CN112851763A - Novel affinity peptide M1 of coronavirus main protease and application thereof - Google Patents
Novel affinity peptide M1 of coronavirus main protease and application thereof Download PDFInfo
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Abstract
The invention discloses a new type affinity peptide M1 of coronavirus main protease and its application, screening out affinity peptide M1 of coronavirus through phage display technique, the affinity peptide and main protease M of coronavirus SARS-CoV-2proHas stronger affinity, can be used as a lead compound of an anti-coronavirus medicament, and further experimental verification shows that the gold nanoparticles modified by the affinity peptide M1 can be used for auxiliary detection of coronavirusM1 provides reference for further research and development of novel vaccines and anti-coronavirus medicines for resisting coronavirus, and has important significance for preventing and controlling the pandemic of coronavirus.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to an affinity peptide M1 of a novel coronavirus main protease and application thereof.
Background
Coronaviruses (CoVs) are enveloped, non-segmented, single-stranded, positive-strand RNA viruses belonging to the family Coronaviridae (Coronaviridae) of the order Nervirales (Nidovirales) and the subfamily of the orthocoronaviruses (Orthoconaviridae), which are widespread in nature, susceptible to humans and a wide variety of animals, whose natural hosts include humans and other mammals, such as cattle, pigs, dogs, cats, mice and bats, etc., Coronaviruses are one of the major pathogenic pathogens responsible for respiratory infections, and can be divided into 4 groups, i.e., alpha, beta, gamma and delta Coronaviruses (Fehr A R, Perlman S. Coronavir: an virons: 2015 viruses of the respiratory and pathogenesis [ J ]. Coronavir, birds: 1-23.), where alpha-and beta-Coronaviruses only infect humans and mammals, and can generally cause respiratory diseases and diseases of the mammalian respiratory system, but some of them can also infect mammals. Currently, 6 human coronaviruses have been identified, including HCoV-29E and HCoV-NL63 of the genus α, HCoVOC43, HCoV-HKU1 of the genus β, Severe acute respiratory syndrome-associated coronavirus (SARS-CoV), and Zhongdong respiratory syndrome-associated coronavirus (MERS-CoV).
The disease caused by coronavirus infection seriously threatens human life and health, and along with the increase of global population mobility and the characteristics of strong pathogenicity and easy genetic variation of the virus, the coronavirus becomes a global health threat. After coronavirus infects host cell, it generally goes through multiple stages of adsorption, penetration, uncoating, nucleic acid replication, transcription translation and packaging to prevent any one processAll of which can cause coronavirus replication to be affected or inhibited, the most effective drugs for inhibiting coronavirus are those which act on the virus-adsorbing host cell or on the nucleic acid replication stage, in which the Main protease (M) of coronaviruspro/3CLpro) Is an important potential drug target and is important for inhibiting the replication of coronavirus. At present, the detection method for coronavirus is mainly nucleic acid detection, and due to sampling, sample preservation and other reasons, "false positive" may exist, and besides symptomatic treatment and supportive treatment, an effective drug for specifically treating coronavirus still lacks, so research and development of a more rapid and reliable detection method and an anti-coronavirus drug with a definite target and a brand-new structure type are urgently needed to ensure capability of preventing infection of coronavirus and dealing with outbreak of related infectious diseases.
The invention screens out the affinity peptide M1 of coronavirus through phage display technology, which is associated with the main protease M of coronavirus SARS-CoV-2proHas strong affinity with the target protein MproThe affinity peptide can be used as a lead compound of an anti-coronavirus drug, and is applied to the development of an anti-coronavirus drug or a vaccine, and further experimental verification shows that the gold nanoparticles modified by the affinity peptide M1 can be used for auxiliary detection of coronavirus.
Disclosure of Invention
The invention aims to provide a novel affinity peptide M1 of coronavirus main protease and application thereof, wherein the affinity peptide has better affinity with coronavirus, and the affinity peptide is found for the first time and provides reference for researching and developing novel effective anti-coronavirus vaccines and medicaments.
The above object of the present invention is achieved by the following technical solutions:
in a first aspect of the invention, an affinity peptide that specifically binds to coronaviruses is provided.
Further, the sequence of the affinity peptide is shown as SEQ ID NO. 1;
preferably, said affinity peptide specifically binds to the coronavirus main protease Mpro；
Preferably, the coronavirus is SARS-CoV-2.
