CN112876540A - Application of affinity peptide in preparation of anti-coronavirus drugs - Google Patents

Application of affinity peptide in preparation of anti-coronavirus drugs Download PDF

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CN112876540A
CN112876540A CN202110178816.3A CN202110178816A CN112876540A CN 112876540 A CN112876540 A CN 112876540A CN 202110178816 A CN202110178816 A CN 202110178816A CN 112876540 A CN112876540 A CN 112876540A
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coronavirus
affinity peptide
preparing
cov
sars
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CN112876540B (en
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王晨轩
于兰兰
王若楠
许海燕
温涛
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Institute of Basic Medical Sciences of CAMS
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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Abstract

The invention discloses an application of an affinity peptide in preparing an anti-coronavirus medicament. The affinity peptide disclosed by the invention comprises a sequence shown as SEQ ID NO.1 or a variant thereof. The invention also discloses application of the affinity peptide in detection of coronavirus.

Description

Application of affinity peptide in preparation of anti-coronavirus drugs
Technical Field
The invention relates to the technical field of biology, in particular to application of an affinity peptide in preparation of an anti-coronavirus medicine.
Background
Infectious diseases caused by viruses are a serious threat to human life and health. Statistically, more than 80% of infectious diseases are caused by viruses. Viral diseases have been implicated in various areas of clinical medicine, and many non-infectious diseases have been found to be associated with viral infections, and some viruses are also associated with certain neoplastic and autoimmune diseases. Coronaviruses are a large group of viruses widely existing in nature, are a large family of zoonosis viruses, and can infect human, mouse, pig, cat, dog, bat and avian vertebrates. To date, approximately 17 different coronavirus strains have been discovered, of which 6 infect humans. At present, the detection method for coronavirus is mainly nucleic acid detection, but the method may generate false positive due to sampling, sample preservation, kit quality and the like. In addition to symptomatic treatment, there is currently a lack of effective agents for the specific treatment of coronaviruses.
After infection of a host cell by a virus, the virus typically undergoes multiple stages of adsorption, penetration, uncoating, nucleic acid replication, transcriptional translation, and packaging, and the prevention of any one of these processes can result in the affected or inhibited replication of the virus. In general, the most effective antiviral drugs are inhibited by acting on the virus-adsorbing host cells or the nucleic acid replication stages, and thus screening for antiviral drugs is mainly focused on these two stages.
With the continuous development and progress of genomics and proteomics, more and more scientific research make internal disorder or usurp found that in most cases, not structural macromolecules such as proteins but small peptide molecules with small molecular weight and relatively simple structure play an important role in life activities. Based on the characteristics of simple structure, stable property, no immunogenicity, easy synthesis and modification and the like of the polypeptide, the polypeptide is gradually favored by researchers in the aspect of drug design. The polypeptide has small molecular weight, can be effectively combined to the active site of target protein to play a certain function, and is easy to absorb and not easy to degrade in intestines and stomach as a medicine molecule. Therefore, the polypeptide which can be combined with the virus and inhibit the virus infection is screened out, and the polypeptide can be applied to the detection and treatment of the coronavirus.
Disclosure of Invention
The invention provides an affinity peptide combined with coronavirus, and provides a new direction for the development of anti-coronavirus medicines.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides an affinity peptide comprising the sequence shown in SEQ ID No.1 or a variant thereof.
The term "peptide" refers to a polymer of two or more amino groups covalently linked by peptide bonds. The term "polypeptide" is used interchangeably with "affinity peptide" in the present invention and refers to a peptide consisting of a plurality of amino acids. Variants of the polypeptides described in the present invention are polypeptides that have been modified at one or more amino acid residues, but still retain the biological activity of the affinity peptide. Variants may be produced by any method, including but not limited to the following: error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, GSSM, or any combination of these methods.
Further, the affinity peptide specifically binds to coronavirus.
Further, the affinity peptide specifically binds to coronavirus S protein.
Further, the affinity peptide specifically binds to the Receptor Binding Domain (RBD) of the coronavirus S protein.
The term "specifically binds" or similar terms means that the two molecules form a complex that is relatively stable under physiological conditions. Specific binding is characterized by high affinity and low to medium capacity. Nonspecific binding typically has low affinity and moderate to high capacity. Generally, when the affinity constant K. Higher than 106M-1Or preferably above 108M-1Binding is considered specific. If desired, non-specific binding is reduced by changing the binding conditions without substantially affecting specific binding. The above conditions are well known in the art and those skilled in the art can select suitable conditions using conventional techniques. The conditions are typically defined by antibody concentration, solution ionic strength, temperature, time to bind, concentration of non-relevant molecules (e.g., serum albumin, milk casein), and the like.
