Artificial virus receptors and their applications

Abstract

The present invention discloses an artificial virus receptor and its application, which belongs to the field of biotechnology. The artificial virus receptor of the present invention includes modules A, B, C, D, E, F, G, and H from the amino terminus to the carboxyl terminus. Module A is a signal peptide of type I membrane protein; module B is a customized virus binding domain for capturing target viruses; modules C and E are linkers; module D is a spacer region for forming the skeleton of a customized artificial virus receptor to maintain a certain length outside the cell; module F is a transmembrane domain for realizing cell surface display of the artificial virus receptor; module G is an intracellular domain, which has the function of maintaining the stability of the artificial virus receptor; module H is a tag protein. The present invention paves the way for the rapid construction of in vitro and in vivo infection systems of viruses that are difficult to culture, especially high-risk coronaviruses, and for downstream prospective research, which will help to further advance the life cycle research of many difficult-to-culture viruses and promote the development of antiviral treatments and vaccines.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to an artificial virus receptor designed for a specific virus based on engineering transformation and its application. Background Art

[0002] For many important viruses, the unknown identity of their receptors and the lack of ideal infection models severely hinder scientific research on these viruses. These include the lack of effective viral culture methods, the difficulty in studying viral transmission and pathogenic mechanisms, and the lack of effective methods for drug and vaccine screening and evaluation. Elucidating the functional receptors of viruses and establishing transgenic cell and animal infection models are key to overcoming these bottlenecks. However, the success of identifying natural viral receptors is unpredictable, and infection models established based on susceptible cell lines and animal models with unknown receptors have significant limitations, such as difficulty in improving efficiency and the inability to genetically engineer them for studies in other cell lines and animals.

[0003] Therefore, the targeted design of artificial viral receptors that can effectively mediate the invasion of specific viruses is an alternative strategy for constructing specific susceptible cell and animal models. Artificial receptor design has been successfully applied in the field of immunotherapy, especially chimeric antigen receptors (CARs). However, the design of artificial viral receptors for the purpose of establishing viral infection systems has not been reported. The functional viral receptors of many important viruses are still unknown, and the lack of convenient infection systems has limited in-depth research on these viruses, such as coronaviruses, which have diverse receptor binding domain (RBD) structures and receptor selection. In addition to identifying natural receptors, another approach to establishing effective infection models is to design functional receptors to support viral entry, but attempts to achieve this goal are still rare. At the same time, in order to design artificial viral receptors and realize their multiple and complex functions, many issues still need to be considered, including supporting specific binding to viral surface proteins and triggering downstream entry events such as enzymatic cleavage, endocytosis, and membrane fusion / penetration.

[0004] In virological research, antibody-dependent enhancement (ADE) describes a phenomenon in which antibodies mediate an increase in disease severity through different mechanisms. One representative mechanism is based on the interaction between the virus and the antibody after forming a complex with the Fc receptor (FcR) and entering the cell through binding and uptake. This phenomenon is particularly prominent in viruses of the Flaviviridae family, and there are also some reports in the Coronaviridae family. However, in many cases, because cells expressing Fc receptors are not the main target cells for viral infection, coupled with some unknown factors, the virus often does not complete its life cycle after entering the cell based on this mechanism and produces infectious progeny viruses. Inspired by this phenomenon, the present invention systematically analyzes the structural and functional characteristics of viral functional receptors and proposes a chimeric receptor design strategy based on highly specific virus-receptor interactions.

[0005] The present invention proposes a new concept of customized viral receptor (CVR) design, that is, a strategy to construct functional viral receptors by assembling modular molecular elements (including viral binding domains, leader signal peptide sequences, linker sequences, transmembrane motifs, intracellular domains and other molecular elements). The present invention obtains a compatible receptor molecular chassis by testing a variety of artificial chimeric proteins, and realizes customization of functional artificial receptor molecules for different viruses by replacing the viral binding domain modules targeting specific epitopes of different viral surface proteins. By testing the functionality of a series of receptors, the present invention has developed a universal modular receptor framework for a variety of coronaviruses, and is applicable to the design of artificial receptors for other types of viruses. Summary of the Invention

[0006] The present invention aims to overcome the shortcomings of existing technologies by providing a customized artificial viral receptor and its applications. This invention paves the way for the rapid construction of in vitro and in vivo infection models for difficult-to-cultivate viruses, particularly novel, high-risk coronaviruses, and for downstream, prospective research. The application of this invention will further advance research on the lifecycles of numerous difficult-to-cultivate viruses and significantly promote the development of antiviral therapies and vaccines.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] A customized artificial viral receptor, the structural diagram of its primary protein structure is as follows Figure 1As shown, from the amino terminus to the carboxyl terminus, it includes: modules A, B, C, D, E, F, G, and H. The module A is a signal peptide of a type I membrane protein (single-pass transmembrane protein), which is used to display the protein on the cell membrane surface. The module B is a customized virus binding domain for capturing the target virus. The module C is a linker 1, which is used to link module B and module D. The module D is a spacer, which is used to form the skeleton of a customized artificial virus receptor to maintain a certain length outside the cell. The module E is a linker 2, which is used to link module D and module F. The module F is a transmembrane domain, including but not limited to a single-pass transmembrane helix of a type I membrane protein and a single-pass transmembrane helix of a type II membrane protein. In the present invention, the cell surface display of the artificial virus receptor is mainly achieved by module F. The module G is an intracellular domain, which has the function of maintaining the stability of the artificial virus receptor. The module H is a tag protein, preferably a 3×flag tag, for detection. Among them, modules C, D, and G are not necessary modules to maintain receptor function. Therefore, in some cases, the customized artificial viral receptor does not contain at least one of modules C, D, and G.

[0009] In some embodiments, the amino acid sequence of module A of the customized artificial viral receptor is selected from the following sequences:

[0010] >CD5 signal peptide: MPMGSLQPLATLYLLGMLVASVLA(SEQ ID NO.1);

[0011] >IgG kappa signal peptide: METDTLLLWVLLLWVPGSTGD (SEQ ID NO. 2).

[0012] In some embodiments, module B of the customized artificial viral receptor is derived from a known antibody truncated.

[0013] In some embodiments, module B of the customized artificial viral receptor is a scFV form modified based on a known antibody.

[0014] In some embodiments, module B of the customized artificial viral receptor is derived from a computer-aided designed binding protein.

[0015] In some embodiments, module B of the customized artificial viral receptor is derived from traditional antibodies and / or nanobodies selected by immunization and / or in vitro panning.

