WO2021189245A1 - Bifunctional fusion protein binding to coronavirus, preparation method therefor, and application thereof - Google Patents

Bifunctional fusion protein binding to coronavirus, preparation method therefor, and application thereof Download PDF

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WO2021189245A1
WO2021189245A1 PCT/CN2020/080859 CN2020080859W WO2021189245A1 WO 2021189245 A1 WO2021189245 A1 WO 2021189245A1 CN 2020080859 W CN2020080859 W CN 2020080859W WO 2021189245 A1 WO2021189245 A1 WO 2021189245A1
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coronavirus
fusion protein
bifunctional fusion
ace2
binds
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PCT/CN2020/080859
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French (fr)
Chinese (zh)
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王皓
钱卫珠
张大鹏
李晶
陶静
李伟
李继阳
张浔敏
李云峰
袁秀珍
焦树阁
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泰州迈博太科药业有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • 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
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/515—Complete light chain, i.e. VL + CL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention discloses a bifunctional fusion protein that binds to coronavirus, and its preparation method and application.
  • 2019-novel coronavirus (2019-nCoV, SARS-CoV-2) is an enveloped, positively charged single-stranded RNA virus, belonging to the genus ⁇ -coronavirus, a subgenus of Sarbecovirus.
  • SARS-CoV-2 2019-nCoV, SARS-CoV-2
  • most patients will develop symptoms such as fever, cough, vomiting and diarrhea, mild pneumonia, and some patients will develop severe pneumonia and the condition is serious Patients may suffer from acute respiratory failure, shock, and even death.
  • ACE2 is a homologue of angiotensin-converting enzyme. It is a type I transmembrane protein composed of 805 amino acids. It has two domains, namely the amino-terminal catalytic domain and the carboxy-terminal domain. The catalytic structure contains 1 With a single active site-zinc metalloprotein domain, ACE2 can be expressed in a variety of tissues such as the human heart, kidney, lung, liver, testis, and intestine.
  • ACE2 The role of ACE2 in the course of new coronary pneumonia, in addition to being a receptor for virus binding, ACE2 also participates in mediating the immune process.
  • ACE2 in cells can be further increased after the new coronavirus infection, which is more conducive to virus infection (Wang,PH(2020).IncreasingHostCellularReceptor-Angiotensin-ConvertingEnzyme2(ACE2)ExpressionbyCoronavirusmayFacilitate2019-nCoV Infection.bioRxiv.).
  • Angiotensin-converting enzyme 2 (ACE2) is a double-edged sword: in the process of virus infection, it is the membrane-bound receptor of the new coronavirus, which is related to the fusion and invasion process of the virus and host cell; in influenza virus, SARS In the case of inflammation and acute lung injury, ACE2 promotes the conversion of angiotensin II to angiotensin 1-7, inhibits the NF- ⁇ B signal mediated by angiotensin II, thereby avoiding the cytokine storm caused by a large number of inflammatory signals and slowing down Course of disease.
  • ACE2 Angiotensin-converting enzyme 2
  • Interleukin-6 is the main factor causing the cytokine storm. After infection with SARS-CoV-2 (2019-nCoV), activated pathogenic T cells will produce granulocytes-macrophages Colony stimulating factor (GM-CSF) and IL-6. GM-CSF can activate CD14+CD16+ inflammatory monocytes and further produce more cytokines, including IL-6. IL-6 plays an indispensable part in this positive feedback loop and ultimately contributes to the loss of control of the immune system.
  • SARS-CoV-2 2019-nCoV
  • GM-CSF Colony stimulating factor
  • IL-6 can activate CD14+CD16+ inflammatory monocytes and further produce more cytokines, including IL-6.
  • IL-6 plays an indispensable part in this positive feedback loop and ultimately contributes to the loss of control of the immune system.
  • SARS-CoV-2 (2019-nCoV) uses angiotensin-converting enzyme 2 (ACE2) as the entry receptor, which is the same as the entry receptor used by the SARS coronavirus. Both coronaviruses pass through the virion.
  • the S protein (SPIKE protein) (virus spike glycoprotein) binds to ACE2, the viral membrane and the cell membrane are fused, and then the RNA virus will replicate its genes in the cell and finally produce new virus particles, which will be secreted to Infect other cells.
  • SPIKE protein virus spike glycoprotein
  • RBD receptor-binding domain
  • SARS virus S protein Spike protein
  • This binding domain has been shown to be the key domain for the virus to bind to the ACE2 receptor during cell entry.
  • the use of this binding domain has been shown to be able to Effectively prevent SARS from entering the cell proliferation.
  • ACE2-Fc coronavirus S protein
  • ACE2-Fc strategy a potential limitation of the ACE2-Fc strategy is that the increase in extracellular ACE2 levels may have unknown effects on the human body. It has been found that the human body can secrete a small amount of extracellular ACE2, so it is not unprecedented that this extracellular domain enters the blood circulation. . (Shao Z, Shrestha K, Borowski AG, et al: Increasing serum soluble angiotensin-converting enzyme2 activity after intensive medical therapy is associated with better prognosis in acute decompensated heart failure.J Card Fail.2013-610(9) ).
  • rhACE2 active recombinant human ACE2
  • ACE2 supplementation in humans can reshape ACE2/ACE Balance (Khan,A.,Benthin,C.,Zeno,B.,Albertson,TE,Boyd,J.,Christie,JD,...&Hardes,K.(2017).A pilot clinical trial of recombinant human angiotensin- converting enzyme 2 in acute respiratory distress syndrome.Critical Care,21(1),234.Treml,B.,Neu,N.,Kleinsasser,A.,Gritsch,C.,Finsterwalder,T.,Geiger,R..&Loeckinger ,A.(2010).Recombinant angiotensin-converting enzyme 2 improve pulmonary blood flow and oxygenation in lipopolysaccharide-induced lung infection in piglets.Critical care medicine,38(2),596-601.), ACE2 can reduce lung inflammation Potential, while having tolerable safety.
  • antiviral S glycoprotein IgG disrupts the response of macrophages and causes severe acute lung injury.
  • S-IgG can significantly reduce the virus titer, it causes lung damage in the early stages of infection, mainly by eliminating the macrophage response and TGF- ⁇ production in wound healing, and at the same time promoting the inflammatory cytokine IL-8, and The production of MCP1 and the accumulation of inflammatory macrophages.
  • Anti-spike IgG causes severe acute lung infection by skewing macrophage responses during acute SARS-CoV infection. [J]. JCI insight, 2019, 4(4): e123158.).
  • Patent US11667640 discloses a composition capable of neutralizing the binding molecules of SARS coronavirus (SARS-CoV).
  • the combination group contains at least two SARS-related coronaviruses (SARS-CoV) and possessing SARS-CoV. Neutralizing active immunoglobulin.
  • an antibody that can bind to ASRS coronavirus is disclosed, and it is disclosed that the antibody binds to amino acids 318-510 of the S protein (Spike protein) of SARS-CoV.
  • the drug described in this patent is a composition
  • the antibodies that make up the combination are all antibodies against the amino acids at positions 318-510 of the S protein (Spike protein) that bind to SARS-CoV, even if each The specific binding sites of these antibodies are the different amino acids in positions 318-510, and they all have the disadvantage of mutual influence.
  • compositions are macromolecular proteins, and due to the characteristics of their spatial structure, there will be interactions between antibodies when the antibodies and antigens are combined with each other.
  • the composition provided in this patent is all the S protein that binds to SARS-CoV.
  • the amino acids at positions 318-510 of (Spike protein), when combined with the virus, will affect the effect of the antibody in neutralizing the SARS-CoV coronavirus.
  • the present invention provides a bifunctional fusion protein that specifically binds to coronavirus.
  • the bifunctional fusion protein that specifically binds to the coronavirus provided by the present invention has the functions of anti-SARS-CoV and anti-SARS-CoV-2, and has the function of Fc, the ability to treat virus-infected cells, and it also has anti-virus and anti-SARS-CoV-2 functions. Reduce lung inflammation and reduce the effect of cytokine storm.
  • the bifunctional fusion protein that specifically binds to coronaviruses has a wide range of neutralizing effects, and at the same time has the ability to neutralize animal-related SARS-CoV (SARS-like CoV).
  • CoV or SARS-CoV-2 similar virus also plays a neutralizing role in the treatment of viruses, and can also be used for the treatment of viruses that have been found to use ACE2 as the receptor and the treatment of new types of viruses that have not been discovered that use ACE2 as the receptor in the future It also has a therapeutic effect on lung injury diseases mediated by the ACE2 axis.
  • the bifunctional fusion protein that specifically binds to coronavirus provided by the present invention has binding regions at both ends that respectively bind to different epitopes of the coronavirus S protein (Spike protein), because the bifunctional fusion protein that specifically binds to coronavirus has The special structure produces steric hindrance after binding to the coronavirus S protein (Spike protein), which blocks other binding sites where the coronavirus S protein (Spike protein) can bind to the host cell.
  • the protein (Spike protein) binds to other ways to invade host cells, such as the CD147 receptor binding pathway (the coronavirus S protein binds to the receptor CD147 on the host cell to invade the host cell).
  • the bifunctional fusion protein that specifically binds to coronaviruses provided by the present invention can block the combination of coronaviruses to host cells in multiple ways, more effectively reduce the invasion and infection of coronaviruses, and more effectively resist viruses and reduce lung inflammation , Reduce the cytokine storm, more effectively treat the disease caused by the coronavirus.
  • the bifunctional fusion protein that specifically binds to coronaviruses has a structure diagram as shown in FIG.
  • the bifunctional fusion protein that specifically binds to the coronavirus provided by the present invention, wherein the Fab end of the anti-SARS-CoV or SARS-CoV-2 antibody can be combined with the S protein (Spike protein) of the coronavirus, and the binding site Unlike the binding site of ACE2, it does not compete with ACE2 to bind to the RBD of the coronavirus.
  • the Fab end of the anti-SARS-CoV or SARS-CoV-2 antibody can synergistically enhance the binding of ACE2 and the coronavirus RBD, enhance antiviral activity, and avoid possible virus enhancement.
  • the bifunctional fusion protein that specifically binds to the coronavirus provided by the present invention can not only bind to the coronavirus at one end of ACE2, but also protect the ACE2 on the cell surface from being bound by the coronavirus, and does not affect the normal function of ACE2.
  • Active ACE2 may be reduced without causing a cytokine storm, avoiding the production of inflammatory factors, reducing inflammation, blocking the ACE2 binding pathway of coronavirus and normal cells, and reducing the ACE2 reduction caused by Inflammation, avoid pneumonia caused by the ACE2 pathway, reduce the symptoms of pneumonia in people infected with coronavirus, and reduce the effect of cytokine storm.
  • the bifunctional fusion protein that specifically binds to the coronavirus as described above, after binding to the coronavirus S protein (Spike protein) through different binding sites, due to its special structure, produces a steric hindrance effect.
  • the S protein can block other binding sites that bind to the host cell, blocking other ways that the coronavirus invades the host cell through the S protein binding, such as the CD147 receptor binding pathway (coronavirus S protein and the receptor CD147 on the host cell Binding, thereby invading host cells).
  • the bifunctional fusion protein that specifically binds to the coronavirus provided by the present invention can target the target molecule to the virus by its antibody portion, and can also reduce the risk of blood pressure drop caused by the use of ACE2 alone.
  • ACE2 combines with the S1 and S2 junctions of SARS coronavirus (Aiping Wu, Peihua Niu, Lulan Wang, et.al. Mutations, Recombination and Insertion in the Evolution of 2019-nCoV.bioRxiv March 02, 2020).
  • the bifunctional fusion protein that specifically binds to coronaviruses provided by the present invention has the characteristics of blocking the ACE2 pathway of coronaviruses, and has stronger advantages than using ACE2-Fc alone. Therefore, the present invention The provided bifunctional fusion protein that specifically binds to the coronavirus can more effectively deal with the mutant virus strain of the coronavirus.
  • the bifunctional fusion protein that specifically binds to the coronavirus provided by the present invention can be effectively used as a drug inventory for the treatment of SARS and SARS-CoV-2 (2019-nCoV) and similar virus outbreaks in the future, and it can also deal with any future through ACE2 Receptor to other new viruses that enter the cell. At the same time, it can also be used as a passive immune protective agent for people at risk for the prevention of SARS and SARS-CoV-2 related diseases.
  • the invention discloses a bifunctional fusion protein that specifically binds to a coronavirus, and the bifunctional fusion protein respectively binds to different binding sites of the coronavirus.
  • the bifunctional fusion protein that specifically binds to the coronavirus as described above, and the coronavirus has a binding site for Spike protein.
  • the bifunctional fusion protein that specifically binds to the coronavirus as described above, and the coronavirus has an ACE2 binding site.
  • the bifunctional fusion protein that specifically binds to the coronavirus as described above binds to the ACE2 binding site of the coronavirus and at the same time binds to the Spike protein of the coronavirus.
