WO2021212568A1 - 新型冠状病毒特异性抗原肽及其用途 - Google Patents

新型冠状病毒特异性抗原肽及其用途 Download PDF

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WO2021212568A1
WO2021212568A1 PCT/CN2020/090064 CN2020090064W WO2021212568A1 WO 2021212568 A1 WO2021212568 A1 WO 2021212568A1 CN 2020090064 W CN2020090064 W CN 2020090064W WO 2021212568 A1 WO2021212568 A1 WO 2021212568A1
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polypeptide
sars
protein
cov
sequence
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French (fr)
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王雅楠
杨衡
高美玲
陈春峰
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苏州方科生物科技有限公司
苏州系统医学研究所
中国人民解放军军事科学院军事医学研究院
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms

Definitions

  • the present disclosure belongs to the field of biotechnology. Specifically, the present disclosure relates to an antigenic peptide of SARS-CoV-2 virus, and the use of the foregoing antigenic peptides in the preparation of COVID-19 vaccines, and the preparation of drugs for preventing and treating COVID-19.
  • the new coronavirus belongs to the genus B coronavirus, a linear single-stranded RNA (ssRNA) virus. Its genome is about 29903 nucleotides in length and contains a total of 10 genes. Since January 10, 2020, the first SARS-CoV-2 genome sequence data was released, and since then, several new coronavirus genome sequences isolated from patients have been released. On January 22, 2020, the Genome Science Data Center officially released the 2019 Novel Coronavirus Resource Library.
  • the sequence similarity between the 2019 novel coronavirus (SARS-CoV-2) and the SARS virus that broke out in 2003 is 80% similar to the Bat SARS-like coronavirus isolate bat collected from domestic bats in February 2017 -SL-CoVZC45 genome sequence has the highest similarity, with a similarity of 88%.
  • SARS-CoV-2 2019 novel coronavirus
  • SARS-CoVZC45 the sequence similarity between the 2019 novel coronavirus
  • 6 institutions around the world have published 13 novel coronavirus genome sequences on the "Global Shared Influenza Virus Database GISAID".
  • the new coronavirus is an enveloped positive-strand RNA virus, containing a 30kb genome and four structural proteins, namely spike protein (S), envelope protein (E), membrane protein (M ) And nucleocapsid protein (N).
  • S protein regulates the attachment of the virus to the receptor on the target host cell.
  • E protein is to assemble viruses and act as ion channels; M protein and E protein play a role in virus assembly and participate in the biosynthesis of new virus particles; N protein forms a ribonucleoprotein complex with viral RNA.
  • the surface spike glycoprotein (S protein) of the new coronavirus is responsible for attaching to the host cell through the interaction with the host cell surface receptor (ACE2).
  • S protein exists in the form of homotrimer, each monomer contains more than 1200 amino acids.
  • RBD receptor binding domain
  • RBM receptor binding motif
  • SARS-CoV-2 enters the cell through the surface ACE2 cell receptor, its surface antigen is presented to B cells through the antigen, which stimulates the differentiation of B cells into memory B cells and plasma cells, and the plasma cells secrete anti-SARS-CoV-2 specific antibodies Neutralize the virus. Therefore, finding antigen peptides that can specifically activate the body's immune system and stimulate the B cell immune response to produce neutralizing antibodies has a key role. Therefore, we screened out specific antigen peptides on the surface of the novel coronavirus through the model, and verified the effectiveness of the novel coronavirus antigen peptides by combining structural biology analysis, clinical data detection, and experiments, so as to achieve the purpose of timely and accurate diagnosis of the novel coronavirus. .
  • the present disclosure provides the following technical solutions.
  • polypeptide wherein the polypeptide is a polypeptide shown in any one of the following (i)-(ii):
  • polypeptide is a polypeptide encoded by the sequence shown in SEQ ID NO: 2;
  • the polypeptide is a polypeptide in which one or more amino acids are substituted, repeated, deleted or added on the basis of the polypeptide shown in (i).
  • polypeptide according to (1) wherein the polypeptide has at least 90% homology with the polypeptide encoded by the sequence shown in SEQ ID NO: 2; preferably, at least 95% or more Homology.
  • kit for detecting SARS-CoV-2 infection or COVID-19 contains the polypeptide according to any one of (1) to (3).
  • composition or vaccine wherein the pharmaceutical composition or vaccine contains the polypeptide according to any one of (1) to (3).
  • a method of treating SARS-CoV-2 infection or suffering from COVID-19 comprising administering to the patient the polypeptide according to any one of (1)-(3) or according to (5) ) The pharmaceutical composition or vaccine.
  • the antigen peptide obtained in the present disclosure has good accuracy and specificity for the new coronavirus, and can be used for the detection of SARS-CoV-2.
  • the antigen peptide obtained in the present disclosure has a good inhibitory effect on SARS-CoV-2, and can be used for the treatment of diseases caused by SARS-CoV-2.
  • Figure 1 shows the preliminary screening of specific antigen peptides by Elisa
  • FIG. 1 shows the 3-D structure of SARS-CoV-2S protein
  • FIG. 3 shows the sequence homology alignment of S672-691
  • Figure 4 shows the positive serum detected by Elisa
  • FIG. 5 shows the results of the neutralization experiment
  • Figure 6 shows the results of the polypeptides inhibiting SARS-CoV-2 true virus and pseudovirus infections
  • FIG. 7 shows that the test result of nucleic acid detection by the kit is positive serum
  • Fig. 8 shows that the nucleic acid test result of Elisa is negative serum
  • Figure 9 shows statistical changes in antibody detection levels of infected patients during hospitalization
  • Figure 10 shows the sensitivity and accuracy (POS/NEG) of the S672-691 polypeptide in detecting SARS-CoV-2-IgG/IgM: POS represents a positive nucleic acid test, and NEG represents a negative nucleic acid test.
  • FIG. 11 shows that the S672-691 polypeptide immunizes mice and can stimulate the production of antibodies.
  • Figure 12 shows that the antibody produced by mice immunized with S672-691 polypeptide has an inhibitory effect on the SARS-CoV-2 virus.
  • Figure 13 shows the Western blot detection results of S2 specific antibodies.
  • Figure 14 shows the role of TMPRSS2 in S protein cleavage.
  • Figure 15 shows that the S672-691 polypeptide can effectively inhibit SARS-CoV-2 pseudovirus infection in HEK293 cells co-transfected with TMPRSS2 and ACE2.
  • the term "about” means: a value includes the standard deviation of the error of the device or method used to determine the value.
  • the selected/optional/preferred “numerical range” includes both the numerical endpoints at both ends of the range and all natural numbers covered by the numerical endpoints relative to the aforementioned numerical endpoints.
  • SARS-CoV-2 also known as “2019-nCoV”
  • 2019-nCoV means the 2019 novel coronavirus.
  • COVID-19 means Corona Virus Disease 2019, or “New Coronary Pneumonia” for short, refers to pneumonia caused by the 2019 Novel Coronavirus (SARS-CoV-2) infection .
  • SARS-CoV-2 2019 Novel Coronavirus
  • amino acid mutation includes “substitution, duplication, deletion or addition of one or more amino acids.”
  • mutation refers to a change in the amino acid sequence. In a specific embodiment, the term “mutation” refers to "substitution.”
  • the "mutation" of the present disclosure may be selected from “conservative mutations".
  • the term “conservative mutation” refers to a mutation that can normally maintain the function of a protein. Representative examples of conservative mutations are conservative substitutions.
  • the term "conservative substitution” involves replacing an amino acid residue with an amino acid residue having a similar side chain.
  • the art has defined families of amino acid residues with similar side chains, and include those with basic side chains (such as lysine, arginine, and histidine), acidic side chains (such as aspartic acid and glutamic acid). ), non-polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine), non-polar side chains (such as alanine, valine) Acid, leucine, isoleucine, proline, phenylalanine, methionine and tryptophan), ⁇ -branched chains (e.g. threonine, valine and isoleucine) and aromatic side chains (E.g. tyrosine, phenylalanine, tryptophan and histidine).
