CN112592390A - Novel coronavirus specific antigen peptide and use thereof - Google Patents

Abstract

The present disclosure relates to a novel coronavirus specific antigen peptide and use thereof. Specifically, the disclosure relates to a polypeptide, which is an antigenic peptide of SARS-CoV-2 virus, and the application of the antigenic peptide in disease diagnosis, preparing COVID-19 vaccine, and preparing medicaments for preventing and treating COVID-19.

Description

Novel coronavirus specific antigen peptide and use thereof

Technical Field

The present disclosure belongs to the field of biotechnology. Specifically, the disclosure relates to an antigenic peptide of SARS-CoV-2 virus and the application of the antigenic peptide in preparing COVID-19 vaccine and medicament for preventing and treating COVID-19.

Background

The novel coronavirus (SARS-CoV-2) belongs to the genus coronavirus B, linear single-stranded RNA (ssRNA) virus. The genome is about 29903 nucleotides in total and contains 10 genes. Since 10/1/2020, the first SARS-CoV-2 genome sequence data was published, and thereafter a plurality of genome sequences of novel coronaviruses isolated from patients were published in succession. In 22 months 1 in 2020, the genome science data center formally releases 2019 a novel coronavirus resource library. Through data analysis, the genome sequence of the 2019 novel coronavirus (SARS-CoV-2) has 80% similarity with the SARS virus outbreak in 2003, and has the highest similarity with the genome sequence of Bat SARS-like coronavirus isolate Bat-SL-CoVZC45 collected from bats in China in month 2 of 2017, and the similarity is 88%. By 30 months and 1 of 2020, there have been 13 new coronavirus sequences published by 6 organizations worldwide on the "global shared influenza virus database GISAID".

The novel coronavirus (SARS-CoV-2) is an enveloped positive-strand RNA virus containing a 30kb genome and four structural proteins, i.e., spike protein (S), envelope protein (E), membrane protein (M), and nucleocapsid protein (N). The S protein regulates viral attachment to receptors on the target host cell. The function of the E protein is to assemble the virus and act as an ion channel; the M protein together with the E protein plays a role in virus assembly and is involved in the biosynthesis of new viral particles; the N protein forms a ribonucleoprotein complex with viral RNA. The surface spike glycoprotein (S protein) of the novel coronavirus is responsible for attachment to host cells through interaction with host cell surface receptor (ACE 2). The S protein exists as a homotrimer with 1200 more amino acids per monomer. In the S protein of SARS-CoV-2, a small domain containing residues 306-575 was identified as the Receptor Binding Domain (RBD), in which the 439-508 residue, called the Receptor Binding Motif (RBM), directly mediates the interaction with ACE 2. The full-length structure of the new crown receptor ACE2 has been resolved by the West lake university West Strength laboratory using cryo-electron microscopy. The binding capacity of the key spike protein (S protein) of the novel coronavirus to the human cell receptor protein ACE2 protein has been found to be much higher than that of the SARS virus, which explains in part why the novel coronavirus is much more infectious than the SARS virus. With the aid of cryoelectron microscopy, scientists were able to better observe the structure of the novel coronavirus S protein and its interaction with the ACE2 protein. The research finds that a foundation is laid for further analyzing the three-dimensional structure of the full-length ACE2 and the S protein complex of the new coronavirus, and more clues are provided for understanding the new coronavirus infecting cells. And the analysis of the full-length structure of the ACE2 is helpful for providing important structural biological data support for the research and development of subsequent vaccines and antiviral drugs. Will help to understand the structural basis and functional characteristics of coronavirus entering target cells, and has important role in finding and optimizing inhibitors for blocking entry into cells.

At the initial stage of infection with COVID-19, patients show no obvious symptoms, but have extremely high infectivity, can be transmitted by people through contact transmission, droplet transmission and the like, and can be killed by serious patients. However, for viral infectious diseases, the available drugs are very limited, and vaccines are one of the most effective means. However, due to the emerging and unpredictable nature of emerging infections, most emerging and virulent infectious diseases do not have an effective vaccine stock.

Prior researchers used computational tools from structural biology and machine learning to identify SARS-CoV-2T cell and B cell epitopes based on viral protein antigen presentation and antibody binding properties. These epitopes can be used to develop more effective vaccines and to identify neutralizing antibodies. 405 viral peptides with good antigen expression scores have been identified in the human MHC-I and MHC-II alleles, and two potential neutralizing B cell epitopes were found in the vicinity of the SARS-CoV-2 Spike protein receptor binding domain (440-.

Disclosure of Invention

Problems to be solved by the invention

Since SARS-CoV-2 enters the cell via the surface ACE2 cell receptor, its surface antigen is presented to the B cell via antigen, stimulating the differentiation of the B cell into memory B cell and plasma cell, which secretes anti-SARS-CoV-2 specific antibody neutralizing virus. Therefore, the finding of antigenic peptides which can specifically activate the immune system of the body and stimulate the immune response of B cells so as to generate neutralizing antibodies has a key role. Therefore, the novel coronavirus surface specific antigen peptide is screened out through a model, and the effectiveness of the novel coronavirus antigen peptide is verified through combination structure biological analysis, clinical data detection and experiments, so that the aim of timely and accurately diagnosing the novel coronavirus is fulfilled.

Means for solving the problems

The present disclosure provides the following technical solutions.

(1) A polypeptide, wherein the polypeptide is a polypeptide represented by any one of (i) to (ii) below:

(i) the polypeptide is shown as SEQ ID NO: 2;

(ii) the polypeptide is a polypeptide obtained by substituting, repeating, deleting or adding one or more amino acids on the basis of the polypeptide shown in (i).