[0016] In some embodiments, module C of the customized artificial viral receptor is a restriction endonuclease site left at the nucleic acid level for cloning, such as AgeI, BamHI, KpnI, etc., whose corresponding amino acid sequences are TG, GS, and GT. Furthermore, the amino acid sequence of module C is TGGS, GSGS, or GTGS.

[0017] In some embodiments, the module D of the customized artificial viral receptor is an antibody human FC segment or different truncations of an antibody human FC segment, and its amino acid sequence is preferably selected from the following sequences:

[0018] human FC:APLEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGA(SEQ ID NO.3);

[0019] truncated human FC 1: APLEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK(SEQ ID NO.4);

[0020] truncated human FC 2: APLEPKSSDKTHTCPPCPA (SEQ ID NO. 5).

[0021] In some embodiments, the module D of the customized artificial viral receptor is a truncation of the backbone of a type I membrane protein, such as the Neck domain of ACE2 or the linker domain of ACE2, and its amino acid sequence is preferably selected from the following sequences:

[0022] ACE2 Neck domain: QSIKVRISLKSALGDKAYEWNDNEMYLFRSSVAYAMRQYFLKVK NQMILFGEEDVRVANLKPRISFNFFVTAPKNVSDIIPRTEVEKAIRMSRSRINDAFRLNDNSLE FLGIQPTLGPPNQPPVS(SEQ ID NO.6);

[0023] ACE2 Linker domain: IQPTLGPPNQPPVS (SEQ ID NO.7).

[0024] In some embodiments, the amino acid sequence of module E of the customized artificial viral receptor is: GSGT.

[0025] In some embodiments, the module F of the customized artificial viral receptor is a single transmembrane helix of a type I membrane protein or a single transmembrane helix of a type II membrane protein, and its amino acid sequence is preferably selected from the following sequences:

[0026] IL2RαTM: VAVAGCVFLLISVLLLSGLTW (SEQ ID NO. 8);

[0027] ACE2TM: IWLIVFGVVMGVIVVGIVILI (SEQ ID NO. 9);

[0028] LDLRTM: ALSIVLPIVLLVFLCLGVFLLWKNW (SEQ ID NO. 10);

[0029] CD3 zeta™: LCYLLDGILFIYGVILTALF (SEQ ID NO. 11).

[0030] In some embodiments, the module G of the customized artificial viral receptor is an intracellular domain of a membrane protein, such as ACE2, LDLR, IL2Rα, CD3, or an element EPM that stably expresses on the cell surface, and its amino acid sequence is preferably selected from the following sequences:

[0031] IL2Rα Cytosol domain: QHRWRKSRRTI (SEQ ID NO.12);

[0032] ACE2 Cytosol domain: FTGIRDRKKKNKARSGENPYASIDISKGENNPGFQNTDDVQTS F(SEQID NO.13);

[0033] LDLR Cytosol domain: RLKNINSINDFDNPVYQKTTEDEVHICHNQDGYSYPSRQMVSL EDDVA(SEQ ID NO.14);

[0034] CD3 zeta Cytosol domain: LRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKR RGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTAT KDTYDALHMQALPPR (SEQ ID NO. 15).

[0035] EPM (endocytosis prevention motif): ALPGNPDHREMGETLPEEVGEYRQPSGGSVPVSPGPPSGLEPTSSSPY (SEQ ID NO. 16).

[0036] In some embodiments, the amino acid sequence of module H of the customized artificial viral receptor is: DYKDHDG DYKDHDIDYKDDDDK (SEQ ID NO. 17).

[0037] The application of the customized artificial viral receptor in establishing a coronavirus infection system / model can be achieved by the following steps: establishing a susceptible cell line overexpressing the customized artificial viral receptor through plasmid transfection, lentiviral packaging, and then transduction. The former is a transiently expressing susceptible cell line, while the latter is a stably expressing susceptible cell line. This establishes a coronavirus infection system / model.

[0038] The application of the customized artificial viral receptor in the evaluation of neutralizing antibodies. The application can be achieved by the following steps: establishing a susceptible cell line that overexpresses the customized artificial viral receptor through plasmid transfection, lentiviral packaging, and then transduction. The neutralizing antibody is incubated with the coronavirus. The antibody concentration, incubation time, and temperature can be adjusted accordingly. The neutralizing antibody is then added to the culture medium and co-cultured with the susceptible cell line. The neutralizing ability of the neutralizing antibody is evaluated by the infection effect. After the outbreak, scientific or medical workers around the world developed or screened a large number of antibodies against SARS-CoV-2. Among them, some neutralizing antibodies that recognize the conserved region of the viral S protein were found; they retain the ability to neutralize mutant strains and have neutralizing ability against other coronaviruses in the same family, reflecting a certain broad spectrum. The present invention can be used to establish infection systems / models for other viruses and evaluate the neutralizing ability of these antibodies against these viruses. This provides an effective response strategy for dealing with sudden outbreaks of newly emerging viruses with unknown receptors.

[0039] The use of the customized artificial viral receptor in supporting coronavirus infection and proliferation can be achieved by: establishing a susceptible cell line overexpressing the customized artificial viral receptor through plasmid transfection, lentiviral packaging, and then transduction. The coronavirus infection system / model is then inoculated with the actual coronavirus to achieve viral amplification.

[0040] The use of the customized artificial viral receptor in coronavirus rescue can be achieved by the following steps: establishing a susceptible cell line overexpressing the customized artificial viral receptor through plasmid transfection, lentiviral packaging, and then transduction. Electroporation of coronavirus genomic RNA (obtained by in vitro transcription kit) in a coronavirus infection system / model; or transfection of a coronavirus infectious clone plasmid in a coronavirus infection system / model to rescue the coronavirus.

[0041] The application of the customized artificial viral receptor in a novel coronavirus animal infection model can be achieved by the following steps: using genetic engineering to replace the reading frame of the natural viral receptor with the customized artificial viral receptor, or knocking it into the genome of mice or other model animals based on a specific promoter to achieve systemic or tissue-specific constitutive or inducible expression, making the transgenic animals susceptible to specific viruses, and using it to study the infection and pathogenic mechanisms of specific viruses, evaluate the effectiveness of specific vaccines or antiviral drugs, or other applications.

