of the invention, the screening method provided by the application can complete screening of a plurality of potential candidate functional polypeptides for a large number of target proteins (the daily flux can reach 1000 samples, and compared with the fragment-based drug design in the modern drug design method, the screening method has the advantage of high flux) in a short time by utilizing the polypeptide chip technology, and has the characteristics of low cost, high flux, high candidate polypeptide binding rate, high accuracy and the like.
In particular, aiming at SARS-CoV-2 spike protein, the polypeptide which can competitively reduce or inhibit the combination of S protein and ACE2 protein can be screened out with high flux, thereby weakening or inhibiting the infection effect of new coronavirus to human body.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:
fig. 1 shows SEQ ID NO: 7, a schematic representation of the specific binding of the polypeptide to the S protein; and
fig. 2 shows SEQ ID NO: 8 and S protein.
Detailed Description
It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail with reference to examples.
Interpretation of terms:
the polypeptide chip is a chip based on a substrate material, the chip comprises the characteristics of pre-designed quantity, positions and sequences, one characteristic is a cluster of polypeptides with the same sequence, the polypeptide sequences between the characteristics are different frequently, and the characteristics form a high-density polypeptide array.
The polypeptide chip technology is a detection technology based on a polypeptide chip, and is characterized in that a plurality of polypeptides on the polypeptide chip are contacted with a sample, then, each characteristic signal (specifically, a fluorescence image carrying each characteristic signal) on the polypeptide chip is acquired by a fluorescence detection technology image acquisition technology, and then, the signal intensity of each characteristic in the chip is output, namely, the detection result data of the polypeptide chip. And a sample detection signal is output, and the analysis of an object to be detected in a sample combined with the polypeptide on the polypeptide chip, the analysis of the sample and the like can be realized based on the sample detection signal output by the polypeptide chip detection result data.
As mentioned in the background, the existing drug screening methods based on pathogen target protein have the problems of low throughput and low screening efficiency, and in the exemplary embodiment of the present application, a polypeptide screening method based on the target protein is provided, which comprises: detecting the target protein with the fluorescent marker by utilizing a polypeptide chip technology to obtain a polypeptide set with different binding signal strengths; sequencing the polypeptide sequences in the polypeptide collection according to the binding signal intensity; selecting a predetermined number of polypeptide sequences which are ranked at the top as candidate polypeptides of the target protein.
It should be noted that, the predetermined number of polypeptide sequences ranked at the top can be the polypeptide sequences ranked at the top N, where N is a positive integer greater than or equal to 1; the polypeptide can also be arranged in M or more and less than N, wherein M and N are positive integers of 1 or more, and M is less than N.
The screening method can complete screening of a plurality of potential polypeptide drugs for a plurality of target proteins in a short time by utilizing a polypeptide chip technology, and has the characteristics of low cost, high flux, high binding rate of candidate functional polypeptides, high accuracy (the screened peptide fragments can be specifically bound with the target proteins, and the accuracy is higher compared with other methods for screening lead compounds), and the like.
For example, the detection of 24 samples can be realized simultaneously by using a V13 chip from Health Tell company, each V13 chip has 24 repeated polypeptide arrays, each array has 131712 polypeptides (features), and the polypeptides can be respectively combined with proteins in a sample to be detected to capture protein distribution information in the sample. The 131712 polypeptides are polypeptide sequences formed by 5-13 random amino acid combinations without deviation, and can achieve 99.9% of diversity coverage of tetramer (4 peptides) and 48.3% of diversity coverage of pentamer (pentapeptides). And the polypeptide chip technology platform of Health Tell company can detect 24 chips at the same time at present.
In a preferred embodiment, the detecting the target protein with fluorescent label by polypeptide chip technology to obtain a collection of polypeptides with different binding signal intensities comprises: diluting the fluorescence-labeled target protein at multiple concentrations to obtain multiple samples to be detected at different concentrations; and respectively incubating a plurality of samples to be detected with the polypeptide chip, thereby obtaining a polypeptide set with different binding signal intensities with the target protein under different concentrations.
Diluting the fluorescence-labeled target protein into multiple concentrations for polypeptide chip detection, screening out the polypeptides which show strong binding signals with the target protein under multiple different concentrations, and further according to the increase along with the dilution times, if the binding signal strength does not become lower along with the increase of the dilution concentrations, namely the strong binding signals are always shown under different concentrations, the specific binding of the peptide fragment and the target protein can be proved.
The target protein is fluorescently labeled before detection, so that the diluted target protein with different concentrations is respectively incubated with the polypeptide chip to obtain a polypeptide set with different binding signal intensities with the target protein under different concentrations, and a fluorescence intensity signal is not required to be obtained by incubation with a second antibody with fluorescence.
In other embodiments, such as the polypeptide chip technology of Health Tell corporation, the target protein does not have a fluorescent label, and the sample to be tested containing the target protein needs to be incubated with the polypeptide chip to obtain a first incubation product; and then, incubating the first incubation product with a fluorescent secondary antibody to obtain a polypeptide set combined with the target protein and having different signal intensities.