(2) The polypeptide of (1), wherein the polypeptide has a sequence relative to the sequence set forth in SEQ ID NO: 2, having at least 90% homology; preferably, there is at least 95% homology or greater.

(3) The polypeptide according to any one of (1) to (2), wherein the number of amino acid residues in the polypeptide is 20.

(4) A kit for detecting whether a SARS-CoV-2 infection or a COVID-19 infection is present, wherein the kit contains the polypeptide according to any one of (1) to (3).

(5) A pharmaceutical composition or vaccine, wherein the polypeptide according to any one of (1) to (3) is contained in the pharmaceutical composition or vaccine.

(6) Use of the polypeptide according to any one of (1) to (3) for the preparation of a reagent for detecting whether it is infected with SARS-CoV-2 or has COVID-19.

(7) Use of a polypeptide according to any one of (1) to (3) in the manufacture of a medicament for the treatment or prevention of infection by SARS-CoV-2 or having COVID-19.

(8) A method of treating a SARS-CoV-2 infected or having COVID-19, wherein the method comprises administering to a patient a polypeptide according to any one of (1) to (3) or a pharmaceutical composition or vaccine according to (5).

ADVANTAGEOUS EFFECTS OF INVENTION

In one embodiment, the antigenic peptides obtained by the present disclosure have good accuracy and specificity for novel coronaviruses, and can be used for the detection of SARS-CoV-2.

In another embodiment, the antigenic peptide obtained by 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.

Drawings

FIG. 1 shows Elisa preliminary screening of specific antigenic peptides;

FIG. 2 shows the 3-D structure of SARS-CoV-2S protein;

FIG. 3 shows an S672-691 sequence homology alignment

FIG. 4 shows Elisa test positive sera;

FIG. 5 shows a graph of neutralization experiment results;

FIG. 6 shows the results of experiments in which polypeptides inhibit infection by SARS-CoV-2 euvirus and pseudovirus;

FIG. 7 shows a positive serum for the detection of nucleic acid by the kit;

FIG. 8 shows sera negative for the detection of nucleic acids by Elisa;

FIG. 9 shows statistical changes in antibody detection levels in infected patients during hospitalization;

FIG. 10 shows sensitivity and accuracy (POS/NEG) for detection of SARS-CoV-2 IgG/IgM by the S672-691 polypeptide, POS representing positive nucleic acid detection and NEG representing negative nucleic acid detection;

FIG. 11 shows that the S672-691 polypeptide immunizes mice and elicits antibody production;

FIG. 12 shows that antibodies raised against SARS-CoV-2 virus by immunization of mice with the S672-691 polypeptide;

FIG. 13 shows the results of Western blot detection of an antibody specific to S2;

figure 14 shows the role of TMPRSS2 in S protein cleavage;

FIG. 15 shows that the S672-691 polypeptide is effective in inhibiting SARS-CoV-2 pseudovirus infection of HEK293 cells co-transfected with TMPRSS2 and ACE 2.

Detailed Description

Definition of

The terms "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification can mean "one," but can also mean "one or more," at least one, "and" one or more than one.

As used in the claims and specification, the terms "comprising," "having," "including," or "containing" are intended to be inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

Throughout this specification, the term "about" means: a value includes the standard deviation of error for the device or method used to determine the value.

Although the disclosure supports the definition of the term "or" as merely an alternative as well as "and/or," the term "or" in the claims means "and/or" unless expressly indicated to be merely an alternative or a mutual exclusion between alternatives.

When used in the claims or specification, the term "range of values" is selected/preferred to include both the end points of the range and all natural numbers subsumed within the middle of the end points of the range with respect to the aforementioned end points of values.

As used in this disclosure, the term "SARS-CoV-2", also known as "2019-nCoV", means a 2019 novel coronavirus.

As used in this disclosure, the term "COVID-19" means a novel coronavirus pneumonia (Corona Virus Disease 2019), abbreviated as "new Corona pneumonia", and refers to pneumonia caused by 2019 infection with a novel coronavirus (SARS-CoV-2).

As used in this disclosure, the term "amino acid mutation" includes "substitution, duplication, deletion or addition of one or more amino acids". In the present disclosure, the term "mutation" refers to an alteration in the amino acid sequence. In a specific embodiment, the term "mutation" refers to "substitution".

In one embodiment, a "mutation" of the present disclosure may be selected from a "conservative mutation". In the present disclosure, the term "conservative mutation" refers to a mutation that can normally maintain the function of a protein. A representative example of conservative mutations is conservative substitutions.

As used in this disclosure, the term "conservative substitution" refers to the replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art and include those having basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), beta-branches (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine).

As used in this disclosure, "conservative substitutions" typically exchange one amino acid for one or more sites in a protein. Such substitutions may be conservative. As a substitution regarded as a conservative substitution, specifically, examples thereof include substitution of Ala for Ser or Thr, substitution of Arg for Gln, His or Lys, substitution of Asn for Glu, Gln, Lys, His or Asp, substitution of Asp for Asn, Glu or Gln, substitution of Cys for Ser or Ala, substitution of Gln for Asn, Glu, Lys, His, Asp or Arg, substitution of Glu for Gly, Asn, Gln, Lys or Asp, substitution of Gly for Pro, substitution of His for Asn, Lys, Gln, Arg or Tyr, substitution of Ile for Leu, Met, Val or Phe, substitution of Leu for Ile, Met, Val or Phe, substitution of Lys for Asn, Glu, Gln, His or Arg, substitution of Met for Ile, Leu, Val or Phe, substitution of Phe for Trp, Tyr, Met, Ile or Leu, substitution of Ser for Thr or Ala, substitution of Thr for Ser or Ala, substitution of Trp for Phe or Tyr, substitution of Tyr for His, Phe or Trp for Val, and substitution of Met or Met for Met or Ile. Furthermore, conservative mutations include naturally occurring mutations due to individual differences in the origin of the gene, differences in strain, species, and the like.