Coronaviruses described in the present invention include (but are not limited to): human coronavirus 229E (HCoV-229E), human coronavirus OC43(HCoV-OC43), severe acute respiratory syndrome coronavirus (SARS-CoV), human coronavirus NL63(HCoV-NL63), human coronavirus HKU1(HCoV-HKU1), middle east respiratory syndrome coronavirus (MERS-CoV), canine coronavirus (CCoV), feline coronavirus (FeCoV), Rat Coronavirus (RCV), Porcine Epidemic Diarrhea Virus (PEDV), transmissible gastroenteritis virus (TGEV).
In a second aspect of the invention, an affinity peptide derivative is provided.
Further, the derivatives of the affinity peptide include modified products of the affinity peptide of the first aspect of the present invention, variants obtained by adding and/or substituting one or more amino acids to the affinity peptide of the first aspect of the present invention, and conjugates of the affinity peptide of the first aspect of the present invention and other substances.
Further, the modified product includes the modified product of the affinity peptide according to the first aspect of the present invention after methylation modification, myristoylation modification, PEG modification, fluorine modification, biotin modification, fluorescence labeling modification, cyclization modification, carboxylation modification, fatty acid modification, acetylation modification, phosphorylation modification, glycosylation modification, amidation modification or other known polypeptide modification;
further, the variants obtained after the amino acid addition and/or substitution include variants obtained after the amino acid addition and/or substitution is performed at any position of the amino terminus, the carboxyl terminus or the interior of the affinity peptide according to the first aspect of the present invention;
preferably, said variant is obtained by adding and/or substituting 1 or more unrelated amino acids at the amino terminus, and/or the carboxy terminus, and/or at any internal position of the affinity peptide according to the first aspect of the invention.
Further, the unrelated amino acids include (but are not limited to): tyrosine, glycine, cysteine, tryptophan, glutamine, methionine, and threonine.
Further, the amino acids to be added and/or substituted include natural amino acids and unnatural amino acids.
Further, the conjugates of the affinity peptide of the first aspect of the present invention and other substances include conjugates obtained by coupling the affinity peptide of the first aspect of the present invention with a carrier protein, an exogenous polypeptide, and a drug;
preferably, the carrier proteins include (but are not limited to): serum albumin, hemocyanin, and chicken ovalbumin;
preferably, the exogenous polypeptides include (but are not limited to): fc region, signal peptide, polypeptide tag;
more preferably, said Fc region is selected from the group consisting of: IgA Fc region, IgD Fc region, IgG Fc region, IgE Fc region, IgM Fc region;
more preferably, the polypeptide marker is selected from the group consisting of: flag marker, strep marker, polyhistidine marker, VSV-G marker, influenza hemagglutinin marker, c-Myc marker;
preferably, the medicament includes (but is not limited to): bisphosphonates, iridoid compounds;
more preferably, the bisphosphonates include (but are not limited to): alendronic acid, ibandronate, zoledronic acid;
more preferably, the iridoid compounds include (but are not limited to): geniposide, genipin-gentiobioside, geniposide and geniposide.
In a third aspect, the invention provides a nucleic acid molecule.
Further, said nucleic acid molecule encodes an affinity peptide according to the first aspect of the invention or encodes an affinity peptide derivative according to the second aspect of the invention.
In a fourth aspect of the invention, an expression vector is provided.
Further, the expression vector comprises the nucleic acid molecule according to the third aspect of the present invention.
Further, the vector refers to a molecule that can carry the inserted polynucleotide into a host cell.
Further, the vector includes (but is not limited to): liposomes, biocompatible polymeric micelles, including natural and synthetic polymers; a lipoprotein; a polypeptide; a polysaccharide; a lipopolysaccharide; enveloping the artificial virus; metal particles; as well as bacteria, viruses, such as baculoviruses, adenoviruses and retroviruses, bacteriophages, cosmids, plasmids, fungal vectors and other recombinant vectors commonly used in the art for the expression of various eukaryotic and prokaryotic hosts, possibly for gene therapy, and for the expression of simple proteins.
In a fifth aspect, the invention provides a host cell.
Further, the host cell comprises a nucleic acid molecule according to the third aspect of the invention or comprises an expression vector according to the fourth aspect of the invention.