  • the bifunctional fusion protein that specifically binds to the coronavirus as described above has the amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO: 14 that binds to the ACE2 binding site of the coronavirus.
  • the bifunctional fusion protein that specifically binds to the coronavirus as described above has the heavy chain amino acid sequence shown in SEQ ID NO: 6 that binds to the Spike protein of the coronavirus and the heavy chain amino acid sequence shown in SEQ ID NO: 2 The amino acid sequence of the light chain is shown.
  • a composition disclosed in the present invention includes the above-mentioned bifunctional fusion protein that specifically binds to coronavirus and pharmaceutically acceptable excipients.
  • the present invention discloses a method for preparing a bifunctional fusion protein that specifically binds to a coronavirus, comprising the following steps: a) Gene synthesis of the above-mentioned gene sequence of the bifunctional fusion protein that specifically binds to a coronavirus, and preparation containing the bifunctional fusion Protein gene expression vector; b) transfecting the expression vector to host cells, and culturing the host cells and expressing the bifunctional fusion protein; c) separation and purification of the bifunctional fusion protein.
  • the method for preparing a bifunctional fusion protein that specifically binds to a coronavirus as described above is characterized in that the host cell is a eukaryotic mammalian cell CHO cell.
  • the method for preparing a bifunctional fusion protein that specifically binds to a coronavirus as described above is characterized in that the host cell is cultured using a serum-free and animal-derived component-free medium.
  • the invention discloses an application of a bifunctional fusion protein that specifically binds to a coronavirus, including the application of the above-mentioned bifunctional fusion protein that specifically binds to a coronavirus in the preparation of anti-coronavirus drugs.
  • the application of the bifunctional fusion protein that specifically binds to the coronavirus as described above is characterized in that the coronavirus is a SARS-CoV related coronavirus, a MERS-CoV related coronavirus, or a SARS-CoV-2 related virus.
  • Figure 1 Schematic diagram of the bifunctional fusion protein that specifically binds to coronavirus
  • FIG. 1 Construction diagram of the full-length expression vector of CoV022 light chain
  • FIG. 1 Construction diagram of CoV022 heavy chain variable region cloning vector
  • FIG. 1 Construction diagram of CoV022 heavy chain full-length expression vector
  • FIG. 1 Construction diagram of CoV022 heavy chain variable region (Knob) cloning vector
  • FIG. 1 Construction diagram of CoV022 heavy chain full length (Knob) expression vector
  • Figure 14 Construction diagram of a single plasmid of CoV022 light chain and heavy chain full length (Knob) expression vector
  • FIG. 1 SDS-PAGE detection map of CHO host cells expressing ACE2-609-Fc, CoV022, ACE2-609-Fc/CoV022 bifunctional fusion protein;
  • FIG. 17 ELISA detection map of the binding ability of CoV022, ACE2-609-Fc, ACE2-609-Fc/CoV022 bifunctional fusion protein and SARS-CoV-2 Spike protein;
  • FIG. 18 ELISA detection map of the binding ability of CoV022, ACE2-615-Fc, ACE2-615-Fc/CoV022 bifunctional fusion protein and SARS-CoV-2 Spike protein.
  • CoV022 is an antibody that binds to coronavirus S protein (Spike protein).
  • the full-gene synthesis CoV022 light chain full-length cloning vector was digested with HindIII and EcoR1, and the pL101 expression vector was digested at the same time.
  • the target fragment of about 750bp and the expression vector fragment of about 9kb were recovered separately, and the two were ligated with T4 DNA ligase. Transformation, a small amount of plasmid extraction and double digestion with HindIII and EcoR1 for identification.
  • the full length of the light chain of CoV022 has the nucleotide sequence shown in SEQ ID NO: 1, and the amino acid sequence shown in SEQ ID NO: 2.
  • the full-gene synthesis CoV022 heavy chain variable region cloning vector is digested with HindIII and NheI, and the IgG1 constant region gene cloning vector is double digested at the same time.
  • the target fragment of about 450 bp and the cloning vector fragment of about 4 kb are recovered separately, and the two are used T4 DNA Ligase ligation and transformation, a small amount of plasmid extraction and double digestion with HindIII and NheI identification.
  • the CoV022 heavy chain full-length cloning vector was double digested with HindIII and EcoR1, and the pL102 expression vector was double digested at the same time.
  • the target fragment of about 1500 bp and the expression vector fragment of about 5 kb were recovered separately, and the two were ligated and transformed with T4 DNA ligase.
  • the plasmid was extracted in a small amount and identified by double digestion with HindIII and EcoR1.
  • the agarose concentration is 1%
  • the arrow shows the target fragment of about 1500bp
  • right 1 DL2000 DNA Marker
  • right 2 ⁇ -HindIII digest DNA Marker.
  • the full length of the CoV022 heavy chain has the nucleotide sequence shown in SEQ ID NO: 3 and the amino acid sequence shown in SEQ ID NO: 4.
  • CoV022 light chain full-length expression vector and CoV022 heavy chain full-length expression vector were digested with SalI and NotINotI, respectively, and a 9kb light chain full-length expression fragment and a 4kb heavy chain full-length expression vector fragment were recovered respectively. Ligation and transformation with T4 DNA ligase, a small amount of plasmid extraction and double enzyme digestion with SalI and NotINotI for identification.
  • the agarose concentration is 1%
  • the arrow shows the light chain full-length expression vector fragment of about 9kb and the heavy chain full-length target fragment of about 4kb
  • right 1 DL2000 DNA Marker
  • right 2 ⁇ - HindIII digest DNA Marker.
  • the full-gene synthesis ACE2-609 cloning vector was digested with HindIII and NheI, and the Fc/pGEM-T cloning vector was digested with double digestion.
  • the target fragment of about 2.0kb and the Fc cloning vector fragment of about 3.7kb were recovered separately, and the two were used T4 DNA ligase ligation and transformation, a small amount of plasmid extraction and double digestion with HindIII and NheI identification.
  • the agarose concentration is 1%
  • the arrow shows the target fragment of about 2.0kb
  • right 1 DL2000 DNA Marker
  • right 2 ⁇ -HindIII digest DNA Marker.
  • the ACE2-609-Fc/pGEM-T cloning vector was digested with HindIII and EcoR1, and the pL101 expression vector was digested at the same time.
  • the target fragments of about 2.7kb and the expression vector fragments of about 9kb were respectively recovered by glue, and the two were connected with T4 DNA Enzyme ligation and transformation, a small amount of plasmid extraction and double digestion with HindIII and EcoR1 for identification.
  • ACE2-609-Fc has the nucleotide sequence shown in SEQ ID NO: 7 and the amino acid sequence shown in SEQ ID NO: 8.
  • the agarose concentration is 1%
  • the arrow shows the target fragment of about 2.7kb
  • right 1 DL2000 DNA Marker
  • right 2 ⁇ -HindIII digest DNA Marker.
  • the ACE2-615 cloning vector for full gene synthesis was digested with HindIII and NheI, and the Fc/pGEM-T cloning vector was double digested at the same time.
  • the target fragment of about 2.0kb and the Fc cloning vector fragment of about 3.7kb were recovered separately, and the two were used T4 DNA ligase ligation and transformation, a small amount of plasmid extraction and double digestion with HindIII and NheI identification.
  • the agarose concentration is 1%
  • the arrow shows the target fragment of about 2.0kb
  • right 1 DL2000 DNA Marker
  • right 2 ⁇ -HindIII digest DNA Marker.
  • the ACE2-615-Fc/pGEM-T cloning vector was digested with HindIII and EcoR1, and the pL101 expression vector was digested at the same time.
  • the target fragment of about 2.7 kb and the expression vector fragment of about 9 kb were recovered separately, and the two were ligated with T4 DNA Enzyme ligation and transformation, a small amount of plasmid extraction and double digestion with HindIII and EcoR1.
  • ACE2-615-Fc has the nucleotide sequence shown in SEQ ID NO: 9 and the amino acid sequence shown in SEQ ID NO: 10.
  • the agarose concentration is 1%
  • the arrow shows the target fragment of about 2.7kb
  • right 1 DL2000 DNA Marker
  • right 2 ⁇ -HindIII digest DNA Marker.
  • the ACE2-609-Fc cloning vector was double-cut with HindIII and EcoR1, and the pL101 expression vector was double-cut at the same time.
  • the target fragment of about 2.7 kb and the expression vector fragment of about 9 kb were respectively recovered, and the two were ligated and transformed with T4 DNA ligase.
  • a small amount of plasmid was extracted and identified with HindIII and EcoR1 double enzyme digestion.
  • ACE2-609-Fc (Fc in Hole) has the nucleotide sequence shown in SEQ ID NO: 11, and the amino acid sequence shown in SEQ ID NO: 12.
  • the agarose concentration is 1%
  • the arrow shows the target fragment of about 2.7kb
  • right 1 DL2000 DNA Marker
  • right 2 ⁇ -HindIII digest DNA Marker.
  • the whole gene synthesis ACE2-615 fragment was double digested with HindIII and EcoR1, and the pL101 expression vector was double digested at the same time.
  • the target fragment of about 2.0 kb and the expression vector fragment of about 9 kb were recovered separately, and the two were ligated and transformed with T4 DNA ligase.
  • the plasmid was extracted in a small amount and identified by double digestion with HindIII and EcoR1.
  • ACE2-615-Fc (Fc in Hole) has the nucleotide sequence shown in SEQ ID NO: 13 and the amino acid sequence shown in SEQ ID NO: 14.
  • the agarose concentration is 1%
  • the arrow shows the target fragment of about 2.0kb
  • right 1 DL2000 DNA Marker
  • right 2 ⁇ -HindIII digest DNA Marker.
  • the full-gene synthesis CoV022 heavy chain variable region cloning vector is digested with HindIII and NheI, and the IgG1 constant region cloning vector (Knob) is double digested at the same time.
  • the agarose concentration is 1%
  • the arrow shows the target fragment of about 450bp
  • right 1 DL2000 DNA Marker
  • left 1 ⁇ -HindIII digest DNA Marker.
  • the CoV022 heavy chain full-length (Knob) cloning vector was digested with HindIII and EcoR1, and the pL102 expression vector was digested at the same time.
  • the target fragment of about 1500bp and the expression vector fragment of about 5kb were recovered separately, and the two were ligated with T4 DNA ligase After transformation, a small amount of plasmids were extracted and identified by double digestion with HindIII and EcoR1.
  • the full length of the CoV022 heavy chain (Knob) has the nucleotide sequence shown in SEQ ID NO: 5 and the amino acid sequence shown in SEQ ID NO: 6.
  • the agarose concentration is 1%
  • the arrow shows the target fragment of about 1500bp
  • right 1 DL2000 DNA Marker
  • right 2 ⁇ -HindIII digest DNA Marker.
  • CoV022 light chain full-length expression vector and CoV022 heavy chain full-length (knob) expression vector were digested with SalI and NotI, respectively, and a 9kb full-length light chain full-length expression fragment and a 4kb full-length heavy chain full-length expression vector fragment were recovered respectively.
  • the two were ligated and transformed with T4 DNA ligase, a small amount of plasmid was extracted and identified by double enzyme digestion with SalI and NotI.
  • the agarose concentration is 1%
  • the arrow shows the light chain full-length expression vector fragment of about 9kb and the heavy chain full-length target fragment of about 4kb
  • right 1 DL2000 DNA Marker
  • right 2 ⁇ - HindIII digest DNA Marker.
  • the CoV022, ACE2-609-Fc, ACE2-609-Fc/CoV022 bifunctional protein expression vectors were purified by column, and the obtained plasmids were transiently transfected into the host cell Expi CHO-S cells, and the feed medium was added for continuous culture 8 For ⁇ 12 days, use a serum-free, animal-derived component-free medium for host cell culture and target protein expression; collect the host cell culture supernatant, and use a Protein A affinity chromatography column for separation and purification.
  • the CoV022, ACE2-615-Fc, ACE2-615-Fc/CoV022 bifunctional protein expression vectors were purified by column, and the obtained plasmids were transiently transfected into the host cell Expi CHO-S cells, and the feed medium was added for continuous culture 8 ⁇ 12 days, collect the host cell culture supernatant, use Protein A affinity chromatography column for separation and purification.
  • the sample sequence of Figure 15 is : The samples in lanes 1 and 2 are ACE2-609-Fc, the samples in lanes 3 and 4 are CoV022, and the samples in lanes 5 and 6 are ACE2-609-Fc/CoV022; the order of the samples in Figure 16 is: the samples in lanes 1, 2 are ACE2- 615-Fc, the samples in lanes 3 and 4 are CoV022, and the samples in lanes 5 and 6 are ACE2-615-Fc/CoV022.
