WO2022073360A1 - 构建新型冠状病毒疫苗的方法及其应用 - Google Patents

构建新型冠状病毒疫苗的方法及其应用 Download PDF

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WO2022073360A1
WO2022073360A1 PCT/CN2021/101458 CN2021101458W WO2022073360A1 WO 2022073360 A1 WO2022073360 A1 WO 2022073360A1 CN 2021101458 W CN2021101458 W CN 2021101458W WO 2022073360 A1 WO2022073360 A1 WO 2022073360A1
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protein
novel coronavirus
receptor binding
binding region
amino acid
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French (fr)
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刘远东
孟薇
陈晴晴
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广州东盛生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells

Definitions

  • the present invention relates to the field of biotechnology, in particular, to a method for constructing a novel coronavirus vaccine, more specifically, to the receptor binding region of the novel coronavirus S protein, the novel coronavirus S protein, nucleic acid molecules, expression vectors, recombinant viruses, vaccine.
  • Attenuated/inactivated vaccines there are five main types of vaccines: attenuated/inactivated vaccines, recombinant protein vaccines, viral vector vaccines, mRNA vaccines and DNA vaccines. These vaccines are designed based on the original protein or nucleic acid sequence of the virus.
  • Coronaviruses belong to the Coronaviridae family of the order Nidovirus, and are enveloped single-stranded, positive-stranded RNA viruses, which are divided into three genera, ⁇ , ⁇ and ⁇ . Coronavirus particles are spherical or irregular in shape, with an envelope and a size of 80-120nm.
  • the 5' end of its genome has a cap structure, followed by 6-10 open reading frames (Open reading frames, ORFs).
  • the first reading frame occupying 2/3 of the genome encodes replicase, and the other 1/3 of the genome mainly encodes structural proteins, generally including spike protein (spike, S), small envelope protein (envelope, E), envelope protein (membrance, M), nucleocapsid (N).
  • the E protein and M protein are mainly involved in the assembly process of the virus, and the N protein wraps the genome to form a nucleoprotein complex.
  • Coronaviruses mainly mediate virus invasion through the binding of Spike glycoprotein (S glycoprotein) to host cell receptors and determine viral tissue or host tropism.
  • S glycoprotein Spike glycoprotein
  • the spike glycoprotein (S protein) is a large trimeric transmembrane glycoprotein that forms a special corolla structure on the virus surface.
  • S protein which recognizes host cell receptors and mediates membrane fusion, is essential for viral particle entry into cells and is a key factor in viral infection of host cells.
  • the isoelectric point of the local amino acid residues in the new coronavirus protein structure is too high, and the dissociation degree and charge are high under the pH conditions in vivo. These proteins are too toxic in vivo as vaccines and have poor safety.
  • An object of the present invention is to propose a method for a novel coronavirus vaccine with high immunogenicity and low toxicity.
  • the invention proposes a method for constructing a novel coronavirus vaccine.
  • the method comprises: (a) performing amino acid substitution on the receptor binding domain (RBD, Receptor binding domain) of the wild-type novel coronavirus S protein, so as to obtain a candidate S-RBD sequence, wherein Compared with the receptor binding region of the wild-type novel coronavirus S protein, the candidate S-RBD sequence has a reduced isoelectric point; (b) based on the candidate S-RBD sequence and the wild-type novel coronavirus The three-dimensional structure of the receptor binding region of the S protein determines the target S-RBD sequence; (c) constructing a novel coronavirus vaccine based on the target S-RBD sequence.
  • the amino acid change of the receptor binding region of the new coronavirus S protein will lead to changes in the species tropism and infection characteristics of the virus.
  • the isoelectric point can reduce the degree of dissociation of the receptor-binding region of the new coronavirus S protein under pH conditions in vivo and reduce its toxicity. Selecting appropriate amino acid sites and candidate replacement amino acids can ensure that the S protein is not changed or less changed. Or the three-dimensional structure of the receptor binding region, thereby achieving the purpose of retaining the immunogenicity of the new coronavirus while reducing the toxicity of the new coronavirus.
  • the novel coronavirus vaccine includes at least one selected from the group consisting of mRNA vaccine, DNA vaccine, protein recombinant vaccine, inactivated vaccine, attenuated vaccine, and viral vector vaccine.
  • part of the amino acids in the receptor binding region of the wild-type new coronavirus S protein are replaced to reduce the toxicity of the new coronavirus without affecting its three-dimensional structure, retaining its immunogenicity, and Preparation of a new coronavirus vaccine.
  • the type of new coronavirus vaccine is not particularly limited, as long as the receptor binding region of the modified new coronavirus S protein can be produced in the organism, and it is immunogenic and can stimulate the organism to produce corresponding antibodies.
  • step (a) in step (a), it further includes: (a-1) determining the basic amino acid in the receptor binding region of the wild-type novel coronavirus S protein; (a-2) using the candidate amino acid, at least one said basic amino acid is substituted so as to lower the isoelectric point of said at least one basic amino acid site in order to obtain said candidate S-RBD sequence.
  • the basic amino acids include arginine, lysine, and histidine, and their isoelectric points are: 10.76, 9.74, and 7.59, respectively, and the specific information of the basic amino acids is shown in Table 1 The specific information of other amino acids with isoelectric points less than 7 is shown in Table 2.
  • the amino acid to be replaced is replaced with a candidate amino acid, and the isoelectric point of the candidate amino acid is lower than the isoelectric point of the replaced amino acid, thereby ensuring the dissociation degree of the amino acid sequence after the replacement Lower than the original sequence, thereby reducing the dissociation degree of the receptor binding region of the new coronavirus S protein in the organism, reducing the binding ability of the receptor (such as ACE2) in the organism, thereby reducing the toxicity of the new coronavirus .
  • the receptor such as ACE2
  • a basic amino acid enrichment region is further determined, and in step (a-2), at least one of the basic amino acid enrichment regions of the basic amino acid enrichment region is further determined.
  • amino acid substitutions According to the method of the embodiment of the present invention, all basic amino acids in the receptor binding region of the new coronavirus S protein can be replaced, or the basic amino acids in the receptor binding region of the new coronavirus S protein can be randomly selected for replacement without pertinence, Substituted with other amino acids with lower isoelectric points, the inventor found through a lot of research that the basic amino acid-enriched region in the receptor binding region of the new coronavirus S protein was selected, and the basic amino acids in the basic amino acid-enriched region were selected. Substitution can more efficiently reduce the affinity of the new coronavirus with receptors (such as ACE2) in the organism.
  • receptors such as ACE2
  • the basic amino acid-rich region contains at least two connected basic amino acids, and the two connected basic amino acids are separated by no more than three amino acids.
  • an area with dense basic amino acids is defined as a basic amino acid-enriched area, and the interval between two connected basic amino acids in the basic amino acid-enriched area is no more than three amino acids.
  • the short peptide formed by the amino acid-enriched region has a higher degree of dissociation in vivo, and then replacing the basic amino acid in the basic amino acid-enriched region that meets the above description can more efficiently reduce the novel coronavirus or its S protein receptor.
  • the degree of dissociation of the binding region in the organism thereby reducing the affinity of the novel coronavirus or its S protein receptor binding region protein with the receptor in the organism.
  • the basic amino acid-rich region includes the amino acid sequence shown in SEQ ID NO: 1 and/or the amino acid sequence shown in SEQ ID NO: 2.
  • step (a-2) at least one arginine is replaced with histidine.
  • the isoelectric point of arginine is 10.76
  • the isoelectric point of histidine is 7.59.
  • step (b) it further includes: (b-1) establishing a three-dimensional structure of the candidate S-RBD sequence; (b-2) applying the obtained data obtained in step (b-1) to The three-dimensional structure is compared with the three-dimensional structure of the receptor binding region of the wild-type novel coronavirus S protein in order to obtain a root mean square deviation value; (b-3) selecting the candidate whose root mean square deviation value is less than a predetermined threshold
  • the S-RBD sequence is used as the target S-RBD sequence.
