Monoclonal antibodies targeting the novel coronavirus spike protein and their applications

Abstract

The present application discloses a single-chain antibody against the S protein of the new coronavirus, which is obtained by screening through phage display library technology. The monoclonal antibody S-pro-ab-750 of the present application has been identified by ELISA to specifically bind to the S protein of the new coronavirus SARS-CoV-2. The virus inhibition rate of the original concentration of S-pro-ab-750 antibody is 84%, and 25μL of antibody can neutralize 205TCID50 new coronavirus pseudovirus. The present application uses the spleen cells of immunized mice to construct a phage single-chain antibody display library, expanding research and diagnosis resources. The mouse-derived single-chain antibody with high binding affinity to the S protein of the new coronavirus screened in the present application can be used for virus detection and diagnosis.

Description

[0001] This application is a divisional application of the invention patent entitled "A monoclonal antibody against the spike protein of the new coronavirus and its application", application number: "202210183112.X", and the application date of the parent case is February 27, 2022. Technical Field

[0002] This application relates to the field of biomedicine, and in particular to monoclonal antibodies and applications against the novel coronavirus. Background Art

[0003] The spike protein (also known as S protein or S protein) on the envelope of the novel coronavirus is the most important protein in the process of infecting cells. The spike protein consists of two subunits, S1 and S2. The receptor binding domain (RBD) in S1 interacts with the angiotensin-converting enzyme II (ACE2) molecule, determining the host range and specificity of the virus; S2 contains the basic elements required for the membrane fusion process, enabling the fusion of virus and cells. The development of neutralizing antibodies against the spike protein is currently an effective means of combating the novel coronavirus. Single-chain antibodies (scFv) are composed of the variable regions of the heavy chain and light chain of an antibody, connected by a peptide chain. Due to their small molecular weight, strong penetrating power, and short half-life, they play an important role and have broad application prospects in the clinical diagnosis, treatment, and prevention of diseases.

[0004] Chinese patent CN 112940111A utilizes a nanoantibody library to target the Spike S1+S2 ECD of the 2019-nCoV, generating a nanoantibody against the novel coronavirus through four rounds of panning. ELISA testing demonstrates high affinity for both the Spike S1+S2 ECD and Spike RBD targets of the 2019-nCoV. Chinese patent CN 113402603A utilizes phage display technology to screen antibody libraries targeting the novel coronavirus S1 protein. Through four rounds of solid-phase panning, the patent discloses an avian monoclonal single-chain antibody (scFv) against the novel coronavirus S1 protein. This scFv specifically binds to the S1 subunit of the novel coronavirus spike protein and exhibits limited viral recognition. Chinese patent CN 113264998A discloses a humanized monoclonal antibody against the novel coronavirus S1 protein. This patent constructs a phage display scFv library using B cells isolated from the peripheral blood of recovered COVID-19 patients. The library was screened against the SARS-CoV-2 S1 protein, and the monoclonal antibodies obtained after three rounds of panning were identified. Flow cytometry analysis revealed strong affinity for the S1 protein and strong neutralizing activity against the SARS-CoV-2 pseudovirus.

[0005] Although many antibodies against the new coronavirus have been screened, in the face of the continuous mutation of the virus and the many problems encountered in actual product development, we need broader antibody library resources and antibodies with higher affinity to meet the challenges. Summary of the Invention

[0006] In response to the problems of the prior art, the present application provides a monoclonal antibody against the novel coronavirus S protein and its preparation method and application.

[0007] The first aspect of the present application is to provide a monoclonal antibody against the novel coronavirus S protein, which can specifically bind to the novel coronavirus spike protein (for example, monoclonal antibodies named S-pro-ab-750, S-pro-ab-753).

[0008] By technical means well known to those skilled in the art, for example, the amino acid sequence of the CDR region of the above antibody sequence is analyzed by the VBASE2 database. It will be understood by those skilled in the art that the CDR region of an antibody is responsible for the binding specificity of the antibody to the antigen. In the case of known antibody heavy chain and light chain variable region sequences, there are currently several methods for determining the CDR region of an antibody, including the Kabat, IMGT, Chothia and AbM numbering systems. However, the application of each definition of the CDR of an antibody or its variants will be within the scope of the terms defined and used herein. Given the variable region amino acid sequence of the antibody, those skilled in the art can generally determine which residues comprise a specific CDR, without relying on any experimental data outside the sequence itself. The following lists the appropriate amino acid residues of the CDRs defined by the IMGT CDR numbering system as a comparison.