  • Example 3 Detection of the binding ability of the bifunctional fusion protein that specifically binds to the coronavirus and the coronavirus
  • the ELISA method detects the binding ability of the bifunctional fusion protein that specifically binds to the coronavirus and the coronavirus protein:
  • Figure 17 shows the ELISA detection of CoV022, ACE2-609-Fc, ACE2-609-Fc/CoV022 bifunctional protein and SARS-CoV-2 S protein (Spike protein) Binding ability;
  • Figure 18 shows the binding ability of CoV022, ACE2-615-Fc, ACE2-615-Fc/CoV022 bifunctional protein and SARS-CoV-2 S protein (Spike protein) detected by ELISA.
  • Example 4 Detection of the binding ability of the bifunctional fusion protein that specifically binds to the coronavirus and the coronavirus
  • SPR surface plasmon resonance technology
  • SPR method detects ACE2-609-Fc, ACE2-615-Fc, CoV022, ACE2-609-Fc/CoV022, ACE2-615-Fc/CoV022 bifunctional fusion protein and SARS-CoV-2 S Protein affinity results table .

Abstract

Disclosed in the present invention is a bifunctional fusion protein binding to a coronavirus (CoV). Binding areas of two ends of the bifunctional fusion protein respectively bind to different epitopes of a Spike (S) protein of the CoV, and meanwhile, the bifunctional fusion protein has anti-SARS-CoV and anti-SARS-CoV-2 functions, and the function of binding to ACE2. The bifunctional fusion protein binding to the CoV provided by the present invention has the capability of treating virally-infected cells, and further has the effects of antivirus, reducing pulmonary inflammations, reducing cytokine storms, and treating pulmonary injuries.

Description

一种结合冠状病毒的双功能融合蛋白、其制备方法及应用A dual-function fusion protein binding to coronavirus, its preparation method and application 技术领域Technical field
本发明属于生物技术领域，具体地，本发明公开了一种结合冠状病毒的双功能融合蛋白、其制备方法及应用。The present invention belongs to the field of biotechnology. Specifically, the present invention discloses a bifunctional fusion protein that binds to coronavirus, and its preparation method and application.
背景技术Background technique
2019-新型冠状病毒(2019 novel coronavirus，2019-nCoV，SARS-CoV-2)，是一种有包膜的、带正电荷的单链RNA病毒，属于β冠状病毒属，Sarbecovirus亚属。人感染SARS-CoV-2(2019-nCoV，SARS-CoV-2)后，大部分患者会出现发热、咳嗽、呕吐和腹泻、轻症肺炎等症状，还有部分患者会出现重症肺炎，病情严重者可出现急性呼吸衰竭、休克、甚至死亡。2019-novel coronavirus (2019-nCoV, SARS-CoV-2) is an enveloped, positively charged single-stranded RNA virus, belonging to the genus β-coronavirus, a subgenus of Sarbecovirus. After humans are infected with SARS-CoV-2 (2019-nCoV, SARS-CoV-2), most patients will develop symptoms such as fever, cough, vomiting and diarrhea, mild pneumonia, and some patients will develop severe pneumonia and the condition is serious Patients may suffer from acute respiratory failure, shock, and even death.
在SARS-CoV-2(2019-nCoV，SARS-CoV-2)感染及发病的整个过程中，人体内一种重要的金属蛋白酶-血管紧张素转换酶2(angiotensin converting enzyme 2，ACE2)发挥了重要作用(Lu R，Zhao X，Li j，et al.Genomic characterization and epidemiology of 2019 novel coronavirus：implications for virus origins and receptor binding[J].Lancet，2020.[Epub ahead of print])。In the entire process of SARS-CoV-2 (2019-nCoV, SARS-CoV-2) infection and pathogenesis, an important metalloproteinase in the human body-angiotensin converting enzyme 2 (ACE2) plays Important role (Lu R, Zhao X, Li j, et al. Genomic characterization and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding[J]. Lancet, 2020. [Epub ahead of print]).
ACE2是血管紧张素转换酶的一种同源物，是由805个氨基酸构成的Ⅰ型跨膜蛋白，有2个结构域，分别为氨基末端催化结构域和羧基末端结构域，催化结构含有1个活性位点-锌金属蛋白结构域，ACE2可在人体心脏、肾脏、肺脏、肝脏、睾丸和肠等多种组织中表达。ACE2 is a homologue of angiotensin-converting enzyme. It is a type I transmembrane protein composed of 805 amino acids. It has two domains, namely the amino-terminal catalytic domain and the carboxy-terminal domain. The catalytic structure contains 1 With a single active site-zinc metalloprotein domain, ACE2 can be expressed in a variety of tissues such as the human heart, kidney, lung, liver, testis, and intestine.
ACE2在新冠肺炎病程中的作用，除了作为病毒结合的受体外，ACE2还参与调解免疫过程。研究者发现的肺腺癌组织中ACE2表达上升，提示该类患者可能具有更大的新冠病毒易感性，并可能通过miR-125b-5p-ACE2-IL6轴，进一步可能引起细胞因子风暴和肺炎加重的现象。同时，在新冠病毒感染后可以进一步增加细胞ACE2的表达，从而更利于病毒的感染(Wang,P.H.(2020).Increasing Host Cellular Receptor—Angiotensin-Converting Enzyme 2(ACE2)Expression by Coronavirus may Facilitate 2019-nCoV Infection.bioRxiv.)。The role of ACE2 in the course of new coronary pneumonia, in addition to being a receptor for virus binding, ACE2 also participates in mediating the immune process. The researchers found that the expression of ACE2 in lung adenocarcinoma tissues increased, suggesting that such patients may be more susceptible to the new coronavirus, and may pass the miR-125b-5p-ACE2-IL6 axis, which may further cause cytokine storm and exacerbation of pneumonia The phenomenon. At the same time, the expression of ACE2 in cells can be further increased after the new coronavirus infection, which is more conducive to virus infection (Wang,PH(2020).IncreasingHostCellularReceptor-Angiotensin-ConvertingEnzyme2(ACE2)ExpressionbyCoronavirusmayFacilitate2019-nCoV Infection.bioRxiv.).
血管紧张素转化酶2(ACE2)是一把双刃剑：在病毒感染过程中，它是新冠病毒的膜上结合受体，与病毒和宿主细胞的融合、入侵过程有关；在流感病毒、SARS引起炎症和急性肺损伤的情况下，ACE2促进血管紧张素II转化为血管紧张素1-7，抑制血管紧张素II介导的NF-κB信号，从而避免大量炎症信号引发的细胞因子风暴，减缓病程。Angiotensin-converting enzyme 2 (ACE2) is a double-edged sword: in the process of virus infection, it is the membrane-bound receptor of the new coronavirus, which is related to the fusion and invasion process of the virus and host cell; in influenza virus, SARS In the case of inflammation and acute lung injury, ACE2 promotes the conversion of angiotensin II to angiotensin 1-7, inhibits the NF-κB signal mediated by angiotensin II, thereby avoiding the cytokine storm caused by a large number of inflammatory signals and slowing down Course of disease.
前期研究发现，一旦被SARS-CoV结合，宿主细胞表面的ACE2量、mRNA水平 和酶活性均会发生显著下降。尽管这可以减少病毒入侵，但也可能加重炎症反应。Previous studies have found that once SARS-CoV is bound, the amount of ACE2, mRNA level and enzyme activity on the surface of the host cell will all decrease significantly. Although this can reduce viral invasion, it may also aggravate the inflammatory response.
白介素-6(interleukin-6；IL-6)正是引起细胞因子风暴的主要因素，感染SARS-CoV-2(2019-nCoV)后，激活的病原性T细胞就会产生粒细胞-巨噬细胞集落刺激因子(GM-CSF)和IL-6。GM-CSF可以激活CD14+CD16+炎性单核细胞，进一步产生更多的细胞因子，包括IL-6。IL-6在这个正反馈循环中扮演不可或缺的一份子，最终促成免疫系统的失控。Interleukin-6 (IL-6) is the main factor causing the cytokine storm. After infection with SARS-CoV-2 (2019-nCoV), activated pathogenic T cells will produce granulocytes-macrophages Colony stimulating factor (GM-CSF) and IL-6. GM-CSF can activate CD14+CD16+ inflammatory monocytes and further produce more cytokines, including IL-6. IL-6 plays an indispensable part in this positive feedback loop and ultimately contributes to the loss of control of the immune system.
此时，肺中大量的免疫细胞和组织液会阻塞肺泡和毛细血管之间的气体交换，从而导致急性呼吸窘迫综合征。一旦形成细胞因子风暴，免疫系统在清除病毒的同时，会杀死肺中的许多正常细胞，从而严重破坏肺的通气功能，进而导致患者死亡At this time, a large number of immune cells and tissue fluid in the lung will block the gas exchange between the alveoli and capillaries, resulting in acute respiratory distress syndrome. Once a cytokine storm is formed, the immune system will kill many normal cells in the lungs while clearing the virus, thereby severely destroying the ventilation function of the lungs, leading to death of the patient
研究发现肺癌患者ACE2表达上调，新冠肺炎易感性上升；但ACE2在新冠肺炎病程不同阶段的角色不同，尽管其介导病毒的入侵，但维持ACE2的正常表达对避免细胞因子风暴有重要作用。(Long Chen,et al.Lung Adenocarcinoma Patients Own Higher Risk of SARS-CoV-2 Infection.Preprints.26Feb,2020.)Studies have found that the expression of ACE2 in patients with lung cancer is up-regulated and the susceptibility of new coronary pneumonia to increase; but ACE2 has different roles in different stages of the course of new coronary pneumonia. Although it mediates virus invasion, maintaining the normal expression of ACE2 plays an important role in avoiding cytokine storms. (Long Chen, et al. Lung Adenocarcinoma Patients Own Higher Risk of SARS-CoV-2 Infection.Preprints. 26Feb, 2020.)
SARS-CoV-2(2019-nCoV)使用血管紧张素转化酶2(ACE2)作为进入的受体，这与SARS冠状病毒用于进入的受体相同，这两种冠状病毒都是通过病毒体上的S蛋白(SPIKE蛋白)(病毒spike糖蛋白)与ACE2结合，病毒膜和细胞膜融合，随后RNA病毒将在细胞内复制其基因，并最终产生新的病毒颗粒，这些病毒颗粒将被分泌，以感染其他细胞。(Zhou P，Yang XL，Wang XG，et al：Discovery of a novel coronavirus associated with the recent pneumonia outbreak in humans and its potential bat origin.BioRxiv.2020.01.22.914852)。SARS-CoV-2 (2019-nCoV) uses angiotensin-converting enzyme 2 (ACE2) as the entry receptor, which is the same as the entry receptor used by the SARS coronavirus. Both coronaviruses pass through the virion. The S protein (SPIKE protein) (virus spike glycoprotein) binds to ACE2, the viral membrane and the cell membrane are fused, and then the RNA virus will replicate its genes in the cell and finally produce new virus particles, which will be secreted to Infect other cells. (Zhou P, Yang XL, Wang XG, et al: Discovery of a novel coronavirus associated with the recent pneumonia outbreak in humans and its potential bat origin. BioRxiv.2020.01.22.914852).
RBD(receptor-binding domain)SARS病毒S蛋白(Spike蛋白)的小受体结合域，该结合域已被证明是病毒进入细胞过程中结合ACE2受体的关键域，使用该结合域已被证明能有效阻止SARS进入细胞增殖。(Wong SK，Li W，Moore MJ，et al：A193-amino acid fragment of the SARS coronavirus S protein efficiently binds angiotensin-converting enzyme2.J Biol Chem.2004；279(5):3197-3201)。RBD (receptor-binding domain) The small receptor binding domain of SARS virus S protein (Spike protein). This binding domain has been shown to be the key domain for the virus to bind to the ACE2 receptor during cell entry. The use of this binding domain has been shown to be able to Effectively prevent SARS from entering the cell proliferation. (Wong SK, Li W, Moore MJ, et al: A193-amino acid fragment of the SARS coronavirus S protein efficiently binds angiotensin-converting enzyme 2.J Biol Chem. 2004; 279(5): 3197-3201).
在冠状病毒治疗的方法中，若使用结合ACE2蛋白的抗体，会与人体内表达ACE2的正常细胞结合，引起正常组织的炎症反应。使用可以与冠状病毒S蛋白(Spike蛋白)相结合的ACE2-Fc，可以阻止冠状病毒与ACE2的结合，进一步阻止冠状病毒进入细胞。In the treatment of coronavirus, if an antibody that binds to the ACE2 protein is used, it will bind to normal cells expressing ACE2 in the human body and cause an inflammatory response in normal tissues. The use of ACE2-Fc, which can be combined with the coronavirus S protein (Spike protein), can prevent the combination of the coronavirus and ACE2, and further prevent the coronavirus from entering the cell.
