WO2021180232A1 - 抗原表位多肽的筛选方法及装置 - Google Patents

抗原表位多肽的筛选方法及装置 Download PDF

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WO2021180232A1
WO2021180232A1 PCT/CN2021/080636 CN2021080636W WO2021180232A1 WO 2021180232 A1 WO2021180232 A1 WO 2021180232A1 CN 2021080636 W CN2021080636 W CN 2021080636W WO 2021180232 A1 WO2021180232 A1 WO 2021180232A1
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region
epitope
screening
peptide
value
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French (fr)
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李英睿
王健
郑汉城
刘兵行
燕鸣琛
曹嘉敏
李振宇
沈凌浩
李丹妮
丁秋霞
孙静
梁志颖
王俊
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珠海碳云智能科技有限公司
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Priority to US17/911,143 priority Critical patent/US20230136600A1/en
Priority to EP21767268.2A priority patent/EP4119946A4/en
Publication of WO2021180232A1 publication Critical patent/WO2021180232A1/zh

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    • 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
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    • 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
    • G01N33/6878—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids in eptitope analysis
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    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
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Definitions

  • the present invention relates to the field of molecular immunology, in particular to a method and device for screening epitope polypeptides.
  • coronavirus pneumonia Corona Virus Disease 2019, COVID-19
  • SARS-CoV-2 severe Acute Respiratory Syndrome Coronavirus 2
  • SARS-CoV-2 severe Acute Respiratory Syndrome Coronavirus 2
  • Polypeptide vaccine is a vaccine prepared by chemical synthesis according to the known or predicted amino acid sequence of a certain epitope in the pathogen antigen gene. Polypeptide vaccines are chemically synthesized, there is no problem of virulence recovery or incomplete inactivation, and specific epitopes can be selected, so it has become a popular research point for vaccine development today. In many fields including tumor vaccines, there are A number of studies have been published, and related clinical trials are also in progress.
  • the main purpose of the present invention is to provide a method and device for screening epitope polypeptides, so as to provide polypeptides for this new type of virus and develop corresponding polypeptide products.
  • a method for screening epitopes of coronaviruses includes: predicting epitopes using all proteomic sequences of the target coronavirus to obtain predicted epitope regions; and using polypeptides
  • the chip technology screens out the peptides that are differentially responsive to the target coronavirus infection positive serum sample and the control serum sample, and records them as differential peptides; compares the differential peptides with all the proteomic sequences of the target coronavirus to obtain the first A conserved motif region; from the predicted epitope region and the first conserved motif region, the region that meets the epitope screening conditions is selected to obtain the antigen epitope; wherein the epitope screening conditions include the non-phosphorylated region of the target coronavirus And/or extramembranous area.
  • the epitope prediction is performed by using all the proteomic sequences of the target coronavirus, and the predicted epitope region is obtained including: using all the proteomic sequences of the target coronavirus, using a variety of different methods to predict the epitope, and the screening length is 8-20, preferably 10-15 amino acid epitopes, to obtain candidate predicted epitopes; according to the epitopes that can be presented by HLA in a specific population and/or hydrophobicity, the candidate predicted epitopes are screened to obtain predictions Epitope region; preferably, the epitopes that can be presented by HLA in the Chinese population are screened from the candidate predicted epitopes, and/or the epitopes whose hydrophobicity is higher than the first hydrophobic threshold are removed from the candidate predicted epitopes to obtain The epitope region is predicted; preferably, an epitope with a hydrophobicity higher than the first hydrophobic threshold refers to an epitope with a hydrophobic amino acid ratio greater than 45%
  • the difference peptide includes: selecting the target coronavirus infection positive serum sample and negative control serum Samples and other lung disease control serum samples, other lung diseases refer to lung diseases caused by virus infections other than the target coronavirus; using immunological characterization methods, the positive serum samples, negative control serum samples and other lung diseases are compared
  • the serum sample is combined with the peptide array chip to obtain the signal value of the binding peptide response; for each binding peptide, calculate the p value when there is a difference between the signal value of the positive serum sample and the signal value of the negative control serum sample, and record it as The first p value, and calculate the p value when the signal value of the positive serum sample is different from the signal value of the control serum sample of other lung diseases, and record it as the second p value; keep all the first p value and the second p value At the same time, the binding peptides that meet the
  • log10 conversion is performed on the signal value of the bound peptide, and the converted log value is used as the feature, and the p-value of each feature when there is a difference between the positive serum sample and the negative control serum sample is calculated by one-tailed T test.
  • the p-value is corrected by multiple hypothesis testing to obtain the first p-value; at the same time, the p-value of the corresponding feature when there is a difference between the positive serum sample and the control serum sample of other lung diseases, and the multiple hypothesis test of the p-value
  • the correction is recorded as the second p value; the binding peptides that meet the first p value less than the difference threshold and the second p value less than the difference threshold are screened to obtain the difference peptide.
  • obtaining the first conserved motif region includes: taking a single amino acid as a unit, calculating the signal value of the binding peptide that covers the amino acid and matches the amino acid
  • the distribution of p2 values between the sample and the control serum samples of other lung diseases is different.
  • the distribution of the p1 value is significantly lower than the p2 value.
  • the hydrophobicity is lower than the second hydrophobicity
  • the threshold region refers to the region where the hydrophobic amino acid ratio is less than or equal to 45% and the hydrophobicity score is less than or equal to 3; preferably, the differential peptide is a differential peptide that can be completely aligned to the entire protein group sequence of the target coronavirus.
  • the screening method further includes: comparing the differential peptide with the protein sequence of the coronavirus family to obtain the first conserved motif region.
  • Two conserved motif regions preferably, comparing the differential peptides with the protein sequence of the coronavirus family, and obtaining the second conserved motif region includes: comparing the differential peptides with the protein sequence of the coronavirus family, and from Among the regions that can be compared, the region where each amino acid position satisfies the following regional screening conditions is selected as the second conserved motif region: among all the differential peptides covering amino acids, the proportion of differential peptides that can be matched with amino acids meets the match Ratio threshold; preferably, the matching ratio threshold is greater than or equal to 75%.
  • the epitope screening conditions also include at least one of the following: (a) overlap with the second conserved motif region; (b) the comparison score with the human proteome sequence is lower than the comparison threshold; (c) meet the following requirements Performance indicators: 1) The number of different peptides covered is ⁇ 3; 2) Hydrophilicity meets the hydrophilic threshold; 3) Accessibility scores, Beta turn and multiple comparison scores are all in the top 100; comparison scores are low
  • the comparison threshold refers to a/b ⁇ 0.8, where a is the matching score of the sequence of the region to be screened and the human proteomic sequence, and b is the sequence of the region to be screened and the entire proteomic sequence of the target coronavirus.
  • the matching score of the alignment preferably, the region that meets the epitope screening conditions is selected from the predicted epitope region and the first conserved motif region, and the obtained epitope includes: combining the predicted epitope region and the first conserved motif Regions are merged according to at least one of the following merging conditions: 1) The two regions are in a containment relationship; 2) The two regions are predicted to be epitope regions by at least two different methods to obtain the first candidate epitope region; from the first candidate The region of the epitope region that overlaps with the second conserved motif region is selected as the second candidate epitope region; the region of the second candidate epitope region whose alignment score with the human proteome sequence is lower than the alignment threshold is selected from the second candidate epitope region, As the third candidate epitope region; screening and retaining the non-phosphorylated region and/or extramembrane region in the target coronavirus proteome sequence from the third candidate epitope region as the fourth candidate epitope region; according to accessibility , Beta turn, hydrophilicity,
  • a coronavirus epitope screening device comprising: an epitope prediction module for predicting the epitope using all the proteomic sequences of the target coronavirus, Obtain the predicted epitope region; the differential peptide screening module is used to screen out the peptides with differential response between the positive serum sample and the control serum sample infected by the target coronavirus by using peptide chip technology, and record it as the differential peptide; the first region screening Module, used to compare the differential peptides with all the proteomic sequences of the target coronavirus to obtain the first conserved motif region; the third region screening module is used to select the predicted epitope region and the first conserved motif region Screening regions that can meet the epitope screening conditions to obtain antigen epitopes: wherein, the epitope screening conditions include non-phosphorylated regions and/or extramembrane regions of the target coronavirus.
  • the epitope prediction module includes: a first screening module for candidate epitopes, which is used to use all the proteomic sequences of the target coronavirus to predict epitopes by using a variety of different methods, and the screening length is 8-20, preferably 10-15 amino acid epitopes to obtain candidate predicted epitopes; the second candidate epitope screening module is used to predict candidate epitopes based on the epitopes and/or hydrophobicity that can be presented by HLA in a specific population After screening, the predicted epitope region is obtained.
  • a first screening module for candidate epitopes which is used to use all the proteomic sequences of the target coronavirus to predict epitopes by using a variety of different methods, and the screening length is 8-20, preferably 10-15 amino acid epitopes to obtain candidate predicted epitopes
  • the second candidate epitope screening module is used to predict candidate epitopes based on the epitopes and/or hydrophobicity that can be presented by HLA in a specific population
  • the second candidate epitope screening module includes: a population epitope screening module, which is used to screen the epitopes that can be presented by HLA in the Chinese population from the candidate predicted epitopes; and/or, the hydrophobic screening module, which uses To remove the epitope whose hydrophobicity is higher than the first hydrophobic threshold from the candidate predicted epitope, to obtain the predicted epitope region; preferably, the epitope whose hydrophobicity is higher than the first hydrophobic threshold means that the proportion of hydrophobic amino acids is greater than 45% and Epitopes with a hydrophobicity score greater than 3.
  • the differential peptide screening module includes: the first screening module includes: a sample selection unit for selecting positive serum samples, negative control serum samples, and other lung disease control serum samples for target coronavirus infection, and other lung disease indicators Purpose: Pulmonary diseases caused by viral infections other than coronavirus; signal acquisition unit, used to use immunological characterization methods to combine positive serum samples, negative control serum samples and other lung disease control serum samples with peptide array chips to obtain The signal value of the binding peptide response; the differential peptide screening unit is used to calculate the p value when there is a difference between the signal value of the positive serum sample and the signal value of the negative control serum sample for each binding peptide, which is recorded as the first A p value, and calculate the p value when the signal value of the positive serum sample is different from the signal value of the control serum sample of other lung diseases, and record it as the second p value; keep all the first p value and the second p value at the same time The binding peptides that meet the difference threshold are combined to obtain the difference peptide
  • the differential peptide screening unit includes: a signal conversion subunit for log10 conversion of the signal value of the bound peptide; Test, calculate the p value of each feature when there is a difference between the positive serum sample and the negative control serum sample, and perform multiple hypothesis test correction on the p value to obtain the first p value; at the same time, calculate the corresponding feature in the positive serum sample and other The p value when there is a difference between the lung disease control serum samples, and the p value is corrected by multiple hypothesis testing, and it is recorded as the second p value; the first p value is less than the difference threshold and the second p value is less than the difference threshold at the same time. Binding peptides to obtain differential peptides.
  • the first region screening module includes: a conservative site screening module, which is used to calculate the signal value of the binding peptide that covers the amino acid and matches the amino acid in the unit of a single amino acid, and the signal value of the positive serum sample and the negative control serum sample is different. At the same time, calculate the distribution of the p2 value of the positive serum sample and the negative control serum sample for the signal value of the binding peptide that covers the amino acid and does not match the amino acid.
  • a conservative site screening module which is used to calculate the signal value of the binding peptide that covers the amino acid and matches the amino acid in the unit of a single amino acid, and the signal value of the positive serum sample and the negative control serum sample is different. At the same time, calculate the distribution of the p2 value of the positive serum sample and the negative control serum sample for the signal value of the binding peptide that covers the amino acid and does not match the amino acid.
  • the distribution of the p1 value is significantly lower than the distribution of the p2 value of amino acids It is the first conserved site; the first conserved motif screening module is used to compare the different peptides with the entire proteomic sequence of the target coronavirus, and select the first conserved site from the regions that can be compared And the hydrophobicity is lower than the second hydrophobic threshold area, so as to obtain the first conserved motif region; preferably, the hydrophobicity lower than the second hydrophobic threshold area means that the proportion of hydrophobic amino acids is less than or equal to 45% and the hydrophobicity score is less than or equal to The region of 3; preferably the differential peptide is a differential peptide that can be completely aligned to the entire proteomic sequence of the target coronavirus.
  • the screening device further includes a second region screening module.
  • the second region screening module includes: an alignment module for comparing the differential peptides with the protein sequence of the coronavirus family, and the second conservative motif screening The module is used to select the region where each amino acid position meets the following regional screening conditions from the regions that can be compared, as the second conserved motif region: the differential peptide that can match the amino acid among all the differential peptides covering amino acids The ratio of the segment meets the matching ratio threshold.
  • the matching ratio threshold is greater than or equal to 75%.
  • the epitope screening conditions in the third region screening module 50 further include at least one of the following: (a) overlap with the second conserved motif region; (b) the comparison score with the human proteome sequence is lower than the comparison Threshold; (c) Meet the following performance indicators: 1) The number of different peptides covered is ⁇ 3; 2) Hydrophilicity meets the hydrophilic threshold; 3) Accessibility score, Beta turn angle and multiple comparison scores are all Located in the top 100; where the comparison score is lower than the comparison threshold means a/b ⁇ 0.8, where a is the matching score of the sequence of the region to be screened and the human proteome sequence, and b is the sequence of the region to be screened The matching score of the alignment with all the proteomic sequences of the target coronavirus.
  • the third region screening module includes: a merging module for merging the predicted epitope region and the first conserved motif region according to at least one of the following merging conditions: 1) the two regions have an inclusion relationship; 2) two The region is predicted to be an epitope region by at least two different methods, and the first candidate epitope region is obtained; the overlap screening module is used to screen the region that overlaps the second conserved motif region from the first candidate epitope region as the first candidate epitope region.
  • Two candidate epitope regions Two candidate epitope regions; an alignment screening module, used to screen the region from the second candidate epitope region whose comparison score with the human proteome sequence is lower than the first threshold, as the third candidate epitope region; non-phosphorylated And extra-membrane region screening module, used to screen and retain the non-phosphorylated region and extra-membrane region in the target coronavirus proteome sequence from the third candidate epitope region, as the fourth candidate epitope region; integrated sorting module, used According to the accessibility, beta turn, hydrophilicity, the number of different peptides covered, and the results of multiple comparisons, the fourth candidate epitope region is comprehensively sorted and then selected to obtain the epitope of the target coronavirus.
  • the device also includes a mutation removal module, which is used to remove regions containing mutations from the regions selected after the comprehensive ranking module to obtain the epitope of the target coronavirus.
  • a storage medium includes a stored program, wherein the device where the storage medium is located is controlled to perform any of the above-mentioned methods for screening coronavirus epitopes when the program is running.
  • a processor which is used to run a program, wherein any of the above-mentioned methods for screening coronavirus epitopes is executed when the program is running.
  • a batch of peptides specifically related to coronavirus infection are obtained through innovative screening of peptide chip technology, and these peptides can be used to prepare antigens and antibodies.
  • Relevant testing reagents such as, kits, and related vaccine products such as peptide vaccines, nucleic acid vaccines, protein recombinant vaccines, etc., so as to provide more powerful tools for preventing and controlling the infection and prevalence of such viruses.
  • Figure 1 shows a schematic flow chart of a method for screening coronavirus epitopes in a preferred embodiment of the present application.
  • Figure 2A and Figure 2B respectively show the activity of neutralizing antibodies produced by the new coronavirus from sera obtained from mice immunized with different monopeptides.
  • Figure 2A shows the results of the microscopic examination, and
  • Figure 2B shows the statistics. result.
  • Figures 3A, 3B, and 3C show the changes over time of the antibody signal corresponding to each polypeptide in mice immunized with combination 1, combination 2 and combination 3, respectively.
  • Figures 4A and 4B respectively show the activity of the neutralizing antibody produced by the new coronavirus from the serum obtained from mice immunized with different combination 1, combination 2 and combination, and Figure 4A shows the result of microscopic examination. 4B shows the statistical results.
  • Figures 5A to 5J respectively show the changes over time of the antibody signal corresponding to the 4 polypeptides of each mix after the mice are immunized with Mix1 to Mix10.
  • Figures 6A to 6F show the antibodies produced at different time points after the 7 peptides were co-immunized with each adjuvant.
  • Fig. 7 is a block diagram of the hardware structure of an epitope polypeptide screening method according to an embodiment of the present invention.
  • Figure 8 shows a schematic structural diagram of a screening device for epitope polypeptides in a preferred embodiment of the present application.
  • the "new coronary pneumonia” or COVID-19 in this application refers to the disease that a patient develops after being infected with the SARS-Cov-2 virus (also called the new coronavirus in this application), that is, the new type of coronavirus pneumonia.
  • Epitope Also known as antigenic determinant, it is a special chemical group with a certain composition and structure on the surface or other parts of an antigenic substance molecule, and a structure that can specifically bind to the corresponding antibody or sensitized lymphocyte.
  • T cell epitopes are generally not located on the surface of antigen molecules.
  • the antigen must be processed by antigen-presenting cells into small molecular peptides and combined with MHC molecules in order to be recognized by TCR. T cells can only recognize processed epitopes.
  • B cell epitopes can exist on the surface of antigen molecules and can be directly recognized by B cells without processing. In this application, it refers to one or more peptides that are predicted or screened to specifically bind to an antibody.
  • Polypeptide In this application, it refers to any peptide that is predicted or screened to specifically bind to antibodies or sensitized lymphocytes.
  • Polypeptide-carrier protein conjugate In this application, it refers to an antigen formed by coupling a polypeptide and a carrier protein.
  • One carrier protein can be coupled to one or more polypeptides. When multiple polypeptides are coupled, multiple polypeptides have the same amino acid sequence. According to the differences in the physical and chemical properties of the specific coupled polypeptide sequences, the types of specific carrier proteins, and the differences in coupling methods, the number of polypeptides coupled on each carrier protein is different.
  • 2 ⁇ 50 more preferably 3 to 45, 5 to 40, 5 to 35, 5 to 30, 8 to 30, 10 to 30, 12 to 30, 15 to 30; or, more preferably 6 to 36, 8 to 32, 10 to 28, 10 to 26, 10 to 24, 10 to 22, 10 to 20, 10 to 18, 10 to 16, and 10 to 15 Any of the cases.
  • Antigen refers to all substances that can induce an immune response in the body. That is, it can be specifically not combined with antigen receptors (TCR/BCR) on the surface of T/B lymphocytes, activate T/B cells, make them proliferate and differentiate, produce immune response products (sensitized lymphocytes or antibodies) and can A substance that specifically binds to the corresponding product in or out of the body. Therefore, antigens have two important characteristics: immunogenicity and immunoreactivity.
  • the antigen in this application refers to a complete antigen with immunogenicity formed after a polypeptide hapten is coupled with a carrier protein, which can be a polypeptide-carrier protein conjugate formed by coupling a polypeptide of a single amino acid sequence with a carrier protein; or A polypeptide-carrier protein conjugate composition formed by coupling polypeptides with multiple different amino acid sequences and a carrier protein.
  • Vaccine usually refers to the ability to have both immunogenicity and reactogenicity.
  • Immunogenicity refers to the ability to stimulate the body to produce an immune response, that is, to stimulate the body to produce specific immune cells, so that immune cells are activated, proliferated, differentiated, and finally produced
  • the ability of an immune effector substance to specific antibodies or sensitized lymphocytes, and reactogenicity refers to the ability to specifically bind to antibodies or sensitized lymphocytes induced by it.
  • Polypeptide vaccine In order to improve the immunogenicity of polypeptides to stimulate the body to produce specific antibodies or sensitized lymphocytes, the polypeptide antigens are usually immunized with adjuvants. Commonly used adjuvants include: aluminum hydroxide adjuvant, Corynebacterium parvum, lipopolysaccharide, cytokine or alum and so on. Freund's complete adjuvant and Freund's incomplete adjuvant are the most common adjuvants in animal immunization.
  • the peptide chip technology is a detection technology based on the peptide chip, which uses the contact between a wide variety of peptides on the peptide chip and the sample, and then uses the image acquisition technology to collect each characteristic signal on the peptide chip (specifically, it can be expressed as a fluorescent image carrying each characteristic signal ), and then output the signal intensity of each feature in the chip, that is, the detection result data of the polypeptide chip.
  • the sample detection signal output based on the polypeptide chip detection result data can realize the analysis of the analyte in the sample bound to the polypeptide on the polypeptide chip, and the analysis of the sample.
  • Motif in biology, is a mathematical statistical model based on data, typically a sequence (Sequence), can also be a structure, is the sequence prediction of a specific group (group), for example, a DNA sequence can be Defined as the transcription factor binding site, that is, the sequence tends to be bound by the transcription factor.
  • group a DNA sequence can be Defined as the transcription factor binding site, that is, the sequence tends to be bound by the transcription factor.
  • sequence motifs can be defined as protein sequences belonging to a given protein family.
  • a simple motif can be, for example, a pattern, and this pattern is shared by all members of the group.
  • ROC curve a curve reflecting the relationship between sensitivity and specificity.
  • the X-axis on the abscissa is 1-specificity, which is also called false positive rate.
  • AUC Absolute Under Curve
  • the existing epitope screening methods combined with unique peptide chip technology, a batch of epitopes related to coronavirus family proteins have been screened out, and Part of it is the epitope specific to the new coronavirus.
  • the corresponding polypeptide antigens, detection kits, polypeptide antibodies, polypeptide vaccines, recombinant vaccines and other related products can be prepared, as well as genetic vaccines or protein recombinant vaccines and other related products that are further developed using these polypeptide sequences.
  • the prevention and control of coronavirus-related diseases and/or new coronary pneumonia provides more ideas and methods.
  • a polypeptide is provided, and the polypeptide is selected from any one of the peptide fragments shown in SEQ ID NO: 1 to SEQ ID NO: 154 shown in Table 1.
  • an epitope is provided, and the epitope includes any one or more of SEQ ID NO: 1 to SEQ ID NO: 154 shown in Table 1.
  • the above-mentioned epitope includes SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 41 To any one or more of SEQ ID NO: 154.
  • the polypeptides shown in SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 31, SEQ ID NO: 35 are obtained through at least two peptide chip screenings, and therefore have higher potential application value as antigen epitopes.
  • polypeptides as epitopes specifically recognized by B cells or T cells, can be prepared into polypeptide vaccines to stimulate the body to produce specific antibodies or sensitized lymphocytes (immunogenicity).
  • adjuvants are often added to stimulate the body to produce helper T cells and further induce B cell immune responses.
  • a separate polypeptide can also be used to stimulate the body to produce an immune response.
  • polypeptides can also be prepared into antigens to stimulate the body to produce antibodies.
  • the use of carrier proteins with many epitopes is beneficial to stimulate helper T cells and further induce B cell immune responses.
  • a polypeptide-carrier protein conjugate is also provided.
  • the polypeptide-carrier protein conjugate includes any one of the above-mentioned polypeptides and a carrier protein coupled to the polypeptide.
  • a polypeptide-carrier protein conjugate is usually used as an antigen to detect antibodies, or as an antigen to prepare antibodies by immunizing animals. Since the above-mentioned polypeptides can specifically recognize coronaviruses, especially SARS-CoV-2 viruses, such polypeptide-carrier protein conjugates can specifically recognize antibodies against coronaviruses, especially SARS-CoV-2 viruses. .
  • a specific suitable carrier protein can be selected to form the polypeptide-carrier protein conjugate.
  • the carrier protein in this application includes but is not limited to BSA (bovine serum albumin), OVA (ovalbumin), KLH (keyhole limpet hemocyanin) or CS (casein).
  • BSA bovine serum albumin
  • OVA ovalalbumin
  • KLH keyhole limpet hemocyanin
  • CS casein
  • amino acid sequence composition of different polypeptides in order to facilitate coupling with the carrier protein, it is necessary to couple with the carrier protein through a linking sequence (also called a linker or linker).
  • the connecting sequence is preferably CGSG.
  • the number of peptides that can be coupled to each carrier protein is also different according to the physical and chemical properties of the amino acids of the peptides, the different carrier proteins used, and the different coupling methods. Considering the efficiency of coupling and the ability to recognize and bind antibodies, it is preferable to couple 2-50 polypeptides per carrier protein, more preferably 3 to 45, 5 to 40, 5 to 35, 5 to 30, 8 to 30, 10 to 30, 12 to 30, 15 to 30; or, more preferably 6 to 36, 8 to 32, 10 to 28, 10 to 26, 10 to 24, Any one of 10-22, 10-20, 10-18, 10-16, and 10-15.
