WO2022032660A1 - 新型冠状病毒rbd融合蛋白 - Google Patents

新型冠状病毒rbd融合蛋白 Download PDF

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WO2022032660A1
WO2022032660A1 PCT/CN2020/109295 CN2020109295W WO2022032660A1 WO 2022032660 A1 WO2022032660 A1 WO 2022032660A1 CN 2020109295 W CN2020109295 W CN 2020109295W WO 2022032660 A1 WO2022032660 A1 WO 2022032660A1
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rbd
cov
novel coronavirus
coronavirus sars
fusion protein
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PCT/CN2020/109295
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French (fr)
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方丽娟
张敬
石剑
王鑫
罗芳
周迟
雷传飞
周鹏飞
肖庚富
潘晓彦
龚睿
张哲�
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武汉友微生物技术有限公司
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Priority to PCT/CN2020/109295 priority Critical patent/WO2022032660A1/zh
Priority to CN202080104145.XA priority patent/CN116096736A/zh
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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
    • 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
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins

Definitions

  • the present invention relates to the field of biomedicine, in particular, to novel coronavirus RBD fusion protein, RBD protein dimer and preparation and application thereof.
  • the core principle of vaccines is to allow the immune system to recognize the target virus in advance, especially the structure that is unique to the target virus and plays a core function, so as to generate a high-quality immune response, thereby generating immunity to it.
  • the current strategies mainly include nucleic acid vaccines (RNA and DNA vaccines), adenovirus vector vaccines, recombinant protein vaccines, inactivated vaccines, and attenuated virus vaccines. Different types of vaccines have different characteristics, advantages and disadvantages.
  • Nucleic acid vaccines and adenovirus vector vaccines rely on the human body to express virus-related proteins to generate an immune response, while recombinant protein vaccines, inactivated virus vaccines and attenuated virus vaccines directly use viral proteins.
  • Recombinant protein vaccines are prepared by expressing part of the functional genes of the new coronavirus in cells or microorganisms, and then purifying them.
  • recombinant protein vaccines are highly safe, especially related vaccines designed based on the RBD of the new coronavirus spike protein receptor binding domain, whose expression proteins are controllable, and theoretically there is no risk of DNA and genome integration. The process is complex, the technical difficulty is relatively high, and the immunogenicity is often weak, and adjuvants need to be added to improve the immunogenicity.
  • RBD derived from SARS-CoV-2 can induce highly potent antibody responses in immunized animals, including mice and horses (Xiaoyan Pan, Pengfei Zhou, et al. Immunoglobulin fragment F(ab')2 again RBD potently neutralizes SARS- CoV-2 in vitro. Antiviral Research 2020).
  • the induced antibody can neutralize the novel coronavirus SARS-CoV-2 and inhibit the virus from infecting Vero-E6 cells. This suggests that the RBD sequence in the spike protein of the novel coronavirus SARS-CoV-2 can induce a highly potent neutralizing antibody response and can be developed into an effective and safe subunit vaccine for the prevention of COVID-19.
  • the spike protein (S protein) of the new coronavirus SARS-CoV-2 contains two subunits S1 and S2.
  • S1 mainly contains a receptor binding region (RBD, the sequence source is Genbank: QHR63260.2), which can specifically bind to the receptor-angiotensin-converting enzyme ACE2 of target cells.
  • the present inventors found that by using the cleavage site of the RBD fusion protease of the novel coronavirus SARS-CoV-2 and a tag favorable for dimer formation, the expression level and purity of the RBD fusion protein of the present invention were significantly improved.
  • the present inventors found that whether it is a wild-type RBD or a mutant RBD, by interacting with, for example, an Fc fragment that does not contain cysteine in the hinge region or an Fc fragment that does not contain a hinge region (as shown in SEQ ID NO: 13) sequence) fusion, significantly improve the expression and purity of RBD fusion protein, improve the effect of protease digestion (for example, improve the recovery rate of target protein, reduce the production of impurity proteins), reduce the proportion of high polymers, and the obtained fusion protein is digested by protease. , higher concentrations of RBD dimers can be obtained.
  • the RBD protein dimer has better binding activity to ACE2.
  • the fusion protein and the further formed RBD dimer can be used for vaccines for preventing novel coronavirus infection or immunizing animals to obtain neutralizing antibodies, and for protection of people who are newly exposed to novel coronavirus.
  • the present invention relates to the following aspects:
  • the RBD fusion protein of the novel coronavirus SARS-CoV-2 the sequence of which comprises the RBD sequence of the novel coronavirus SARS-CoV-2 (preferably, the RBD sequence comprises and is located according to the position numbering of the sequence described in SEQ ID NO: 1 as cysteine at position 220, and has activity in binding the human ACE2 receptor; more preferably the RBD sequence comprises the amino acid sequence shown in any one of SEQ ID NOs: 1-8), a protease cleavage site, and has A tag that favors dimer formation.
  • the group consisting of a sequence comprising HHHHHH) and an Fc fragment, wherein the Fc fragment does not contain cysteine in the hinge region preferably the Fc fragment is an Fc fragment derived from human IgG, murine IgG or horse IgG, more preferably , the Fc fragment does not comprise a hinge region, further more preferably the Fc fragment comprises the amino acid sequence shown in any one of SEQ ID NOs: 13-16, 29
  • the leucine zipper is from c-JUN Or c-FOS protein, more preferably, the C-terminus of the leucine zipper is further linked with His, Flag, C-myc or HA tag.
  • the RBD fusion protein of the novel coronavirus SARS-CoV-2 according to any one of claims 1-3, wherein the protease is selected from thrombin, enterokinase, TEV protease or HRC-3C protease; preferably, the The amino acid sequence of the thrombin, enterokinase, TEV protease or HRC-3C protease cleavage site is shown in any one of SEQ ID NOs: 17-20; more preferably, the amino acid sequence of the protease cleavage site is the same as the There are no repeats in the amino acid sequence in the RBD sequence or the Fc fragment.
  • the RBD dimer of the novel coronavirus SARS-CoV-2 characterized in that according to the position numbering of the RBD monomer sequence shown in SEQ ID NO: 1, the cysteines at positions 18 and 43 of the monomer RBD, Cysteines 61 and 114, cysteines at positions 73 and 207, and cysteines at positions 162 and 170, respectively, form intrachain disulfide bonds, and the cysteines at position 220 of the two monomeric RBDs form an intrachain disulfide bond, respectively.
  • Form interchain disulfide bonds between, preferably, the monomer RBD sequence comprises the amino acid sequence shown in any one of SEQ ID NO: 1 and 5-8, more preferably, the two monomers of the dimer
  • the body RBD sequence is the same.
  • the RBD dimer is prepared by the method of the following 8.
  • the method for preparing the RBD dimer of the novel coronavirus SARS-CoV-2 of claim 6, comprising cleaving the RBD fusion protein of the novel coronavirus SARS-CoV-2 of any one of claims 1-5 with a protease , preferably, the method further comprises purifying the RBD fusion protease cleavage product, preferably, the purification comprises chromatography (eg affinity chromatography, ion exchange chromatography).
  • a vector comprising the polynucleotide of claim 9.
  • a host cell comprising the polynucleotide of claim 9 or the vector of claim 10.
  • Vaccine preferably for preventing the infection of novel coronavirus, it is characterized in that comprising the RBD fusion protein of the novel coronavirus SARS-CoV-2 described in any one of claim 1-5, the novel coronavirus described in claim 6 The RBD dimer of the virus SARS-CoV-2, or the polynucleotide of claim 9.
  • a method for preventing or treating a novel coronavirus SARS-CoV-2 infection or obtaining a neutralizing antibody against the novel coronavirus SARS-CoV-2 comprising adding the novel coronavirus SARS-CoV-2 according to any one of claims 1 to 5.
  • - RBD fusion protein of CoV-2, the RBD dimer of the novel coronavirus SARS-CoV-2 of claim 6, or the polynucleotide of claim 9 to immunize an animal to obtain antiviral serum, the animal Preferably it is a human or non-human mammal, more preferably a horse.