The coronaviruses described in the present invention can be divided into four genera: α, β, γ, δ. Alphacoronaviruses include, but are not limited to, canine coronavirus (CCoV), feline coronavirus (FeCoV), human coronavirus 229E (HCoV-229E), Porcine Epidemic Diarrhea Virus (PEDV), transmissible gastroenteritis virus (TGEV), human coronavirus NL63(NL or New Haven); the genus Beta coronavirus includes, but is not limited to, bovine coronavirus (BCoV), canine respiratory coronavirus (CRCoV), human coronavirus OC43(HCoV-OC43), Mouse Hepatitis Virus (MHV), porcine Hemagglutinating Encephalomyelitis Virus (HEV), Rat Coronavirus (RCV), HCoV-HKU1, Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2), middle east respiratory syndrome coronavirus (MERS-CoV); gamma coronaviruses include, but are not limited to, Infectious Bronchitis Virus (IBV), turkey coronavirus (Bluecomb virus), pheasant coronavirus, guinea fowl coronavirus; delta genus coronaviruses include, but are not limited to, Bulbul coronavirus (BuCoV), Thrush coronavirus (ThCoV), Munia coronavirus (MuCoV), porcine coronavirus (PorCov) HKU 15.
In a specific embodiment of the invention, the coronavirus is SARS-CoV-2.
In a second aspect, the invention provides the use of an affinity peptide according to the first aspect of the invention, which is at least one of (a1) - (a 7):
(a1) as a vector targeting coronaviruses;
(a2) as a vector targeting the functional component to the coronavirus;
(a3) as a vector targeting drugs to coronaviruses;
(a4) preparing a vector targeting the coronavirus;
(a5) preparing a vector targeting the functional component to the coronavirus;
(a6) preparing a vector for targeting a drug to a coronavirus;
(a7) preparing the vaccine for resisting the coronavirus.
Further, the coronavirus is SARS-CoV-2.
In a third aspect, the present invention provides a conjugate comprising an affinity peptide according to the first aspect of the present invention and an additional substance.
Further, the substance comprises a labeling group, a modification, or any combination thereof.
Further, the labeling group comprises a radioactive element, a fluorescent group and a high-absorptivity chromophore.
The term "fluorophore" as used herein includes molecules, proteins, colloids, quantum dots and collections of atoms that form structures that are fluorescent. Fluorophores include, but are not limited to, dihydrorhodamine 123, tetramethylrhodamine-6-maleimide, tetramethylrhodamine-5-maleimide, 5-indolacetylaminofluorescein, fluorescein-5 maleimide, 5-fluorescein isothiocyanate cadaverine, G sulforhodamine G, 7-hydroxy-4-methylcoumarin, 3-cyano-7-hydroxycoumarin, fluorescein disodium salt, fluorescein, 5' fluorescein phosphoramidate, tetramethylrhodamine-6-isothiocyanate, 6-carboxy-X-rhodamine succinimidyl ester, 5-carboxy-X-rhodamine succinimidyl ester, 6-carboxy-X-rhodamine, 5-carboxytetramethylrhodamine succinimidyl ester, 5-carboxyrhodamine-6-imide ester, 5-carboxyrhodamine-N-acylester, 5-carboxymethylester, 5-iodorhodamine-N-acylimide ester, 5-carboxymethyliodorhodamine-N-acylimide ester, 5-carboxymethyliodonium-N-carbonyl, 6-carboxytetramethylrhodamine, 5-carboxytetramethylrhodamine, 6-carboxyrhodamine 6G, fluorescein isothiocyanate, 6-carboxyfluorescein succinimidyl ester, 5-carboxyfluorescein, 6-carboxyfluorescein, rhodamine B, rhodamine 6G, 7-amino-4-methylcoumarin.
Furthermore, the modifier comprises a nano material, a fluorescent material, enzymes and biotin.
The term "nanomaterial" refers to a material having at least one dimension in three dimensions in the nanometer size (1-100nm) or composed of them as a basic unit.
The nano material of the invention comprises metal nano particles.
Further, the metal nanoparticles include noble metal nanoparticles.
Further, the noble metal nanoparticles are gold nanoparticles.
Further, the additional substance is directly linked to the affinity peptide or is coupled, conjugated or fused to the affinity peptide via a linker.
Further, the other substance is coupled, conjugated or fused to the affinity peptide via a linker.
Linkers described herein are well known in the art and include, but are not limited to, linkers comprising one or more (e.g., 1, 2, 3, 4, or 5) amino acids (e.g., cysteine (Cys) or serine (Ser)) or amino acid derivatives (e.g., Ahx, β -Ala, GABA, or Ava), or PEG, and the like.
In a specific embodiment of the invention, the linker is Cys.
In a fourth aspect, the invention provides the use of an affinity peptide according to the first aspect of the invention or a conjugate according to the third aspect of the invention, wherein the affinity peptide is at least one of (b1) - (b 2):
(b1) preparing a product for detecting coronavirus;
(b2) preparing products for diagnosing coronavirus related diseases.
Further, the product comprises a kit and a chip.
In the present invention, the kit further comprises a container, instructions for use, a positive control, a negative control, a buffer, an auxiliary agent or a solvent, and instructions for use with the kit, wherein the instructions describe how to use the kit for detection, and how to use the detection results to determine the development of a disease and select a treatment regimen.