  • the three-dimensional structure of the candidate S-RBD sequence and the three-dimensional structure of the receptor binding region of the wild-type new coronavirus S protein are respectively constructed, and the two three-dimensional structures are compared, and the three-dimensional structure of the wild-type new coronavirus is selected.
  • the candidate S-RBD sequence with the closest three-dimensional structure of the receptor binding region of the virus S protein is used as the final sequence, which can ensure that the three-dimensional structure of the final sequence is the closest to the wild type, and minimize the S-RBD sequence other than the degree of dissociation.
  • the change of the characteristics can further ensure the immunogenicity of the virus carrying the above S-RBD sequence, so that the vaccine has higher immunogenicity after being injected into the body, and at the same time, the toxicity is reduced.
  • the predetermined threshold does not exceed 1.93, for example, does not exceed 1.2, preferably does not exceed 1.1, such as 1.
  • the S-RBD sequence screened by the above threshold has a close three-dimensional structure to the wild-type sequence, which can ensure the immunogenicity of the novel coronavirus carrying the screened S-RBD sequence to the greatest extent.
  • the present invention proposes a receptor binding region of the novel coronavirus S protein.
  • the receptor binding region of the novel coronavirus S protein has a reduced isoelectric point compared with the receptor binding region of the wild-type novel coronavirus S protein, and the novel coronavirus S protein has a reduced isoelectric point.
  • the three-dimensional structure of the receptor binding region of , and the three-dimensional structure of the receptor binding region of the wild-type novel coronavirus S protein have a root mean square deviation value that does not exceed a predetermined threshold.
  • the receptor binding region of the new coronavirus S protein according to the embodiment of the present invention has a weaker receptor binding ability, so that the novel coronavirus carrying the embodiment of the present invention has a weaker receptor binding ability.
  • the novel coronavirus in the receptor binding region of the coronavirus S protein has reduced receptor binding ability and lower toxicity in the organism, but at the same time, the receptor binding region of the novel coronavirus S protein in the embodiment of the present invention is similar to the wild-type novel coronavirus.
  • the three-dimensional structure of the receptor binding region of the S protein is similar, and other properties of the protein are not affected, so that other properties of the novel coronavirus carrying the receptor binding region of the novel coronavirus S protein of the embodiment of the present invention are not affected, especially It is the immunogenicity in the living body. After entering the living body, it can normally cause the immune response of the living body and produce a large number of antibodies.
  • the predetermined threshold value is 1.93, such as not exceeding 1.2, preferably not exceeding 1.1, such as 1.
  • the receptor binding region of the novel coronavirus S protein when the root mean square deviation value between its three-dimensional structure and the three-dimensional structure of the receptor binding region of the wild-type novel coronavirus S protein does not exceed the above-mentioned threshold, Its three-dimensional structure is similar to the three-dimensional structure of the receptor binding region of the wild-type novel coronavirus S protein, which can ensure that the immunogenicity of the novel coronavirus carrying the receptor binding region of the novel coronavirus S protein of the embodiment of the present invention is not affected. Influence.
  • At least one basic amino acid in the receptor binding region of the novel coronavirus S protein according to an embodiment of the present invention is replaced by Amino acids with lowered isoelectric points.
  • the basic amino acids, such as histidine, arginine, and lysine, in the receptor binding region of the wild-type novel coronavirus S protein according to the embodiment of the present invention can be reduced after being replaced with amino acids with a lower isoelectric point.
  • the degree of dissociation of the polypeptide in the receptor binding region of the S protein in the organism reduces its binding ability to the receptor in the organism.
  • the basic amino acids in the basic amino acid-enriched region in the receptor binding region of the novel coronavirus S protein are replaced, and one, several or all basic amino acids can also be randomly replaced.
  • At least the amino acid sequence shown in SEQ ID NO: 1 and/or the amino acid sequence shown in SEQ ID NO: 2 is at least One arginine was replaced with histidine.
  • the receptor binding region of the S protein of the novel coronavirus according to the embodiment of the present invention after the arginine in the above two sequences is replaced by histidine with a lower isoelectric point, the polypeptide segment formed by the above two sequences is in biological The degree of dissociation in vivo was significantly reduced, and the three-dimensional structure was similar to that of the wild type, and other properties were not significantly affected except for the reduced degree of dissociation.
  • sequence of the receptor binding region of the novel coronavirus S protein is shown in SEQ ID NO:3 and/or SEQ ID NO:4.
  • the sequence of the receptor binding region of the wild-type novel coronavirus S protein is shown in SEQ ID NO:5.
  • the present invention proposes a novel coronavirus S protein.
  • the novel coronavirus S protein contains the receptor binding region of the novel coronavirus S protein proposed in the second aspect of the present invention.
  • the present invention provides a nucleic acid molecule encoding the receptor binding region of the novel coronavirus S protein proposed in the second aspect of the present invention or the novel coronavirus proposed in the fourth aspect of the present invention.
  • Coronavirus S protein According to an embodiment of the present invention, the nucleic acid molecule is RNA or DNA.
  • the nucleic acid molecule can form the receptor binding region of the novel coronavirus S protein proposed in the second aspect of the present invention, the novel coronavirus S protein proposed in the fourth aspect of the present invention, and carry The novel coronavirus of the above-mentioned S protein, and the novel coronavirus carrying the receptor binding region of the above-mentioned S protein.
  • the present invention provides an expression vector carrying the nucleic acid molecule proposed in the fourth aspect of the present invention.
  • the expression vector can be expressed in cells, bacteria, yeast, or organisms that are capable of infecting humans, mice, rats, rabbits, camels, alpacas, horses, donkeys, mules, etc. Organisms of the novel coronavirus.
  • the present invention provides a recombinant virus carrying the nucleic acid molecule proposed in the fourth aspect of the present invention.
  • the present invention provides a vaccine.
  • the vaccine contains at least one of the following: the receptor binding region of the novel coronavirus S protein proposed in the second aspect of the present invention; the novel coronavirus S protein proposed in the third aspect of the present invention; The nucleic acid molecule proposed in the fourth aspect of the present invention; the expression vector proposed in the fifth aspect of the present invention; and the expression vector recombinant virus proposed in the sixth aspect of the present invention.
  • 1 is a three-dimensional structural model diagram of the RBD of SARS-CoV-2 according to an embodiment of the present invention
  • FIG. 2 is a three-dimensional structural model diagram of the RBD of SARS-CoV according to an embodiment of the present invention
  • FIG. 3 is a three-dimensional structural model diagram of the S protein receptor binding region of the wild-type novel coronavirus according to Embodiment 1 and Embodiment 2 of the present invention using Software 1;
  • Fig. 4 is the three-dimensional structural model diagram of the novel coronavirus S protein receptor binding region after the transformation according to the embodiment of the present invention 1 using software 1;
  • Fig. 5 is the three-dimensional structural model comparison diagram of the wild-type and modified novel coronavirus S protein receptor binding region according to the embodiment of the present invention 1 using software 2, wherein Fig. a is the wild-type novel coronavirus S protein receptor binding region The three-dimensional structural model of , Figure b is the three-dimensional structural model of the modified novel coronavirus S protein receptor binding region;
  • Fig. 6 is the isoelectric point result before amino acid substitution according to the embodiment of the present invention 1;
  • Fig. 7 is the isoelectric point result after the amino acid substitution according to the embodiment of the present invention 1;
  • Fig. 8 is the three-dimensional structural model diagram of the novel coronavirus S protein receptor binding region after the transformation according to the embodiment of the present invention 2 using software 1;
  • FIG. 9 is a comparison diagram of the three-dimensional structural model of the wild-type and modified new coronavirus S protein receptor binding regions according to Example 2 of the present invention using software 2, wherein FIG. c is the three-dimensional RBD protein of wild-type SARS-CoV-2. Conformation model, Figure d is the three-dimensional conformation model of the RBD protein of the modified SARS-CoV-2;
  • Figure 10 is the isoelectric point result before amino acid substitution according to Example 2 of the present invention.