  • the Fc fragment is an Fc fragment derived from human IgG, preferably the sequence shown in SEQ ID NO: 13.
  • the Fc fragment is an Fc fragment derived from horse IgG, preferably the sequence shown in SEQ ID NO: 15.
  • conservative amino acid substitutions can generally be described as amino acid substitutions in which amino acid residues are replaced by those of similar chemical structure Another amino acid residue is replaced with little or no effect on the function, activity, or other biological property of the polypeptide.
  • conservative amino acid substitutions are well known in the art, for example, from WO 04/037999, GB-A-3357 768, WO 98/49185, WO 00/46383 and WO 01/09300; and can be based on WO 04/ 037999 and the related teachings of WO 98/49185 and other references cited therein, such alternative (preferred) types and/or combinations were selected.
  • Constant amino acid substitutions are those in which amino acid residues are replaced with amino acid residues having similar side chains. Families of amino acid residues with similar side chains have been defined in the art and include basic side chains (eg lysine, arginine, histidine), acidic side chains (eg aspartic acid, glutamic acid) ), uncharged polar side chains (eg, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (eg, alanine , valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), side chains of beta-branched chains (eg, threonine, valine, isoleucine amino acid) and aromatic side chains (eg tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains eglysine, arginine, histidine
  • non-essential amino acid residues of an immunoglobulin polypeptide are preferably replaced by other amino acid residues from the same side chain family.
  • a string of amino acids may be replaced by a structurally similar string of amino acids that differ in sequence and/or composition of side chain families.
  • Non-limiting examples of conservative amino acid substitutions are provided in the table below, where a similarity score of 0 or higher indicates a conservative substitution between the two amino acids.
  • the conservative substitution is preferably one in which one amino acid within the following groups (a)-(e) is replaced by another amino acid residue within the same group: (a) a small Aliphatic, non-polar or weakly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (c) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, Ile, Val and Cys; and (e) ) Aromatic residues: Phe, Tyr and Trp.
  • Particularly preferred conservative substitutions are as follows: Ala for Gly or Ser; Arg for Lys; Asn for Gln or His; Asp for Glu; Cys for Ser; Gln for Asn; Glu for Asp; Replace Gly with Ala or with Pro; His with Asn or with Gln; Ile with Leu or with Val; Leu with Ile or with Val; Lys with Arg, with Gln or with Glu; Met Replace with Leu, with Tyr or with Ile; Phe with Met, with Leu or with Tyr; Ser with Thr; Thr with Ser; Trp with Tyr; Tyr with Trp; and/or Phe into Val, replace with Ile or replace with Leu.
  • FIG. 1 Schematic representation of the recombinant fusion protein RBD-Fc.
  • Figure 2 RBD-His SDS-PAGE non-reducing electrophoresis detection map.
  • Figure 2A Ni affinity chromatography sample, lanes 1-3 are samples collected in separate tubes for the same elution peak;
  • Figure 2B cation exchange chromatography sample, lane 1 is protein marker, lanes 2-10 are from low salt to high, respectively Different components of salt elution.
  • FIG. 3 RBD-Fc SDS-PAGE electrophoresis detection map.
  • Figure 3A lane 1, RBD-TFc non-reducing; lane 2, RBD-YFc non-reducing; lane 3, RBD-TFc reducing; lane 4, RBD-YFc reducing; lane 5, RBD-eqIgG1Fc non-reducing; lane 6, RBD - eqIgG1Fc reduced; lane 7, RBD-eqIgG4Fc non-reduced; lane 8, RBD-eqIgG4Fc reduced; MK, protein marker.
  • Figure 3B lane 1, RBD L455I F456V-YFc non-reducing; lane 2, RBD G476S-YFc non-reducing; lane 3, RBD V483A-YFc non-reducing; lane 4, RBD S494P-YFc non-reducing; lane 5, RBD L455I F456V -YFc reduction; lane 6, RBD G476S-YFc reduction; lane 7, RBD V483A-YFc reduction; lane 8, RBD S494P-YFc reduction; MK, protein marker.
  • FIG. 1 Non-reducing electrophoresis detection of RBD-TFc and RBD-YFc by enzyme digestion SDS-PAGE. Lane 1, before digestion with RBD-TFc; Lane 2, after digestion with RBD-TFc; Lane 3, before digestion with RBD-YFc; Lane 4, after digestion with RBD-YFc.
  • Figure 6 Identification of untagged products RBD TFc and RBD YFc .
  • Figure 6A SDS-PAGE electrophoresis detection, lane 1, RBD TFc non-reduction; lane 2, RBD YFc non-reduction; lane 3, RBD TFc reduction; lane 4, RBD YFc reduction;
  • Figure 6B HPLC-SEC detection chart;
  • Figure 6C mass spectrometry molecular weight detection.
  • FIG. Enzymatic SDS-PAGE non-reducing electrophoresis detection of horse Fc-tagged RBD. Lane 1, before digestion with RBD-eqIgG1Fc; Lane 2, after digestion with RBD-eqIgG1Fc; Lane 3, before digestion with RBD-eqIgG4Fc; Lane 4, after digestion with RBD-eqIgG4Fc.
  • Figure 8 RBD eqG1Fc identification.
  • Figure 8A SDS-PAGE electrophoresis detection, lane 1, RBD eqG1Fc non-reduction; lane 2, RBD eqG1Fc reduction;
  • Figure 8B HPLC-SEC detection chart;
  • Figure 8C mass spectrometry molecular weight detection chart.
  • FIG. 12 Schematic representation of the structure of the RBD YFc dimer.
  • the RBD protein sequence comes from Genbank: QHR63260.2.
  • the RBD gene fragment is obtained by whole gene synthesis, and it is constructed into the polyclonal restriction sites of the eukaryotic expression vector (such as pcDNA3.1 vector, Invitrogen Company), and placed in the Add a signal peptide (such as CD33 signal peptide, IL2 signal peptide, human albumin HSA signal peptide, etc.) to its N-terminus, and add a protein tag (such as His, Flag, HA, myc or Fc tag, etc.) to its C-terminus to facilitate purification. .
  • a signal peptide such as CD33 signal peptide, IL2 signal peptide, human albumin HSA signal peptide, etc.
  • a protein tag such as His, Flag, HA, myc or Fc tag, etc.
  • protease cleavage site coding sequence was added between the downstream of the RBD gene and the upstream of the tag gene, including but not limited to thrombin (Sigma), enterokinase (New England Biolabs), TEV protease (Invitrogen), or HRV3C protease ( Novagen).
  • the protease cleavage site and eg the Fc tag are linked by a linker peptide.
  • the specific structure of the fusion protein is shown in Figure 1 .
  • the specific sequence information of the constructs is shown in Table 1.
  • RBD is a domain in the S protein of SARS-CoV-2 novel coronavirus, located in the region of amino acid residues 319-541 of the entire S protein (SEQ ID NO: 1).
  • the plasmid was extracted according to the conventional plasmid extraction method and used for chemical transfection of 293 (ATCC) or CHO-S (Gibco) cells.
  • the transfected cells were cultured for 7-10 days in a 37°C, 5% CO 2 shaker with suspension shaking. The supernatant was harvested by centrifugation at 3000 ⁇ g and filtered through a 0.22 ⁇ m filter.
  • the RBD-His feed liquid was harvested and subjected to Ni column affinity chromatography, and the expression level was less than 1 mg/L, and the sample was detected by SDS-PAGE.
  • the sample after Ni column affinity chromatography has impurity components and low purity, so cation exchange chromatography was used for purification, and the sample was eluted linearly with low salt (5mM NaCl) on the column and high salt (500mM NaCl).
  • the different components eluted from low-salt to high-salt during the process were collected in separate tubes and detected by SDS-PAGE, as shown in Figure 2B.