[0009] The heavy chain variable region (VH) of the antibody S-pro-ab-750 is:

[0010] Heavy chain complementarity determining region 1 (H-CDR1): GYSFTTYW (SEQ ID NO: 2)

[0011] Heavy chain complementarity determining region 2 (H-CDR2): IDPSDSVI (SEQ ID NO: 4)

[0012] Heavy chain complementarity determining region 3 (H-CDR3): ARLDSTGYPTWFLY (SEQ ID NO: 6)

[0013] The light chain variable region (VL) of the antibody S-pro-ab-750 is:

[0014] Light chain complementarity determining region 1 (L-CDR1): QSVDYNGISY (SEQ ID NO: 9)

[0015] Light chain complementarity determining region 2 (L-CDR2): TAS (SEQ ID NO: 11)

[0016] Light chain complementarity determining region 3 (L-CDR3): QQNIEDPLT (SEQ ID NO: 13)

[0017] The heavy chain variable region (VH) of the antibody S-pro-ab-753 is:

[0018] Heavy chain complementarity determining region 1 (H-CDR1): GFNIKDTY (SEQ ID NO: 16)

[0019] Heavy chain complementarity determining region 2 (H-CDR2): IDPANGNT (SEQ ID NO: 18)

[0020] Heavy chain complementarity determining region 3 (H-CDR3): ASPRALLLRYYAMDY (SEQ ID NO: 20)

[0021] The light chain variable region (VL) of the antibody S-pro-ab-753 is:

[0022] Light chain complementarity determining region 1 (L-CDR1): QSVDYDGDSY (SEQ ID NO: 23)

[0023] Light chain complementarity determining region 2 (L-CDR2): AAS (SEQ ID NO: 25)

[0024] Light chain complementarity determining region 3 (L-CDR3): QQSNEDPFT (SEQ ID NO: 27)

[0025] The framework region H-FR of the heavy chain variable region and the framework region L-FR of the light chain variable region of S-pro-ab-750 are as follows: the sequence of H-FR1 is shown in SEQ NO: 1, the sequence of H-FR2 is shown in SEQ NO: 3, the sequence of H-FR3 is shown in SEQ NO: 5, the sequence of H-FR4 is shown in SEQ NO: 7, the sequence of L-FR1 is shown in SEQ NO: 8, the sequence of H-FR2 is shown in SEQ NO: 10, the sequence of L-FR3 is shown in SEQ NO: 12, and the sequence of L-FR4 is shown in SEQ NO: 14.

[0026] H-FR1:QVQLQQSGPQLVRPGASVKISCKTS(SEQ ID NO:1)

[0027] H-FR2:MHWVKQRPGQGLEWIGM(SEQ ID NO:3)

[0028] H-FR3:RLNQKFKDKATLTVNKSSSTAYMQLSSPPTSEDSAVYYC(SEQ ID NO:5)

[0029] H-FR4:WGQGTLVTVSS(SEQ ID NO:7)

[0030] L-FR1:DIVLTQSPASLAVSLGQRATIFCRAS(SEQ ID NO:8)

[0031] L-FR2:IHWFQQKPGQPPKLLIF(SEQ ID NO:10)

[0032] L-FR3:NLESGVPARFSGSGSSESDFTLTIDPVEADDAATYYC(SEQ ID NO:12)

[0033] L-FR4:FGAGTKLELK(SEQ ID NO:14)

[0034] The framework region H-FR of the heavy chain variable region and the framework region L-FR of the light chain variable region of S-pro-ab-753 are as follows: the sequence of H-FR1 is shown in SEQ NO:15, the sequence of H-FR2 is shown in SEQ NO:17, the sequence of H-FR3 is shown in SEQ NO:19, the sequence of H-FR4 is shown in SEQ NO:21, the sequence of L-FR1 is shown in SEQ NO:22, the sequence of H-FR2 is shown in SEQ NO:24, the sequence of L-FR3 is shown in SEQ NO:26, and the sequence of L-FR4 is shown in SEQ NO:28.