[0042] The present invention offers the following advantages and benefits over existing technologies: It provides a functional artificial viral receptor based on a modular framework, with comparable or superior transduction capabilities to its native receptor. Using this receptor, an infection system can be established, enabling the rescue and isolation of authentic viruses that are difficult to culture using traditional methods. This invention demonstrates for the first time the feasibility of successfully rescuing a coronavirus with no known receptor and no traditional susceptible cells in transgenic cells independent of its natural receptor. This means that even if the natural receptor is unknown and traditional susceptible cells are unavailable, this system can be used to isolate the virus from a sample containing virions. Alternatively, even if a sample containing the virus is unavailable, the virus can be rescued using reverse genetics as long as the complete genome sequence is available. Furthermore, novel transgenic animals can be constructed for evaluating viral infection mechanisms and antiviral strategies. This invention paves the way for the rapid construction of novel in vitro and in vivo infection systems for difficult-to-culture viruses, particularly high-risk coronaviruses, and for downstream, prospective research. It will help further advance the study of the life cycles of numerous difficult-to-culture viruses and significantly advance the development of antiviral therapies and vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 Schematic diagram of the primary structure of the artificial viral receptor.

[0044] Figure 2 Preliminary exploration of the artificial viral receptor skeleton. A: Cartoon diagram of exploring different truncated forms of the ACE2 molecule and replacing different structural domains. B: Figure 1 Western blotting in A detects the expression of various truncated and replaced forms of ACE2. C: FCS was used to detect the differences in the ability of different forms of skeletons displayed on the cell surface to bind to SARS-CoV-2 RBD. D, E: Figure 1 Different forms of the skeleton in C mediate the invasion of SARS-CoV-2 pseudovirus. The pseudovirus carries EGFP and fLuc genes. The successful infection of SARS-CoV-2 pseudovirus is characterized by immunofluorescence (D), and the intensity of luciferase is measured to determine the ability of different forms of the skeleton to mediate infection (E). F: The EPM motif can stabilize the cell surface distribution of CVR. The above figure shows the detection of CVR cell surface distribution by immunofluorescence after adding the EPM sequence. G: EPM can further improve the mediating ability of CVR to SARS-CoV-2 pseudovirus. H: Western detection of the expression of spacer molecules of different lengths on the artificial receptor skeleton. I: Comparison of the ability of artificial receptors carrying spacers of different lengths in F to mediate viral infection.

[0045] Figure 3 Exploration of receptor function by VBD. A, B: Schematic animation of molecules replacing different forms of viral binding domains (A), replaced with virtually designed small peptides, Nbs, ScFvs, and Fabs, respectively, and their ability to mediate viral entry (B); C: Flow cytometric analysis of the expression, binding, neutralization, and SARS-CoV-2 entry-mediating abilities of 24 artificial receptors targeting the SARS-CoV-2 RBD region. Receptor expression, binding, neutralization, and entry-mediating abilities were normalized in descending order of mediating ability, and a heat map was plotted. D: Schematic diagram of different antibodies recognizing various epitopes of the SARS-CoV-2 Spike protein. E: Comparison of the viral entry-mediating abilities of artificial receptors recognizing different epitopes. Artificial receptor expression was detected by flow cytometry and a heat map was plotted. Mediating abilities were measured by measuring luciferase intensity in infected cells and a heat map was plotted.

[0046] Figure 4CVRs resemble natural receptor-mediated entry pathways. A: Artificial virus receptors specifically mediate entry of the corresponding viruses. The figure shows five different coronavirus pseudoviruses, SARS-CoV, SARS-CoV-2, MERS-CoV, NL63-CoV, and 229E-CoV, infecting cells with ACE2, LCB1, S2Nb24, DPP4, mNb1, APN, and 2C5. Green fluorescence signals are detected after viral infection. B: Susceptible cell lines established from different cell types (BHK21, 293T, A549, and Tb1-Lu) mediate entry of SARS-CoV-2 pseudoviruses. The figure shows the green fluorescence signals after infection. C: Animated schematic diagram of a membrane fusion assay based on a dual cleavage protein (DSP) complementation assay. D, E: SARS-CoV-2 S, MERS-CoV S, and 229E-CoV S proteins mediate membrane fusion through the natural receptors ACE2, DPP4, and APN, as well as the corresponding artificial virus receptors LCB1, S2Nb24, mNb1, and 2A5. D shows the green fluorescence signal after fusion, and E shows the luciferase activity assay. F: The artificial viral receptor performs comparable to its natural receptor in evaluating viral entry inhibitors. The figure shows the IC50 of each inhibitor for neutralizing SARS-CoV-2 viral entry in cell lines expressing different receptors. G: Comparison of the receptor function of the artificial viral receptor with several reported SARS-CoV-2 entry factors or alternative receptors / coreceptors.

[0047] Figure 5 Application of artificial viral receptors in neutralizing antibody evaluation. A: Inhibitory activity of SARS-CoV-2 protective antiserum against 293T cells expressing ACE2, LCB1-CVR, and S2Nb24-CVR. B: Phylogenetic tree analysis of eight betacoronaviruses with unknown receptors. C: Through nanobody screening, artificial receptor-susceptible cell lines were designed and constructed that mediate invasion of HKU31, HKU5, HKU1, HKU9, ZJ2013, HKU3, RmYN02, and ZC45. Luciferase activity was measured 10-16 hours after infection. D: Breadth of the cross-reactivity spectrum of broadly neutralizing antibodies against betacoronaviruses.

[0048] Figure 6Artificial receptors support the proliferation of true coronavirus infection and the rescue of VSV-HKU3 S-GFP chimeric viruses. A: Artificial virus receptors mediate the entry of true coronaviruses SARS-CoV-2, 229E-CoV, and MHV-A59, respectively. B: Technical roadmap for the rescue of VSV chimeric HKU3 S protein viruses. C, D: Successful rescue of rVSV-HKU3S-GFP chimeric viruses using the above method. C: After transfection, EGFP signal was observed and gradually increased over time. D: P0 supernatant was centrifuged and inoculated into Caco-2-S2Nb27 cells, successfully infecting them. E: Three days after infection, the virus was harvested and inoculated into new Caco-2-S2Nb27 cells at a 1:5 dilution. The virus was passaged for 10 consecutive generations. Virus passage was recorded using EGFP and anti-HKU3 S antibodies. F: Dynamic replication curves of the HKU3 chimeric virus in cell lines expressing different artificial virus receptors over time. Set up three replicate wells, collect the culture supernatant every 12 hours, and replace with fresh medium. Take 20 μL of the viral supernatant from each time period to detect the viral copy number.