In a sixth aspect of the invention, a pharmaceutical composition is provided.
Further, the pharmaceutical composition comprises the affinity peptide of the first aspect of the invention, the affinity peptide derivative of the second aspect of the invention, the nucleic acid molecule of the third aspect of the invention, the expression vector of the fourth aspect of the invention;
further, the pharmaceutical composition can also comprise a pharmaceutically acceptable carrier and/or an auxiliary material.
The pharmaceutically acceptable carriers and/or excipients contained in the pharmaceutical composition of the present invention are carriers and/or excipients commonly used in formulations, and the carriers and/or excipients include pharmaceutically acceptable carriers, diluents, fillers, binders and other excipients, depending on the administration mode and the designed dosage form. Suitable pharmaceutically acceptable carriers and/or adjuvants are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).
Further, the administration mode of the pharmaceutical composition comprises oral administration and non-oral administration.
Further, the non-oral administration includes (but is not limited to): intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, local administration and transdermal administration.
Further, the appropriate dose of the pharmaceutical composition varies depending on factors such as the formulation method, the administration method, the age, body weight, sex, morbid state, diet, administration time, administration route, excretion rate, and reaction sensitivity of the patient, and the skilled physician can easily determine the dose to be used for obtaining the desired therapeutic effect or effective prophylactic effect.
Further, the pharmaceutical composition is formulated using pharmaceutically acceptable carriers and/or excipients according to a method that can be easily performed by those skilled in the art, and thus can be prepared in a unit volume form or in a multi-volume container. The dosage form may be in the form of a solution, suspension or emulsion in an oily or aqueous medium or in the form of an extract, powder, granule, tablet, capsule or gel (e.g., hydrogel), and may further comprise a dispersing or stabilizing agent.
In a seventh aspect the invention provides a product for detecting coronaviruses.
Further, the product comprises the affinity peptide according to the first aspect of the present invention, the affinity peptide derivative according to the second aspect of the present invention, the nucleic acid molecule according to the third aspect of the present invention, the expression vector according to the fourth aspect of the present invention, the host cell according to the fifth aspect of the present invention;
preferably, the product comprises a kit, a test strip and a chip.
Furthermore, the kit also comprises a container, an instruction for use, a positive control substance, a negative control substance, a buffer, an auxiliary agent or a solvent, wherein the instruction for use describes how to use the kit for detecting the coronavirus, how to use the detection result for judging and the like.
An eighth aspect of the invention provides a method for non-diagnostic and treatment-oriented detection of coronaviruses.
Further, the method comprises performing the detection using the affinity peptide according to the first aspect of the present invention, the affinity peptide derivative according to the second aspect of the present invention, the nucleic acid molecule according to the third aspect of the present invention, the expression vector according to the fourth aspect of the present invention, the host cell according to the fifth aspect of the present invention, and the product according to the seventh aspect of the present invention.
Further, the coronavirus is preferably SARS-CoV-2.
Further, the method of detection includes (but is not limited to): plasmon resonance assay, enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, and luminescence immunoassay.
In an embodiment of the present invention, the method of detection is a plasmon resonance assay.
As an example of non-diagnostic and therapeutic purposes, the methods of the invention can be used in the study of the infection and pathogenesis of coronavirus in vitro.
According to a ninth aspect of the invention there is provided use of an affinity peptide according to the first aspect of the invention.
Further, the application comprises the application of any one of the following aspects:
(1) use in the preparation of an affinity peptide derivative according to the second aspect of the invention;
(2) use in the manufacture of a pharmaceutical composition according to the sixth aspect of the invention;
(3) use in the manufacture of a product according to the seventh aspect of the invention.
Further, the application in preparing the pharmaceutical composition of the sixth aspect of the invention includes application in preparing a pharmaceutical composition for resisting coronavirus, and application in preparing a pharmaceutical composition for resisting coronavirus-related diseases.
Further, the coronavirus related diseases include (but are not limited to): chronic bronchitis, otitis media, pneumonia, myocarditis, frontal sinusitis, acute respiratory distress syndrome, diarrhea, pleural effusion, metabolic acidosis, blood coagulation dysfunction, septic shock.
Further, the coronavirus is preferably SARS-CoV-2.