Further, the coronavirus is SARS-CoV-2.
In a fifth aspect, the present invention provides a pharmaceutical composition comprising an affinity peptide according to the first aspect of the present invention.
The pharmaceutical composition of the invention can also comprise an effective amount of a medicament for resisting the coronavirus or treating the coronavirus-related diseases and a pharmaceutically acceptable carrier and/or auxiliary material.
The term "effective amount" as used herein, refers to an amount that has a therapeutic effect or is required to produce a therapeutic effect in a subject being treated. For example, a therapeutically or pharmaceutically effective amount of a drug refers to the amount of drug required to produce the desired therapeutic effect, which can be reflected in the results of clinical trials, model animal studies, and/or in vitro studies. The pharmaceutically effective amount will depend on several factors including, but not limited to, the characteristics of the subject (e.g., height, weight, sex, age, and medical history), and the severity of the disease.
The medicine composition and the medicine for resisting coronavirus or treating coronavirus related diseases can be prepared into separate preparations for combined application, and the two medicines can also be prepared into a preparation for application in the form of a composition.
The carrier and/or adjuvant includes pharmaceutically acceptable carrier, diluent, filler, binder and other excipients, which depend on the administration mode and the designed dosage form. Therapeutically inert inorganic or organic carriers known to those skilled in the art include, but are not limited to, lactose, corn starch or derivatives thereof, talc, vegetable oils, waxes, fats, polyols (e.g., polyethylene glycol, water, sucrose, ethanol, glycerol), various preservatives, lubricants, dispersants, flavoring agents, wetting agents, sweeteners, flavorants, emulsifiers, suspending agents, preservatives, antioxidants, colorants, stabilizers, salts, buffers, and the like, which may also be added.
Suitable pharmaceutically acceptable carriers and/or adjuvants are described in detail in Remington's Pharmaceutical Sciences (19th ed.,1995) as needed to aid in the stability of the formulation or to enhance the activity or its bioavailability or to produce an acceptable mouthfeel or odor in the case of oral administration, and the formulations which may be used in such Pharmaceutical compositions may be in the form of their original compounds as such, or optionally in the form of their pharmaceutically acceptable salts. The pharmaceutical composition thus formulated may be administered by any suitable means known to those skilled in the art, as desired, by administering a safe and effective amount of the drug of the present invention to a human.
The appropriate dose of the pharmaceutical composition of the present invention can be prescribed in various ways depending on factors such as the method of preparation, the mode of administration, the age, body weight, sex, disease state, diet, time of administration, route of administration, excretion rate and reaction sensitivity of the patient, and a skilled physician can easily determine the prescription and the dose of administration effective for the desired treatment or prevention.
In a sixth aspect, the present invention provides an affinity peptide according to the first aspect of the present invention or a pharmaceutical composition according to the fifth aspect of the present invention, wherein the affinity peptide is at least one of (c1) - (c 2):
(c1) preparing an anti-coronavirus medicament;
(c2) preparing the medicine for treating the diseases related to the coronavirus.
The coronavirus related diseases described in the present invention include, but are not limited to, acute respiratory distress syndrome, sepsis, septic shock, metabolic acidosis, procoagulant dysfunction, bronchitis, pneumonia, diarrhea, myocarditis, neonatal necrotizing enteritis, multiple sclerosis, otitis media.
Further, the coronavirus is SARS-CoV-2.
In a seventh aspect, the invention provides a nucleic acid encoding an affinity peptide according to the first aspect of the invention.
According to an eighth aspect of the invention there is provided a product for detecting or diagnosing a coronavirus related disease, said product comprising an affinity peptide according to the first aspect of the invention or a conjugate according to the third aspect of the invention or a nucleic acid according to the seventh aspect of the invention.
Further, the product comprises a chip and a kit.
Further, the coronavirus is SARS-CoV-2.
In a ninth aspect, the invention provides a method of preparing a vaccine against coronavirus which method comprises purifying the viral S protein using an affinity peptide according to the first aspect of the invention.
Further, the coronavirus is SARS-CoV-2.
In a tenth aspect, the invention provides a method for the detection of coronaviruses for non-diagnostic purposes, said method comprising detecting a sample using an affinity peptide according to the first aspect of the invention or a conjugate according to the third aspect of the invention or a nucleic acid according to the seventh aspect of the invention or a product according to the eighth aspect of the invention.
Samples described herein include blood, plasma, serum, spinal fluid, urine, sweat, saliva, tears, breast aspirate, prostatic fluid, semen, vaginal secretion, stool, cervical scrape, cells, amniotic fluid, ocular fluid, mucus, respiratory moisture, animal tissue, cell lysate, tumor tissue, hair, skin, oral scrape, nail, bone marrow, cartilage, infectious proteins, bone meal, cerumen, or combinations thereof.