The polypeptides bound to the target protein at different concentrations are sorted according to the binding signal from strong to weak, and the specific sorting operation mode is not limited as long as the polypeptides are sorted according to the binding signal strength. In a preferred embodiment, ordering the polypeptide sequences in the collection of polypeptides by binding signal strength comprises: and calculating the signal intensity value of the corresponding characteristic of each polypeptide sequence in the polypeptide set combined with the target protein at each concentration, and sequencing according to the signal intensity value.
In order to obtain more accurate signal intensities, the mean or median value of the signal intensities of the corresponding features of each of the polypeptide sequences in the collection of polypeptides that bind to the target protein is calculated and ranked according to the mean or median value in a further preferred embodiment. More preferably, the signal intensity values for the corresponding features of each polypeptide sequence at each concentration are log10 transformed and sorted by mean or median of the transformed log10 values. And the log10 value is converted, and the absolute value of the data is reduced, so that the calculation is convenient.
It should be noted that the main purpose of the log10 transformation is to optimize the value distribution, which is only a preferred way to normalize the specific values of the signal strength values, and other transformation ways to normalize the values may be used (e.g., taking logarithms (e.g., ln, log2, log20, etc.) from the original signal strength values, or taking quadratic calculations based on log10 (e.g., reciprocal of log 10), or using quantile transformation or Rank Guass, etc.
In the above step of rank screening candidate polypeptides satisfying the predetermined number, the screening criteria are: 1) ranking all top at multiple concentrations; 2) not in the top-ranked positions of the blank and standard controls; 3) ranked top by log mean.
In a preferred embodiment, selecting a predetermined number of top-ranked polypeptide sequences as candidate polypeptides for a target protein comprises: selecting as a first candidate set polypeptide sequences having log-value orderings all leading by a first predetermined number over a set proportion of a concentration range (e.g., at least 70%, 75%, 80%, 85%, 90%, or 95% or more); removing a second preset number of polypeptide sequences with the log value ordering being equal to that in the blank control and the standard control from the first candidate set to obtain a second candidate set; calculating the mean value of log values of each peptide fragment sequence in the second candidate set under different concentrations; and sorting each peptide segment in the second candidate set according to the average value, and selecting a third predetermined number of the polypeptide sequences as candidate polypeptides.
In the above preferred embodiment, the specific values of the first predetermined amount, the second predetermined amount and the third predetermined amount are not limited, and can be set reasonably according to the specific screening condition of the specific target protein. However, it should be noted that there is a gradually decreasing trend among the three. For example, if the first predetermined number is 500; the second predetermined number is 100, the third predetermined number is 50, 40, 30, 20, 10, 9, or 8, or any number between 10-50.
The screening method of the present application is applicable to target proteins of various pathogens, preferably proteins of viral origin. The virus includes, but is not limited to, any one of respiratory tract infection viruses. Preferably, the respiratory infection virus includes, but is not limited to, any one of influenza virus, parainfluenza virus, syncytial virus, coronavirus, metapneumovirus and adenovirus. Preferably, the coronavirus includes any one of SARS-CoV, SARS-CoV-2 and MERS-CoV.
From the viewpoint of indication, the target protein against which the screening method of the present application can be directed includes, but is not limited to, any one of the following: spike protein of SARS-CoV-2, spike protein of SARS-CoV, AKT protein kinase (AKT protein kinase), Bromodomain-containing protein (Bromodemains conjugation protein), Glucagon receptor (Glucaon receptor), Syk tyrosine kinase (Syk tyrosine kinase), TrkA receptor (TrkA receptor), TLR-4(TLR-4), CD40 ligand receptor (CD40 ligand receptor), mTOR complex 2(mTOR complex 2), G protein-coupled receptor 44(G-protein linker-44), Beta amyloid protein (Beta amyloid synthase), phosphate receptor 1(Sphingosine-1-phosphate linker-1), Hsp 90, PDE 10, oxalic acid VR1 (MAP 6754), Cyclin-dependent glycoprotein-9 (Cyclin-dependent kinase-9), cell surface glycoprotein alpha kinase (CD 57) and cell surface glycoprotein alpha kinase (CD) 8), cell surface glycoprotein alpha kinase (CD kinase-34-alpha kinase), Tau aggregation (Tau aggregation) and inhibitor of apoptosis proteins (apoptosis protein inhibitor).
The same applies to tumor and rare disease targets. The tumor and its related target protein include, but are not limited to, any one of the following: lung cancer target Voltage-gated calcium ion channels (Voltage-gated calcium channels), gastric cancer target Estrogen receptors (Estrogen receptors), colorectal cancer target Epidermal growth factor receptors erbB1 (epidermalogenreceptor erbB1), liver cancer target cyclooxygenase (cycloxygenes) and Beta adrenergic receptors (Beta adrenergic receptors), breast cancer target Glucocorticoid receptors (Glucocorticoid receptors), esophageal cancer target Gamma-aminobutyric acid A receptors (Gamma-aminobutyryc A receptors), thyroid cancer target Stem cell growth factor receptors (Stem cell growth factor receptors) and Vascular endothelial growth factor receptor 2 (Vaselendorphyrin growth factor receptor 2), cervical cancer Interferon alpha/Beta receptors (hormone/Beta receptors), brain cancer target and pancreatic cancer (pancreatic cancer) cancers and pancreatic cancer cancers.