  • basic side chains such as lysine, arginine, and histidine
  • substitutions generally exchange an amino acid at one or more positions in a protein. This substitution can be conservative. As the substitutions considered as conservative substitutions, specifically, the substitution of Ala to Ser or Thr, the substitution of Arg to Gln, His, or Lys, the substitution of Asn to Glu, Gln, Lys, His or Asp, the substitution of Asp to Asn, Glu or Gln substitution, Cys to Ser or Ala, Gln to Asn, Glu, Lys, His, Asp or Arg, Glu to Gly, Asn, Gln, Lys or Asp, Gly to Pro Replacement, His to Asn, Lys, Gln, Arg or Tyr, Ile to Leu, Met, Val or Phe, Leu to Ile, Met, Val or Phe, Lys to Asn, Glu, Gln, His or Arg substitution, Met to Ile, Leu, Val or Phe, Phe to Trp, Tyr, Met, Ile or Leu
  • sequence identity or “percent identity” in the comparison of two nucleic acids or polypeptides refers to when using a nucleotide or amino acid residue sequence comparison algorithm or as measured by visual inspection, When comparing and aligning with the greatest correspondence, they are the same or have a certain percentage of the same sequence. That is to say, the identity of a nucleotide or amino acid sequence can be defined by the following ratio, which is a way to maximize the number of identical nucleotides or amino acids in two or more nucleotide or amino acid sequences. In addition, gaps are added as needed to compare the number of nucleotides or amino acids that are identical during the comparison, and the ratio of the total number of nucleotides or amino acids in the comparison part.
  • sequence identity between two or more polynucleotides or polypeptides can be determined by the following method: aligning the nucleotide or amino acid sequence of the polynucleotide or polypeptide and aligning the aligned Score the number of positions containing the same nucleotide or amino acid residue in the polynucleotide or polypeptide, and compare it with the number of positions containing different nucleotide or amino acid residues in the aligned polynucleotide or polypeptide .
  • Polynucleotides may differ at one position, for example, by containing different nucleotides or missing nucleotides.
  • Polypeptides may differ at one position, for example, by containing different amino acids or missing amino acids.
  • Sequence identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of amino acid residues in the polynucleotide or polypeptide.
  • percent identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of nucleotide or amino acid residues in the polynucleotide or polypeptide and multiplying by 100.
  • two or more sequences or subsequences when using sequence comparison algorithms or measuring by visual inspection to compare and align with the greatest correspondence, two or more sequences or subsequences have at least 40%, 50%, 60% %, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the "sequence of nucleotide or amino acid residues" Identity” or "Percent Identity”.
  • Identity or “Percent Identity”.
  • Percent Identity The judgment/calculation of "sequence identity” or “percent identity” can be based on any suitable region of the sequence.
  • the sequence is substantially the same over the entire length of any one or two compared biopolymers (i.e., nucleic acids or polypeptides).
  • the new coronavirus infects human respiratory epithelial cells through the molecular mechanism of S-protein and human ACE2 interaction.
  • the S protein of the new coronavirus is initiated by TMPRSS2.
  • the S protein regulates the attachment of the coronavirus to the receptor on the target host cell, and plays an important role in binding to the host cell to produce interaction. Therefore, we screened out seven antigenic peptides by studying the specific epitopes of the S protein on the surface of the new coronavirus.
  • amino acid positions of the antigen peptide in the S protein are located at 25-39 (PPAYTNSFTRGVYYP), 672-691 (ASYQTQTNSPRRARSVASQS), 764-778 (NRALTGIAVEQDKNT), 907-921 (NGIGVTQNVLYENQK), 1155-1169 (YFKNHTSPDVDLGDI), 1198 -1212 (IDLQELGKYEQYIKW), 1257-1271 (DEDDSEPVLKGVKLH), the specific sequence is shown in Table 1 below.
  • an antigen peptide that can clearly detect the antibody level in the serum of patients with COVID-19, and is significantly different from normal people.
  • This sequence is located at the 672-691 position of the S protein, and the specific amino acid sequence is ASYQTQTNSPRRARSVASQS.
  • the sequence alignment found that it is located at the TMPRSS2 position of the S protein. And according to the existing research data, this site has an important role in activating the S protein, so it has important research significance.
  • polypeptide epitopes that may have antigenicity in the obtained S protein.
  • polypeptide screening process we found a sequence that can detect the antibody difference between the patient and the normal person. For this polypeptide sequence, we have expanded further research.
  • this polypeptide is located at the TMPRSS2 site (672-691) structure of the S protein.
  • SEQ ID NO: 1 shows the amino acid sequence of positions 25-39 of the coronavirus S protein
  • SEQ ID NO: 2 shows the amino acid sequence of positions 672-691 of the coronavirus S protein
  • SEQ ID NO: 3 shows the amino acid sequence of positions 764-778 of the S protein of coronavirus
  • SEQ ID NO: 4 shows the amino acid sequence of positions 907-921 of the coronavirus S protein
  • SEQ ID NO: 5 shows the amino acid sequence at positions 1155-1169 of the coronavirus S protein
  • SEQ ID NO: 6 shows the 1198-1212 amino acid sequence of the coronavirus S protein
  • SEQ ID NO: 7 shows the amino acid sequence of positions 1257-1271 of the coronavirus S protein.
  • the peptide sequence is synthesized by GenScript Biotechnology, the purity is ⁇ 85%, the gross weight is 4mg, and each 1mg is divided into 1 tube.
  • TMB-ELISA color developing solution (Thermo Scientific, Lot#TK2666052)
  • Figure 1 shows the preliminary screening of specific antigen peptides by Elisa.
  • 2, 3, 7, 17 are the sera of patients with new crown positive
  • MES is the negative control group with only added peptides and no serum
  • background is the background with only serum and no peptides added.
  • H-1 and H-2 are normal human serum, and draw a graph based on the OD450 readings of the IgG results.
  • SARS-CoV-2S protein 3-D structure annotates B cell epitopes
  • FIG. 2 shows the 3-D structure diagram of the SARS-CoV-2S protein.
  • the protein S25-39 is annotated in blue
  • the protein S672-691 is annotated in green
  • the protein S764-778 is annotated in red
  • the protein S907-921 is annotated in purple.
  • the potential Furin and TMPRSS2 cleavage sites in the S protein of coronavirus are between the S1 and S2 domains, and the insertion mutation occurs at amino acid 681 of the SARS-CoV-2S protein.
  • the sequence of S672-691 and human, bat SARS- Like CoVs and MERS have been compared for homology, and the software used for the comparison is MEGA7.
  • Figure 3 shows the potential Fruin and TMPRSS2 positions of the SARS-CoV-2S protein, as well as the homology between the S672-691 sequence and human and bat SRARS-like CoVs and MERS. It can be seen that the homology is not high, so S672-691 protein is likely to be a specific detection site for SARS-CoV-2.
  • Figure 4 shows the positive sera tested by Elisa. After synthesizing the S672-691 antigen peptide sequence, the Elisa experiment was designed in vitro to detect serum samples with positive nucleic acid test results. Each group of experiments set up a serum background control group and a random antigen peptide sequence control group. It can be found that in the positive serum, the levels of IgG and IgM detection antibodies are basically relatively high. The antibody level of antigen peptide S25-39 and the background control group are basically the same, at a very low antibody level. The sera we collected are all samples taken from patients with COVID-19 when they went to the hospital for treatment. A positive nucleic acid test result indicates the presence of COVID-19 in the body during the nucleic acid test.
  • the operation is performed by trained experimental operators. Before the experimental operation, change clothes in the clean area (wear disposable sterile clothes, change work shoes, wear masks, hats, disposable medical latex gloves) before entering the experimental area Inside, carry out experimental operations.