The tenth aspect of the invention provides the use of an affinity peptide derivative according to the second aspect of the invention or the use of a nucleic acid molecule according to the third aspect of the invention or an expression vector according to the fourth aspect of the invention or a host cell according to the fifth aspect of the invention.
Further, the application comprises the application of any one of the following aspects:
(1) use in the manufacture of a pharmaceutical composition according to the sixth aspect of the invention;
(2) use in the manufacture of a product according to the seventh aspect of the invention.
In an eleventh aspect the invention provides a method of preparing a vaccine against coronavirus.
Further, the method comprises purifying coronavirus M using the affinity peptide of the first aspect of the inventionproA protein.
Further, the coronavirus is preferably SARS-CoV-2.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terms used in the specification of the present invention are only for describing specific embodiments and are not intended to limit the present invention, and furthermore, some terms are explained as follows.
The term "modification" as used herein refers to any change made to an affinity peptide, such as a change in the length of the peptide, a change in the amino acid sequence, a change in the chemical structure, a co-translational modification or a post-translational modification of the peptide. In some cases, the peptides of the invention comprise one or more modified amino acid residues. The type of modification is well known in the art.
The term "treatment" as used herein refers to the treatment of a human or animal (e.g., as applied by a veterinarian) in which some desired therapeutic effect is achieved, e.g., inhibition of the progression of a condition (including reduction in the rate of progression, cessation of progression), amelioration of the condition, and cure of the condition. Treatment as a prophylactic measure (e.g., prophylaxis) is also included. The use of a patient who has not yet developed a condition but who is at risk of developing the condition is also encompassed by the term "treatment".
The terms "subject" and "patient," as used herein, generally refer to a human subject and are used interchangeably. The subject may be male or female and may be of any ethnic or ethnic group including, but not limited to, caucasian, african, asian, hispanic, indian, etc. The subject may be of any age, including neonates, infants, toddlers, children, adolescents, adults, and the elderly. Subjects can also include animal subjects, particularly mammalian subjects, such as canines, felines, bovines, caprines, equines, ovines, porcines, rodents (e.g., rats and mice), lagomorphs, primates (including non-human primates), and the like, for the purpose of development of veterinary or medical drugs.
The term "natural amino acids" as used herein refers to those amino acids encoded by the genetic code, as well as those amino acids that are later modified, such as hydroxyproline, gamma-carboxyglutamic acid, and O-phosphoserine. Naturally occurring alpha-amino acids include (but are not limited to): alanine (Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (gin), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), and combinations thereof. Stereoisomers of natural amino acids include (but are not limited to): d-alanine (D-Ala), D-cysteine (D-Cys), D-aspartic acid (D-Asp), D-glutamic acid (D-Glu), D-phenylalanine (D-Phe), D-histidine (D-His), dileucine (D-Ile), D-arginine (D-Arg), D-lysine (D-Lys), D-leucine (D-Leu), D-methionine (D-Met), D-asparagine (D-Asn).
The term "unnatural amino acid," as used herein, includes (but is not limited to): amino acid analogs, amino acid mimetics, synthetic amino acids, M' -modified lysines, and methyl amino acids in either the L-or D-configuration that function in a manner similar to natural amino acids. An unnatural amino acid is not encoded by the genetic code and can, but need not, have the same basic structure as a natural amino acid. Such unnatural amino acids include (but are not limited to): 2-aminofatty acid, 3-aminofatty acid, beta-alanine, beta-aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, piperidinecarboxylic acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, 2, 4-diaminobutyric acid, desmosine, 2' -diaminopimelic acid, 2, 3-diaminopropionic acid, N-ethylglycine, N-ethylaspartamide, hydroxylysine, isohydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, iso-isoleucine, N-methylglycine, N-methylisoleucine, 6-N-methyllysine, N-methylvaline, N-methylisoleucine, N-methyllysine, N, Norvaline, norleucine or ornithine.
The invention has the advantages and beneficial effects that:
(1) the invention reports an affinity peptide M1 for the first time, wherein the affinity peptide M1 can specifically bind to coronavirus and can be used for auxiliary detection of coronavirus.
(2) The affinity peptide provided by the invention provides reference for further research and development of related novel vaccines and anti-coronavirus medicines for resisting coronavirus, and has important significance for preventing and controlling the pandemic of coronavirus.