  • an antigen which includes a polypeptide-carrier protein conjugate, or a combination of multiple different polypeptide-carrier protein conjugates, wherein the polypeptide-carrier
  • the protein conjugate is any of the above-mentioned polypeptide-carrier protein conjugates.
  • the polypeptides coupled to the carrier protein are all polypeptides with the same amino acid sequence, that is, the same polypeptide is coupled to the same carrier protein.
  • the polypeptide-carrier protein conjugate has a single epitope when used as an antigen.
  • the antigen when detecting the presence of virus antibodies in the serum as an antigen, the antigen may be an antigen with a single epitope, or an antigen with multiple epitopes at the same time.
  • a polypeptide-carrier protein conjugate coupled with different polypeptide sequences is used as an antigen in the form of a composition, a variety of epitopes can be generated.
  • the polypeptide of sequence A is coupled with BSA to obtain an A-BSA conjugate
  • the polypeptide of sequence B is coupled with BSA to obtain a B-BSA conjugate
  • the polypeptide of sequence C is coupled with OVA to obtain a C-OVA conjugate.
  • the antigens containing these three polypeptide-carrier protein conjugates have three epitopes: A, B, and C. If the antigen includes only one of the three polypeptide-carrier protein conjugates, it has only one epitope.
  • a coronavirus antibody detection kit which includes any one of the above antigens.
  • the epitopes in these antigens are derived from any of the above-mentioned polypeptides, and the above-mentioned polypeptides are in the known coronavirus protein family. Therefore, the kit containing the antigen can be used to accurately and specifically target the coronavirus, especially SARS. -CoV-2 infection patients are identified and diagnosed.
  • kits can be prepared into a variety of different types of detection kits according to specific needs.
  • most of the peptide antigens in the kit are pre-coated antigens.
  • the pre-coated antigen is coated on the solid-phase carrier; the specific pre-coated solid-phase carrier is rationally designed according to needs.
  • the solid phase carrier includes an enzyme-labeled plate (mostly made of polystyrene), a membrane carrier or microspheres; further preferably, the membrane carrier includes a nitrocellulose membrane (the most widely used), a glass cellulose membrane or a nylon membrane More preferably, the membrane carrier is also coated with a positive control substance, and the polypeptide-carrier protein conjugate and the positive control substance are sequentially arranged on the nitrocellulose membrane in the order of detection.
  • the specific matching reagents in the kit are correspondingly different, but the matching reagents can be combined according to the preparation method of the known kit.
  • the above kit also includes at least one of the following: (1) an enzyme-labeled secondary antibody, more preferably an enzyme-labeled secondary antibody is an HRP-labeled secondary antibody (corresponding to an ELISA detection kit); (2) colloidal gold binding pad , The colloidal gold binding pad is coated with a colloidal gold-labeled polypeptide-carrier protein conjugate and a specific binding substance of the positive control (corresponding to the immunocolloidal gold detection kit); (3) the labeling pad, the labeling pad is coated with Fluorescence-labeled microspheres, the microspheres are loaded with the specific binding substance of the positive control substance (corresponding to the immunofluorescence detection kit).
  • the above-mentioned immunocolloidal gold detection kit and immunofluorescence detection kit are relatively more convenient, and only the C line of the positive control and the T line of the test sample need to be established.
  • the positive control pre-coated at the C line of the positive control, as long as it can be combined with the specific binding substance with the detection label carried by the serum chromatography process of the sample to be tested.
  • the specific antigen or antibody is not particularly limited.
  • the positive control substance is selected from mouse immunoglobulin, human immunoglobulin, goat immunoglobulin or rabbit immunoglobulin, and correspondingly, the specific binding substance of the positive control substance is selected from anti-mouse immunoglobulin, anti-human immunoglobulin Immunoglobulin, anti-goat immunoglobulin or anti-rabbit immunoglobulin.
  • the aforementioned anti-mouse immunoglobulin may be goat anti-mouse immunoglobulin or rabbit anti-mouse immunoglobulin, or other immunizable animal anti-mouse immunoglobulin.
  • anti-human immunoglobulin, anti-goat immunoglobulin or anti-rabbit immunoglobulin can also be anti-immunoglobulins from different species according to the different animals being immunized.
  • the aforementioned immunoglobulin may be any one of IgM, IgG, IgA, IgD, or IgE.
  • These anti-immunoglobulin antibodies can be monoclonal antibodies or polyclonal antibodies.
  • the size of the microtiter plate used is also different, and it can be reasonably selected from 12 to 384-well microtiter plates.
  • the coating of polypeptide-carrier protein conjugates in each well The amount of quilt is also different.
  • the coating amount of the polypeptide-carrier protein conjugate in each well is preferably 0.1-32 ⁇ g, preferably 0.2-30 ⁇ g, 0.3-30 ⁇ g, 0.4-28 ⁇ g, 0.6-25 ⁇ g, 0.6 ⁇ 24 ⁇ g, 0.7 ⁇ 24 ⁇ g, 0.7 ⁇ 22 ⁇ g, or 0.7 ⁇ 20 ⁇ g, more preferably 0.7 ⁇ 19 ⁇ g, 0.7 ⁇ 18 ⁇ g, 0.7 ⁇ 17 ⁇ g, 0.7 ⁇ 16 ⁇ g, 0.7 ⁇ 15 ⁇ g, 0.7 ⁇ 14 ⁇ g, 0.7 ⁇ 13 ⁇ g or 0.7 ⁇ 12 ⁇ g; more preferably 0.8-19 ⁇ g, 0.8-18 ⁇ g, 0.8-17 ⁇ g, 0.8-16 ⁇ g, 0.8-15 ⁇ g, 0.8-14 ⁇ g, 0.8-13 ⁇ g, 0.8-12 ⁇ g, 0.8-11 ⁇ g, 0.8-10 ⁇ g, 0.8-9 ⁇ g, 0.8 ⁇ 8 ⁇ g, 0.8 ⁇ 7 ⁇ g, 0.8 ⁇ 6 ⁇ g, 0.8 ⁇ 5 ⁇ g,
  • the coating amount of the polypeptide-carrier protein conjugate on the membrane carrier is also different, preferably 0.8-8 ⁇ g/cm, more preferably 0.8-7 ⁇ g/cm, 0.8-6 ⁇ g/ cm, 0.8 ⁇ 5 ⁇ g/cm, 0.8 ⁇ 4 ⁇ g/cm, 0.8 ⁇ 3 ⁇ g/cm, 0.8 ⁇ 2 ⁇ g/cm, 0.8 ⁇ 1.8 ⁇ g/cm, 0.8 ⁇ 1.7g, 0.8 ⁇ 1.6 ⁇ g/cm, 0.8 ⁇ 1.5 ⁇ g/ cm, 0.8 ⁇ 1.4 ⁇ g/cm or 0.8 ⁇ 1.2 ⁇ g/cm.
  • the application of any of the above-mentioned polypeptides or epitopes in the preparation of drugs related to the treatment of diseases caused by coronaviruses is also provided.
  • the coronavirus is SARS-CoV-2.
  • the relevant polypeptide vaccine can be prepared by chemical synthesis.
  • the nucleic acid encoding the above-mentioned polypeptide can be obtained by recombining the gene, thereby obtaining a genetic vaccine. Therefore, the above-mentioned drugs can be antibodies or vaccines.
  • the antibodies can be monoclonal antibodies or polyclonal antibodies.
  • the vaccines can be polypeptide vaccines or genetic vaccines.
  • the above-mentioned drug is also provided.
  • the drug may be an antibody or a vaccine.
  • the antibody is obtained by immunizing an animal with the above-mentioned antigen.
  • the vaccine is a polypeptide vaccine or a gene vaccine.
  • the polypeptide vaccine includes Table 1 Any one or more of the polypeptides in the genetic vaccine contains a nucleic acid encoding any one or more of the polypeptides in Table 1.
  • the polypeptide is selected from any one or more of SEQ ID NO: 1 to SEQ ID NO: 40; more preferably, the polypeptide is selected from SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 31, Any one or more of SEQ ID NO: 35 and SEQ ID NO: 36, these 5 polypeptides are obtained through at least two independent peptide chip screenings, and therefore have a greater potential as a vaccine in terms of probability.
  • the above-mentioned antibody is obtained by using a polypeptide-carrier protein conjugate as an antigen to immunize animals.
  • Commonly used immunized animals include mammals such as rats, mice, goats or rabbits.
  • the obtained antibody may be a monoclonal antibody or a polyclonal antibody.
  • the vaccine can be a polypeptide vaccine.
  • the polypeptide vaccine can be obtained by chemical synthesis according to the polypeptide sequence, or can be obtained by recombinant expression in vitro by genetic engineering means, and then obtained by enzyme digestion and purification. Gene vaccines are designed to contain nucleic acids encoding the target polypeptides through genetic engineering methods, and the nucleic acids are expressed to produce polypeptides with epitope effects.
  • a method for prevention or treatment of pneumonia caused by coronavirus comprises: administering a preventive effective amount of an anti-coronavirus drug to the subject, the drug being the above A vaccine in medicine; the treatment method includes: administering a therapeutically effective amount of an anti-coronavirus medicine to the subject, and the medicine is an antibody in the above-mentioned medicine.
  • the aforementioned coronavirus is SARS-CoV-2.
  • a polypeptide composition in order to further enhance the immune response of the polypeptide itself to body stimulation, in a preferred embodiment, includes the SEQ ID NO shown in Table 1: 1 to at least two of the peptides shown in SEQ ID NO: 154.
  • the polypeptide composition includes at least any one of the peptide fragments shown in SEQ ID NO: 1 to SEQ ID NO: 40.
  • the polypeptide composition includes at least any one of SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 31, SEQ ID NO: 35, and SEQ ID NO: 36.
  • the above-mentioned polypeptide composition can be mixed in a physical form to form a composition according to different research and development requirements such as vaccine or antibody preparation, or a long-chain polypeptide composition can be formed in the form of a chemical bond.
  • the specific peptide sequence, the number and sequence of the peptides to be connected can be adjusted reasonably according to actual needs.
  • two are used for connection, and the specific connection method can be realized by connecting arms (for example, it can be glycine or lysine) and the like.
  • the polypeptide composition includes one or more of the first peptide set, wherein the first peptide set includes SEQ ID NO: 1-4, 6-8, 11, 13-17, Peptides shown as 20-25, 27-30, 32-33, 35-36 and 39-40.
  • the peptides in the first peptide collection above show stronger sequence specificity of the new coronavirus. The preparation of vaccines based on these peptides facilitates the acquisition of vaccines specific to the new coronavirus.
  • the polypeptide composition includes one or more of the second peptide set, and the second peptide set includes SEQ ID NO: 5, 9, 10, 12, 18, 19, 26, 31 , 34, 37, and 38.
  • the peptides in the second peptide collection show stronger sequence conservation of the coronavirus. The preparation of vaccines based on these polypeptides facilitates the acquisition of broad-spectrum vaccines against coronaviruses.
  • the polypeptide composition in addition to one or more of the first peptide set, also includes one or more polypeptides from the second peptide set.
  • Vaccines are prepared based on the peptides in the above two sets, in order to obtain vaccines with stronger immunogenicity against a variety of coronaviruses.
  • the polypeptide composition can also be formed according to the combination of T cell epitopes and B cell epitopes, which is convenient for enhancing the immune effect. Specifically, among the above 40 polypeptides, T cell epitopes or B cell epitopes can be distinguished based on multiple epitope prediction software.
  • the polypeptide composition includes polypeptides derived from the same protein and/or different proteins. More preferably, there are no more than two polypeptides derived from the same protein in the polypeptide composition. Further preferably, the polypeptide composition is selected from One of the following combinations:
  • Combination 1 SEQ ID NO: 28, SEQ ID NO: 6, SEQ ID NO: 13 and SEQ ID NO: 18;
  • Combination 2 SEQ ID NO: 27, SEQ ID NO: 14, SEQ ID NO: 5, and SEQ ID NO: 17;
  • Combination 3 SEQ ID NO: 32, SEQ ID NO: 4, SEQ ID NO: 10, and SEQ ID NO: 23;
  • Combination 4 SEQ ID NO: 25, SEQ ID NO: 3, SEQ ID NO: 34, and SEQ ID NO: 40;
  • Combination 5 SEQ ID NO: 30, SEQ ID NO: 8, SEQ ID NO: 37, and SEQ ID NO: 21;
  • Combination 6 SEQ ID NO: 2, SEQ ID NO: 11, SEQ ID NO: 33, and SEQ ID NO: 19;
  • Combination 7 SEQ ID NO: 1, SEQ ID NO: 15, SEQ ID NO: 12, and SEQ ID NO: 29;
  • Combination 8 SEQ ID NO: 26, SEQ ID NO: 35, SEQ ID NO: 38, and SEQ ID NO: 22;
  • Combination 9 SEQ ID NO: 31, SEQ ID NO: 36, SEQ ID NO: 16 and SEQ ID NO: 20;
  • Combination 10 SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 39, and SEQ ID NO: 24;
  • Combination 11 SEQ ID NO: 29, SEQ ID NO: 35, SEQ ID NO: 40, and SEQ ID NO: 20;
  • Combination 12 SEQ ID NO: 3, SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, SEQ ID NO: 29, SEQ ID NO: 33, and SEQ ID NO: 34.
  • polypeptide vaccine in order to further effectively control the infection of humans by coronaviruses, in a preferred embodiment of the present application, a polypeptide vaccine is provided.
  • the polypeptide vaccine includes SEQ ID NO: 1 to SEQ ID NO: 154 shown in Table 1. Any one or more of the peptides shown. These peptides can be used to rationally select specific peptides based on broad-spectrum and/or new coronavirus-specific peptides to form an effective peptide vaccine.
  • the polypeptide vaccine includes a polypeptide including at least any one of the peptides shown in SEQ ID NO: 1 to SEQ ID NO: 40; preferably, the polypeptide vaccine includes at least SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 31, SEQ ID NO: 35, and SEQ ID NO: 36.
  • the polypeptide vaccine includes one or more of the first peptide set, wherein the first peptide set includes SEQ ID NO: 1-4, 6-8, 11, 13-17, 20 -25, 27-30, 32-33, 35-36 and 39-40 peptides.
  • the peptides in the first peptide collection above show stronger sequence specificity of the new coronavirus.
  • the polypeptide vaccine includes one or more of the second peptide set, and the second peptide set includes SEQ ID NO: 5, 9, 10, 12, 18, 19, 26, 31, Peptides shown in 34, 37 and 38.
  • the peptides in the second peptide collection show stronger sequence conservation of the coronavirus.
  • the polypeptide vaccine in addition to one or more of the first peptide set, also includes one or more of the second peptide set.
  • Vaccines are prepared based on the peptides in the above two sets, in order to obtain vaccines with stronger immunogenicity against a variety of coronaviruses.
  • coronavirus broad-spectrum peptides to prepare vaccines is helpful for the development of universal vaccines against coronaviruses, so as to achieve the prevention of different coronavirus infections.
  • Vaccines prepared with peptides specific to the new coronavirus can specifically target the new coronavirus.
  • T cells T cells
  • epitopes from B cells it is also possible to combine epitopes derived from T cells and epitopes from B cells to form a polypeptide vaccine, so that the combined polypeptide vaccine is convenient to enhance the immune effect.
  • T cell epitopes or B cell epitopes can be distinguished based on multiple epitope prediction software.
  • the polypeptide vaccine includes polypeptides derived from different proteins. More preferably, there are no more than two polypeptides derived from the same protein in the polypeptide vaccine. Further preferably, the polypeptide vaccine is selected from any combination of the following polypeptides:
  • Combination 1 SEQ ID NO: 28, SEQ ID NO: 6, SEQ ID NO: 13 and SEQ ID NO: 18;
  • Combination 2 SEQ ID NO: 27, SEQ ID NO: 14, SEQ ID NO: 5, and SEQ ID NO: 17;
  • Combination 3 SEQ ID NO: 32, SEQ ID NO: 4, SEQ ID NO: 10, and SEQ ID NO: 23;
  • Combination 4 SEQ ID NO: 25, SEQ ID NO: 3, SEQ ID NO: 34, and SEQ ID NO: 40;
  • Combination 5 SEQ ID NO: 30, SEQ ID NO: 8, SEQ ID NO: 37, and SEQ ID NO: 21;
  • Combination 6 SEQ ID NO: 2, SEQ ID NO: 11, SEQ ID NO: 33, and SEQ ID NO: 19;
  • Combination 7 SEQ ID NO: 1, SEQ ID NO: 15, SEQ ID NO: 12, and SEQ ID NO: 29;
  • Combination 8 SEQ ID NO: 26, SEQ ID NO: 35, SEQ ID NO: 38, and SEQ ID NO: 22;
  • Combination 9 SEQ ID NO: 31, SEQ ID NO: 36, SEQ ID NO: 16 and SEQ ID NO: 20;
  • Combination 10 SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 39, and SEQ ID NO: 24;
  • Combination 11 SEQ ID NO: 29, SEQ ID NO: 35, SEQ ID NO: 40, and SEQ ID NO: 20;
  • Combination 12 SEQ ID NO: 3, SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, SEQ ID NO: 29, SEQ ID NO: 33, and SEQ ID NO: 34.
  • the quality of each polypeptide can be set reasonably according to the immunogenicity.
  • the mass of each polypeptide is 0.1-1 mg, more preferably 0.25-0.5 mg.
  • the polypeptide can be coupled to a carrier protein.
  • the polypeptide vaccine further includes a carrier protein, and polypeptides derived from different proteins are coupled with the carrier protein to form a polypeptide-carrier protein conjugate. After mixing the polypeptides of any combination according to a reasonable mass ratio, a polypeptide mixture is formed, and the polypeptide mixture is coupled with the same carrier protein at the same time, thereby obtaining a polypeptide-carrier protein conjugate coupled with multiple polypeptide sequences.
  • the specific type of carrier protein is not limited, including but not limited to bovine serum albumin, ovalbumin, keyhole limpet hemocyanin or casein.
  • the polypeptide can also be coupled to the carrier protein through a linking sequence, and the linking sequence is preferably CGSG.
  • the polypeptide vaccine is an injection, and preferably the injection further includes an adjuvant. More preferably, the volume of the adjuvant in the injection is equal to the volume of 50-100 ⁇ g polypeptide-carrier protein conjugate.
  • the polypeptide combination (mixture) or the conjugate formed by coupling the polypeptide combination and the carrier can be stored in the form of a solid powder before being mixed with an adjuvant to immunize the body.
  • immunization it can be formulated into a liquid, and an equal volume of adjuvant can be added to form an injection solution for immunization.
  • adjuvant can also be directly combined with adjuvant to prepare a vaccine in liquid form.
  • any peptide in the polypeptide vaccine is a modified peptide.
  • the modified peptide segment is to add 1-4 hydrophilic amino acids at the N-terminal, C-terminal or NC ends; preferably, the aqueous amino acid is Glu, Lys, Ser or Gly; preferably, 1-4 hydrophilic
  • the sex amino acid is selected from any one of the following: Glu-Glu, Lys-Lys or Ser-Gly-Ser.
  • any peptide in the polypeptide vaccine can be a cysteine modified peptide. Specifically, it includes but is not limited to adding cysteine to the N-terminal, C-terminal or NC ends of the peptide, or adding cysteine in the middle of the peptide chain of the peptide.
  • cysteine is added in the middle of the peptide chain of the peptide
  • one or more cysteine can be inserted in the middle of the peptide chain (that is, inserted between two amino acid residues), or one or more cysteine can be inserted into the middle of the peptide chain.
  • the acid is connected in the middle of the peptide chain in the form of a branch (that is, as a side chain of a certain amino acid in the middle of the peptide chain).
  • polypeptide in the above-mentioned polypeptide vaccine can be in the form of a single peptide or a combination of multiple peptides.
  • the above-mentioned polypeptide vaccine includes multiple peptide segments, and the multiple peptide segments exist in tandem.
  • At least One peptide segment is connected in series 1 to 5 times, preferably 1 to 3 times; more preferably, multiple peptide segments are connected in series via a connecting arm; further preferably, the connecting arm is selected from glycine, lysine, AEA ((2-Aminoethoxy) acetic acid, 2-aminoethoxyacetic acid), Ava(5-Aminovaleric Acid, 5-aminovaleric acid), ANP(3-Amino-3-(2-nitrophenyl)propanoic acid, 3-amino-3(2- Nitrobenzene) propionic acid), ⁇ -alanine, GABA (4-Aminobutyric Acid) or PEG (polyethylene glycol). Polyethylene glycol-linked peptides can not only improve solubility, but also protect the peptides from being cleaved by proteolytic enzymes and increase the half-life of biological activity.
  • AEA ((2-Aminoethoxy) acetic acid, 2-aminoeth
  • the application of any of the above-mentioned polypeptides in the preparation of a vaccine for the treatment of diseases caused by coronaviruses is also provided.
  • the coronavirus is SARS-CoV-2.
  • the vaccine includes any one of the peptides shown in SEQ ID NO: 1 to SEQ ID NO: 40; more preferably, includes SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 31 , SEQ ID NO: 35 and SEQ ID NO: 36.
  • the vaccine includes one or more of the first peptide set, wherein the first peptide set includes SEQ ID NO: 1-4, 6-8, 11, 13-17, 20-25 , 27-30, 32-33, 35-36 and 39-40.
  • the vaccine includes one or more of the second peptide set, and the second peptide set includes SEQ ID NO: 5, 9, 10, 12, 18, 19, 26, 31, 34, 37 And the peptide shown in 38.
  • the vaccine includes at least one of the first peptide set and at least one of the second peptide set.
  • the present application also provides a nucleic acid vaccine, which comprises a nucleic acid, which encodes any of the above-mentioned polypeptides or polypeptide compositions.
  • the nucleic acid vaccine may be a DNA vaccine or an RNA vaccine; more preferably, it is an mRNA vaccine.
  • the present application also provides a recombinant protein vaccine, the recombinant protein vaccine comprising any one or more peptides in SEQ ID NO: 1 to SEQ ID NO: 154.
  • the recombinant protein vaccine is any one or more peptides from SEQ ID NO: 1 to SEQ ID NO: 40, more preferably SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 31, SEQ ID NO: 35 and SEQ ID NO: 36 are recombined with 4-6 histidines or 4 Gly and 1 Ser (that is, 4 Gly and 1 Ser are connected in sequence) to form a protein vaccine.
  • a method for preventing a disease caused by a coronavirus comprises: administering a preventively effective amount of any of the above-mentioned polypeptide vaccines, gene vaccines or recombinant protein vaccines to the subject. Also provided is a treatment method for a disease caused by a coronavirus, the treatment method comprising: administering to a subject a therapeutically effective amount of any of the aforementioned antibodies.
  • the aforementioned coronavirus is SARS-CoV-2.
  • an antigen epitope screening method is provided. As shown in FIG. 1, the screening method includes:
  • Step S101 using all the proteomic sequences of the target coronavirus to perform epitope prediction to obtain the predicted epitope region;
  • Step S102 using peptide chip technology to screen out peptides with differential responses between the positive serum samples and the control serum samples infected with the target coronavirus, and record them as differential peptides;
  • Step S103 comparing the differential peptide segment with all the proteomic sequences of the target coronavirus to obtain the first conserved motif region;
  • Step S104 screening the regions that meet the epitope screening conditions from the predicted epitope region and the first conserved motif region to obtain the epitope; wherein, the epitope screening conditions include the non-phosphorylated region and/or the target coronavirus Extramembranous area.
  • Washing buffer means: adding 1ml tween-20 to 1L PBS, and mixing it is the washing buffer.
  • HRP-labeled goat anti-human IgM secondary antibody from Sigma, catalog number: A6907.
  • HRP-labeled goat anti-human IgG secondary antibody from Abcam, catalog number: ab97225.
  • the first part Take SARS-CoV-2 virus as an example to illustrate the method of screening epitopes (ie polypeptides).
  • the method for screening antigenic epitopes is usually based on the target protein sequence, using public software for bioinformatics prediction or selection based on existing knowledge.
  • the method adopted has the following improvements: 1) Use multiple software for comprehensive prediction and evaluation to avoid the deviation of a single software; 2) Use the Chinese population high-frequency HLA database to help select candidate regions suitable for the Chinese population ; 3) In the face of protein structure and function information is not clear, large-scale screening of the entire viral protein group sequence instead of selecting only specific protein sequences for screening in order to reduce the amount of calculation, so it is easy to find viral proteins All possible candidate areas on the group.