  • change clothes in the clean area wear disposable sterile clothes, change work shoes, wear masks, hats, disposable medical latex gloves
  • inhibition rate [1-(average luminous intensity of the sample group-average CC of the blank control)/(average luminous intensity of the negative group VC-average of the blank control CC)]*100%.
  • Figure 6 shows the results of an experiment for the polypeptide to inhibit virus infection. It can be seen from Figure 6 that when the polypeptide dose is 1 ⁇ g, the virus infection can be significantly inhibited, and the difference with the control group is statistically significant.
  • enzyme conjugate add 100 ⁇ l of enzyme conjugate to each well.
  • Stop add 50 ⁇ l stop solution to each well to stop the reaction. Shake gently to mix.
  • Reading value Set the microplate reader at a wavelength of 450nm, and measure the OD value of each well. The value should be read within 30 minutes after terminating the reaction.
  • Fig. 7 shows that the test result of the kit for detecting nucleic acid is positive serum.
  • kits for detecting serum antibodies against some key proteins of the new coronavirus S protein and N protein have developed kits for detecting serum antibodies against some key proteins of the new coronavirus S protein and N protein. From the comparative test results in Figure 7, it is also clear that this specific antigen peptide can detect serum antibodies. Antibodies raised against the new coronavirus.
  • Fig. 8 shows that the result of the nucleic acid test by Elisa is negative serum.
  • Specific antibodies can also be detected in the serum of patients whose nucleic acid test results are negative, and the antigen peptide has a relatively high sensitivity in negative serum through statistical analysis. It shows that although the virus is cleared from the body after the patient is infected with the new crown pneumonia virus, the antibody does exist.
  • Figure 9 shows the changes in serum antibody levels of the five patients we tracked after the diagnosis of new coronary pneumonia and during hospitalization, as well as the comprehensive clinical scores based on the disease.
  • the S672-691 protein can detect changes in the patient's serum antibody levels during treatment, and the trend is basically the same as the clinical comprehensive score. This means that our peptide can sensitively detect the level of antibodies in patients, and it can also reflect the immune status of SARS-CoV-2 through this antibody level.
  • Figure 10 shows SARS-CoV-2-IgG (POS/NEG): POS represents a positive nucleic acid test, and NEG represents a negative nucleic acid test.
  • Figure 9 shows SARS-CoV-2-IgM (POS/NEG): POS represents a positive nucleic acid test, and NEG represents a negative nucleic acid test.
  • the sensitivity and specificity are calculated.
  • the inventors tested 50 cases of COVID-19 patients with positive nucleic acid, 114 cases of COVID-19 patients with negative nucleic acid, and 47 cases of normal people, and statistically calculated SARS-CoV-2-IgG and SARS-CoV-2-IgM.
  • the Elisa experiment can efficiently and sensitively detect the antibodies in the sample. Therefore, the Elisa experiment was carried out in vitro to detect the serum samples of patients with new coronavirus infection and normal people to verify the sensitivity and accuracy of the peptides.
  • the statistical results according to the ROC curve are as follows:
  • the antigen peptide detects the SARS-CoV-2-IgG level in the serum of 47 cases of normal people and 50 cases of new crown patients (nucleic acid test positive):
  • the detection sensitivity is 94%, and the specificity is 87.23%
  • the detection sensitivity is 86%, and the specificity is 100%
  • the antigen peptide was tested for the levels of SARS-CoV-2-IgG in the serum of 47 cases of normal people and 114 cases of new crown patients (nucleic acid test negative):
  • the detection sensitivity is 91.23%, and the specificity is 87.23%
  • This antigen peptide detects the level of SARS-CoV-2-IgM in the serum of 47 cases of normal people and 50 cases of new crown patients (nucleic acid test positive):
  • the detection sensitivity is 94%, and the specificity is 78.72%
  • the detection sensitivity is 88%, and the specificity is 80.85%
  • This antigen peptide detects the level of SARS-CoV-2-IgM in the serum of 47 cases of normal people and 114 cases of new crown patients (negative nucleic acid test):
  • the detection sensitivity is 87.72%, and the specificity is 74.47%
  • Each mouse is immunized with 50 ⁇ g polypeptide. Under aseptic conditions, add 50 ⁇ g of polypeptide to 50 ⁇ l with enzyme-free water, add 50 ⁇ l of adjuvant to prepare 100 ⁇ l of polypeptide mixture, and place on ice for later use.
  • Each mouse uses a 1 ml sterile syringe to subcutaneously inject 100 ⁇ l of the peptide mixture into the abdomen.
  • One injection each on March 17, March 26, and April 2.
  • FIG. 11 shows that the S672-691 polypeptide can obviously stimulate the immune response in mice and produce antibodies.
  • mice immunized with S672-691 polypeptide has inhibitory effect on SARS-CoV-2 pseudovirus
  • Figure 12 shows that the antibody produced by mice immunized with S672-691 polypeptide has an inhibitory effect on the SARS-CoV-2 virus.
  • CST Rabbit anti- ⁇ -actin
  • MP mouse anti-coronavirus spike2
  • Figure 13 shows the Western blot detection results of S2 specific antibodies.
  • S2 specific antibodies Two main bands were specifically detected in the protein extracts infected with SARS-CoV-2, but they were not infected. Not detected in Vero cells, these two bands represent the full-length spike protein and the S2 fragment that may be cleaved at the potential TMRPSS2 cleavage site.
  • HEK293 cells were plated on a 48-well plate at a density of 1*10 5 and cultured overnight. Add camostat mesylate (MCE, HY-13512), nafamostat mesylate (MCE, HY-B0190A) and BSA or Biotin coupled 672-691 peptide to the culture medium for 2 hours. Then HEK293cells were co-transfected with GFP-labeled SARS-COV-2-S plasmid (GenScript, C1043FB180-1) and Tmprss2 expression plasmid (OriGene, RC208677). The culture medium was replaced 16 hours after transfection and the culture was continued for 24 hours.
  • MCE camostat mesylate
  • MCE nafamostat mesylate
  • BSA Biotin coupled 672-691 peptide
  • Spike protein was identified by SDS-polyacrylamide gel electrophoresis and Western blot. The primary antibody was incubated with Rabbit anti-beta-actin (CST, 8457S, 1:2000) and mouse anti-eGFP (AtaGenix, 539191227, 1:1000).
  • FIG 14 shows the role of TMPRSS2 in S protein cleavage.
  • TMRPSS2 overexpression of TMRPSS2 was significantly increased, and TMRPSS2 inhibitors such as camostat or nafamostat and the synthetic S672-691 polypeptide inhibited the cleavage of S protein.
  • HEK293T cells were plated in a 24-well plate at a density of 2 ⁇ 10 5 /well. After overnight culture, they were transferred to the corresponding hACE2 and TMPRSS2 expression plasmids as shown in the figure. After 24 hours of transfection, the corresponding concentration of S672-691 was added. Polypeptide, after 2 hours of pretreatment, it was infected with SARS-CoV-2 pseudovirus. After 24 hours of infection, the level of pseudovirus in the cell was detected by measuring the luciferase reporter experiment, and the data was analyzed to calculate the effect of S672-691 polypeptide on SARS-CoV-2. Suppressive effect of pseudovirus into host cell.
  • Figure 15 shows that the S672-691 polypeptide and the antibody stimulated by the polypeptide can effectively inhibit the SARS-CoV-2 pseudovirus infection of HEK293 cells co-transfected with TMPRSS2 and ACE2.
  • TMRPSS2 and ACE2 are co-transfected with HEK293 cells, it significantly increases SARS-CoV-2 pseudovirus infection, S672-691 polypeptide can inhibit this infection.