Drawings
Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1 shows a schematic diagram of the screening process, a result diagram of the blue spots of phage obtained by screening, and a partial display diagram of the sequencing-related results;
FIG. 2 shows the M1 polypeptide and the target protein MproBinding dissociation kinetics profile;
FIG. 3 shows M3 and M4 polypeptides and target protein MproBinding dissociation kinetics profile, wherein, panel a: m3, panel B: m4;
FIG. 4 shows M1 polypeptide-modified gold nanoparticle pairs MproGraph of the results of response evaluation of/BSA protein, where graph a: mproAnd B, drawing: a BSA protein;
FIG. 5 shows M3 and M4 polypeptide modified gold nanoparticle pairsproGraph of the results of response evaluation of/BSA protein, whichIn (1), A is as follows: M3-MproAnd B, drawing: M3-BSA protein, Panel C: M4-MproAnd D, drawing: M4-BSA protein.
Detailed Description
The present invention is further illustrated below with reference to specific examples, which are intended to be illustrative only and are not to be construed as limiting the invention. As will be understood by those of ordinary skill in the art: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents. The following examples are examples of experimental methods not indicating specific conditions, and the detection is usually carried out according to conventional conditions or according to the conditions recommended by the manufacturers.
EXAMPLE 1 viral Master protease M of SARS-CoV-2proPreparation of affinity peptides of (4)
1. Experimental Material
The Ph.D. -12 phage display peptide library kit was purchased from New England Biolabs, Inc. Interaction kinetics analysis and affinity determination a ForteBio Octet instrument was used. The experimental reagent and the experimental operation method are carried out according to the use instructions of the product.
2. Experimental methods
Obtaining virus main proteinase M of SARS-CoV-2 by phage display technologyproThe polypeptide library of the display system of pIII using M13 phage, which library comprises 109A plurality of different polypeptide sequences. The process of adsorption-elution-amplification is repeated for 3-5 rounds to obtain the phage capable of binding with the target protein. Extracting the DNA of the bacteriophage, completing sequencing, analyzing to obtain a polypeptide sequence capable of being combined with a target protein, and further synthesizing a related high-affinity peptide sequence (a specific screening process schematic diagram is shown in figure 1), wherein the detailed experimental steps are as follows:
phage display experiments
Day one
(1) With 0.1M NaHCO3Solution (pH 8.6) was prepared to give M10-100. mu.g/mLproA protein molecule solution;
(2) adding 1.5mL of the solution prepared in the step (1) into a plate, and repeatedly rotating until the surface is completely wet;
(3) incubating overnight at 4 ℃ with gentle shaking or storing in a wet box at 4 ℃ for later use;
the next day
(4) Inoculating ER2738 into 10mL LB + Tet (tetracycline) medium, which will be used for titration in step (11), within 5-10 hours, and if the eluted phage are to be amplified on the same day (see step (12)), inoculating 20mL LB medium into a 25mL Erlenmeyer flask containing ER2738, incubating both cultures with vigorous shaking at 37 ℃, and incubating the titration culture until the desired requirements are met; the 20mL culture was carefully monitored so that it did not exceed the log-early stage (OD600 ═ 0.01-0.05) for 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)3Solution (pH 8.6), 35mg BSA was added to give a 5mg/mL BSA solution, i.e. blocking solution), and incubated at 4 ℃ for at least 1 h. Adding 2mL of confining liquid into a plate, and shaking for 2h at 4 ℃;
(6) after the sealing is completed, the sealing solution is discarded. Using TBST (TBS + 0.1% [ v/v ] Tween-20) to wash each plate for 6 times, and rotating repeatedly to ensure that the bottom and the side of the well are washed quickly and avoid plate drying;
(7) dilute 1X 10 with 1mL TBST11Phage (10. mu.L, 100X dilution), pipetted onto the coated plate, and gently shaken at room temperature for 10-60 min.