As used in this disclosure, the term "sequence identity" or "percent identity" in a comparison of two nucleic acids or polypeptides refers to the identity or a specific percentage number of identical sequences when compared and aligned for maximum correspondence as measured using nucleotide or amino acid residue sequence comparison algorithms or by visual inspection. That is, the identity of nucleotide or amino acid sequences can be defined by the ratio of the number of nucleotides or amino acids that are identical when aligned with gaps added as necessary, to the number of nucleotides or amino acids that are identical in two or more nucleotide or amino acid sequences in such a manner as to maximize the number of nucleotides or amino acids that are identical.

As used in the present disclosure, sequence identity between two or more polynucleotides or polypeptides may be determined by: the nucleotide or amino acid sequences of the polynucleotides or polypeptides are aligned and the number of positions in the aligned polynucleotides or polypeptides containing the same nucleotide or amino acid residue is scored and compared to the number of positions in the aligned polynucleotides or polypeptides containing different nucleotide or amino acid residues. Polynucleotides may differ at one position, for example, by containing different nucleotides or deleting nucleotides. Polypeptides may differ at one position, for example, by containing different amino acids or deleting 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. For example, percent identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of nucleotides or amino acid residues in the polynucleotide or polypeptide and multiplying by 100.

Illustratively, in the present disclosure, two or more sequences or subsequences have "sequence identity" or "percent identity" of at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% nucleotide or amino acid residues when compared and aligned for maximum correspondence as measured using a sequence comparison algorithm or by visual inspection. The determination/calculation of "sequence identity" or "percent identity" can be based on any suitable region of the sequence. For example, a region of at least about 10 residues, a region of at least about 15 residues, a region of at least about 18 residues, a region of at least about 20 residues in length. In certain embodiments, the sequences are substantially identical over the entire length of either or both of the biopolymers (i.e., nucleic acids or polypeptides) to be compared.

Studies have shown that the novel coronavirus infects human airway epithelial cells through a molecular mechanism of interaction of the S-protein with human ACE2, and in addition, the S protein of the novel coronavirus is initiated by TMPRSS 2. The S protein regulates the attachment of coronaviruses to receptors on target host cells, and plays an important role in binding to host cells to produce interactions. Therefore, we can screen seven antigenic peptides by researching the specific epitope of the S protein on the surface of the novel coronavirus. Based on the amino acid position of the antigenic peptide in the S protein, the antigenic peptides are respectively positioned at 25-39(PPAYTNSFTRGVYYP), 672-691 (ASYQTQTNSPRRARSVASQS), 764-778(NRALTGIAVEQDKNT), 907-921 (NGIGVTQNVLYENQK), 1155-1169(YFKNHTSPDVDLGDI), 1198-1212 (IDLQELGKYEQYIKW) and 1257-1271(DEDDSEPVLKGVKLH), and the specific sequences are shown in the following table 1.

TABLE 1 antigenic peptide sequences

After obtaining the epitope, we performed immunogenicity testing studies of the antigenic peptide. We synthesized all these antigenic peptides in vitro and initially screened serum samples from new coronary patients as well as normal humans using the enzyme-linked immunosorbent assay Elisa. According to the nucleic acid detection result in sampling, the serum sample of the new coronary patient is divided into two groups of positive nucleic acid detection and negative nucleic acid detection.

In a specific embodiment, by primary screening of the antigenic peptide, we screened an antigenic peptide that was significantly detectable in serum of a new coronary patient and was significantly different from normal humans. The sequence is located at position 672-691 of the S protein, the specific amino acid sequence is ASYQTQTNSPRRARSVASQS, and the sequence is found to be located at the position TMPRSS2 of the S protein by sequence alignment. And the existing research data show that the site has an important function of starting the S protein, so that the site has important research significance.

The human body will produce corresponding anti-viral antibodies about the second to third weeks after infection with the virus. We collected a large number of clinical samples of SARS-CoV-2 infection, classified these samples, and recorded the individual information and health indicators of the collected serum samples in detail, including the date of infection, date of sampling, medical history, etc.

By synthesizing the obtained antigenic peptide sequence, we firstly screen the serum of a patient infected by the novel coronavirus in vitro with an Elisa experiment. According to the detection results, the serum samples of patients with higher and lower antibody detection levels are independently taken out for virus neutralization experiments, and then the neutralization effect of the detected antibodies on viruses is verified. Meanwhile, the experiment of inhibiting virus infection by the polypeptide is carried out, and compared with other research medicines, the polypeptide has an obvious inhibiting effect on virus infection when the dosage of the polypeptide reaches 2 mu g.

The polypeptide epitope which is possibly antigenic in the obtained S protein is synthesized, a sequence is found in the screening process of the polypeptide, the antibody difference between a patient and a normal person can be detected, and further research is carried out aiming at the polypeptide sequence.

Through sequence alignment, we know that the polypeptide is located in TMPRSS2 site (672-691) of the S protein.