  • Figure 11 is the isoelectric point result after amino acid substitution according to Example 2 of the present invention.
  • Figure 12 shows the results of the ELISA detection of antigen specificity according to Example 3 of the present invention.
  • first and second are only used for descriptive purposes, and should not be construed as indicating or implying relative importance or implying the number of indicated technical features. Thus, a feature delimited with “first”, “second” may expressly or implicitly include at least one of that feature.
  • plurality means at least two, such as two, three, etc., unless otherwise expressly and specifically defined.
  • the terms “installed”, “connected”, “connected”, “fixed” and other terms should be understood in a broad sense, for example, it may be a fixed connection or a detachable connection , or integrated; it can be a mechanical connection or an electrical connection; it can be directly connected or indirectly connected through an intermediate medium, it can be the internal connection of two elements or the interaction relationship between the two elements, unless otherwise specified limit.
  • installed may be a fixed connection or a detachable connection , or integrated; it can be a mechanical connection or an electrical connection; it can be directly connected or indirectly connected through an intermediate medium, it can be the internal connection of two elements or the interaction relationship between the two elements, unless otherwise specified limit.
  • the present invention proposes a method for constructing a novel coronavirus vaccine.
  • the method comprises: (a) performing amino acid substitutions on the receptor binding region of the wild-type novel coronavirus S protein, so as to obtain a candidate S-RBD sequence, which is compatible with the wild-type novel coronavirus Compared with the receptor binding region of the S protein, the candidate S-RBD sequence has a reduced isoelectric point; (b) based on the candidate S-RBD sequence and the receptor binding region of the wild-type new coronavirus S protein The three-dimensional structure of the target S-RBD sequence is determined; (c) based on the target S-RBD sequence, a novel coronavirus vaccine is constructed.
  • an amino acid with a lower isoelectric point than the basic amino acid to be replaced is selected to replace it, and the replaced S-RBD sequence is compared with that of the wild-type new coronavirus.
  • the three-dimensional structure of the receptor binding region of the S protein was compared, and the S-RBD sequence similar to the three-dimensional structure of the receptor binding region of the wild-type new coronavirus S protein was selected as the final sequence, and a new coronavirus vaccine was constructed.
  • the novel coronavirus vaccine includes at least one selected from the group consisting of mRNA vaccine, DNA vaccine, protein recombinant vaccine, inactivated vaccine, attenuated vaccine, and viral vector vaccine.
  • the vaccine can be in the form of mRNA, or in the form of DNA.
  • the proteins expressed by the mRNA vaccine and the DNA vaccine in vivo carry at least the receptor binding region of the modified new coronavirus S protein, which is compatible with the in vivo protein.
  • the receptor affinity is reduced, but the three-dimensional configuration is not affected; protein recombinant vaccines carry at least the receptor binding region of the modified new coronavirus S protein; inactivated vaccines contain receptors carrying the modified new coronavirus S protein.
  • Inactivated 2019-nCoV in the body binding region, or a receptor binding region fragment carrying the modified 2019-nCoV S protein; the attenuated vaccine and viral vector vaccine also carry at least the receptor binding of the modified 2019-nCoV S protein Area.
  • step (a) in step (a), it further includes: (a-1) determining the basic amino acid in the receptor binding region of the wild-type novel coronavirus S protein; (a-2) using the candidate amino acid, at least one said basic amino acid is substituted so as to lower the isoelectric point of said at least one basic amino acid site in order to obtain said candidate S-RBD sequence.
  • the candidate amino acids are not limited to amino acids with an isoelectric point less than 7.
  • the amino acid to be replaced is determined to be arginine
  • its isoelectric point is 10.76
  • lysine and histidine are It can also be used as its candidate amino acid to replace arginine with lysine and histidine, which can reduce the dissociation degree of the sequence where the replaced amino acid is located in the organism, thereby reducing the receptor binding region of the new coronavirus S protein.
  • the affinity of receptor binding in an organism is determined to be arginine, its isoelectric point is 10.76, lysine and histidine are It can also be used as its candidate amino acid to replace arginine with lysine and histidine, which can reduce the dissociation degree of the sequence where the replaced amino acid is located in the organism, thereby reducing the receptor binding region of the new coronavirus S protein.
  • the affinity of receptor binding in an organism is determined to be arginine, its isoelectric point is 10.76
  • lysine and histidine are It can also be used as its candidate amino acid to replace
  • step (b) it further includes: (b-1) establishing a three-dimensional structure of the candidate S-RBD sequence; (b-2) applying the obtained data obtained in step (b-1) to The three-dimensional structure is compared with the three-dimensional structure of the receptor binding region of the wild-type novel coronavirus S protein in order to obtain a root mean square deviation value; (b-3) selecting the candidate whose root mean square deviation value is less than a predetermined threshold
  • the S-RBD sequence is used as the target S-RBD sequence.
  • the three-dimensional structure of the candidate S-RBD sequence and the three-dimensional structure of the receptor binding region of the wild-type new coronavirus S protein are respectively constructed, and the two three-dimensional structures are compared, and the three-dimensional structure of the wild-type new coronavirus is selected.
  • the candidate S-RBD sequence with the closest three-dimensional structure of the receptor binding region of the virus S protein is used as the final sequence, which can ensure that the three-dimensional structure of the final sequence is the closest to the wild type, and minimize the S-RBD sequence other than the degree of dissociation.
  • the change of the characteristics can further ensure the immunogenicity of the virus carrying the above S-RBD sequence, so that the vaccine has higher immunogenicity after being injected into the body, and at the same time, the toxicity is reduced.
  • the predetermined threshold does not exceed 1.93, for example, does not exceed 1.2, preferably does not exceed 1.1, such as 1.
  • the determination of the threshold value is determined based on the three-dimensional structure RMSD of the atypical pneumonia virus SARS-CoV and the novel coronavirus SARS-CoV-2.
  • SARS-CoV-2 and SARS-CoV belong to the same genus of viruses with similar structures, and SARS-CoV has a lower receptor affinity than SARS-CoV-2.
  • the mutated SARS-CoV-2 S-RBD protein is similar to wild-type SARS-CoV-2.
  • the 2RBD protein difference should be smaller than the RMSD value of the RBD of SARS-Cov-2 and the RMSD of SARS-Cov RBD in order to ensure the structural similarity between the two and to recognize the AEC2 receptor.
  • software 1 https://swissmodel.expasy.org/interactive
  • software 2 http://superpose.wishartlab.com/
  • RMSD root mean square deviation
  • the RBD of SARS-CoV-2 has two basic amino acid-enriched regions , the RBD of SARS-CoV has one, and the RBD of MERS-CoV has no basic amino acid-enriched region. Based on the above characteristics, it can also be shown that SARS-CoV-2 has the strongest infectivity in the process of virus infection of susceptible people, and It has the same receptor as SARS-CoV, both of which are ACE2, and the protein structure of the two is also similar.
  • the present invention also proposes a receptor binding region of a novel coronavirus S protein, a novel coronavirus S protein, a nucleic acid molecule, an expression vector, a recombinant virus, and a vaccine, which have been It is described in the Summary of the Invention and will not be repeated here.
  • Example 1 NRKRIS (SEQ ID NO: 1) ⁇ NHKHIS (SEQ ID NO: 6)
  • the arginine in the sequence of SEQ ID NO: 1 was replaced by histidine to obtain the sequence of SEQ ID NO: 6, using software 1 (https://swissmodel.expasy .org/interactive) respectively carry out the three-dimensional structure modeling of the RBD sequences (SEQ ID NO: 5, SEQ ID NO: 3) carrying the two, and the three-dimensional structure model is shown in Figure 3 and Figure 4, wherein Figure 3 is the three-dimensional conformation model of the RBD protein of the wild-type SARS-CoV-2, and Figure 4 is the three-dimensional conformation model of the RBD protein of the modified SARS-CoV-2.