  • the screening method of this application innovatively uses peptide chip technology, which is specifically embodied in: 1) Adopting unique peptide chip technology (that is, using a large number of peptides synthesized on a silicon-based chip to bind to the antibody in the test sample. Partially obtain the immune characterization of the subject sample) Carry out real data high-throughput auxiliary screening of the peptides that are differentially expressed between the new coronary pneumonia and the control sample; 2) Find the differentially expressed peptides and compare them to the new coronavirus proteome sequence According to the improved method, determine the "high confidence conserved sites" (see step (3) for specific definitions), 3) Screen epitope candidate regions based on the motif of "high confidence conserved sites” .
  • this application uses multiple software to predict the sequence and region presented by T cells and B cells.
  • the SARS-Cov-2 protein sequence (GeneBank MN908947) was obtained from NCBI, with a total length of 9703 amino acids and a total of 10 open reading frames (ORF) in the genome.
  • the proteomic sequence was predicted for the affinity of MHC-1 and MHC-2, and the results of each software were combined and comprehensively evaluated based on the high-frequency HLA comparison results of Chinese people obtained from 85 experiments recorded in the Allele Frequency Net Database (AFND) database. 2391 highly reliable virus epitope regions.
  • the length of the filter is set to 10-15 AA.
  • the peptides of this length range have better affinity prediction effects when using software to predict the affinity of peptides.
  • peptides of this length have lower synthesis cost and less difficulty, and it is easy to obtain better purity (peptide length The longer, the higher the synthesis cost, the greater the difficulty of synthesis, and the lower the purity).
  • the length of the screening peptide is not limited to 10-15 AA, and in some other embodiments, it can also be set to 8-20, 8-18 or 8-16 AA.
  • differential peptides are obtained. By comparing the differential peptides to influenza virus, common cold-related virus and more coronavirus protein sequences, it is finally determined that these differential peptides are all coronavirus family sequence-related peptides.
  • the idea of screening is that the first step is to compare F and H to screen out the characteristics of peptides. Such characteristics correspond to the increase in the antibody concentration caused by the disease, but the antibodies found are not necessarily COVID-19-specific antibodies. It may be due to the increase in antibodies caused by lung infections and other factors; in the second step, by comparing F and T, it is possible to find antibodies specific to new coronary pneumonia compared with other lung diseases.
  • the V13 chip (produced by Health Tell, model P/N:600001 V13 Slides) was used for sample detection according to the standard process, and the signal value of 125,509 peptides of the V13 chip was obtained, and the signal value of each peptide was said to be a feature.
  • the value range is 0 ⁇ 65535, and the original data is converted by log10. We assume that new coronary pneumonia will cause an increase in specific antibody signals.
  • the above screening process can also be performed based on the original data of the signal value of the peptide.
  • the signal value of the positive serum sample and the signal value of the negative control serum sample are different, it is recorded as the first difference, and the positive serum is calculated at the same time.
  • the difference between the signal value of the sample and the signal value of other lung disease control serum samples is recorded as the second difference; all binding peptides that meet the first difference and the second difference while meeting the threshold are retained to obtain the target difference peptide part.
  • T test is a commonly used statistical method in the detection of differential protein expression. It evaluates whether a protein is differentially expressed in two samples by combining variable data between samples. But because the sample size is usually small, the estimation of the overall variance is not very accurate, so the test power of the T test will be reduced, and if the T test is used multiple times, the number of false positives will be significantly increased.
  • the above differential peptides were compared using BLASTp with the protein sequences of various pathogenic microorganisms such as coronavirus, influenza virus, common cold-related virus, pneumonia-related bacteria, mycoplasma and chlamydia published in existing databases: the results showed that 443 of them were Two different peptides can be directly compared to the new coronavirus protein group with a higher bit score, with a threshold of 14.
  • the comparison result of more than two overlapping comparison results is called a 2CCR region (2 The above polypeptide continuous coverage area), of which 861 differential peptides are located in the 2CCR region of the new coronavirus protein group. It shows that these differential signal peptides almost all come from the immune response related to the new coronavirus.
  • the above comparison score is obtained according to the rules of BLASTp.
  • BLASTp has multiple modes adapted to different scenarios.
  • the "short sequence alignment" mode is selected.
  • the threshold of 14 is our further screening (or verification) of the above 864 differential peptides that have different responses between patients with new coronary pneumonia and healthy people and other pneumonia patients. That is, the input is already the 864 differential peptides obtained in the previous step, of which 443 can be directly compared to the protein sequence of the new coronavirus (high score), which also shows that the results obtained by the screening method in the previous step are reliable of. For these 443 differential peptides, the high comparison score here proves to some extent that these differential peptides are indeed from the new coronavirus.
  • a 96-well plate is a detection unit. Make an experimental design before the start of the experiment. According to the number of test samples, the number of blank controls and the number of standard products, calculate the number of chips that need to be used and determine the chip number and sample layout.
  • a total of 4 chips are used, and the chip codes are 001752_01, 001752_02, 001752_03, and 001752_04, respectively.
  • Each chip is equipped with 2 standard products (std) and 1 blank control (blk), and the rest are test samples.
  • the 8 holes shown in bold are four samples with two replicates (ie F573 and F573', F574 and F574', F575 and F575', and F577 and F577'). The same number is the same sample. .
  • Standards, blank controls and test samples are randomly distributed on all chips used, as shown in Table 2.
  • Serum or plasma samples use 1% D-mannitol-containing PBST solution in a 96-well deep-well plate, after two 25-fold dilutions, to obtain a 625-fold diluted sample plate to be tested for use;
  • a 2nM fluorescent secondary antibody solution was prepared with a PBST solution containing 0.75% casein (Casein), 40 ⁇ L/well was added to the assay cassette, and placed on a constant temperature shaker and incubated for 1 hour with shaking.
  • the chips in the assay cassette are disassembled, cleaned, dried, and then assembled into an imaging cassette (imaging cassette), and placed into the ImageXpress micro4 imager of Molecular Device for scanning and imaging.
  • imaging cassette imaging cassette
  • ImageXpress micro4 imager of Molecular Device for scanning and imaging.
  • TIFF image file is obtained for each test sample, which is the original data.
  • the GPR5 file contains all the information of a sample and the fluorescence intensity information of all the features.
  • Control is calculated, and all peptides covering this amino acid are divided into two groups: match or mismatch with this amino acid, and judge the matched group and unmatched group peptide p If the p-value of the peptide in the matched group is significantly lower than that of the unmatched group (when comparing the distribution of p-values between the two groups, the "Wilcoxon signed rank test" is used, and the threshold for testing significance is P ⁇ 0.05), the amino acid at this position is judged as a highly reliable conservative site.
  • the region where 443 different peptides that can be directly mapped (Map) to the new coronavirus protein group is used as the motif region, and selected according to the high-confidence conservative sites, and finally a total of 136 motifs are obtained.
  • Align 864 differential peptides to the sequence of the coronavirus family take a single amino acid as a unit, and use the sites that cover this amino acid with a match rate of more than 75% as trusted conserved sites to obtain 350 motif regions.
  • coronavirus family includes 1,600 types of coronaviruses, some of which are listed as follows: Bat Hp-betacoronavirus/Zhejiang2013; Betacoronavirus England 1; Betacoronavirus Erinaceus/VMC/DEU/2012; Betacoronavirus HKU24; Bovinecoronavirus; Humancoronavirus HKU1; Humancoronavirus HKU1; Humancoronavirus HKU1; Humancoronavirus ;Middle East respiratory syndrome coronavirus; Murine hepatitis virus; Pip peptide chip rellus batcoronavirus HKU5; Rabbitcoronavirus HKU14; Ratcoronavirus Parker; Rousettus batcoronavirus; Rousettus batcoronavirus; Rousettus batcoronavirus HKU9; SARylvirusScoronavirus HKU9;
  • the peptide numbers, sequences and basic attributes are shown in Table 1, and the first 40 in Table 1 are selected as follow-up vaccine peptides. See Table 3 below for details.
  • Table 1 shows the physical and chemical properties of 154 polypeptide sequences (including the above 40 polypeptide sequences), and the species source of all sequences is SARS-CoV-2.
  • Table 3 shows the physical and chemical properties of 154 polypeptide sequences (including the above 40 polypeptide sequences), and the species source of all sequences is SARS-CoV-2.
  • S protein surface glycoprotein, also known as S protein
  • pp1ab orf1ab polyprotein
  • Membrane glycoprotein membrane glycoprotein, also known as M protein
  • Nucleocapsid phosphoprotein also known as N protein
  • the software predicts the epitope region and obtains 800 candidate epitope regions; screening these (ie, the above-mentioned combined 800 candidate epitope regions) the peptides of the V13 chip can cover and overlap with the 350 motif regions in (4) As a candidate for vaccine peptides, a total of 728 candidate regions were obtained.
  • BlastP comparison score (BitScore) is greater than 14 The number must be at least 3);
  • the accessibility and Beta corner scores are sorted from high to low; and then the best selection is based on the results of multiple comparisons.
  • 11 regions located in pp1ab are preferentially selected, of which 2 regions are specific to the new coronavirus, 9 regions are broad-spectrum coronaviruses; 19 of the S protein (12 regions are specific to the new coronavirus, 7 are coronavirus Virus broad-spectrum), 6 N protein (5 regions are specific for new coronavirus, 1 is coronavirus broad-spectrum), 2 M protein (specific) and 2 ORF7a (1 specific , 1 broad-spectrum), a total of 40 peptides (a total of 29 specific and 11 broad-spectrum, see Table 3 for details).
  • a step of removing regions containing mutations may also be included. This step is optional. In the case where the mutation type is determined to be contained, a certain mutation region may also be included.
  • the above comprehensive ranking based on the accessibility, beta turn, hydrophilicity, HT peptide coverage, and multiple comparison results of these 431 regions is based on the following considerations: Since the SARS-CoV-2 proteome has a total of 10 proteins, Among them, pplab protein is the longest, and its length is more than ten times that of other proteins (such as S/N protein, etc.). Therefore, most of these 431 regions are located in the sequence of pplab protein. Taking into account the different biological meanings of each protein, the regions of the few other proteins are also selected first. In addition to the mutations in 4 regions, the other screening indicators meet the requirements. Therefore, when considering the design of a vaccine against the mutant strains, these regions can also be used as candidate peptides.
  • the BLASTp comparison score between the sequence of the above-mentioned 728 candidate regions and the human proteome sequence is divided by the BLASTp comparison score between the sequence of the 728 candidate regions and the new coronavirus.
  • the threshold value of the obtained value is 0.8, that is, the removal is greater than 0.8 Candidate area.
  • the score is based on the degree of match.
  • BLASTp is a widely used comparison software provided by NCBI, and Bitscore is the score given by the software; similar software includes DIAMOND, Muscle, and ClustalW.
  • the candidate vaccine peptides are produced by chemical synthesis.
  • the peptide quality control requires that the HPLC-MS purity is above 98% and the endotoxin content is not higher than 1EU/mg to ensure that the peptides comply with animal experiments.
  • Polypeptide products that have passed quality control undergo biological verification and effectiveness screening through in vivo animal experiments.
  • To young healthy mice with complete immune system function subcutaneous injections are given and blood samples are drawn regularly for peptide chip detection, and the difference in peptide sequence signal strength of the specific peptide chip corresponding to the designed vaccine peptide is analyzed to evaluate the immunogenicity .
  • the mouse endpoint serum will also be used for live virus neutralization experiments (CPE method) to evaluate the neutralizing effect of antibodies in mice after immunization.
  • CPE method live virus neutralization experiments
  • Polypeptides synthesized based on this method can be used alone or in combination, or can be coupled with proteins, and can also be used as a compatibility with different reagents.
  • the specific scheme is illustrated by the following examples.
  • the 40 peptides shown in SEQ ID NO: 1 to SEQ ID NO: 40 obtained above were directly synthesized by a third-party company (unmodified).
  • Customized 8-12AA peptide formulations a total of 40 customized peptides are shown in the following table; each 50mg, divided into 5mg/piece of formulations, a total of 10; purity is greater than or equal to 98%, sterilized and freeze-dried under GMP cleanliness requirements.
  • Glu-Glu, Lys-Lys or Ser- Synthesis is performed after Gly-Ser.
  • the peptide in order to better achieve directional coupling, can be modified with cysteine, including but not limited to adding cysteine at the N-terminus, C-terminus, or both N and C ends at the same time. Or add cysteine in the middle of the peptide segment.
  • cysteine in the middle of the peptide chain of the peptide, one or more cysteine can be inserted in the middle of the peptide chain, or one or more cysteine can be branched in the middle of the peptide chain.
  • ICX_ID icx_16, 24, 32, 35, 37 (corresponding to SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 36, SEQ ID NO. 34 and SEQ ID NO. 21) for single peptide vaccine Validity verification.
  • the peptide powder synthesized under the above conditions is dissolved in PBS solution, and diluted to prepare a peptide solution with a final concentration of 2 mg/ml.
  • each group of mice was injected with 100 ⁇ g polypeptide on the 0th, 14th, and 28th day according to the grouping, and each mouse was injected 3 times with a total of 300ug polypeptide.
  • the peptide solution was mixed with an equal volume of adjuvant MF59 (AddaVax, Invivo Gen), and then administered by subcutaneous injection through the gastrocnemius muscle of the mouse's legs using a micro syringe.
  • the adjuvant-only group used the same method and frequency of administration as the peptide experimental group for the experiment.
  • the 35th day after the mice received immunization was used as the end point of the experiment. The mice were sacrificed, blood was collected, and the supernatant was separated and prepared.
  • mice serum is performed at 1:10, 1:20, 1:40, 1:80, 1:160, 1:320, 1:640, 1: 1280 serial dilutions.
  • the solution containing 100 TCID50 viruses and the serum of each dilution were mixed in equal amounts, and incubated in a 37°C water bath for 1 hour.
  • the incubated virus serum mixture was added to a 96-well culture plate pre-inoculated with vero cells, and cultured in an environment of 37°C and 5% CO 2. Cytopathic effect (CPE) was observed every day after inoculation, and the final result was judged on the 7th day.
  • CPE Cytopathic effect
  • the serum obtained from mice immunized with a single peptide can neutralize the new coronavirus.
  • the antibodies in the mice injected with icx_32 have the best neutralization effect, 40% of them produced neutralizing antibodies, the highest neutralization titer reached 1:640, and the geometric average neutralization titer was 1:160.
  • the 40 polypeptide powders were dissolved and diluted to a final concentration of 2 mg/ml polypeptide solution with PBS solution.
  • 50 ⁇ g of each peptide in each group combination was mixed to form a mixture containing 200 ⁇ g polypeptide as the peptide solution for the first injection of mice.
  • 25 ⁇ g of each polypeptide was mixed to form a 100 ⁇ g mixture as a polypeptide solution for the second and third injections of mice.
  • Each group of mice was injected with the first, second, and third injections respectively on the 0th, 7th, and 14th day according to the grouping.
  • the peptide solution was mixed with the equal volume of the adjuvant Imject Alum Adjuvant (Thermo Fisher Scientific), and then the micro-syringe was used for injection.
  • the gastrocnemius muscles of the mice's legs were administered by subcutaneous injection.
  • the adjuvant-only group used the same method and frequency of administration as the peptide experimental group for the experiment.
  • the peptide chip detection technology is used to detect the blood samples of patients with new crown infection, patients with new crown pneumonia cured and uninfected healthy people, and comparative analysis to obtain the immune characteristics of new crown virus-specific antibodies based on peptide chips and corresponding analysis Model.
  • the mouse serum sample was detected by peptide chip detection.
  • the experiment used 10 ⁇ L mouse serum sample, which was initially incubated and combined with the chip, and then anti-mouse IgG antibody and fluorescent antibody were successively added for incubation and combination. After the incubation is completed, the sample will be loaded into the imager to perform fluorescence signal imaging and extract the characteristic fluorescence intensity value. Standardize the original fluorescence intensity, obtain a data matrix, and compare and analyze the pre-experimental data to identify whether the characteristics of the peptide binding site on the mouse serum and the peptide chip are the characteristics of the new crown-specific antibody.
  • Pattern 1 Continuously increasing sequence at any time (Pattern1)
  • Mode 2 Sequence rising at any time and remain stable after D13 (Pattern2)
  • Pattern2 Sequence rising at any time and remain stable after D13
  • step 4 The two peptide chip peptide collections obtained in step 2 and step 3 are subjected to Fisher's exact test.
  • Figure 3A shows the combination 1
  • Figure 3B shows the antibody signals corresponding to the four polypeptides in combination 2
  • Figure 3C shows the antibody signals corresponding to the four polypeptides in combination 3. It can be seen that the three combinations can stimulate the immunity of mice and increase the level of antibodies in the body.
  • the antibody signal corresponding to the peptide vaccine has a certain degree of increase at different time points in the three combinations.
  • mice serum is performed at 1:10, 1:20, 1:40, 1:80, 1:160, 1:320, 1:640, 1: 1280 serial dilutions.
  • the solution containing 100 TCID50 viruses and the serum of each dilution were mixed in equal amounts, and incubated in a 37°C water bath for 1 hour.
  • the incubated virus serum mixture was added to a 96-well culture plate pre-inoculated with vero cells, and cultured in a 37°C, 5% CO 2 environment. Cytopathic effect (CPE) was observed every day after inoculation, and the final result was judged on the 7th day.
  • CPE Cytopathic effect
  • the serum obtained from mice immunized with polypeptides can all have neutralizing activity against the new coronavirus.
  • the antibodies in mice injected with combination 2 and combination 3 have a better neutralizing effect.
  • 75% produced neutralizing antibodies, the highest neutralizing titer reached 1:640, and the geometric average neutralizing titer was 1:403.
  • 50% produced neutralizing antibodies, the highest neutralizing titer reached 1:1280, and the geometric average neutralizing titer was 1:640.
  • the trend between the neutralization effect groups is consistent with the score in 2.2c) Table 8. The results proved that the new coronavirus vaccine peptide assisted by the peptide chip design can neutralize the new coronavirus.
  • Hemocyanin keyhole limpet hemocyanin, KLH
  • KLH keyhole limpet hemocyanin
  • reaction buffer pH 6.0
  • 0.1M-MES and 0.5M-NaCl dilute KLH to 1mg/mL with reaction buffer, and take 1mL for use.
  • mice Female Balb/c mice aged 5 to 6 weeks were randomly divided into 12 groups, of which 10 groups were peptide-KLH experimental groups (combinations 1-10), group 1 was a separate KLH group with unconjugated peptides, and group 1 was simple Adjuvant group, 5 mice in each group.
  • the 40 polypeptide powders were dissolved and diluted to a final concentration of 2 mg/ml polypeptide solution with PBS solution.
  • 50 ⁇ g of each peptide in each combination was mixed to form a mixture containing 200 ⁇ g of peptide as the peptide solution for the first injection of mice. Thereafter, in each combination Take 25 ⁇ g of each polypeptide and mix it to form 100 ⁇ g of the mixture as the polypeptide solution for the second and third injections of mice.
  • Each group of mice was injected with the first, second, and third injections respectively on the 0th, 7th, and 14th day according to the grouping.
  • the peptide solution was mixed with the equal volume of the adjuvant Imject Alum Adjuvant (Thermo Fisher Scientific), and then the micro-syringe was used for injection.
  • the gastrocnemius muscles of the mice's legs were administered by subcutaneous injection.
  • the KLH single control group was injected with an equal amount of KLH and an equal volume of Imject Alum Adjuvant with the peptide-KLH group for injection, while the simple adjuvant group only used the same volume of Imject Alumn original concentration solution as the final solution of the peptide experimental group for injection.
  • the KLH single control group and the simple adjuvant group used the same method and frequency of administration as the polypeptide experimental group for experiments.
  • the polypeptide chip detection method consistent with that in the second embodiment is used to detect the immunized mouse serum.
  • the vaccine immunogenicity scoring system based on peptide chip evaluates 10 combinations.
  • the evaluation method is the same as that in the second implementation.
  • the evaluation results are shown in the following table:
  • ICX ID: icx_16 (SEQ ID NO: 20), 21 (SEQ ID NO: 29), 24 (SEQ ID NO: 22), 32 (SEQ ID NO: 36), 33 (SEQ ID NO: 3), 35 (SEQ ID NO: 34) and 37 (SEQ ID NO: 21) are combined into 7 peptides and different adjuvants for compatibility screening and verification.
  • AddaVax also denoted as MF59, InvivoGen
  • Imject Alumn Thermo Scientific
  • Alhydrogel InvivoGen
  • Adju-Phos InvivoGen
  • Novavax also denoted as MA103A, Mai Ke Kang
  • MA103B also recorded as positively charged, Mai Ke Kang
  • female Balb/c mice aged 5 to 6 weeks were randomly divided into 12 groups, of which 6 groups were experimental groups combining 7 peptides and different adjuvants, and 6 groups were simple adjuvant groups, with 5 mice in each group.
  • the 7 peptide powders were dissolved and diluted to a peptide solution with a final concentration of 2 mg/ml with PBS solution.
  • 30 ⁇ g of each polypeptide in each group combination was mixed to form a mixture containing 210 ⁇ g polypeptide as the polypeptide solution for the first, second, and third injections of mice.
  • the mice of each group were injected with the first, second and third injections respectively on the 0th, 7th and 14th day according to the grouping.
  • the peptide solution was mixed with the corresponding adjuvant of equal volume, and then the mice were subcutaneously passed through the gastrocnemius muscles of the legs with a micro-injector.
  • the adjuvant-only group used the same method and frequency of administration as the peptide experimental group for the experiment.
  • Blood samples from the tail vein of mice were collected before the immunization of mice in each experimental group (day 0) and on the 7th and 14th days after the initial immunization (days 7 and 14 were before injection).
  • the amount of blood samples collected at each time point was approximately 100-200 ⁇ L, separate and prepare serum for peptide chip detection.
  • the 21st day after the mice were immunized was used as the end of the experiment. The mice were sacrificed, blood was collected and the supernatant was separated and used for the neutralization experiment.
  • the polypeptide chip detection method consistent with that in the second embodiment is used to detect the immunized mouse serum.
  • NA means: not applicable, not applicable.
  • the vaccine immunogenicity scoring system based on peptide chip evaluates each combination, and the evaluation results are shown in the following table:
  • the adjuvants MA103A and MA103B have a better compatibility with 7 peptides. Therefore, 7 peptides and MA103A or MA103B have the potential to be compatible with vaccine formulations.
  • the data processing part of the technical solution of the present application can be embodied in the form of a software product, and the computer software product can be stored in a storage medium, such as ROM/RAM, magnetic disk, optical disk, etc., including several instructions.
  • a computer device which may be a personal computer, a server, or a network device, etc. executes the various embodiments of the application or the methods of some parts of the embodiments.
  • FIG. 7 is a block diagram of the hardware structure of the terminal of a method for screening an epitope polypeptide according to an embodiment of the present invention.
  • the terminal may include one or more (only one is shown in FIG. 7) processor 102 (the processor 102 may include, but is not limited to, a processing device such as a microprocessor MCU or a programmable logic device FPGA) and The memory 104 is used to store data.
  • the aforementioned terminal may further include a transmission device 106 and an input/output device 108 for communication functions.
  • FIG. 7 is only for illustration, and it does not limit the structure of the foregoing terminal.
  • the terminal may also include more or fewer components than shown in FIG. 7, or have a different configuration from that shown in FIG. 7.
  • the memory 104 may be used to store computer programs, for example, software programs and modules of application software, such as the computer programs corresponding to the method for determining the formula of intestinal probiotic supplements in the embodiment of the present invention, and the processor 102 is stored in the memory 104 by running The computer program to perform various functional applications and data processing, that is, to achieve the above-mentioned methods.
  • the memory 104 may include a high-speed random access memory, and may also include a non-volatile memory, such as one or more magnetic storage devices, flash memory, or other non-volatile solid-state memory.
  • the memory 104 may further include a memory remotely provided with respect to the processor 102, and these remote memories may be connected to the terminal through a network. Examples of the aforementioned networks include, but are not limited to, the Internet, corporate intranets, local area networks, mobile communication networks, and combinations thereof.
  • the transmission device 106 is used to receive or send data via a network.
  • the above-mentioned specific examples of the network may include a wireless network provided by the communication provider of the terminal.
  • the transmission device 106 includes a network adapter (Network Interface Controller, NIC for short), which can be connected to other network devices through a base station so as to communicate with the Internet.