[0035] H-FR1:QVQLQQSGAELVKPGASVKLSCTAS(SEQ ID NO:15)

[0036] H-FR2:IHWVKQRPEQGLEWIGR(SEQ ID NO:17)

[0037] H-FR3:KYDPNFQGKATITADTSSNTAYLHLSSLTSEDTAVYYC(SEQ ID NO:19)

[0038] H-FR4:WGQGTSVTVSS(SEQ ID NO:21)

[0039] L-FR1:DIVLTQSPASLAVSLGQRATISCKAS(SEQ ID NO:22)

[0040] L-FR2:MNWYQQKPGQPPKLLIY(SEQ ID NO:24)

[0041] L-FR3:NLESGIPARFSGSGSGTDFTLTVNPVEADDVATYYC(SEQ ID NO:26)

[0042] L-FR4: FGSGTKLEIK(SEQ ID NO:28)

[0043] In some embodiments, the antibody comprises: (1) a heavy chain variable region hypervariable region CDR. The HCDR comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO. 2 or 16; HCDR2 comprises the amino acid sequence shown in SEQ ID NO. 4 or 18; and HCDR3 comprises the amino acid sequence shown in SEQ ID NO. 6 or 20. (2) a light chain variable region hypervariable region CDR. The LCDR comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO. 9 or 23; LCDR2 comprises the amino acid sequence shown in SEQ ID NO. 11 or 25; and LCDR3 comprises the amino acid sequence shown in SEQ ID NO. 13 or 27.

[0044] In some embodiments, the antibody further comprises a framework region FR of a heavy chain variable region and a framework region FR of a light chain variable region. The framework region FR of the heavy chain variable region comprises HFR1, HFR2, HFR3, and HFR4, wherein HFR1 comprises the amino acid sequence of SEQ ID NO.1 or 15; HFR2 comprises the amino acid sequence of SEQ ID NO.3 or 17; HFR3 comprises the amino acid sequence of SEQ ID NO.5 or 19; and HFR4 comprises the amino acid sequence of SEQ ID NO.7 or 21. The framework region FR of the light chain variable region comprises LFR1, LFR2, LFR3, and LFR4, wherein LFR1 comprises the amino acid sequence of SEQ ID NO.8 or 22; LFR2 comprises the amino acid sequence of SEQ ID NO.10 or 24; LFR3 comprises the amino acid sequence of SEQ ID NO.12 or 26; and LFR4 comprises the amino acid sequence of SEQ ID NO.14 or 28.

[0045] In some embodiments, the antibody comprises: (1) a heavy chain variable region comprising or consisting of the amino acid sequence shown in SEQ ID NO: 29 or 31, and (2) a light chain variable region comprising or consisting of the amino acid sequence shown in SEQ ID NO: 30 or 32.

[0046] The second aspect of the present application is to provide an isolated nucleic acid molecule encoding any one of the above-mentioned monoclonal antibodies.

[0047] The third aspect of the present application is to provide an expression vector comprising the above-mentioned nucleic acid molecule.

[0048] The expression vector in this application refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a murine scFv can be inserted and the scFv can be expressed. The vector can be used to transform, transduce, or transfect host cells so that the genetic material it carries can be expressed in the host cells. The types of vectors include bacteria, bacteriophages, yeast, plant cell viruses, mammalian cell viruses (such as adenoviruses, retroviruses), or other vectors well known in the art. In addition to containing an origin of replication, the expression vector should also contain a marker gene and other translation regulatory elements.

[0049] The fourth aspect of the present application relates to a host cell, which contains the above-mentioned nucleic acid molecule or the above-mentioned expression vector.

[0050] Host cells for expressing monoclonal antibodies can be prokaryotic cells, such as bacterial cells; lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples include: Escherichia coli; fungal cells, such as yeast; plant cells; insect cells such as Drosophila S2 or Sf9; and animal cells such as CHO, COS, 293 cells, or Bowes melanoma cells.

[0051] The fifth aspect of the present application is to provide a method for screening anti-novel coronavirus monoclonal antibodies, the method comprising the following steps:

[0052] a) Antigen immunization of mice.

[0053] b) Design primers to obtain VH and VL, and connect them to obtain scFv gene.

[0054] c) Obtaining a recombinant phagemid: Connecting the single-chain antibody gene fragment to the phagemid expression vector.

[0055] d) Construction of phage antibody bacterial library.

[0056] e) Amplifying the phage antibody library.

[0057] f) Screening and obtaining monoclonal antibodies against the new coronavirus.

[0058] The mouse immune antigen in step a) is S trimeric protein, which can induce a stronger immune response and produce a higher titer of neutralizing antibodies compared to immunizing mice with S monomer protein.

[0059] The mouse immunization in step a) may be carried out with an adjuvant. The adjuvant used may be an adjuvant known or commonly used in the art, such as aluminum salt, Freund's adjuvant, etc.