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Abstract

提供一种新型冠状病毒特异性抗原肽及其用途。具体提供一种多肽，所述多肽为SARS-CoV-2病毒的抗原肽，及其前述抗原肽在疾病诊断、制备COVID-19疫苗、制备预防、治疗COVID-19的药物中的用途。

Description

新型冠状病毒特异性抗原肽及其用途 技术领域
本公开属于生物技术领域。具体来说，本公开涉及一种SARS-CoV-2病毒的抗原肽，及其前述抗原肽在制备COVID-19疫苗、制备预防、治疗COVID-19的药物中的用途。
背景技术
新型冠状病毒(SARS-CoV-2)属于乙型冠状病毒属，线性单链RNA(ssRNA)病毒。其基因组全长约29903个核苷酸，共包含10个基因。自2020年1月10日，第一个SARS-CoV-2基因组序列数据被公布，此后陆续有多个从患者身上分离的新型冠状病毒的基因组序列发布。2020年1月22日，基因组科学数据中心正式发布2019新型冠状病毒资源库。经数据分析，2019新型冠状病毒(SARS-CoV-2)与2003年爆发的SARS病毒基因组序列相似度为80％，与2017年2月从国内的蝙蝠中采集到的Bat SARS-like coronavirus isolate bat-SL-CoVZC45基因组序列相似性最高，相似度为88％。截止到2020年1月30日，全球已有6家机构在“全球共享流感病毒数据库GISAID”上发布了13例新型冠状基因组序列。
新型冠状病毒(SARS-CoV-2)是一种包膜的正链RNA病毒，含有30kb的基因组和四种结构蛋白，即刺突蛋白(S)、包膜蛋白(E)、膜蛋白(M)和核衣壳蛋白(N)。S蛋白调节病毒对目标宿主细胞上受体的附着。E蛋白质的功能是组装病毒并充当离子通道；M蛋白与E蛋白一起在病毒组装中发挥作用，并参与新病毒颗粒的生物合成；N蛋白与病毒RNA形成核糖核蛋白复合物。新型冠状病毒的表面刺状糖蛋白(S蛋白)负责通过与宿主细胞表面受体(ACE2)的相互作用附着到宿主细胞上。S蛋白以同型三聚体的形式存在，每个单体含有1200多个氨基酸。在SARS-CoV-2的S蛋白中，一个含有306-575残基的小结构域被鉴定为受体结合域(RBD)，其中被称为受体结合基序(RBM)的439-508残基直接介导了与ACE2的相互作用。利用冷冻电镜技术，西湖大学周强实验室已经解析出了新冠受体ACE2的全长结构。已经发现新型冠状病毒的关键刺突蛋白(S蛋白)与人体细胞的受体蛋白ACE2蛋白的结合能力要远高于SARS病毒，这部分解释了为什么新型冠状病毒传染性要比SARS病毒强得多。借助冷冻电镜技术，科学家能够更好地观察新型冠状病毒S蛋白的结构，以及它与ACE2蛋白之间的相互作用。这一研究发现为进一步解析全长ACE2和新冠病毒的S蛋白复合物的三维结构奠定了基础，将为理解新冠病毒侵染细胞，提供更多线索。而对ACE2全长结构的解析，有助于为后续疫苗和抗病毒药物的研发，提供重要的结构生物学数据支撑。将有助于理解冠状病毒进入靶细胞的结构基础和功能特征，对发现和优化阻断进入细胞的抑制剂有重要作用。
在感染COVID-19初期，患者表现症状不明显，但具有极高的传染性，可通过接触传播，飞沫传播等方式人传人，严重者可导致死亡。但是对于病毒性传染病，可应用的药物非常有限，疫苗是最有效的手段之一。然而由于新发传染病的新发及不可预见性，大多数新发和烈性传染病并无有效的疫苗储备。
前期有科研人员使用来自结构生物学和机器学习的计算工具来识别基于病毒蛋白抗原呈现和抗体结合特性的SARS-CoV-2T细胞和B细胞表位。这些表位可用于开发更有效的疫苗和鉴定中和抗体。已经在人类MHC-I和MHC-II等位基因中鉴定了405个抗原表达分数良好的病毒肽，并在SARS-CoV-2Spike蛋白受体结合域附近发现了两个潜在的中和B细胞表位(440-460和494-506)。然而，目前还没有针对SARS-CoV-2或任何形式的冠状病毒的批准疫苗。
发明内容
发明要解决的问题
由于SARS-CoV-2通过表面ACE2细胞受体进入细胞，其表面抗原通过抗原递呈给B细胞，刺激B细胞分化为记忆B细胞和浆细胞，浆细胞分泌抗SARS-CoV-2特异性抗体中和病毒。因此找到能特异性激活机体免疫系统，并且能刺激B细胞免疫应答从而产生中和抗体的抗原肽具有关键作用。因此，我们通过模型筛选出关于新型冠状病毒表面特异性抗原肽，通过结合结构生物学分析，临床数据检测，实验验证新型冠状病毒抗原肽的有效性，从而达到及时准确的诊断新型冠状病毒的目的。
用于解决问题的方案
本公开提供了如下技术方案。
(1)一种多肽，其中，所述多肽为如下(i)-(ii)任一项所示的多肽：
(i)所述多肽为如SEQ ID NO：2所示的序列编码的多肽；
(ii)所述多肽为在(i)所示的多肽的基础上，取代、重复、缺失或添加一个或多个氨基酸的多肽。
(2)根据(1)所述的多肽，其中，所述多肽相对于如SEQ ID NO：2所示的序列编码的多肽，具有至少90％的同源性；优选的，具有至少95％以上的同源性。
(3)根据(1)-(2)任一项所述的多肽，其中，所述多肽的氨基酸残基的个数为20个。
(4)一种检测是否被SARS-CoV-2感染或患有COVID-19的试剂盒，其中，所述试剂盒中含有根据(1)-(3)任一项所述的多肽。
(5)一种药物组合物或疫苗，其中，所述药物组合物或疫苗中含有根据(1)-(3)任一项所 述的多肽。
(6)根据(1)-(3)任一项所述的多肽在制备用于检测是否被SARS-CoV-2感染或患有COVID-19的试剂中的用途。
(7)根据(1)-(3)任一项所述的多肽在制备用于治疗或预防被SARS-CoV-2感染或患有COVID-19的药物中的用途。
(8)一种治疗被SARS-CoV-2感染或患有COVID-19的方法，其中，所述方法包括向患者施用根据(1)-(3)任一项所述的多肽或根据(5)所述的药物组合物或疫苗。
发明的效果
在一个实施方式中，本公开得到的抗原肽对于新型冠状病毒具有良好的准确性和特异性，能够用于SARS-CoV-2的检测。
在另一个实施方式中，本公开得到的抗原肽对于SARS-CoV-2具有良好的抑制作用，能够用于SARS-CoV-2所导致的疾病的治疗。
附图说明
图1表示Elisa初步筛选特异性抗原肽；
图2表示SARS-CoV-2S蛋白3-D结构；
图3表示S672-691序列同源性比对
图4表示Elisa检测阳性血清；
图5示出中和实验结果图；
图6示出多肽抑制SARS-CoV-2真病毒和假病毒感染实验的结果；
图7示出试剂盒检测核酸检测结果呈阳性血清；
图8示出Elisa检测核酸检测结果呈阴性血清；
图9示出统计感染病人在住院治疗过程中抗体检测水平变化；
图10示出S672-691多肽检测SARS-CoV-2-IgG/IgM灵敏度和准确性(POS/NEG):POS代表核酸检测阳性，NEG代表核酸检测阴性。
图11示出S672-691多肽免疫小鼠并且能激发产生抗体。
图12示出S672-691多肽免疫小鼠产生的抗体对SARS-CoV-2病毒具有抑制作用。