  • the transmission device 106 may be a radio frequency (Radio Frequency, referred to as RF) module, which is used to communicate with the Internet in a wireless manner.
  • RF Radio Frequency
  • This embodiment provides a screening device for epitope polypeptides. As shown in FIG. 8, the screening device includes:
  • the epitope prediction module 10 is used to perform antigen epitope prediction using all the proteomic sequences of the target coronavirus to obtain the predicted epitope region;
  • the differential peptide screening module 30 is used to screen out peptides that have a differential response between the positive serum sample and the control serum sample infected by the target coronavirus by using peptide chip technology, and record them as differential peptides;
  • the first region screening module 50 is used to compare the differential peptide fragments with all the proteomic sequences of the target coronavirus to obtain the first conserved motif region;
  • the third region screening module 70 is used to screen regions that can meet the following epitope screening conditions from the predicted epitope region and the first conserved motif region to obtain the epitope polypeptide:
  • the epitope screening conditions include the non-phosphorylated region and/or the extramembrane region of the target coronavirus.
  • the epitope prediction module includes: a first screening module for candidate epitopes, which is used to use all the proteomic sequences of the target coronavirus to predict epitopes by using a variety of different methods, and the screening length is 8-20, preferably 10-15 amino acid epitopes to obtain candidate predicted epitopes; the second candidate epitope screening module is used to predict candidate epitopes based on the epitopes and/or hydrophobicity that can be presented by HLA in a specific population After screening, the predicted epitope region is obtained.
  • a first screening module for candidate epitopes which is used to use all the proteomic sequences of the target coronavirus to predict epitopes by using a variety of different methods, and the screening length is 8-20, preferably 10-15 amino acid epitopes to obtain candidate predicted epitopes
  • the second candidate epitope screening module is used to predict candidate epitopes based on the epitopes and/or hydrophobicity that can be presented by HLA in a specific population
  • the second screening module for candidate epitopes includes: a population epitope screening module, which is used to screen out the epitopes that can be presented by HLA in the Chinese population from the candidate predicted epitopes; and/or the hydrophobic screening module, using To remove the epitope whose hydrophobicity is higher than the first hydrophobic threshold from the candidate predicted epitope, to obtain the predicted epitope region; preferably, the epitope whose hydrophobicity is higher than the first hydrophobic threshold means that the proportion of hydrophobic amino acids is greater than 45% and Epitopes with a hydrophobicity score greater than 3.
  • the differential peptide screening module includes: the first screening module includes: a sample selection unit for selecting positive serum samples, negative control serum samples, and other lung disease control serum samples for the target coronavirus infection, and other lung disease indicators Purpose Pulmonary diseases caused by virus infections other than coronavirus; signal acquisition unit, used to adopt the method of polypeptide chip, combine positive serum samples, negative control serum samples and other lung disease control serum samples with polypeptide array chip to obtain The signal value of the binding peptide response; the differential peptide screening unit is used to calculate the p value when there is a difference between the signal value of the positive serum sample and the signal value of the negative control serum sample for each binding peptide, which is recorded as the first A p value, and calculate the p value when the signal value of the positive serum sample is different from the signal value of the control serum sample of other lung diseases, and record it as the second p value; keep all the first p value and the second p value at the same time The binding peptides that meet the difference threshold are combined to obtain
  • the differential peptide screening unit includes: a signal conversion subunit for log10 conversion of the signal value of the bound peptide; Test, calculate the p value of each feature when there is a difference between the positive serum sample and the negative control serum sample, and perform multiple hypothesis test correction on the p value to obtain the first p value; at the same time, calculate the corresponding feature in the positive serum sample and other The p value when there is a difference between the lung disease control serum samples, and the p value is corrected by multiple hypothesis testing, and it is recorded as the second p value; the first p value is less than the difference threshold and the second p value is less than the difference threshold at the same time. Binding peptides to obtain differential peptides.
  • the first region screening module includes: a conservative site screening module, which is used to calculate the signal value of the binding peptide that covers the amino acid and matches the amino acid with a single amino acid as a unit, and there is a difference between the positive serum sample and the negative control serum sample. At the same time, calculate the distribution of the p2 value of the positive serum sample and the negative control serum sample for the signal value of the binding peptide that covers the amino acid and does not match the amino acid.
  • a conservative site screening module which is used to calculate the signal value of the binding peptide that covers the amino acid and matches the amino acid with a single amino acid as a unit, and there is a difference between the positive serum sample and the negative control serum sample.
  • the distribution of the p1 value is significantly lower than the distribution of the p2 value of amino acids It is the first conserved site; the first conserved motif screening module is used to compare the different peptides with the entire proteomic sequence of the target coronavirus, and select the first conserved site from the regions that can be compared And the hydrophobicity is lower than the second hydrophobic threshold area, so as to obtain the first conserved motif region; preferably, the hydrophobicity lower than the second hydrophobic threshold area means that the proportion of hydrophobic amino acids is less than or equal to 45% and the hydrophobicity score is less than or equal to The region of 3; preferably the differential peptide is a differential peptide that can be completely aligned to the entire proteomic sequence of the target coronavirus.
  • the screening device further includes a second region screening module.
  • the second region screening module includes: an alignment module for comparing the differential peptides with the protein sequence of the coronavirus family, and the second conservative motif screening The module is used to select the region where each amino acid position meets the following regional screening conditions from the regions that can be compared, as the second conserved motif region: the differential peptide that can match the amino acid among all the differential peptides covering amino acids The ratio of the segment meets the matching ratio threshold.
  • the matching ratio threshold is greater than or equal to 75%.
  • the epitope screening conditions in the third region screening module 50 further include at least one of the following: (a) overlap with the second conserved motif region; (b) the comparison score with the human proteome sequence is lower than the comparison Threshold; (c) Meet the following performance indicators: 1) The number of different peptides covered is ⁇ 3; 2) Hydrophilicity meets the hydrophilic threshold; 3) Accessibility score, Beta turn angle and multiple comparison scores are all Located in the top 100; where the comparison score is lower than the comparison threshold means a/b ⁇ 0.8, where a is the matching score of the sequence of the region to be screened and the human proteome sequence, and b is the sequence of the region to be screened The matching score of the alignment with all the proteomic sequences of the target coronavirus.
  • the third region screening module includes: a merging module for merging the predicted epitope region and the first conserved motif region according to at least one of the following merging conditions: 1) the two regions have an inclusion relationship; 2) two The region is predicted to be an epitope region by at least two different methods, and the first candidate epitope region is obtained; the overlap screening module is used to screen the region that overlaps the second conserved motif region from the first candidate epitope region as the first candidate epitope region.
  • a merging module for merging the predicted epitope region and the first conserved motif region according to at least one of the following merging conditions: 1) the two regions have an inclusion relationship; 2) two The region is predicted to be an epitope region by at least two different methods, and the first candidate epitope region is obtained; the overlap screening module is used to screen the region that overlaps the second conserved motif region from the first candidate epitope region as the first candidate epitope region.
  • Two candidate epitope regions Two candidate epitope regions; an alignment screening module, used to screen the region from the second candidate epitope region whose comparison score with the human proteome sequence is lower than the first threshold, as the third candidate epitope region; non-phosphorylated And extramembrane region screening module, used to screen and retain the non-phosphorylated region and extramembrane region in the target coronavirus proteomic sequence from the third candidate epitope region, as the fourth candidate epitope region; integrated sorting module, used After comprehensively sorting the fourth candidate epitope region according to the accessibility, beta turn, hydrophilicity, the number of different peptides, and the multiple comparison results, the epitope polypeptide of the target coronavirus is obtained.
  • the device further includes a mutation removal module, which is used to remove regions containing mutations from the regions selected after the comprehensive ranking module to obtain the epitope polypeptide of the target coronavirus.
  • a mutation removal module which is used to remove regions containing mutations from the regions selected after the comprehensive ranking module to obtain the epitope polypeptide of the target coronavirus.
  • the target coronavirus is SARS-CoV-2.
  • a storage medium includes a stored program, wherein the device where the storage medium is located is controlled to perform any of the above-mentioned methods for screening epitope polypeptides when the program is running.
  • a processor is provided, and the processor is used to run a program, wherein any one of the above-mentioned methods for screening epitope polypeptides is executed when the program is running.