Rare diseases and their targets include, but are not limited to, any of the following: hemophilia target coagulation factor X (coagulation factor X), Prothrombin (Prothrombin) and Fibrinogen (Fibrinogen), pituitary adenoma target Glucocorticoid Receptor (glucoportic Receptor) and Dopamine D2 Receptor (Dopamine D2 Receptor), Duchenne type dystrophia target Glucocorticoid Receptor (glucoportic Receptor) and Voltage-gated calcium channel (Voltage-gated calcium channel), spinocerebellar ataxia-ataxia Erythropoietin Receptor (osteoproportin Receptor), Potassium channel (Potassium channel) and Peroxisome proliferation-activating Receptor (Peroxisome-activated Receptor gamma), autosomal dominant nephrotic Epidermal growth factor Receptor B1(Epidermal growth factor B1), tyrosine-2 Receptor B2-tyrosine kinase (tyrosine-kinase B2-gene) and factor B2-reductase B), primary dystonia target cyclooxygenase (cycloxygenases) and Glucagon-like peptide 1receptor (Glucagon-like peptide 1receptor), myasthenia gravis target Acetylcholinesterase (Acetylcholinesterase) and 5-hydroxytryptamine receptor (Serotonin 3(5-HT3) receptor), Cushing syndrome target Glucocorticoid receptor (Glucocorticoid receptor), HMG-CoA reductase (HMG-CoA reductase) and Lanosterol 14 alpha demethylase (lanocorticoid 14 alpha-congenital dehydrogenase), adrenocorticotropin-proliferator Glucocorticoid receptor (Glucocorticoid target), Mineralocorticoid receptor (Mineralocorticoid receptor) and adrenocorticoid releasing factor receptor 1 (Glucocorticoid-Corticotropin receptor 1), homophenylalaninemia target Phenylalanine hydroxylase (Phenylalanine hydroxylase target), multiple sclerosis (glucoronidase receptor), and multiple sclerosis (Glucocorticoid receptor) with multiple sclerosis, Cyclooxygenase-2 (Cyclooxogene-2) and Cyclooxygenase-1 (Cyclooxogene-1), and, idiopathic hypogonadotropic hypogonadism-targeted Estrogen receptors (Estrogen receptors), Androgen receptors (Androgen receptors), and Gonadotropin-releasing hormone (Gonadotropin-releasing hormone).
In a preferred embodiment, the target protein is a protein derived from SARS-CoV-2; preferably, the target protein is the spike protein of SARS-CoV-2; more preferably, the target protein is a recombinant protein of the receptor binding region of the spike protein of SARS-CoV-2. The spike protein is screened for the ability to inhibit or attenuate the binding of SARS-CoV-2 to human cells, thereby reducing or preventing infection of the human.
In the step of diluting the target protein into a plurality of different concentrations, the specific diluted concentration can be reasonably set according to different target proteins. In a preferred embodiment, the target protein is a recombinant protein of the receptor binding region of the spike protein of SARS-CoV-2, and the diluting the fluorescently labeled target protein with a plurality of concentration gradients to obtain a plurality of test samples with different concentrations comprises: the recombinant protein with the fluorescent label is respectively expressed according to the weight ratio of 1:500, 1:1000, 1:5000, 1:10000, 1:50000 and 1: 500000 and 1: diluting at 5000000 concentration to obtain multiple samples to be detected with different concentrations.
To further confirm that the candidate polypeptide being screened is inhibiting infection of a host cell (e.g., human epithelial cell) by a pathogen (e.g., SARS-CoV-2), in a preferred embodiment, after obtaining the candidate polypeptide, the screening method further comprises performing further validation on the candidate peptide fragment to determine the peptide fragment of interest: based on the synthesized candidate polypeptide, the candidate polypeptide is functionally verified to determine the peptide fragment of interest. The target peptide fragment refers to the first A peptide fragments with optimal performance obtained after verification and comprehensive judgment, wherein A is a natural number. Any existing validation method capable of validating a candidate polypeptide is suitable for use in the present application. Preferably, functionally validating the candidate polypeptide to determine the peptide fragment of interest comprises: performing in vitro binding experimental verification on the candidate polypeptide and the target protein to determine the binding effect (for example, by binding the synthesized candidate polypeptide and the target protein with fluorescence and further confirming the binding effect by checking the fluorescence intensity); or incubating the candidate polypeptide with SARS-CoV-2 and the host cell for cell level experimental verification (for example, counting the infection rate of the cell to verify the inhibition effect or degree of the candidate polypeptide on the binding of SARS-CoV-2 to the host cell).
In a preferred embodiment, before the polypeptide chip technology detection of the target protein with fluorescent label, the screening method further comprises: selecting a target protein according to the indication; and carrying out fluorescence labeling on the target protein to obtain the target protein with the fluorescence label.