但是，ACE2-Fc策略的一个潜在局限性是细胞外ACE2水平的增加可能对人体产生未知的影响，已发现人体可分泌少量的细胞外ACE2，因此该细胞外结构域进入血液循环并非是前所未有的。(Shao Z，Shrestha K，Borowski AG，et al：Increasing serum soluble angiotensin-converting enzyme2 activity after intensive medical therapy is associated with better prognosis in acute decompensated heart failure.J Card Fail.2013；19(9):605-610)。将 具有活性的重组人ACE2(rhACE2)蛋白应用到酸损伤ACE2基因敲除小鼠和野生型小鼠后，急性肺损伤症状如肺水肿等得到改善(Imai,Y.,Kuba,K.,Rao,S.,Huan,Y.,Guo,F.,Guan,B.,...&Crackower,M.A.(2005).Angiotensin-converting enzyme 2 protects from severe acute lung failure.Nature,436(7047),112-116.)。在动物体内以及Ⅱ期临床研究对确诊的急性呼吸窘迫综合征(ARDS)患者应用rhACE2注射治疗后发现，AngⅡ水平快速下降，Ang1-7水平回升，初步显示在人体补充ACE2能够重塑ACE2/ACE平衡(Khan,A.,Benthin,C.,Zeno,B.,Albertson,T.E.,Boyd,J.,Christie,J.D.,...&Hardes,K.(2017).A pilot clinical trial of recombinant human angiotensin-converting enzyme 2 in acute respiratory distress syndrome.Critical Care,21(1),234.Treml,B.,Neu,N.,Kleinsasser,A.,Gritsch,C.,Finsterwalder,T.,Geiger,R..&Loeckinger,A.(2010).Recombinant angiotensin-converting enzyme 2 improves pulmonary blood flow and oxygenation in lipopolysaccharide-induced lung injury in piglets.Critical care medicine,38(2),596-601.)，ACE2具有降低肺部炎症的潜力，同时具有可以耐受的安全性。However, a potential limitation of the ACE2-Fc strategy is that the increase in extracellular ACE2 levels may have unknown effects on the human body. It has been found that the human body can secrete a small amount of extracellular ACE2, so it is not unprecedented that this extracellular domain enters the blood circulation. . (Shao Z, Shrestha K, Borowski AG, et al: Increasing serum soluble angiotensin-converting enzyme2 activity after intensive medical therapy is associated with better prognosis in acute decompensated heart failure.J Card Fail.2013-610(9) ). After the active recombinant human ACE2 (rhACE2) protein was applied to acid-damaged ACE2 knockout mice and wild-type mice, the symptoms of acute lung injury such as pulmonary edema were improved (Imai, Y., Kuba, K., Rao ,S.,Huan,Y.,Guo,F.,Guan,B.,...&Crackower,MA(2005).Angiotensin-converting enzyme 2 protections from severe acute lung failure.Nature,436(7047),112- 116.). After rhACE2 injection in patients with diagnosed acute respiratory distress syndrome (ARDS) in animals and in phase Ⅱ clinical studies, it was found that AngⅡ levels dropped rapidly and Ang1-7 levels recovered. It was preliminarily shown that ACE2 supplementation in humans can reshape ACE2/ACE Balance (Khan,A.,Benthin,C.,Zeno,B.,Albertson,TE,Boyd,J.,Christie,JD,...&Hardes,K.(2017).A pilot clinical trial of recombinant human angiotensin- converting enzyme 2 in acute respiratory distress syndrome.Critical Care,21(1),234.Treml,B.,Neu,N.,Kleinsasser,A.,Gritsch,C.,Finsterwalder,T.,Geiger,R..&Loeckinger ,A.(2010).Recombinant angiotensin-converting enzyme 2 improve pulmonary blood flow and oxygenation in lipopolysaccharide-induced lung infection in piglets.Critical care medicine,38(2),596-601.), ACE2 can reduce lung inflammation Potential, while having tolerable safety.
在急性SARS-CoV感染过程中抗病毒S糖蛋白IgG扰乱巨噬细胞反应而导致严重的急性肺损伤。使用S-IgG尽管可以明显降低病毒滴度，却在感染的早期阶段引起肺损伤，主要是通过消除伤口愈合的巨噬细胞反应和TGF-β产生，同时促进炎性细胞因子IL-8，和MCP1的产生和炎性巨噬细胞的积累。(Anti-spike IgG causes severe acute lung injury by skewing macrophage responses during acute SARS-CoV infection.[J].JCI insight,2019,4(4):e123158.)。During acute SARS-CoV infection, antiviral S glycoprotein IgG disrupts the response of macrophages and causes severe acute lung injury. Although the use of S-IgG can significantly reduce the virus titer, it causes lung damage in the early stages of infection, mainly by eliminating the macrophage response and TGF-β production in wound healing, and at the same time promoting the inflammatory cytokine IL-8, and The production of MCP1 and the accumulation of inflammatory macrophages. (Anti-spike IgG causes severe acute lung infection by skewing macrophage responses during acute SARS-CoV infection. [J]. JCI insight, 2019, 4(4): e123158.).
在SARS(Severe Acute Respiratory Syndrome，严重急性呼吸综合征)爆发后，出现多种治疗方法，同时也有针对SARS的药物生产，其中也包含抗体类药物。在专利US11667640中公开了一种能够中和SARS冠状病毒(SARS-CoV)的结合分子的组合物，该组合组中包含至少两种能够结合SARS相关冠状病毒(SARS-CoV)并具有SARS-CoV中和活性的免疫球蛋白。After the SARS (Severe Acute Respiratory Syndrome) outbreak, a variety of treatment methods appeared. At the same time, SARS drugs were produced, including antibody drugs. Patent US11667640 discloses a composition capable of neutralizing the binding molecules of SARS coronavirus (SARS-CoV). The combination group contains at least two SARS-related coronaviruses (SARS-CoV) and possessing SARS-CoV. Neutralizing active immunoglobulin.
在专利US11667640中，公开了可以与ASRS冠状病毒相结合的抗体，并且公开了所述的抗体是结合SARS-CoV的S蛋白(Spike蛋白)的第318-510位的氨基酸。虽然该件专利中所述的药物是一种组合物，但是组成该组合的抗体，都是针对结合SARS-CoV的S蛋白(Spike蛋白)的第318-510位的氨基酸的抗体，即使每一种抗体的具体结合位点是第318-510位中的不同氨基酸，均会存在彼此间相互影响的弊端。In patent US11667640, an antibody that can bind to ASRS coronavirus is disclosed, and it is disclosed that the antibody binds to amino acids 318-510 of the S protein (Spike protein) of SARS-CoV. Although the drug described in this patent is a composition, the antibodies that make up the combination are all antibodies against the amino acids at positions 318-510 of the S protein (Spike protein) that bind to SARS-CoV, even if each The specific binding sites of these antibodies are the different amino acids in positions 318-510, and they all have the disadvantage of mutual influence.
由于抗体属于大分子蛋白质，并且由于其空间结构的特点，在抗体与抗原相互结合时，会存在抗体间的相互影响，该件专利所提供的组合物，均是结合在SARS-CoV的S蛋白(Spike蛋白)的第318-510位的氨基酸，在与病毒相结合时，会影响抗体中和SARS-CoV冠状病毒的作用效果。Since antibodies are macromolecular proteins, and due to the characteristics of their spatial structure, there will be interactions between antibodies when the antibodies and antigens are combined with each other. The composition provided in this patent is all the S protein that binds to SARS-CoV. The amino acids at positions 318-510 of (Spike protein), when combined with the virus, will affect the effect of the antibody in neutralizing the SARS-CoV coronavirus.
至2019-新型冠状病毒(2019 novel coronavirus，2019-nCoV，SARS-CoV-2)爆发以来，多方科学家在研究SARS-CoV-2的感染途径，中国科学家陈志南及其团队，经研究发现SARS-CoV-2可以通过CD147的新途径侵入宿主细胞：S蛋白(Spike蛋白)与宿主细胞上的受体CD147结合，从而介导病毒入侵(Zhinan.Chen，Ping.Zhu，et al：SARS-CoV-2 invades host cells via a novel route:CD147-spike protein，BioRxiv)。Since the outbreak of 2019-novel coronavirus (2019-nCoV, SARS-CoV-2), many scientists have been studying the infection route of SARS-CoV-2. Chinese scientist Chen Zhinan and his team have discovered SARS-CoV through research -2 can invade host cells through a new way of CD147: S protein (Spike protein) binds to the receptor CD147 on the host cell to mediate virus invasion (Zhinan. Chen, Ping. Zhu, et al: SARS-CoV-2 invades host cells via a novel route: CD147-spike protein, BioRxiv).
发明内容Summary of the invention
为了解决上述现有技术存在的技术问题，本发明提供一种特异性结合冠状病毒的双功能融合蛋白。In order to solve the above-mentioned technical problems in the prior art, the present invention provides a bifunctional fusion protein that specifically binds to coronavirus.
本发明所提供的特异性结合冠状病毒的双功能融合蛋白，具有抗SARS-CoV及抗SARS-CoV-2的功能，并具有Fc功能效应，治疗病毒感染细胞的能力，同时还具有抗病毒及降低肺部炎症、降低细胞因子风暴的作用。The bifunctional fusion protein that specifically binds to the coronavirus provided by the present invention has the functions of anti-SARS-CoV and anti-SARS-CoV-2, and has the function of Fc, the ability to treat virus-infected cells, and it also has anti-virus and anti-SARS-CoV-2 functions. Reduce lung inflammation and reduce the effect of cytokine storm.
本发明所提供的特异性结合冠状病毒的双功能融合蛋白，具有广泛的中和作用，同时具有中和动物相关的SARS-CoV(SARS-like CoV)的能力，在未被发现的与SARS-CoV或SARS-CoV-2相似病毒的治疗中也起到中和作用，还可以用于现在已发现以ACE2为受体的病毒治疗以及未被发现的以ACE2为受体的未来新型病毒的治疗，对于由ACE2轴介导的肺部损伤性疾病也具有治疗作用。The bifunctional fusion protein that specifically binds to coronaviruses provided by the present invention has a wide range of neutralizing effects, and at the same time has the ability to neutralize animal-related SARS-CoV (SARS-like CoV). CoV or SARS-CoV-2 similar virus also plays a neutralizing role in the treatment of viruses, and can also be used for the treatment of viruses that have been found to use ACE2 as the receptor and the treatment of new types of viruses that have not been discovered that use ACE2 as the receptor in the future It also has a therapeutic effect on lung injury diseases mediated by the ACE2 axis.
本发明所提供的特异性结合冠状病毒的双功能融合蛋白，其两端结合区域分别结合冠状病毒S蛋白(Spike蛋白)的不同表位，由于所述特异性结合冠状病毒的双功能融合蛋白具有特殊的结构，在与冠状病毒S蛋白(Spike蛋白)结合后，产生空间位阻效应，将冠状病毒的S蛋白(Spike蛋白)可与宿主细胞结合的其他结合位点封闭，对冠状病毒经S蛋白(Spike蛋白)结合入侵宿主细胞的其他途径阻断，例如：CD147受体结合途径(冠状病毒S蛋白与宿主细胞上的受体CD147结合，从而入侵宿主细胞)。The bifunctional fusion protein that specifically binds to coronavirus provided by the present invention has binding regions at both ends that respectively bind to different epitopes of the coronavirus S protein (Spike protein), because the bifunctional fusion protein that specifically binds to coronavirus has The special structure produces steric hindrance after binding to the coronavirus S protein (Spike protein), which blocks other binding sites where the coronavirus S protein (Spike protein) can bind to the host cell. The protein (Spike protein) binds to other ways to invade host cells, such as the CD147 receptor binding pathway (the coronavirus S protein binds to the receptor CD147 on the host cell to invade the host cell).
本发明所提供的特异性结合冠状病毒的双功能融合蛋白能够多途径阻断冠状病毒对宿主细胞的结合，更加有效地降低冠状病毒的入侵、感染，及更有效地抗病毒、降低肺部炎症、降低细胞因子风暴，更有效地治疗因冠状病毒所引起的病症。The bifunctional fusion protein that specifically binds to coronaviruses provided by the present invention can block the combination of coronaviruses to host cells in multiple ways, more effectively reduce the invasion and infection of coronaviruses, and more effectively resist viruses and reduce lung inflammation , Reduce the cytokine storm, more effectively treat the disease caused by the coronavirus.
本发明所提供的特异性结合冠状病毒的双功能融合蛋白，其结构图如图1所示，具有抗SARS-CoV或SARS-CoV-2的冠状病毒结合端，且具有ACE2的一端。The bifunctional fusion protein that specifically binds to coronaviruses provided by the present invention has a structure diagram as shown in FIG.
本发明所提供的特异性结合冠状病毒的双功能融合蛋白，其中抗SARS-CoV或SARS-CoV-2抗体的Fab端可以与冠状病毒的S蛋白(Spike蛋白)相结合，并且该结合位点与ACE2的结合位点不同，不会与ACE2竞争性结合冠状病毒的RBD。抗SARS-CoV或SARS-CoV-2抗体的Fab端可以协同增强ACE2和冠状病毒RBD的结合，增强抗病毒活性，同时避免可能的病毒增强作用。The bifunctional fusion protein that specifically binds to the coronavirus provided by the present invention, wherein the Fab end of the anti-SARS-CoV or SARS-CoV-2 antibody can be combined with the S protein (Spike protein) of the coronavirus, and the binding site Unlike the binding site of ACE2, it does not compete with ACE2 to bind to the RBD of the coronavirus. The Fab end of the anti-SARS-CoV or SARS-CoV-2 antibody can synergistically enhance the binding of ACE2 and the coronavirus RBD, enhance antiviral activity, and avoid possible virus enhancement.