  • the isoelectric point calculation tool (Peptide Analyzing Tool) to calculate the isoelectric points of the above-mentioned two RBD proteins, the isoelectric point results before the amino acid substitution are shown in Figure 6, and the isoelectric point results after the amino acid substitution are shown in Figure 7. The results showed that the isoelectric point was 12.5 before the amino acid substitution, and the isoelectric point was 10.1 after the amino acid substitution, and the isoelectric point was significantly reduced.
  • the isoelectric point calculation tool (Peptide Analyzing Tool) to calculate the isoelectric points of the above two RBD proteins, the isoelectric point results before amino acid substitution are shown in Figure 10, and the isoelectric point results after amino acid substitution are shown in Figure 11. The results showed that the isoelectric point was 12.5 before the amino acid substitution, and the isoelectric point was 10.6 after the amino acid substitution, and the isoelectric point was significantly reduced.
  • Example 3 ELISA experiment to verify the immunogenicity of RBD antibody after base substitution to wild-type RBD antigen
  • the expression vectors were constructed with the corresponding nucleic acid sequences of SEQ ID NO: 1 and SEQ ID NO: 6, respectively, and the corresponding proteins were expressed, and the obtained recombinant proteins were named WT-RBD and M6-RBD, respectively.
  • mice (strain BALB/c, female, 6-8 weeks) were immunized with WT-RBD and M6-RBD as antigens, respectively, and corresponding antibodies were obtained.
  • the first injection on the 1st day, 1 ⁇ g, 5 ⁇ g, 10 ⁇ g of recombinant protein WT-RBD and M6-RBD were mixed with 0.2 ⁇ g, 1 ⁇ g, and 2 ⁇ g of aluminum adjuvant, and the volume was made up to 200 ⁇ l with PBS buffer. Rat abdominal cavity. At the same time, 200 ⁇ l PBS buffer was used as a control;
  • each mouse was immunized for the second time according to the above method, and 1 ⁇ g, 5 ⁇ g and 10 ⁇ g of recombinant protein WT-RBD and M6-RBD and 0.2 ⁇ g, 1 ⁇ g and 2 ⁇ g of aluminum adjuvant were taken respectively.
  • 200 ⁇ l PBS buffer was used as a control;
  • Coating Dilute the recombinant protein WT-RBD to 0.01 ⁇ g/ml with PBS buffer (0.02M, pH 7.4), add 100 ⁇ l/well to the ELISA plate, and coat overnight at 4°C;
  • wash the plate discard the liquid in the well, add 0.5% Triton X-100 washing solution, 300 ⁇ l/well, wash 5 times, 1 minute each time;
  • Blocking Add 2.0% BSA blocking solution at 200 ⁇ l/well, place at room temperature for 90 minutes to block, discard the blocking solution, and wash 5 times, 1 minute each time;
  • Color development add 100 ⁇ l of TMB color development solution to each well, and develop color at room temperature for about 15 minutes in the dark;
  • Termination Add 50 ⁇ l of 2M sulfuric acid to each well to stop the reaction;
  • the microplate reader reads the absorbance value at 450nm.

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Abstract

提供一种构建新型冠状病毒疫苗的方法，包括：(a)对野生型新型冠状病毒S蛋白的受体结合区进行氨基酸置换，以便获得候选S-RBD序列，其中，与野生型新型冠状病毒S蛋白的受体结合区相比，候选S-RBD序列具有降低的等电点；(b)基于候选S-RBD序列与野生型新型冠状病毒S蛋白的受体结合区的三维结构，确定目标S-RBD序列；(c)基于目标S-RBD序列，构建新型冠状病毒疫苗。

Description

构建新型冠状病毒疫苗的方法及其应用
优先权信息
本申请请求2020年10月10日向中国国家知识产权局提交的、专利申请号为202011079455.9的专利申请的优先权和权益，并且通过参照将其全文并入此处。
技术领域