The most reliable results are possible when processing samples in a laboratory environment. For example, a sample may be taken from a subject in a doctor's office and then sent to a hospital or commercial medical laboratory for further testing. However, in many cases, it may be desirable to provide immediate results at the clinician's office or to allow the subject to perform the test at home. In some cases, the need for testing that is portable, prepackaged, disposable, ready to use by the subject without assistance or guidance, etc., is more important than a high degree of accuracy. In many cases, especially in the case of physician visits, it may be sufficient to perform a preliminary test, even a test with reduced sensitivity and/or specificity. Thus, assays provided in product form can involve detecting and measuring relatively small amounts of biomarkers to reduce the complexity and cost of the assay.
Further, the detection method comprises enzyme-linked immunosorbent assay, plasma resonance assay, radioimmunoassay, fluorescent immunodetection and luminescent immunodetection.
In a specific embodiment of the present invention, the detection method is a plasmon resonance test.
Further, the coronavirus is SARS-CoV-2.
The invention has the advantages and excellent effects that:
the invention discovers for the first time that an affinity peptide can be used for detecting coronavirus, and the affinity peptide comprises a sequence shown as SEQ ID NO.1 or a variant thereof.
The invention discloses a method for detecting coronavirus for non-diagnosis and treatment purposes.
The invention discloses the application of the affinity peptide in the treatment of anti-coronavirus or related diseases thereof.
Drawings
FIG. 1 is a schematic diagram of the screening process, the experimental result of the blue spots of the screened phage, and a partial display of the sequencing-related results.
FIG. 2 is a graph showing the kinetics of dissociation of the binding of R1 polypeptide to the target protein RBD.
FIG. 3 is a graph showing the results of an experiment for evaluating the response of R1 polypeptide-modified gold nanoparticles to RBD/BSA protein and FBS. Wherein, the graph A is a graph of the SPR peak change of the gold nanoparticles added with different volumes of RBD (13ug/mL), the graph B is a graph of the SPR peak change of the gold nanoparticles added with different volumes of BSA (20ug/mL) protein solution, the graph C is a graph of the SPR peak change of the gold nanoparticles added with different volumes of FBS solution, and the graph D is a graph comparing the response effect of the RBD protein and the BSA protein on the gold nanoparticles.
FIG. 4 is a graph showing the results of cytotoxicity experiments for detecting R1 polypeptide by CCK 8.
FIG. 5 is a graph showing the experimental results of the ability of the R1 polypeptide to inhibit pseudovirus infection. The concentrations of the polypeptides added from left to right were classified into 0, 0.003. mu.M, 0.01. mu.M, 0.03. mu.M, and 0.1. mu.M.
Detailed Description
The following detailed description of embodiments of the present application will be made with reference to the accompanying drawings. It should be understood that the detailed description and specific examples, while indicating the present application, are given by way of illustration and explanation only, and are not intended to limit the present application.
Experimental materials:
ph.d. -12 phage display peptide library kit purchased from New England Biolabs, inc; the CCK8 kit is a 500T kit of Japan Dojindo company; pseudovirus system and HEK293T-ACE2 cells were purchased from Ghman Biotechnology (Shanghai) Inc. under the product name SARS-CoV-2 Spike Pseudotyped Virus (GFP-Luciferase) (1E6TU/mL/293T ACE 2); ForteBio Octet instrument.
Example 1 screening of affinity peptide for SARS-CoV-2 RBD protein by phage display technology
1. Experimental procedure
As shown in FIG. 1, the experiment used a polypeptide library of M13 phage, pIII display system, which includes 109A plurality of different polypeptide sequences; repeating the process of adsorption-elution-amplification for 3-5 rounds to obtain phage capable of binding with target protein (RBD); extracting the DNA of the phage, completing sequencing, analyzing to obtain a polypeptide sequence capable of being combined with the target protein, and further synthesizing a related high-affinity peptide sequence. The method comprises the following specific steps:
phage display experiment
Day one
(1) A solution of 10-100. mu.g/ml LRBD protein molecules was prepared with 0.1M NaHCO3(pH 8.6).
(2) Add 1.5mL of the above solution to the dish and rotate repeatedly until the surface is completely wet.
(3) In a wet box, incubate overnight at 4 ℃ with gentle shaking or store in a wet box at 4 ℃ until use.
The next day
(4) ER2738 was inoculated into 10mL LB + Tet (Tetracycline) medium. This culture will be used for titration in step 11 and may be used within 5-10 hours. If the eluted phage are to be amplified on the same day (see step 12), 20mL of LB medium should also be inoculated into a 250mL Erlenmeyer flask containing ER 2738. Both cultures were incubated at 37 ℃ with vigorous shaking. Incubating, titrating and culturing until needed; the 20mL culture was carefully monitored so that it did not exceed the log early stage (OD 6000.01-0.05) for use in step 12.
(5) And (3) sealing: the plate was inverted on a clean paper towel, the coating solution was removed, and the plate was filled with Blocking Solution (BSA) (blocking solution preparation: 7mL of 0.1M NaHCO)3(pH 8.6), 35mg of BSA was added to give 5mg/mL BSA as blocking solution), and incubated at 4 ℃ for at least 1 h. Add 2mL of blocking solution to the dish and shake for 2h at 4 ℃.