[0060] In step a), the ratio of S protein to adjuvant component in the mouse immunization is 1 to 5:1.

[0061] The mice are immunized with 10-100 μg of the first immunization dose in step a), and the booster dose is generally 20%-50% of the first immunization dose.

[0062] The primers designed in step b) include a heavy chain variable region 5' primer and a heavy chain variable region 3' primer, and a kappa chain variable region 5' primer and a 3' primer.

[0063] The primers designed in step b) introduce restriction enzyme sites, including but not limited to SfiI and NotI.

[0064] The sixth aspect of the present application is to provide a method for preparing anti-novel coronavirus monoclonal antibodies, characterized in that the method comprises the following steps:

[0065] a) Obtaining an expression vector: Clone the anti-novel coronavirus scFv gene into an expression vector to construct a recombinant expression vector.

[0066] The expression vector in this application refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a murine scFv can be inserted and the scFv can be expressed. The vector can be used to transform, transduce, or transfect host cells so that the genetic material elements it carries are expressed in the host cells. The types of vectors include bacterial materials, bacteriophages, yeast materials, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art. In principle, any vector can be used as long as it can replicate and be stable in the host. In addition to containing an origin of replication, the expression vector may also contain a marker gene and other translation regulatory elements.

[0067] b) Obtaining transformed cells: Transform host cells with the recombinant eukaryotic expression vector described in step a).

[0068] Host cells for expressing scFv antibodies can be prokaryotic cells, such as bacterial cells; lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells, such as yeast; plant cells; insect cells such as Drosophila S2 or Sf9; and animal cells such as CHO, COS, 293 cells, or Bowes melanoma cells.

[0069] c) Cultivate and induce the recombinant strain to express.

[0070] d) Obtaining anti-novel coronavirus monoclonal antibodies. Extract the plasmid from c) and use it to transfect cells. After incubation, collect the supernatant to obtain antibodies.

[0071] The seventh aspect of the present application is to provide a method for purifying antibody protein:

[0072] a) Purify the antibody supernatant using a nickel column.

[0073] b) Use a desalting column to replace the eluent.

[0074] The eighth aspect of this application is to provide the use of antibodies or expression vectors based on the novel coronavirus S protein as described above in the preparation of reagents for detecting and / or treating novel coronavirus infection.

[0075] Compared with the prior art, this application adopts the above technical solution, which has the following technical effects:

[0076] This application uses the novel coronavirus spike protein to immunize mice to establish a murine phage antibody library. The spike protein is then used for plate-coating and panning. After ELISA validation of the potential antibodies obtained, the resulting positive antibodies are co-incubated with a novel coronavirus pseudovirus to identify their neutralizing activity. The newly screened antibodies are able to specifically recognize and target the novel coronavirus S protein. The inhibition rate against the pseudovirus can reach up to 84% at the original concentration, demonstrating good neutralizing activity and making them suitable for the detection of novel coronaviruses and the development of treatment and prevention products. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Figure 1 The indirect ELISA method is used to detect the specific antibody titer of mouse serum.

[0078] Figure 2 The relevant primer sequences are shown.

[0079] Figure 3 The results of agarose gel electrophoresis after PCR amplification of VH and VL are shown.

[0080] Figure 4 The results of agarose gel electrophoresis of VH and VL overlap extension PCR products are shown.

[0081] Figure 5 The plasmid map of the pCANTAB-5E vector is shown.

[0082] Figure 6 Agarose gel electrophoresis of the PCR product scFv and the double-enzyme digestion product of the vector plasmid pCANTAB-5E is shown.

[0083] Figure 7 The sequencing results of positive clones are shown.

[0084] Figure 8 The results of ELISA testing of the affinity of different polyclonal antibodies to S protein are shown.

[0085] Figure 9 The results of ELISA testing of the affinity of different monoclonal clones for S protein are shown.

[0086] Figure 10 The results of WB detection of antibody expression in the supernatant are shown.

[0087] Figure 11 The results of silver staining identification of antibody supernatant purification are shown.

[0088] Figure 12 The results of silver staining and antibody desalting are shown.

[0089] Figure 13 Antibody S-pro-ab-750 inhibition curves of the virus are shown.

[0090] Figure 14 Antibody S-pro-ab-753 inhibition curves of the virus are shown. DETAILED DESCRIPTION

[0091] Specific implementation methods: The present application will be further described below in conjunction with specific examples. The described embodiments are part of the embodiments of the present application, rather than all of the embodiments. It should be understood that the following examples are given to provide a complete disclosure and explanation of how to use the methods and compositions of the present application to general professionals in the technical field to which the present application belongs, and are not intended to limit the scope of the present application. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present application.