本发明涉及生物技术领域，具体地，涉及构建新型冠状病毒疫苗的方法，更具体地，涉及新型冠状病毒S蛋白的受体结合区、新型冠状病毒S蛋白、核酸分子、表达载体、重组病毒、疫苗。
背景技术
目前，疫苗主要有五大类：减活/灭活疫苗、蛋白重组疫苗、病毒载体疫苗、mRNA疫苗和DNA疫苗，这些疫苗都是根据病毒原始蛋白或核酸序列设计。
冠状病毒(Coronaviruses，CoV)属于尼多病毒目冠状病毒科，为有包膜的单股、正链RNA病毒，分为α，β和γ三个属。冠状病毒粒子呈球形或不规则形，有囊膜，大小为80-120nm。其基因组的5'端带有帽子结构，其后包含6-10个开放阅读框(Open reading frames，ORFs)。占据基因组2/3的第一个阅读框编码复制酶，基因组的另1/3主要编码结构蛋白，一般包括纤突蛋白(spike，S)、小包膜蛋白(envelope，E)、囊膜蛋白(membrance，M)、核蛋白(nucleocapsid，N)。E蛋白和M蛋白主要参与病毒的装配过程，N蛋白包裹基因组形成核蛋白复合体，
冠状病毒主要通过刺突糖蛋白(Spike glycoprotein,S glycoprotein)与宿主细胞受体结合介导病毒的入侵并决定病毒组织或宿主嗜性。刺突糖蛋白(S蛋白)是一类很大的三聚体跨膜糖蛋白，其在病毒表面形成特殊的花冠结构。S蛋白可识别宿主细胞受体并介导膜融合，对于病毒颗粒进入细胞至关重要，是病毒感染宿主细胞的关键因子。
新型冠状病毒蛋白结构中局部氨基酸残基等电点过高，在体内pH条件下解离度高、带电荷高。这些蛋白作为疫苗在体内会产生过高的毒性，安全性差。
因此，研发一种制备在生物体内毒性低的新型冠状病毒疫苗的方法及疫苗很有必要。
发明内容
本发明的一个目的在于提出一种免疫原性高、毒性低的新型冠状病毒疫苗的方法。
在本发明的第一方面，本
发明提出了一种构建新型冠状病毒疫苗的方法。根据本发明的实施例，所述方法包括：(a)对野生型新型冠状病毒S蛋白的受体结合区(RBD,Receptor binding domain)进行氨基酸置换，以便获得候选S-RBD序列，其中，与所述野生型新型冠状病毒S蛋白的受体结 合区相比，所述候选S-RBD序列具有降低的等电点；(b)基于所述候选S-RBD序列与所述野生型新型冠状病毒S蛋白的受体结合区的三维结构，确定目标S-RBD序列；(c)基于所述目标S-RBD序列，构建新型冠状病毒疫苗。根据本发明实施例的方法，新型冠状病毒S蛋白的受体结合区的氨基酸变化会导致病毒的种属嗜性和感染特性的变化，选择对该区域的氨基酸进行改造，降低S-RBD序列的等电点，可以降低新型冠状病毒S蛋白的受体结合区域在生物体内pH条件下的解离度，降低其毒性，选择合适的氨基酸位点和候选置换氨基酸可以保证不改变或者少改变S蛋白或者受体结合区的三维结构，进而达到保留新型冠状病毒的免疫原性，同时降低新型冠状病毒的毒性的目的。
另外，根据本发明上述实施例的方法，还可以具有如下附加的技术特征：
根据本发明的实施例，所述新型冠状病毒疫苗包括选自mRNA疫苗、DNA疫苗、蛋白重组疫苗、灭活疫苗、减活疫苗、病毒载体疫苗的至少之一。根据本发明实施例的方法，对野生型新型冠状病毒S蛋白的受体结合区内的部分氨基酸进行置换，以降低新型冠状病毒的毒性，同时不影响其三维结构，保留其免疫原性，并制备新型冠状病毒疫苗。新型冠状疫苗的种类不受特别限制，只要在生物体内能够产生改造后的新型冠状病毒S蛋白的受体结合区，并且具有免疫原性，可以刺激生物体产生相应抗体即可。
根据本发明的实施例，在步骤(a)中，进一步包括：(a-1)确定所述野生型新型冠状病毒S蛋白的受体结合区中的碱性氨基酸；(a-2)利用候选氨基酸，对至少一个所述碱性氨基酸进行置换，以便降低所述至少一个碱性氨基酸位点的等电点，以便获得所述候选S-RBD序列。根据本发明实施例的方法，所述碱性氨基酸包括精氨酸、赖氨酸、组氨酸，其等电点分别为：10.76、9.74、7.59，所述碱性氨基酸的具体信息如表1所示，其他等电点小于7的氨基酸具体信息如表2所示。当确定需要被置换的氨基酸后，利用候选氨基酸对需要被置换的氨基酸进行置换，被候选氨基酸的等电点低于被置换的氨基酸的等电点，进而保证置换之后的氨基酸序列的解离度低于原序列，进而使新型冠状病毒S蛋白的受体结合区在生物体内的解离度降低，使其与生物体内的受体(例如ACE2)的结合能力降低，从而降低新型冠状病毒的毒性。
根据本发明的实施例，在步骤(a-1)中，进一步确定碱性氨基酸富集区，在步骤(a-2)中，对所述碱性氨基酸富集区的至少一个所述碱性氨基酸进行置换。根据本发明实施例的方法，可以将新型冠状病毒S蛋白受体结合区的所有碱性氨基酸进行置换，或者没有针对性地随机选择新型冠状病毒S蛋白受体结合区的碱性氨基酸进行置换，置换为等电点较低的其他氨基酸，发明人经过大量的研究发现，选择新型冠状病毒S蛋白受体结合区内的碱性氨基酸富集区，对碱性氨基酸富集区内的碱性氨基酸进行置换可以更高效地降低新型冠状病毒与生物体内的受体(如ACE2)的亲和力。
根据本分发明的实施例，所述碱性氨基酸富集区含有至少两个相连的碱性氨基酸，所述两个相连的碱性氨基酸之间相隔不超过三个氨基酸。根据本发明实施例的方法，将碱性氨基酸密集的区域定义为碱性氨基酸富集区，碱性氨基酸富集区内的两个相连的碱性氨基酸之间相隔不超过三个氨基酸，碱性氨基酸富集区所形成的短肽在生物体内的解离度更高，进而替换符合上述描述的碱性氨基酸富集区内的碱性氨基酸可以更高效地降低新型冠状病毒或者其S蛋白受体结合区在生物体内的解离度，进而降低新型冠状病毒或者其S蛋白受体结合区蛋白与生物体内受体的亲和力。
根据本发明的实施例，所述碱性氨基酸富集区包括如SEQ ID NO:1所示氨基酸序列和/或如SEQ ID NO:2所示氨基酸序列。
碱性氨基酸富集区1氨基酸序列：NRKRIS(SEQ ID NO:1)；
碱性氨基酸富集区2氨基酸序列：RLFRKSNLK(SEQ ID NO:2)。
根据本发明的实施例，在步骤(a-2)中，将至少一个精氨酸置换为组氨酸。根据本发明实施例的方法，精氨酸的等电点为10.76，组氨酸的等电点为7.59，使用组氨酸替换精氨酸可以降低前述碱性氨基酸富集区短肽的解离度，进而降低其与生物体内受体的结合能力。
根据本发明的实施例，在步骤(b)中，进一步包括：(b-1)建立所述候选S-RBD序列的三维结构；(b-2)将步骤(b-1)中获得的所述三维结构与所述野生型新型冠状病毒S蛋白的受体结合区的三维结构进行比较，以便获得均方根偏差值；(b-3)选择均方根偏差值小于预定阈值的所述候选S-RBD序列作为目标S-RBD序列。根据本发明实施例的方法，分别构建候选S-RBD序列的三维结构及野生型新型冠状病毒S蛋白的受体结合区的三维结构，并将两个三维结构进行比较，选择与野生型新型冠状病毒S蛋白的受体结合区的三维结构最为接近的候选S-RBD序列作为最终序列，可以保证最终序列的三维结构与野生型最为相近，尽量减少S-RBD序列除解离度之外的其他特征的改变，进而保证携带上述S-RBD序列的病毒的免疫原性，使疫苗注射进体内后具有较高的免疫原性，同时毒性降低。
根据本发明的实施例，所述预定阈值不超过1.93，例如不超过1.2，优选不超过1.1，例如为1。根据本发明实施例的方法，采用上述阈值所筛选出的S-RBD序列与野生型序列具有接近的三维结构，可以最大程度保证携带筛选出的S-RBD序列的新型冠状病毒的免疫原性。
在本发明的第二方面，本发明提出了一种新型冠状病毒S蛋白的受体结合区。根据本发明的实施例，所述新型冠状病毒S蛋白的受体结合区与野生型新型冠状病毒S蛋白的受体结合区相比，具有降低的等电点，并且所述新型冠状病毒S蛋白的受体结合区的三维结构与野生型新型冠状病毒S蛋白的受体结合区的三维结构之间具有不超过预定阈值的均方根偏差值。根据本发明实施例的新型冠状病毒S蛋白的受体结合区与野生型新型冠状病毒 的S蛋白受体结合区相比，具有更弱的受体结合能力，使携带有本发明实施例的新型冠状病毒S蛋白的受体结合区的新型冠状病毒与生物体内的受体结合能力降低，毒性更低，但同时本发明实施例的新型冠状病毒S蛋白的受体结合区与野生型新型冠状病毒的S蛋白受体结合区的三维结构相近，不会影响蛋白的其他特性，使携带有本发明实施例的新型冠状病毒S蛋白的受体结合区的新型冠状病毒的其他特性不受影响，特别是在生物体内的免疫原性，在进入生物体内后，能够正常引起生物体的免疫反应，产生大量抗体。