本发明所提供的特异性结合冠状病毒的双功能融合蛋白，其ACE2一端不仅可以结合冠状病毒，还可以保护细胞表面的ACE2不被冠状病毒结合，不影响ACE2的正常作用，同时补充一定的具有活性的ACE2，有可能降低且不会引起细胞因子风暴，避免产生炎性因子，起到减少炎症产生的功效，具有阻断冠状病毒与正常细胞的ACE2结合途径，还可以减轻因ACE2降低而引起的炎症，避免因ACE2途径引起的肺炎，减轻冠状病毒感染者的肺炎症状，降低细胞因子风暴作用。The bifunctional fusion protein that specifically binds to the coronavirus provided by the present invention can not only bind to the coronavirus at one end of ACE2, but also protect the ACE2 on the cell surface from being bound by the coronavirus, and does not affect the normal function of ACE2. Active ACE2 may be reduced without causing a cytokine storm, avoiding the production of inflammatory factors, reducing inflammation, blocking the ACE2 binding pathway of coronavirus and normal cells, and reducing the ACE2 reduction caused by Inflammation, avoid pneumonia caused by the ACE2 pathway, reduce the symptoms of pneumonia in people infected with coronavirus, and reduce the effect of cytokine storm.
如上所述的特异性结合冠状病毒的双功能融合蛋白，通过不同的结合位点与冠状病毒S蛋白(Spike蛋白)结合后，由于其具有特殊的结构，产生空间位阻效应，将冠状病毒的S蛋白可与宿主细胞结合的其他结合位点封闭，将冠状病毒经S蛋白结合入侵宿主细胞的其他途径阻断，例如：CD147受体结合途径(冠状病毒S蛋白与宿主细胞上的受体CD147结合，从而入侵宿主细胞)。The bifunctional fusion protein that specifically binds to the coronavirus as described above, after binding to the coronavirus S protein (Spike protein) through different binding sites, due to its special structure, produces a steric hindrance effect. The S protein can block other binding sites that bind to the host cell, blocking other ways that the coronavirus invades the host cell through the S protein binding, such as the CD147 receptor binding pathway (coronavirus S protein and the receptor CD147 on the host cell Binding, thereby invading host cells).
而且，本发明所提供的特异性结合冠状病毒的双功能融合蛋白，其抗体部分可以使目标分子靶向病毒，还可以降低因单独使用ACE2所引起的血压降低的风险。ACE2与SARS冠状病毒的S1、S2交界处结合(Aiping Wu,Peihua Niu,Lulan Wang,et.al.Mutations,Recombination and Insertion in the Evolution of 2019-nCoV.bioRxiv March 02,2020)。Moreover, the bifunctional fusion protein that specifically binds to the coronavirus provided by the present invention can target the target molecule to the virus by its antibody portion, and can also reduce the risk of blood pressure drop caused by the use of ACE2 alone. ACE2 combines with the S1 and S2 junctions of SARS coronavirus (Aiping Wu, Peihua Niu, Lulan Wang, et.al. Mutations, Recombination and Insertion in the Evolution of 2019-nCoV.bioRxiv March 02, 2020).
目前对中国武汉、中国深圳、中国香港、法国的SARS-CoV-2病毒进行检测，发现SARS-CoV-2的RBD(receptor-binding domain)存在突变的现象，但这些突变的病毒株均具有较强的ACE2结合能力(Junxian Ou，Zhonghua Zhou，et al：RBD mutations from circulating SARS-CoV-2 strains enhance the structure stability and infectivity of the spike protein.bioRxiv)。正如上所述本发明所提供的特异性结合冠状病毒的双功能融合蛋白具有的特性，具有阻断冠状病毒ACE2途径的功能，并且较单独使用ACE2-Fc具有较强的优势，所以，本发明所提供的特异性结合冠状病毒的双功能融合蛋白能够更有效地应对冠状病毒的突变病毒株。At present, the SARS-CoV-2 virus in Wuhan, China, Shenzhen, China, Hong Kong, and France has been tested, and it has been found that the RBD (receptor-binding domain) of SARS-CoV-2 has mutations, but these mutated virus strains have relatively high levels. Strong ACE2 binding ability (Junxian Ou, Zhonghua Zhou, et al: RBD mutations from circulating SARS-CoV-2 strains enhancement the structure stability and infectivity of the spike protein.bioRxiv). As mentioned above, the bifunctional fusion protein that specifically binds to coronaviruses provided by the present invention has the characteristics of blocking the ACE2 pathway of coronaviruses, and has stronger advantages than using ACE2-Fc alone. Therefore, the present invention The provided bifunctional fusion protein that specifically binds to the coronavirus can more effectively deal with the mutant virus strain of the coronavirus.
本发明所提供的特异性结合冠状病毒的双功能融合蛋白，可以有效的用作治疗SARS和SARS-CoV-2(2019-nCoV)及未来相似病毒爆发的药物库存，也可以应对任何将来通过ACE2受体进入细胞的其他新型病毒。同时也可以作为危险人群的被动免疫保护剂，用于SARS和SARS-CoV-2相关疾病的预防。The bifunctional fusion protein that specifically binds to the coronavirus provided by the present invention can be effectively used as a drug inventory for the treatment of SARS and SARS-CoV-2 (2019-nCoV) and similar virus outbreaks in the future, and it can also deal with any future through ACE2 Receptor to other new viruses that enter the cell. At the same time, it can also be used as a passive immune protective agent for people at risk for the prevention of SARS and SARS-CoV-2 related diseases.
本发明公开的一种特异性结合冠状病毒的双功能融合蛋白，所述双功能融合蛋白分别结合冠状病毒的不同结合位点。The invention discloses a bifunctional fusion protein that specifically binds to a coronavirus, and the bifunctional fusion protein respectively binds to different binding sites of the coronavirus.
如上所述的特异性结合冠状病毒的双功能融合蛋白，所述的冠状病毒具有Spike蛋白的结合位点。The bifunctional fusion protein that specifically binds to the coronavirus as described above, and the coronavirus has a binding site for Spike protein.
如上所述的特异性结合冠状病毒的双功能融合蛋白，所述的冠状病毒具有ACE2结合位点。The bifunctional fusion protein that specifically binds to the coronavirus as described above, and the coronavirus has an ACE2 binding site.
如上所述的特异性结合冠状病毒的双功能融合蛋白，所述双功能融合蛋白结合冠状病毒的ACE2结合位点，同时结合冠状病毒的Spike蛋白。The bifunctional fusion protein that specifically binds to the coronavirus as described above, the bifunctional fusion protein binds to the ACE2 binding site of the coronavirus and at the same time binds to the Spike protein of the coronavirus.
如上所述的特异性结合冠状病毒的双功能融合蛋白，所述双功能融合蛋白具有与冠状病毒的ACE2结合位点相结合的SEQ ID NO：12或SEQ ID NO：14所示的氨基酸序列。The bifunctional fusion protein that specifically binds to the coronavirus as described above has the amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO: 14 that binds to the ACE2 binding site of the coronavirus.
如上所述的特异性结合冠状病毒的双功能融合蛋白，所述双功能融合蛋白具有与冠状病毒的Spike蛋白相结合的SEQ ID NO：6所示的重链氨基酸序列及SEQ ID NO：2所示的轻链氨基酸序列。The bifunctional fusion protein that specifically binds to the coronavirus as described above has the heavy chain amino acid sequence shown in SEQ ID NO: 6 that binds to the Spike protein of the coronavirus and the heavy chain amino acid sequence shown in SEQ ID NO: 2 The amino acid sequence of the light chain is shown.
本发明公开的一种组合物，包括上述特异性结合冠状病毒的双功能融合蛋白及药学上可接受的赋形剂。A composition disclosed in the present invention includes the above-mentioned bifunctional fusion protein that specifically binds to coronavirus and pharmaceutically acceptable excipients.
本发明公开的一种特异性结合冠状病毒的双功能融合蛋白的制备方法，包含以下步骤：a)基因合成上述特异性结合冠状病毒的双功能融合蛋白的基因序列，制备含有所述双功能融合蛋白基因的表达载体；b)将所述表达载体转染至宿主细胞，并进行宿主细胞的培养、及所述双功能融合蛋白的表达；c)所述双功能融合蛋白的分离、纯化。The present invention discloses a method for preparing a bifunctional fusion protein that specifically binds to a coronavirus, comprising the following steps: a) Gene synthesis of the above-mentioned gene sequence of the bifunctional fusion protein that specifically binds to a coronavirus, and preparation containing the bifunctional fusion Protein gene expression vector; b) transfecting the expression vector to host cells, and culturing the host cells and expressing the bifunctional fusion protein; c) separation and purification of the bifunctional fusion protein.
如上所述的特异性结合冠状病毒的双功能融合蛋白的制备方法，其特征在于，所述宿主细胞为真核哺乳动物细胞CHO细胞。The method for preparing a bifunctional fusion protein that specifically binds to a coronavirus as described above is characterized in that the host cell is a eukaryotic mammalian cell CHO cell.
如上所述的特异性结合冠状病毒的双功能融合蛋白的制备方法，其特征在于，所述的宿主细胞的培养使用无血清、无动物源组分的培养基培养。The method for preparing a bifunctional fusion protein that specifically binds to a coronavirus as described above is characterized in that the host cell is cultured using a serum-free and animal-derived component-free medium.
本发明公开的一种特异性结合冠状病毒的双功能融合蛋白的应用，包含如上所述的特异性结合冠状病毒的双功能融合蛋白在制备抗冠状病毒药物中的应用。The invention discloses an application of a bifunctional fusion protein that specifically binds to a coronavirus, including the application of the above-mentioned bifunctional fusion protein that specifically binds to a coronavirus in the preparation of anti-coronavirus drugs.
如上所述的特异性结合冠状病毒的双功能融合蛋白的应用，所述的冠状病毒具有ACE2感染途径。The application of the bifunctional fusion protein that specifically binds to the coronavirus as described above, and the coronavirus has an ACE2 infection pathway.
如上所述的特异性结合冠状病毒的双功能融合蛋白的应用，其特征在于，所述的冠状病毒为SARS-CoV相关冠状病毒、MERS-CoV相关冠状病毒、或SARS-CoV-2相关病毒。The application of the bifunctional fusion protein that specifically binds to the coronavirus as described above is characterized in that the coronavirus is a SARS-CoV related coronavirus, a MERS-CoV related coronavirus, or a SARS-CoV-2 related virus.
附图说明Description of the drawings
图1、特异性结合冠状病毒的双功能融合蛋白结构示意图；Figure 1. Schematic diagram of the bifunctional fusion protein that specifically binds to coronavirus;
图2、CoV022轻链全长表达载体构建图；Figure 2. Construction diagram of the full-length expression vector of CoV022 light chain;
图3、CoV022重链可变区克隆载体构建图；Figure 3. Construction diagram of CoV022 heavy chain variable region cloning vector;
图4、CoV022重链全长表达载体构建图；Figure 4. Construction diagram of CoV022 heavy chain full-length expression vector;
图5、CoV022轻链、重链全长单个质粒表达载体构建图；Figure 5. CoV022 light chain and heavy chain full-length single plasmid expression vector construction diagram;
图6、ACE2-609-Fc融合蛋白克隆载体构建图；Figure 6. ACE2-609-Fc fusion protein cloning vector construction diagram;
图7、ACE2-609-Fc融合蛋白表达载体构建图；Figure 7. ACE2-609-Fc fusion protein expression vector construction diagram;
图8、ACE2-615-Fc融合蛋白克隆载体构建图；Figure 8. ACE2-615-Fc fusion protein cloning vector construction diagram;
图9、ACE2-615-Fc融合蛋白表达载体构建图；Figure 9. ACE2-615-Fc fusion protein expression vector construction diagram;
图10、ACE2-609-Fc融合蛋白(Fc in Hole)表达载体构建图；Figure 10. ACE2-609-Fc fusion protein (Fc in Hole) expression vector construction diagram;
图11、ACE2-615-Fc融合蛋白(Fc in Hole)表达载体构建图；Figure 11. ACE2-615-Fc fusion protein (Fc in Hole) expression vector construction diagram;
图12、CoV022重链可变区(Knob)克隆载体构建图；Figure 12. Construction diagram of CoV022 heavy chain variable region (Knob) cloning vector;
图13、CoV022重链全长(Knob)表达载体构建图；Figure 13. Construction diagram of CoV022 heavy chain full length (Knob) expression vector;
图14、CoV022轻链、重链全长(Knob)表达载体单个质粒构建图；Figure 14. Construction diagram of a single plasmid of CoV022 light chain and heavy chain full length (Knob) expression vector;
图15、CHO宿主细胞表达ACE2-609-Fc、CoV022、ACE2-609-Fc/CoV022双功能融合蛋白的SDS-PAGE检测图谱；Figure 15. SDS-PAGE detection map of CHO host cells expressing ACE2-609-Fc, CoV022, ACE2-609-Fc/CoV022 bifunctional fusion protein;
图16、CHO宿主细胞表达ACE2-615-Fc、CoV022、ACE2-615-Fc/CoV022双功能融合蛋白的SDS-PAGE检测图谱；Figure 16. SDS-PAGE detection map of CHO host cells expressing ACE2-615-Fc, CoV022, ACE2-615-Fc/CoV022 bifunctional fusion protein;
图17、CoV022、ACE2-609-Fc、ACE2-609-Fc/CoV022双功能融合蛋白与SARS-CoV-2 Spike蛋白结合能力的ELISA检测图谱；Figure 17. ELISA detection map of the binding ability of CoV022, ACE2-609-Fc, ACE2-609-Fc/CoV022 bifunctional fusion protein and SARS-CoV-2 Spike protein;
图18、CoV022、ACE2-615-Fc、ACE2-615-Fc/CoV022双功能融合蛋白与SARS-CoV-2 Spike蛋白结合能力的ELISA检测图谱。Figure 18. ELISA detection map of the binding ability of CoV022, ACE2-615-Fc, ACE2-615-Fc/CoV022 bifunctional fusion protein and SARS-CoV-2 Spike protein.