[0092] Example 1 Construction of phage antibody library against SARS-COV-2 spike protein

[0093] 1.1 Immunization of mice

[0094] The new coronavirus S protein antigen was emulsified with aluminum salt adjuvant before use, with the ratio of antigen to adjuvant being 3:1. For the first immunization, 50 μg of emulsified antigen was injected subcutaneously at 3 points on the back of the mouse. The control group immunized mice with PBS plus adjuvant. Three weeks later, the first booster immunization was performed, and 25 μg of emulsified antigen was injected subcutaneously at 3 points on the back of the mouse. Three weeks later, the second booster immunization was performed, and 25 μg of emulsified antigen was injected subcutaneously at 3 points on the back of the mouse. Ten days after the last booster immunization, blood was collected from the orbital cavity to separate the serum for ELISA to detect the antibody titer. Two days later, the mice were killed and the spleen was taken to separate the spleen cells.

[0095] 1.2ELISA detection of serum antibody titer

[0096] Dilute the SARS-CoV-2-RBD protein to 0.2 μg / mL and coat the plate with PBST (containing 0.05% Tween-20) overnight at 4°C. Wash the plate three times with PBST. Add blocking solution and block at room temperature for 2 hours, then wash the plate three times with PBST. Dilute the mouse serum in a gradient manner, add 100 μL of serum to the plate and incubate at room temperature for 1.5 hours. Set up a control group and wash the plate three times with PBST. Add 100 μL of goat anti-mouse antibody (1:2000 dilution) and incubate at room temperature for 1.5 hours, then wash the plate three times with PBST. Add the colorimetric solution and incubate at room temperature for 10 minutes. Add the stop solution to terminate the colorimetric reaction, and measure the absorbance at 450 nm on a microplate reader. The specific antibody titer of mouse serum was detected by indirect ELISA. The mouse serum was diluted 500, 1000, 20000, 40000, 80000, 160000, and 320000 times respectively. Mice immunized with PBS plus adjuvant were set as negative control group, and non-immunized mice were set as blank control group. The absorbance value was detected. If the absorbance value was greater than 2.1 times that of the PBS well, it was considered positive. The results are shown in the attached figure. Figure 1 As shown in Figure 2, the serum antibody titer of mice in group 1 was the highest after immunization, with a final antibody titer of 3.2×10 5 .

[0097] 1.3 Isolation of mouse splenocytes

[0098] Kill the mice by dislocating the neck, remove the spleen tissue, and rinse it in RPMI-1640 medium. Transfer the spleen to a 70μm cell strainer and grind the spleen thoroughly. Add PBS to rinse the strainer several times until the solution changes from red to white. Centrifuge at 400×g for 5 minutes, discard the supernatant, and add PBS to resuspend the cells. Centrifuge at 400×g for 5 minutes, discard the supernatant, add 5mL of red blood cell lysis buffer to resuspend the cells, and incubate at room temperature for 5 minutes. Add PBS to terminate the reaction, centrifuge at 400×g for 5 minutes, discard the supernatant, add RPMI-1640 medium to resuspend the cells, and count them with trypan blue staining. Take 1×10 7 cells.

[0099] 1.4 Total RNA extraction from mouse spleen cells

[0100] Centrifuge at 400×g for 5 minutes, discard the supernatant, and take the cell pellet. Add 1mL RZ lysis buffer to resuspend the cells. Add chloroform, shake for 15 seconds, and let it stand at room temperature for 3 minutes. Centrifuge at 12000rpm for 10 minutes, separate the sample into three layers, and transfer the colorless aqueous phase to a new tube. Add 0.5 times the volume of anhydrous ethanol, transfer to the adsorption column, and centrifuge for 1 minute. Add deproteinization solution and centrifuge for 1 minute. Add rinse solution, let it stand at room temperature for 2 minutes, centrifuge for 1 minute, and repeat this step. Place the adsorption column in a collection tube and centrifuge for 2 minutes to remove the residual liquid. Open the cover of the adsorption column and ventilate and dry it for 2 minutes, transfer it to a new 1.5mL EP tube, add ddH2O to elute, let it stand at room temperature for 2 minutes, centrifuge for 1 minute, collect the RNA sample and perform reverse transcription experiment immediately.