  • This application can be used in many general or special computing system environments or configurations. For example: personal computers, server computers, handheld devices or portable devices, tablet devices, multi-processor systems, microprocessor-based systems, set-top boxes, programmable consumer electronic devices, network PCs, small computers, large computers, including Distributed computing environment for any of the above systems or equipment, etc.
  • modules or steps of this application can be implemented in a general computing device, and they can be concentrated on a single computing device or distributed on a network composed of multiple computing devices.
  • they can be implemented with program codes executable by a computing device, so that they can be stored in a storage device for execution by the computing device, or they can be made into individual integrated circuit modules, or Multiple modules or steps are made into a single integrated circuit module to achieve. In this way, this application is not limited to any specific combination of hardware and software.
  • the present invention has at least the following beneficial effects:
  • a batch of peptides specifically related to coronavirus infection are obtained through innovative screening of peptide chip technology, and these peptides can be used to prepare antigens and antibodies.
  • Relevant testing reagents such as, kits, and related vaccine products such as peptide vaccines, nucleic acid vaccines, protein recombinant vaccines, etc., so as to provide more powerful tools for preventing and controlling the infection and prevalence of such viruses.

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Abstract

一种抗原表位多肽的筛选方法及装置。该筛选方法包括：利用目的冠状病毒的全部蛋白组序列进行抗原表位预测，得到预测表位区域；利用多肽芯片技术筛选出对目的冠状病毒感染的阳性血清样本与对照血清样本中存在差异响应的多肽，记为差异肽段；将差异肽段与目的冠状病毒的全部蛋白组序列进行比对，获得第一保守基序区域；从预测表位区域和第一保守基序区域中筛选出满足表位筛选条件的区域，得到抗原表位；其中，表位筛选条件包括位于目的冠状病毒的非磷酸化区域和/或膜外区域。通过利用成本低、通量高的多肽芯片技术筛选获得一批与冠状病毒感染特异性相关的多肽，为此类病毒的防控提供更有力的多肽产品。

Description

抗原表位多肽的筛选方法及装置 技术领域
本发明涉及分子免疫学领域，具体而言，涉及一种抗原表位多肽的筛选方法及装置。
背景技术
由新型冠状病毒“SARS-CoV-2”(Severe Acute Respiratory Syndrome Coronavirus 2)感染所致的新型冠状病毒肺炎(Corona Virus Disease 2019，COVID-19)目前正在全球肆虐。截止到2020年12月11日，在全球范围内SARS-CoV-2感染病例已达70714214例，其中死亡病例为1588277例。由于疫情发展迅速，而且尚未发现特效药，故以预防感染为目的的新冠特异性疫苗成为降低感染率、抑制疫情恶化的希望。
传统疫苗包括减毒活疫苗、灭活疫苗等，制备过程需要使用病毒株，虽然免疫原性较高但存在病毒返祖的可能性，具有潜在致病危险，故安全性相对较低。近年多种新型疫苗包括DNA重组疫苗及合成多肽疫苗等陆续出现。但由于DNA重组疫苗常用的载体为腺病毒、痘苗病毒、SV40病毒等，目前对于该类载体的体内安全性仍存在一定疑虑，故对开发更具安全性的新一代疫苗依然存在很大的需求。多肽疫苗是依照病原体抗原基因中已知或预测的某段抗原表位的氨基酸序列，通过化学合成方法制备的疫苗。多肽疫苗由化学合成，不存在毒力回升或灭活不全的问题，且可以选定特定的抗原表位，故成为现今疫苗开发的热门研究点，在包括肿瘤疫苗内的多个领域，已有多项研究发表，有关临床试验也在进行中。
如前述，针对目前全球大流行的新型冠状病毒肺炎，急需要开发出相应的疫苗，尤其是多肽疫苗。
发明内容
本发明的主要目的在于提供一种抗原表位多肽的筛选方法及装置，以提供针对该类新型病毒的多肽进而开发相应的多肽产品。
根据本申请的第一个方面，提供了一种冠状病毒抗原表位的筛选方法，该筛选方法包括：利用目的冠状病毒的全部蛋白组序列进行抗原表位预测，得到预测表位区域；利用多肽芯片技术筛选出对目的冠状病毒感染的阳性血清样本与对照血清样本中存在差异响应的肽段，记为差异肽段；将差异肽段与目的冠状病毒的全部蛋白组序列进行比对，获得第一保守基序区域；从预测表位区域和第一保守基序区域中筛选出满足表位筛选条件的区域，得到抗原表位；其中，表位筛选条件包括位于目的冠状病毒的非磷酸化区域和/或膜外区域。
进一步地，利用目的冠状病毒的全部蛋白组序列进行抗原表位预测，得到预测表位区域包括：利用目的冠状病毒的全部蛋白组序列，采用多种不同方法进行抗原表位预测，并筛选长度为8～20个，优选10～15个氨基酸的表位，得到候选预测表位；根据特定人群中的HLA 所能递呈的表位和/或亲疏水性，对候选预测表位进行筛选，得到预测表位区域；优选地，从候选预测表位中筛选出中国人群中HLA所能递呈的表位，和/或从候选预测表位中去除疏水性高于第一疏水阈值的表位，得到预测表位区域；优选地，疏水性高于第一疏水阈值的表位指疏水性氨基酸占比大于45％且疏水性得分大于3的表位。
进一步地，利用免疫表征的方法筛选出对目的冠状病毒感染的阳性血清样本与对照血清样本存在差异响应的肽段，记为差异肽段包括：选取目的冠状病毒感染的阳性血清样本、阴性对照血清样本和其他肺部疾病对照血清样本，其他肺部疾病指目的冠状病毒之外的病毒感染引起的肺部疾病；采用免疫表征的方法，将阳性血清样本、阴性对照血清样本和其他肺部疾病对照血清样本与多肽阵列芯片结合，获得结合肽段响应的信号值；针对每个结合肽段，计算阳性血清样本的信号值与阴性对照血清样本的信号值之间存在差异时的p值，记为第一p值，同时计算阳性血清样本的信号值与其他肺部疾病对照血清样本的信号值存在差异时的p值，记为第二p值；保留所有符合第一p值和第二p值同时满足差异阈值的结合肽段，从而得到差异肽段；优选差异阈值为<0.05。
进一步地，对结合肽段的信号值进行log10转换，以转换后的log值为特征，通过单尾T检验，计算各特征在阳性血清样本与阴性对照血清样本之间存在差异时的p值，并对p值进行多重假设检验校正，得到第一p值；同时计算相应的特征在阳性血清样本与其他肺部疾病对照血清样本之间存在差异时的p值，并对p值进行多重假设检验校正，记为第二p值；筛选同时满足第一p值小于差异阈值且第二p值小于差异阈值的结合肽段，从而得到差异肽段。
进一步地，将差异肽段与目的冠状病毒的全部蛋白组序列进行比对，获得第一保守基序区域包括：以单个氨基酸为单位，计算覆盖氨基酸且与氨基酸匹配的结合肽段的信号值在阳性血清样本与在阴性对照血清样本和其他肺部疾病对照血清样本存在差异的p1值的分布，同时计算覆盖氨基酸且与氨基酸不匹配的结合肽段的信号值在阳性血清样本与在阴性对照血清样本和其他肺部疾病对照血清样本存在差异的p2值的分布，p1值的分布显著低于p2值的分布氨基酸为第一保守位点；将差异肽段与目的冠状病毒的全部蛋白组序列进行比对，并从能够比对上的区域中选择具有第一保守位点且疏水性低于第二疏水阈值的区域，从而获得第一保守基序区域；优选地，疏水性低于第二疏水阈值的区域指疏水性氨基酸占比小于等于45％且疏水性得分小于等于3的区域；优选差异肽段为能够完全比对到目的冠状病毒的全部蛋白组序列上的差异肽段。
进一步地，在从预测表位区域和第一保守基序区域中筛选出满足表位筛选条件的区域之前，筛选方法还包括：将差异肽段与冠状病毒家族的蛋白序列进行比对，获得第二保守基序区域；优选地，将差异肽段与冠状病毒家族的蛋白序列进行比对，获得第二保守基序区域包括：将差异肽段与冠状病毒家族的蛋白序列进行比对，并从能够比对上的区域中选择各氨基酸位点均满足如下区域筛选条件的区域，作为第二保守基序区域：覆盖氨基酸的所有差异肽段中，能够与氨基酸匹配的差异肽段的比例满足匹配比例阈值；优选地，匹配比例阈值大于等于75％。
进一步地，表位筛选条件还包括如下至少之一：(a)与第二保守基序区域相重叠；(b)与人类蛋白组序列比对得分低于比对阈值；(c)满足如下多个性能指标：1)差异肽段覆盖条数≥3；2)亲水性满足亲水阈值；3)可及性分值、Beta转角及多重比对分值均位于前100；比对得分低于比对阈值指a/b≤0.8，其中，a为待筛选区域的序列与人类蛋白组序列进行比对的匹配度得分，b为待筛选区域的序列与目的冠状病毒的全部蛋白组序列进行比对的匹配度得分；优选地，从预测表位区域和第一保守基序区域中筛选出满足表位筛选条件的区域，得到抗原表位包括：将预测表位区域和第一保守基序区域根据如下至少之一的合并条件进行合并：1)两区域间为包含关系；2)两区域至少被两种不同方法预测为抗原表位区域，得到第一候选表位区域；从第一候选表位区域中筛选与第二保守基序区域重叠的区域，作为第二候选表位区域；从第二候选表位区域中筛选与人类蛋白组序列的比对得分低于比对阈值的区域，作为第三候选表位区域；从第三候选表位区域中筛选并保留目的冠状病毒蛋白组序列中的非磷酸化区域和/或膜外区域，作为第四候选表位区域；根据可及性、beta转角、亲水性、差异肽段的覆盖条数及多重比对结果，对第四候选表位区域进行综合排序后择优选择，得到目的冠状病毒的抗原表位；更优选地，在择优选择之后，筛选方法还包括去除包含突变的区域；优选地，目的冠状病毒为SARS-CoV-2。
根据本申请的第十四个方面，提供了一种冠状病毒抗原表位的筛选装置，该筛选装置包括：表位预测模块，用于利用目的冠状病毒的全部蛋白组序列进行抗原表位预测，得到预测表位区域；差异肽段筛选模块，用于利用多肽芯片技术筛选出对目的冠状病毒感染的阳性血清样本与对照血清样本存在差异响应的肽段，记为差异肽段；第一区域筛选模块，用于将差异肽段与目的冠状病毒的全部蛋白组序列进行比对，获得第一保守基序区域；第三区域筛选模块，用于从预测表位区域和第一保守基序区域中筛选能够满足表位筛选条件的区域，得到抗原表位：其中，表位筛选条件包括位于目的冠状病毒的非磷酸化区域和/或膜外区域。
进一步地，表位预测模块包括：候选表位第一筛选模块，用于利用目的冠状病毒的全部蛋白组序列，采用多种不同方法进行抗原表位预测，并筛选长度为8～20个，优选10～15个氨氨基酸的表位，得到候选预测表位；候选表位第二筛选模块，用于根据特定人群中的HLA所能递呈的表位和/或亲疏水性，对候选预测表位进行筛选，得到预测表位区域。
进一步地，候选表位第二筛选模块包括：人群表位筛选模块，用于从候选预测表位中筛选出中国人群中HLA所能递呈的表位；和/或，疏水性筛选模块，用于从候选预测表位中去除疏水性高于第一疏水阈值的表位，得到预测表位区域；优选地，疏水性高于第一疏水阈值的表位指疏水性氨基酸占比大于45％且疏水性得分大于3的表位。
进一步地，差异肽段筛选模块包括：第一筛选模块包括：样本选择单元，用于选取目的冠状病毒感染的阳性血清样本、阴性对照血清样本和其他肺部疾病对照血清样本，其他肺部疾病指目的冠状病毒之外的病毒感染引起的肺部疾病；信号获取单元，用于采用免疫表征的方法，将阳性血清样本、阴性对照血清样本和其他肺部疾病对照血清样本与多肽阵列芯片结合，获得结合肽段响应的信号值；差异肽段筛选单元，用于针对每个结合肽段，计算阳性血清样本的信号值与阴性对照血清样本的信号值之间存在差异时的p值，记为第一p值，同时计算阳性血清样本的信号值与其他肺部疾病对照血清样本的信号值存在差异时的p值，记为 第二p值；保留所有符合第一p值和第二p值同时满足差异阈值的结合肽段，从而得到差异肽段；优选差异阈值为<0.05。
进一步地，差异肽段筛选单元包括：信号转换子单元，用于对结合肽段的信号值进行log10转换；差异肽段筛选子单元，用于以转换后的log值为特征，通过单尾T检验，计算各特征在阳性血清样本与阴性对照血清样本之间存在差异时的p值，并对p值进行多重假设检验校正，得到第一p值；同时计算相应的特征在阳性血清样本与其他肺部疾病对照血清样本之间存在差异时的p值，并对p值进行多重假设检验校正，记为第二p值；筛选同时满足第一p值小于差异阈值且第二p值小于差异阈值的结合肽段，从而得到差异肽段。
进一步地，第一区域筛选模块包括：保守位点筛选模块，用于以单个氨基酸为单位，计算覆盖氨基酸且与氨基酸匹配的结合肽段的信号值在阳性血清样本和阴性对照血清样本存在差异的p1值的分布，同时计算覆盖氨基酸且与氨基酸不匹配的结合肽段的信号值在阳性血清样本和阴性对照血清样本存在差异的p2值的分布，p1值的分布显著低于p2值的分布氨基酸为第一保守位点；第一保守基序筛选模块，用于将差异肽段与目的冠状病毒的全部蛋白组序列进行比对，并从能够比对上的区域中选择具有第一保守位点且疏水性低于第二疏水阈值的区域，从而获得第一保守基序区域；优选地，疏水性低于第二疏水阈值的区域指疏水性氨基酸占比小于等于45％且疏水性得分小于等于3的区域；优选差异肽段为能够完全比对到目的冠状病毒的全部蛋白组序列上的差异肽段。
进一步地，筛选装置还包括第二区域筛选模块，优选地，第二区域筛选模块包括：比对模块，用于将差异肽段与冠状病毒家族的蛋白序列进行比对，第二保守基序筛选模块，用于从能够比对上的区域中选择各氨基酸位点均满足如下区域筛选条件的区域，作为第二保守基序区域：覆盖氨基酸的所有差异肽段中，能够与氨基酸匹配的差异肽段的比例满足匹配比例阈值。
进一步地，匹配比例阈值大于等于75％。
进一步地，第三区域筛选模块50中的表位筛选条件还包括如下至少之一：(a)与第二保守基序区域相重叠；(b)与人类蛋白组序列比对得分低于比对阈值；(c)满足如下多个性能指标：1)差异肽段覆盖条数≥3；2)亲水性满足亲水阈值；3)可及性分值、Beta转角及多重比对分值均位于前100；其中，比对得分低于比对阈值指a/b≤0.8，其中，a为待筛选区域的序列与人类蛋白组序列进行比对的匹配度得分，b为待筛选区域的序列与目的冠状病毒的全部蛋白组序列进行比对的匹配度得分。
进一步地，第三区域筛选模块包括：合并模块，用于将预测表位区域和第一保守基序区域根据如下至少之一的合并条件进行合并：1)两区域间为包含关系；2)两区域至少被两种不同方法预测为抗原表位区域，得到第一候选表位区域；重叠筛选模块，用于从第一候选表位区域中筛选与第二保守基序区域重叠的区域，作为第二候选表位区域；比对筛选模块，用于从第二候选表位区域中筛选与人类蛋白组序列的比对得分低于第一阈值的区域，作为第三候选表位区域；非磷酸化及膜外区域筛选模块，用于从第三候选表位区域中筛选并保留目的冠状病毒蛋白组序列中的非磷酸化区域和膜外区域，作为第四候选表位区域；综合排序模块， 用于根据可及性、beta转角、亲水性、差异肽段的覆盖条数及多重比对结果，对第四候选表位区域进行综合排序后择优选择，得到目的冠状病毒的抗原表位。
进一步地，装置还包括突变去除模块，用于将综合排序模块后择优选择的区域中去除包含突变的区域，得到目的冠状病毒的抗原表位。
根据本发明的第三个方面，提供了一种存储介质，存储介质包括存储的程序，其中，在程序运行时控制存储介质所在设备执行上述任一种冠状病毒抗原表位的筛选方法。
根据本发明的第四个方面，提供了一种处理器，处理器用于运行程序，其中，程序运行时执行上述任一种冠状病毒抗原表位的筛选方法。
应用本发明的技术方案，通过创新性地结合多肽芯片技术筛选获得一批与冠状病毒感染(尤其是SARS-Cov-2病毒感染)特异性相关的多肽，这些多肽能够被用来制备抗原、抗体、试剂盒等相关检测试剂以及多肽疫苗、核酸疫苗、蛋白重组疫苗等相关疫苗产品，从而为防控此类病毒的传染和流行提供更有力的工具。
附图说明
构成本申请的一部分的说明书附图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。在附图中：
图1示出了本申请的一种优选的实施例中的冠状病毒抗原表位的筛选方法的流程示意图。
图2A和图2B分别示出了不同单肽免疫的小鼠所获得的血清对新冠活病毒产生的中和抗体的活性，其中，图2A显示的是显微镜下检测结果，图2B显示的是统计结果。
图3A、图3B及图3C分别示出了组合1、组合2及组合3免疫的小鼠中每条多肽对应的抗体信号随时间变化情况。
图4A和图4B分别示出了不同组合1、组合2和组合免疫的小鼠所获得的血清对新冠活病毒产生的中和抗体的活性，其中，图4A显示的是显微镜下检测结果，图4B显示的是统计结果。
图5A至图5J分别示出了Mix1至Mix10免疫小鼠后，每个mix的4条多肽对应的抗体信号随时间变化情况。
图6A至图6F示出了7肽分别与每一种佐剂共同免疫小鼠后，在不同时间点上产生的抗体情况。
图7是本发明实施例的一种抗原表位多肽的筛选方法的硬件结构框图。
图8示出了本申请一种优选实施例中抗原表位多肽的筛选装置的结构示意图。
具体实施方式
需要说明的是，在不冲突的情况下，本申请中的实施例及实施例中的特征可以相互组合。下面将结合实施例来详细说明本发明。
术语解释：
本申请中的“新冠肺炎”或COVID-19均是指感染SARS-Cov-2病毒(本申请中也叫新冠病毒)后患者所出现的疾病，即新型冠状病毒肺炎。
抗原表位：又称抗原决定簇(antigenic determinant)，是抗原物质分子表面或其他部位具有一定组成和结构的特殊化学基团，能够与相应抗体或致敏淋巴细胞发生特异结合的结构。在免疫应答过程中，T细胞的抗原受体TCR和B细胞的抗原受体BCR所识别的表位具有不同特点，分别被称为T细胞表位和B细胞表位。T细胞表位一般不位于抗原分子表面，须由抗原呈递细胞将抗原加工处理为小分子多肽并与MHC分子结合，才能被TCR识别。T细胞仅能识别经加工处理的表位。而B细胞表位可存在抗原分子表面，不须经加工处理，即可直接被B细胞所识别。本申请中指预测的或者筛选的能够与抗体特异性结合的一条或多条肽段。
多肽：本申请中指预测的或者筛选的能够与抗体或致敏淋巴细胞特异性结合的任意一条肽段。
多肽-载体蛋白偶联物：本申请中指一条多肽与载体蛋白偶联形成的抗原，其中，一个载体蛋白可以偶联一条或多条多肽，多条多肽偶联时，多条多肽具有相同的氨基酸序列。根据具体偶联的多肽序列的理化性质的差异、具体载体蛋白的种类的不同以及偶联方法的不同，每个载体蛋白上所偶联的多肽的条数有所差异，本申请中优选2～50条，更优选为3～45条、5～40条、5～35条、5～30条、8～30条、10～30条、12～30条、15～30条；或者，更优选为6～36条、8～32条、10～28条、10～26条、10～24条、10～22条、10～20条、10～18条、10～16条及10～15条中的任意一种情况。
抗原：指所有能够诱导机体发生免疫应答的物质。即能够被T/B淋巴细胞表面的抗原受体(TCR/BCR)特异性是不与结合，活化T/B细胞，使之增殖分化，产生免疫应答产物(致敏淋巴细胞或抗体)并能与相应产物在体内外发生特异性结合的物质。因此，抗原具备两方面的重要特性：免疫原性和免疫反应性。本申请中的抗原指多肽半抗原与载体蛋白偶联之后形成的具有免疫原性的完全抗原，可以为单一氨基酸序列的多肽与载体蛋白偶联形成的多肽-载体蛋白偶联物；也可以为具有多种不同氨基酸序列的多肽与载体蛋白偶联形成的多肽-载体蛋白偶联物的组合物。
疫苗：通常指具有免疫原性和反应原性两方面的能力，免疫原性指能够刺激机体产生免疫应答的性能，即刺激机体产生特定的免疫细胞，使免疫细胞活化、增殖、分化，最终产生免疫效应物质特异性抗体或致敏淋巴细胞的能力，而反应原性指与其诱导产生的抗体或致敏淋巴细胞特异性结合的能力。
多肽疫苗：为了提高多肽的免疫原性，以刺激机体产生特异性抗体或致敏性淋巴细胞，通常会将多肽抗原与佐剂配伍免疫。常用的佐剂包括：氢氧化铝佐剂、短小棒状杆菌、脂多糖、细胞因子或明矾等。弗氏完全佐剂和弗氏不完全佐剂是动物免疫中最常见的佐剂。
多肽芯片技术是基于多肽芯片的检测技术，其利用多肽芯片上的种类繁多的多肽与样本的接触，然后利用图像采集技术采集多肽芯片上各个特征信号(具体可表现为携带各个特征信号的荧光图像)，进而输出芯片中每个特征的信号强度，即多肽芯片检测结果数据。基于多肽芯片检测结果数据输出的样本检测信号，可实现对与多肽芯片上的多肽结合的样本中的待测物的分析，样本的分析等。
Motif：基序，在生物学中是一个基于数据的数学统计模型，典型的是一段序列(Sequence)，也可以是一个结构，是特定的组(group)的序列预测，例如，一个DNA序列可以定义为转录因子结合位点，也就是序列倾向于被转录因子结合。对蛋白质来说，序列基序(Sequence motif)可以被定义为蛋白质序列属于一个给定的蛋白质家族。一个简单的motif可以是，比如一个模式(pattern)，而这个模式被这个组(group)中的所有成员共享。
ROC曲线：反映敏感性与特异性之间关系的曲线。横坐标X轴为1-特异性，也称为假阳性率，X轴越接近零准确率越高；纵坐标Y轴称为敏感度，也称为真阳性率，Y轴越大代表敏感度越好。根据曲线位置，把整个图划分为两部分，曲线下方部分的面积被称为AUC(Area Under Curve)，用来表示预测准确性，AUC值越高，表明预测准确率越高。曲线越接近左上角(X越小，Y越大)，预测准确率越高。
如背景技术部分所提到的，新出现的冠状病毒，如SARS-Cov-2，因具有很强的传染性而在全球迅速蔓延，且目前没有针对性的特效药，因而尽快获得相应的疫苗是防控疫情恶化和后续复发的关键。因此，本申请也从疫苗开发的角度开展了相关研究，并在该研究结果的基础上，提出了本申请的技术方案。本申请从寻找新型冠状病毒特异性的抗原表位着手，通过已有的抗原表位的筛选方法基础上结合独特的多肽芯片技术，筛选出了一批冠状病毒家族蛋白相关的抗原表位，并且部分是新型冠状病毒特异性的抗原表位。根据这些表位对应的多肽序列可以制备相应的多肽抗原、检测试剂盒、多肽抗体、多肽疫苗以及重组疫苗等相关产品，以及利用这些多肽序列进一步开发的基因疫苗或蛋白重组疫苗等相关产品，为冠状病毒相关疾病和/或新冠肺炎的防控提供更多的思路和手段。
在一种优选的实施例中，提供了一种多肽，该多肽选自表1所示的SEQ ID NO：1至SEQ ID NO：154所示的肽段中的任意一条。
在一种优选的实施例中，提供了一种抗原表位，该抗原表位包括表1所示的SEQ ID NO：1至SEQ ID NO：154中的任意一条或多条。
表1：
Figure PCTCN2021080636-appb-000001
Figure PCTCN2021080636-appb-000002
Figure PCTCN2021080636-appb-000003
Figure PCTCN2021080636-appb-000004
Figure PCTCN2021080636-appb-000005
在一种更优选的实施例中，上述抗原表位包括SEQ ID NO：25、SEQ ID NO：28、SEQ ID NO：31、SEQ ID NO：35及SEQ ID NO：36及SEQ ID NO：41至SEQ ID NO：154中的任意一条或多条。其中，SEQ ID NO：25、SEQ ID NO：28、SEQ ID NO：31、SEQ ID NO：35所示多肽经过至少两次多肽芯片筛选得到，因而能够作为抗原表位的潜在应用价值更高。
上述多肽作为B细胞或T细胞特异性识别的抗原表位，可以被制备成多肽疫苗，刺激机体产生特异性抗体或致敏淋巴细胞(免疫原性)。在免疫机体时，为了更好的刺激免疫反应，常添加佐剂刺激机体产生辅助性T细胞，进一步诱导B细胞免疫反应。当然，也可以使用单独的多肽进行免疫机体刺激产生免疫反应。
上述多肽也可以被制备成抗原，刺激机体产生抗体。为了更好的刺激激起充分的免疫反应(即免疫原性很低)，采用带有很多抗原表位的载体蛋白有利于刺激辅助性T细胞，进一步诱导B细胞免疫反应。
因此，在一种优选的实施例中，还提供了一种多肽-载体蛋白偶联物，该多肽-载体蛋白偶联物包括上述任意一条多肽以及与该条多肽偶联的载体蛋白。这样的多肽-载体蛋白偶联物通常作为抗原来检测抗体，或者作为抗原通过免疫动物来制备抗体。由于上述多肽能够特异性地识别冠状病毒，尤其是SARS-CoV-2病毒，因此，这样的多肽-载体蛋白偶联物能够特异性识别冠状病毒的抗体，尤其是SARS-CoV-2病毒的抗体。
根据多肽-载体蛋白偶联物制备的需求，可以选择具体合适的载体蛋白来形成多肽-载体蛋白偶联物。本申请中的载体蛋白包括但不仅限于BSA(牛血清蛋白)、OVA(卵清白蛋白)、KLH(钥孔血蓝蛋白)或CS(酪蛋白)。根据不同多肽的氨基酸序列组成，为了便于与载体蛋白偶联，通过需要通过连接序列(又叫连接子或linker)与载体蛋白偶联。本申请中，连接序列优选CGSG。
根据多肽氨基酸的理化性质、所用的载体蛋白的不同及偶联方式的不同，每个载体蛋白所能偶联上的多肽的条数也有所不同。从偶联的效率及对抗体识别结合能力综合考虑，优选每个载体蛋白偶联2～50条多肽，更优选为3～45条、5～40条、5～35条、5～30条、8～30条、10～30条、12～30条、15～30条；或者，更优选为6～36条、8～32条、10～28条、10～26条、10～24条、10～22条、10～20条、10～18条、10～16条及10～15条中的任意一种情况。
在一种优选的实施例中，还提供了一种抗原，该抗原包括一种多肽-载体蛋白偶联物，或者多种不同的多肽-载体蛋白偶联物的组合物，其中，多肽-载体蛋白偶联物为上述任一种多肽-载体蛋白偶联物。