As mentioned above, the target protein is selected for the corresponding indication according to the diseases caused by different pathogens. Target proteins were selected according to the indication, in the case of COVID-19, with 5 essential genes, directed against 4 structural proteins, nucleoprotein (N), viral envelope (E), matrix protein (M) and spike protein (S), and RNA-dependent RNA polymerase (RdRp). The nucleoprotein (N) wraps the RNA gene to form a nucleocapsid, the nucleocapsid surrounds the viral envelope (E), and the matrix protein (M), the spike protein (S) and other proteins are embedded in the viral envelope. Spike proteins enter cells by binding to angiotensin converting enzyme 2 (ACE-2). When the novel coronavirus is isolated and cultured in vitro, the novel coronavirus can be found in human respiratory epithelial cells within about 96 hours, and the isolation and culture in VeroE6 and Huh-7 cell lines take about 4-6 days. Coronavirus is sensitive to ultraviolet rays and heat, lipid solvents such as ether, 75% ethanol, chlorine-containing disinfectant, peracetic acid, chloroform and the like can effectively inactivate the virus at the temperature of 56 ℃ for 30 minutes, and chlorhexidine cannot effectively inactivate the virus. Thus, any of the above proteins of SARS-CoV-2, preferably the spike protein thereof, and more preferably the receptor binding domain recombinant protein of the spike protein thereof, can be selected as the target protein.
The specific operation of carrying out fluorescence labeling on the target protein can be carried out by using a commercially available fluorescence labeling kit, and the specific selection of the fluorescence labeling is not limited as long as the fluorescence labeling with the recognizable signal intensity on the target protein can be carried out. For example, GFP, RFP, FITC, CY3, CY5, luminol, isoluminol, etc. can be mentioned.
The advantageous effects of the present application will be further described with reference to specific examples.
EXAMPLE 1 screening of the Polypeptides
In this example, screening of candidate polypeptides having resistance to the novel coronavirus was performed by using the receptor binding region (RBD, i.e., amino acids 319 to 541 of S protein) of spike protein (S) as a target protein. The specific screening steps are as follows:
1. for the novel coronavirus S-RBD protein (recombinant protein, GenScript Co., Ltd.; specific sequence) SEQ ID NO: 9) and carrying out fluorescence labeling.In particular adopting a fluorescence labeling kit (Alexa)
555Protein Labeling Kit (A20174)), the specific process is as follows.
(1) The operation flow of the S-RBD protein fluorescence labeling experiment is as follows:
a. preparing 1M sodium carbonate buffer solution: 1ml of distilled water was added to a component B glass bottle (84mg of sodium carbonate), and mixed well to dissolve it.
b. Protein dilution: if the protein concentration is greater than 2mg/mL, the protein is diluted to 2mg/mL (e.g., 1mg protein in 0.5mL 1 XPBS or 0.1M sodium carbonate buffer).
c. Adjusting the pH value: add 50. mu.L of 1M sodium carbonate buffer (from step a) to 0.5mL of 1 XPBS containing protein; the final pH value is 7.5-8.3. This step can be skipped if the protein has been placed in sodium carbonate buffer (pH 8.0-8.3) or PBS (pH 8.0).
d. Preparing a labeling reaction mixed solution: the protein solution (from step c) was added to a glass vial containing the reactive dye (component A). Covering the bottle cap, and gently inverting or slightly blowing and beating the mixture through a gun head to mix the mixture evenly. The reaction mixture was stirred to avoid foaming.
e. And (3) incubation labeling reaction: magnetically stir at low speed for 1 hour at room temperature. Care was taken to avoid foaming of the solution.
Note that: since it takes 15 minutes to prepare the purification column, the purification column can be filled at the time of the labeling reaction of the step e.
(2) Experimental operational flow for purifying labeled S-RBD protein:
a. preparation of purification column/purification resin: the purification column was assembled and the purification resin (component C) was added to the top 3 cm. Add 1 XPBS to check whether the buffer was able to flow through. If buffer flow is blocked, the resin is removed, the glass frit is cleaned and refilled with resin/buffer. The excess buffer solution needs to be drained before the labeled protein is added.
b. Configuration 1X elution buffer: 10 Xelution buffer (component D) was diluted 10-fold with distilled water at room temperature. Normally, the amount of single purification is not more than 10 mL.
c. Protein purification: the funnel was removed. Pouring the reaction liquid onto the resin of the purification column; wait for the reaction solution to be melted into the resin. The proteins were eluted with elution buffer or 1X PBS and all remaining solution was collected and retained.
d. DOL (degree of labeling) was calculated and labeled protein was stored.
2. Performing concentration gradient dilution on the new crown S-RBD protein which is marked with fluorescence, and detecting through a polypeptide chip and S-
RBD interaction specific peptide fragments, and the fluorescence signal of the peptide fragments is reduced along with the increase of dilution.
The polypeptide chip detection method comprises the following steps:
1) sample preparation
The experimental concentration of the S-RBD protein which is subjected to the fluorescent labeling is set as a sample to be detected with 7 concentration gradients (serial dilution of 1:500, 1:1000, 1:5000, 1:10000, 1:50000, 1: 500000 and 1: 5000000) for later use.
2) Hydration and assembly of chips
The chip is placed in a chip hydration tool, ultrapure water is added to submerge the chip, and the chip is hydrated for 20min at 55 +/-5 rpm/min on an orbital shaker. Then spraying isopropanol on the surface of the chip, and putting the chip into a centrifugal machine for centrifugal drying. The dried chips were assembled into assay cassettes according to the positions designed for the experiment.