  • Lane 2 is the highest purity component, with a band size of 50KD, which is similar to RBD.
  • the C-terminal protein tag of RBD is replaced with other tags, including but not limited to Flag tag, HA tag and c-Myc tag.
  • the corresponding RBD fusion tag proteins are RBD-Flag, RBD-HA and RBD-myc respectively.
  • the constructed expression After the plasmid was transiently transfected into 293 or CHO-S cells, and cultured in suspension and shaking for 7-10 days, the protein expression level of the harvested feed liquid was similar to that of RBD-His, and the expression level was less than 1 mg/L. The protein yield was not more than 20% after tag-corresponding affinity chromatography. It can be seen that in mammalian expression systems, the expression level and purification yield are low when a short peptide protein tag is fused to the C-terminus of RBD.
  • the protein expression solution of RBD fused Fc tag was harvested and subjected to protein A affinity chromatography.
  • the protein after one-step affinity purification was analyzed by SDS-PAGE.
  • the band size was about 110KD, which was fused with RBD Fc tag
  • HPLC-SEC high-performance size exclusion chromatography
  • RBD-YFc9, RBD-YFc10 and RBD-YFc11 were not significantly different from RBD-YFc.
  • the expression level and purity of RBD-epIG1Fc1 were not significantly different from RBD-epIG1Fc. There were no significant differences in expression level and purity between RBD-epIG4Fc1 and RBD-epIG4Fc.
  • the Fc tag was excised by protease to obtain an unlabeled RBD protein.
  • the specific method is as follows: Add the corresponding protease to the RBD fusion Fc protein purified by affinity chromatography. , incubate under specific conditions according to the requirements of the instructions, and then obtain RBD protein through the flow-through mode of protein A or protein G affinity chromatography, which is an unlabeled product. The digestion effect of the RBD fusion Fc protein before and in the digestion sample was detected by SDS-PAGE, and the recovery rate of the digestion and purification of the RBD protein was calculated, and the final unlabeled RBD protein was detected.
  • ACE2 protein construct a eukaryotic expression plasmid (the vector is pcDNA3.1) of the protein hACE2-Fc (SEQ ID NO: 24) fused with human ACE2 extracellular domain and Fc, and transiently transfer the plasmid to 293 or CHO- In S cells, the supernatant was harvested after culturing for 7-10 days, and purified by ProteinA affinity chromatography to obtain hACE2-Fc protein.
  • the optimized construct, RBD-YFc has a significantly better enzyme cleavage effect than the pre-optimized RBD-TFc.
  • Table 3 the optimized RBD-YFc, RBD-YFc1-RBD-YFc11 and RBD-eqIgG1Fc digestion and purification recovery rates were significantly improved compared with the pre-optimized RBD-TFc.
  • the optimized RBD-YFc has better digestion effect than RBD-TFc, and can improve the recovery rate of target protein by 20-30%.
  • the unlabeled products RBD TFc and RBD YFc were identified, and they were consistent in terms of purity (Figure 6A and 6B) and molecular weight ( Figure 6C), and mass spectrometry molecular weight detection RBD protein contained monomer (31KD) and dimer (62KD) two forms, the results are consistent with the results of SDS-PAGE and SEC.
  • the binding activity to hACE2-Fc FIG. 9
  • the binding activity of the unlabeled product RBD YFc to hACE2-Fc was higher than that of RBD TFc .
  • RBD-TFc RBD TFc 58.6%
  • RBD-YFc RBD TFc 82.3%
  • RBD-YFc1 RBD YFc1 78.8%
  • RBD-YFc2 RBD YFc2 79.3%
  • RBD-YFc3 RBD YFc3 77.5%
  • RBD-YFc4 RBD YFc4 77.4%
  • RBD-YFc5 RBD YFc5 78.9%
  • RBD-YFc6 RBD YFc6 79.2%
  • RBD-YFc7 RBD YFc7 78.9%
  • RBD-YFc8 RBD YFc8 77.36%
  • RBD-YFc9 RBD YFc9
  • RBD-YFc9 76.9%
  • RBD-YFc10 RBD YFc10 80.1%
  • RBD-YFc11 RBD YFc11
  • the recovery rate of RBD target protein the final amount of RBD protein/(RBD-Fc protein amount ⁇ 50%).
  • RBD fusion Fc proteins constructed using different protease cleavage sites, such as RBD-YFc (thrombin), RBD-YFc1 (enterokinase), RBD-YFc2 (TEV protease) and RBD-YFc3 (HRV-3C protease), enzymes
  • the cleavage effect is better than that of RBD-TFc containing the corresponding cleavage site before optimization (that is, the thrombin cleavage site in RBD-TFc is replaced with enterokinase cleavage site, TEV protease cleavage site, HRV-3C protease cleavage site cleavage site), the recovery rate of RBD target protein was significantly improved, and after enzyme digestion , affinity chromatography and ion exchange chromatography, the purity, molecular weight and There was no significant difference in the activity of binding to hACE2-Fc.
  • RBD fusion Fc proteins constructed from different regions of S protein, such as RBD-YFc6, RBD-YFc7 and RBD-YFc8, after enzyme digestion and affinity chromatography to obtain unlabeled products RBD YFc6 , RBD YFc7 and RBD YFc8 through non-reduction
  • the purity detected by SDS-PAGE is shown in Table 4.
  • the ratio of RBD YFc6 dimer is more than 80%
  • the ratio of RBD YFc7 dimer is less than 30%
  • the ratio of polymer (molecular weight over 90kD) and monomer is more than 30%
  • RBD YFc8 has only monomer.
  • RBD-YFc9-11 which is different from the linking peptide of RBD-YFc, obtained unlabeled products RBD YFc9 , RBD YFc10 and RBD YFc11 after enzyme digestion and affinity chromatography with nearly 90% dimer components ( Figure 13).
  • RBD unlabeled products High polymer ratio dimer ratio Monomer ratio RBD TFc 0 65% 35% RBD YFc 0 90% 10% RBD YFc6 0 81% 19% RBD YFc7 31% 29% 40% RBD YFc8 0 0 100% RBD YFc9 O 87% 13% RBD YFc10 0 88% 12% RBD YFc11 0 89% 11%
  • RBD YFc7 nor RBD YFc8 contain the cysteine located at position 220 according to the position number of SEQ ID NO; 1
  • the RBD eqG4Fc obtained after RBD- eqIgG4Fc digestion also has a dimeric protein with a molecular weight of 62kD and a monomer with a molecular weight of 31kD, and the Biacore detection has the same activity as RBD eqG1Fc .
  • the label-free product RBD YFc (that is, the label has been cleaved by protease) was further eluted by a high-salt (500mM NaCl) linear gradient by cation exchange chromatography (eg, Capto SP ImpRes from GE), and the eluted fraction was subjected to SDS- PAGE detection, as shown in Figure 10, allows efficient separation of monomers and dimers.
  • Collected and merged lanes 2-4 were RBD YFc monomer fractions, collected and merged lanes 7-12 were RBD YFc dimer fractions, and tested for purity by HPLC-SEC. The purity of the monomer and dimer fractions was over 97%, respectively. .
  • the amino acid residue coding of the RBD YFc monomer is shown in Table 5.
  • the RBD YFc dimer was digested with Chymotrypsin (Sigma) and analyzed by mass spectrometry. It was found that the cysteine 220 of the two monomers formed a pair of interchain disulfide bonds. This indicates that the RBD YFc dimer is covalently linked by two RBD YFc monomers through an interchain disulfide bond formed by a pair of cysteine 220.
  • the C-terminus is fused with a tag that is conducive to dimer formation (such as an Fc tag derived from IgG), preferably in the RBD Add a protease cleavage site between the tag and optimize the connecting peptide and hinge region between the protease cleavage site and the tag to construct an RBD-enzyme cleavage site-tag fusion protein, which is in mammalian cells.