[0049] Figure 7 Transgenic mice based on artificial receptors support infection with the new coronavirus. A: Schematic diagram of the construction of transgenic mice with artificial viral receptors. As shown in the figure, the target gene replaces the coding frame of mACE2 in situ. B: After dissecting the mice, the lung tissue was lysed and sampled, and the expression of the artificial receptor LCB1 was detected by Western blot. C: Animal infection experimental process. On day 0, the virus was inoculated through the nasal cavity. The hACE2 group dissected the mice 5 days after infection. The artificial receptor LCB1 group dissected the mice on the 3rd, 5th and 7th days, and the lung tissue cells were isolated to detect the viral load (n=3). D: The viral load of the lung tissue of each group of mice after infection was detected by qPCR. E: Pathological staining of lung tissue after infection. DETAILED DESCRIPTION

[0050] The following examples are intended to further illustrate the present invention but should not be construed as limiting the present invention. Any other changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principles of the present invention are considered equivalent substitutions and are included within the scope of protection of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0051] The methods involved in the following embodiments:

[0052] (1) Cell culture

[0053] 293T (CRL-3216) cells, VERO E6 (CRL-1586) cells, A549 (CCL-185) cells, BHK-21 (CCL-10) cells, Caco2 (HTB-37) cells, and the bat epithelial cell line Tb 1Lu (CCL-88) were purchased from the American Type Culture Collection (ATCC). All cells were maintained in DMEM supplemented with 10% FBS, 1 mM sodium pyruvate, and 50 IU / mL of the dual-antibody and cultured at 37°C in a 5% CO2 cell culture incubator.

[0054] (2) Plasmid construction

[0055] The coding nucleotides of receptors such as ACE2, DPP4, APN, AXL, NRP1, ASGPR1, KREMEN1, TMPRSS2, SR-B1, CEACAM1A and different forms of artificial receptors were digested by EcoR I and Not I and ligated into the lentiviral vector pLVX-EF1α-Receptor-IRES-puro (Addgene, Catalog #134665), which has a 3×Flag tag (DYKDHD-G-DYKDHD-I-DYKDDDDK) at the C-terminus. The nucleic acid sequences encoding the 229E-CoV S protein (AF344189.1), HKU3-CoV S protein (DQ022305.2), HKU5-CoV S protein (YP_001039962.1), HKU31 S protein (QGA70692.1), SARS-CoV-2 S protein (YP_009724390.1), MERS-CoV S protein (YP_009047204.1), and MHV-CoV S protein (AY_497328.1) were humanized and constructed in the pCAGGS vector. To improve the packaging efficiency of VSV pseudovirus, the C-terminal 13-15 amino acids of these S proteins were deleted (corresponding to the C-terminal 18 amino acids of SARS-CoV-2 S). The coronavirus RBD and / or S1 proteins were extracted from the above plasmids, and the N-terminus was connected to the CD5 secretion signal peptide (MPMGSLQPLATLYLLGMLVASVL). Among them, HKU5-CoV RBD (aa385-586), HKU31-CoV RBD (aa366-575), SARS-CoV-2 RBD (aa331-524), MERS-CoV RBD (aa377-588), HKU3-CoV S1 (aa20-655), ZJ2013-CoV S1 (aa20-665)... The C-terminus was fused with a twin-strep tag and a 3×Flag tag for expression; SARS-CoV-2 RBD (aa331-524), MERS-CoV The C-terminus of RBD (aa377-588) was fused with the hFC tag, twin-strep tag, and 3×Flag tag (WSHPQFEKGGGSGGGSGGSAWSHPQFEK-GGGRS-DYKDHDGDYKDHDIDYKDDDDK).

[0056] (3) Packaging of pseudoviruses and lentiviruses

[0057] Based on pVSV-eGFP-dG (addgene, Catalog #31842), a firefly luciferase (fLuc) reporter gene was introduced between the M gene and the L gene, and the VSV-dG-GFP-fLuc tool virus carrying the dual reporter gene was successfully rescued by reverse genetics. When the 293T cells grew to a confluence of more than 90%, they were suspended and transfected with the corresponding coronavirus S protein, and fresh culture medium was replaced after 4-6 hours. 36 hours after transfection, the culture medium was replaced after 4 hours of infection with the VSV-dG-GFP-fLuc tool virus, and VSV G monoclonal antibody I1 was added to neutralize the residual VSV tool virus. After 24 hours, the culture supernatant and the corresponding coronavirus pseudovirus were collected by centrifugation and stored at -80°C.

[0058] Lentiviral packaging. 293T cells were transfected with the lentiviral vector (pLVX-EF1a-Puro, Genewiz), the third-generation packaging plasmid pMD2G (Addgene catalog no. 12259), and psPAX2 (Addgene catalog no. 12260) at a ratio of 2:1:1 using Lip2000 transfection reagent (Biosharp, BL623B). The medium was changed after 4-6 hours. The culture supernatant was collected by centrifugation at 24 and 48 hours and stored at -80°C.

[0059] (4) Pseudovirus invasion experiment

[0060] 24 hours after transfection, cells or stably transfected cells were digested with trypsin and incubated with the corresponding coronavirus pseudovirus (1×10 5 TCID 50 / well, or the same genome equivalent) were mixed and plated in 96-well plates (5×10 4 After 16 hours of infection, GFP images were collected using a fluorescence microscope (Mshot, MI52-N), and intracellular luciferase activity was measured using the Bright-Glo Luciferase Assay Kit (Promega, E2620). The data were collected using a microplate reader (SpectraMax iD3, Molecular Devices) or a GloMax 20 / 20 Luminometer (Promega).

[0061] (5) Pseudovirus neutralization experiment

[0062] For the neutralization test of serum from recovered patients or vaccine recipients, pseudovirus (2×10 5 TCID 50 / well) and 50-fold diluted serum were incubated at 37°C for 30 min. For the neutralization experiment of monoclonal antibodies, the antibodies were serially diluted and each dilution was incubated with pseudovirus (2×105 TCID 50 / well) were incubated at 37°C for 30 min. Then, the cells were mixed and plated in a 96-well plate (2×10 4 16 h after infection, luciferase activity was measured using a Bright-Glo Luciferase Assay Kit (Promega). Data were collected using a microplate reader (SpectraMax iD3, Molecular Devices).