The serum of the new coronary patient, which is detected to be positive in nucleic acid detection through an Elisa experiment, has a higher antibody level for specific antigen peptide detection, and the result is consistent with the detection result of a commercial kit for detecting the serum, such as novel coronavirus S protein, N protein and the like, and the detection rate is relatively high. Meanwhile, a random sequence is set as a contrast, two indexes of IgG and IgM of the antigen peptide are detected, and the IgG and IgM detection results have correlation through analysis. According to the detection result of the polypeptide, patient serum with higher antibody level, patient serum with lower antibody level and normal human serum are selected to be used for a pseudovirus neutralization experiment. And verifying the accuracy of antibody detection according to the result of the neutralization experiment. Meanwhile, the polypeptide is used for inhibiting pseudovirus infected cells, and the inhibition effect can be obviously observed.

In the technical scheme of the disclosure, the meanings represented by the numbers of the nucleotide and amino acid sequence table in the specification are as follows:

SEQ ID NO: 1 represents the amino acid sequence of 25 th to 39 th positions of coronavirus S protein;

SEQ ID NO: 2 shows the amino acid sequence at the 672-691 position of the coronavirus S protein;

SEQ ID NO: 3 represents the amino acid sequence of the position 764-778 of the coronavirus S protein;

SEQ ID NO: 4 shows the amino acid sequence of the S protein of the coronavirus at the 907-;

SEQ ID NO: 5 shows the amino acid sequence of the coronavirus S protein at the position 1155-1169;

SEQ ID NO: 6 shows the amino acid sequence of the S protein of the coronavirus at the 1198-1212 position;

SEQ ID NO: 7 shows the amino acid sequence of the coronavirus S protein at position 1257-1271.

"methods in general Biology in the art" in the present disclosure can be referred to corresponding methods described in publications such as "Current Protocols in Molecular Biology, Wiley publication", "Molecular Cloning, A Laboratory Manual, Cold spring harbor Laboratory publication", and the like.

Examples

Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

All reagents and starting materials used in this disclosure are commercially available unless otherwise indicated.

Example 1a

Synthesis of epitopes

Seven polypeptide epitopes which can have antibody reaction are synthesized and positioned at the amino acids 25-39, 672-691, 764-778, 907-921, 1155-1169, 1198-1212 and 1257-1271 of the coronavirus S protein respectively.

The synthesis method of the antigen peptide comprises the following steps:

the polypeptide sequence is synthesized by Kisry biotechnology, the purity is more than or equal to 85 percent, the gross weight is 4mg, and each 1mg is separately packaged into 1 tube.

Example 1b

Elisa primary screening specific antigen peptide

Enzyme-linked immunosorbent assay Elisa experimental procedure:

1. MES powder (SIGMA, Lot # SLBZ3485) was formulated with ddH2O into a MES buffer of 0.1M, pH ═ 6.0; EDC (C8H17N3, Thermo Scientific, Lot # TB257918) was diluted to 10mg/ml with ddH 2O.

2. The polypeptide was diluted to 4. mu.g/ml with 0.1M MES buffer.

3. Setting negative control in the micro-porous plate, and adding 10 mul EDC solution and 50 mul polypeptide solution into each hole; the remaining wells were filled with 10. mu.l EDC solution and 50. mu.l MES buffer. And (5) lightly shaking and mixing. Placing the plate with pressure sensitive adhesive strip at 4 deg.C overnight or at room temperature for more than two hours.

4. Removing the non-drying adhesive tape, sucking off the liquid in the holes, adding 300 mu l of ddH2O into each hole, standing for 2 minutes, discarding the liquid, and patting dry the plate. Repeat the above step 2 times.

5. Blocking solutions of 1% BSA (10 XPBST: Solambio, Cat # P1033-500; BSA: Solambio, Cat # A8020) were prepared using 1XPBST, 200. mu.l of blocking solution was added to each well and blocked for 1 hour at room temperature.

6. Discard the well liquid and pat the plate dry. The serum to be tested was diluted with blocking solution at a ratio of 1:500, and 100. mu.l of the diluted serum was added to each well and reacted at room temperature for 1 hour.

7. Discard the liquid in the wells, add 300. mu.l of 1XPBST to each well, stand for 2 minutes, discard the liquid, and pat the plate dry. Repeat the above step 2 times.

8. HRP-labeled Goat Anti-Human IgG (Cwbio, Cat # CW0169S) was applied to the cells using a blocking solution at a ratio of 1: diluted at 5000, 100. mu.l/well, and reacted at room temperature for 40 minutes.

9. Repeat procedure 7, wash plate 5 times with 1 XPBST.

10. 100. mu.l of TMB-ELISA developing solution (Thermo Scientific, Lot # TK2666052) was added to each well, and the reaction was carried out for 5-15 minutes in the absence of light.

11. 2M H per well₂SO₄The reaction was stopped with 50. mu.l of the solution.

12. The OD value of each well was measured by setting the wavelength of the microplate reader to 450nm, and the value was read within 30 minutes after the termination of the reaction.

The results of the experiment are shown in FIG. 1. FIG. 1 shows Elisa primary screening specific antigen peptides, in FIG. 1, 2, 3, 7, 17 are serum of new coronary positive patients, MES is negative control group with only polypeptide and no serum, background is background control group with only serum and no polypeptide, H-1 and H-2 are normal human serum, and the graph is drawn according to IgG result OD450 reading value.