图13示出S2特异性抗体的Western blot检测结果。
图14示出TMPRSS2在S蛋白裂解中的作用。
图15示出S672-691多肽能有效抑制TMPRSS2和ACE2共转染HEK293细胞的SARS-CoV-2假病毒感染。
具体实施方式
定义
当在权利要求和/或说明书中与术语“包含”联用时，词语“一(a)”或“一(an)”可以指“一个”，但也可以指“一个或多个”、“至少一个”以及“一个或多于一个”。
如在权利要求和说明书中所使用的，词语“包含”、“具有”、“包括”或“含有”是指包括在内的或开放式的，并不排除额外的、未引述的元件或方法步骤。
在整个申请文件中，术语“约”表示：一个值包括测定该值所使用的装置或方法的误差的标准偏差。
虽然所公开的内容支持术语“或”的定义仅为替代物以及“和/或”，但除非明确表示仅为替代物或替代物之间相互排斥外，权利要求中的术语“或”是指“和/或”。
当用于权利要求书或说明书时，选择/可选/优选的“数值范围”既包括范围两端的数值端点，也包括相对于前述数值端点而言，所述数值端点中间所覆盖的所有自然数。
如本公开所使用的，术语“SARS-CoV-2”，也被称为“2019-nCoV”，其含义为2019新型冠状病毒。
如本公开所使用的，术语“COVID-19”的含义为新型冠状病毒肺炎(Corona Virus Disease 2019)，简称“新冠肺炎”，是指2019新型冠状病毒(SARS-CoV-2)感染导致的肺炎。
如本公开所使用的，术语“氨基酸突变”，包括“取代、重复、缺失或添加一个或多个氨基酸”。在本公开中，术语“突变”是指氨基酸序列的改变。在一个具体的实施方式中，术语“突变”是指“取代”。
在一个实施方式中，本公开的“突变”可以选自“保守突变”。在本公开中，术语“保守突变”是指可正常维持蛋白质的功能的突变。保守突变的代表性例子为保守置换。
如本公开所使用的，术语“保守置换”涉及用具有类似侧链的氨基酸残基替换氨基酸残基。本领域已经定义了具有类似侧链的氨基酸残基家族，并且包括具有碱性侧链(例如赖氨酸、精氨酸和组氨酸)、酸性侧链(例如天冬氨酸和谷氨酸)、不带电极性侧链(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、和半胱氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、蛋氨酸和色氨酸)、β-支链(例如苏氨酸、缬氨酸和异亮氨酸)和芳香侧链(例如酪氨酸、苯丙氨酸、色氨酸和组氨酸)。
如本公开所使用的，“保守置换”通常在蛋白质的一个或多个位点上交换一种氨基酸。这种取代可以是保守的。作为被视作保守置换的置换，具体而言，可以举出Ala向Ser或Thr的置换、Arg向Gln、His或Lys的置换、Asn向Glu、Gln、Lys、His或Asp的置换、Asp向Asn、Glu或Gln的置换、Cys向Ser或Ala的置换、Gln向Asn、Glu、Lys、His、Asp或Arg的置换、Glu向Gly、Asn、Gln、Lys或Asp的置换、Gly向Pro的置换、His向Asn、Lys、Gln、Arg或Tyr的置换、Ile向Leu、Met、Val或Phe的置换、Leu向Ile、Met、Val或Phe的置换、Lys向Asn、Glu、Gln、His或Arg的置换、Met向Ile、Leu、Val或Phe的置换、Phe向Trp、Tyr、Met、Ile或Leu的置换、Ser向Thr或Ala的置换、Thr向Ser或Ala的置换、Trp向Phe或Tyr的置换、Tyr向His、Phe或Trp的置换、及Val向Met、Ile或Leu的置换。此外，保守突变还包括起因于基因所来源的个体差异、株、种的差异等天然产生的突变。
如本公开所使用的，在两种核酸或多肽比较中的术语“序列同一性”或“同一性百分比”，是指当使用核苷酸或氨基酸残基序列比较算法或通过目视检查测量，以最大的对应性进行比较和比对时，它们是相同的或具有相同序列特定百分比数。也就是说，核苷酸或者氨基酸序列的同一性可以利用下述比例来定义，该比例是将两个或多个核苷酸或氨基酸序列按照一致的核苷酸或氨基酸数达到最大的方式，并根据需要加入空位来进行比对时一致的核苷酸数或氨基酸数，在比对部分的全部核苷酸或氨基酸数中的比例。
如本公开所使用的，两个或更多个多核苷酸或多肽之间的序列同一性可通过以下方法测定：将多核苷酸或多肽的核苷酸或氨基酸序列对准且对经对准的多核苷酸或多肽中含有相同核苷酸或氨基酸残基的位置数目进行评分，且将其与经对准的多核苷酸或多肽中含有不同核苷酸或氨基酸残基的位置数目进行比较。多核苷酸可例如通过含有不同核苷酸或缺失核苷酸而在一个位置处不同。多肽可例如通过含有不同氨基酸或缺失氨基酸而在一个位置处不同。序列同一性可通过用含有相同核苷酸或氨基酸残基的位置数目除以多核苷酸或多肽中氨基酸残基的总数来计算。举例而言，可通过用含有相同核苷酸或氨基酸残基的位置数目除以多核苷酸或多肽中核苷酸或氨基酸残基的总数且乘以100来计算同一性百分比。
示例性的，在本公开中，当使用序列比较算法或通过目视检查测量以最大的对应性进行比较和比对时，两个或多个序列或子序列具有至少40％、50％、60％、70％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％核苷酸或氨基酸残基的“序列同一性”或“同一性百分比”。“序列同一性”或“同一性百分比”的判断/计算可以基于序列任何合适的区域上。例如，长度至少约10个残基的区域、至少约15个残基的区域，至少约18个残基的区域，至少约20个残基的区域。在某些实施方案中，所述序列在任一或两个相比较的生物聚合物(就是核酸或多肽)的整个长度上基本相 同。
研究表明新型冠状病毒是通过S-蛋白与人ACE2互作的分子机制，来感染人的呼吸道上皮细胞，此外，新型冠状病毒的S蛋白由TMPRSS2启动。S蛋白调节冠状病毒对目标宿主细胞上受体的附着，在结合到宿主细胞产生相互作用时具有重要作用。因此我们通过对新型冠状病毒表面S蛋白进行特异性抗原表位进行研究，筛选出七条抗原肽。根据抗原肽在S蛋白的氨基酸位点，分别定位在25-39(PPAYTNSFTRGVYYP)，672-691(ASYQTQTNSPRRARSVASQS)，764-778(NRALTGIAVEQDKNT)，907-921(NGIGVTQNVLYENQK)，1155-1169(YFKNHTSPDVDLGDI)，1198-1212(IDLQELGKYEQYIKW)，1257-1271(DEDDSEPVLKGVKLH),具体序列如下表1所示。
表1抗原肽序列
Figure PCTCN2020090064-appb-000001
在得到抗原表位后，我们进行了抗原肽的免疫原性检测研究。我们在体外将这些抗原肽全部合成，使用酶联免疫吸附实验Elisa初步筛选了新冠患者以及正常人的血清样本。根据采样时的核酸检测结果我们将新冠患者血清样本分为核酸检测阳性和阴性两个组别。