3) Incubation binding of sample to chip
The diluted sample was added to the assembled chip at 90. mu.L/well, and incubated on a constant temperature shaker for 1 hour with shaking.
4) Sample cleaning
The assay cassette is placed in a plate washer for washing.
5) Imaging
The chip in the assay cassette is assembled into an imaging cassette after being disassembled, cleaned and dried, and then the imaging cassette is put into an ImageXpress micro 4 imager of Molecular Device company for scanning imaging. And finally, obtaining a TIFF picture file as the original data by each detection sample.
In this embodiment, the polypeptide chip technology platform is used to capture signals of the target protein and the polypeptide chip set, and then the signals are converted to obtain data. The instrumentation used in the polypeptide chip technology includes polypeptide chips (such as Health toll V13 chip, model P/N:600001V13 Slides), fluorescence imaging devices (such as molecular Device Image Xpress Micro-4), chip centrifuges (such as Labnet C1303T-230V), plate washers (such as BioTek Instruments, 405TSUVS), 96-well plate orbital shakers (such as Thermo scientific, 88880026), and thermostatic mixers (such as Eppendorf Thermomixer C).
4. Data pre-processing
1) And extracting fluorescence intensity values of the features and outputting 1 GPR5 data file and 1 corn images file. The GPR5 file contains all information for a sample and fluorescence intensity information for all features.
2) Fluorescence intensity information for features was extracted from GPR5 data files for all samples, yielding a raw fluorescence intensity (FG) data matrix. Then, the data of each sample are subjected to logarithmic transformation to obtain an LFG (log-transformed formed) data matrix, and the Z-score normalization processing is carried out to obtain an NLFG (normalized and log-transformed formed) data matrix. This step also generates a sample chip information file that includes information such as the sample array location, the chip number used, etc.
5. Quality control
The quality control of the sample and the system is qualified by the quality control method carried by Health Tell.
6. Screening for polypeptide sequences that specifically bind to S-RBD protein: and after the background signal is eliminated, the signal peptide fragments which are close to each other under different concentrations are sorted according to the signal intensity without supersaturation.
6.1) converting each signal intensity into a log10 value, and then calculating the median of log10 values of three technical repetitions feature by feature for each concentration of S-RBD protein samples (three technical repetitions of each sample);
6.2) for the median sample of each concentration, convert the values to descending order (descending order: desblending, for features with the same value, using method min (lowest rank in group));
6.3) for a peptide, if the rank ordering of a peptide in the concentration of more than 70 percent is within the top 100, the peptide is included into a candidate;
6.4) removing irrelevant samples (i.e. the peptide fragment at rank top 5000 in the blank, standard) from all candidate peptide fragments;
6.5) sorting the peptide segments according to rank mean value, and selecting the peptide segments with the top rank.
7. The sequence of the candidate polypeptide screened by the method is as follows:
SEQ ID NO：1：YAYEYVFFSE；
SEQ ID NO：2：PFFFFEG；
SEQ ID NO：3：QVVEVFWLFD；
SEQ ID NO：4：NYVFFFEG；
SEQ ID NO：5：RLEFLFLFE；
SEQ ID NO：6：QYLFFLEG；
SEQ ID NO：7：PVFLVFPQRG；
SEQ ID NO：8：QSLFLEVFS。
example 2 polypeptide validation
And (3) comparing all the candidate peptide fragments with a target protein (S protein) by utilizing blast software, and selecting bit score >14 as a threshold capable of being compared, thereby showing that the peptide fragments have certain similarity with the target protein sequence. Based on the results, the above peptide fragments were further annotated. Depending on the specific requirements, the results are adjusted (e.g., if one wishes to add one or two "sequences that can be aligned to the target protein", it can be selected from the alignment results even if the ranking is not the highest).
Based on the S protein sequence alignment analysis, the similarity of the candidate polypeptide and the S protein sequence is calculated, and based thereon, the candidate polypeptide sequences can be classified into different types:
a) the peptide segment (2) matched with the S protein sequence is similar, namely the corresponding polypeptide is a potential linear epitope of the S protein, or the scientificity of the combination with the S protein can be explained;
b) peptide fragments that do not match the sequence of the S protein, but have high signal intensity (the remaining 6), may be nonlinear binding epitopes of the protein, or may be derived from unknown binding epitopes.
Example 3
The verification result of the known binding site of the polypeptide sequence on the S-RBD protein and the excavation of the potential binding site
In peptides that bind to S-RBD protein and have signal intensities ranked in the top order, if the binding force of these peptides to S-RBD protein is weak or the signal intensity of these peptides to non-S-RBD protein specifically binds to the peptides, the signal intensities of these peptides become weaker with concentration after the concentration of S-RBD protein is decreased, making the ranking in the bottom order relative to other peptides. On the contrary, if the signal intensity of the peptide fragment is not weakened along with the reduction of the concentration of the S-RBD protein and is reflected to be kept forward in the sequencing of the peptide fragment, the specific binding of the peptide fragment and the S-RBD protein can be verified, and the binding force is strong.