需要说明的是，上述多肽-载体蛋白偶联物中，载体蛋白上所偶联的多肽均为具有同一氨基酸序列的多肽，也就是说，同一载体蛋白上偶联的是同一种多肽，这样的多肽-载体蛋白偶联物作为抗原时具有单一的抗原表位。而在某些实施例中，作为抗原来检测血清中是否存在病毒抗体时，抗原可以是具有单一抗原表位的抗原，也可以是同时具有多种抗原表位的抗原。当将偶联不同多肽序列的多肽-载体蛋白偶联物以组合物的形式作为抗原时，就可以产生多种抗原表位。比如，序列A的多肽与BSA偶联得到A-BSA偶联物，而序列B的多肽与BSA偶联得到B-BSA偶联物，序列C的多肽与OVA偶联得到C-OVA偶联物，则包含这三种多肽-载体蛋白偶联物的抗原则具有A，B，C三种抗原表位。若抗原仅包括三种多肽-载体蛋白偶联物中的一种，则仅具有一种抗原表位。
在一种优选的实施例中，还提供了一种冠状病毒抗体检测试剂盒，该试剂盒包括上述任意一种抗原。这些抗原中的抗原表位来源于上述任一种多肽，而上述多肽在已知冠状病毒蛋白家族中都有，因而采用包含该抗原的试剂盒准确地、特异性地对冠状病毒，尤其是SARS-CoV-2感染患者进行鉴别和诊断。
上述试剂盒，根据具体需要可以制备成多种不同类型的检测试剂盒。但从方便检测，便于判断检测结果的角度考虑，试剂盒中的多肽抗原多为预包被抗原。优选地，预包被抗原包被于固相载体上；具体的预包被的固相载体根据需要合理设计。更优选，固相载体包括酶标板(多为聚苯乙烯材料的)、膜载体或微球；进一步优选地，膜载体包括硝酸纤维素膜(使用最广泛)、玻璃纤维素膜或尼龙膜，更进一步优选地，膜载体上还包被有阳性对照物，多肽-载体蛋白偶联物和阳性对照物按检测顺序在硝酸纤维素膜上依次设置。
根据试剂盒的具体检测方法的不同，试剂盒中具体的配套试剂也相应有所不同，但均可以根据已知试剂盒的配制方式进行组合配套试剂。优选地，上述试剂盒中还包括以下至少之一：(1)酶标二抗，更优选酶标二抗为HRP标记的二抗(对应于ELISA检测试剂盒)；(2)胶体金结合垫，胶体金结合垫上包被有胶体金标记的多肽-载体蛋白偶联物和阳性对照物的特异性结合物(对应于免疫胶体金检测试剂盒)；(3)标记垫，标记垫上包被有荧光标记的微球，微球上负载有阳性对照物的特异性结合物(对应于免疫荧光检测试剂盒)。
上述免疫胶体金检测试剂盒和免疫荧光检测试剂盒检测相对更方便，只需要建立阳性对照的C线和检测样本的T线即可。阳性对照的C线处预包被的阳性对照物，只要是能够随着待测样本的血清层析过程携带过来的带有检测标记的特异性结合物结合即可，对具体的阳性对照物的具体抗原或抗体并无特殊限定。优选地，上述阳性对照物选自鼠免疫球蛋白、人免疫球蛋白、羊免疫球蛋白或兔免疫球蛋白，相应地，阳性对照物的特异性结合物选自抗鼠免疫球蛋白、抗人免疫球蛋白、抗羊免疫球蛋白或抗兔免疫球蛋白。
上述抗鼠免疫球蛋白根据免疫的对象的不同，可以是羊抗鼠的免疫球蛋白或兔抗鼠的免疫球蛋白，或者是其他可免疫的动物抗鼠的免疫球蛋白。同样地，抗人免疫球蛋白、抗羊免疫球蛋白或抗兔免疫球蛋白也可以根据免疫动物的不同，是不同物种来源的抗免疫球蛋白。上述免疫球蛋白可以是IgM、IgG、IgA、IgD或IgE中的任意一种。这些抗免疫球蛋白抗体可以是单克隆抗体或多克隆抗体。
上述试剂盒中，根据所需要检测的样本数量的多少，所使用的酶标板的规格也有所不同，可以在12～384孔酶标板中合理选择。预包被的酶标板中，根据不同多肽-载体蛋白偶联物中抗原表位的不同，或者针对不同发病阶段的检测对象的不同，每个孔中对多肽-载体蛋白偶联物的包被量也有所差异。本申请的某些实施例中，优选每个孔中多肽-载体蛋白偶联物的包被量为0.1～32μg，优选为0.2～30μg,、0.3～30μg、0.4～28μg、0.6～25μg、0.6～24μg,、0.7～24μg、0.7～22μg、或0.7～20μg，更优选为0.7～19μg、0.7～18μg、0.7～17μg、0.7～16μg、0.7～15μg、0.7～14μg、0.7～13μg或0.7～12μg；进一步优选为0.8～19μg、0.8～18μg、0.8～17μg、0.8～16μg、0.8～15μg、0.8～14μg、0.8～13μg、0.8～12μg、0.8～11μg、0.8～10μg、0.8～9μg、0.8～8μg、0.8～7μg、0.8～6μg、0.8～5μg、0.8～4μg、0.8～3μg、0.8～2μg、0.8～1.8μg、0.8～1.7g、0.8～1.6μg、0.8～1.5μg、0.8～1.4μg或0.8～1.2μg。
类似地，多肽-载体蛋白偶联物在膜载体上(比如，硝酸纤维素膜)的包被量也不同，优选为0.8～8μg/cm，更优选为0.8～7μg/cm，0.8～6μg/cm、0.8～5μg/cm、0.8～4μg/cm、0.8～3μg/cm、0.8～2μg/cm、0.8～1.8μg/cm、0.8～1.7g、0.8～1.6μg/cm、0.8～1.5μg/cm、0.8～1.4μg/cm或0.8～1.2μg/cm。
在一种优选的实施例中，还提供了上述任多肽或抗原表位在制备治疗冠状病毒引起的疾病相关的药物中的应用。在一些优选实施例中，冠状病毒为SARS-CoV-2。比如，利用含有这些多肽或抗原表位的多肽-载体蛋白偶联物作为抗原去免疫动物，从而制备特异性抗体。或者根据本申请所提供的相关的抗原表位，可以通过化学合成的方式制备相关多肽疫苗。或者通过重组基因获得编码上述多肽的核酸，从而获得基因疫苗。因此，上述药物可以是抗体或疫苗，抗体可以是单克隆抗体，也可以是多克隆抗体，疫苗可以是多肽疫苗，也可以是基因疫苗。
相应地，在一种优选的实施例中，还提供了上述药物，该药物可以是抗体或疫苗，抗体通过上述的抗原免疫动物得到，疫苗为多肽疫苗或基因疫苗，其中，多肽疫苗包含表1中的任意一种或多种多肽，基因疫苗包含编码表1中任意一种或多种多肽的核酸。优选地，多肽选自SEQ ID NO：1至SEQ ID NO：40中的任意一种或多种；更优选，多肽选自SEQ ID NO：25、SEQ ID NO：28、SEQ ID NO：31、SEQ ID NO：35及SEQ ID NO：36中的任意一条或多条，这5条多肽经过至少两次独立的多肽芯片筛选得到，因而从概率上而言作为疫苗的潜在可能性更大。
需要说明的是，上述抗体通过多肽-载体蛋白偶联物作为抗原去免疫动物得到，常用的免疫动物包括大鼠、小鼠、山羊或兔等哺乳动物。根据抗原中所含有的多肽-载体蛋白的种类的不同，所得抗体可以是单克隆抗体，也可以是多克隆抗体。疫苗可以是多肽疫苗，多肽疫苗可以根据多肽序列通过化学合成得到，也可以通过基因工程手段体外重组表达后经酶切、纯 化获得。基因疫苗通过基因工程手段设计包含编码目的多肽的核酸，使核酸表达而产生具有抗原表位作用的多肽。
在一种优选的实施例中，还提供了一种冠状病毒引起的肺炎的预防或治疗方法，其中，预防法包括：向受试者给予预防有效量的抗冠状病毒的药物，该药物为上述药物中的疫苗；治疗方法包括：向受试者给予治疗有效量的抗冠状病毒的药物，该药物为上述药物中的抗体。
优选地，上述冠状病毒为SARS-CoV-2。
在本申请中，为了进一步增强多肽本身对机体刺激而产生的免疫反应，在一种优选的实施例中，提供了一种多肽组合物，该多肽组合物包括表1所示的SEQ ID NO：1至SEQ ID NO：154所示的肽段中的至少两条。
在某些优选的实施例中，该多肽组合物中至少包括SEQ ID NO：1至SEQ ID NO：40所示的肽段中的任意一条。优选地，该多肽组合物中至少包括SEQ ID NO：25、SEQ ID NO：28、SEQ ID NO：31、SEQ ID NO：35及SEQ ID NO：36中的任意一条。
上述多肽组合物，可以根据疫苗或抗体制备等不同研发要求，以物理形式混合形成组合物，或者以化学键连接的形式形成长链多肽形式的组合物。具体连接的肽段序列，条数及先后顺序可以根据实际需要进行合理调整。优选采用两条进行连接，具体连接的方式可以通过连接臂(比如，可以是甘氨酸或赖氨酸)等来实现。
在一些优选的实施例中，多肽组合物包括第一肽段集合中的一条或多条，其中，第一肽段集合包括SEQ ID NO：1-4、6-8、11、13-17、20-25、27-30、32-33、35-36及39-40所示的肽段。上述第一肽段集合中的肽段表现出更强的新型冠状病毒序列特异性。基于这些多肽来制备疫苗，便于获得特异性针对新冠病毒的疫苗。
在另一些优选的实施例中，多肽组合物包括第二肽段集合中的一条或多条，第二肽段集合包括SEQ ID NO：5、9、10、12、18、19、26、31、34、37及38所示的肽段。第二肽段集合中的肽段表现出更强的冠状病毒序列保守性。基于这些多肽来制备疫苗，便于获得针对冠状病毒的广谱性的疫苗。
在某些实施例中，多肽组合物除了包括第一肽段集合中的一条或多条外，还包括第二肽段集合中的一条或多肽。基于上述两个集合中的肽段来制备疫苗，以期获得针对多种冠状病毒的，免疫原性更强的疫苗。
在一些实施例中，还可以根据T细胞表位与B细胞表位进行组合的方式来形成多肽组合物，这样便于增强免疫效果。具体的，上述40条多肽中，来源于T细胞表位或B细胞表位可以根据多个表位预测软件来进行区别。
在一些实施例中，多肽组合物包括源于同一蛋白和/或不同蛋白的多肽，更优选地，多肽组合物中来源于同一蛋白的多肽不超过两条，进一步优选地，多肽组合物选自如下组合之一：
组合1：SEQ ID NO：28、SEQ ID NO：6、SEQ ID NO：13和SEQ ID NO：18；
组合2：SEQ ID NO：27、SEQ ID NO：14、SEQ ID NO：5和SEQ ID NO：17；
组合3：SEQ ID NO：32、SEQ ID NO：4、SEQ ID NO：10和SEQ ID NO：23；
组合4：SEQ ID NO：25、SEQ ID NO：3、SEQ ID NO：34和SEQ ID NO：40；
组合5：SEQ ID NO：30、SEQ ID NO：8、SEQ ID NO：37和SEQ ID NO：21；
组合6：SEQ ID NO：2、SEQ ID NO：11、SEQ ID NO：33和SEQ ID NO：19；
组合7：SEQ ID NO：1、SEQ ID NO：15、SEQ ID NO：12和SEQ ID NO：29；
组合8：SEQ ID NO：26、SEQ ID NO：35、SEQ ID NO：38和SEQ ID NO：22；
组合9：SEQ ID NO：31、SEQ ID NO：36、SEQ ID NO：16和SEQ ID NO：20；
组合10：SEQ ID NO：7、SEQ ID NO：9、SEQ ID NO：39和SEQ ID NO：24；
组合11：SEQ ID NO：29、SEQ ID NO：35、SEQ ID NO：40和SEQ ID NO：20；
组合12：SEQ ID NO：3、SEQ ID NO：20、SEQ ID NO：21和SEQ ID NO：22、SEQ ID NO：29、SEQ ID NO：33和SEQ ID NO：34。
为进一步有效控制冠状病毒对人类的感染，在本申请一种优选的实施例中，提供了一种多肽疫苗，该多肽疫苗包括表1所示的SEQ ID NO：1至SEQ ID NO：154所示的肽段中的任意一条或多条。利用这些多肽可以根据广谱性和/或新冠病毒特异性的肽段，合理选择特定的肽段来形成有效的多肽疫苗。
在一些优选的实施例中，多肽疫苗包括多肽至少包括SEQ ID NO：1至SEQ ID NO：40所示的肽段中的任意一条；优选地，多肽疫苗中至少包括SEQ ID NO：25、SEQ ID NO：28、SEQ ID NO：31、SEQ ID NO：35及SEQ ID NO：36中的任意一条。
在一些优选的实施例中，多肽疫苗包括第一肽段集合中的一条或多条，其中，第一肽段集合包括SEQ ID NO：1-4、6-8、11、13-17、20-25、27-30、32-33、35-36及39-40所示的肽段。上述第一肽段集合中的肽段表现出更强的新型冠状病毒序列特异性。
在另一些优选的实施例中，多肽疫苗包括第二肽段集合中的一条或多条，第二肽段集合包括SEQ ID NO：5、9、10、12、18、19、26、31、34、37及38所示的肽段。第二肽段集合中的肽段表现出更强的冠状病毒序列保守性。
在某些实施例中，多肽疫苗除了包括第一肽段集合中的一条或多条外，还包括第二肽段集合中的一条或多肽。基于上述两个集合中的肽段来制备疫苗，以期获得针对多种冠状病毒的，免疫原性更强的疫苗。
采用冠状病毒广谱性的多肽来制备疫苗，有助于开发针对冠状病毒通用型的疫苗，从而实现对不同的冠状病毒感染的预防。采用新型冠状病毒特异性的多肽所制备的疫苗能够特异性地针对新冠病毒。
在一些实施例中，还可以将来源于T细胞的表位与B细胞的表位进行组合的方式来形成多肽疫苗，这样组合而成的多肽疫苗便于增强免疫效果。具体的，上述40条多肽中，来源于T细胞表位或B细胞表位可以根据多个表位预测软件来进行区别。
在一些实施例中，多肽疫苗包括来源于不同蛋白的多肽，更优选地，多肽疫苗中来源于同一蛋白的多肽不超过两条，进一步优选地，多肽疫苗选自如下任一组合多肽：
组合1：SEQ ID NO：28、SEQ ID NO：6、SEQ ID NO：13和SEQ ID NO：18；
组合2：SEQ ID NO：27、SEQ ID NO：14、SEQ ID NO：5和SEQ ID NO：17；
组合3：SEQ ID NO：32、SEQ ID NO：4、SEQ ID NO：10和SEQ ID NO：23；
组合4：SEQ ID NO：25、SEQ ID NO：3、SEQ ID NO：34和SEQ ID NO：40；
组合5：SEQ ID NO：30、SEQ ID NO：8、SEQ ID NO：37和SEQ ID NO：21；
组合6：SEQ ID NO：2、SEQ ID NO：11、SEQ ID NO：33和SEQ ID NO：19；
组合7：SEQ ID NO：1、SEQ ID NO：15、SEQ ID NO：12和SEQ ID NO：29；
组合8：SEQ ID NO：26、SEQ ID NO：35、SEQ ID NO：38和SEQ ID NO：22；
组合9：SEQ ID NO：31、SEQ ID NO：36、SEQ ID NO：16和SEQ ID NO：20；
组合10：SEQ ID NO：7、SEQ ID NO：9、SEQ ID NO：39和SEQ ID NO：24；
组合11：SEQ ID NO：29、SEQ ID NO：35、SEQ ID NO：40和SEQ ID NO：20；
组合12：SEQ ID NO：3、SEQ ID NO：20、SEQ ID NO：21和SEQ ID NO：22、SEQ ID NO：29、SEQ ID NO：33和SEQ ID NO：34。
上述任一多肽组合中，每种多肽的质量可以根据免疫原性强弱来合理设定。在某些优选的实施例中，每种多肽的质量为0.1～1mg，更优选为0.25～0.5mg。这些多肽按照合理优化的质量比进行物理混合后，根据混合后的多肽组合的体积，等体积混合佐剂后即可形成多肽疫苗对实验动物或人体进行免疫。
为了进一步提高多肽疫苗的免疫原性，可以将多肽与载体蛋白偶联。在某些优选的实施例中，多肽疫苗还包括载体蛋白，来源于不同蛋白的多肽与该载体蛋白偶联形成多肽-载体蛋白偶联物。将上述任一种组合的多肽按合理的质量比混合后，形成多肽混合物，该多肽混合物同时与同一种载体蛋白偶联，从而获得偶联有多种多肽序列的多肽-载体蛋白偶联物。载体蛋白的具体种类不限，包括但不仅限于牛血清蛋白、卵清白蛋白、钥孔血蓝蛋白或酪蛋白。在某些实施例中，多肽还可以通过连接序列与载体蛋白偶联，连接序列优选为CGSG。
进一步优选地，该多肽疫苗为注射液，优选注射液还包括佐剂，更优选地，该注射液中佐剂的体积与50～100μg多肽-载体蛋白偶联物的体积相等。
需要说明的是，为了便于储存，上述多肽组合(混合物)或者多肽组合与载体偶联而成的偶联物，在与佐剂混合对机体免疫之前，可以以固态粉末的形式保存。可以在免疫时，配制成液体，并加入等体积的佐剂形成注射液进行免疫。当然，也可以直接与佐剂制备成液体形式的疫苗。
为进一步提高某些肽段的亲和性，在一些优选的实施例中，多肽疫苗中的任一条肽段为修饰肽段。优选地，修饰肽段为在N端、C端或者NC两端添加1-4个亲水性氨基酸；优选地，水性氨基酸为Glu、Lys、Ser或Gly；优选地，1-4个亲水性氨基酸选自如下任意一种：Glu-Glu、Lys-Lys或Ser-Gly-Ser。
在某些实施例中，为了更好地实现对肽段的定向偶联，多肽疫苗中的任一条肽段可以为半胱氨酸修饰的肽段。具体地，包括但不限于在肽段的N端、C端或者NC两端添加，亦或者在肽段的肽链中间添加半胱氨酸。当在肽段的肽链中间添加半胱氨酸时，一个或多个半胱氨酸可以插入肽链中间(即插入两个氨基酸残基之间)，也可以将一个或多个半胱氨酸以支链形式连接于肽链的中间(即作为肽链中间的某个氨基酸的侧链)。
上述多肽疫苗中的多肽可以是单一肽段的形式，也可以是多条肽段的组合形式。为了进一步提高多肽疫苗的免疫原性和免疫反应性，在本申请一种优选的实施例中，上述多肽疫苗包括多条肽段，多条肽段以串联形式存在，优选地，多肽疫苗中至少一条肽段串联1～5次，优选1～3次；更优选地，多条肽段经连接臂串联连接；进一步优选地，连接臂选自甘氨酸、赖氨酸、AEA((2-Aminoethoxy)acetic acid，2-氨基乙氧基乙酸)、Ava(5-Aminovaleric Acid，5-氨基戊酸)、ANP(3-Amino-3-(2-nitrophenyl)propanoic acid，3-氨基-3(2-硝基苯)丙酸)、β-丙氨酸、GABA(4-Aminobutyric Acid)或PEG(聚乙二醇)。经过聚乙二醇连接的多肽，既可以提高溶解度，又可以保护多肽不被蛋白水解酶酶切，提高生物活性的半衰期。
在一种优选的实施例中，还提供了上述任一多肽在制备治疗冠状病毒引起的疾病的疫苗中的应用。优选地，冠状病毒为SARS-CoV-2。
在一些优选实施例中，疫苗包括SEQ ID NO：1至SEQ ID NO：40所示的肽段中的任意一条；更优选包括SEQ ID NO：25、SEQ ID NO：28、SEQ ID NO：31、SEQ ID NO：35及SEQ ID NO：36中的任意一条。
在一些优选实施例中，疫苗包括第一肽段集合中的一条或多条，其中，第一肽段集合包括SEQ ID NO：1-4、6-8、11、13-17、20-25、27-30、32-33、35-36及39-40所示的肽段。
在一些优选实施例中，疫苗包括第二肽段集合中的一条或多条，第二肽段集合包括SEQ ID NO：5、9、10、12、18、19、26、31、34、37及38所示的肽段。
在一些优选实施例中，疫苗包括第一肽段集合中的至少一条和第二肽段集合中的至少一条。
在一种优选的实施例中，本申请还提供了一种核酸疫苗，该核酸疫苗包括核酸，该核酸编码上述任一种多肽或多肽组合物。具体地，该核酸疫苗可以是DNA疫苗或RNA疫苗；更优选为mRNA疫苗。
在一种优选的实施例中，本申请还提供了一种重组蛋白疫苗，该重组蛋白疫苗包含SEQ ID NO：1至SEQ ID NO：154中任一条或多条肽段。优选地，该重组蛋白疫苗是SEQ ID NO：1至SEQ ID NO：40中任一条或多条肽段，更优选为SEQ ID NO：25、SEQ ID NO：28、SEQ ID NO：31、SEQ ID NO：35及SEQ ID NO：36中的任意一条与4～6个组氨酸或4个Gly 1个Ser(即4个Gly及1个Ser顺序连接)重重组而成的蛋白疫苗。
在一种优选的实施例中，提供了一种冠状病毒引起的疾病的预防方法，该预防方法包括：向受试者给予预防有效量的上述任一种多肽疫苗、基因疫苗或重组蛋白疫苗。还提供了一种冠状病毒引起的疾病的治疗方法，该治疗方法包括：向受试者给予治疗有效量的上述任一种抗体。
优选地，上述冠状病毒为SARS-CoV-2。
在一种优选的实施例中，提供了一种抗原表位的筛选方法，如图1所示，该筛选方法包括：
步骤S101，利用目的冠状病毒的全部蛋白组序列进行抗原表位预测，得到预测表位区域；
步骤S102，利用多肽芯片技术筛选出对目的冠状病毒感染的阳性血清样本与对照血清样本中存在差异响应的肽段，记为差异肽段；
步骤S103，将差异肽段与目的冠状病毒的全部蛋白组序列进行比对，获得第一保守基序区域；
步骤S104，从预测表位区域和第一保守基序区域中筛选出满足表位筛选条件的区域，得到抗原表位；其中，表位筛选条件包括位于目的冠状病毒的非磷酸化区域和/或膜外区域。
下面将结合更详细的实施例来进行说明本申请的方案及其有益效果。
需要说明的是，以下实施例中用到的抗体、试剂及耗材等，若无特殊说明，均为市售商业化产品。
PBS溶液，如无特殊说明，均指浓度为10mM的磷酸缓冲液(pH＝7.4)，来源TaKaRa，货号：T900。多肽抗原稀释液：50mM碳酸盐缓冲液(pH＝9.6)。洗涤缓冲液(PBST)是指：1L PBS中加入1ml tween-20，混匀后即为洗涤缓冲液。HRP标记山羊抗人IgM二抗：来源于Sigma，货号：A6907。HRP标记山羊抗人IgG二抗：来源于Abcam，货号：ab97225。
第一部分：以SARS-CoV-2病毒为例，来说明抗原表位(也即多肽)的筛选方法。
现有技术中，筛选抗原表位的方法通常是根据目的蛋白序列，利用公开的软件进行生物信息学预测或根据已有知识进行选择。而在本申请中，所采用的方法具有以下改进点：1) 采用多种软件进行综合预测评估，避免单一软件的偏差；2)使用了中国人群高频率HLA数据库帮助选择适合中国人群的候选区域；3)在面对蛋白结构和功能信息不太明了的情况下，对病毒蛋白组序列整体进行了大规模筛选而非为了减少计算量仅选择特定蛋白的序列来进行筛选，因而便于找到病毒蛋白组上所有可能的候选区域。此外，本申请的筛选方法创新性地采用了多肽芯片技术，具体体现在：1)采用独有的多肽芯片技术(即使用合成在硅基芯片上的大量多肽与试验样本中的抗体结合，无偏地获得受试样本的免疫表征)进行真实数据高通量辅助筛选出新冠肺炎与对照样本差异性表达的肽段；2)找出差异表达的肽段比对到新冠病毒蛋白组序列中的区域，根据改进的方法确定“高可信保守位点”(具体定义见第(三)步骤)，3)基于“高可信保守位点”的基序(motif)筛选抗原表位候选区域。
以下筛选方法的各步骤具体示例如下：
(一)本申请基于公开的病毒蛋白序列，调用多个软件预测了T细胞和B细胞提呈序列和区域。
从NCBI获取SARS-Cov-2蛋白序列(GeneBank MN908947)，全长为9703个氨基酸，基因组共计10个开放阅读框(ORF)，我们使用了NetMHCpan4.0、IEDB_recommonded、mixMHCpred、COBEpro等软件对病毒全蛋白组序列进行了MHC-1和MHC-2亲和力预测，综合各软件结果并依据Allele Frequency Net Database(AFND)数据库中记录的85个实验得到的中国人高频率HLA比对结果进行综合评价，获得2391个高可信的病毒抗原表位区域。
为提高亲水性，优选去除其中疏水性过高(指疏水性氨基酸占比大于45％且疏水性得分大于3)的区域，得到1123个抗原表位区域。本实施例中，设置筛选的长度是10-15个AA。采用该长度范围的多肽在利用软件预测肽段的亲和力时，具有更好的亲和力预测效果，且该长度的多肽，合成成本较低，难度比较小，也容易获得更好的纯度(肽段长度越长，合成成本越高，合成难度越大，纯度也越低)。
需要说明的是，筛选肽段的长度并不局限于10-15个AA，在其他某些实施例中，也可以设置为8-20个、8-18个或8-16个AA。
(二)基于健康人群样本和新冠感染患者样本的多肽芯片技术平台的检测数据，获得差异肽段。通过将差异肽段比对到流感病毒、普通感冒相关病毒和更多冠状病毒蛋白序列，最终确定这些差异肽段均为冠状病毒家族序列相关肽段。
搜集70份新冠肺炎血清样本(F)、5份健康人血清样本(H)和5份其他肺部疾病血清样本(T)，采用多肽芯片技术筛选新冠肺炎血清样本相对于健康人血清样本和其他肺部疾病血清样本差异表达的抗体所对应的肽段。
筛选的思路是，第一步，通过F与H的对比，可以筛选出在多肽特征，此类特征对应着疾病带来的抗体浓度上升，但是找出的抗体不一定是新冠特异的抗体，也可能是由于肺部感染等因素引起的抗体提高；第二步，通过对比F与T，可以找出新冠肺炎相比其他肺部疾病特异性的抗体，但是由于疾病状态下抗体表达比较复杂，且受T样本数量限制，新冠与其他肺 部疾病的比较可能会容易错误的找到一些非特异性的多肽；最后通过将第一步与第二步中找出的特征肽段取交集，从而得到具有高准确性的新冠特异肽段。
差异肽段筛选的基本操作流程如下：
采用V13芯片(由Health Tell公司生产，型号为P/N:600001 V13 Slides)依照标准流程进行样本检测，获得V13芯片125，509个肽段的信号值，称每个肽段信号值为特征，其值域为0～65535，将原始数据进行log10转换。我们假设新冠肺炎会造成特定抗体信号的升高，因此，通过单尾的T检验，计算每个特征，F相比T升高的p值，并进行多重假设检验校正(同一数据集有超过2次以上的假设检验时，需要进行多重假设检验校正)，记为p_FT_BH；同时，计算每个特征F相比于H升高的p值，并进行多重假设检验校正，记为p_FH_BH；筛选所有同时满足p_FT_BH<0.05且p_FH_BH<0.05的特征肽段，作为目标肽段。筛选得到与健康人群和其他肺炎人群存在显著响应差异的特征肽段864个。
需要说明的是，上述筛选过程也可以基于肽段的信号值的原始数据进行，当阳性血清样本的信号值与阴性对照血清样本的信号值的差，记为第一差值，同时计算阳性血清样本的信号值与其他肺部疾病对照血清样本的信号值的差，记为第二差值；保留所有符合第一差值和第二差值同时满足阈值的结合肽段，从而得到目的差异肽段。
此时的阈值应该是保证阳性大于阴性，且阳性大于其他疾病的，可以是0，也可以是较小特征的一定比例(比如110％-300％)。比如，阳性比其他疾病的信号值大，例如阳性是x，其他疾病是y，则要求x>y，但有的时候希望检测的标准更严格一些，也可以设置为x>ay，而a＝1.1～3等。
上述步骤中，先找出差异肽段，对每个肽段值需要在不同组样本的信号间两两比较，一共有125，509个信号肽段，则需要比较125，509次，则多重假设检验校正的n＝125，509；每个肽段的差异p值通过多重假设检验校正，得到q值，根据q值判断在哪两组之间存在差异。
通过T检验等统计学方法对每个蛋白进行p值计算。T检验是差异蛋白表达检测中常用的统计学方法，通过合并样本间可变的数据，来评价某一个蛋白在两个样本中是否有差异表达。但由于通常样本量较少，从而对总体方差的估计不很准确，所以T检验的检验效能会降低，并且如果多次使用T检验会显著增加假阳性的次数。
比如，当某个蛋白的p值小于0.05(5％)时，我们通常认为这个蛋白在两个样本中的表达是有差异的。但仍有5％的概率，这个蛋白不是差异蛋白，那么我们就错误例否认了原假设(在两个样本之间没有差异表达)，导致了假阳性的产生(出错的概率为5％)。如果检验一次，出错的概率是5％；检测10000次，出错的次数就是500次，即额外多出了500次差异的结论，即实际没有差异。为了控制假阳性的次数，就需要对p值进行多重检验校正，提高阈值。