另外，根据本发明上述实施例的新型冠状病毒S蛋白的受体结合区，还可以具有如下附加的技术特征：
根据本发明的实施例，所述预定阈值为1.93，例如不超过1.2，优选不超过1.1，例如为1。根据本发明实施例的新型冠状病毒S蛋白的受体结合区，其三维结构与野生型新型冠状病毒S蛋白的受体结合区的三维结构之间的均方根偏差值不超过上述阈值时，其三维结构与野生型新型冠状病毒S蛋白的受体结合区的三维结构相近，能够保证携带有本发明实施例的新型冠状病毒S蛋白的受体结合区的新型冠状病毒的免疫原性不受影响。
根据本发明的实施例，与所述野生型新型冠状病毒S蛋白的受体结合区相比，根据本发明实施例的新型冠状病毒S蛋白的受体结合区的至少一个碱性氨基酸被置换为等电点降低的氨基酸。根据本发明实施例的野生型新型冠状病毒S蛋白的受体结合区内的碱性氨基酸，如组氨酸、精氨酸、赖氨酸，被替换为等电点更低的氨基酸后可以降低S蛋白受体结合区多肽在生物体内的解离度，降低其与生物体内受体的结合能力。根据本发明实施例的新型冠状病毒S蛋白的受体结合区内碱性氨基酸富集区的碱性氨基酸被替换，也可以随机替换一个、几个或者全部的碱性氨基酸。
根据本发明的实施例，与所述野生型新型冠状病毒S蛋白的受体结合区相比，如SEQ ID NO:1所示氨基酸序列和/或如SEQ ID NO:2所示氨基酸序列的至少一个精氨酸被置换为组氨酸。根据本发明实施例的新型冠状病毒S蛋白的受体结合区，上述两个序列中的精氨酸被等电点更低的组氨酸代替后，上述两个序列所形成的多肽段在生物体内的解离度明显降低，并且三维结构与野生型相似，除解离度降低外，其他特性并未受明显影响。
根据本发明的实施例，所述新型冠状病毒S蛋白的受体结合区的序列如SEQ ID NO:3和/或SEQ ID NO:4所示。
Figure PCTCN2021101458-appb-000001
Figure PCTCN2021101458-appb-000002
所述野生型新型冠状病毒S蛋白的受体结合区的序列如SEQ ID NO:5所示。
Figure PCTCN2021101458-appb-000003
在本发明的第三方面，本发明提出了一种新型冠状病毒S蛋白。根据本发明的实施例，所述新型冠状病毒S蛋白含有在本发明的第二方面所提出的新型冠状病毒S蛋白的受体结合区。
在本发明的第四方面，本发明提出了一种核酸分子，所述核酸分子编码本发明第二方面所提出的新型冠状病毒S蛋白的受体结合区或本发明第四方面所提出的新型冠状病毒S蛋白。根据本发明的一个实施例，所述核酸分子为RNA或DNA。
根据本发明的实施例，所述核酸分子可以在生物体内形成本发明第二方面所提出的新型冠状病毒S蛋白的受体结合区、本发明第四方面所提出的新型冠状病毒S蛋白、携带上述S蛋白的新型冠状病毒、携带上述S蛋白受体结合区的新型冠状病毒。
在本发明的第五方面，本发明提出了一种表达载体，所述表达载体携带本发明第四方面所提出的核酸分子。根据本发明的实施例，所述表达载体可以在细胞、细菌、酵母、生物体内表达，所述生物体为人、小鼠、大鼠、兔子、骆驼、羊驼、马、驴、骡等能够感染新型冠状病毒的生物体。
在本发明的第六方面，本发明提出了一种重组病毒，所述重组病毒携带本发明第四方面所提出的核酸分子。
在本发明的第七方面，本发明提出了一种疫苗。根据本发明的实施例，所述疫苗含有下列的至少之一：在本发明第二方面提出的新型冠状病毒S蛋白的受体结合区；在本发明第三方面提出的新型冠状病毒S蛋白；在本发明第四方面提出的核酸分子；在本发明第五方面提出的表达载体；和在本发明第六方面提出的表达载体重组病毒。
表1：碱性氨基酸信息表
中文名 缩写 符号 等电点
精氨酸 Arg R 10.76
赖氨酸 Lys K 9.74
组氨酸 His H 7.59
表2：其它氨基酸信息表
中文名 缩写 符号 等电点
甘氨酸 Gly G 6.06
丙氨酸 Ala A 6.11
缬氨酸 Val V 6
亮氨酸 Leu L 6.01
异亮氨酸 Ile I 6.05
苯丙氨酸 Phe F 5.49
色氨酸 Trp W 5.89
酪氨酸 Tyr Y 5.64
天冬氨酸 Asp D 2.85
天冬酰胺 Asn N 5.41
谷氨酸 Glu E 3.15
谷氨酰胺 Gln Q 5.65
甲硫氨酸 Met M 5.74
丝氨酸 Ser S 5.68
苏氨酸 Thr T 5.6
半胱氨酸 Cys C 5.05
脯氨酸 Pro P 6.3
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：
图1是根据本发明实施例的SARS-CoV-2的RBD三维结构模型图；
图2是根据本发明实施例的SARS-CoV的RBD三维结构模型图；
图3是利用软件1根据本发明实施例1、实施例2的野生型新型冠状病毒S蛋白受体结合区的三维结构模型图；
图4是利用软件1根据本发明实施例1的改造后的新型冠状病毒S蛋白受体结合区的三维结构模型图；
图5是利用软件2根据本发明实施例1的野生型和改造后的新型冠状病毒S蛋白受体结合区的三维结构模型对比图，其中图a为野生型新型冠状病毒S蛋白受体结合区的三维结构模型，图b为改造后的新型冠状病毒S蛋白受体结合区的三维结构模型；
图6是根据本发明实施例1的氨基酸置换前的等电点结果；
图7是根据本发明实施例1的氨基酸置换后的等电点结果；
图8是利用软件1根据本发明实施例2的改造后的新型冠状病毒S蛋白受体结合区的 三维结构模型图；
图9是利用软件2根据本发明实施例2的野生型和改造后的新型冠状病毒S蛋白受体结合区的三维结构模型对比图，其中图c为野生型SARS-CoV-2的RBD蛋白三维构象模型，图d为改造后的SARS-CoV-2的RBD蛋白三维构象模型；
图10是根据本发明实施例2的氨基酸置换前的等电点结果；
图11是根据本发明实施例2的氨基酸置换后的等电点结果；
图12是根据本发明实施例3的ELISA检测抗原特异性实验结果。
发明详细描述
下面详细描述本发明的实施例，所述实施例的示例在附图中示出。下面通过参考附图描述的实施例是示例性的，旨在用于解释本发明，而不能理解为对本发明的限制。
此外，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括至少一个该特征。在本发明的描述中，“多个”的含义是至少两个，例如两个，三个等，除非另有明确具体的限定。
在本发明中，除非另有明确的规定和限定，术语“安装”、“相连”、“连接”、“固定”等术语应做广义理解，例如，可以是固定连接，也可以是可拆卸连接，或成一体；可以是机械连接，也可以是电连接；可以是直接相连，也可以通过中间媒介间接相连，可以是两个元件内部的连通或两个元件的相互作用关系，除非另有明确的限定。对于本领域的普通技术人员而言，可以根据具体情况理解上述术语在本发明中的具体含义。
在本发明的第一方面，本发明提出了一种构建新型冠状病毒疫苗的方法。根据本发明的实施例，所述方法包括：(a)对野生型新型冠状病毒S蛋白的受体结合区进行氨基酸置换，以便获得候选S-RBD序列，其中，与所述野生型新型冠状病毒S蛋白的受体结合区相比，所述候选S-RBD序列具有降低的等电点；(b)基于所述候选S-RBD序列与所述野生型新冠状病毒S蛋白的受体结合区的三维结构，确定目标S-RBD序列；(c)基于所述目标S-RBD序列，构建新型冠状病毒疫苗。根据本发明实施例的方法，确定需要被置换的氨基酸后，选择比需要被置换的碱性氨基酸等电点低的氨基酸对其进行置换，对置换后的S-RBD序列与野生型新冠状病毒S蛋白的受体结合区的三维结构进行比较，选择与野生型新冠状病毒S蛋白的受体结合区的三维结构相似的S-RBD序列作为最终序列，并构建新型冠状病毒疫苗。