Example 2

SARS-CoV-2S protein 3-D structure labeling B cell epitope

SARS-CoV-2 whole genome sequence (NC-045512) was downloaded in NCBI GeneBank, and then S protein full length sequence was extracted for structural simulation. We submitted the S protein sequence in SWISS-MODEL online server, obtained SARS-CoV-2S protein structure using SARS coronavirus S protein (PDB ID:6VSB) as template, and performed epitope tag on PYMOL.

FIG. 2 shows the structure diagram of SARS-CoV-2S protein 3-D, proteins S25-39, proteins S672-691, proteins S764-778, and proteins S907-921, all of which are labeled in FIG. 2.

Example 3

S672-691 sequence homology alignment

The potential Furin and TMPRSS2 cleavage sites in coronavirus S protein are between the S1 and S2 domains, the insertion mutation occurs at amino acid 681 of the SARS-CoV-2S protein, and we performed homology alignment of the S672-691 sequence with human, bat SARS-like coms and MERS, using the software MEGA7.

FIG. 3 shows the positions of potential Fruin and TMPRSS2 sites of SARS-CoV-2S protein, and the homology between the S672-691 sequence and human and bat SRARS-like CoVs, MERS, which is not high, so that the S672-691 protein is likely to be a specific detection site for SARS-CoV-2.

Example 4

Serum with positive nucleic acid detection result detected by Elisa

ELISA procedure as in example 1b

FIG. 4 shows Elisa test positive sera. After synthesizing an S672-691 antigen peptide sequence, an Elisa experiment is designed in vitro, and a serum sample with positive nucleic acid detection results is detected. Each experiment was set with a serum background control and a random antigenic peptide sequence control. It can be found that the levels of IgG and IgM detection antibodies are substantially higher in the positive serum. The antibody level of the antigen peptide S25-39 and the background control group is basically consistent and is at a very low antibody level. The collected serum is a sample when a new corona patient is hospitalized after the new corona patient is ill, wherein the positive nucleic acid detection result indicates that the new corona virus exists in the body when the nucleic acid detection is carried out.

Example 5

Neutralization test

5.1 preparation before experiment

5.1.1 equilibration reagents

Taking out the reagent (pancreatin, DMEM complete medium) stored at 2-8 deg.C, allowing to equilibrate at room temperature for more than 30 minutes

5.1.2 operator

The experimental operation is carried out by trained experimental operators, and before the experimental operation, changing clothes (wearing disposable aseptic clothes, changing work shoes, wearing a mask, a cap and disposable medical latex gloves) in the clean area can enter the experimental area for the experimental operation.

5.2 Experimental procedures

5.2.1 inactivating the serum (or plasma) to be detected in 56 deg.C water bath for 30min, centrifuging at 6000g for 3min, and transferring the supernatant to a 1.5ml centrifuge tube for use.

5.2.2 taking 96-well plate, adding DMEM complete medium (1% double antibody, 25mM HEPES, 10% FBS) 150. mu.l/well in column 2 (cell control CC, see Table 2), adding DMEM complete medium 100. mu.l/well in columns 3-11 (column 3 is virus control VV, column 4-11 is sample well), and adding DMEM complete medium 42.5. mu.l/well in B4-B11 well.

5.2.3 plasma sample 1 (7.5. mu.l) … … was added to wells B4 and B5 and so on, and plasma sample 4 (7.5. mu.l) was added to wells B10 and B11.

5.2.4 adjusting the multi-channel pipettor to 50 mul, gently and repeatedly blowing and sucking the liquid in the B4-B11 holes for 6-8 times, fully and uniformly mixing, then transferring 50 mul of liquid to the corresponding C4-C11, sucking and discarding 50 mul of liquid, and referring to the sample adding sequence in Table 2.

TABLE 2 sample application sequence

5.2.5 dilution of pseudovirus to 2x10 with DMEM complete Medium⁴TCID50/ml (diluted by dilution factor provided) was added to 50. mu.l per well in columns 3-11 to give a pseudovirus content of 1x 10 per well³A hole.

5.2.6 Place the above 96-well plates in a cell incubator (37 ℃, 5% CO)₂) Incubate for 1 hour.

5.2.7 when the incubation time is half an hour, taking out the Huh-7 cells prepared in advance in the incubator (the confluence rate is 80% -90%), taking a T75 culture bottle as an example, removing the culture medium in the bottle, adding 5ml of PBS buffer solution to clean the cells, pouring off the PBS, adding 3ml of 0.25% pancreatin-EDTA to immerse the cells for digestion for 1 minute, pouring off the pancreatin, placing the cells in the cell incubator for digestion for 5 minutes, slightly beating the side wall of the culture bottle to make the cells fall off, adding 10ml of culture medium to neutralize the pancreatin, blowing for several times, transferring the cells to a centrifuge tube, centrifuging for 5 minutes at 210g, pouring off the supernatant, completely suspending the cells with 10ml of DMEM, counting the cells, diluting the cells to 5 x10 with the DMEM complete culture medium, and counting the cells⁵One per ml.

5.2.8 incubations to 1 hour, add 100. mu.l cells per well in 96-well plates, 5 x10 cells per well⁴And (4) respectively.

5.2.9 gently shaking the 96-well plate to disperse cells uniformly in the well, placing the 96-well plate in a cell culture box at 37 deg.C and 5% CO₂Culturing for 20-28 hours.

5.2.1020-28 hours later, the 96-well plate is taken out from the cell culture box, 150 μ l of supernatant is sucked from each sample loading hole by a multi-channel pipette, then 100 μ l of luciferase detection reagent is added, and the reaction is carried out for 2min at room temperature in a dark place.