  • a tag that is conducive to dimer formation such as an Fc tag derived from IgG
  • the molecular weight of the RBD dimer is 62kD, which is covalently linked between two RBD monomers through a pair of interchain disulfide bonds (specifically, the 220th cysteine shown in Table 5 encodes the formation of two Sulfur bond), and there are four pairs of intrachain disulfide bonds in each RBD monomer (see Figure 12), respectively: (1) the disulfide between the 18th cysteine and the 43rd cysteine bond, (2) a disulfide bond between cysteine 61 and cysteine 114, (3) a disulfide bond between cysteine 73 and cysteine 207, (4) The disulfide bond between cysteine 162 and cysteine 170, see Table 5 for the sequence code.
  • RBD-mIgG2aFc, RBD-JUN and RBD-FOS obtained RBD dimer fractions after purification, digestion, affinity chromatography and ion-exchange chromatography, with RBD dimerization shown in Figure 12 The same sequence, structure and binding activity to hACE2-Fc.

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Abstract

提供新型冠状病毒RBD融合蛋白，RBD蛋白二聚体及其制备和用于预防新型冠状病毒SARS-CoV-2感染的应用。

Description

新型冠状病毒RBD融合蛋白 技术领域
本发明涉及生物医药领域，具体而言，涉及新型冠状病毒RBD融合蛋白，RBD蛋白二聚体及其制备和应用。
背景技术
由新型冠状病毒SARS-CoV-2感染所导致的肺炎在全世界多个国家出现大规模感染事件，粗病死率约2.3％，对人类的生命健康和生活造成极大不利影响，引起巨大社会恐慌，对经济造成沉重损失。目前，全世界范围内均无特效药用于临床救治因SARS-CoV-2所致的肺炎。因此，迫切需要开发有效的和安全的疫苗用于保护受威胁的人群。
截止2020年5月22日，据WHO统计，全球已有10种新冠疫苗开展临床试验(其中5种来自中国)，114种开展临床前研究。疫苗的核心原理是提前让免疫系统识别目标病毒，特别是目标病毒特有的并且发挥核心功能的结构，以产生高质量免疫应答，从而对其产生免疫力。目前的策略主要有核酸类疫苗(RNA和DNA疫苗)，腺病毒载体疫苗，重组蛋白疫苗，灭活疫苗，减毒病毒疫苗等。不同种类的疫苗有不同特点和优劣势。核酸疫苗，腺病毒载体疫苗依赖人体表达病毒相关蛋白产生免疫应答，而重组蛋白疫苗，灭活病毒疫苗以及减毒病毒疫苗直接使用病毒蛋白。重组蛋白疫苗是将新冠病毒的部分功能基因在细胞或微生物中大量表达，经过纯化制备而成。从原理上来讲，重组蛋白疫苗安全性高，特别是基于新冠病毒刺突蛋白受体结合结构域RBD设计的相关疫苗，其表达蛋白可控，并且理论上没有DNA与基因组整合等风险，但制备工艺复杂，技术难度较大，且往往免疫原性较弱，需要添加佐剂提高免疫原性。
来源于SARS-CoV-2的RBD可以在免疫动物，包括小鼠和马中诱导高度有效的抗体反应(Xiaoyan Pan，Pengfei Zhou，et al.Immunoglobulin fragment F(ab’)2 against RBD potently neutralizes SARS-CoV-2 in vitro.Antiviral Research 2020)。所述诱导抗体可中和新型冠状病毒SARS-CoV-2并抑制病毒感染Vero-E6细胞。这表明，新型冠状病毒SARS-CoV-2的突起蛋白中的RBD序列可以诱导高度有效的中和性抗体反应，并且可以被开发成为用于预防COVID-19的有效的和安全的亚单位疫苗。
发明内容
新型冠状病毒SARS-CoV-2的突起蛋白(spike protein，S蛋白)，含有两个亚基S1和S2。其中S1主要包含有受体结合区(RBD，序列来源为Genbank：QHR63260.2)， 其可以特异性结合靶细胞的受体-血管紧张肽转变酶ACE2。
本发明人发现通过使用将新型冠状病毒SARS-CoV-2的RBD融合蛋白酶的酶切位点和有利于形成二聚体的标签，显著了提高本发明RBD融合蛋白的表达量和纯度。进一步地，本发明人发现不论是野生型RBD，还是突变型RBD，通过与例如铰链区不含半胱氨酸的Fc片段或不含铰链区的Fc片段(如SEQ ID NO：13所示的序列)融合，显著提高RBD融合蛋白的表达量和纯度，提高蛋白酶酶切效果(例如提高目标蛋白回收率、减少杂蛋白产生)，减少高聚体比例，并且获得的融合蛋白经蛋白酶酶切后，可以获得更高浓度的RBD二聚体。所述RBD蛋白二聚体具有更好结合ACE2的活性。所述融合蛋白以及进一步形成的RBD二聚体可以用于预防新型冠状病毒感染的疫苗或免疫动物获得中和性抗体，以及用于初接触新型冠状病毒人群的防护。
具体地，本发明涉及以下几个方面：
1.新型冠状病毒SARS-CoV-2的RBD融合蛋白，其顺序包含新型冠状病毒SARS-CoV-2的RBD序列(优选所述RBD序列包含按照SEQ ID NO：1所述序列的位置编号定位为位置220的半胱氨酸，且具有结合人ACE2受体的活性；更优选地RBD序列包含如SEQ ID NO：1-8任一项所示的氨基酸序列)，蛋白酶酶切位点，和有利于二聚体形成的标签。
2.权利要求1的新型冠状病毒SARS-CoV-2的RBD融合蛋白，其中所述有利于二聚体形成的标签选自亮氨酸拉链(优选SEQ ID NO：30或31所示序列中不包含HHHHHH的序列)和Fc片段组成的组，其中所述Fc片段不包含铰链区的半胱氨酸(优选所述Fc片段为来源于人IgG、鼠IgG或马IgG的Fc片段，更优选地，所述Fc片段不包含铰链区，进一步更优选所述Fc片段包含如SEQ ID NO：13-16、29任一项所示的氨基酸序列)，优选地所述亮氨酸拉链来自c-JUN或c-FOS蛋白，更优选地，所述亮氨酸拉链C端还连接有His、Flag、C-myc或HA标签。
3.权利要求1或2的新型冠状病毒SARS-CoV-2的RBD融合蛋白，其中所述蛋白酶酶切位点和标签通过连接肽连接，优选地，所述连接肽为柔性肽，更优选地，所述连接肽选自SEQ ID NO：26，27或28。
4.权利要求1-3任一项所述的新型冠状病毒SARS-CoV-2的RBD融合蛋白，其中所述蛋白酶选自凝血酶、肠激酶、TEV蛋白酶或HRC-3C蛋白酶；优选地，所述凝血酶、肠激酶、TEV蛋白酶或HRC-3C蛋白酶酶切位点的氨基酸序列如SEQ ID NO：17-20任一项所示；更优选所述蛋白酶酶切位点的氨基酸序列与所述RBD序列或Fc片段中的氨基酸序列不存在重复。
5.权利要求1-4任一项所述的新型冠状病毒SARS-CoV-2的RBD融合蛋白，其中 所述融合蛋白的N端还包含信号肽，优选地，所述信号肽的氨基酸序列包含如SEQ ID NO：21-23任一项所示的氨基酸序列。