[0101] 1.5 Reverse transcription and cDNA synthesis

[0102] Prepare the following mixture in a PCR tube: 6 μL of ddH2O, 4 μL of 4× gDNA Wiper Mix, and 4 μL of template RNA. Incubate in a PCR instrument at 42°C for 2 minutes. Add 4 μL of 5× HiScript II and perform reverse transcription in a PCR instrument at 50°C for 15 minutes, followed by 85°C for 5 seconds. Store the cDNA template at -20°C.

[0103] 1.6 PCR amplification of VH and VL

[0104] The mouse cDNA obtained by reverse transcription in the previous step was used as a template, and the upstream and downstream primer pairs are shown in Table 1 (for details, see Figure 2 ), PCR amplification reaction was performed according to the reaction system and procedure in Table 2 and Table 3. After the PCR reaction, the product was subjected to 1.5% agarose gel electrophoresis detection. The VH and VL amplification results are shown in the attached Figure 3 As shown, the band size is between 300 and 350 bp, which is consistent with the theoretical value. The product was purified using the Novozymes DNA purification kit. After the overlap extension PCR was completed, the spliced ​​product was recovered by gel electrophoresis. The splicing results were detected by 1.5% agarose gel electrophoresis. Figure 4 As shown, the band size is approximately 750 bp, consistent with the theoretical value. The concentration of the spliced ​​product was measured by Qubit and was 67.6 ng / μL. The product was stored at -20°C.

[0105] Table 1 Primer pairs

[0106]

[0107] Table 2 PCR amplification reaction system

[0108]

[0109] Table 3 PCR amplification reaction program

[0110]

[0111]

[0112] 1.7 Double digestion of scFv and pCANTAB-5E vector

[0113] The pCANTAB-5E vector plasmid was purchased from Miaoling Biological Company. The specific map is attached. Figure 5 .

[0114] After obtaining the plasmid, transform E. coli TG1 and select a single colony. Inoculate the colony into 2×YT medium and shake at 37°C for 14 hours. Extract the plasmid and measure its concentration using a Qubit assay, which is 768 ng / μL. Store the product at -20°C.

[0115] Prepare the double enzyme digestion reaction system according to Table 4. First, add SfiI endonuclease and react in a metal bath at 50℃ for 2h. Then add NotI-HF endonuclease and react in a metal bath at 37℃ for 2h. After the digestion is completed, perform 1% agarose gel electrophoresis to identify the digestion results. Cut the target band and perform gel recovery reaction. The gel recovery product was detected by 1% agarose gel electrophoresis. The results are shown in the attached figure. Figure 6 , consistent with the theoretical value. Concentrations were measured using a Qubit assay, with vector concentrations of 10 ng / μL and spliced ​​product concentrations of 30 ng / μL. Gel-recovered products were stored at -20°C.

[0116] Table 4 Double enzyme digestion reaction system

[0117]

[0118] 1.8 Connection Reorganization

[0119] Carry out the ligation reaction according to Table 5. After mixing, place the system in a metal bath at 25°C for 5 hours. Store the recombinant product at -20°C.

[0120] Table 5 Ligation reaction system

[0121]

[0122]

[0123] 1.9 Electroporation library construction

[0124] Thaw the electroporation competent bacterial solution TG1 on ice. Pre-cool a 0.1 cm electroporation cuvette. Add 2 μL of DNA recombinant product and incubate on ice for 1 minute. Transfer the recombinant product and bacterial solution mixture to the electroporation cuvette and pulse at 1.8 kV for 4-5 ms. Immediately add 1 mL of 37°C preheated SOC medium. Incubate on a shaker at 37°C for 1 hour. Take 10 μL of the bacterial solution and perform a 10-fold gradient dilution (10 -1 ~10 -6 ), take 100 μL of the diluted bacterial solution and spread it on SOB plates, and invert and culture at 37°C overnight. On the second day, count the number of colonies on the plate and calculate the library capacity. The bacterial library capacity is about 1.2×10 7 The positive clones were sent for sequencing, and the sequencing results were compared to detect library diversity. The results are shown in the attached Figure 7 As shown, the sequences are all different and approximately 750 bp in length, containing the complete sequence of the scFv gene. This demonstrates that the scFv gene has been successfully linked to the pCANTAB-5E plasmid vector, indicating successful construction of the phage antibody library. The remaining bacterial liquid was plated on SOB plates. On the second day, the colonies were resuspended in 2×YT medium, glycerol was added to a final concentration of 20%, and the cells were aliquoted into EP tubes and stored in a -80°C freezer. This constituted the phage antibody bacterial library.