(6) The blocking solution was discarded as described in step 5. Each plate was washed 6 times rapidly with TBST (TBS + 0.1% [ v/v ] Tween-20). Repeated rotation ensures that both the bottom and the sides of the well are washed. The washing should be rapid, avoiding plate drying.
(7) Dilute 1X 10 with 1ml TBST11Phage (10uL, 100X dilution). Pipetted onto the coated plate and gently shaken at room temperature for 10-60 min.
(8) The unbound phage was decanted by patting it down on a clean paper towel.
(9) The plates were washed 10 times with TBST as per step 6. One clean towel at a time was used to prevent cross-contamination.
(10) Elution of bound phage: bound phage were eluted with 1mL of elution buffer (0.2M glycine-hydrochloric acid (pH 2.2)). Gently shaking for no more than 10min, transferring the eluate into a microfuge tube, and neutralizing with 150 μ L of 1M Tris-HCl (pH 9.1).
(11) A small amount of eluate (20. mu.L) was taken for titer determination.
(12) The remaining eluate was amplified by adding it to the 20ml ER2738 culture in step 4 and incubated with vigorous shaking at 37 ℃ for 4.5 h.
(13) The culture was transferred to a centrifuge tube and centrifuged at 12000g for 10min at 4 ℃. The supernatant was transferred to a new tube and then centrifuged.
(14) 80% of the supernatant was transferred to a new tube and 1/6 volumes of 20% PEG/2.5M NaCl were added to it. Phage were precipitated at 4 ℃ for at least 2 hours or overnight.
The third day
(15) Centrifuge at 14000rpm for 15min at 4 ℃, discard the supernatant, briefly re-spin the tube, and pipette off the remaining supernatant.
(16) The pellet was suspended with 1ml of LTBS and transferred to a microfuge tube and the remaining cells were pelleted by centrifugation at 14000rpm for 5min at 4 ℃.
(17) The supernatant was transferred to a new microfuge tube and precipitated by adding 1/6 volumes of 20% PEG/2.5M NaCl. Incubate on ice for 15-60 min. Centrifugation was carried out at 14000rpm for 10min at 4 ℃ to discard the supernatant, centrifugation was carried out again, and the remaining supernatant was removed by a micropipette.
(18) The pellet was suspended with 200. mu. LTBS and microcentrifuged for 1min to precipitate any remaining insoluble material. Transferring the supernatant to a new tube, namely the amplified eluent.
(19) The titer of the phage was measured.
(20) And (4) carrying out a second round of screening on the coated plate in the same step 1-3.
Day four and day five
(21) In step 19, blue plaques are counted from the plate and the phage titer is determined, which should be at 1013–1014pfu/ml. The calculation corresponds to 1 × 1011-2×1011phage volume of pfu. If the titer is too low, a value corresponding to 10 can be used in the screening9phage volume of pfu.
(22) And (3) carrying out a second round of panning: the steps 4-18 were repeated using a calculated amount of the first round amplification eluate as input phage, and the Tween 20 concentration in the washing step was increased to 0.5% (v/v).
(23) The second round of screening amplification eluents were titrated.
(24) Coating the plate to carry out a third round of screening, and carrying out the same steps as 1-3.
Day six
(25) Performing a third panning: steps 4-10 were repeated using the same input titer as the first round (step 7), using the second round amplified eluate, again using 0.5% Tween in the wash step.
(26) The unamplified third round of eluate was titrated on LB/IPTG/Xgal plates as per step 11. No third round of eluate was amplified. The titer of the unamplified third eluate was determined. It is not necessary to amplify the third round of the screen eluate if the fourth round of screening is not performed. Blue plaques at titer determination can be used for sequencing: the incubation time of the plate should not be longer than 18 h. The remaining eluate was stored at 4 ℃ for at least one week.
Second, measuring the titer of the phage
(1) The ER2738 clone was inoculated in 5-10 mL LB medium and incubated with shaking for 4-8 h (log-intermediate, OD600 of about 0.5).
(2) When the cells grow, the top agar is put into a microwave oven to be melted and is subpackaged into sterile culture tubes, 3mL of each tube is used, and the cells are reserved at 45 ℃.
(3) A piece of LB/IPTG/Xgal plate was preheated at 37 ℃ for at least 1h at the desired dilution until ready for use.
(4) Preparation in LB from 10 to 103Multiple phage dilution series, final volume 1 mL.
(5) When the cultures in step 1 reached mid-log phase, they were aliquoted into microfuge tubes at 200. mu.L per tube.
(6) Add 10. mu.l of each phage dilution to each tube, add only one dilution per tube, vortex quickly, and incubate at room temperature for 1-5 min.
(7) One infected cell was transferred at a time to a culture tube containing 45 ℃ top agar and briefly vortexed.