The S-RBD protein of the new coronavirus mediates entry of the new coronavirus into cells for infection by binding to angiotensin converting enzyme 2(ACE2) on host cells. When the polypeptide is combined with the S-RBD protein, the S-RBD protein cannot be combined with the ACE2 receptor or the binding force is weakened, so that the infection of the virus to host cells is blocked or reduced, and the polypeptide has the function of competitively inhibiting the virus infection.
The specific design verification test is as follows: 7 dilution concentrations (dilution times are respectively 500, 1000, 5000, 10000, 50000, 500000 and 5000000) are designed for the concentration of the S-RBD protein, and a blank control group and a positive standard substance control group are additionally designed. The test results are shown in Table 1, and the candidate polypeptides remain prostate in the ranking of all peptide fragment signals with increasing dilution of concentration, but do not decrease with increasing concentration gradient. The ranking of the same peptide stretch signal was significantly different in the blank control and the standard control compared to the S-RBD binding signal, indicating that these polypeptides bind strongly specifically to the S-RBD protein.
Wherein the binding sites of the polypeptides PVFLVFPQRG and QSLFLEVFS to the S-RBD are shown in Table 1 and FIGS. 1 and 2, and the binding sites of the polypeptides to the S protein are respectively marked in FIGS. 1 and 2 (the two polypeptides shown in the figure are different in binding direction to the S protein and thus appear to have different structures, GLN6 in FIG. 1 indicates that the amino acid at the position 6 indicated by the arrow is glutamine, and PRO60 in FIG. 2 indicates that the amino acid at the position 60 indicated by the arrow is proline).
Table 1: ranking binding strength of candidate polypeptides at different dilution concentrations of known binding site and potential binding site of S-RBD protein
As can be seen from the above table of the sequence of the binding strength, the 8 polypeptide sequences have stronger binding force with the S protein than the control.
In another embodiment, the peptide fragment of the present invention is prepared into an aqueous solution as an experimental group, a positive control and a negative control are set, and the aqueous solution is mixed with SARS-CoV-2 pseudovirus at ambient temperature for incubation, and the incubated solution is inoculated into a cell prepared in advance for incubation, i.e., a pseudovirus neutralization experiment is performed. Neutralization experiments show that the polypeptide of the invention can block infection of host cells by pseudoviruses to a certain extent. The blocking results can be used to further evaluate the ability of each peptide fragment to resist SARS-CoV-2 infection.
As can be seen from the description of the above examples, the present application can rapidly and efficiently screen a plurality of potential functional polypeptides against target proteins by using polypeptide chip technology, and the polypeptides can competitively reduce or inhibit the binding of S protein and ACE2 protein, thereby weakening or inhibiting the infection effect of new coronavirus on human body. On the basis of the candidate polypeptide, the screened target polypeptide can be further confirmed to be prepared into a medicament (such as an anti-infection preparation).
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
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Claims (10)
1. A method for screening a polypeptide based on a target protein, the method comprising:
detecting the target protein with the fluorescent marker by utilizing a polypeptide chip technology to obtain a polypeptide set with different binding signal strengths;
ordering the polypeptide sequences in said collection of polypeptides according to said binding signal strengths;
selecting a predetermined number of the polypeptide sequences ranked at the top as candidate polypeptides for the target protein.
2. The screening method of claim 1, wherein detecting the target protein with fluorescent label by polypeptide chip technology to obtain a collection of polypeptides with different binding signal intensities comprises:
diluting the fluorescence-labeled target protein at multiple concentrations to obtain multiple samples to be detected at different concentrations;
and respectively incubating the multiple samples to be detected with the polypeptide chip, thereby obtaining a polypeptide set with different binding signal intensities with the target protein under different concentrations.
3. The screening method of claim 2, wherein ranking the polypeptide sequences in the collection of polypeptides according to the binding signal strengths comprises:
calculating a signal intensity value of a corresponding feature of each polypeptide sequence in the polypeptide set bound to the target protein at each concentration, and sorting according to the signal intensity values;
preferably, the mean or median of the signal intensity of the corresponding features of each polypeptide sequence in the collection of polypeptides that bind to the target protein at each concentration is calculated and ranked according to the mean or median;
more preferably, the signal intensity values for the corresponding characteristic of each of said polypeptide sequences at each concentration are log10 transformed and sorted by mean or median of the transformed log10 values.
4. The screening method of claim 3, wherein selecting a predetermined number of the top-ranked polypeptide sequences as candidate polypeptides for the target protein comprises:
selecting as a first candidate set a first predetermined number of polypeptide sequences, all ordered in a set ratio of concentration ranges;
removing a second predetermined number of polypeptide sequences from the first candidate set that are top ranked in both the blank control and the standard control to obtain a second candidate set;
calculating the average value of each peptide fragment sequence in the second candidate set under different concentrations;
and sequencing each peptide segment in the second candidate set according to the average value, and selecting a third predetermined number of the polypeptide sequences ranked at the top as the candidate polypeptides.