6.新型冠状病毒SARS-CoV-2的RBD二聚体，其特征在于按照SEQ ID NO：1所示RBD单体序列的位置编号，单体RBD的位置18和43的半胱氨酸、位置61和114的半胱氨酸、位置73和207的半胱氨酸以及位置162和170的半胱氨酸分别形成链内二硫键，两个单体RBD的位置220的半胱氨酸之间形成链间二硫键，优选地，所述单体RBD序列包含如SEQ ID NO：1和5-8任一项所示的氨基酸序列，更优选地，所述二聚体的两个单体RBD序列相同。优选地，所述RBD二聚体由下述8的方法制备得到。
7.制备权利要求1-5任一项所述新型冠状病毒SARS-CoV-2的RBD融合蛋白的方法，包括依次顺序连接新型冠状病毒SARS-CoV-2的RBD序列，蛋白酶酶切位点和有利于二聚体形成的标签，优选地，所述蛋白酶酶切位点和所述标签通过连接肽连接。
8.制备权利要求6所述新型冠状病毒SARS-CoV-2的RBD二聚体的方法，包括用蛋白酶切割权利要求1-5任一项所述新型冠状病毒SARS-CoV-2的RBD融合蛋白，优选地，所述方法还包括对RBD融合蛋白酶切产物进行纯化，优选地，所述纯化包括层析(例如亲和层析、离子交换层析)。
9.多核苷酸，其特征在于编码权利要求1-5任一项所述的新型冠状病毒SARS-CoV-2的RBD融合蛋白或权利要求6所述的新型冠状病毒SARS-CoV-2的RBD二聚体。
10.载体，其包含权利要求9所述的多核苷酸。
11.宿主细胞，其包含权利要求9所述的多核苷酸或权利要求10所述的载体。
12.疫苗，优选用于预防新型冠状病毒的感染，其特征在于包含权利要求1-5任一项所述的新型冠状病毒SARS-CoV-2的RBD融合蛋白、权利要求6所述的新型冠状病毒SARS-CoV-2的RBD二聚体，或权利要求9所述的多核苷酸。
13.权利要求1-5任一项所述的新型冠状病毒SARS-CoV-2的RBD融合蛋白、权利要求6所述的新型冠状病毒SARS-CoV-2的RBD二聚体，或权利要求9所述的多核苷酸，其用于预防人类感染新型冠状病毒SARS-CoV-2，或用于免疫动物以获得抗病毒血清，所述动物优选是哺乳动物，更优选马，优选所述抗病毒血清可用于预防或治疗人类感染新型冠状病毒SARS-CoV-2。
14.预防或治疗新型冠状病毒SARS-CoV-2感染或获得针对新型冠状病毒SARS-CoV-2的中和抗体的方法，其包含将权利要求1-5任一项所述的新型冠状病毒SARS-CoV-2的RBD融合蛋白、权利要求6所述的新型冠状病毒SARS-CoV-2的RBD二聚体，或权利要求9所述的多核苷酸免疫动物以获得抗病毒血清，所述动物优选是人或非人哺乳动物，更优选马。
在本发明的一些实施方式中，所述Fc段为来源于人IgG的Fc片段，优选地为SEQ ID NO：13所示的序列。
在本发明的一些实施方式中，所述Fc段为来源于马IgG的Fc片段，优选为SEQ ID NO：15所示的序列。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
在本发明中涉及的术语具备本领域技术人员理解的常规含义。在本技术领域内使用和/或可接受的情况下，一个术语有两个或两个以上定义时，本文使用的术语的定义用于包括所有的含义。
在确定两种氨基酸序列之间的序列同一性程度时，专业技术人员可以考虑所谓的“保守”氨基酸替换，其通常可以描述为这样的氨基酸替换，即，其中氨基酸残基被具有相似化学结构的另一种氨基酸残基替换，并且其对所述多肽的功能、活性或其它生物学特性几乎没有或者基本上没有影响。这种保守氨基酸替换是本领域内公知的，例如，从WO 04/037999，GB-A-3357 768，WO 98/49185，WO 00/46383和WO 01/09300中可知；并且可以基于WO 04/037999以及WO 98/49185和其中所引用的其它参考文献的相关教导而选择这种替换的(优选)类型和/或结合。
“保守氨基酸替换”是其中氨基酸残基被具有类似侧链的氨基酸残基替换。具有类似侧链的氨基酸残基家族已在本领域中定义，其包括碱性侧链(例如赖氨酸、精氨酸、组氨酸)，酸性侧链(例如天冬氨酸，谷氨酸)，不带电荷的极性侧链(例如，甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸)，非极性侧链(例如，丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、蛋氨酸、色氨酸)，β-支链的侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳族侧链(例如酪氨酸、苯丙氨酸、色氨酸、组氨酸)。因此，免疫球蛋白多肽的非必需氨基酸残基优选被来自相同侧链家族的其他氨基酸残基替换。在另一些实施方案中，一串氨基酸可被结构上类似的氨基酸串替换，后者在顺序上和/或侧链家族的组成上不同。
在下表中提供了的保守性氨基酸替换的非限制性实例，其中相似性得分为0或更高表示在这两个氨基酸之间有保守替换。
  C G P S A T D E N Q H K R V M I L F Y W
W -8 -7 -6 -2 -6 -5 -7 -7 -4 -5 -3 -3 2 -6 -4 -5 -2 0 0 17
Y 0 -5 -5 -3 -3 -3 -4 -4 -2 -4 0 -4 -5 -2 -2 -1 -1 7 10  
F -4 -5 -5 -3 -4 -3 -6 -5 -4 -5 -2 -5 -4 -1 -0 1 2 9    
L -6 -4 -3 -3 -2 -2 -4 -3 -3 -2 -2 -3 -3 2 4 2 6      
I -2 -3 -2 -1 -1 0 -2 -2 -2 -2 -2 -2 -2 4 2 5        
M -5 -3 -2 -2 -1 -1 -3 -2 0 -1 -2 0 0 2 6          
V -2 -1 -1 -1 0 0 -2 -2 -2 -2 -2 -2 -2 4            
R -4 -3 0 0 -2 -1 -1 -1 0 1 2 3 6              
K -5 -2 -1 0 -1 0 0 0 1 1 0 5                
H -3 -2 0 -1 -1 -1 1 1 2 3 6                  
Q -5 -1 0 -1 0 -1 2 2 1 4                    
N -4 0 -1 1 0 0 2 1 2                      
E -5 0 -1 0 0 0 3 4                        
D -5 1 -1 0 0 0 4                          
T -2 0 0 1 1 3                            
A -2 1 1 1 2                              
S 0 1 1 1                                
P -3 -1 6                                  
G -3 5                                    
C 12                                      
在一些实施方案中，所述保守替换优选地是这样的替换，即，其中下列组(a)-(e)内的一个氨基酸被同组内的另一氨基酸残基替换：(a)小的脂肪族、非极性或弱极性的残基：Ala，Ser，Thr，Pro和Gly；(b)极性、带负电荷的残基及其(不带电荷的)酰胺：Asp，Asn，Glu和Gln；(c)极性、带正电荷的残基：His，Arg和Lys；(d)大的脂肪族、非极性残基：Met，Leu，Ile，Val和Cys；以及(e)芳族残基：Phe，Tyr和Trp。