在一个具体的实施方式中，通过对抗原肽的初步筛选，我们筛选出一条能明显检测到新冠患者血清中的抗体水平的抗原肽，并且和正常人存在明显差异。这条序列位于S蛋白的672-691位点处，具体氨基酸序列为ASYQTQTNSPRRARSVASQS，通过序列比对发现刚好位于S蛋白的TMPRSS2位点。并且根据已有研究数据表明，该位点具有启动S蛋白的重要作用，因此具有重要的研究意义。
人体在感染病毒之后会在第二周至三周左右产生相应的抗病毒抗体。我们收集了大量感染SARS-CoV-2临床样本，并对这些样本进行分类，并对被收集血清样品的个体信息和健康指标进行详细的记录，包括感染日期，采样日期,病史等等。
通过合成得到的抗原肽序列，我们首先在体外用Elisa实验对新型冠状病毒感染病人血清进行筛 查。根据检测结果，我们分别将抗体检测水平比较高，比较低的患者血清样本单独取出进行病毒中和实验，进而验证检测到的抗体对于病毒的中和效果。同时，我们也进行了多肽抑制病毒感染的实验，与其他研究类药物相比较，在多肽剂量达到2μg时，对病毒感染具有明显的抑制作用。
我们将得到的S蛋白中可能具有抗原性的多肽表位进行合成，在多肽的筛选过程中发现有一条序列能检测出患者与正常人之间的抗体差异，针对这条多肽序列我们又展开了进一步研究。
通过序列比对，我们得知这条多肽位于S蛋白的TMPRSS2位点(672-691)结构。
我们通过Elisa实验检测到核酸检测呈阳性的新冠患者血清对于特异性抗原肽检测呈比较高的抗体水平，并且结果与商品化的用于检测血清的新型冠状病毒S蛋白，N蛋白等试剂盒检测结果一致，并且检出率相对较高。同时我们设置了一条随机序列作为对照，检测抗原肽的IgG和IgM两个指标，经分析，IgG和IgM检测结果具有相关性。我们根据多肽检测结果，挑选出抗体水平比较高的病人血清，抗体水平比较低的病人血清以及正常人血清去做假病毒中和实验。根据中和实验结果来验证抗体检测的准确性。同时我们使用多肽抑制假病毒感染细胞，能明显观察到抑制作用。
在本公开的技术方案中，说明书核苷酸和氨基酸序列表的编号所代表的含义如下所示：
SEQ ID NO：1所示的是冠状病毒S蛋白第25-39位的氨基酸序列；
SEQ ID NO：2所示的是冠状病毒S蛋白第672-691位的氨基酸序列；
SEQ ID NO：3所示的是冠状病毒S蛋白第764-778位的氨基酸序列；
SEQ ID NO：4所示的是冠状病毒S蛋白第907-921位的氨基酸序列；
SEQ ID NO：5所示的是冠状病毒S蛋白第1155-1169位的氨基酸序列；
SEQ ID NO：6所示的是冠状病毒S蛋白第1198-1212位的氨基酸序列；
SEQ ID NO：7所示的是冠状病毒S蛋白第1257-1271位的氨基酸序列。
本公开中的“本领域的常规生物学方法”，可以参见“最新分子生物学实验方法汇编(Current Protocols in Molecular Biology，Wiley出版)”，“分子克隆实验指南(Molecular Cloning：A Laboratory Manual，冷泉港实验室出版)”等公开出版物中记载的相应方法。
实施例
本公开的其他目的、特征和优点将从以下详细描述中变得明显。但是，应当理解的是，详细描述和具体实施例(虽然表示本公开的具体实施方式)仅为解释性目的而给出，因为在阅读该详细说明后，在本公开的精神和范围内所作出的各种改变和修饰，对于本领域技术人员来说将变得显而易见。
除非有特别说明，否则本公开中采用的所有试剂和原料均可以通过商业渠道购买。
实施例1a
抗原表位的合成
我们合成得到七条可能具有抗体反应的多肽表位，分别定位在冠状病毒S蛋白的25-39，672-691，764-778，907-921，1155-1169，1198-1212，1257-1271位氨基酸。
抗原肽的合成方法：
将多肽序列给金斯瑞生物科技合成，纯度≥85％，毛重4mg，每1mg分装1管。
实施例1b
Elisa初步筛选特异性抗原肽
酶联免疫吸附反应Elisa实验步骤：
1.将MES粉末(SIGMA,Lot#SLBZ3485)用ddH 2O配制成0.1M、pH＝6.0的MES buffer；将EDC(C 8H 17N 3，Thermo Scientific,Lot#TB257918)用ddH 2O稀释成10mg/ml。
2.将多肽用0.1M的MES buffer稀释成4μg/ml。
3.在微孔板设阴性对照，每孔中加入10μl EDC溶液和50μl多肽溶液；其余孔中加入10μl EDC溶液和50μl MES buffer。轻轻震动混匀。用不干胶条封板后于4℃过夜或室温放置两个小时以上。
4.去掉不干胶条，吸去孔内液体，每孔加300μl ddH 2O，静置2分钟，弃去液体，拍干板子。再重复以上步骤2次。
5.用1X PBST配制1％BSA的封闭液(10X PBST：Solarbio，Cat#P1033-500；BSA:Solarbio，Cat#A8020)，每孔加入200μl封闭液，室温封闭1小时。
6.弃去孔内液体，拍干板子。将待测血清用封闭液按1:500稀释，每孔加入100μl稀释血清，室温反应1小时。
7.弃去孔内液体，每孔加300μl 1XPBST，静置2分钟，弃去液体，拍干板子。再重复以上步骤2次。
8.将HRP标记的Goat Anti-Human IgG(Cwbio，Cat#CW0169S)用封闭液按1：5000稀释，每孔100μl，室温反应40分钟。
9.重复操作步骤7，用1XPBST洗板5次。
10.每孔加入TMB-ELISA显色液(Thermo Scientific,Lot#TK2666052)100μl，避光反应5-15分钟。
11.每孔加入2M H 2SO 4溶液50μl终止反应。
12.将酶标仪设定波长450nm测量各孔OD值，应在终止反应后30分钟内读值。
实验结果如图1所示。图1表示Elisa初步筛选特异性抗原肽，图1中2，3，7，17为新冠阳性患者血清，MES为只加多肽不加血清的阴性对照组，background为只加血清不加多肽的背景对照组，H-1和H-2为正常人血清，根据IgG结果OD450读值画图。
实施例2
SARS-CoV-2S蛋白3-D结构标注B细胞表位
在NCBI GeneBank下载SARS-CoV-2全基因组序列(NC_045512)，然后提取S蛋白全长序列进行结构模拟。我们在SWISS-MODEL在线服务器提交了S蛋白序列，以SARS coronavirus S蛋白(PDB ID:6VSB)为模板获得了SARS-CoV-2S蛋白结构，在PYMOL上进行表位标注。
图2表示SARS-CoV-2S蛋白3-D结构图，蛋白S25-39标注为蓝色，蛋白S672-691标注为绿色，蛋白S764-778标注为红色，蛋白S907-921标注为紫色。
实施例3
S672-691序列同源性比对
冠状病毒S蛋白中潜在的Furin和TMPRSS2切割位点在S1和S2结构域之间，插入突变发生在SARS-CoV-2S蛋白氨基酸681位点，并且我们对S672-691序列和human，bat SARS-like CoVs以及MERS进行了同源性比对，比对使用的软件是MEGA7.