另外，根据本发明上述实施例的方法，还可以具有如下附加的技术特征：
根据本发明的实施例，所述新型冠状病毒疫苗包括选自mRNA疫苗、DNA疫苗、蛋白重组疫苗、灭活疫苗、减活疫苗、病毒载体疫苗的至少之一。根据本发明实施例的方法， 疫苗可以为mRNA形式，DNA形式，mRNA疫苗和DNA疫苗在体内表达出的蛋白中至少携带改造后的新型冠状病毒S蛋白的受体结合区，其与生物体内的受体亲和力降低，但三维构型并未受到影响；蛋白重组疫苗中至少携带改造后的新型冠状病毒S蛋白的受体结合区；灭活疫苗中含有携带改造后的新型冠状病毒S蛋白的受体结合区的灭活新型冠状病毒，或者携带改造后的新型冠状病毒S蛋白的受体结合区片段；减活疫苗与病毒载体疫苗中也至少携带改造后的新型冠状病毒S蛋白的受体结合区。
根据本发明的实施例，在步骤(a)中，进一步包括：(a-1)确定所述野生型新型冠状病毒S蛋白的受体结合区中的碱性氨基酸；(a-2)利用候选氨基酸，对至少一个所述碱性氨基酸进行置换，以便降低所述至少一个碱性氨基酸位点的等电点，以便获得所述候选S-RBD序列。根据本发明实施例的方法，候选氨基酸并不限定于等电点小于7的氨基酸，例如，当确定要置换的氨基酸为精氨酸时，其等电点为10.76，赖氨酸和组氨酸也可以作为其候选氨基酸，利用赖氨酸和组氨酸对精氨酸进行置换，可以降低被置换氨基酸所在序列在生物体内的解离度，进而降低新型冠状病毒S蛋白的受体结合区与生物体内受体结合的亲和力。
根据本发明的实施例，在步骤(b)中，进一步包括：(b-1)建立所述候选S-RBD序列的三维结构；(b-2)将步骤(b-1)中获得的所述三维结构与所述野生型新型冠状病毒S蛋白的受体结合区的三维结构进行比较，以便获得均方根偏差值；(b-3)选择均方根偏差值小于预定阈值的所述候选S-RBD序列作为目标S-RBD序列。根据本发明实施例的方法，分别构建候选S-RBD序列的三维结构及野生型新型冠状病毒S蛋白的受体结合区的三维结构，并将两个三维结构进行比较，选择与野生型新型冠状病毒S蛋白的受体结合区的三维结构最为接近的候选S-RBD序列作为最终序列，可以保证最终序列的三维结构与野生型最为相近，尽量减少S-RBD序列除解离度之外的其他特征的改变，进而保证携带上述S-RBD序列的病毒的免疫原性，使疫苗注射进体内后具有较高的免疫原性，同时毒性降低。
根据本发明的实施例，所述预定阈值不超过1.93，例如不超过1.2，优选不超过1.1，例如为1。根据本发明实施例的方法，所述阈值的确定基于非典型肺炎病毒SARS-CoV与新型冠状病毒SARS-CoV-2的三维结构RMSD确定。SARS-CoV-2与SARS-CoV属于同属病毒，结构相似，并且SARS-CoV比SARS-CoV-2的受体亲和力低，突变后的SARS-CoV-2S-RBD蛋白与野生型SARS-CoV-2RBD蛋白差异应该小于SARS-Cov-2的RBD与SARS-Cov RBD的RMSD值，才能保证两者之间结构相似性，都能识别AEC2受体。
根据本发明的具体实施例，利用软件1(https://swissmodel.expasy.org/interactive)分别对SARS-CoV-2的RBD与SARS-CoV的RBD蛋白序列从头建模，生成三维结构文件，再利用软件2(http://superpose.wishartlab.com/)进行计算，并对两个三维结构进行比较，选择 均方根偏差值(RMSD)的阈值不大于SARS-CoV-2的RBD与SARS-CoV的RBD之间比较的RMSD值，通过计算两者之间RMSD＝1.93。
根据本发明的一个具体实施例，比较SARS-CoV、中东呼吸综合症冠状病毒MERS-CoV、SARS-CoV-2的RBD蛋白序列发现SARS-CoV-2的RBD有两个碱性氨基酸富集区，SARS-CoV的RBD有一个，MERS-CoV的RBD没有碱性氨基酸富集区，基于上述特征，在病毒感染易感人群过程中也可以显示，SARS-CoV-2的感染力最强，并且其与SARS-CoV的受体相同，均为ACE2，二者蛋白结构也相似。
在swissmodel服务器上将SARS-CoV-2和SARS-CoV的RBD序列从头三维建模，SARS-CoV-2的RBD三维图如附图1所示，SARS-CoV的RBD三维图如附图2所示，用在线SuperPose Version 1.0计算两个蛋白三维结构RMSD，RMSD越小，说明两个蛋白三维结构越相似，空间重叠性越高。计算得到SARS-CoV-2和SARS-CoV的RBD序列的RMSD值为1.93，二者结构极为相似，二者的受体也同为ACE2。
按照上述方法比较SARS-CoV-2和MERS-CoV的RBD蛋白三维结构的RMSD，计算结果为10.66，RMSD值过大，进一步说明SARS-CoV-2和MERS-CoV的RBD蛋白结构不同。
上述结果说明，通过RMSD值判断并筛选合适的候选RBD序列的方法是可行的。
基于上述方法，本发明还提出了一种新型冠状病毒S蛋白的受体结合区、一种新型冠状病毒S蛋白、一种核酸分子、一种表达载体、一种重组病毒、一种疫苗，已在发明内容中描述，在此不再赘述。
下面参考具体实施例，对本发明进行描述，需要说明的是，这些实施例仅仅是描述性的，而不以任何方式限制本发明。
实施例1：NRKRIS(SEQ ID NO:1)→NHKHIS(SEQ ID NO:6)
针对野生型新型冠状病毒S蛋白受体结合区，将SEQ ID NO:1序列中的精氨酸置换为组氨酸，得到SEQ ID NO:6序列，利用软件1(https://swissmodel.expasy.org/interactive)分别对携带有二者的RBD序列(SEQ ID NO:5，SEQ ID NO:3)进行三维结构建模，三维结构模型如附图3、附图4所示，其中图3为野生型SARS-CoV-2的RBD蛋白三维构象模型，图4为改造后的SARS-CoV-2的RBD蛋白三维构象模型。再利用软件2(http://superpose.wishartlab.com/)再次对两个三维结构进行比较，如附图5所示，其中图a为野生型SARS-CoV-2的RBD蛋白三维构象模型，图b为改造后的SARS-CoV-2的RBD蛋白三维构象模型。同时利用该软件计算得到二者的RMSD值为0.04，说明二者结构非常相似，说明氨基酸置换后并未影响蛋白的三维结构，进而不会影响携带上述氨基酸置换后的RBD序列的新型冠状病毒的免疫原性。
再利用等电点计算工具(Peptide Analyzing Tool)计算上述两者RBD蛋白的等电点，氨基酸置换前的等电点结果如附图6所示，氨基酸置换后的等电点结果如图7所示，结果可知，在氨基酸置换前，等电点为12.5，氨基酸置换后等电点为10.1，等电点显著降低。
实施例2：RLFRKSNLK(SEQ ID NO:2)→HLFHKSNLK(SEQ ID NO:7)
针对野生型新型冠状病毒S蛋白的受体结合区，将SEQ ID NO:2序列中的精氨酸置换为组氨酸，得到SEQ ID NO:7序列，按照实施例1所述的方法，分别对携带有二者的RBD序列(SEQ ID NO:5，SEQ ID NO:4)构建三维构象模型，如附图3、附图8所示，其中图3为野生型SARS-CoV-2的RBD蛋白三维构象模型，图8为改造后的SARS-CoV-2的RBD蛋白三维构象模型。再利用软件2(http://superpose.wishartlab.com/)再次对两个三维结构进行比较，如附图9所示，其中图c为野生型SARS-CoV-2的RBD蛋白三维构象模型，图d为改造后的SARS-CoV-2的RBD蛋白三维构象模型。同时利用该软件计算得到二者的RMSD值为0.04，说明二者结构非常相似，说明氨基酸置换后并未影响蛋白的三维结构，进而不会影响携带上述氨基酸置换后的RBD序列的新型冠状病毒的免疫原性。