具体实施例Specific embodiment
实施例1、特异性结合冠状病毒的双功能融合蛋白的基因合成Example 1. Gene synthesis of bifunctional fusion protein specifically binding to coronavirus
(1)、结合冠状病毒S蛋白(Spike蛋白)的抗体的表达载体构建(1) Construction of expression vector of antibody binding to coronavirus S protein (Spike protein)
CoV022为结合冠状病毒S蛋白(Spike蛋白)的抗体。CoV022 is an antibody that binds to coronavirus S protein (Spike protein).
CoV022轻链全长表达载体构建：Construction of CoV022 light chain full-length expression vector:
全基因合成CoV022轻链全长克隆载体用HindIII及EcoR1双酶切，同时双酶切pL101表达载体，分别胶回收750bp左右目的片段及9kb左右表达载体片段，将两者用T4 DNA连接酶连接并转化，质粒小量抽提并用HindIII及EcoR1双酶切鉴定。The full-gene synthesis CoV022 light chain full-length cloning vector was digested with HindIII and EcoR1, and the pL101 expression vector was digested at the same time. The target fragment of about 750bp and the expression vector fragment of about 9kb were recovered separately, and the two were ligated with T4 DNA ligase. Transformation, a small amount of plasmid extraction and double digestion with HindIII and EcoR1 for identification.
CoV022轻链全长具有SEQ ID NO：1所示核苷酸序列，SEQ ID NO：2所示氨基酸序列。The full length of the light chain of CoV022 has the nucleotide sequence shown in SEQ ID NO: 1, and the amino acid sequence shown in SEQ ID NO: 2.
如图2所示：琼脂糖浓度为1％，箭头所示为约750bp的目的片段右1：DL2000 DNA Marker，右2：λ-HindIII digest DNA Marker。As shown in Figure 2: The agarose concentration is 1%, and the arrow shows the target fragment of about 750bp. Right 1: DL2000 DNA Marker, right 2: λ-HindIII digest DNA Marker.
CoV022重链全长克隆载体构建：Construction of CoV022 heavy chain full-length cloning vector:
全基因合成CoV022重链可变区克隆载体用HindIII及NheI双酶切，同时双酶切IgG1恒定区基因克隆载体，分别胶回收450bp左右目的片段及4kb左右克隆载体片段，将两者用T4 DNA连接酶连接并转化，质粒小量抽提并用HindIII及NheI双酶切鉴定。The full-gene synthesis CoV022 heavy chain variable region cloning vector is digested with HindIII and NheI, and the IgG1 constant region gene cloning vector is double digested at the same time. The target fragment of about 450 bp and the cloning vector fragment of about 4 kb are recovered separately, and the two are used T4 DNA Ligase ligation and transformation, a small amount of plasmid extraction and double digestion with HindIII and NheI identification.
如图3所示：琼脂糖浓度为1％，箭头所示为约450bp的目的片段，右1：DL2000 DNA Marker，左1：λ-HindIII digest DNA Marker。As shown in Figure 3: The agarose concentration is 1%, the arrow shows the target fragment of about 450bp, right 1: DL2000 DNA Marker, left 1: λ-HindIII digest DNA Marker.
CoV022重链全长表达载体构建：Construction of CoV022 heavy chain full-length expression vector:
将CoV022重链全长克隆载体用HindIII及EcoR1双酶切，同时双酶切pL102表达载体，分别胶回收1500bp左右目的片段及5kb左右表达载体片段，将两者用T4 DNA连接酶连接并转化，质粒小量抽提并用HindIII及EcoR1双酶切鉴定。The CoV022 heavy chain full-length cloning vector was double digested with HindIII and EcoR1, and the pL102 expression vector was double digested at the same time. The target fragment of about 1500 bp and the expression vector fragment of about 5 kb were recovered separately, and the two were ligated and transformed with T4 DNA ligase. The plasmid was extracted in a small amount and identified by double digestion with HindIII and EcoR1.
如图4所示：琼脂糖浓度为1％，箭头所示为约1500bp的目的片段，右1：DL2000 DNA Marker，右2：λ-HindIII digest DNA Marker。As shown in Figure 4: the agarose concentration is 1%, the arrow shows the target fragment of about 1500bp, right 1: DL2000 DNA Marker, right 2: λ-HindIII digest DNA Marker.
CoV022重链全长具有SEQ ID NO：3所示核苷酸序列，SEQ ID NO：4所示氨基酸序列。The full length of the CoV022 heavy chain has the nucleotide sequence shown in SEQ ID NO: 3 and the amino acid sequence shown in SEQ ID NO: 4.
CoV022轻链、重链全长单个质粒表达载体构建：Construction of a single full-length plasmid expression vector for CoV022 light chain and heavy chain:
将CoV022轻链全长表达载体及CoV022重链全长表达载体分别用SalI及NotINotI双酶切，分别胶回收9kb左右轻链全长表达片段及4kb左右重链全长表达载体片段，将两者用T4 DNA连接酶连接并转化，质粒小量抽提并用SalI及NotINotI双酶切鉴定。The CoV022 light chain full-length expression vector and CoV022 heavy chain full-length expression vector were digested with SalI and NotINotI, respectively, and a 9kb light chain full-length expression fragment and a 4kb heavy chain full-length expression vector fragment were recovered respectively. Ligation and transformation with T4 DNA ligase, a small amount of plasmid extraction and double enzyme digestion with SalI and NotINotI for identification.
如图5所示：琼脂糖浓度为1％，箭头所示为约9kb的轻链全长表达载体片段及4kb左右的重链全长目的片段，右1：DL2000 DNA Marker，右2：λ-HindIII digest DNA Marker。As shown in Figure 5: the agarose concentration is 1%, the arrow shows the light chain full-length expression vector fragment of about 9kb and the heavy chain full-length target fragment of about 4kb, right 1: DL2000 DNA Marker, right 2: λ- HindIII digest DNA Marker.
(2)与冠状病毒的ACE2结合位点结合的融合蛋白表达载体构建(2) Construction of a fusion protein expression vector that binds to the ACE2 binding site of coronavirus
a)、ACE2-609-Fc融合蛋白表达载体构建a) Construction of ACE2-609-Fc fusion protein expression vector
ACE2-609-Fc融合蛋白克隆载体构建：Construction of ACE2-609-Fc fusion protein cloning vector:
全基因合成ACE2-609克隆载体用HindIII及NheI双酶切，同时双酶切Fc/pGEM-T克隆载体，分别胶回收2.0kb左右目的片段及3.7kb左右Fc克隆载体片段，将两者用T4 DNA连接酶连接并转化，质粒小量抽提并用HindIII及NheI双酶切鉴定。The full-gene synthesis ACE2-609 cloning vector was digested with HindIII and NheI, and the Fc/pGEM-T cloning vector was digested with double digestion. The target fragment of about 2.0kb and the Fc cloning vector fragment of about 3.7kb were recovered separately, and the two were used T4 DNA ligase ligation and transformation, a small amount of plasmid extraction and double digestion with HindIII and NheI identification.
如图6所示：琼脂糖浓度为1％，箭头所示为约2.0kb的目的片段，右1：DL2000 DNA Marker，右2：λ-HindIII digest DNA Marker。As shown in Figure 6: the agarose concentration is 1%, the arrow shows the target fragment of about 2.0kb, right 1: DL2000 DNA Marker, right 2: λ-HindIII digest DNA Marker.
ACE2-609-Fc融合蛋白表达载体构建：Construction of ACE2-609-Fc fusion protein expression vector:
将ACE2-609-Fc/pGEM-T克隆载体用HindIII及EcoR1双酶切，同时双酶切pL101表达载体，分别胶回收2.7kb左右目的片段及9kb左右表达载体片段，将两者用T4 DNA连接酶连接并转化，质粒小量抽提并用HindIII及EcoR1双酶切鉴定。The ACE2-609-Fc/pGEM-T cloning vector was digested with HindIII and EcoR1, and the pL101 expression vector was digested at the same time. The target fragments of about 2.7kb and the expression vector fragments of about 9kb were respectively recovered by glue, and the two were connected with T4 DNA Enzyme ligation and transformation, a small amount of plasmid extraction and double digestion with HindIII and EcoR1 for identification.
ACE2-609-Fc具有SEQ ID NO：7所示核苷酸序列，SEQ ID NO：8所示氨基酸序列。ACE2-609-Fc has the nucleotide sequence shown in SEQ ID NO: 7 and the amino acid sequence shown in SEQ ID NO: 8.
如图7所示：琼脂糖浓度为1％，箭头所示为约2.7kb的目的片段，右1：DL2000 DNA Marker，右2：λ-HindIII digest DNA Marker。As shown in Figure 7: the agarose concentration is 1%, the arrow shows the target fragment of about 2.7kb, right 1: DL2000 DNA Marker, right 2: λ-HindIII digest DNA Marker.
b)、ACE2-615-Fc融合蛋白表达载体构建b) Construction of ACE2-615-Fc fusion protein expression vector
ACE2-615-Fc融合蛋白克隆载体构建：Construction of ACE2-615-Fc fusion protein cloning vector:
全基因合成ACE2-615克隆载体用HindIII及NheI双酶切，同时双酶切Fc/pGEM-T克隆载体，分别胶回收2.0kb左右目的片段及3.7kb左右Fc克隆载体片段，将两者用T4 DNA连接酶连接并转化，质粒小量抽提并用HindIII及NheI双酶切鉴定。The ACE2-615 cloning vector for full gene synthesis was digested with HindIII and NheI, and the Fc/pGEM-T cloning vector was double digested at the same time. The target fragment of about 2.0kb and the Fc cloning vector fragment of about 3.7kb were recovered separately, and the two were used T4 DNA ligase ligation and transformation, a small amount of plasmid extraction and double digestion with HindIII and NheI identification.
如图8所示：琼脂糖浓度为1％，箭头所示为约2.0kb的目的片段，右1：DL2000 DNA Marker，右2：λ-HindIII digest DNA Marker。As shown in Figure 8: the agarose concentration is 1%, the arrow shows the target fragment of about 2.0kb, right 1: DL2000 DNA Marker, right 2: λ-HindIII digest DNA Marker.
ACE2-615-Fc融合蛋白表达载体构建：Construction of ACE2-615-Fc fusion protein expression vector:
将ACE2-615-Fc/pGEM-T克隆载体用HindIII及EcoR1双酶切，同时双酶切pL101表达载体，分别胶回收2.7kb左右目的片段及9kb左右表达载体片段，将两者用T4 DNA连接酶连接并转化，质粒小量抽提并用HindIII及EcoR1双酶切鉴定。The ACE2-615-Fc/pGEM-T cloning vector was digested with HindIII and EcoR1, and the pL101 expression vector was digested at the same time. The target fragment of about 2.7 kb and the expression vector fragment of about 9 kb were recovered separately, and the two were ligated with T4 DNA Enzyme ligation and transformation, a small amount of plasmid extraction and double digestion with HindIII and EcoR1.
ACE2-615-Fc具有SEQ ID NO：9所示核苷酸序列，SEQ ID NO：10所示氨基酸序列。ACE2-615-Fc has the nucleotide sequence shown in SEQ ID NO: 9 and the amino acid sequence shown in SEQ ID NO: 10.
如图9所示：琼脂糖浓度为1％，箭头所示为约2.7kb的目的片段，右1：DL2000 DNA Marker，右2：λ-HindIII digest DNA Marker。As shown in Figure 9: the agarose concentration is 1%, the arrow shows the target fragment of about 2.7kb, right 1: DL2000 DNA Marker, right 2: λ-HindIII digest DNA Marker.