(8) The front side of the sample is downwards shot on a clean paper towel, and the bacteriophage without combination is poured off;
(9) the plates were washed 10 times with TBST according to step (6), using one clean paper towel each time to prevent cross contamination;
(10) elution of bound phage: the bound phage were eluted with 1mL of elution buffer (0.2M glycine-hydrochloric acid (pH 2.2)), gently shaken for no more than 10min, the eluate was transferred to a microcentrifuge tube and neutralized with 150 μ L of 1M Tris-HCl (pH 9.1);
(11) a small amount of eluate (20. mu.L) was taken to determine titer;
(12) amplifying the remaining eluate by adding it to 20mL of ER2738 culture in step (4), incubating with vigorous shaking at 37 ℃ for 4.5 h;
(13) transferring the culture into a centrifuge tube, centrifuging at 4 ℃ and 12000g for 10min, transferring the supernatant into a new test tube, and then centrifuging again under the same conditions;
(14) transferring 80% of the supernatant to a new tube, and adding 1/6 volume of 20% PEG/2.5M NaCl solution thereto, and precipitating the phage at 4 ℃ for at least 2 hours or overnight;
the third day
(15) Centrifuging at 14000rpm at 4 ℃ for 15min, discarding the supernatant, briefly re-rotating the tube, and removing the remaining supernatant with a pipette;
(16) the sediment was suspended with 1mL TBS and transferred to a microcentrifuge tube, and the residual cells were pelleted by centrifugation at 14000rpm for 5min at 4 ℃;
(17) transferring the supernatant to a new microcentrifuge tube, precipitating by adding 1/6 volume of 20% PEG/2.5M NaCl solution, incubating on ice for 15-60min, centrifuging at 4 deg.C and 14000rpm for 10min, discarding the supernatant, centrifuging again, and removing the residual supernatant with a micropipette;
(18) suspending the precipitate with 200 μ L TBS, microcentrifuging for 1min to precipitate any residual insoluble material, and transferring the supernatant to a new tube as the amplified eluate;
(19) measuring the titer of the phage;
(20) coating the plate for a second round of screening;
day four and day five
(21) In step (19), the blue plaques are counted from the plate and the phage titer is determined, which should be at 1013-1014Around pfu/mL, the calculation corresponds to 1X 1011-2×1011The phage volume of pfu, if the titer is too low, can be used at screening corresponding to 109phage volume of pfu;
(22) and (3) carrying out a second round of panning: using a calculated amount of the first round amplification eluate as input phage and increasing the Tween 20 concentration in the washing step to 0.5% (v/v);
(23) determining the titer of the second round of screening amplification eluents;
(24) coating the plate to carry out a third round of screening;
day six
(25) Performing a third panning: repeating steps (4) - (10) using the same input titer as the first round (step (7)) and the second round of amplified eluate, again using 0.5% Tween in the wash step;
(26) titrating the unamplified third round of eluate on an LB/IPTG/Xgal plate according to step (11) (without amplifying the third round of eluate, determining the titer of the unamplified third round of eluate; without amplifying the third round of eluate if the fourth round of screening is not performed; the titer-determined blue plaques can be used for sequencing: the incubation time of the plate is not longer than 18 h. the remaining eluate is stored at 4 ℃ for at least one week.
Phage titer determination
(1)5-10mL LB medium was inoculated with the ER2738 clone and incubated with shaking for 4-8h (mid-log, OD600 of about 0.5);
(2) when cells grow, melting top agar in a microwave oven, subpackaging into sterile culture tubes with 3mL of each tube, and storing at 45 ℃ for later use;
(3) preheating an LB/IPTG/Xgal plate at the desired dilution for at least 1h at 37 ℃ until ready for use;
(4) preparation in LB from 10 to 103Multiple dilutions of phage, final volume 1 mL;
(5) when the culture in the step (1) reaches the middle logarithmic phase, subpackaging the culture into microcentrifuge tubes with 200 mu L of each tube;
(6) adding 10 μ L of each phage dilution to each tube, adding only one dilution to each tube, rapidly vortexing, and incubating at room temperature for 1-5 min;
(7) one infected cell at a time was transferred to a culture tube containing top agar at 45 ℃ and briefly vortexed;
(8) immediately pouring the culture of step (7) onto pre-warmed LB/IPTG/Xgal plates, gently tilting and rotating the plates to spread the top agar evenly;
(9) cooling the plate for 5min, and performing inverted culture at 37 ℃ overnight;
(10) plaques were counted on a plate of approximately 100 plaques, and each number was multiplied by the dilution factor of the plate, at a phage titer of every 10. mu.L plaque forming units (pfu).