[0063] (6) Western-blot

[0064] The cell culture supernatant was discarded, the cells were washed once with PBS, and incubated with RIPA lysis buffer at 4°C for 15 minutes. The lysate mixture was transferred to a 1.5 mL EP tube and centrifuged at 12,000 rpm at 4°C for 10 minutes. The supernatant was removed, 5× SDS loading buffer was added, and the cells were boiled at 95°C for 5 minutes for sample preparation. After SDS-PAGE electrophoresis, the membrane was transferred to a PVDF membrane (GenScript), blocked with 5% skim milk at room temperature for 1 hour, and incubated with the primary antibody (1:10,000 dilution) at 4°C overnight. The next day, the membrane was washed four times with PBST (5 minutes each), incubated with the secondary antibody (1:10,000 dilution) at room temperature for 1 hour, and then washed four times with PBST (5 minutes each). The membrane was exposed to the colorimetric solution.

[0065] (7) Flow cytometry determination of the binding ability of various nanobodies to SARS2-RBD

[0066] 25 artificial receptors constructed based on the SARS-CoV RBD nanobody were transiently transfected into 293T cells. 24 hours after transfection, the cells were treated with cold 5mM EDTA solution to disperse them into single cells. Recombinant SARS-CoV-RBD fused to mFC (expressed using a mammalian expression system) was added to allow the viral RBD to bind to the nanobody on the artificial receptor. The cells were incubated on ice for 1 hour, washed three times with PBS, and centrifuged at 1000 rpm for 10 minutes each time. The wash solution was discarded. The artificial receptors were labeled with anti-hFC Alex 488, and the SARS-CoV-RBD was labeled with anti-mFC Alex 647. After a one-hour incubation, the cells were washed three times with PBS and analyzed on an immunoassay. The ratio of the mean fluorescence intensity of Alex 647-positive cells to the mean fluorescence intensity of Alex 488-positive cells represented the binding ability of the nanobody to the viral RBD.

[0067] (8) Immunofluorescence

[0068] The transfected cells were fixed with paraformaldehyde for 10 minutes at room temperature, permeabilized with 0.1% Triten-X100 for 10 minutes at room temperature, blocked with 1% BSA at 37°C for 1 hour, and incubated with SARS2-RBD-mFC at 37°C for 1 hour. After washing three times with PBS, the SARS2-RBD protein was labeled with anti-mFC 594 antibody, incubated at 37°C for 1 hour, and then washed three times with PBS. Hoechst 33342 (diluted 1:5000 in HBSS) was added, the nuclei were stained, and the cells were photographed.

[0069] (9) Reverse genetics rescue of VSV with HKU3 S capable of multiple rounds of replication

[0070] The pVSV-eGFP-HKU3 S plasmid was constructed by inserting the HKU3 S gene between the M and L genes using the Mlu I and Not I restriction sites based on pVSV-eGFP-dG (Addgene, Catalog #31842). VVT7-infected BHK21 cells were then transfected with pVSV N, pVSVP, pVSV G, pVSV L, and pVSV-eGFP-HKU3 S at a ratio of 3:5:8:1:5. The medium was changed 4-6 hours after transfection, and the supernatant was collected by centrifugation after 48 hours. VVT7 was filtered through a 0.22 μM filter and then infected into a susceptible HKU3 cell line (Caco-2 stably expressing the S2Nb27 artificial receptor). After 48 hours, the culture supernatant was collected to obtain the VSV-eGFP-HKU3S virus.

[0071] (10) Cell membrane fusion experiment

[0072] Cell membrane fusion experiments were performed using bimolecular complementation technology on BHK-21 cells stably expressing different receptors. The cells were divided into two aliquots, one of which was transfected with a plasmid containing the viral S protein and an N-terminal truncated version of RLuc (amino acids 1-155) fused to an N-terminal truncated version of GFP (amino acids 1-157), while the other aliquot was transfected with a plasmid containing the viral S protein and a C-terminal truncated version of GFP (amino acids 158-231) fused to an C-terminal truncated version of RLuc (amino acids 156-311). Twelve hours after transfection, the two aliquots were digested and mixed, then plated in 96-well plates (8 × 10 4 24 hours after transfection, Hoechst 33342 (1:5000 dilution in HBSS) was added to stain the nuclei at 37°C for 30 min and the cells were photographed. Live cell luciferase substrate (EnduRen live cell substrate, Promega E6481) was added and incubated at 37°C for 1 hour. Detection and data collection were performed using a GloMax 20 / 20 Luminometer (Promega).

[0073] (11) Protein expression and purification:

[0074] The RBD (S1) proteins of HKU3, 229E, RmYN02, HUK5, SARS-CoV-2, and MERS carry a CD5 signal peptide (MPMGSLQPLATLYLLGMLVASVL) at the N-terminus and a twin-strep tag and a 3×Flag tag (WSHPQFEKGGGSGGGSGGSAWSHPQFEK-GGGRS-DYKDHDGDYKDHDIDYKDDDDK) at the C-terminus. After being cloned into the pCAGGS vector, 293T cells were transfected using GeneTwin transfection reagent (Biomed, TG101-01). Four hours after transfection, the supernatant was replaced with fresh SMM 293-TII serum-free medium (Sino Biological, M293TII), and the supernatant was collected every two days for a total of 5-6 days. Proteins were captured using Strep-Tactin XT 4Flow high capacity resin (IBA, 2-5030-002). Unbound proteins were washed away with buffer W (100mM Tris / HCl, pH 8.0, 150mM NaCl, 1mM EDTA) and eluted with buffer BXT (100mM Tris / HCl, pH 8.0, 150mM NaCl, 1mM EDTA, 50mM biotin). The eluted proteins were exchanged for PBS using a concentrator tube, quantified by BCA assay, and aliquoted and stored frozen at -80°C. MHV-A59-NTD (S1) protein fused with a twin-strep tag and a 3×Flag tag was expressed in sf9 insect cells using a bac-to-bac expression system and purified using the same method as above.

[0075] (12) Screening and identification of nanoantibodies

[0076] Nanobodies specific for different viral spikes were screened through phage display. The phage nanobody library used was purchased from Chengdu Apec. In the first round of panning, MagStrep "type 3" XT beads (IBA) were loaded with antigen, and bound phage were eluted with 50mM biotin. In the second round of panning, MagStrep "type 3" XT beads or immunotubes were loaded with antigen, and phage in the immunotubes were eluted with 100mM triethanolamine. After two rounds of panning, 90 single clones were selected for ELISA analysis.

[0077] Plates were coated (Corning) with 2 μg / mL target antigen (dissolved in PBS) at 4°C for 12 h (overnight coating is also possible), washed three times with 0.1% PBST, blocked with 3% skim milk (PBS) for 2 h, and washed three times with 0.1% PBST. Phage supernatant was then allowed to bind for 1 h, washed five times with 0.1% PBST, and anti-M13 antibody (Sino Biological) was allowed to bind for 1 h. The plates were washed seven times with 0.1% PBST, and positive clones were detected using a colorimetric solution.