5. The screening method according to any one of claims 1 to 4, wherein the target protein is a protein derived from a virus; or the target protein is the target protein of the tumor;
preferably, the virus is selected from any one of respiratory tract infection viruses; more preferably, the respiratory infection virus comprises any one of influenza virus, parainfluenza virus, syncytial virus, coronavirus, metapneumovirus and adenovirus; more preferably, the coronavirus includes any one of SARS-CoV, SARS-CoV-2 and MERS-CoV;
preferably, the tumor is selected from any one of: lung cancer, gastric cancer, colorectal cancer, liver cancer, breast cancer, esophageal cancer, thyroid cancer, cervical cancer, brain cancer, and pancreatic cancer; more preferably, the target protein for lung cancer is a voltage-gated calcium channel; the target protein of the gastric cancer is an estrogen receptor; the target protein of the colorectal cancer is an epidermal growth factor receptor erbB 1; the target protein of the liver cancer is cyclooxygenase or beta adrenergic receptor; the target protein of the breast cancer is a glucocorticoid receptor; the target protein of the esophageal cancer is a gamma-aminobutyric acid A receptor; the target protein of the thyroid cancer is a stem cell growth factor receptor or a vascular endothelial growth factor receptor 2; the cervical cancer target protein is an interferon alpha/beta receptor; the target protein of the brain cancer is a glucocorticoid receptor; the target protein of the pancreatic cancer is cyclooxygenase.
6. The screening method according to any one of claims 1 to 4, wherein the target protein is any one of: spike protein of SARS-CoV-2, spike protein of SARS-CoV, AKT protein kinase, bromodomain-containing protein, glucagon receptor, Syk tyrosine kinase, TrkA receptor, TLR-4, CD40 ligand receptor, mTOR complex 2, G protein-coupled receptor 44, beta amyloid protein, phosphate receptor 1, Hsp 90, PDE 10, oxalate VR1, cyclin-dependent enzyme 9, nucleoprotein alpha, MAP kinase, t-cell surface glycoprotein CD8, tau-polymerization and apoptosis protein inhibitors; preferably, the target protein is a recombinant protein of the receptor binding region of the spike protein of SARS-CoV-2; or
The target protein is a target protein of a rare disease;
preferably, the rare disease is selected from any one of: hemophilia, pituitary adenoma, Duchenne muscular dystrophy, spinocerebellar ataxia, autosomal dominant polycystic kidney disease, primary dystonia, myasthenia gravis, cushing's syndrome, congenital adrenal cortical hyperplasia, hyperphenylalaninemia, multiple sclerosis, early onset muscular dystrophy, and idiopathic hypogonadotropic hypogonadism;
more preferably, the target protein of hemophilia is any one of coagulation factor X, prothrombin and fibrinogen; the target protein of the pituitary adenoma is a glucocorticoid receptor or a dopamine D2 receptor; the Duchenne muscular dystrophy target protein is a glucocorticoid receptor or a voltage-gated calcium channel; the target protein of spinocerebellar ataxia is any one of an erythropoietin receptor, a potassium ion channel and a peroxisome proliferation activation receptor; the target protein of the autosomal dominant polycystic kidney disease is any one of an epidermal growth factor receptor erbB1, a receptor protein tyrosine kinase erbB-2 and a nuclear factor kappa B; the target protein of the primary dystonia is a cyclooxygenase or glucagon-like peptide 1 receptor; the target protein of the myasthenia gravis is acetylcholinesterase or a 5-hydroxytryptamine receptor; the target protein of the cushing syndrome is any one of glucocorticoid receptor, HMG-CoA reductase and lanosterol 14 alpha demethylase; the target protein of the congenital adrenal cortical hyperplasia is any one of a glucocorticoid receptor, a mineralocorticoid receptor and a corticoid releasing factor receptor 1; the target protein of the hyperphenylalaninemia is phenylalanine hydroxylase; the target protein of the multiple sclerosis is a glucocorticoid receptor; the target protein of the early type muscular dystrophy is any one of glucocorticoid receptor, cyclooxygenase-2 and cyclooxygenase-1; the target protein of the idiopathic hypogonadotropic hypogonadism is any one of an estrogen receptor, an androgen receptor and a gonadotropin-releasing hormone receptor.
7. The screening method of claim 6, wherein the target protein is a recombinant protein of the receptor binding region of the spike protein of SARS-CoV-2, and the diluting the fluorescently labeled target protein with a plurality of concentration gradients to obtain a plurality of test samples with different concentrations comprises:
the recombinant protein of the fluorescent label is respectively expressed according to the weight ratio of 1:500, 1:1000, 1:5000, 1:10000, 1:50000 and 1: 500000 and 1: diluting at 5000000 concentration to obtain multiple samples to be detected with different concentrations.
8. The screening method of claim 5, wherein after obtaining the candidate polypeptide, the screening method further comprises:
based on the synthesized candidate polypeptide, the candidate polypeptide is further validated to determine a peptide fragment of interest.
9. The screening method of claim 7, wherein the further validating the candidate polypeptide to determine the peptide fragment of interest comprises:
carrying out in-vitro binding experiment verification on the candidate polypeptide and the target protein, and determining the target peptide segment according to a verification result; and/or
And (3) incubating the candidate polypeptide with the SARS-CoV-2 and the host cell for cell experiment verification, and determining the target peptide segment according to the verification result.