特别优选的保守替换如下：Ala替换成Gly或替换成Ser；Arg替换成Lys；Asn替换成Gln或替换成His；Asp替换成Glu；Cys替换成Ser；Gln替换成Asn；Glu替换成Asp；Gly替换成Ala或替换成Pro；His替换成Asn或替换成Gln；Ile替换成Leu或替换成Val；Leu替换成Ile或替换成Val；Lys替换成Arg，替换成Gln或替换成Glu；Met替换成Leu，替换成Tyr或替换成Ile；Phe替换成Met，替换成Leu或替换成Tyr；Ser替换成Thr；Thr替换成Ser；Trp替换成Tyr；Tyr替换成Trp；和/或Phe替换成Val，替换成Ile或替换成Leu。
附图说明
图1.重组融合蛋白RBD-Fc的示意图。
图2.RBD-His SDS-PAGE非还原电泳检测图。图2A，Ni亲和层析样品，泳道1-3为同一洗脱峰的分管收集样品；图2B，阳离子交换层析样品，泳道1为蛋白Marker，泳道2-10分别为从低盐向高盐洗脱的不同组分。
图3.RBD-Fc SDS-PAGE电泳检测图。图3A，泳道1，RBD-TFc非还原；泳道2，RBD-YFc非还原；泳道3，RBD-TFc还原；泳道4，RBD-YFc还原；泳道5，RBD-eqIgG1Fc非还原；泳道6，RBD-eqIgG1Fc还原；泳道7，RBD-eqIgG4Fc非还原；泳道8，RBD-eqIgG4Fc还原；MK，蛋白Marker。图3B，泳道1，RBD L455I F456V-YFc非还原；泳道2，RBD G476S-YFc非还原；泳道3，RBD V483A-YFc非还原；泳道4，RBD  S494P-YFc非还原；泳道5，RBD L455I F456V-YFc还原；泳道6，RBD G476S-YFc还原；泳道7，RBD V483A-YFc还原；泳道8，RBD S494P-YFc还原；MK，蛋白Marker。
图4.RBD-Fc HPLC-SEC检测图。
图5.RBD-TFc和RBD-YFc的酶切SDS-PAGE非还原电泳检测。泳道1，RBD-TFc酶切前；泳道2，RBD-TFc酶切后；泳道3，RBD-YFc酶切前；泳道4，RBD-YFc酶切后。
图6.无标签产品RBD TFc和RBD YFc的鉴定。图6A，SDS-PAGE电泳检测，泳道1，RBD TFc非还原；泳道2，RBD YFc非还原；泳道3，RBD TFc还原；泳道4，RBD YFc还原；图6B，HPLC-SEC检测图；图6C，质谱分子量检测。
图7.马Fc标签RBD的酶切SDS-PAGE非还原电泳检测。泳道1，RBD-eqIgG1Fc酶切前；泳道2，RBD-eqIgG1Fc酶切后；泳道3，RBD-eqIgG4Fc酶切前；泳道4，RBD-eqIgG4Fc酶切后。
图8.RBD eqG1Fc鉴定。图8A，SDS-PAGE电泳检测，泳道1，RBD eqG1Fc非还原；泳道2，RBD eqG1Fc还原；图8B，HPLC-SEC检测图；图8C，质谱分子量检测图。
图9.不同RBD-Fc经酶切、亲和层析后的无标签产品与hACE2-Fc结合的Biacore检测图。
图10.RBD YFc经过阳离子交换层析洗脱的不同组分的非还原SDS-PAGE和考马斯亮蓝染色图。
图11.RBD YFc二聚体(A)和单体(B)分别与hACE2-Fc结合的Biacore检测图。
图12.RBD YFc二聚体的结构示意图。
图13.不同RBD-Fc经酶切、亲和层析后的无标签产品RBD YFc9，RBD YFc10和RBD YFc11的非还原和还原SDS-PAGE考马斯亮蓝染色图。
具体实施方式
下面结合附图对本发明的具体实施方式做详细的说明。本发明能够以很多不同于在此描述的其它方式来实施，本领域技术人员可以在不违背本发明内涵的情况下做类似改进，因此本发明的保护范围以权利要求书为准，不受下面公开的具体实施的限制。
实施例1：融合蛋白结构设计
RBD蛋白序列来源于Genbank：QHR63260.2，通过全基因合成得到RBD基因片段，将其构建至真核表达载体(如pcDNA3.1载体，Invitrogen公司)的多克隆酶切位点之间，并在其N端加信号肽(如CD33信号肽，IL2信号肽，人白蛋白HSA信号肽等)，在其C端加蛋白标签(如His、Flag、HA、myc或Fc标签等)，以利于纯化。此外，在 RBD基因下游与标签基因上游之间加入蛋白酶酶切位点编码序列，蛋白酶包括但不限于凝血酶(Sigma)、肠激酶(New England Biolabs)、TEV蛋白酶(Invitrogen)、或HRV3C蛋白酶(Novagen)。任选地，蛋白酶酶切位点和例如Fc标签之间通过连接肽连接。所述融合蛋白结构具体如图1所示。构建物的具体序列信息如表1。
RBD为SARS-CoV-2新型冠状病毒的S蛋白中的一段结构域，位于整个S蛋白的第319-541位氨基酸残基区域(SEQ ID NO：1)。有研究报道选取不同区域的RBD进行单独的或者与Fc融合表达，以获得重组RBD蛋白，如选取S蛋白第319-545位残基(SEQ ID NO：2)进行外源表达(文献：Jingyun Yang，Wei Wang，Zimin Chen，et al.A vaccine targeting the RBD of the S protein of SARS-CoV-2 induces protective immunity.Nature 2020.)，或者选取S蛋白第331-524位残基(SEQ ID NO：3)融合Fc进行外源表达(文献：Hongjing Gu，Qi Chen，Guan Yang，et al.Adaptation of SARS-CoV-2 in BALB/c mice for testing vaccine efficacy.Science 2020.)，或者选取S蛋白第319-537位残基(SEQ ID NO：4)进行外源表达(文献：Lianpan Dai，Tianyi Zheng，Kun Xu，et al.A universal design of betacoronavirus vaccines against COVID-19，MERS and SARS.Cell 2020.)。针对上述不同区域RBD序列，均进行了融合Fc的构建，见表1的RBD-YFc6，RBD-YFc7和RBD-YFc8，其中RBD区域分别对应S蛋白第319-545位、第331-524位和第319-537位残基。
Figure PCTCN2020109295-appb-000001
Figure PCTCN2020109295-appb-000002
实施例2：不同标签RBD蛋白的表达与纯化
按常规的质粒提取方法进行质粒的提取，并用于化学转染293(ATCC)或CHO-S(Gibco)细胞。转染后的细胞在37℃、5％CO 2摇床中悬浮震荡培养7-10天。通过3000×g离心收获上清并用0.22μm滤膜过滤。
RBD-His料液收获和进行Ni柱亲和层析，表达量小于1mg/L，并对该样品进行SDS-PAGE检测。如图2A所示，Ni柱亲和层析后的样品成份杂、纯度低，因此采用阳离子交换层析进行精纯，样品低盐(5mM NaCl)挂柱高盐(500mM NaCl)线性洗脱，并对过程中从低盐向高盐洗脱的不同组份进行分管收集和SDS-PAGE检测，如图2B所示，其中的泳道2，为纯度最高组分，条带大小为50KD，与RBD-His的理论分子量相符，但是样品纯化收率极低，不超过20％。同样的，RBD-His1的表达料液进行与前述相同的Ni柱和阳离子交换层析纯化过程，获得的蛋白纯度和条带大小也是一样的，且纯化收率无显著差异。说明使用不同的信号肽进行RBD融合His标签的蛋白表达，在表达水平及纯化收率方面并无显著差异，且蛋白表达和收率较低。