将以上差异肽段使用BLASTp与已有数据库中公开的冠状病毒属、流感病毒属、普通感冒相关病毒、肺炎相关细菌、支原体和衣原体等多种致病微生物蛋白序列进行比对：结果显示其中443个差异肽段能以较高的比对得分(Bit score)被直接比对到新冠病毒蛋白组，阈值 14；将比对结果中2个以上重叠的比对结果称为一个2CCR区(2个以上多肽连续覆盖区)，其中有861个差异肽段位于新冠病毒蛋白组的2CCR区域中。说明这些差异信号肽段几乎全部来自新冠病毒相关的免疫反应。
上述比对得分是按BLASTp的规则比对得到，BLASTp有适应不同场景的多种模式，本实施例中选择的是“短序列比对”模式。阈值14是我们对以上在新冠肺炎患者中与在健康人群和其他肺炎患者中存在差异响应的864个差异肽段做的进一步筛选(或者说是验证)。即输入的已经是前一步得到的864个差异肽段，其中的443个能直接被(高分值)比对到新冠病毒的蛋白序列上，这也表明前一步的筛选方法得到的结果是可靠的。对于这443个差异肽段来说，此处的高比对分值从某种程度上证明这些差异肽段确实来自新冠病毒。
上述多肽芯片技术数据产生的详细过程如下：
1.实验设计
一个96孔板为一个检测单位。在实验开始前做好实验设计，根据检测样本数、设置的空白对照数和标准品数，计算需要使用的芯片数目并确定芯片编号和样本排版方式。
表2：
Figure PCTCN2021080636-appb-000006
本实施例中，共使用4张芯片，芯片编码分别为001752_01，001752_02，001752_03和001752_04。每个芯片设置了2个标准品(std)和1个空白对照(blk)，剩余为检测样本。粗体显示的8个孔位为4例样本分别做了两个重复(即F573和F573’、F574和F574’、F575和F575’，以及F577和F577’)，同一数字编号的为同一例样本。标准品、空白对照和检测样本随机分布在所有使用的芯片上，具体如表2所示。
2.实验流程
1)样本制备
血清或者血浆样本用含1％D-甘露醇(D-mannitol)的PBST溶液，于96孔深孔板中，经两次25倍稀释，得到625倍稀释的待测样本板备用；
2)芯片的水化和组装
将芯片置于芯片水化用具中，加入超纯水没过芯片，在轨道摇床上55±5rpm/min，水化20min。然后用异丙醇喷洒芯片表面后将芯片放入离心机离心干燥。干燥好的芯片按照实验设计的位置组装成分析盒(assay cassette)。
3)样本与芯片孵育结合
将稀释好的样本按照90μL/孔加入组装好的芯片上，置于恒温振荡仪上振荡孵育1小时。
4)样本清洗
将assay cassette置于洗板机进行清洗。
5)荧光二抗孵育
用含0.75％的酪蛋白(Casein)的PBST溶液配制2nM的荧光二抗溶液，按40μL/每孔加入到assay cassette中，置于恒温振荡仪上振荡孵育1小时。
6)二抗清洗
同步骤3)。
7)成像
将assay cassette中的芯片进行拆卸、清洗、干燥后组装进成像盒(imaging cassette)，放入Molecular Device公司的ImageXpress micro4成像仪进行扫描成像。最终每个检测样本得到一张TIFF图片文件，即为原始数据。
3.数据预处理
1)提取特征的荧光强度数值，输出1个GPR5数据文件和1个corner images文件。其中，GPR5文件包含了一个样本的所有信息和所有特征的荧光强度信息。
2)从所有样本的GPR5数据文件中提取特征的荧光强度信息，生成原始荧光强度(FG，foreground)数据矩阵。然后对每个样本的数据分别进行对数转换得到LFG(log-transferred foreground)数据矩阵、进行z-score的标准化处理获得NLFG(normalized and log-transferred foreground)数据矩阵。该步骤还会生成一个样本芯片信息文件，该文件包括了样本阵列位置、所用芯片编号等信息。
4.质控
通过Health Tell自带的质控方法对样本和系统进行质控，是合格的。
(三)基于差异肽段、所有肽段与新冠病毒序列的比对结果构建统计模型，获得motif(基序)高可信保守位点。
将V13芯片的所有125，509个肽段用BLASTp比对到新冠病毒的蛋白组序列上，以单个氨基酸为单位，计算覆盖此氨基酸的所有肽段的p值，p值由肽段在两组(新冠肺炎VS.对照)间信号差异计算得到，将覆盖此氨基酸的所有肽段分为两组：与此氨基酸匹配(match)或不 匹配(mismatch)，判断匹配组和不匹配组肽段p值的分布(分布即pattern)，若匹配组肽段p值显著低于不匹配组(两组p值的分布比较的时候，采用的是“Wilcoxon符号秩检验”，检验显著性的阈值是P<0.05)，则将此位置氨基酸判定为一个高可信保守位点。
将443个能够直接比对(Map)到新冠病毒蛋白组的差异肽段所在区域作为基序(motif)区域，并根据其中的高可信保守位点进行了选择，最终共获得136个基序区域，计算这些区域的疏水性，去除疏水性过高的区域(即：疏水性氨基酸占比小于等于45％且疏水性得分小于等于3的区域，疏水性得分的计算方法可参考文献A simple method for displaying the hydropathic character of a protein.(Kyte J，Doolittle RF.))，剩余114个基序区域。
(四)基于差异肽段与冠状病毒家族序列的比对结果，获得motif(基序)的可信保守位点。
将864个差异肽段比对到冠状病毒属家族序列上，以单个氨基酸为单位，将覆盖此氨基酸的差异肽段的匹配(Match)率超过75％的位点作为可信保守位点，获得350个基序区域。
上述冠状病毒家族共包括1600种冠状病毒，部分冠状病毒列举如下：Bat Hp-betacoronavirus/Zhejiang2013；Betacoronavirus England 1；Betacoronavirus Erinaceus/VMC/DEU/2012；Betacoronavirus HKU24；Bovine coronavirus；Human coronavirus HKU1；Human coronavirus OC43；Middle East respiratory syndrome coronavirus；Murine hepatitis virus；Pip多肽芯片rellus bat coronavirus HKU5；Rabbit coronavirus HKU14；Rat coronavirus Parker；Rousettus bat coronavirus；Rousettus bat coronavirus HKU9；SARS coronavirus；Tylonycteris bat coronavirus HKU4。
(五)基于上述基序位点和预测的提呈区域，挑选出最终特异性肽段(即抗原表位)。
按照上述步骤筛选了三次，第一次筛选出24条，第二次筛选出97(其中有2条与第一批的24条中的2条重复)，第三次筛选出了40条(与第二批有5条重复)，共计154条，肽段编号、序列和基本属性见表1，并选取表1中的前40条作为后续疫苗肽，具体见下表3。
表3：
Figure PCTCN2021080636-appb-000007
Figure PCTCN2021080636-appb-000008
需要说明的是，前述表1中显示了154条多肽序列(包含上述40条多肽序列)的理化性质，所有序列的物种来源均为SARS-CoV-2。在表1或表3中，
表面糖蛋白：surface glycoprotein，又称S蛋白；
pp1ab：orf1ab polyprotein；
膜糖蛋白：membrane glycoprotein，又称M蛋白；
核衣壳磷蛋白：nucleocapsid phosphoprotein，又称N蛋白；
特异：指仅在SARS-cov-2中存在的序列；
广谱：指冠状病毒家族蛋白共有的序列。
需要说明的是，前述表1中，分子量、残基数、等电点、平均疏水性等性能参数均是都是通过软件预测的，具体参见网址：https://biopython.org/。
具体操作如下：
将(一)中得到的1123个抗原表位区域与(三)中得到的114个基序区域进行连接和合并，合并标准为：1)两区域间为包含关系；或2)两区域被不同软件预测为抗原表位区域，获得800个候选表位区域；筛选这些(即上述合并的800个候选表位区域)区域中V13芯片肽段能够覆盖并且与(四)中350个基序区域重叠的位置区域，作为疫苗肽段的候选，共得到728个候选区域。
将这728个候选区域的序列与人类蛋白质组序列进行比对，比对得分低于0.8的区域共计540个被保留；筛选其中新冠状病毒蛋白组的非磷酸化区域和膜外部分得到431个区域；将这些区域进行可及性、beta转角、亲水性、HT肽段覆盖数、多重比对结果进行综合排序，综合排序具体为：
首先，筛选差异肽段覆盖条数≥3的区域，覆盖到的条件为：BlastP比对分值(BitScore)大于14(即指满足比对分值大于14且覆盖某区域的差异肽段的条数至少有3条)；
其次，去除亲水性低于亲水阈值(或疏水性高于第一疏水阈值，第一疏水阈值的涵义与前述疏水阈值的涵义相同)的区域；
然后，将可及性和Beta转角分值按照从高到低排序；进而根据多重比对结果择优选择。
其中，优先选取位于pp1ab的11个区域，其中2个区域是新冠病毒特异的，9个区域是冠状病毒广谱的；S蛋白的19个(12个区域是新冠病毒特异的，7个是冠状病毒广谱的)、N蛋白的6个(5个区域是新冠病毒特异的，1个是冠状病毒广谱的)、M蛋白的2个(特异的)及ORF7a的2个(1个特异的，1个广谱的)，共计40个肽段(共计29个特异的，11个广谱的，具体见表3)。
最后，根据需要，还可以包括去除包含突变的区域的步骤。该步骤为可选步骤，在确定含有该变异类型的情况下，也可以包含某一个突变的区域。
上述根据这431个区域的可及性、beta转角、亲水性、HT肽段覆盖数、多重比对结果进行综合排序时，基于如下考虑：由于SARS-CoV-2蛋白质组共有10个蛋白，其中pplab蛋白最长，其长度是其他蛋白(如S/N蛋白等)的十几倍，故，这431个区域里，大部分也是位于pplab蛋白序列的。考虑到各个蛋白的生物学意义不同，所以就把为数不多的其他蛋白的区域也先都选了。其中有4个区域除了含有突变外，其余各筛选指标都满足要求，因此，在考虑设计针对存在变异株的疫苗时，这些区域也可以作为候选肽段。
将上述728个候选区域的序列与人类蛋白质组序列进行BLASTp比对的比对得分，除以728个候选区域的序列与新冠病毒的BLASTp比对得分，所得数值的阈值为0.8，即去除大于0.8的候选区域。得分是根据匹配程度打分。BLASTp是NCBI提供的一个广泛使用的比对软件，Bitscore是该软件给的得分；类似的软件有DIAMOND，Muscle，ClustalW。
第二部分：对上述筛选的疫苗肽段进行化学合成及生物学验证
在获得潜在疫苗肽段序列信息后，候选疫苗多肽通过化学合成方法生产，多肽质控要求HPLC-MS纯度在98％以上以及内毒素含量不高于1EU/mg，以保证多肽符合进行动物体内实验要求。通过质控的多肽制品通过动物体内实验进行生物学验证及有效性筛选。对免疫系统功能完全的青壮年健康小鼠，以皮下注射方式给药并定期抽取血样进行多肽芯片检测，分析与所设计的疫苗肽段对应的特异性多肽芯片多肽序列信号强度差异评估免疫原性。同时，小鼠终点血清亦会用于进行活病毒中和实验(CPE法)，评估免疫后小鼠内抗体的中和效果。
基于本方法合成的多肽可单独使用或组合使用，或可与蛋白偶联使用，亦可以与不同试剂作为配伍使用。具体方案由以下实施例说明。
多肽疫苗肽的合成
1)疫苗肽优化设计：
a.在不破坏抗原上述疫苗肽核心位点的前提下，将疫苗肽两端的疏水性氨基酸进行规避；
b.计算规避后疫苗肽的疏水性，
2)疫苗制备：
对前述获得的SEQ ID NO：1至SEQ ID NO：40所示的40条肽段，委托第三方公司直接合成(未经修饰)。
定制8-12AA肽制剂：共计40条定制肽如下表；每条50mg，分装成5mg/支的制剂共10支；纯度大于等于98％，GMP洁净度要求下除菌冻干。
在其他实施例中，根据各具体序列的性质，如需提高肽段的水溶性，可将肽段的N端、C端或者N、C两端同时添加Glu-Glu、Lys-Lys或Ser-Gly-Ser后再进行合成。
在其他实施例中，为了更好地实现定向偶联，可对肽段进行半胱氨酸修饰，包括但不限于在N端、C端或者N、C两端同时添加半胱氨酸，亦或者在肽段的链段中间添加半胱氨酸。在肽段的肽链中间添加半胱氨酸时，可以在肽链中间插入一个或多个半胱氨酸，或者将一个或多个半胱氨酸以支链形式连接于肽链的中间。
实施例一 单肽疫苗有效性验证
一、实验操作
1.免疫小鼠
挑选ICX_ID：icx_16、24、32、35、37(分别对应于SEQ ID NO.20、SEQ ID NO.22、SEQ ID NO.36、SEQ ID NO.34和SEQ ID NO.21)进行单肽疫苗有效性验证。实验采用5至6周龄雌性Balb/c小鼠随机分成6组，其中5组为单肽实验组(即分别对应icx_16、24、32、35、37)，1组为单纯佐剂组，每组5只小鼠。
按上述条件合成的多肽粉末使用PBS溶液溶解，稀释配制成终浓度为2mg/ml的多肽溶液。对于多肽实验组，每组小鼠根据分组在第0、14、28天各注射100μg多肽，每只小鼠共注射3次合计300ug多肽。注射时将多肽溶液与等体积佐剂MF59(AddaVax，Invivo Gen)混合，再使用微量注射器经小鼠两腿腓肠肌以皮下注射方式给药。单纯佐剂组中则仅使用与多肽实验组终溶液等体积的MF59原浓度溶液进行注射。单纯佐剂组使用与多肽实验组同等给药方式及给药频次进行实验。小鼠接受免疫后的第35天作为实验终点，处死小鼠，采血并分离制备上清。
2.小鼠血清体外活病毒中和实验
对新冠活病毒进行稀释度为10 -1-10 -10的连续10倍稀释，将稀释后的病毒分别接种到96孔培养板中，每个稀释度接种一纵排共8孔。每孔中加入细胞悬液进行共培养5天后显微镜下统计出现细胞病变(Cytopathic effect，CPE)孔数，获得病毒半数细胞培养物感染量TCID50。
对小鼠血清进行56℃ 30分钟补体灭活，灭活后小鼠血清进行1：10、1：20、1：40、1：80、1：160、1：320、1：640、1：1280梯度稀释。将含有100个TCID50病毒的溶液与各稀释度的血清等量混合，置于37℃水浴中培养1小时。将孵育后的病毒血清混合液加入到预先接种vero细胞的96孔培养板中，置于37℃、5％CO 2环境中培养。接种后每天观察细胞病变(cytopathic effect，CPE)，第7天判定最后结果。
二、实验结果
如图2A、2B和下表所示，进行单肽免疫的小鼠所获得的血清均能对新冠活病毒有中和活性。其中，注射icx_32的小鼠体内抗体具有最优的中和作用，有40％产生中和抗体，最高中和效价达到1：640，几何平均中和效价为1：160。结果证明，通过多肽芯片辅助设计的新冠疫苗肽可对新冠病毒起到中和作用。
表4：
单肽疫苗 中和抗体阳性率(％) 最高中和效价 几何平均中和效价(GMT)
icx_16 20 1：160 1：160
icx_24 20 1：80 1：80
icx_32 40 1：640 1：160
icx_35 20 1：40 1：40
icx_37 40 1：80 1：80
实施例二 组合多肽疫苗有效性验证
一、实验操作
1.免疫小鼠
按下表中选取12条多肽组成3个多肽组合。实验采用5至6周龄雌性Balb/c小鼠随机分成4组，其中3组为多肽组合实验组(组合1、2、3)，1组为单纯佐剂组，每组5只小鼠。
用PBS溶液将40条多肽粉末分别溶解稀释至终浓度为2mg/ml的多肽溶液。对于多肽组合实验组，按照分组信息，每一组组合内每条多肽各取50μg混合组成共含200μg多肽的混合液作为小鼠第一次注射用多肽溶液，其后，每一组组合内每条多肽各取25μg混合组成100μg混合液作为小鼠第二、三次注射用多肽溶液。每组小鼠根据分组在第0、7、14天分别对应注射第一、二、三针，注射时将多肽溶液与等体积佐剂Imject Alum Adjuvant(Thermo Fisher Scientific)混合，再使用微量注射器经小鼠两腿腓肠肌以皮下注射方式给药。单纯佐剂组中则仅使用与多肽实验组终溶液等体积的Imject Alumn原浓度溶液进行注射。单纯佐剂组使用与多肽实验组同等给药方式及给药频次进行实验。
在各实验组小鼠接受免疫之前(第0天)及初次免疫之后的第7、14、21天(第7、14天为注射前)采集小鼠尾静脉血样，每个时间点采集血样量为约100-200μL，分离制备血清，以备进行多肽芯片检测。小鼠接受免疫后的第28天作为实验终点，处死小鼠，采血并分离制备上清作中和实验使用。
表5：
Figure PCTCN2021080636-appb-000009
2.多肽芯片检测多肽免疫原性
2.1实验操作
在前置实验中，利用多肽芯片检测技术对新冠感染者、新冠肺炎治愈患者及未感染健康人的血液样本进行检测，对比分析获得基于多肽芯片的新冠病毒特异性抗体的免疫特征以及相应的分析模型。利用前置实验方法，利用多肽芯片检测对小鼠血清样本进行检测，实验采用小鼠10μL血清样本，与芯片进行初步孵育结合，其后相继加入抗小鼠IgG抗体、荧光抗体进行孵育结合。完成孵育后，样本会装载入成像仪进行荧光信号成像及提取特征的荧光强度数值。对原始荧光强度进行标准化处理，获得数据矩阵，与前置实验数据进行对比分析，鉴定小鼠血清与多肽芯片上多肽结合位点特征是否为新冠特异性抗体特征。
2.2结果分析
a).疫苗肽特异性信号排序：
1.根据疫苗肽注射时间序列构建两个疫苗特异反应模式：模式一：随时序持续上升(Pattern1)；模式二：随时序上升并在D13后保持稳定(Pattern2)；
2.使用Spearman相关系数确定符合以上两种模式的信号，定义为A时序反应肽段集合；
3.对于每个疫苗肽分别计算与之有序列相似性的多肽芯片信号肽段集合B(疫苗特异性肽段集合)，序列相似性定义为多肽芯片信号肽BLASTp比对到疫苗肽设计区域，并且Bit Score>＝14，比对区域与疫苗肽交集长度超过该疫苗肽设计长度的1/2；
4.将第2步和第3步所得的两个多肽芯片肽段集合进行Fisher精确检验。
结果见下表。
表6：
Figure PCTCN2021080636-appb-000010
b).95分位数分析方法:对每一组混合多肽进行单条多肽的多肽芯片特异性信号进行分析，对每一组内单只小鼠的单条多肽的特异性信号变化及其组内中位值按时间顺序进行描绘。具体方法如下：
1.提取a方法第3步获得的每个疫苗肽的序列相似性多肽芯片肽段的整体信号；
2.计算该整体信号的每个时间点的上95分位数；
3.绘制每个小鼠的95分位数变迁图，以及计算每个时间点组内中位值并绘图。
结果如图3A至图3C所示(图中S+数字所表示的是实验小鼠的编号，即各组合中，每条多肽免疫的5只小鼠的编号)，图3A显示的是组合1中的4条多肽对应的抗体信号，图3B显示的是组合2中的4条多肽对应的抗体信号，图3C显示的是组合3中的4条多肽对应的抗体信号。从中可以看出，3个组合均能刺激小鼠免疫，提高体内抗体水平，与多肽疫苗对应的抗体信号在3个组合中均在不同时间点有一定程度的上升。
c).基于多肽芯片的免疫原性评价
基于上述a)及b)分析数据分布及其特征，设计基于多肽芯片的疫苗免疫原性评分体系(见表7)，评价结果见表8：
表7：
Figure PCTCN2021080636-appb-000011
Figure PCTCN2021080636-appb-000012
表8：
Figure PCTCN2021080636-appb-000013
3.小鼠血清体外活病毒中和实验
3.1与前一实施例一致，对新冠活病毒进行稀释度为10 -1-10 -10的连续10倍稀释，将稀释后的病毒分别接种到96孔培养板中，每个稀释度接种一纵排共8孔。每孔中加入细胞悬液进行共培养5天后显微镜下统计出现细胞病变(Cytopathic effect，CPE)孔数，获得病毒半数细胞培养物感染量TCID50。
对小鼠血清进行56℃ 30分钟补体灭活，灭活后小鼠血清进行1：10、1：20、1：40、1：80、1：160、1：320、1：640、1：1280梯度稀释。将含有100个TCID50病毒的溶液与各稀释度的血清等量混合，置于37℃水浴中培养1小时。将孵育后的病毒血清混合液加入到预先接种vero细胞的96孔培养板中，置于37℃、5％CO 2环境中培养。接种后每天观察细胞病变(cytopathic effect，CPE)，第7天判定最后结果。
3.2实验结果
如图4A、4B及下表所示，进行多肽免疫的小鼠所获得的血清均能对新冠活病毒有中和活性。其中，注射组合2及组合3的小鼠体内抗体具有较优的中和作用。注射多肽组合2的小鼠中，有75％产生中和抗体，最高中和效价达到1：640，几何平均中和效价为1：403。注射多肽组合3的小鼠中，有50％产生中和抗体，最高中和效价达到1：1280，几何平均中和效价为1：640。中和效果组间的趋势与2.2c)表8中的评分一致。结果证明，通过多肽芯片辅助设计的新冠疫苗肽可对新冠病毒起到中和作用。
表9：
组合 中和抗体阳性率(％) 最高中和效价 几何平均中和效价(GMT)
组合1 25 1：40 1：40
组合2 75 1：640 1：403
组合3 50 1：1280 1：640
实施例三 多肽偶联蛋白疫苗有效性验证
一、实验操作
1.多肽偶联KLH制备
血蓝蛋白(keyhole limpet hemocyanin，KLH)为软体动物、节肢动物(蜘蛛和甲壳虫)的血淋巴中发现的一种游离的蓝色呼吸色素，有较高免疫原性，为最常被选用的载体蛋白。按下表10中选取40条多肽组成10个多肽组合，其中Mix4与Mix9分别与实施列二中组合1及组合2一致。每组多肽分别与KLH进行偶联，多肽-KLH偶联实验步骤如下：
1)使用0.1M-MES及0.5M-NaCl配制reaction buffer(pH6.0)，将KLH用reaction buffer稀释至1mg/mL，取1mL备用。
2)向1中的溶液中加入终浓度为2mM的EDC(0.4mg)，5mM的sulfo-NHS(1.1mg)
3)混合均匀后室温反应15min
4)向反应液中加入终浓度为20mM的β-巯基乙醇(1.4ul)以终止EDC的反应，室温孵育10min。
5)将置于PBS中的半抗原(多肽)加入活化的KLH蛋白溶液中，室温反应2h。
6)加入终浓度为10mM的羟胺(hydroxylamine)或者20-50mM Tris,lysine等终止反应。
2.免疫小鼠
实验采用5至6周龄雌性Balb/c小鼠随机分成12组，其中10组为多肽-KLH实验组(组合1-10)，1组为未偶联多肽的单独KLH组，1组为单纯佐剂组，每组5只小鼠。
用PBS溶液将40条多肽粉末分别溶解稀释至终浓度为2mg/ml的多肽溶液。对于多肽-KLH实验组，按照分组信息，每一组组合内每条多肽各取50μg混合组成共含200μg多肽的混合液作为小鼠第一次注射用多肽溶液，其后，每一组组合内每条多肽各取25μg混合组成 100μg混合液作为小鼠第二、三次注射用多肽溶液。每组小鼠根据分组在第0、7、14天分别对应注射第一、二、三针，注射时将多肽溶液与等体积佐剂Imject Alum Adjuvant(Thermo Fisher Scientific)混合，再使用微量注射器经小鼠两腿腓肠肌以皮下注射方式给药。KLH单独对照组使用与多肽-KLH组中等量的KLH与等体积的Imject Alum Adjuvant混合进行注射，单纯佐剂组中则仅使用与多肽实验组终溶液等体积的Imject Alumn原浓度溶液进行注射。KLH单独对照组及单纯佐剂组使用与多肽实验组同等给药方式及给药频次进行实验。
在各实验组小鼠接受免疫之前(第0天)及初次免疫之后的第7、14、21天(第7、14天为注射前)采集小鼠尾静脉血样，每个时间点采集血样量为约100-200μL，分离制备血清，以备进行多肽芯片检测。小鼠接受免疫后的第28天作为实验终点，处死小鼠，采血并分离制备上清作中和实验使用。
表10：
分组 多肽1 多肽2 多肽3 多肽4
Mix1 icx_7 icx_19 icx_9 icx_36
Mix2 icx_34 icx_22 icx_11 icx_10
Mix3 icx_17 icx_2 icx_6 icx_29
Mix4 icx_39 icx_33 icx_35 icx_14
Mix5 icx_8 icx_18 icx_27 icx_37
Mix6 icx_30 icx_5 icx_4 icx_1
Mix7 icx_38 icx_25 icx_15 icx_21
Mix8 icx_26 icx_31 icx_28 icx_24
Mix9 icx_3 icx_32 icx_23 icx_16
Mix10 icx_13 icx_40 icx_12 icx_20
2.多肽芯片检测多肽免疫原性
2.1实验操作
本实施例采用与实施列二中一致的多肽芯片检测方法对接受免疫后的小鼠血清进行检测。
2.1实验操作
2.2结果分析
a).疫苗肽特异性信号排序：方法与实施例二一致，结果如下表。
表11：
Figure PCTCN2021080636-appb-000014
Figure PCTCN2021080636-appb-000015
b).95分位数分析方法：方法与实施例二一致。
结果如图5A至图5J所示(图中S+数字所表示的是实验小鼠的编号，即各组合中，每条多肽免疫的3只小鼠的编号)，图5A至图5J分别示出了每个mix中的4条多肽对应的抗体信号。从中可以看出，10个组合均能刺激小鼠免疫，提高体内抗体水平，与多肽疫苗对应的抗体信号在10个组合中均在不同时间点有一定程度的上升。
c).基于多肽芯片的免疫原性评价
基于多肽芯片的疫苗免疫原性评分体系对10个组合进行评价，评价方法与实施列二一致，评价结果见下表：
表12：
Figure PCTCN2021080636-appb-000016
Figure PCTCN2021080636-appb-000017
根据上述分组总分进行排序，排序结果见下表。
表13：
排名 组合 分组总计
1 Mix3 75.2
2 Mix6 74.7
3 Mix2 70.8
4 Mix7 67.9
5 Mix9 61.6
6 Mix4 59.5
7 Mix1 55.3
8 Mix8 52.1
9 Mix10 48.2
10 Mix5 44.5
由于其中Mix4与Mix9分别与实施列二中组合1及组合2一致，根据实施列二结果显示Mix4及Mix9具有中和效果。基于Mix3、Mix6、Mix2、Mix7排名比Mix4和Mix9靠前，因而推测也具有潜在有效性，可用于疫苗开发。
实施例四 多肽疫苗及佐剂配伍有效性验证
一、实验操作
1.免疫小鼠