Sequencing of extracted DNA
(1) ER2738 in LB overnight, 1: 100 dilution, 1 mL/tube, one plaque per tube;
(2) puncturing the blue plaque by the gun head, and putting the blue plaque into the culture tube;
(3) shaking and incubating for 4.5-5h at 37 ℃;
(4) transferring the culture into a 1.5mL centrifuge tube, and centrifuging for 30s at 14000 rpm;
(5) sucking the supernatant into a new centrifuge tube, and centrifuging for 30s at 14000 rpm;
(6) sucking 80% of the supernatant into a new centrifugal tube, and storing at 4 ℃;
(7) adding 200 μ L PEG/NaCl into 500 μ L, mixing, and standing at room temperature for 10-20 min.
(8) Centrifuging at 14000rpm for 10min at 4 deg.C, discarding the supernatant, centrifuging again, and discarding the supernatant;
(9) resuspending with 100 μ L iodide, shaking vigorously, adding 250 μ L anhydrous ethanol, and standing at room temperature for 10-20 min;
(10) centrifuging at 14000rpm for 10min at 4 deg.C, discarding the supernatant, adding 500 μ L70% ethanol, centrifuging again at 14000rpm for 10min at 4 deg.C;
(11) discarding the supernatant, and drying in the sun;
(12) after resuspension with 30. mu.L of TE Buffer, the mixture was frozen at-20 ℃ and sequenced.
3. Results of the experiment
M screened by phage display technologyproThe affinity peptides M1, M3, M4 and their sequences of the proteins are shown in Table 1, respectively.
TABLE 1M obtained by screening by phage display technologyproAffinity peptides and sequences of proteins
Example 2 affinity and interaction kinetics detection of affinity peptides with target proteins
1. Experimental methods
Detection of affinity peptides M1, M3, M4 and target protein M by biofilm interference technique (BLI)proThe magnitude of the affinity of the interaction between the two, and the kinetics of the binding and dissociation between the polypeptide and the target protein. M1 polypeptide was modified at C-terminus with Biotin (Biotin-GG-GDAH TRPP WSAW-NH)2) The control group of M3 and M4 was treated with M1 and plotted against different concentrations of M immobilized on the SA-modified sensorproThe association and dissociation kinetic curve of the interaction is obtained by calculating the dissociation equilibrium constant K of the interaction through the association and dissociation kinetic curveDThe detailed experimental procedure is as follows:
(1) immersing a biosensor with Streptavidin (Streptavidin, SA) modified on the surface into PBS buffer solution for balancing for at least 10 min;
(2) immersing the sensor in PBS buffer solution to obtain a baseline;
(3) the sensor is immersed in a known concentration of immobilized solution (M1/M3/M4 polypeptide solution), and the biotinylated polypeptide in the solution binds to the biosensor surface, so that the surface film layer is changed. This step allows the polypeptide to bind to the SA biosensor.
(4) The cured sensor was immersed in PBS buffer for baseline.
(5) Immersing the cured biosensor in a solution containing MproIn the sample solution of protein, polypeptide and MproThe proteins bind to each other resulting in an increase in the thickness of the membrane layer. In this step MproThe protein concentrations were 900nM, 300nM, and 100 nM.
(6) Immersing the sensor bound with the antibody to be detected in a buffer solution for dissociation, MproThe shedding of proteins from the biosensor surface results in a reduction in the thickness of the membrane layer.
(7) The affinity peptides M1, M3, M4 and M are obtained by fitting through real-time monitoring of the thickness of the biological film layer of the biosensor in the experimental processproKinetic constants for protein interactions.
2. Results of the experiment
M1 polypeptide and target protein MproThe binding dissociation kinetics curve is shown in FIG. 2, the results of the binding dissociation kinetics and affinity analysis of the affinity peptide interaction with the target protein are shown in Table 2, and the control group of affinity peptides M3 and M4 and the target protein MproThe binding dissociation kinetics curves are shown in FIGS. 3A-B, and the results show that compared with the affinity peptide M3 (K) selected by phage display technologyD＝(4.34±0.05)E-9M)、M4(KD(7.53 ± 0.43) E-9M), affinity peptides M1 with M)proHigh affinity of protein, KDIn E-10M (9.09 +/-0.28), the M1 polypeptide is expected to be a lead compound of an anti-coronavirus drug due to the strong affinity.