(8) The culture from the previous step was immediately poured onto pre-warmed LB/IPTG/Xgal plates. The plate was gently tilted and rotated to spread the top agar evenly.
(9) The plate was cooled for 5min and incubated at 37 ℃ in an inverted format overnight.
(10) Plaques were counted on a plate of approximately 100 plaques. Each number was multiplied by the dilution factor of the plate, at a phage titer of every 10. mu.L plaque forming units (pfu).
Thirdly, extracting DNA sequencing
(1) ER2738 in LB overnight, 1: 100 dilutions, 1mL per tube, one plaque per tube.
(2) The blue plaques were punctured by the tip into the above culture tubes.
(3) Incubated at 37 ℃ for 4.5-5h with shaking.
(4) The culture was transferred to a 1.5ml centrifuge tube and centrifuged at 14000rpm for 30S.
(5) The supernatant was aspirated into a new centrifuge tube and centrifuged at 14000rpm for 30S.
(6) 80% of the supernatant was aspirated into a new centrifuge tube and stored at 4 ℃.
(7) Adding 200 μ L of LPEG/NaCl into 500 μ L of the mixture, uniformly mixing, and standing at room temperature for 10-20 min.
(8) Centrifuge at 14000rpm for 10min at 4 ℃ and discard the supernatant. Then centrifuged, and the supernatant was discarded.
(9) Resuspend with 100. mu.L iodide, shake vigorously, add 250. mu.l absolute ethanol, and stand at room temperature for 10-20 min.
(10) Centrifuge at 14000rpm for 10min at 4 ℃ and discard the supernatant. 500 μ L of 70% ethanol was added. Then, the mixture was centrifuged at 14000rpm for 10min at 4 ℃.
(11) Discard the supernatant and dry in the sun.
(12) Resuspend with 30 μ LTE Buffer. Freezing and storing at-20 ℃. And (5) sequencing.
2. Results of the experiment
The high affinity peptide sequences are shown in table 1.
TABLE 1 RBD protein affinity peptides and their sequences screened by phage display technology
Target protein Name of affinity peptide Affinity peptide sequences
RBDprotein R1 DVDVLIKYQFSF-NH2
Example 2 affinity and interaction kinetics detection of affinity peptides with target proteins
1. Experimental procedure
The size of the affinity of the interaction between the affinity peptide and the target protein is detected by a Bio-layer interference technique (BLI) technique, and the kinetic process of the binding and dissociation between the polypeptide and the target protein is studied. Modifying Biotin (Biotin-GG-DVDVLIKYQFSF-NH2) at the C terminal of R1 polypeptide, fixing the R1 polypeptide on an SA modified sensor, drawing an association and dissociation kinetic curve when the R1 polypeptide interacts with RBDs with different concentrations, and then calculating a dissociation equilibrium constant K of the interaction of the R1 polypeptide and the RBDs by the association and dissociation kinetic curveD. The method comprises the following specific steps:
(1) the biosensor with the surface modified with Streptavidin (Streptavidin, SA) is immersed in PBS buffer solution for balancing for at least 10 min.
(2) The sensor was immersed in PBS buffer to obtain a baseline.
(3) The sensor is then immersed in a known concentration of a solidifying solution (R1 polypeptide solution) in which biotinylated polypeptides bind to the biosensor surface, causing a change in the surface film layer. This step allows the R1 polypeptide to bind to the SA biosensor.
(4) The cured sensor was immersed in PBS buffer for baseline.
(5) The immobilized biosensor is immersed in a sample solution containing RBD protein, and the thickness of the membrane layer is increased due to the mutual combination of the R1 polypeptide and the RBD protein. The concentration of RBD protein in this step was 900nM,300nM,100 nM.
(6) When the sensor combined with the antibody to be detected is immersed in the buffer solution for dissociation, the RBD protein falls off from the surface of the biosensor, so that the thickness of the film is reduced.
(7) And (3) fitting to obtain a kinetic constant of the interaction of the R1 affinity peptide and the RBD protein by monitoring the thickness of the biological film layer of the biosensor in real time in the experimental process.
2. Results of the experiment
The R1 polypeptide obtained by screening by using phage display technology has the strongest affinity with RBD protein, and KDAt (9.52. + -. 1.83) E-11And M. The binding and dissociation kinetics curves of the R1 polypeptide when it interacted with different concentrations of RBD are shown in fig. 2, and the results of the binding and dissociation kinetics and affinity analysis are shown in table 2.
Table 2 binding dissociation kinetics and affinity analysis of the interaction of affinity peptides with target proteins.