[0125] Example 2 Enrichment, screening and identification of phage antibody library

[0126] 2.1 Phage antibody library amplification and purification

[0127] Inoculate 1 mL of frozen bacterial suspension into 2×YT medium and culture at 37°C with shaking until the OD600 is 0.5. Add helper phage M13K07 (helper phage:bacteria = 20:1) and culture at 37°C with shaking for 1 hour. Add Kana (final concentration 50 μg / mL) and IPTG (final concentration 0.2 μM) and culture at 30°C with shaking overnight. Centrifuge the suspension at 8000 × g for 15 minutes and remove the precipitate. Add 1 / 4 the volume of pre-chilled PEG / NaCl, incubate on ice for 30 minutes, centrifuge at 12000 × g for 10 minutes, and discard the supernatant. Resuspend the pellet in 1 mL of PBS and store at -80°C.

[0128] Take 10 μL of purified phage to measure titer: add XL1-Blue bacterial solution to 2×YT medium and culture on a shaker at 37°C until OD600 reaches 0.5. Add PBS to dilute the phage 10-fold. -1 ~10 -8 Add diluted phage to the bacterial solution and incubate at 37°C for 30 min. Apply to 2×YT-Amp plates and invert and incubate at 37°C overnight. Count the number of colonies on the second day to calculate the titer. The titer of the purified phage antibody library is 1×10 10 pfu / mL.

[0129] 2.2 Screening

[0130] Coating: In the first round of panning, coat each well with 2 μg of recombinant SARS-CoV-2-S protein; in the second round, coat each well with 500 ng of protein; and in the third round, coat each well with 200 ng of protein. Dissolve the protein in PBS and add it to the ELISA plate and incubate at 4°C overnight. Wash the plate three times with PBST. Block with blocking buffer at room temperature for 2 hours and wash the plate three times with PBST. Add phage antibody supernatant, incubate at room temperature for 1 hour, and wash the plate ten times with PBST. Elute the phage with trypsin (10 μg / mL) and incubate at 37°C for 30 minutes. Transfer the eluate to a 5 mL EP tube.

[0131] Take TG1 bacterial solution and add it to 2×YT medium. Incubate on a shaker at 37°C until OD600 reaches 0.5. Add TG1 bacterial solution to the eluted phage, incubate at 37°C for 30 minutes, and incubate on a shaker at 37°C for 30 minutes. Add 2×YT medium and 10×GA, and incubate on a shaker at 37°C until OD600 reaches 0.5. Add helper phage, incubate at 37°C for 30 minutes, and incubate on a shaker at 37°C for 30 minutes. Centrifuge at 3000×g for 5 minutes and discard the supernatant. Add 2×YT-AK medium and incubate on a shaker at 30°C overnight. Centrifuge at 3000×g for 5 minutes, transfer the supernatant to a new EP tube for the next round of screening, and store in a -80°C refrigerator.

[0132] 2.3 ELISA identification of recombinant phage antibodies

[0133] The recombinant SARS-CoV-2-S protein was added to PBS and diluted to a concentration of 1.33 μg / mL to coat the ELISA plate at 4°C overnight. Blocking solution was added and blocked at room temperature for 2 hours. 200 μL of phage antibody supernatant solution was added and incubated at room temperature for 1.5 hours. A blank control group was set up, and PBS and culture medium were used as negative control groups. Anti-M13 antibody (1:200 dilution) was added and incubated at room temperature for 1.5 hours. The color development solution was added and incubated at 37°C for 20 minutes. 50 μL of stop solution was added to terminate the color development reaction, and the absorbance was detected at 450 nm on a microplate reader. The OD value of the positive clone was plotted using GraphPad Prism, and the results are shown in the attached figure. Figure 8 As shown, 22 different antibody polyclonal strains were shown to have a certain affinity for SARS-CoV-2-S protein.

[0134] 2.4 Picking positive monoclonal strains

[0135] Pick the five positive clones with the highest absorbance values ​​and infect them with TG1 bacterial solution with an OD600 of 0.5. Incubate at 37°C for 30 minutes, apply to 2×YT-Amp plates, and invert and culture at 37°C overnight. Pick 8 single clones from each plate, add 2×YT medium, and culture on a shaker at 37°C until the OD600 reaches 0.5. Add helper phage, incubate at 37°C for 30 minutes, and culture on a shaker at 37°C for 30 minutes. Centrifuge at 3000×g for 5 minutes and discard the supernatant. Add 2×YT-AK medium and culture on a shaker at 30°C overnight. Take 1 mL of the overnight culture from each tube and store it at -80°C. Centrifuge the remaining culture at 3000×g for 5 minutes, transfer the supernatant to an EP tube and store it at -80°C.