TABLE 2 binding dissociation kinetics and affinity analysis of the interaction of affinity peptides with target proteins
Example 3 validation of the ability of the affinity peptide M1 to respond to the target protein
1. Experimental methods
Introducing cysteine into the C end of the affinity peptide to enable the polypeptide to be combined on the surface of the gold nanoparticle through Au-S bonds. 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 detailed experimental procedure is as follows:
(1) preparation of gold nanoparticles
Adding 150mL of sodium citrate solution (2.2mM) into a 250mL round-bottom flask, placing the flask in a water bath at 90 ℃, magnetically stirring at 400rpm, adding 1mL of chloroauric acid solution (25mM), adding 1mL of 60mM sodium citrate solution and 1mL of 25mM chloroauric acid solution after reacting for 30min, and cooling to room temperature after reacting for 30min to obtain a gold nanoparticle solution;
(2) polypeptide recognition Mpro
Taking 200 mu L of the gold nanoparticle solution, respectively adding 20 mu L of M1, M3 and M4 polypeptide (0.5mg/mL), uniformly mixing, placing in a water bath at 30 ℃ for 30min, then centrifuging at 12000rpm for 5min, discarding supernatant, and adding 200 mu L of water to resuspend precipitate. Adding M with different concentrations into 200 μ L of the above solutionproAnd BSA, and performing ultraviolet visible near infrared absorption spectrum measurement.
2. Results of the experiment
The experimental result shows that the gold nanoparticles with the surface modified with M1 polypeptide are added with MproAfter the protein, the SPR peak gradually shifts to a short wave (blue shift), the degree of blue shift shows a certain concentration dependence (see fig. 4A), and the blue shift is not common, while under the same experimental conditions, the addition of Bovine Serum Albumin (BSA) does not change the position of the SPR peak (see fig. 4B); the affinity peptides M3 and M4 screened by the phage display technology can generate the shift of SPR peak position due to the addition of BSA, thereby affecting the detection result (see figures 5A-D), namely, the gold nanoparticles modified by the M1 polypeptide can not generate the shift of SPR peak position due to the addition of BSA, therefore, the gold nanoparticles modified by the M1 polypeptide can show the effect on MproThe specific response of the protein further indicates that the M1 polypeptide can be used for auxiliary detection of coronavirus.
The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.
Claims (10)
1. An affinity peptide specifically binding to coronavirus, wherein the sequence of the affinity peptide is shown as SEQ ID NO. 1;
preferably, said affinity peptide specifically binds to the coronavirus main protease Mpro；
Preferably, the coronavirus is SARS-CoV-2.
2. An affinity peptide derivative comprising a modified product of the affinity peptide of claim 1, a variant of the affinity peptide of claim 1 obtained by addition and/or substitution of one or more amino acids, and a conjugate of the affinity peptide of claim 1 with another substance.
3. A nucleic acid molecule encoding the affinity peptide of claim 1 or encoding the affinity peptide derivative of claim 2.
4. An expression vector comprising the nucleic acid molecule of claim 3.
5. A host cell comprising the nucleic acid molecule of claim 3 or comprising the expression vector of claim 4.
6. A pharmaceutical composition comprising the affinity peptide of claim 1, the affinity peptide derivative of claim 2, the nucleic acid molecule of claim 3, the expression vector of claim 4;
preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and/or adjuvant.
7. A product for detecting coronaviruses, comprising the affinity peptide of claim 1, the affinity peptide derivative of claim 2, the nucleic acid molecule of claim 3, the expression vector of claim 4, the host cell of claim 5;
preferably, the product comprises a kit, a test strip and a chip.
8. A method for the non-diagnostic and therapeutic detection of coronaviruses, comprising the use of the affinity peptide according to claim 1, the affinity peptide derivative according to claim 2, the nucleic acid molecule according to claim 3, the expression vector according to claim 4, the host cell according to claim 5, or the product according to claim 7.
9. The use of an affinity peptide according to claim 1, wherein the use comprises any of the following:
(1) use of the affinity peptide derivative of claim 2;
(2) use in the preparation of a pharmaceutical composition according to claim 6;
(3) use in the preparation of a product according to claim 7.
10. Use of the affinity peptide derivative of claim 2 or the nucleic acid molecule of claim 3 or the expression vector of claim 4 or the host cell of claim 5, wherein said use comprises use of any one of:
(1) use in the preparation of a pharmaceutical composition according to claim 6;
(2) use in the preparation of a product according to claim 7.
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