10. The screening method of claim 1, wherein prior to detecting the fluorescently labeled target protein using polypeptide chip technology, the screening method further comprises:
selecting the target protein according to the indication;
and carrying out fluorescence labeling on the target protein to obtain the target protein with the fluorescence label.
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| CN113484521A (en) * | 2021-05-19 | 2021-10-08 | 南京医科大学第二附属医院 | ACE2 protein biochip and preparation method and application thereof |
| CN114606345A (en) * | 2022-01-18 | 2022-06-10 | 中国中医科学院医学实验中心 | Preparation method, detection method and kit of protein thermal stability detection chip |
| WO2022202816A1 (en) * | 2021-03-22 | 2022-09-29 | ペプチエイド株式会社 | Peptide and peptide-containing composition |
| WO2023202237A1 (en) * | 2022-04-18 | 2023-10-26 | 深圳碳云智肽药物科技有限公司 | Polypeptide targeting hdac5, and use thereof in the preparation of formulation for treating or preventing individual tumors |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1903878A (en) * | 2005-07-26 | 2007-01-31 | 复旦大学 | Anti SARS virus human source nature antibody IgG Fab section |
| KR20100074743A (en) * | 2008-12-24 | 2010-07-02 | 충북대학교 산학협력단 | High-throughput screening method for antibody mimic peptides using protein chip |
| KR20110118231A (en) * | 2010-04-23 | 2011-10-31 | 한국기초과학지원연구원 | Method for screening unknown binding partners using fluorescence protein-labeled singleton gene library |
| CN102924571A (en) * | 2012-10-29 | 2013-02-13 | 复旦大学 | Rabies virus glycoprotein and nucleoprotein antigen epitope polypeptides, and screening and identification method and application thereof |
| WO2013087789A1 (en) * | 2011-12-13 | 2013-06-20 | Glykos Finland Ltd. | Antibody isoform arrays and methods thereof |
| CN105659087A (en) * | 2013-06-13 | 2016-06-08 | 比奥德赛公司 | Method of screening candidate biochemical entities targeting a target biochemical entity |
| CN111499692A (en) * | 2020-06-16 | 2020-08-07 | 国家纳米科学中心 | Polypeptide of targeting novel coronavirus COVID-19 and application thereof |
| CN111849994A (en) * | 2020-03-31 | 2020-10-30 | 厦门大学 | Aptamer of SARS-CoV-2S protein or RBD protein and application thereof |
-
2020
- 2020-12-11 CN CN202011447276.6A patent/CN112630444B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1903878A (en) * | 2005-07-26 | 2007-01-31 | 复旦大学 | Anti SARS virus human source nature antibody IgG Fab section |
| KR20100074743A (en) * | 2008-12-24 | 2010-07-02 | 충북대학교 산학협력단 | High-throughput screening method for antibody mimic peptides using protein chip |
| KR20110118231A (en) * | 2010-04-23 | 2011-10-31 | 한국기초과학지원연구원 | Method for screening unknown binding partners using fluorescence protein-labeled singleton gene library |
| WO2013087789A1 (en) * | 2011-12-13 | 2013-06-20 | Glykos Finland Ltd. | Antibody isoform arrays and methods thereof |
| CN102924571A (en) * | 2012-10-29 | 2013-02-13 | 复旦大学 | Rabies virus glycoprotein and nucleoprotein antigen epitope polypeptides, and screening and identification method and application thereof |
| CN105659087A (en) * | 2013-06-13 | 2016-06-08 | 比奥德赛公司 | Method of screening candidate biochemical entities targeting a target biochemical entity |
| CN111849994A (en) * | 2020-03-31 | 2020-10-30 | 厦门大学 | Aptamer of SARS-CoV-2S protein or RBD protein and application thereof |
| CN111499692A (en) * | 2020-06-16 | 2020-08-07 | 国家纳米科学中心 | Polypeptide of targeting novel coronavirus COVID-19 and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| SHRAVAN B. RATHOD等: "Peptide modelling and screening against human ACE2 and spike glycoprotein RBD of SARS-CoV-2", IN SILICO PHARMACOLOGY, vol. 8, no. 3, 9 November 2020 (2020-11-09), pages 1 - 10 * |
| 李敬等: "冠状病毒抑制剂研究的药物化学策略", 药学学报, vol. 55, no. 4, 26 February 2020 (2020-02-26), pages 537 - 553 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022202816A1 (en) * | 2021-03-22 | 2022-09-29 | ペプチエイド株式会社 | Peptide and peptide-containing composition |
| CN113484521A (en) * | 2021-05-19 | 2021-10-08 | 南京医科大学第二附属医院 | ACE2 protein biochip and preparation method and application thereof |
| CN114606345A (en) * | 2022-01-18 | 2022-06-10 | 中国中医科学院医学实验中心 | Preparation method, detection method and kit of protein thermal stability detection chip |
| CN114606345B (en) * | 2022-01-18 | 2024-02-27 | 中国中医科学院医学实验中心 | Preparation method, detection method and kit of protein thermal stability detection chip |
| WO2023202237A1 (en) * | 2022-04-18 | 2023-10-26 | 深圳碳云智肽药物科技有限公司 | Polypeptide targeting hdac5, and use thereof in the preparation of formulation for treating or preventing individual tumors |
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