(3)ACE2-609-Fc、ACE2-615-Fc融合蛋白与CoV022双功能融合蛋白表达载体构建(Knob in Hole)(3) ACE2-609-Fc, ACE2-615-Fc fusion protein and CoV022 bifunctional fusion protein expression vector construction (Knob in Hole)
ACE2-609-Fc融合蛋白(Fc in Hole)表达载体构建：Construction of ACE2-609-Fc fusion protein (Fc in Hole) expression vector:
将ACE2-609-Fc克隆载体用HindIII及EcoR1双酶切，同时双酶切pL101表达载体，分别胶回收2.7kb左右目的片段及9kb左右表达载体片段，将两者用T4 DNA连接酶连接并转化，质粒小量抽提并用HindIII及EcoR1双酶切鉴定。The ACE2-609-Fc cloning vector was double-cut with HindIII and EcoR1, and the pL101 expression vector was double-cut at the same time. The target fragment of about 2.7 kb and the expression vector fragment of about 9 kb were respectively recovered, and the two were ligated and transformed with T4 DNA ligase. , A small amount of plasmid was extracted and identified with HindIII and EcoR1 double enzyme digestion.
ACE2-609-Fc(Fc in Hole)具有SEQ ID NO：11所示核苷酸序列，SEQ ID NO：12所示氨基酸序列。ACE2-609-Fc (Fc in Hole) has the nucleotide sequence shown in SEQ ID NO: 11, and the amino acid sequence shown in SEQ ID NO: 12.
如图10所示：琼脂糖浓度为1％，箭头所示为约2.7kb的目的片段，右1：DL2000 DNA Marker，右2：λ-HindIII digest DNA Marker。As shown in Figure 10: the agarose concentration is 1%, the arrow shows the target fragment of about 2.7kb, right 1: DL2000 DNA Marker, right 2: λ-HindIII digest DNA Marker.
ACE2-615-Fc融合蛋白(Fc in Hole)表达载体构建Construction of ACE2-615-Fc fusion protein (Fc in Hole) expression vector
全基因合成ACE2-615片段用HindIII及EcoR1双酶切，同时双酶切pL101表达载体，分别胶回收2.0kb左右目的片段及9kb左右表达载体片段，将两者用T4 DNA连接酶连接并转化，质粒小量抽提并用HindIII及EcoR1双酶切鉴定。The whole gene synthesis ACE2-615 fragment was double digested with HindIII and EcoR1, and the pL101 expression vector was double digested at the same time. The target fragment of about 2.0 kb and the expression vector fragment of about 9 kb were recovered separately, and the two were ligated and transformed with T4 DNA ligase. The plasmid was extracted in a small amount and identified by double digestion with HindIII and EcoR1.
ACE2-615-Fc(Fc in Hole)具有SEQ ID NO：13所示核苷酸序列，SEQ ID NO：14所示氨基酸序列。ACE2-615-Fc (Fc in Hole) has the nucleotide sequence shown in SEQ ID NO: 13 and the amino acid sequence shown in SEQ ID NO: 14.
如图11所示：琼脂糖浓度为1％，箭头所示为约2.0kb的目的片段，右1：DL2000 DNA  Marker，右2：λ-HindIII digest DNA Marker。As shown in Figure 11: the agarose concentration is 1%, the arrow shows the target fragment of about 2.0kb, right 1: DL2000 DNA Marker, right 2: λ-HindIII digest DNA Marker.
CoV022重链全长克隆载体构建(Knob)：Construction of CoV022 heavy chain full-length cloning vector (Knob):
全基因合成CoV022重链可变区克隆载体用HindIII及NheI双酶切，同时双酶切IgG1恒定区克隆载体(Knob)，分别胶回收450bp左右目的片段及4kb左右克隆载体片段，将两者用T4 DNA连接酶连接并转化，质粒小量抽提并用HindIII及NheI双酶切鉴定。The full-gene synthesis CoV022 heavy chain variable region cloning vector is digested with HindIII and NheI, and the IgG1 constant region cloning vector (Knob) is double digested at the same time. T4 DNA ligase ligation and transformation, a small amount of plasmid extraction and double digestion with HindIII and NheI for identification.
如图12所示：琼脂糖浓度为1％，箭头所示为约450bp的目的片段，右1：DL2000 DNA Marker，左1：λ-HindIII digest DNA Marker。As shown in Figure 12: the agarose concentration is 1%, the arrow shows the target fragment of about 450bp, right 1: DL2000 DNA Marker, left 1: λ-HindIII digest DNA Marker.
CoV022重链全长表达载体构建(Knob)：Construction of CoV022 heavy chain full-length expression vector (Knob):
将CoV022重链全长(Knob)克隆载体用HindIII及EcoR1双酶切，同时双酶切pL102表达载体，分别胶回收1500bp左右目的片段及5kb左右表达载体片段，将两者用T4 DNA连接酶连接并转化，质粒小量抽提并用HindIII及EcoR1双酶切鉴定。The CoV022 heavy chain full-length (Knob) cloning vector was digested with HindIII and EcoR1, and the pL102 expression vector was digested at the same time. The target fragment of about 1500bp and the expression vector fragment of about 5kb were recovered separately, and the two were ligated with T4 DNA ligase After transformation, a small amount of plasmids were extracted and identified by double digestion with HindIII and EcoR1.
CoV022重链全长(Knob)具有具有SEQ ID NO：5所示核苷酸序列，SEQ ID NO：6所示氨基酸序列。The full length of the CoV022 heavy chain (Knob) has the nucleotide sequence shown in SEQ ID NO: 5 and the amino acid sequence shown in SEQ ID NO: 6.
如图13所示：琼脂糖浓度为1％，箭头所示为约1500bp的目的片段，右1：DL2000 DNA Marker，右2：λ-HindIII digest DNA Marker。As shown in Figure 13: the agarose concentration is 1%, the arrow shows the target fragment of about 1500bp, right 1: DL2000 DNA Marker, right 2: λ-HindIII digest DNA Marker.
CoV022轻链、重链全长(Knob)表达载体单个质粒构建：Construction of a single plasmid of CoV022 light chain and heavy chain full-length (Knob) expression vector:
将CoV022轻链全长表达载体及CoV022重链全长(knob)表达载体分别用SalI及NotI双酶切，分别胶回收9kb左右轻链全长表达片段及4kb左右重链全长表达载体片段，将两者用T4 DNA连接酶连接并转化，质粒小量抽提并用SalI及NotI双酶切鉴定。The CoV022 light chain full-length expression vector and CoV022 heavy chain full-length (knob) expression vector were digested with SalI and NotI, respectively, and a 9kb full-length light chain full-length expression fragment and a 4kb full-length heavy chain full-length expression vector fragment were recovered respectively. The two were ligated and transformed with T4 DNA ligase, a small amount of plasmid was extracted and identified by double enzyme digestion with SalI and NotI.
如图14所示：琼脂糖浓度为1％，箭头所示为约9kb的轻链全长表达载体片段及4kb左右的重链全长目的片段，右1：DL2000 DNA Marker，右2：λ-HindIII digest DNA Marker。As shown in Figure 14: the agarose concentration is 1%, the arrow shows the light chain full-length expression vector fragment of about 9kb and the heavy chain full-length target fragment of about 4kb, right 1: DL2000 DNA Marker, right 2: λ- HindIII digest DNA Marker.
实施例2、特异性结合冠状病毒的双功能融合蛋白的制备Example 2. Preparation of bifunctional fusion protein specifically binding to coronavirus
(1)ACE2-609-Fc/CoV022特异性结合冠状病毒的双功能融合蛋白的制备：(1) Preparation of bifunctional fusion protein of ACE2-609-Fc/CoV022 specifically binding to coronavirus:
将CoV022、ACE2-609-Fc、ACE2-609-Fc/CoV022双功能蛋白的表达载体进行柱纯，获得的质粒进行瞬时转染至宿主细胞Expi CHO-S细胞，添加补料培养基连续培养8～12天，使用无血清、无动物源组分的培养基，进行宿主细胞培养、目标蛋白质的表达；收集宿主细胞培养液上清，使用Protein A亲和层析柱分离、纯化。The CoV022, ACE2-609-Fc, ACE2-609-Fc/CoV022 bifunctional protein expression vectors were purified by column, and the obtained plasmids were transiently transfected into the host cell Expi CHO-S cells, and the feed medium was added for continuous culture 8 For ~12 days, use a serum-free, animal-derived component-free medium for host cell culture and target protein expression; collect the host cell culture supernatant, and use a Protein A affinity chromatography column for separation and purification.
SDS-PAGE确认纯化后CoV022、ACE2-609-Fc、ACE2-609-Fc/CoV022双功能蛋白分子量。SDS-PAGE confirmed the molecular weight of the purified CoV022, ACE2-609-Fc, ACE2-609-Fc/CoV022 bifunctional protein.
(2)ACE2-615-Fc/CoV022特异性结合冠状病毒的双功能融合蛋白的制备：(2) Preparation of bifunctional fusion protein of ACE2-615-Fc/CoV022 specifically binding to coronavirus:
将CoV022、ACE2-615-Fc、ACE2-615-Fc/CoV022双功能蛋白的表达载体进行柱纯， 获得的质粒进行瞬时转染至宿主细胞Expi CHO-S细胞，添加补料培养基连续培养8～12天，收集宿主细胞培养液上清，使用Protein A亲和层析柱分离、纯化。The CoV022, ACE2-615-Fc, ACE2-615-Fc/CoV022 bifunctional protein expression vectors were purified by column, and the obtained plasmids were transiently transfected into the host cell Expi CHO-S cells, and the feed medium was added for continuous culture 8 ~12 days, collect the host cell culture supernatant, use Protein A affinity chromatography column for separation and purification.
SDS-PAGE确认纯化后CoV022、ACE2-615-Fc、ACE2-615-Fc/CoV022双功能蛋白分子量。SDS-PAGE confirmed the molecular weight of the purified CoV022, ACE2-615-Fc, ACE2-615-Fc/CoV022 bifunctional protein.
CoV022、ACE2-609-Fc、ACE2-615-Fc、ACE2-609-Fc/CoV022、ACE2-615-Fc/CoV022的SDS-PAGE电泳图谱分别见图15、图16，其中图15的样品顺序为：1、2泳道样品为ACE2-609-Fc，3、4泳道样品为CoV022，5、6泳道样品为ACE2-609-Fc/CoV022；图16的样品顺序为：1、2泳道样品为ACE2-615-Fc，3、4泳道样品为CoV022，5、6泳道样品为ACE2-615-Fc/CoV022。The SDS-PAGE electrophoresis patterns of CoV022, ACE2-609-Fc, ACE2-615-Fc, ACE2-609-Fc/CoV022, and ACE2-615-Fc/CoV022 are shown in Figure 15 and Figure 16, respectively. The sample sequence of Figure 15 is : The samples in lanes 1 and 2 are ACE2-609-Fc, the samples in lanes 3 and 4 are CoV022, and the samples in lanes 5 and 6 are ACE2-609-Fc/CoV022; the order of the samples in Figure 16 is: the samples in lanes 1, 2 are ACE2- 615-Fc, the samples in lanes 3 and 4 are CoV022, and the samples in lanes 5 and 6 are ACE2-615-Fc/CoV022.
实施例3、特异性结合冠状病毒的双功能融合蛋白与冠状病毒的结合能力检测Example 3. Detection of the binding ability of the bifunctional fusion protein that specifically binds to the coronavirus and the coronavirus
ELISA检测法检测特异性结合冠状病毒的双功能融合蛋白与冠状病毒蛋白的结合能力：The ELISA method detects the binding ability of the bifunctional fusion protein that specifically binds to the coronavirus and the coronavirus protein:
使用包被液将SARS-CoV-2 S蛋白(Spike蛋白)加入酶标板，37℃包被1小时；弃除包被液，加入封闭液，350μl/孔，37℃封闭1.5小时；用稀释液逐级稀释CoV022、ACE2-609-Fc、ACE2-609-Fc/CoV022双功能蛋白，及ACE2-615-Fc、ACE2-615-Fc/CoV022双功能蛋白，加入酶标板内，37℃水浴1小时；洗板机洗涤5-7次；用稀释液稀释兔抗人-HRP加入酶标板，37℃水浴1小时；弃上清，洗板机洗涤5-7次；加入TMB显色液避光显色，加入等体积终止液终止显色，迅速混匀。Use the coating solution to add SARS-CoV-2 S protein (Spike protein) to the ELISA plate, and coat at 37°C for 1 hour; discard the coating solution, add blocking solution, 350μl/well, and block at 37°C for 1.5 hours; dilute with Dilute CoV022, ACE2-609-Fc, ACE2-609-Fc/CoV022 bifunctional protein, and ACE2-615-Fc, ACE2-615-Fc/CoV022 bifunctional protein step by step with the solution, add it to the microplate, 37℃ water bath 1 hour; wash with a plate washer for 5-7 times; dilute the rabbit anti-human-HRP with the diluent and add it to the ELISA plate, bath at 37°C for 1 hour; discard the supernatant, and wash with the plate washer for 5-7 times; add TMB color developing solution Avoid light for color development, add an equal volume of stop solution to stop color development, and mix quickly.