[0078] Example 1 Functional analysis of ACE2 receptor

[0079] We decomposed ACE2 into different structural components based on function and structure, including the head domain, neck domain, spacer domain, and transmembrane domain, to investigate the ACE2 motifs and domains necessary for the efficient entry of SARS-CoV-2 ( Figure 2 A). Cryo-electron microscopy (Cryo-EM) structures show that the interaction of SARS-CoV-2 RBD is participated by the α1 and α2 helices of the ACE2 protease domain (also known as the head domain) and the β3 and β4 sheet linker regions. However, the importance of other components for receptor function is unclear. We first tried to see whether the virus receptor function could be maintained after replacing the ACE2 head and neck domains with a variety of computer-aided designed small virus binding proteins (peptides of about 60 amino acids). We constructed 10 ACE2 head domain and head and neck domain replacement chimeras (LCB1-8 and AHB1-2, amino acid sequences are shown in Table 1) and tested their ability to support SARS-CoV-2 RBD binding and pseudovirus entry. The results showed that these small binding proteins can replace the head and neck domain to varying degrees, among which LCB1 has the highest replacement efficiency. Subsequently, we designed 10 chimeric proteins LCB1 carrying or partially carrying ACE2 sequences. The ACE2 sequences were sequentially truncated (Del 1-Del 5, amino acid sequences are shown in Table 1) or replaced by non-ACE2 sequences (Rep 1-Rep 5, amino acid sequences are shown in Table 1), such as the human IgG Fc domain, transmembrane motifs and epitope tags ( Figure 2 A). The expression levels of all chimeras were detectable, with some small molecular weight proteins expressed weakly ( Figure 2 B). Effective binding of SARS-CoV-2 RBD-hFC fusion protein can be detected by flow cytometry or immunofluorescence on 293T cells stably expressing these receptors ( Figure 2 C). Except for the smallest construct D5, most chimeric proteins retained the ability to support SARS-CoV-2 pseudovirus entry to some extent, as shown by the efficiency of GFP after cell infection ( Figure 2 D) and luciferase ( Figure 2E) intensity. The structure of the smallest functional structure D2 consists of only five functional motifs / domains: a cell surface localization signal peptide (SP), a virus binding domain (VBD), a spacer sequence (spacer) and a transmembrane domain (TMD), as well as a C-terminal tag. These motifs / domains constitute a type I transmembrane chimeric protein of only 110 residues. R3 does not carry a sequence derived from ACE2, and its highly efficient receptor function indicates that the virus receptor function can still be achieved after all sequences of ACE2 are replaced. In addition, R5, which carries the DPP4 transmembrane domain, has a topological structure of a type II transmembrane protein and can also effectively support viral invasion ( Figure 2 A). We further inserted an EPM sequence into the C-terminus of the receptor to stabilize the cell surface and expression distribution of the receptor, thereby enhancing its ability to mediate viral invasion ( Figure 2 FG).

[0080] We noted the importance of the length of the spacer connecting the VBD and TMD, as constructs with an IgG Fc domain supported higher invasion efficiency than constructs with a short or no spacer. Therefore, we tested the effect of spacers with different immunoglobulin domains and the presence or absence of dimerization on invasion efficiency, including hinge-only constructs and constructs containing 1-3 immunoglobulin domains (LCB1 0*IgG, LCB1 1*IgG, LCB1 2*IgG, LCB1 3*IgG, LCB1 2*IgG (no dimer)) (amino acid sequences are shown in Table 1) ( Figure 2 The results showed that at least two IgG Fc domains are necessary, and dimerization does not seem to contribute significantly to receptor function, as Fc domains with mutations in the dimerization sequence (monomers) can still effectively mediate infection.

[0081] The sequences involved in Example 1 are shown in Table 1 below.

[0082] Table 1

[0083]

[0084]

[0085]

[0086]

[0087]

[0088] Example 2 Effect of VBD on Receptor Function

[0089] To elucidate what type of VBD can support viral binding and entry into cells, we used Figure 2The CVR-R1 in A serves as a prototype framework for the design of custom viral receptors (CVRs). We explored other VBD types and formats besides virtually designed small peptides, such as nanobodies (Nb), single-chain variable domains (ScFv), and antigen-binding fragments (Fab). Figure 3 As shown in Figure A, we compared eight CVRs that recognize SARS-CoV-2, carrying two small peptides (LCB1-hFC, LCB2-hFC), Nbs (S2Nb17-hFC, S2Nb24-hFC), ScFv (SCFV-S2H14-hFC, SCFV-S2X35-hFC) and Fabs (CB6-VH-hFC, CB6-VL, REGN-VH-hFC, REGN-VL). The amino acid sequences are shown in Table 2. The results showed that these antibody fragments can all be used as VBDs for CVR design. Nanobodies have the advantages of small size, compact folding, single-chain structure, and suitability for bio-panning, which are superior to other VBD types ( Figure 3 B).

[0090] To explore the relationship between the binding affinity, neutralization activity, and entry-supporting ability of different VBDs, we tested 25 Nbs targeting the SARS-CoV-2 RBD region (S2Nb1-25, amino acid sequences are shown in Table 2 ), which were combined with hFc and Figure 2 The structural framework of CVR-R in A was fused. Flow cytometry was used to detect the binding efficiency of SARS2-CoV-2-RBD to 293T expressing Nbs-CVR; the neutralization activity of nanobody-hfc fusion protein on 293T-ACE2 cells was determined by SARS2-CoV-2 pseudotype neutralization experiment; 293T cells expressing different nanobody-CVRs were infected with pseudoviruses to evaluate their ability to support viral invasion. Our results show that the ability of VBD to support viral invasion does not correspond to their neutralizing activity or virus binding ability. Nevertheless, all CVRs that support efficient invasion showed strong RBD binding ability ( Figure 3 C).