图3表示SARS-CoV-2S蛋白潜在Fruin和TMPRSS2位点位置，以及S672-691序列与human和bat SRARS-like CoVs，MERS之间的同源性，可以看到同源性并不高，因此S672-691蛋白很有可能作为SARS-CoV-2的特异性检测位点。
实施例4
Elisa检测核酸检测结果呈阳性血清
同实施例1b的ELISA步骤
图4表示Elisa检测阳性血清。合成S672-691抗原肽序列后，在体外设计Elisa实验，检测核酸检测结果呈阳性的血清样本。每组实验都设置了血清背景对照组和随机抗原肽序列对照组。可以发现在阳性血清中，IgG和IgM检测抗体水平基本比较高。而抗原肽S25-39和背景对照组抗体水平基本一致，处于很低的抗体水平。我们收集到的血清均为新冠患者发病后到医院就医时的样本，其中核酸检测结果阳性表示进行核酸检测时体内存在新冠病毒。
实施例5
中和实验
5.1实验前准备
5.1.1平衡试剂
将保存在2-8℃的试剂(胰酶，DMEM完全培养基)取出，至室温平衡，30分钟以上
5.1.2操作人员
由经过培训的实验操作人员进行操作，实验操作前，在清洁区内更衣(穿好一次性无菌衣，换好工作鞋，戴好口罩，帽子，一次性医用乳胶手套)方可进入实验区内，进行实验操作。
5.2实验操作
5.2.1将待检测的血清(或血浆)于56℃水浴灭活30min，6000g离心3min，将上清转移至1.5ml离心管中待用。
5.2.2取96孔板，于第2列(细胞对照CC，见表2)加入DMEM完全培养基(1％双抗，25mM HEPES，10％FBS)150μl/孔，于第3～11列(第3列为病毒对照组VV，第4～11列为样品孔)加入DMEM完全培养基100μl/孔，于B4～B11孔中再加入DMEM完全培养基42.5μl/孔。
5.2.3于B4和B5孔加入血浆样品1(7.5μl)……以此类推，于B10和B11孔加入血浆样品4(7.5μl)。
5.2.4将多道移液器调至50μl，对B4～B11孔中液体轻柔的反复吹吸6～8次充分混匀，然后转移50μl液体至对应的C4～C11中吸弃50μl液体，加样顺序参照表2。
表2样品加样顺序
Figure PCTCN2020090064-appb-000002
5.2.5用DMEM完全培养基将假病毒稀释至2*10 4TCID50/ml(按提供的稀释倍数稀释)，于第3～11列每孔加50μl，使每孔含假病毒的量为1*10 3/孔。
5.2.6将上述96孔板置于细胞培养箱中(37℃，5％CO 2)孵育1小时。
5.2.7当孵育时间至半小时，取出培养箱中事先准备好的Huh-7细胞(汇合率达80％～90％)，以 T75培养瓶为例，吸弃瓶中的培养基，加入5ml PBS缓冲液清洗细胞，倾去PBS后，加入3ml 0.25％胰酶-EDTA，使其浸没细胞消化1分钟，倾去胰酶，置于细胞培养箱中消化5分钟，轻轻拍打培养瓶侧壁使细胞脱落，加入10ml培养基中和胰酶，吹打几次后转移至离心管中，210g离心5分钟，倾去上清，用10ml DMEM完全培养基重悬细胞，细胞计数，用DMEM完全培养基将细胞稀释至5*10 5个/ml。
5.2.8孵育至1小时，向96孔板中每孔加100μl细胞，使每孔细胞为5*10 4个。
5.2.9将96孔板前后左右轻轻晃动，使细胞在孔中分散均匀，将96孔板放入细胞培养箱中，37℃，5％CO 2培养20～28小时。
5.2.10 20～28小时后从细胞培养箱中取出96孔板，用多道移液器从每个上样孔中吸弃150μl上清，然后加入100μl荧光素酶检测试剂，室温避光反应2min。
5.2.11反应结束后，用多道移液器将反应孔中的液体反复吹吸6～8次，使细胞充分裂解，从每孔中吸出150μl液体，加入对应96孔化学发光检测板中，置于化学发光检测仪中读取发光值。
5.2.12计算中和抑制率：抑制率＝[1-(样品组的发光强度均值-空白对照CC均值)/(阴性组的发光强度VC均值-空白对照CC均值)]*100％。
5.2.13根据中和抑制率结果，利用Reed-Muench法计算IC50。图3示出中和实验结果图。从图5可以看出，4号，7号和17号患者血清抗体都对病毒有抑制作用，并且和正常人有很明显的差异。
实施例6
多肽抑制病毒感染实验
按2X10 4个细胞/孔的密度铺Huh7.5细胞于96孔板中，第二天按照图中所示的剂量加入BSA偶联的S672-691多肽，预处理2hr后，按2倍稀释加入SARS-CoV-2假病毒感染液，24hr后收取细胞，测定Lciferase荧光素酶活性(Promega E151A)。
图6示出多肽抑制病毒感染实验的结果。从图6可以看出，当多肽剂量为1μg时能明显抑制病毒感染，与对照组之间的差异具有统计学意义。
实施例7
S蛋白和N蛋白试剂盒检测核酸检测呈阳性血清
1.准备：取出试剂盒(Tarcine，S蛋白IgG：600156；S蛋白IgM：600157；N蛋白IgG：600158；N蛋白IgM：600159)，室温(18-25℃)平衡30分钟。将20倍浓缩洗涤液1用蒸馏水做20倍稀释。将待测样本用样品稀释液在EP管内按1：100提前稀释，混匀后室温放置10分钟。IgG样稀与IgM样稀不可 混用！
2.加样：在微孔板中设阴性对照3孔，每孔100μl；稀释后的待测样本每孔100μl。
3.温育：用不干胶条封板后，37℃温育反应30分钟。
4.洗板：温育后，去掉不干胶条，吸去孔内液体，每孔加300μl洗涤液，静置30秒，弃去液体，拍干板子。再重复以上步骤4次。
5.加入酶联物：每孔加入酶联物100μl。
6.温育：用不干胶条封板后，37℃温育反应20分钟。
7.洗板：重复步骤4。
8.显色：每孔分别加入底物液A、B各50μl，37℃避光温育10分钟。
9.终止：每孔加入终止液50μl，终止反应。轻轻震动混匀。
10.读值：将酶标仪设定在波长450nm，测量各孔OD值。应在终止反应后30分钟内读值。
图7示出试剂盒检测核酸检测结果呈阳性血清。
目前已经有公司针对新型冠状病毒S蛋白和N蛋白一些关键蛋白开发了检测血清抗体的试剂盒，从图7的对比检测结果也能清楚的看到这条特异性抗原肽能检测到血清中的针对新型冠状病毒产生的抗体。
实施例8
Elisa检测核酸检测结果呈阴性血清
同实施例1b的ELISA步骤
图8示出Elisa检测核酸检测结果呈阴性血清。
在核酸检测结果呈阴性的病人血清中也能检测到特异性抗体，并且通过统计分析该抗原肽在阴性血清中也具有比较高的灵敏度。说明病人在感染新冠肺炎病毒发病后虽然体内病毒被清除，但是抗体也是确实存在的。
实施例9
统计感染病人在住院治疗过程中抗体检测水平变化
同实施例1b的ELISA步骤
图9示出我们追踪的五名病人在确诊新冠肺炎之后，住院治疗的过程中，血清中抗体水平变化，以及根据病情的临床综合打分情况。
由图9可以看出，S672-691蛋白能检测出病人在治疗过程中的血清中抗体水平变化，并且趋势和临床综合打分基本一致。这代表我们的这条多肽能很灵敏的检测病人体内的抗体水平，也能够通过这 种抗体水平来反映对SARS-CoV-2的免疫情况。
实施例10
特异性抗原肽灵敏度和特异性检测
我们将检测到的SARS-CoV-2PCR+/-以及正常人的血清中抗体水平进行统计，在GraphPad Prism7.0中进行ROC curve统计分析。
图10示出SARS-CoV-2-IgG(POS/NEG):POS代表核酸检测阳性，NEG代表核酸检测阴性。图9示出SARS-CoV-2-IgM(POS/NEG):POS代表核酸检测阳性，NEG代表核酸检测阴性。
根据ROC曲线统计结果，统计出灵敏度和特异性。发明人共检测了新冠患者核酸阳性50例，新冠患者核酸阴性114例，正常人47例，统计SARS-CoV-2-IgG与SARS-CoV-2-IgM。
Elisa实验能高效灵敏的检测出样本中的抗体，因此在体外大规模开展Elisa实验检测新型冠状病毒感染患者以及正常人的血清样本，验证多肽的灵敏度和准确性。根据ROC曲线统计结果如下：
1.本抗原肽检测正常人47例，新冠患者(核酸检测阳性)50例血清中SARS-CoV-2-IgG的水平：
以0.395为判定值，检测灵敏度为94％，特异性为87.23％