Figure BDA0002940867420000131
Example 3 ability of affinity peptides to respond to target proteins
1. Experimental procedure
Introduction of a cysteine (DVDVLIKYQFSFC-NH) at the C-terminus of the affinity peptide2) And the polypeptide is bonded on the surface of the gold nanoparticle through an S-Au bond. And (3) adding a series of target proteins with concentration gradient in the system, and detecting the SPR peak position of the gold nanoparticles when the proteins are combined with the affinity peptides on the surfaces of the gold nanoparticles. The method comprises the following specific steps:
preparation of gold nanoparticles
Adding 150mL of sodium citrate (2.2mM) into a 250mL round-bottom flask, magnetically stirring at 400rpm, carrying out water bath at 90 ℃, adding 1mL of chloroauric acid (25mM), reacting for 30min, adding 1mL of 60mM sodium citrate and 1mL of 25mM chloroauric acid, reacting for 30min, and cooling to room temperature to obtain the gold nanoparticle solution.
II, polypeptide recognition RBD
Taking 200 mu L of the gold nanoparticle solution, adding 20 mu L R1 polypeptide (0.5mg/mL), uniformly mixing, placing in a water bath at 30 ℃ for 30min, centrifuging at 12000rpm for 5min, discarding supernatant, and adding 200 mu L of water to resuspend precipitate. The solution was taken at 200. mu.L, added with RBD/BSA/FBS at different concentrations, and subjected to ultraviolet-visible near-infrared absorption spectroscopy.
2. The experimental results are as follows:
as shown in fig. 3, when the RBD protein was added to the gold nanoparticles surface-modified with R1 polypeptide, the peak position gradually shifted toward a long wavelength (red shift) and showed concentration dependence. Under the same experimental conditions, although the peak height is changed due to the addition of Fetal Bovine Serum (FBS) (because FBS itself has an absorption peak in the detection wavelength range), the addition of BSA and FBS does not change the position of the SPR peak, that is, the position of the SPR peak is not shifted due to the addition of BSA and FBS in the R1 polypeptide-modified gold nanoparticles. Therefore, the R1 polypeptide modified gold nanoparticles show specific response to RBD protein, and the R1 polypeptide can be applied to detection of viruses.
Example 4 cytotoxicity and efficacy evaluation of affinity peptides
1. Experimental procedure
The cytotoxicity of the R1 polypeptide was tested using CCK8 Kit (Cell Counting Kit-8).
(1) HEK293T (ACE2 high expression) cells were cultured, cells grew about 70% -80%, and cells were plated in 96-well plates, approximately 1 ten thousand cells per well.
(2) The polypeptide R1 was cultured in a culture medium at a concentration of 0.003. mu.M, 0.01. mu.M, 0.03. mu.M, 0.1. mu.M, 0.3. mu.M, 1. mu.M, 3. mu.M, 10. mu.M or 30. mu.M in each well of 96 wells for 48 hours. Add 100. mu.L of complete medium to the blank wells. This experiment had 3 replicates per concentration.
(3) mu.L of CCK-8 kit solution was added to each well and the absorbance OD450 was measured after incubation at 37 ℃ for 2-4 h.
2. Results of the experiment
As shown in FIG. 4, the R1 polypeptide did not exhibit significant cytotoxicity in the detection range of 0-30. mu.M.
Example 5 evaluation of the effectiveness of affinity peptides
1. Experimental procedure
Adopts a SARS-CoV-2 neutralization test technology platform which is established by Jiman biotechnology (Shanghai) Limited company and is based on a pseudovirus system and is safer to operate, expresses SARS-CoV-2S protein on the surface of the pseudovirus, and simultaneously carries GFP fluorescence and Luciferase Luciferase reporter genes. The dosage of pseudovirus is determined by a preliminary experiment, and HEK293T (ACE2 high expression) cells are infected by 0.5 mu L dosage to detect the effect of the R1 polypeptide on inhibiting virus infection. The method comprises the following specific steps:
preliminary experiments on the amount of pseudovirus
(1) HEK293T (ACE2 high expression) cells were cultured, the cells grew about 70% to 80%, and the cells were plated in 96-well plates, approximately 1 ten thousand cells per well.
(2) Each well was added with pseudovirus containing 3. mu.L, 1. mu.L, 0.3. mu.L, 0.1. mu.L, 0.03. mu.L, 0.01. mu.L, 0.003. mu.L and 100. mu.L of the medium, respectively.
(3) After the cells are cultured for 6h, the cells are sucked out, the fresh culture medium is replaced, and the cells are cultured for 48 h.
(4) Cells were blown down with PBS and GFP fluorescence was measured using flow cytometry. Fluorescence efficiencies of 10% -40% are a suitable range of pseudoviral load. The fluorescence rate of 0.3. mu.L/well was 16%, and the fluorescence rate of 1. mu.L/well was 58%. Therefore, a false virus amount of 0.5. mu.L/well was finally selected for the official experiment.
Second, inhibition experiment of R1 affinity peptide on virus infected cell
(1) HEK293T (ACE2 high expression) cells were cultured, the cells grew about 70% to 80%, and the cells were plated in 96-well plates, approximately 1 ten thousand cells per well.
(2) A culture medium containing the polypeptide R1 at a concentration of 600. mu.M, 200. mu.M, 60. mu.M, 20. mu.M, 6. mu.M, 2. mu.M, 0.6. mu.M, 0.2. mu.M, or 0.06. mu.M was prepared.