5.2.11 after the reaction, repeatedly blowing and sucking the liquid in the reaction hole for 6-8 times by using a multi-channel pipette to fully lyse the cells, sucking 150 mul of liquid from each hole, adding the liquid into a corresponding 96-hole chemiluminescence detection plate, and placing the plate in a chemiluminescence detector to read the luminescence value.

5.2.12 calculating neutralization inhibition ratio: the inhibition rate was [1- (mean value of luminescence intensity of sample group-CC mean value of blank control)/(mean value of luminescence intensity of negative group VC-CC mean value of blank control) ]. 100%.

5.2.13 IC50 was calculated by the Reed-Muench method based on the results of neutralization inhibition. Fig. 3 shows a graph of the results of the neutralization experiment. As can be seen from FIG. 5, the serum antibodies of patients No. 4, No. 7 and No. 17 all had inhibitory effect on the virus and were clearly different from those of normal persons.

Example 6

Experiment of inhibiting virus infection by polypeptide

Huh7.5 cells were plated at a density of 2X104 cells/well in 96-well plates, BSA-conjugated S672-691 polypeptide was added the next day in the amount shown in the figure, after 2hr of pretreatment, SARS-CoV-2 pseudovirus infection was diluted 2-fold and cells were harvested 24hr later, and Lciferase luciferase activity was measured (Promega E151A).

FIG. 6 shows the results of experiments in which polypeptides inhibit viral infection. As can be seen from FIG. 6, the polypeptide dose of 1. mu.g significantly inhibited viral infection, and the difference from the control group was statistically significant.

Example 7

Serum positive to nucleic acid detection by S protein and N protein kit

1. Preparing: the kit was removed (Tarcine, S protein IgG: 600156; S protein IgM: 600157; N protein IgG: 600158; N protein IgM: 600159) and equilibrated at room temperature (18-25 ℃) for 30 minutes. The 20-fold concentrated washing solution 1 was diluted 20-fold with distilled water. And (3) diluting the sample to be detected in an EP tube according to the ratio of 1:100, diluting in advance, mixing uniformly, and standing for 10 minutes at room temperature. IgG-like dilutions immiscible with IgM-like dilutions!

2. Sample adding: setting 3 negative control holes in a microplate, wherein each hole is 100 mu l; the diluted sample to be tested is 100 mul per well.

3. And (3) incubation: after sealing with a pressure-sensitive adhesive strip, the reaction was incubated at 37 ℃ for 30 minutes.

4. Washing the plate: after incubation, the non-drying gel strips were removed, the wells were aspirated, 300. mu.l of washing solution was added to each well, allowed to stand for 30 seconds, the liquid was discarded, and the plates were patted dry. Repeat the above steps 4 times.

5. Adding an enzyme conjugate: 100. mu.l of enzyme conjugate was added to each well.

6. And (3) incubation: after sealing with a pressure-sensitive adhesive strip, the reaction was incubated at 37 ℃ for 20 minutes.

7. Washing the plate: and (4) repeating the step.

8. Color development: 50. mu.l of the substrate solution A, B was added to each well, and incubated at 37 ℃ for 10 minutes in the absence of light.

9. And (4) terminating: stop solution (50. mu.l) was added to each well to terminate the reaction. And (5) lightly shaking and mixing.

10. Reading value: the microplate reader was set at a wavelength of 450nm and the OD of each well was measured. The values should be read within 30 minutes after termination of the reaction.

FIG. 7 shows that the detection result of the nucleic acid by the kit is positive serum.

At present, companies develop a kit for detecting serum antibodies against some key proteins of S protein and N protein of the novel coronavirus, and it can be clearly seen from the comparative detection result in fig. 7 that the specific antigen peptide can detect the antibodies generated against the novel coronavirus in the serum.

Example 8

Serum with negative nucleic acid detection result of Elisa detection

ELISA procedure as in example 1b

FIG. 8 shows that the results of the nucleic acid detection by Elisa are negative sera.

The specific antibody can be detected in the serum of a patient with negative nucleic acid detection results, and the antigen peptide has higher sensitivity in the negative serum through statistical analysis. It is shown that although the virus is cleared in vivo after the patients are infected with the new coronavirus, the antibody does exist.

Example 9

Statistics of antibody detection level changes of infected patients during hospitalization

ELISA procedure as in example 1b

Figure 9 shows the changes in antibody levels in serum during hospitalization after the confirmation of new coronary pneumonia, and the clinical overall scoring of the disease, which we tracked.

As can be seen in FIG. 9, the S672-691 protein detected changes in antibody levels in the serum of patients during treatment, and the trend was substantially consistent with the clinical composite score. This indicates that the polypeptide can detect the antibody level in the patient with high sensitivity, and the immune condition to SARS-CoV-2 can be reflected by the antibody level.

Example 10

Specific antigenic peptide sensitivity and specificity detection

We counted the detected SARS-CoV-2PCR +/-and antibody levels in normal human serum and performed ROC curve statistical analysis in GraphPad Prism 7.0.

FIG. 10 shows SARS-CoV-2-IgG (POS/NEG) where POS represents positive nucleic acid detection and NEG represents negative nucleic acid detection. FIG. 9 shows SARS-CoV-2-IgM (POS/NEG) where POS represents positive nucleic acid detection and NEG represents negative nucleic acid detection.

And (5) according to the ROC curve statistical result, counting the sensitivity and specificity. The inventor co-detects 50 positive nucleic acids of the new coronary patients, 114 negative nucleic acids of the new coronary patients and 47 normal people, and counts SARS-CoV-2-IgG and SARS-CoV-2-IgM.