[0091] Next, we explored the association of the binding epitope with the VBD for mediating efficient invasion. We generated CVRs based on 17 well-characterized SARS-CoV-2 neutralizing mAbs, including 12 CTD-binding mAbs (six different RBD epitopes; SCFV-CB6, SCFV-S2E12, SCFV-REGN, SCFV-S2H14, SCFV-CoV555, SCFV-S2H13, SCFV-CoV1404, SCFV-S309, SCFV-S2H97, SCFV-BD-744, SCFV-S2X35, SCFV-S2X259), two NTD-binding mAbs (SCFV-S2L20, SCFV-S2M28), and three S2-binding mAbs (SCFV-S2P6, SCFV-B6, SCFV-76E1) (amino acid sequences are shown in Table 2), which recognize specific neutralizing epitopes. The results showed that the VBD recognized by CTD was superior to the VBDs recognizing NTD and S2 in supporting viral invasion. However, the acceptable CTD epitopes are not limited to the receptor binding surface involved in ACE2, such as epitope V, a relatively conserved broad-spectrum neutralizing epitope ( Figure 3 D).

[0092] Table 2

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105] Example 3 CVR is similar to the natural receptor-mediated invasion pathway

[0106] To test whether CVR supports entry of coronaviruses with different receptors, we prepared antibodies that can mediate entry of MERS-CoV and 229E-CoV (amino acid sequences are shown in Table 3), whose natural receptors are DPP4, APN, and CEACAM1a, respectively. VHH library), the optimal CVR selected can support the efficient entry of multiple viruses ( Figure 4 A).

[0107] By testing the entry of five different coronavirus pseudoviruses, SARS-CoV, SARS-CoV-2, MERS-CoV, NL63-CoV, and 229E-CoV, into 293T cells, the specificity of CVRs and ACE2-mediated entry into cells was compared. As expected, CVRs specifically supported the entry of their target viruses but not other viruses, while S2Nb24 could cross-recognize a conserved epitope in SARS-CoV and a SARS-related virus, thus also supporting SARS-CoV infection ( Figure 4 A). In addition, CVRs can also mediate the entry of SARS-CoV-2 and MERS-CoV into various cells such as BHK-21, 293T, A549 and Tb 1lu. The above results show that CVRs can support efficient and specific viral entry in cells of different host and tissue origins similar to natural receptors ( Figure 4 B).

[0108] We next investigated whether the CVR-mediated entry pathway resembles infection via the natural receptor. Based on the dual cleavage protein (DSP) complementation assay ( Figure 4 C), we first demonstrated that expression of CVRs can induce cell-cell membrane fusion as effectively as their natural receptors. 12-24 hours after transfection, we observed the expression of GFP ( Figure 4 D) and Ranilla luciferase ( Figure 4 We then assessed whether CVR-mediated invasion could be similarly disrupted by inhibitors of different viral entry steps, such as protease cleavage, fusion, and endocytic vesicle acidification ( Figure 4F). The results of inhibition experiments based on cells expressing CVR and ACE2 were similar, indicating that the CVR infection system can be used to screen and evaluate inhibitors of viruses that lack viable infection models. In addition, we compared the receptor functions of CVR with those of several reported SARS-CoV-2 invasion factors or alternative receptors / co-receptors: SCARB1, NRP1, KREMEN1, CD147 (Basigin), ASGPR1, AXL, CLEC4M (CD209L). The results showed that in the absence of ACE2 expression, receptors other than ACE2 could not support viral infection as effectively as CVR (LCB1) ( Figure 4 G).

[0109] Table 3

[0110]

[0111]

[0112] Example 4 CVR-based antibody neutralization assay

[0113] Next, we explored whether the CVR infection system could be used to evaluate neutralizing antibodies, especially antisera and broadly neutralizing antibodies. We first compared the inhibitory activity of SARS-CoV-2 protective antisera against 293T cells expressing ACE2, LCB1-CVR (LCB1-hFC in Table 2) and S2Nb24-CVR (S2Nb24 in Table 2), which were collected from individuals recovering from COVID-19 and vaccinated. LCB1 recognizes a very similar epitope of the receptor binding motif (RBM) on the viral spike protein as ACE2, while S2Nb24 binds to an epitope that is different from the classic RBM region. Overall, the serum neutralization data based on the three infection systems showed similar inhibition spectra, while S2Nb24-CVR was slightly different. This result suggests that the CVR-based system can be used to evaluate the effectiveness of humoral immunity. Although the differences in the binding interface between artificial receptors and natural receptors may affect the sensitivity and accuracy of the test to some extent, the overall efficacy is less affected when evaluating polyclonal antibodies in antisera ( Figure 5 AB).

[0114] Next, we attempted to generate functional CVRs for eight coronaviruses that do not use any known protein receptors (HKU31, HKU5, HKU1, HKU9, ZJ2013, HKU3, RmYN02, and ZC45). Figure 5 C). We screened a series of nanobodies that specifically bind to the putative RBD region (CTD) of coronaviruses and tested the CVRs ( Figure 5B). We ultimately identified seven CVRs (amino acid sequences shown in Table 4) that efficiently supported the entry of eight coronaviruses, increasing entry by 10 compared to control cells without receptor expression. 3 ~10 6 times ( Figure 5 D) These results indicate that CVRs can not only mimic known coronavirus receptors, but more importantly, these CVRs can also support the entry of coronaviruses with unknown receptors into cells.

[0115] In addition, we further investigated the breadth of the cross-reactivity spectrum of broadly neutralizing antibodies against beta-coronaviruses, such as antibodies that recognize the class IV neutralizing epitope RBD (S2H97), the conserved stem helix of the fusion machinery (S2P6,B6), or the conserved fusion peptide (76E1). We tested the neutralizing efficacy of antibodies against a series of coronaviruses without known receptors, including HKU1, HKU3, HKU5, HKU9, HKU31, ZJ2013, ZC45, and RmYN02 ( Figure 5 D). The results showed that S2H97 effectively neutralized HKU3 entry, but had poor activity against ZC45 and RmYN02, both of the B lineage, consistent with the conservation of the recognized epitope sequences. Antibody 76E1, targeting the more conserved fusion peptide region, showed some inhibitory activity against viruses other than HKU1. Antibodies S2P6 and B6, targeting the stem-helix region, exhibited varying degrees of broad-spectrum neutralizing activity against different viruses. These results suggest that novel CVR-based infection systems are powerful tools for evaluating vaccines and neutralizing antibodies, such as antisera and broadly neutralizing antibodies targeting specific regions.