酶标仪读数，检测结果见图17、图18，图17为ELISA检测CoV022、ACE2-609-Fc、ACE2-609-Fc/CoV022双功能蛋白与SARS-CoV-2 S蛋白(Spike蛋白)的结合能力；图18为ELISA检测CoV022、ACE2-615-Fc、ACE2-615-Fc/CoV022双功能蛋白与SARS-CoV-2 S蛋白(Spike蛋白)的结合能力。The reading of the microplate reader, the detection results are shown in Figure 17, Figure 18. Figure 17 shows the ELISA detection of CoV022, ACE2-609-Fc, ACE2-609-Fc/CoV022 bifunctional protein and SARS-CoV-2 S protein (Spike protein) Binding ability; Figure 18 shows the binding ability of CoV022, ACE2-615-Fc, ACE2-615-Fc/CoV022 bifunctional protein and SARS-CoV-2 S protein (Spike protein) detected by ELISA.
由实验结果可知，特异性结合冠状病毒的双功能融合蛋白ACE2-609-Fc/CoV022、ACE2-615-Fc/CoV022均与ARS-CoV-2 S蛋白(Spike蛋白)结合，并且双功能融合蛋白ACE2-609-Fc/CoV022、ACE2-615-Fc/CoV022与ARS-CoV-2 S蛋白(Spike蛋白)的结合能力均较强于CoV022、ACE2-609-Fc或ACE2-615-Fc与ARS-CoV-2 S蛋白(Spike蛋白)的结合能力。It can be seen from the experimental results that the bifunctional fusion proteins ACE2-609-Fc/CoV022 and ACE2-615-Fc/CoV022, which specifically bind to the coronavirus, bind to the ARS-CoV-2 S protein (Spike protein), and the bifunctional fusion protein ACE2-609-Fc/CoV022, ACE2-615-Fc/CoV022 and ARS-CoV-2 S protein (Spike protein) have stronger binding ability than CoV022, ACE2-609-Fc or ACE2-615-Fc and ARS- CoV-2 S protein (Spike protein) binding ability.
实施例4、特异性结合冠状病毒的双功能融合蛋白与冠状病毒的结合能力检测Example 4. Detection of the binding ability of the bifunctional fusion protein that specifically binds to the coronavirus and the coronavirus
使用SPR表面等离子共振技术(surface plamon resonace technology,SPR)检测CoV022、ACE2-609-Fc、ACE2-609-Fc/CoV022双功能融合蛋白、ACE2-615-Fc、ACE2-615-Fc/CoV022双功能融合蛋白与SARS-CoV-2 S Protein亲和力。Use SPR surface plasmon resonance technology (SPR) to detect CoV022, ACE2-609-Fc, ACE2-609-Fc/CoV022 dual function fusion protein, ACE2-615-Fc, ACE2-615-Fc/CoV022 dual function The fusion protein has affinity with SARS-CoV-2 S Protein.
在CM5芯片表面偶联ARS-CoV-2 S Protein，之后检测浓度依次升高的CoV022、ACE2-609-Fc、ACE2-609-Fc/CoV022双功能融合蛋白、ACE2-615-Fc、ACE2-615-Fc/CoV022双功能融合蛋白，评估五个样品和ARS-CoV-2 S Protein的亲和力参数。样品之间进行再生处理。结果使用Biacore仪器进行采集，并使用Biacore Evaluation software进行分析数据分析，评估亲和力参数(KD值)，结果见表1。Couple ARS-CoV-2 S Protein on the surface of CM5 chip, and then detect CoV022, ACE2-609-Fc, ACE2-609-Fc/CoV022 bifunctional fusion protein, ACE2-615-Fc, ACE2-615 with increasing concentrations -Fc/CoV022 dual-function fusion protein to evaluate the affinity parameters of five samples and ARS-CoV-2 S Protein. Regenerate treatment between samples. The results were collected using Biacore instruments, and analyzed data using Biacore Evaluation software to evaluate the affinity parameters (KD values). The results are shown in Table 1.
结果显示双功能融合蛋白ACE2-609-Fc/CoV022、ACE2-615-Fc/CoV022与ARS-CoV-2 S蛋白(Spike蛋白)的亲和力均要高于CoV022、ACE2-609-Fc或ACE2-615-Fc与ARS-CoV-2 S蛋白(Spike蛋白)的亲和力，提示双功能融合蛋白ACE2-609-Fc/CoV022、ACE2-615-Fc/CoV022其具有较好的潜在病毒中和效果。The results show that the affinity of the bifunctional fusion protein ACE2-609-Fc/CoV022, ACE2-615-Fc/CoV022 and ARS-CoV-2 S protein (Spike protein) is higher than that of CoV022, ACE2-609-Fc or ACE2-615 The affinity between Fc and ARS-CoV-2 S protein (Spike protein) suggests that the bifunctional fusion proteins ACE2-609-Fc/CoV022 and ACE2-615-Fc/CoV022 have good potential virus neutralization effects.
表1、SPR法检测ACE2-609-Fc、ACE2-615-Fc、CoV022、ACE2-609-Fc/CoV022、ACE2-615-Fc/CoV022双功能融合蛋白与SARS-CoV-2 S Protein亲和力结果表。Table 1. SPR method detects ACE2-609-Fc, ACE2-615-Fc, CoV022, ACE2-609-Fc/CoV022, ACE2-615-Fc/CoV022 bifunctional fusion protein and SARS-CoV-2 S Protein affinity results table .
表1、SPR法检测结果Table 1. SPR method test results
样品sample KD(M)KD(M)
CoV022CoV022 6.198×10 -9 6.198×10 -9
ACE2-609-FcACE2-609-Fc 1.013×10 -8 1.013×10 -8
ACE2-609-Fc/CoV022ACE2-609-Fc/CoV022 1.276×10 -9 1.276×10 -9
ACE2-615-FcACE2-615-Fc 1.296×10 -8 1.296×10 -8
ACE2-615-Fc/CoV022ACE2-615-Fc/CoV022 1.411×10 -9 1.411×10 -9

Claims (16)

  1. 一种结合冠状病毒的双功能融合蛋白，其特征在于，所述双功能融合蛋白分别结合冠状病毒的不同表位。A bifunctional fusion protein that binds to a coronavirus is characterized in that the bifunctional fusion protein respectively binds to different epitopes of the coronavirus.
  2. 根据权利要求1所述的结合冠状病毒的双功能融合蛋白，其特征在于，所述的冠状病毒具有Spike蛋白。The bifunctional fusion protein binding to coronavirus according to claim 1, wherein the coronavirus has a Spike protein.
  3. 根据权利要求1所述的结合冠状病毒的双功能融合蛋白，其特征在于，所述的冠状病毒具有ACE2结合位点。The bifunctional fusion protein that binds to a coronavirus according to claim 1, wherein the coronavirus has an ACE2 binding site.
  4. 根据权利要求1所述的结合冠状病毒的双功能融合蛋白，其特征在于，所述双功能融合蛋白结合冠状病毒的ACE2结合位点，同时结合冠状病毒的Spike蛋白。The bifunctional fusion protein that binds to the coronavirus according to claim 1, wherein the bifunctional fusion protein binds to the ACE2 binding site of the coronavirus and simultaneously binds to the Spike protein of the coronavirus.
  5. 根据权利要求1所述的结合冠状病毒的双功能融合蛋白，其特征在于，所述双功能融合蛋白具有与冠状病毒的ACE2结合位点相结合的SEQ ID NO：12或SEQ ID NO：14所示的氨基酸序列。The bifunctional fusion protein that binds to the coronavirus according to claim 1, wherein the bifunctional fusion protein has SEQ ID NO: 12 or SEQ ID NO: 14 that binds to the ACE2 binding site of the coronavirus. The amino acid sequence shown.
  6. 根据权利要求1所述的结合冠状病毒的双功能融合蛋白，其特征在于，所述双功能融合蛋白具有与冠状病毒的Spike蛋白结合的SEQ ID NO：6所示的重链氨基酸序列及SEQ ID NO：2所示的轻链氨基酸序列。The bifunctional fusion protein that binds to coronavirus according to claim 1, wherein the bifunctional fusion protein has the heavy chain amino acid sequence shown in SEQ ID NO: 6 and SEQ ID that binds to the Spike protein of the coronavirus. NO: the amino acid sequence of the light chain shown in 2.
  7. 一种组合物，其特征在于，包括权利要求1、4-6所述的结合冠状病毒的双功能融合蛋白及药学上可接受的赋形剂。A composition characterized by comprising the bifunctional fusion protein binding to coronavirus as described in claims 1, 4-6 and a pharmaceutically acceptable excipient.
  8. 一种结合冠状病毒的双功能融合蛋白的制备方法，其特征在于，包含以下步骤：a)基因合成权利要求1、4-6所述的结合冠状病毒的双功能融合蛋白的基因序列，制备含有所述双功能融合蛋白基因的表达载体；b)将所述表达载体转染至宿主细胞，并进行宿主细胞的培养、及所述双功能融合蛋白的表达；c)所述双功能融合蛋白的分离、纯化。A method for preparing a bifunctional fusion protein binding to a coronavirus, which is characterized in that it comprises the following steps: a) Gene synthesis of the gene sequence of the bifunctional fusion protein binding to a coronavirus according to claim 1, 4-6, and preparing a gene sequence containing The expression vector of the bifunctional fusion protein gene; b) transfecting the expression vector into a host cell, and carrying out the cultivation of the host cell and the expression of the bifunctional fusion protein; c) the expression of the bifunctional fusion protein Isolation and Purification.
  9. 根据权利要求8所述的结合冠状病毒的双功能融合蛋白的制备方法，其特征在于，所述宿主细胞为真核哺乳动物细胞CHO细胞。The method for preparing a bifunctional fusion protein binding to coronavirus according to claim 8, wherein the host cell is a eukaryotic mammalian cell CHO cell.
  10. 根据权利要求8所述的结合冠状病毒的双功能融合蛋白的制备方法，其特征在于，所述的宿主细胞的培养使用无血清、无动物源组分的培养基培养。The method for preparing a coronavirus-binding bifunctional fusion protein according to claim 8, wherein the host cell is cultured using a serum-free and animal-derived component-free medium.
  11. 一种结合冠状病毒的双功能融合蛋白的应用，其特征在于，包含权利要求1、4-6、8-10所述的结合冠状病毒的双功能融合蛋白在制备抗冠状病毒药物中的应用。An application of a bifunctional fusion protein that binds to a coronavirus, which is characterized in that it comprises the use of the bifunctional fusion protein that binds to a coronavirus according to claims 1, 4-6, and 8-10 in the preparation of anti-coronavirus drugs.
  12. 一种结合冠状病毒的双功能融合蛋白的应用，其特征在于，包含权利要求7所述的结合冠状病毒的双功能融合蛋白在制备抗冠状病毒药物中的应用。An application of a bifunctional fusion protein that binds to a coronavirus, which is characterized in that it comprises the use of the bifunctional fusion protein that binds to a coronavirus according to claim 7 in the preparation of anti-coronavirus drugs.
  13. 根据权利要求11所述的结合冠状病毒的双功能融合蛋白的应用，其特征在于，所述的冠状病毒具有ACE2感染途径。The use of a bifunctional fusion protein that binds to a coronavirus according to claim 11, wherein the coronavirus has an ACE2 infection route.
  14. 根据权利要求12所述的结合冠状病毒的双功能融合蛋白的应用，其特征在于，所述的冠状病毒具有ACE2感染途径。The use of a bifunctional fusion protein that binds to a coronavirus according to claim 12, wherein the coronavirus has an ACE2 infection route.
  15. 根据权利要求11所述的结合冠状病毒的双功能融合蛋白的应用，其特征在于，所述的冠状病毒为SARS-CoV相关冠状病毒、MERS-CoV相关冠状病毒、或SARS-CoV-2相关病毒。The application of a bifunctional fusion protein that binds to a coronavirus according to claim 11, wherein the coronavirus is a SARS-CoV-related coronavirus, a MERS-CoV-related coronavirus, or a SARS-CoV-2 related virus .
  16. 根据权利要求12所述的结合冠状病毒的双功能融合蛋白的应用，其特征在于，所述的冠状病毒为SARS-CoV相关冠状病毒、MERS-CoV相关冠状病毒、或SARS-CoV-2相关病毒。The application of a dual-function fusion protein that binds to a coronavirus according to claim 12, wherein the coronavirus is a SARS-CoV-related coronavirus, a MERS-CoV-related coronavirus, or a SARS-CoV-2 related virus .
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