RBD的C端蛋白标签更换为其他标签，包括但不限于Flag标签、HA标签和c-Myc标签，对应的RBD融合标签蛋白分别为RBD-Flag、RBD-HA和RBD-myc，所构建的表达质粒瞬时转染至293或CHO-S细胞后，悬浮振荡培养7-10天，收获的料液检测蛋白表达水平与RBD-His类似，表达量均小于1mg/L。进行标签相应的亲和层析后，获得蛋白收率也不超过20％。由此可见，在哺乳动物表达系统中，在RBD的C端融合短肽蛋白标签，表达水平和纯化收率都较低。
RBD融合Fc标签的蛋白表达料液收获并进行蛋白A亲和层析，通过SDS-PAGE分析一步亲和纯化后的蛋白，如图3所示，条带大小约为110KD，与RBD融合Fc标签的理论分子量相符，进一步通过高性能尺寸排阻色谱法(HPLC-SEC)测试其高聚体含量，具体如表2和图4。可以看到，RBD的标签由短肽标签更换为Fc后，表达量和纯度显著提高，而且采用马IgG1Fc标签后的表达量较人Fc标签有明显提高。针对RBD-Fc融合蛋白进行优化，优化前的RBD-TFc的HPLC-SEC纯度为89.43％，而优化后的RBD-YFc纯度提高至95.82％，高聚体比例减少。此外，对于优化后的RBD L455I F456V-YFc、RBD G476S-YFc和RBD V483A-YFc，高聚体比例显著减少，纯度提高5-10％；而优化后的RBD S494P-YFc与优化前的RBD S494P-TFc相比，高聚体比例也显著减少，纯度提高5％。RBD-YFc9，RBD-YFc10和RBD-YFc11的表达水平和纯度跟RBD-YFc之间无显著差异。RBD-epIG1Fc1的表达水平和纯度跟RBD-epIG1Fc之间无显著差异。RBD-epIG4Fc1和表达水平和纯度跟RBD-epIG4Fc之间无显著差异。
表2.不同RBD-Fc的表达量与纯度
构建物 表达量 HPLC-SEC
RBD-TFc 80mg/L 89.43％
RBD-YFc 100mg/L 95.82％
RBD-YFc1 98mg/L 94.56％
RBD-YFc2 95mg/L 95.11％
RBD-YFc3 99mg/L 95.63％
RBD-YFc4 87mg/L 95.02％
RBD-YFc5 88mg/L 94.75％
RBD-YFc6 90mg/L 90.91％
RBD-YFc7 92mg/L 77.78％
RBD-YFc8 89mg/L 94.59％
RBD-YFc9 95mg/L 92.77％
RBD-YFc10 99mg/L 94.64％
RBD-YFc11 99mg/L 93.18％
RBD L455I F456V-YFc 108mg/L 60.70％
RBD G476S-YFc 94mg/L 94.41％
RBD V483A-YFc 97mg/L 95.28％
RBD S494P-TFc 43mg/L 55.00％
RBD S494P-YFc 54mg/L 60.66％
RBD-eqIgG1Fc 180mg/L 89.84％
RBD-eqIgG4Fc 92mg/L 78.13％
实施例3：无标签的RBD制备和鉴定
为消除Fc标签在体内诱导的非特异性抗体以及对RBD的影响，采用蛋白酶切除Fc标签，得到无标签的RBD蛋白，具体方法如下：在亲和层析纯化的RBD融合Fc蛋白中加入相应的蛋白酶，按照说明书要求进行特定条件下的孵育，再通过蛋白A或蛋白G亲和层析的流穿模式获得RBD蛋白，即为无标签产品。通过SDS-PAGE对RBD融合Fc蛋白的酶切前和酶切样品进行酶切效果检测，并统计RBD蛋白的酶切纯化回收率，并对最终的无标签RBD蛋白进行检测。
ACE2蛋白的制备：构建人ACE2胞外结构域与Fc融合的蛋白hACE2-Fc(SEQ ID NO：24)的真核表达质粒(载体为pcDNA3.1)，将该质粒瞬转至293或CHO-S细胞中， 培养7-10天收获上清，用ProteinA亲和层析纯化获得hACE2-Fc蛋白。
如图5中的泳道2和4所示，优化后的构建物RBD-YFc，其酶切效果明显优于优化前的RBD-TFc。如表3所示，优化后的RBD-YFc、RBD-YFc1-RBD-YFc11和RBD-eqIgG1Fc酶切纯化回收率较优化前的RBD-TFc明显提高。并且不论RBD是野生型还是突变型，优化后的RBD-YFc的酶切效果均优于RBD-TFc，可将目标蛋白回收率提高20-30％。对无标签产品RBD TFc和RBD YFc进行鉴定，从纯度(图6A和6B)、分子量(图6C)来说，两者一致，且质谱分子量检测RBD蛋白中含有单体(31KD)和二聚体(62KD)两种形式，该结果与SDS-PAGE及SEC结果一致。从与hACE2-Fc的结合活性(图9)来说，无标签产品RBD YFc与hACE2-Fc的结合活性高于RBD TFc。
表3.RBD回收率统计
构建物 去除Fc后的RBD 目标蛋白回收率
RBD-TFc RBD TFc 58.6％
RBD-YFc RBD YFc 82.3％
RBD-YFc1 RBD YFc1 78.8％
RBD-YFc2 RBD YFc2 79.3％
RBD-YFc3 RBD YFc3 77.5％
RBD-YFc4 RBD YFc4 77.4％
RBD-YFc5 RBD YFc5 78.9％
RBD-YFc6 RBD YFc6 79.2％
RBD-YFc7 RBD YFc7 78.9％
RBD-YFc8 RBD YFc8 77.36％
RBD-YFc9 RBD YFc9 76.9％
RBD-YFc10 RBD YFc10 80.1％
RBD-YFc11 RBD YFc11 79.3％
RBD-eqIgG1Fc RBD eqG1Fc 70.7％
注：由于每毫克RBD-Fc蛋白是由0.5毫克的RBD和0.5毫克Fc标签组成，因此RBD目标蛋白回收率＝最终获得RBD蛋白量/(RBD-Fc蛋白量×50％)。
使用不同的蛋白酶切位点构建的RBD融合Fc蛋白，如RBD-YFc(凝血酶)，RBD-YFc1(肠激酶)，RBD-YFc2(TEV蛋白酶)和RBD-YFc3(HRV-3C蛋白酶)，酶切效果优于优化前含有相应酶切位点的RBD-TFc(即RBD-TFc中的凝血酶酶切位点替换为肠激酶酶切位点、TEV蛋白酶酶切位点、HRV-3C蛋白酶酶切位点)，RBD目标蛋白回收率显著提高，并且在酶切、亲和层析和离子交换层析之后，获得的无标签产品 RBD YFc、RBD YFc1、RBD YFc2和RBD YFc3的纯度、分子量和结合hACE2-Fc的活性无显著差异。
选取S蛋白不同区域构建的RBD融合Fc蛋白，如RBD-YFc6，RBD-YFc7和RBD-YFc8，在酶切、亲和层析之后的获得无标签产品RBD YFc6，RBD YFc7和RBD YFc8通过非还原SDS-PAGE检测纯度见表4。其中RBD YFc6二聚体比例超过80％，RBD YFc7二聚体比例不到30％且高聚体(分子量超过90kD)和单体比例都超过30％，RBD YFc8只有单体。与RBD-YFc的连接肽不同的RBD-YFc9～11，酶切和亲和层析后获得无标签产品RBD YFc9、RBD YFc10和RBD YFc11均有近90％的二聚体组分(图13)。
表4非还原SDS-PAGE检测RBD无标签产品的纯度
RBD无标签产品 高聚体比例 二聚体比例 单体比例
RBD TFc 0 65％ 35％
RBD YFc 0 90％ 10％
RBD YFc6 0 81％ 19％
RBD YFc7 31％ 29％ 40％
RBD YFc8 0 0 100％
RBD YFc9 O 87％ 13％
RBD YFc10 0 88％ 12％
RBD YFc11 0 89％ 11％
注：RBD YFc7和RBD YFc8均不包含按照SEQ ID NO；1的位置编号定位为位置220的半胱氨酸
如图7泳道2和4所示，构建物RBD-eqIgG1Fc的酶切效率明显优于RBD-eqIgG4Fc，且如图8和图9所示，RBD-eqIgG1Fc酶切后获得的RBD eqG1Fc，从纯度、分子量以及hACE2-Fc结合活性来说，与RBD YFc高度一致。RBD-eqIgG4Fc酶切后获得的RBD eqG4Fc也具有分子量为62kD的二聚体蛋白和分子量31kD的单体，Biacore检测与RBD eqG1Fc具有一致的活性。