(3) Preparing pseudovirus solution with corresponding dosage.
(4) Polypeptide and pseudovirus 1: 1 (actual polypeptide concentration halved), incubated at 37 ℃ for 1h and added to a 96-well plate.
(5) After the cells are cultured for 6h, the cells are sucked out, replaced by new culture medium and cultured for 48 h.
(6) Fluorescence images were taken with an EVOS FLAuto2 microscope to observe the degree of inhibition of pseudoviruses by the R1 polypeptide.
2. Results of the experiment
As shown in fig. 5, the R1 polypeptide had inhibitory effect on pseudoviruses: from the fluorescence microscope picture, it can be seen that the fluorescence intensity gradually weakens, and the R1 polypeptide shows the effect of inhibiting virus infection at a certain concentration. The experimental result indicates that the affinity peptide can be applied to antiviral treatment.
The preferred embodiments of the present application have been described in detail with reference to the accompanying drawings, however, the present application is not limited to the details of the above embodiments, and various simple modifications can be made to the technical solution of the present application within the technical idea of the present application, and these simple modifications are all within the protection scope of the present application.
It should be noted that, in the foregoing embodiments, various features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described in the present application.
In addition, any combination of the various embodiments of the present application is also possible, and the same should be considered as disclosed in the present application as long as it does not depart from the idea of the present application.
Sequence listing
<110> institute of basic medicine of Chinese academy of medical sciences
<120> application of affinity peptide in preparation of anti-coronavirus drugs
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Asp Val Asp Val Leu Ile Lys Tyr Gln Phe Ser Phe
1 5 10

Claims (10)

1. An affinity peptide comprising the sequence shown in SEQ ID No.1 or a variant thereof, preferably said affinity peptide specifically binds to coronavirus S protein, preferably said affinity peptide specifically binds to Receptor Binding Domain (RBD) of coronavirus S protein, preferably said coronavirus is SARS-CoV-2.
2. The use of the affinity peptide of claim 1, which is at least one of (a1) - (a 7):
(a1) as a vector targeting coronaviruses;
(a2) as a vector targeting the functional component to the coronavirus;
(a3) as a vector targeting drugs to coronaviruses;
(a4) preparing a vector targeting the coronavirus;
(a5) preparing a vector targeting the functional component to the coronavirus;
(a6) preparing a vector for targeting a drug to a coronavirus;
(a7) preparing a vaccine against coronavirus;
preferably, the coronavirus is SARS-CoV-2.
3. A conjugate comprising the affinity peptide of claim 1 and a further substance, preferably the substance comprises a label, a modifier or any combination thereof, preferably the label comprises a radioactive element, a fluorescent group, a high absorption coefficient chromophore, preferably the modifier comprises a nanomaterial, a fluorescent material, an enzyme, biotin, preferably the nanomaterial comprises a metal nanoparticle, preferably the metal nanoparticle comprises a noble metal nanoparticle, preferably the noble metal nanoparticle is a gold nanoparticle, preferably the further substance is directly linked to the affinity peptide or is coupled, conjugated or fused to the affinity peptide via a linker, preferably the further substance is coupled to the affinity peptide via a linker, Conjugation or fusion, preferably, the linker is cysteine.
4. The affinity peptide of claim 1 or the conjugate of claim 3, wherein the affinity peptide is at least one of (b1) - (b 2):
(b1) preparing a product for detecting coronavirus;
(b2) preparing a product for diagnosing a coronavirus related disease;
preferably, the product comprises a kit and a chip, and preferably, the coronavirus is SARS-CoV-2.
5. A pharmaceutical composition comprising the affinity peptide of claim 1.
6. The affinity peptide of claim 1 or the pharmaceutical composition of claim 5, for use as at least one of (c1) - (c 2):
(c1) preparing an anti-coronavirus medicament;
(c2) preparing a medicament for treating diseases related to coronavirus;
preferably, the coronavirus is SARS-CoV-2.
7. A nucleic acid encoding the affinity peptide of claim 1.
8. A product for detecting coronavirus or diagnosing a coronavirus-related disease, said product comprising an affinity peptide according to claim 1 or a conjugate according to claim 3 or a nucleic acid according to claim 7, preferably said product comprises a chip, a kit, and preferably said coronavirus is SARS-CoV-2.
9. A method for preparing a vaccine against coronavirus comprising purifying coronavirus S protein using the affinity peptide of claim 1, preferably said coronavirus is SARS-CoV-2.
10. A method for non-diagnostic detection of a coronavirus, said method comprising detecting a sample using the affinity peptide of claim 1 or the conjugate of claim 3 or the nucleic acid of claim 7 or the product of claim 8, preferably said detection comprises an enzyme-linked immunosorbent assay, a plasmon resonance assay, a radioimmunoassay, a fluorescent immunoassay, a luminescent immunoassay, preferably said detection is a plasmon resonance assay, preferably said coronavirus is SARS-CoV-2.
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