以0.4925为判定值，检测灵敏度为86％，特异性为100％
2.本抗原肽检测正常人47例，新冠患者(核酸检测阴性)114例血清中SARS-CoV-2-IgG的水平：
以0.3925为判定值，检测灵敏度为91.23％，特异性为87.23％
3.本抗原肽检测正常人47例，新冠患者(核酸检测阳性)50例血清中SARS-CoV-2-IgM的水平：
以0.1905为判定值，检测灵敏度为94％，特异性为78.72％
以0.198为判定值，检测灵敏度为88％，特异性为80.85％
4.本抗原肽检测正常人47例，新冠患者(核酸检测阴性)114例血清中SARS-CoV-2-IgM的水平：
以0.156为判定值，检测灵敏度为87.72％，特异性为74.47％
基于上述结果，统计出如下表3的灵敏度以及特异性：
表3.抗原肽灵敏度和特异性
  灵敏度 特异性
新冠患者核酸阳性-IgG 94％ 87.23％
新冠患者核酸阴性-IgG 91.23％ 87.23％
新冠患者核酸阳性-IgM 94％ 78.72％
新冠患者核酸阴性-IgM 87.72％ 74.47％
上述结果表明，本公开中的抗原肽在检测时，具有良好的灵敏度和特异性。
实施例11
S672-691多肽免疫小鼠
1.每只小鼠免疫50μg多肽。在无菌条件下将50μg多肽用无酶水补到50μl，加入50μl佐剂，配制成100μl的多肽混合液，置于冰上备用。
2.将小鼠腹部毛发用剃毛机剃除。
3.每只小鼠用1ml无菌注射器在腹部皮下注射100μl多肽混合液。3月17、3月26、4月2日各注射一次。
4.4月10日用内径0.9-1.1毫米、壁厚0.1-0.15毫米、管长100毫米的玻璃毛细管对待测小鼠进行眼眶采血，每只小鼠采血100μl。
5.将取出的小鼠血液放进离心机中，4℃、6000g离心10min后吸取上层血清进行ELISA实验，实验步骤同1b。
图11示出S672-691多肽能明显激发小鼠体内的免疫反应并且产生抗体。
实施例12
S672-691多肽免疫小鼠产生的抗体对SARS-CoV-2假病毒具有抑制作用
实验步骤同实施例6。
图12示出S672-691多肽免疫小鼠产生的抗体对SARS-CoV-2病毒具有抑制作用。
实施例13
S2特异性抗体的Western blot检测结果
Vero细胞以1*10 5密度铺于48孔板，培养过夜。感染SARS-CoV-2，以未感染的vero细胞作为阴性对照。分别在感染24小时和48小时后收集细胞加裂解液[50mM tris-HCl(pH＝7.5),150mM NaCl,5mM EDTA,1％NP-40,1mM phenylmethylsulfonyl fluoride(PMSF),and 1×protease inhibitor(Roche)]裂解蛋白。SDS-聚丙烯胺凝胶电泳和Western blot鉴定Spike蛋白，一抗使用Rabbit anti-β-actin(CST，8457S，1:2000)，mouse anti-coronavirus spike2(MP，087204，1:1000)孵育。
图13示出S2特异性抗体的Western blot检测结果，根据S2特异性抗体的Western blot分析，在感染SARS-CoV-2的蛋白提取液中特异性检测到两条主要的条带，但未感染的vero细胞中未检测到，这两条条带分别代表全长的spike蛋白和可能在潜在的TMRPSS2裂解位点被裂解的S2片段。
实施例14
TMPRSS2在S蛋白裂解中的作用
HEK293细胞以1*10 5密度铺于48孔板，培养过夜。在培养液中加入camostat mesylate(MCE， HY-13512)、nafamostat mesylate(MCE，HY-B0190A)和BSA或Biotin偶连的672-691peptide处理2小时。然后在HEK293cells中共转染GFP标签SARS-COV-2-S质粒(GenScript，C1043FB180-1)和Tmprss2表达质粒(OriGene，RC208677)。转染16小时后更换培养液继续培养24小时。收集细胞加裂解液[50mM tris-HCl(pH＝7.5),150mM NaCl,5mM EDTA,1％NP-40,1mM phenyl ethylsulfonylfluoride(PMSF),and 1×protease inhibitor(Roche)]裂解蛋白。SDS-聚丙烯胺凝胶电泳和Western blot鉴定Spike蛋白，一抗使用Rabbit anti-beta-actin(CST，8457S，1:2000),mouse anti-eGFP(AtaGenix，539191227，1:1000)孵育。
图14示出TMPRSS2在S蛋白裂解中的作用，我们在存在或不存在TMRPSS2抑制剂或S672-691多肽的情况下，将表达S蛋白的质粒与对照载体或表达TMRPSS2的质粒共转染。如图14所示，TMRPSS2的过表达显著增加，而TMRPSS2抑制剂如camostat或nafamostat以及合成的S672-691多肽抑制了S蛋白的裂解。
实施例15
S672-691多肽对TMPRSS2和ACE2共转染HEK293细胞的SARS-CoV-2假病毒感染效果的抑制 作用
前一天按2х10 5/孔的密度将HEK293T细胞铺于24孔板中，过夜培养之后按照图中所示转入相应的hACE2和TMPRSS2表达质粒，转染24小时后，加入相应浓度的S672-691多肽，预处理2小时后，感染SARS-CoV-2假病毒，感染24小时后，通过测定荧光素酶报告实验检测细胞内假病毒的水平，分析数据计算S672-691多肽对SARS-CoV-2假病毒进入宿主细胞的抑制效果。
S672-691刺激小鼠产生的相应抗体对TMPRSS2和ACE2共转染HEK293细胞的SARS-CoV-2假病毒感染效果的抑制作用通过抗体中和实验来验证，如上转入相应的hACE2和TMPRSS2表达质粒，转染24小时后，抗体中和实验步骤同实施例6.
图15示出S672-691多肽以及多肽刺激产生的抗体都能有效抑制TMPRSS2和ACE2共转染HEK293细胞的SARS-CoV-2假病毒感染，当TMRPSS2与ACE2共转染HEK293细胞后，显著增强了SARS-CoV-2伪病毒感染，S672-691多肽可以抑制这种感染。
本公开并不旨在限于具体公开的实施方案的范围，提供所述实施方案例如来说明本公开的各方面。从本文的描述和教导，对所述组合物和方法的各种修改将变得明显。可以在不脱离本公开的真正范围和精神的情况下实践这类变化，并且这类变化旨在落入本公开的范围内。

Claims (8)

  1. 一种多肽，其中，所述多肽为如下(1)-(2)任一项所示的多肽：
    (1)所述多肽为如SEQ ID NO：2所示的序列编码的多肽；
    (2)所述多肽为在(1)所示的多肽的基础上，取代、重复、缺失或添加一个或多个氨基酸的多肽。
  2. 根据权利要求1所述的多肽，其中，所述多肽相对于如SEQ ID NO：2所示的序列编码的多肽，具有至少80％的同源性；优选的，具有至少90％以上的同源性。
  3. 根据权利要求1-2任一项所述的多肽，其中，所述多肽的氨基酸残基的个数为20个。
  4. 一种检测是否被SARS-CoV-2感染或患有COVID-19的试剂盒，其中，所述试剂盒中含有根据权利要求1-3任一项所述的多肽。
  5. 一种药物组合物或疫苗，其中，所述药物组合物或疫苗中含有根据权利要求1-3任一项所述的多肽。
  6. 根据权利要求1-3任一项所述的多肽在制备用于检测是否被SARS-CoV-2感染或患有COVID-19的试剂中的用途。
  7. 根据权利要求1-3任一项所述的多肽在制备用于治疗或预防被SARS-CoV-2感染或患有COVID-19的药物中的用途。
  8. 一种治疗被SARS-CoV-2感染或患有COVID-19的方法，其中，所述方法包括向患者施用根据权利要求1-3任一项所述的多肽或根据权利要求5所述的药物组合物或疫苗。
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