实施例4：RBD蛋白与人ACE2的结合活性检测
有大量文献报道，SARS-CoV-2新冠病毒RBD跟人ACE2受体之间具有高结合活性。
无标签产品RBD YFc(即通过蛋白酶切除了标签)进一步通过阳离子交换层析(如填料为GE公司的Capto SP ImpRes)进行高盐(500mM NaCl)线性梯度洗脱，对洗脱组分进行SDS-PAGE检测，如图10所示，可以将单体和二聚体有效分离。收集合并泳道2-4为RBD YFc单体组分，收集合并泳道7-12为RBD YFc二聚体组分，进行HPLC-SEC 纯度检测，单体及二聚体组分的纯度分别超过97％。
分别将RBD YFc单体和二聚体组分进行与hACE2-Fc蛋白的结合活性Biacore检测，如图11所示，RBD YFc二聚体与hACE2-Fc的亲和力为K D＝0.192nM，而RBD YFc单体与hACE2-Fc的亲和力为K D＝15.60nM。RBD YFc二聚体结合hACE2-Fc的能力显著强于RBD YFc单体结合hACE2-Fc的能力。
RBD YFc单体的氨基酸残基编码见表5。RBD YFc二聚体在Chymotrypsin(Sigma)酶解后用质谱进行分析，发现两个单体第220位半胱氨酸形成了一对链间二硫键。这表明RBD YFc二聚体是两个RBD YFc单体通过一对第220位半胱氨酸形成的链间二硫键共价相连。
表5 RBD YFc单体的氨基酸残基编码(SEQ ID NO：1)
Figure PCTCN2020109295-appb-000003
综上，当选择SARS-CoV-2病毒S蛋白RBD区域(例如第319-541位残基)，C端融合有利于二聚体形成的标签(例如来源于IgG的Fc标签)，优选在RBD与标签之间加上蛋白酶酶切位点和优化蛋白酶酶切位点与标签之间的连接肽和铰链区，构建的RBD-酶切位点-标签融合蛋白，所述融合蛋白在哺乳动物细胞中具有良好的表达水平和纯度，并且经过蛋白酶酶切、亲和层析及离子交换层析后，可以获得高回收率的RBD二聚体组分，并且二聚体组分跟hACE2-Fc具有更高的结合活性。所述RBD二聚体的分子量是62kD，是由两个RBD单体之间通过一对链间二硫键共价连接(具体为表5 编码所示第220位半胱氨酸之间形成二硫键)，并且每个RBD单体存在四对链内二硫键(见图12)，分别为：(1)第18位半胱氨酸和第43位半胱氨酸之间的二硫键，(2)第61位半胱氨酸和第114位半胱氨酸之间的二硫键，(3)第73位半胱氨酸和第207位半胱氨酸的二硫键，(4)第162位半胱氨酸和第170位半胱氨酸之间的二硫键，序列编码参见表5。
RBD-mIgG2aFc、RBD-JUN和RBD-FOS(表2)通过纯化、酶切、亲和层析及离子交换层析后获得的RBD二聚体组分，具有与图12所示的RBD二聚体相同的序列、结构以及与hACE2-Fc的结合活性。
Figure PCTCN2020109295-appb-000004
Figure PCTCN2020109295-appb-000005
Figure PCTCN2020109295-appb-000006

Claims (14)

  1. 新型冠状病毒SARS-CoV-2的RBD融合蛋白，其顺序包含新型冠状病毒SARS-CoV-2的RBD序列(优选所述RBD序列包含按照SEQ ID NO：1所述序列的位置编号定位为位置220的半胱氨酸，且具有结合人ACE2受体的活性；更优选地RBD序列包含如SEQ ID NO：1-8任一项所示的氨基酸序列)，蛋白酶酶切位点，和有利于二聚体形成的标签。
  2. 权利要求1的新型冠状病毒SARS-CoV-2的RBD融合蛋白，其中所述有利于二聚体形成的标签选自亮氨酸拉链(优选SEQ ID NO：30或31中不包含HHHHHH的序列)和Fc片段组成的组，其中所述Fc片段不包含铰链区的半胱氨酸(优选所述Fc段为来源于人IgG或马IgG的Fc片段，更优选地，所述Fc片段不包含铰链区，进一步更优选所述Fc片段包含如SEQ ID NO：13-16、29任一项所示的氨基酸序列)，优选地所述亮氨酸拉链来自c-JUN或c-FOS蛋白，更优选地，所述亮氨酸拉链C端还连接有His、Flag、C-myc或HA标签。
  3. 权利要求1或2的新型冠状病毒SARS-CoV-2的RBD融合蛋白，其中所述蛋白酶酶切位点和标签通过连接肽连接，优选地，所述连接肽选自SEQ ID NO：26，27或28。
  4. 权利要求1-3任一项所述的新型冠状病毒SARS-CoV-2的RBD融合蛋白，其中所述蛋白酶选自凝血酶、肠激酶、TEV蛋白酶或HRC-3C蛋白酶；优选地，所述凝血酶、肠激酶、TEV蛋白酶或HRC-3C蛋白酶酶切位点的氨基酸序列如SEQ ID NO：17-20任一项所示；更优选所述蛋白酶酶切位点的氨基酸序列与所述RBD序列或Fc片段中的氨基酸序列不存在重复。
  5. 权利要求1-4任一项所述的新型冠状病毒SARS-CoV-2的RBD融合蛋白，其中所述融合蛋白的N端还包含信号肽，优选地，所述信号肽的氨基酸序列包含如SEQ ID NO：21-23任一项所示的氨基酸序列。
  6. 新型冠状病毒SARS-CoV-2的RBD二聚体，其特征在于按照SEQ ID NO：1所示单体RBD序列的位置编号，单体RBD的位置18和43的半胱氨酸、位置61和114的半胱氨酸、位置73和207的半胱氨酸以及位置162和170的半胱氨酸分别形成链内二硫键，两个单体RBD的位置220的半胱氨酸之间形成链间二硫键，优选地，所述单 体RBD序列包含如SEQ ID NO：1和5-8任一项所示的氨基酸序列，更优选地，所述二聚体的两个单体RBD序列相同。
  7. 制备权利要求1-5任一项所述新型冠状病毒SARS-CoV-2的RBD融合蛋白的方法，包括依次顺序连接新型冠状病毒SARS-CoV-2的RBD序列，蛋白酶酶切位点和有利于二聚体形成的标签，优选地，所述蛋白酶酶切位点和所述标签通过连接肽连接。
  8. 制备权利要求6所述新型冠状病毒SARS-CoV-2的RBD二聚体的方法，包括用蛋白酶切割权利要求1-5任一项所述新型冠状病毒SARS-CoV-2的RBD融合蛋白，优选地，所述方法还包括对RBD融合蛋白酶切产物进行纯化，优选地，所述纯化包括层析(例如亲和层析、离子交换层析)。
  9. 多核苷酸，其特征在于编码权利要求1-5任一项所述的新型冠状病毒SARS-CoV-2的RBD融合蛋白或权利要求6所述的新型冠状病毒SARS-CoV-2的RBD二聚体。
  10. 载体，其包含权利要求9所述的多核苷酸。
  11. 宿主细胞，其包含权利要求9所述的多核苷酸或权利要求10所述的载体。
  12. 疫苗，优选用于预防新型冠状病毒的感染，其特征在于包含权利要求1-5任一项所述的新型冠状病毒SARS-CoV-2的RBD融合蛋白、权利要求6所述的新型冠状病毒SARS-CoV-2的RBD二聚体，或权利要求9所述的多核苷酸。
  13. 权利要求1-5任一项所述的新型冠状病毒SARS-CoV-2的RBD融合蛋白、权利要求6所述的新型冠状病毒SARS-CoV-2的RBD二聚体，或权利要求9所述的多核苷酸，其用于预防人类感染新型冠状病毒SARS-CoV-2，或用于免疫动物以获得抗病毒血清，所述动物优选是哺乳动物，更优选马，优选所述抗病毒血清可用于预防或治疗人类感染新型冠状病毒SARS-CoV-2。
  14. 预防或治疗新型冠状病毒SARS-CoV-2感染或获得针对新型冠状病毒SARS-CoV-2的中和抗体的方法，其包含将权利要求1-5任一项所述的新型冠状病毒SARS-CoV-2的RBD融合蛋白、权利要求6所述的新型冠状病毒SARS-CoV-2的RBD二聚体，或权利要求9所述的多核苷酸免疫动物以获得抗病毒血清，所述动物优选是人或非人哺乳动物，更优选马。
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