再利用等电点计算工具(Peptide Analyzing Tool)计算上述两者RBD蛋白的等电点，氨基酸置换前的等电点结果如附图10所示，氨基酸置换后的等电点结果如图11所示，结果可知，在氨基酸置换前，等电点为12.5，氨基酸置换后等电点为10.6，等电点显著降低。
实施例3：ELISA实验验证碱基置换后的RBD抗体对野生型RBD抗原的免疫原性
实验过程：
构建载体，表达蛋白
分别以SEQ ID NO:1和SEQ ID NO:6对应的核酸序列构建表达载体，并表达相应的蛋白，获得的重组蛋白分别命名为WT-RBD和M6-RBD。
2.制备抗体
按表3的分组情况，分别以WT-RBD和M6-RBD为抗原免疫小鼠(品系BALB/c，雌性，6～8周)，获得对应的抗体。
表3：免疫小鼠分组情况
Figure PCTCN2021101458-appb-000004
第一次注射：第1天，分别取1μg、5μg、10μg的重组蛋白WT-RBD和M6-RBD与0.2μg、1μg、2μg的铝佐剂混合，并用PBS缓冲液补足体积至200μl，注射小鼠腹腔部位。 同时以200μl PBS缓冲液免疫作为对照；
第二次注射：第10天，按上述方法对各只小鼠进行第二次免疫分别取1μg、5μg、10μg的重组蛋白WT-RBD和M6-RBD与0.2μg、1μg、2μg的铝佐剂混合，并用PBS缓冲液补足体积至200μl，注射小鼠腹腔部位。同时以200μl PBS缓冲液免疫作为对照；
收集血清：第15天，各只小鼠分别取脾，收集血清。
3.ELISA实验
基本流程：
包被：用PBS缓冲液(0.02M，pH7.4)将重组蛋白WT-RBD稀释至0.01μg/ml，按100μl/孔加入酶标板中，4℃包被过夜；
洗板：弃去孔内液体，加入0.5％Triton X-100洗涤液，300μl/孔，洗涤5次，每次1分钟；
封闭：按200μl/孔加入2.0％BSA封闭液，室温放置90分钟进行封闭，弃去封闭液，洗涤5次，每次1分钟；
一抗孵育：将收集的血清用含0.2％BSA的PBS缓冲液稀释100倍，加入酶标板中，100μl/孔，每个血清检测3个复孔，37℃恒温孵育1小时；
表4：一抗/对照品清单
Figure PCTCN2021101458-appb-000005
二抗孵育：一抗孵育结束后，每孔加入1:6000HRP标记的羊抗鼠抗体100μl，37℃恒温孵育30分钟；
显色：每孔加入100μl TMB显色液，室温避光显色15分钟左右；
终止：每孔加入50μl 2M硫酸终止反应；
检测：酶标仪读取450nm处的吸光值。
实验结果发现，5μg抗原与1μg佐剂混合的效果最好。5μg抗原与1μg佐剂的果如图12所示，WT-RBD蛋白与抗体WT-RBD-Ab及M6-RBD-Ab均有明显的免疫反应，虽然相比于WT-RBD-Ab，M6-RBD-Ab对WT-RBD的特异性下降15.9％左右，但是依然是明显的阳性反应。此结果表明将精氨酸置换为组氨酸的RBD抗体可以用于检测野生型新冠病毒，即进行碱性氨基酸置换后生产的疫苗可以产生针对新冠病毒有效的抗体。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、置换和变型。

Claims (19)

  1. 一种构建新型冠状病毒疫苗的方法，其特征在于，包括：
    (a)对野生型新型冠状病毒S蛋白的受体结合区进行氨基酸置换，以便获得候选S-RBD序列，其中，与所述野生型新型冠状病毒S蛋白的受体结合区相比，所述候选S-RBD序列具有降低的等电点；
    (b)基于所述候选S-RBD序列与所述野生型新型冠状病毒S蛋白的受体结合区的三维结构，确定目标S-RBD序列；
    (c)基于所述目标S-RBD序列，构建新型冠状病毒疫苗。
  2. 根据权利要求1所述的方法，其特征在于，所述新型冠状病毒疫苗包括选自mRNA疫苗、DNA疫苗、蛋白重组疫苗、灭活疫苗、减活疫苗、病毒载体疫苗的至少之一。
  3. 根据权利要求1所述的方法，其特征在于，在步骤(a)中，进一步包括：
    (a-1)确定所述野生型新型冠状病毒S蛋白的受体结合区中的碱性氨基酸；
    (a-2)利用候选氨基酸，对至少一个所述碱性氨基酸进行置换，以便降低所述至少一个碱性氨基酸位点的等电点，以便获得所述候选S-RBD序列。
  4. 根据权利要求3所述的方法，其特征在于，在步骤(a-1)中，进一步确定碱性氨基酸富集区，在步骤(a-2)中，对所述碱性氨基酸富集区的至少一个所述碱性氨基酸进行置换。
  5. 根据权利要求4所述的方法，其特征在于，所述碱性氨基酸富集区含有至少两个相连的碱性氨基酸，所述两个相连的碱性氨基酸之间相隔不超过三个氨基酸。
  6. 根据权利要求4所述的方法，其特征在于，所述碱性氨基酸富集区包括如SEQ ID NO:1所示氨基酸序列和/或如SEQ ID NO:2所示氨基酸序列。
  7. 根据权利要求4所述的方法，其特征在于，在步骤(a-2)中，将至少一个精氨酸置换为组氨酸。
  8. 根据权利要求1所述的方法，其特征在于，在步骤(b)中，进一步包括：
    (b-1)建立所述候选S-RBD序列的三维结构；
    (b-2)将步骤(b-1)中获得的所述三维结构与所述野生型新型冠状病毒S蛋白的受体结合区的三维结构进行比较，以便获得均方根偏差值；
    (b-3)选择均方根偏差值小于预定阈值的所述候选S-RBD序列作为目标S-RBD序列。
  9. 根据权利要求8所述的方法，其特征在于，所述预定阈值不超过1.93，例如不超过1.2，优选不超过1.1，例如为1。
  10. 一种新型冠状病毒S蛋白的受体结合区，其特征在于，所述新型冠状病毒S蛋白的受体结合区与野生型新型冠状病毒S蛋白的受体结合区相比，具有降低的等电点，并且所述新型冠状病毒S蛋白的受体结合区的三维结构与野生型新型冠状病毒S蛋白的受体结合区的三维结构之间具有不超过预定阈值的均方根偏差值。
  11. 根据权利要求10所述的新型冠状病毒S蛋白的受体结合区，其特征在于，所述预定阈值为1.93，例如不超过1.2，优选不超过1.1，例如为1。
  12. 根据权利要求10所述的新型冠状病毒S蛋白的受体结合区，其特征在于，与所述野生型新型冠状病毒S蛋白的受体结合区相比，至少一个碱性氨基酸被置换为等电点降低的氨基酸。
  13. 根据权利要求10所述的新型冠状病毒S蛋白的受体结合区，其特征在于，与所述野生型新型冠状病毒S蛋白的受体结合区相比，如SEQ ID NO:1所示氨基酸序列和/或如SEQ ID NO:2所示氨基酸序列的至少一个精氨酸被置换为组氨酸。
  14. 根据权利要求10所述的新型冠状病毒S蛋白的受体结合区，其特征在于，所述新型冠状病毒S蛋白的受体结合区的序列包括如SEQ ID NO:3和/或SEQ ID NO:4所示的氨基酸序列。
  15. 一种新型冠状病毒S蛋白，其特征在于，含有权利要求10～14任一项所述的新型冠状病毒S蛋白的受体结合区。
  16. 一种核酸分子，所述核酸分子编码权利要求10～14所述的新型冠状病毒S蛋白的受体结合区或权利要求15所述的新型冠状病毒S蛋白；
    任选地，所述核酸分子为RNA或DNA。
  17. 一种表达载体，所述表达载体携带权利要求16所述的核酸分子。
  18. 一种重组病毒，所述重组病毒携带权利要求16所述的核酸分子。
  19. 一种疫苗，其特征在于，含有下列的至少之一：
    权利要求10～14任一项所述的新型冠状病毒S蛋白的受体结合区；
    权利要求15所述的新型冠状病毒S蛋白；
    权利要求16所述的核酸分子；
    权利要求17所述的表达载体；和
    权利要求18所述的表达载体重组病毒。
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