The Elisa experiment can detect the antibody in the sample efficiently and sensitively, so the Elisa experiment is carried out in vitro on a large scale to detect serum samples of novel coronavirus infected patients and normal people, and the sensitivity and the accuracy of the polypeptide are verified. The statistical results according to the ROC curve are as follows:

1. the antigen peptide detects the SARS-CoV-2-IgG level in 47 normal human cases and 50 new coronary patients (positive for nucleic acid detection):

the detection sensitivity is 94% and the specificity is 87.23% by taking 0.395 as a judgment value

0.4925 is taken as a judgment value, the detection sensitivity is 86 percent, and the specificity is 100 percent

2. The antigen peptide detects the level of SARS-CoV-2-IgG in the serum of 47 normal people and 114 new coronary patients (negative nucleic acid detection):

0.3925 is used as a judgment value, the detection sensitivity is 91.23 percent, and the specificity is 87.23 percent

3. The antigen peptide detects the level of SARS-CoV-2-IgM in 47 normal human cases and 50 new coronary patients (positive for nucleic acid detection):

0.1905 is taken as a judgment value, the detection sensitivity is 94 percent, and the specificity is 78.72 percent

The detection sensitivity is 88 percent and the specificity is 80.85 percent by taking 0.198 as a judgment value

4. The antigen peptide detects the level of SARS-CoV-2-IgM in 47 normal human cases and 114 new coronary patients (negative nucleic acid detection) serum cases:

the detection sensitivity is 87.72 percent and the specificity is 74.47 percent by taking 0.156 as a judgment value

Based on the above results, the following sensitivity and specificity were counted as in table 3:

TABLE 3 antigenic peptide sensitivity and specificity

	Sensitivity of the probe	Specificity of
			Nucleic acid positive-IgG of neocoronary patient	94％	87.23％
Nucleic acid negative-IgG of new coronary patients	91.23％	87.23％
			Nucleic acid positive-IgM of Xinguan patient	94％	78.72％
Nucleic acid negative-IgM of Xinguan patient	87.72％	74.47％

The above results indicate that the antigenic peptides of the present disclosure have good sensitivity and specificity when detected.

Example 11

S672-691 polypeptide immunized mice

1. Each mouse was immunized with 50ug of polypeptide. Under aseptic condition, 50ug of polypeptide is supplemented to 50ul with non-enzyme water, 50ul of adjuvant is added to prepare 100ul of polypeptide mixed solution, and the mixed solution is placed on ice for standby.

2. The abdominal hair of the mouse was shaved with a shaver.

3. Each mouse was injected subcutaneously into the abdomen with 100ul of the polypeptide mixture using a 1ml sterile syringe. The injection is administered once every 3 months 17, 3 months 26, 4 months 2 days.

And 4.4 months and 10 days, carrying out orbital blood collection on the mouse to be detected by using a glass capillary tube with the inner diameter of 0.9-1.1 mm, the wall thickness of 0.1-0.15 mm and the tube length of 100 mm, wherein 100ul of blood is collected from each mouse.

5. And (3) putting the taken mouse blood into a centrifuge, centrifuging at 4 ℃ and 6000g for 10min, and then sucking upper serum to perform an ELISA experiment, wherein the experiment steps are the same as those in the step 1 b.

FIG. 11 shows that the S672-691 polypeptide can significantly stimulate an immune response in mice and produce antibodies.

Example 12

Antibody generated by S672-691 polypeptide immunized mice has inhibition effect on SARS-CoV-2 pseudovirus

The experimental procedure was as in example 6.

FIG. 12 shows that antibodies generated by mice immunized with the S672-691 polypeptide have an inhibitory effect on SARS-CoV-2 virus.

Example 13

Western of S2 specific antibody blob detection result

Vero cells were plated at 1 × 105 density in 48-well plates and cultured overnight. SARS-CoV-2 was infected, and uninfected vero cells were used as negative controls. Cells were harvested 24 hours and 48 hours after infection with lysis buffer [50mM tris-HCl (pH 7.5), 150mM NaCl, 5mM EDTA, 1% NP-40, 1mM phenylmethylsulfonyl fluoride (PMSF), and 1 XProtease inhibitor (Roche) ] lysis protein. SDS-polyacrylamide gel electrophoresis and Western blot to identify Spike protein, primary antibody was incubated with Rabbit anti-beta-actin (CST, 8457S, 1:2000), mouse anti-coronavirus Spike2(MP, 087204, 1: 1000).

FIG. 13 shows the results of Western blot detection of antibodies specific for S2, and based on Western blot analysis of antibodies specific for S2, two major bands were specifically detected in protein extracts infected with SARS-CoV-2, but not in uninfected vero cells, which represent the full-length spike protein and the S2 fragment that may be cleaved at the potential TMRPSS2 cleavage site, respectively.

Example 14

Role of TMPRSS2 in S protein cleavage

HEK293 cells were plated at 1 × 105 density in 48-well plates and cultured overnight. Camostat mesylate (MCE, HY-13512), nafamostat mesylate (MCE, HY-B0190A) and BSA or Biotin coupled-672-691 peptide were added to the culture medium and treated for 2 hours. The GFP-tagged SARS-COV-2-S plasmid (GenScript, C1043FB180-1) and the Tmprss2 expression plasmid (OriGene, RC208677) were then co-transfected in HEK293 cells. Culture medium was changed 16 hours after transfection and culture was continued for 24 hours. The cells were collected and lysed [50mM tris-HCl (pH 7.5), 150mM NaCl, 5mM EDTA, 1% NP-40, 1mM phenyl ethyl sulfone fluoride (PMSF), and 1 × protease inhibitor (Roche) ]. SDS-polyacrylamide gel electrophoresis and Western blot to identify Spike protein, primary antibody was incubated with Rabbit anti-beta-actin (CST, 8457S, 1:2000), mouse anti-eGFP (AtaGenix, 539191227, 1: 1000).