选取ICX ID：icx_16(SEQ ID NO：20)、21(SEQ ID NO：29)、24(SEQ ID NO：22)、32(SEQ ID NO：36)、33(SEQ ID NO：3)、35(SEQ ID NO：34)、37(SEQ ID NO：21)组合成7肽与不同佐剂进行配伍有效性筛选验证。本实验中使用6种佐剂进行筛选，分别为AddaVax(又记为MF59，InvivoGen)、Imject Alumn(Thermo Scientific)、Alhydrogel(InvivoGen)、Adju-Phos(InvivoGen)、Novavax(又记为MA103A，迈科康)、MA103B(又记为带正电，迈科康)。实验采用5至6周龄雌性Balb/c小鼠随机分成12组，其中6组为7肽与不同佐剂组合的实验组，6组为单纯佐剂组，每组5只小鼠。
用PBS溶液将7条多肽粉末分别溶解稀释至终浓度为2mg/ml的多肽溶液。对于7肽组合实验组，按照分组信息，每一组组合内每条多肽各取30μg混合组成共含210μg多肽的混合液作为小鼠第一、二、三次注射用多肽溶液。每组小鼠根据分组在第0、7、14天分别对应注射第一、二、三针，注射时将多肽溶液与等体积对应佐剂混合，再使用微量注射器经小鼠两腿腓肠肌以皮下注射方式给药。单纯佐剂组中则使用PBS替代7肽混合液与等体积的对应佐剂溶液混合后进行注射。单纯佐剂组使用与多肽实验组同等给药方式及给药频次进行实验。
在各实验组小鼠接受免疫之前(第0天)及初次免疫之后的第7、14天(第7、14天为注射前)采集小鼠尾静脉血样，每个时间点采集血样量为约100-200μL，分离制备血清，以备进行多肽芯片检测。小鼠接受免疫后的第21天作为实验终点，处死小鼠，采血并分离制备上清作中和实验使用。
2.多肽芯片检测多肽免疫原性
2.1实验操作
本实施例采用与实施列二中一致的多肽芯片检测方法对接受免疫后的小鼠血清进行检测。
2.2结果分析
a).疫苗肽特异性信号排序：与实施例二一致。结果见下表。
表14：
Figure PCTCN2021080636-appb-000018
Figure PCTCN2021080636-appb-000019
上表中NA表示：not applicable，不适用。
b).95分位数分析方法：方法与实施例二一致。结果见图6A至图6F。各图示出了7肽分别与每一种佐剂共同免疫小鼠后，在不同时间点上产生的抗体情况，7肽按照icx_16、21、24、32、33、35、37的顺序依次展示。其中从图6A至图6F对应的佐剂顺序与上表中1至6的佐剂顺序相同。
c).基于多肽芯片的免疫原性评价
与实施列二一致，基于多肽芯片的疫苗免疫原性评分体系对各组合进行评价，评价结果见下表：
表15：
Figure PCTCN2021080636-appb-000020
Figure PCTCN2021080636-appb-000021
根据上表的评分结果可知，佐剂MA103A及MA103B与7肽配伍效果较佳，因而7肽与MA103A或MA103B有潜在配伍成为疫苗配方的可能性。
需要说明的是，对于前述的各方法实施例，为了简单描述，故将其都表述为一系列的动作组合，但是本领域技术人员应该知悉，本发明并不受所描述的动作顺序的限制，因为依据本发明，某些步骤可以采用其他顺序或者同时进行。其次，本领域技术人员也应该知悉，说明书中所描述的实施例均属于优选实施例，所涉及的动作并不一定是本发明所必须的。
通过以上的实施方式的描述可知，本领域的技术人员可以清楚地了解到本申请可借助软件加必需的检测仪器等硬件设备的方式来实现。基于这样的理解，本申请的技术方案中数据处理的部分可以以软件产品的形式体现出来，该计算机软件产品可以存储在存储介质中，如ROM/RAM、磁碟、光盘等，包括若干指令用以使得一台计算机设备(可以是个人计算机，服务器，或者网络设备等)执行本申请各个实施例或者实施例的某些部分的方法。
第三部分 能够执行上述抗原表位多肽的筛选方法的仪器设备
实施例一
本申请前述实施例所提供的方法可以在终端、计算机终端或者类似的运算装置中执行。以运行在终端上为例，图7是本发明实施例的一种抗原表位多肽的筛选方法的终端的硬件结构框图。如图7所示，终端可以包括一个或多个(图7中仅示出一个)处理器102(处理器102可以包括但不限于微处理器MCU或可编程逻辑器件FPGA等的处理装置)和用于存储数据的存储器104，可选地，上述终端还可以包括用于通信功能的传输设备106以及输入输出设 备108。本领域普通技术人员可以理解，图7所示的结构仅为示意，其并不对上述终端的结构造成限定。例如，终端还可包括比图7中所示更多或者更少的组件，或者具有与图7所示不同的配置。
存储器104可用于存储计算机程序，例如，应用软件的软件程序以及模块，如本发明实施例中的肠道益生菌补剂配方的确定方法对应的计算机程序，处理器102通过运行存储在存储器104内的计算机程序，从而执行各种功能应用以及数据处理，即实现上述的方法。存储器104可包括高速随机存储器，还可包括非易失性存储器，如一个或者多个磁性存储装置、闪存、或者其他非易失性固态存储器。在一些实例中，存储器104可进一步包括相对于处理器102远程设置的存储器，这些远程存储器可以通过网络连接至终端。上述网络的实例包括但不限于互联网、企业内部网、局域网、移动通信网及其组合。
传输设备106用于经由一个网络接收或者发送数据。上述的网络具体实例可包括终端的通信供应商提供的无线网络。在一个实例中，传输设备106包括一个网络适配器(Network Interface Controller，简称为NIC)，其可通过基站与其他网络设备相连从而可与互联网进行通讯。在一个实例中，传输设备106可以为射频(Radio Frequency，简称为RF)模块，其用于通过无线方式与互联网进行通讯。
实施例二
本实施例提供了一种抗原表位多肽的筛选装置，如图8所示，该筛选装置包括：
表位预测模块10，用于利用目的冠状病毒的全部蛋白组序列进行抗原表位预测，得到预测表位区域；
差异肽段筛选模块30，用于利用多肽芯片技术筛选出对目的冠状病毒感染的阳性血清样本与对照血清样本存在差异响应的肽段，记为差异肽段；
第一区域筛选模块50，用于将差异肽段与目的冠状病毒的全部蛋白组序列进行比对，获得第一保守基序区域；
第三区域筛选模块70，用于从预测表位区域和第一保守基序区域中筛选能够满足如下表位筛选条件的区域，得到抗原表位多肽：
其中，表位筛选条件包括位于目的冠状病毒的非磷酸化区域和/或膜外区域。
优选地，表位预测模块包括：候选表位第一筛选模块，用于利用目的冠状病毒的全部蛋白组序列，采用多种不同方法进行抗原表位预测，并筛选长度为8～20个，优选10～15个氨氨基酸的表位，得到候选预测表位；候选表位第二筛选模块，用于根据特定人群中的HLA所能递呈的表位和/或亲疏水性，对候选预测表位进行筛选，得到预测表位区域。
优选地，候选表位第二筛选模块包括：人群表位筛选模块，用于从候选预测表位中筛选出中国人群中HLA所能递呈的表位；和/或，疏水性筛选模块，用于从候选预测表位中去除疏 水性高于第一疏水阈值的表位，得到预测表位区域；优选地，疏水性高于第一疏水阈值的表位指疏水性氨基酸占比大于45％且疏水性得分大于3的表位。
优选地，差异肽段筛选模块包括：第一筛选模块包括：样本选择单元，用于选取目的冠状病毒感染的阳性血清样本、阴性对照血清样本和其他肺部疾病对照血清样本，其他肺部疾病指目的冠状病毒之外的病毒感染引起的肺部疾病；信号获取单元，用于采用多肽芯片的方法，将阳性血清样本、阴性对照血清样本和其他肺部疾病对照血清样本与多肽阵列芯片结合，获得结合肽段响应的信号值；差异肽段筛选单元，用于针对每个结合肽段，计算阳性血清样本的信号值与阴性对照血清样本的信号值之间存在差异时的p值，记为第一p值，同时计算阳性血清样本的信号值与其他肺部疾病对照血清样本的信号值存在差异时的p值，记为第二p值；保留所有符合第一p值和第二p值同时满足差异阈值的结合肽段，从而得到差异肽段；优选差异阈值为<0.05。
优选地，差异肽段筛选单元包括：信号转换子单元，用于对结合肽段的信号值进行log10转换；差异肽段筛选子单元，用于以转换后的log值为特征，通过单尾T检验，计算各特征在阳性血清样本与阴性对照血清样本之间存在差异时的p值，并对p值进行多重假设检验校正，得到第一p值；同时计算相应的特征在阳性血清样本与其他肺部疾病对照血清样本之间存在差异时的p值，并对p值进行多重假设检验校正，记为第二p值；筛选同时满足第一p值小于差异阈值且第二p值小于差异阈值的结合肽段，从而得到差异肽段。
优选地，第一区域筛选模块包括：保守位点筛选模块，用于以单个氨基酸为单位，计算覆盖氨基酸且与氨基酸匹配的结合肽段的信号值在阳性血清样本和阴性对照血清样本存在差异的p1值的分布，同时计算覆盖氨基酸且与氨基酸不匹配的结合肽段的信号值在阳性血清样本和阴性对照血清样本存在差异的p2值的分布，p1值的分布显著低于p2值的分布氨基酸为第一保守位点；第一保守基序筛选模块，用于将差异肽段与目的冠状病毒的全部蛋白组序列进行比对，并从能够比对上的区域中选择具有第一保守位点且疏水性低于第二疏水阈值的区域，从而获得第一保守基序区域；优选地，疏水性低于第二疏水阈值的区域指疏水性氨基酸占比小于等于45％且疏水性得分小于等于3的区域；优选差异肽段为能够完全比对到目的冠状病毒的全部蛋白组序列上的差异肽段。
优选地，筛选装置还包括第二区域筛选模块，优选地，第二区域筛选模块包括：比对模块，用于将差异肽段与冠状病毒家族的蛋白序列进行比对，第二保守基序筛选模块，用于从能够比对上的区域中选择各氨基酸位点均满足如下区域筛选条件的区域，作为第二保守基序区域：覆盖氨基酸的所有差异肽段中，能够与氨基酸匹配的差异肽段的比例满足匹配比例阈值。
优选地，匹配比例阈值大于等于75％。
优选地，第三区域筛选模块50中的表位筛选条件还包括如下至少之一：(a)与第二保守基序区域相重叠；(b)与人类蛋白组序列比对得分低于比对阈值；(c)满足如下多个性能指标：1)差异肽段覆盖条数≥3；2)亲水性满足亲水阈值；3)可及性分值、Beta转角及多重比对 分值均位于前100；其中，比对得分低于比对阈值指a/b≤0.8，其中，a为待筛选区域的序列与人类蛋白组序列进行比对的匹配度得分，b为待筛选区域的序列与目的冠状病毒的全部蛋白组序列进行比对的匹配度得分。
优选地，第三区域筛选模块包括：合并模块，用于将预测表位区域和第一保守基序区域根据如下至少之一的合并条件进行合并：1)两区域间为包含关系；2)两区域至少被两种不同方法预测为抗原表位区域，得到第一候选表位区域；重叠筛选模块，用于从第一候选表位区域中筛选与第二保守基序区域重叠的区域，作为第二候选表位区域；比对筛选模块，用于从第二候选表位区域中筛选与人类蛋白组序列的比对得分低于第一阈值的区域，作为第三候选表位区域；非磷酸化及膜外区域筛选模块，用于从第三候选表位区域中筛选并保留目的冠状病毒蛋白组序列中的非磷酸化区域和膜外区域，作为第四候选表位区域；综合排序模块，用于根据可及性、beta转角、亲水性、差异肽段的覆盖条数及多重比对结果，对第四候选表位区域进行综合排序后择优选择，得到目的冠状病毒的抗原表位多肽。
优选地，装置还包括突变去除模块，用于将综合排序模块后择优选择的区域中去除包含突变的区域，得到目的冠状病毒的抗原表位多肽。
优选地，目的冠状病毒为SARS-CoV-2。
实施例三
在本实施例中，提供了一种存储介质，该存储介质包括存储的程序，其中，在程序运行时控制存储介质所在设备执行上述任一种抗原表位多肽的筛选方法。
实施例四
在本实施例中，提供了一种处理器，该处理器用于运行程序，其中，程序运行时执行上述任一种抗原表位多肽的筛选方法。
本说明书中的各个实施例均采用递进的方式描述，各个实施例之间相同相似的部分互相参见即可，每个实施例重点说明的都是与其他实施例的不同之处。尤其，对于系统实施例而言，由于其基本相似于方法实施例，所以描述的比较简单，相关之处参见方法实施例的部分说明即可。
本申请可用于众多通用或专用的计算系统环境或配置中。例如：个人计算机、服务器计算机、手持设备或便携式设备、平板型设备、多处理器系统、基于微处理器的系统、置顶盒、可编程的消费电子设备、网络PC、小型计算机、大型计算机、包括以上任何系统或设备的分布式计算环境等等。
显然，本领域的技术人员应该明白，上述的本申请的部分模块或步骤可以在通用的计算装置来实现，它们可以集中在单个的计算装置上，或者分布在多个计算装置所组成的网络上，可选地，它们可以用计算装置可执行的程序代码来实现，从而，可以将它们存储在存储装置中由计算装置来执行，或者将它们分别制作成各个集成电路模块，或者将它们中的多个 模块或步骤制作成单个集成电路模块来实现。这样，本申请不限制于任何特定的硬件和软件结合。
需要说明的是，本申请的说明书和权利要求书及上述附图中的术语“第一”、“第二”“第三”等是用于区别类似的对象，而不必用于描述特定的顺序或先后次序。应该理解这样使用的数据在适当情况下可以互换，以便这里描述的本申请的实施例。此外，术语“包括”和“具有”以及他们的任何变形，意图在于覆盖不排他的包含，例如，包含了一系列步骤或单元的过程、方法、系统、产品或设备不必限于清楚地列出的那些步骤或单元，而是可包括没有清楚地列出的或对于这些过程、方法、产品或设备固有的其它步骤或单元。
以上仅为本申请的优选实施例而已，并不用于限制本申请，对于本领域的技术人员来说，本申请可以有各种更改和变化。凡在本申请的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
工业实用性
通过本申请的技术方法，本发明至少有如下有益效果：
应用本发明的技术方案，通过创新性地结合多肽芯片技术筛选获得一批与冠状病毒感染(尤其是SARS-Cov-2病毒感染)特异性相关的多肽，这些多肽能够被用来制备抗原、抗体、试剂盒等相关检测试剂以及多肽疫苗、核酸疫苗、蛋白重组疫苗等相关疫苗产品，从而为防控此类病毒的传染和流行提供更有力的工具。

Claims (19)

  1. 一种抗原表位多肽的筛选方法，其中，所述筛选方法包括：
    利用目的冠状病毒的全部蛋白组序列进行抗原表位预测，得到预测表位区域；
    利用多肽芯片技术筛选出对所述目的冠状病毒感染的阳性血清样本与对照血清样本中存在差异响应的肽段，记为差异肽段；
    将所述差异肽段与所述目的冠状病毒的全部蛋白组序列进行比对，获得第一保守基序区域；
    从所述预测表位区域和所述第一保守基序区域中筛选出满足表位筛选条件的区域，得到所述抗原表位多肽；
    其中，所述表位筛选条件包括位于所述目的冠状病毒的非磷酸化区域和/或膜外区域。
  2. 根据权利要求1所述的筛选方法，其中，利用所述目的冠状病毒的全部蛋白组序列进行抗原表位预测，得到预测表位区域包括：
    利用所述目的冠状病毒的全部蛋白组序列，采用多种不同方法进行抗原表位预测，并筛选长度为8～20个，优选10～15个氨基酸的表位，得到候选预测表位；
    根据特定人群中的HLA所能递呈的表位和/或亲疏水性，对所述候选预测表位进行筛选，得到所述预测表位区域；
    优选地，从所述候选预测表位中筛选出中国人群中HLA所能递呈的表位，和/或从所述候选预测表位中去除疏水性高于第一疏水阈值的表位，得到所述预测表位区域；
    优选地，所述疏水性高于第一疏水阈值的表位指疏水性氨基酸占比大于45％且疏水性得分大于3的表位。
  3. 根据权利要求1所述的筛选方法，其中，利用多肽芯片的方法筛选出对所述目的冠状病毒感染的阳性血清样本与对照血清样本存在差异响应的肽段，记为差异肽段包括：
    选取所述目的冠状病毒感染的阳性血清样本、阴性对照血清样本和其他肺部疾病对照血清样本，所述其他肺部疾病指所述目的冠状病毒之外的病毒感染引起的肺部疾病；
    采用所述多肽芯片技术的方法，将所述阳性血清样本、所述阴性对照血清样本和所述其他肺部疾病对照血清样本与多肽阵列芯片结合，获得结合肽段响应的信号值；
    针对每个所述结合肽段，计算所述阳性血清样本的所述信号值与所述阴性对照血清样本的所述信号值之间存在差异时的p值，记为第一p值，同时计算所述阳性血清样本的所述信号值与所述其他肺部疾病对照血清样本的所述信号值存在差异时的p值，记为第二p值；
    保留所有符合所述第一p值和所述第二p值同时满足差异阈值的结合肽段，从而得到所述差异肽段；
    优选所述差异阈值为<0.05。
  4. 根据权利要求3所述的筛选方法，其中，对所述结合肽段的信号值进行log10转换，以转换后的log值为特征，通过单尾T检验，
    计算各特征在所述阳性血清样本与所述阴性对照血清样本之间存在差异时的p值，并对所述p值进行多重假设检验校正，得到所述第一p值；
    同时计算相应的所述特征在所述阳性血清样本与所述其他肺部疾病对照血清样本之间存在差异时的p值，并对所述p值进行多重假设检验校正，记为所述第二p值；
    筛选同时满足所述第一p值小于所述差异阈值且所述第二p值小于所述差异阈值的所述结合肽段，从而得到所述差异肽段。
  5. 根据权利要求3所述的筛选方法，其中，将所述差异肽段与所述目的冠状病毒的全部蛋白组序列进行比对，获得第一保守基序区域包括：
    以单个氨基酸为单位，计算覆盖所述氨基酸且与所述氨基酸匹配的所述结合肽段的信号值在所述阳性血清样本与在所述阴性对照血清样本和所述其他肺部疾病对照血清样本存在差异的p1值的分布，同时计算覆盖所述氨基酸且与所述氨基酸不匹配的所述结合肽段的信号值在所述阳性血清样本与在所述阴性对照血清样本和所述其他肺部疾病对照血清样本存在差异的p2值的分布，所述p1值的分布显著低于所述p2值的分布所述氨基酸为第一保守位点；
    将所述差异肽段与所述目的冠状病毒的全部蛋白组序列进行比对，并从能够比对上的区域中选择具有所述第一保守位点且疏水性低于第二疏水阈值的区域，从而获得所述第一保守基序区域；
    优选地，所述疏水性低于第二疏水阈值的区域指疏水性氨基酸占比小于等于45％且疏水性得分小于等于3的区域；
    优选所述差异肽段为能够完全比对到所述目的冠状病毒的全部蛋白组序列上的差异肽段。
  6. 根据权利要求3所述的筛选方法，其中，在从所述预测表位区域和所述第一保守基序区域中筛选出满足表位筛选条件的区域之前，所述筛选方法还包括：将所述差异肽段与冠状病毒家族的蛋白序列进行比对，获得第二保守基序区域；
    优选地，将所述差异肽段与冠状病毒家族的蛋白序列进行比对，获得所述第二保守基序区域包括：
    将所述差异肽段与所述冠状病毒家族的蛋白序列进行比对，并从能够比对上的区域中选择各氨基酸位点均满足如下区域筛选条件的区域，作为所述第二保守基序区域：
    覆盖所述氨基酸的所有所述差异肽段中，能够与所述氨基酸匹配的所述差异肽段的 比例满足匹配比例阈值；
    优选地，所述匹配比例阈值大于等于75％。
  7. 根据权利要求6所述的筛选方法，其中，所述表位筛选条件还包括如下至少之一：
    (a)与所述第二保守基序区域相重叠；以及
    (b)与人类蛋白组序列比对得分低于比对阈值；
    (c)满足如下多个性能指标：1)所述差异肽段覆盖条数≥3；2)亲水性满足在亲水阈值范围内；3)可及性分值、Beta转角及多重比对分值均位于前100；
    所述比对得分低于比对阈值指a/b≤0.8，其中，a为待筛选区域的序列与人类蛋白组序列进行比对的匹配度得分，b为所述待筛选区域的序列与所述目的冠状病毒的全部蛋白组序列进行比对的匹配度得分；
    优选地，从所述预测表位区域和所述第一保守基序区域中筛选出满足所述表位筛选条件的区域，得到所述抗原表位多肽包括：
    将所述预测表位区域和所述第一保守基序区域根据如下至少之一的合并条件进行合并：1)两区域间为包含关系；2)两区域至少被两种不同方法预测为抗原表位区域，得到第一候选表位区域；
    从所述第一候选表位区域中筛选与所述第二保守基序区域重叠的区域，作为第二候选表位区域；
    从所述第二候选表位区域中筛选与人类蛋白组序列的比对得分低于所述比对阈值的区域，作为第三候选表位区域；
    从所述第三候选表位区域中筛选并保留所述目的冠状病毒蛋白组序列中的所述非磷酸化区域和/或膜外区域，作为第四候选表位区域；
    根据可及性、beta转角、亲水性、所述差异肽段的覆盖条数及多重比对结果，对所述第四候选表位区域进行综合排序后择优选择，得到所述目的冠状病毒的抗原表位多肽；
    更优选地，在所述择优选择之后，所述筛选方法还包括去除包含突变的区域；
    优选地，所述目的冠状病毒为SARS-CoV-2。
  8. 一种抗原表位多肽的筛选装置，其中，所述筛选装置包括：
    表位预测模块，用于利用目的冠状病毒的全部蛋白组序列进行抗原表位预测，得到预测表位区域；
    差异肽段筛选模块，用于利用多肽芯片技术筛选出对所述目的冠状病毒感染的阳性 血清样本与对照血清样本存在差异响应的肽段，记为差异肽段；
    第一区域筛选模块，用于将所述差异肽段与所述目的冠状病毒的全部蛋白组序列进行比对，获得第一保守基序区域；
    第三区域筛选模块，用于从所述预测表位区域和所述第一保守基序区域中筛选能够满足表位筛选条件的区域，得到所述抗原表位多肽：
    其中，所述表位筛选条件包括位于所述目的冠状病毒的非磷酸化区域和/或膜外区域。
  9. 根据权利要求8所述的筛选装置，其中，所述表位预测模块包括：
    候选表位第一筛选模块，用于利用所述目的冠状病毒的全部蛋白组序列，采用多种不同方法进行抗原表位预测，并筛选长度为8～20个，优选10～15个氨氨基酸的表位，得到候选预测表位；
    候选表位第二筛选模块，用于根据特定人群中的HLA所能递呈的表位和/或亲疏水性，对候选预测表位进行筛选，得到所述预测表位区域。
  10. 根据权利要求9所述的筛选装置，其中，所述候选表位第二筛选模块包括：
    人群表位筛选模块，用于从所述候选预测表位中筛选出中国人群中HLA所能递呈的表位；和/或，
    疏水性筛选模块，用于从所述候选预测表位中去除疏水性高于第一疏水阈值的表位，得到所述预测表位区域；
    优选地，所述疏水性高于第一疏水阈值的表位指疏水性氨基酸占比大于45％且疏水性得分大于3的表位。
  11. 根据权利要求8所述的筛选装置，其中，所述差异肽段筛选模块包括第一筛选模块，所述第一筛选模块包括：
    样本选择单元，用于选取所述目的冠状病毒感染的阳性血清样本、阴性对照血清样本和其他肺部疾病对照血清样本，其他肺部疾病指目的冠状病毒之外的病毒感染引起的肺部疾病；
    信号获取单元，用于采用多肽芯片的方法，将所述阳性血清样本、所述阴性对照血清样本和所述其他肺部疾病对照血清样本与多肽阵列芯片结合，获得结合肽段响应的信号值；
    差异肽段筛选单元，用于针对每个所述结合肽段，计算所述阳性血清样本的信号值与所述阴性对照血清样本的信号值之间存在差异时的p值，记为第一p值，同时计算所述阳性血清样本的信号值与所述其他肺部疾病对照血清样本的信号值存在差异时的p值，记为第二p值；保留所有符合所述第一p值和所述第二p值同时满足差异阈值的所述结合肽段，从而得到所述差异肽段；
    优选所述差异阈值<0.05。
  12. 根据权利要求11所述的筛选装置，其中，所述差异肽段筛选单元包括：
    信号转换子单元，用于对所述结合肽段的信号值进行log10转换；
    差异肽段筛选子单元，用于以转换后的log值为特征，通过单尾T检验，计算各特征在所述阳性血清样本与所述阴性对照血清样本之间存在差异时的p值，并对p值进行多重假设检验校正，得到所述第一p值；同时计算相应的特征在所述阳性血清样本与所述其他肺部疾病对照血清样本之间存在差异时的p值，并对p值进行多重假设检验校正，记为所述第二p值；筛选同时满足所述第一p值小于所述差异阈值且所述第二p值小于所述差异阈值的结合肽段，从而得到所述差异肽段。
  13. 根据权利要求11所述的筛选装置，其中，所述第一区域筛选模块包括：
    保守位点筛选模块，用于以单个氨基酸为单位，计算覆盖氨基酸且与氨基酸匹配的所述结合肽段的信号值在所述阳性血清样本和所述阴性对照血清样本存在差异的p1值的分布，同时计算覆盖氨基酸且与氨基酸不匹配的所述结合肽段的信号值在所述阳性血清样本和所述阴性对照血清样本存在差异的p2值的分布，将所述p1值的分布显著低于所述p2值的分布的氨基酸记为第一保守位点；
    第一保守基序筛选模块，用于将所述差异肽段与所述目的冠状病毒的全部蛋白组序列进行比对，并从能够比对上的区域中选择具有所述第一保守位点且疏水性低于第二疏水阈值的区域，从而获得所述第一保守基序区域；
    优选地，所述疏水性低于第二疏水阈值的区域指疏水性氨基酸占比小于等于45％且疏水性得分小于等于3的区域；
    优选所述差异肽段为能够完全比对到目的冠状病毒的全部蛋白组序列上的差异肽段。
  14. 根据权利要求8所述的筛选装置，其中，所述筛选装置还包括第二区域筛选模块，所述第二区域筛选模块包括：
    比对模块，用于将所述差异肽段与冠状病毒家族的蛋白序列进行比对；
    第二保守基序筛选模块，用于从能够比对上的区域中选择各氨基酸位点均满足如下区域筛选条件的区域，作为第二保守基序区域：覆盖氨基酸的所有差异肽段中，能够与氨基酸匹配的所述差异肽段的比例满足匹配比例阈值；
    优选地，所述匹配比例阈值大于等于75％。
  15. 根据权利要求14所述的筛选装置，其中，所述第三区域筛选模块中的表位筛选条件还包括如下至少之一：(i)与所述第二保守基序区域相重叠；(ii)与人类蛋白组序列比对得分低于比对阈值；(iii)满足如下多个性能指标：1)差异肽段覆盖条数≥3；2)亲水性满 足在亲水阈值范围内；3)可及性分值、Beta转角及多重比对分值均位于前100；其中，比对得分低于比对阈值指a/b≤0.8，其中，a为待筛选区域的序列与人类蛋白组序列进行比对的匹配度得分，b为待筛选区域的序列与目的冠状病毒的全部蛋白组序列进行比对的匹配度得分。
  16. 根据权利要求15所述的筛选装置，其中，所述第三区域筛选模块包括：
    合并模块，用于将所述预测表位区域和所述第一保守基序区域根据如下至少之一的合并条件进行合并：1)两区域间为包含关系；2)两区域至少被两种不同方法预测为抗原表位区域，得到第一候选表位区域；
    重叠筛选模块，用于从所述第一候选表位区域中筛选与所述第二保守基序区域重叠的区域，作为第二候选表位区域；
    比对筛选模块，用于从所述第二候选表位区域中筛选与人类蛋白组序列的比对得分低于第一阈值的区域，作为第三候选表位区域；
    非磷酸化及膜外区域筛选模块，用于从所述第三候选表位区域中筛选并保留所述目的冠状病毒蛋白组序列中的非磷酸化区域和膜外区域，作为第四候选表位区域；
    综合排序模块，用于根据可及性、beta转角、亲水性、所述差异肽段的覆盖条数及多重比对结果，对所述第四候选表位区域进行综合排序后择优选择，得到所述目的冠状病毒的抗原表位多肽。
  17. 根据权利要求16所述的筛选装置，其中，所述筛选装置还包括突变去除模块，用于从所述综合排序模块择优选择的区域中去除包含突变的区域，得到所述目的冠状病毒的抗原表位多肽；
    优选地，所述目的冠状病毒为SARS-CoV-2。
  18. 一种存储介质，其中，所述存储介质包括存储的程序，其中，在所述程序运行时控制所述存储介质所在设备执行权利要求1至7中任意一项所述的抗原表位多肽的筛选方法。
  19. 一种处理器，其中，所述处理器用于运行程序，其中，所述程序运行时执行权利要求1至7中任意一项所述的抗原表位多肽的筛选方法。
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