[0116] Table 4

[0117]

[0118]

[0119] Example 5 Artificial Receptors Support Proliferative Infection of Coronaviruses

[0120] We further tested whether CVRs can mediate infection with real viruses. We first compared the infection of SARS-CoV-2 (corresponding receptors are LCB1-hFC, S2Nb24, and S2Nb17 in Table 2), 229E-CoV (corresponding receptors are 2A5, 2C5, and 4H5 in Table 3), and MHV-A59 (corresponding receptors are 3B8 and 3H6 in Table 5) in 293T cells expressing their natural receptors and optimized CVRs, respectively. Immunofluorescence of viral N antigens showed that CVRs can support real coronavirus infection to the same extent or more strongly than natural receptors ( Figure 6 A).

[0121] Table 5

[0122]

[0123] Example 6 Artificial Receptors Support the Proliferation of Viruses Related to the Lack of Infection Model

[0124] The lack of infection models is a major bottleneck in the study of coronaviruses with potential biosafety risks. However, it is difficult to obtain these viruses due to the lack of susceptible cell isolation and culture. A unique advantage of the CVR-based infection system is that it can support infection with viruses that lack conventional infection systems. In order to demonstrate this advantage and for biosafety reasons, and to avoid directly rescuing highly pathogenic coronaviruses, the present invention constructed a chimeric virus based on a DNA fragment of the VSV viral genome sequence with low biological risk, in which the G protein was replaced by the HKU3-CoV spike protein sequence ( Figure 6 B), the GFP gene sequence was inserted between the HKU3 Spike gene and the L gene. Using engineered CaCo-2 cells expressing HKU3-specific CVRs (S2Nb27 in Table 2 and 9A3 in Table 4), we successfully rescued HKU3-based chimeric viruses ( Figure 6 C). In cells expressing HKU3-CVR, we can effectively amplify the rescued virus, and the proportion of positive cells gradually increases during the infection and amplification process ( Figure 6 D). We also observed an increase in viral RNA copy number in infected cells expressing HKU3-specific CVRs ( Figure 6 F). This virus strain can be stably propagated for at least ten generations, and the deletion of the GFP coding sequence can be observed during long-term propagation ( Figure 6 E and Figure 6 F), resulting in a gradual decrease in the GFP signal, but based on the staining of the HKU3 spike protein, the virus with the deletion mutation is still replicating efficiently, and the sequence of the S protein has not changed significantly during the replication process. Using the chimeric virus with the HKU3 outer membrane carrying GFP, we evaluated the inhibitory effects of a series of viral entry inhibitors and neutralizing antibodies on the HKU3 outer membrane protein chimeric virus. These results show that the new infection system based on CVR has outstanding potential in rescuing, isolating, amplifying and studying coronaviruses that are difficult to culture in traditional cell culture systems.

[0125] Example 7: Transgenic mice based on artificial receptors support infection with the new coronavirus

[0126] We constructed transgenic mice expressing artificial viral receptors and established an animal model of coronavirus infection. Figure 7As shown in a, the gene of the SARS-CoV-2 artificial virus receptor LCB1 (LCB1-hFC in Table 2) was codon-optimized and the mouse ACE2 reading frame was replaced in situ using CRISPER-Cas9 gene editing technology to construct transgenic mice expressing the artificial virus receptor. We first verified the expression of LCB1 in lung tissue. Figure 7 b Western blot. Three groups were set up, namely wild type group, hACE2 in situ expression group and artificial virus receptor LCB1 expression group. Figure 7 c infection process, SARS-CoV-2 virus was inoculated intranasally on day 0 (2x10 7 PFU / mL). Mice were dissected 5 days after infection in the hACE2 group and 3, 5, and 7 days after infection in the LCB1 group, and RNA was extracted from lung tissues. SARS-CoV-2 virus infection in the lungs was then detected by qPCR (n=3). Figure 7 As shown in d, compared with the wild-type group, hACE2 successfully detected viral genome copies on the 5th day, and the LCB1 group successfully detected the virus 3 days after infection, and also detected the presence of the virus on the 5th and 7th days. Figure 7 e. As shown in the figure, we observed significant inflammatory cell infiltration in the lung tissue sections of all groups except the wild-type group. In summary, we constructed transgenic mice expressing artificial viral receptors and demonstrated through viral infection experiments that these mice acquired susceptibility to the new coronavirus and exhibited lung viral infection and inflammatory responses comparable to those of hACE2 transgenic mice, providing a new animal infection model for coronavirus that is independent of native receptor expression.

Claims

1. A customized artificial viral receptor, characterized by: The customized artificial viral receptor is composed of modules A, B, C, D, E, F, G, and H from the amino terminus to the carboxyl terminus. The module A is a signal peptide of type I membrane protein, which is used to display the protein on the cell membrane surface; the module B is a customized virus binding domain, which is used to capture the target virus; the module C is a connector 1; the module D is a spacer region, which is used to form the skeleton of the customized artificial virus receptor; the module E is a connector 2; the module F is a transmembrane domain, including a single transmembrane helix of type I membrane protein or a single transmembrane helix of type II membrane protein, and the cell surface display of the artificial virus receptor is mainly achieved by module F; the module G is an intracellular domain, which has the function of maintaining the stability of the artificial virus receptor; the module H is a tag protein.

2. The customized artificial viral receptor according to claim 1, characterized in that: The customized artificial viral receptor does not contain at least one of modules C, D, and G.

3. The customized artificial viral receptor according to claim 1, wherein: The module B is obtained by a method including known antibody interception, computer-aided design and / or nanobody panning.

4. The customized artificial viral receptor according to claim 1, wherein: The amino acid sequence of module A is selected from SEQ ID NO.1; The amino acid sequence of module D is selected from SEQ ID NO. 3-7; The amino acid sequence of module F is selected from SEQ ID NO.8-11; The amino acid sequence of module G is selected from SEQ ID NO.12-16; The amino acid sequence of module H is selected from SEQ ID NO.

17.

5. The customized artificial viral receptor according to claim 1, wherein: The amino acid sequence of module C is: TGGS, GSGS or GTGS; The amino acid sequence of module E is: GSGT.

6. Use of the customized artificial viral receptor according to any one of claims 1 to 5 in establishing a coronavirus infection model.

7. Use of the customized artificial virus receptor according to any one of claims 1 to 5 in the evaluation of neutralizing antibodies, characterized in that: The application is achieved through the following steps: establishing a susceptible cell line that overexpresses a customized artificial viral receptor by plasmid transfection, lentiviral packaging and then transduction; incubating the neutralizing antibody with the coronavirus, and then adding it to the culture medium and co-culturing it with the susceptible cell line; and evaluating the neutralizing ability of the neutralizing antibody by the infection effect.

8. Use of the customized artificial viral receptor according to any one of claims 1 to 5 in supporting coronavirus infection and proliferation.

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