Figure 14 shows the role of TMPRSS2 in S protein cleavage, we co-transfected a plasmid expressing S protein with a control vector or a plasmid expressing TMPRSS2 in the presence or absence of a TMPRSS2 inhibitor or S672-691 polypeptide. As shown in fig. 14, overexpression of TMRPSS2 was significantly increased, while TMRPSS2 inhibitors such as camostat or nafamostat and synthetic S672-691 polypeptides inhibited cleavage of S protein.

Example 15

SARS-CoV-2 pseudovirus infection effect of S672-691 polypeptide on HEK293 cell co-transfected by TMPRSS2 and ACE2 Inhibition of

HEK293T cells are paved in a 24-well plate according to the density of 2 х 105/well on the previous day, after overnight culture, the cells are transferred into corresponding expression plasmids of hACE2 and TMPRSS2 according to the figure, S672-691 polypeptide with corresponding concentration is added after 24 hours of transfection, SARS-CoV-2 pseudovirus is infected after 2 hours of pretreatment, the level of the pseudovirus in the cells is detected by measuring a luciferase report experiment after 24 hours of infection, and the analysis data calculates the inhibition effect of the S672-691 polypeptide on the SARS-CoV-2 pseudovirus entering the host cells.

The inhibitory effect of the corresponding antibodies produced by S672-691 stimulated mice on the infection effect of SARS-CoV-2 pseudovirus of TMPRSS2 and ACE2 co-transfected HEK293 cells was verified by antibody neutralization experiments, which were performed 24 hours after transfection with the corresponding hACE2 and TMPRSS2 expression plasmids, as described above.

FIG. 15 shows that both S672-691 polypeptide and antibody produced by polypeptide stimulation can effectively inhibit SARS-CoV-2 pseudovirus infection of HEK293 cells co-transfected by TMPRSS2 and ACE2, when TMRPSS2 and ACE2 co-transfect HEK293 cells, SARS-CoV-2 pseudovirus infection is remarkably enhanced, and S672-691 polypeptide can inhibit the infection.

The present disclosure is not intended to be limited in scope by the specifically disclosed embodiments, which are provided, for example, to illustrate aspects of the present disclosure. Various modifications to the compositions and methods will be apparent from the description and teachings herein. Such variations may be practiced without departing from the true scope and spirit of the disclosure, and are intended to fall within the scope of the disclosure.

Sequence listing

<110> Suzhou systematic medical institute

Academy of military medicine, Academy of Military Sciences, PLA

Suzhou Fangke Biological Technology Co., Ltd.

<120> novel coronavirus-specific antigen peptide and use thereof

<130> 6A59-2033466I-3

<150> 202010318504.3

<151> 2020-04-21

<160> 7

<170> SIPOSequenceListing 1.0

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<213> SARS-CoV-2

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1 5 10 15

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<211> 20

<212> PRT

<213> SARS-CoV-2

<400> 2

Ala Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Ser Val

1 5 10 15

Ala Ser Gln Ser

20

<210> 3

<211> 15

<212> PRT

<213> SARS-CoV-2

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Asn Arg Ala Leu Thr Gly Ile Ala Val Glu Gln Asp Lys Asn Thr

1 5 10 15

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<213> SARS-CoV-2

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Asn Gly Ile Gly Val Thr Gln Asn Val Leu Tyr Glu Asn Gln Lys

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Tyr Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile

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<210> 6

<211> 15

<212> PRT

<213> SARS-CoV-2

<400> 6

Ile Asp Leu Gln Glu Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp

1 5 10 15

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Claims (8)

1. A polypeptide, wherein the polypeptide is a polypeptide shown in any one of the following (1) to (2):

(1) the polypeptide is shown as SEQ ID NO: 2;

(2) the polypeptide is a polypeptide obtained by substituting, repeating, deleting or adding one or more amino acids on the basis of the polypeptide shown in (1).

2. The polypeptide of claim 1, wherein the polypeptide has a relative position to the polypeptide as set forth in SEQ ID NO: 2, having at least 80% homology; preferably, there is at least 90% homology or greater.

3. The polypeptide of any one of claims 1-2, wherein the number of amino acid residues in said polypeptide is 20.

4. A kit for detecting whether a patient is infected with SARS-CoV-2 or has COVID-19, wherein the kit contains the polypeptide of any one of claims 1-3.

5. A pharmaceutical composition or vaccine comprising a polypeptide according to any one of claims 1-3.

6. Use of a polypeptide according to any one of claims 1-3 in the preparation of a reagent for detecting whether it is infected with SARS-CoV-2 or has COVID-19.

7. Use of a polypeptide according to any one of claims 1-3 in the manufacture of a medicament for the treatment or prevention of infection by SARS-CoV-2 or having COVID-19.

8. A method of treating a patient infected with SARS-CoV-2 or having COVID-19, wherein the method comprises administering to the patient the polypeptide of any one of claims 1 to 3 or the pharmaceutical composition or vaccine of claim 5.

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