[0136] 2.5 ELISA identification of phage antibody monoclonal

[0137] The recombinant SARS-CoV-2-S protein was added to PBS and diluted to 1.33 μg / mL to coat the ELISA plate at 4°C overnight. Blocking solution was added and blocked at room temperature for 2 hours. 200 μL of phage antibody supernatant solution was added and incubated at room temperature for 1.5 hours, and a control group was set up. Anti-M13 antibody (1:200 dilution) was added and incubated at room temperature for 1.5 hours. The color development solution was added and incubated at 37°C for 20 minutes. 50 μL of stop solution was added to terminate the color development reaction, and the absorbance was detected at 450 nm on an enzyme-labeled instrument. The OD value of the positive monoclonal strain was plotted using GraphPad Prism, and the results are shown in the attached figure. Figure 9 As shown, 13 different antibody monoclonal strains showed a certain affinity for SARS-CoV-2-S protein, and the 10 monoclonal bacterial liquids with the highest absorbance values ​​were picked and sent for sequencing.

[0138] 2.6 Antibody Expression

[0139] The two antibody sequences obtained by sequencing (one antibody was 750 bp long, designated S-pro-ab-750; the other was 753 bp long, designated S-pro-ab-753) were cloned into the pcDNA3.1(+)-C-6His eukaryotic expression vector. The plasmids were transformed into Escherichia coli DH5α, and single colonies were selected and inoculated into LB medium, incubated at 37°C for 12 hours. The plasmids were extracted and the concentrations were measured using a Qubit assay. The pcDNA3.1-S-pro-ab-750 plasmid concentration was 59 ng / μL, and the pcDNA3.1-S-pro-ab-753 plasmid concentration was 184 ng / μL. The plasmids were stored at -20°C.

[0140] 293T cells were cultured at 5 × 10 6Cells were seeded at a density of 1000 cells / mL in a T-25cm2 culture flask. When the cell confluence reached 85%, the culture medium was replaced. Take two EP tubes, add 20μL P3000, 10μg plasmid and DME / F-12 culture medium to tube A (make up to a final volume of 250μL); add 242μL DME / F-12 culture medium and 7.5μL Lipofactamine3000 to tube B. Mix the solutions in tubes A and B, incubate at room temperature for 10 minutes, add to the cell culture flask, and culture in a cell culture incubator at 37°C and 5% CO2. Collect the culture supernatant 48h and 72h after transfection, centrifuge at 400×g for 5 minutes to remove the cell pellet, transfer the supernatant to an EP tube, and store at -80°C.

[0141] 2.7 Western blotting to identify antibody expression in the supernatant

[0142] Mix the antibody supernatant with 5×SDS-PAGE protein loading buffer and boil in a metal bath at 99°C for 10 minutes to denature the protein. Prepare a 12% lower gel and spot the protein marker and sample. Electrophorese the upper gel at 80V for 15 minutes and the lower gel at 120V for 50 minutes. Cut off the excess gel and cut the filter paper and NC membrane. Immerse the filter paper, NC membrane and gel in transfer buffer, transfer to the transfer instrument, and transfer at 15V for 20 minutes. Add blocking solution (5% skim milk powder dissolved in PBST) and block on a shaker at room temperature for 2 hours. Dilute the His-tag antibody at 1:10000 and incubate at 4°C overnight. Add ECL colorimetric solution to the NC membrane to detect the target protein. The results are shown in the attached figure. Figure 10 As shown, both antibodies were expressed at 48h and 72h, with a protein size of about 35KD. The two bands of the antibodies in lanes 1 and 2 may be dimer structures.

[0143] 2.8 Nickel column purification of antibody supernatant

[0144] Equilibrate the nickel column with 600 μL of equilibration buffer and centrifuge at 2900 rpm for 2 minutes. Add 600 μL of antibody supernatant in portions and centrifuge at 1600 rpm for 5 minutes. Collect the aliquots and transfer them to EP tubes. Wash twice with 600 μL of wash buffer and centrifuge at 2900 rpm for 2 minutes. Transfer the column to a new 1.5 mL EP tube and elute twice with 300 μL of elution buffer. Centrifuge at 2900 rpm for 2 minutes. Collect the eluate and store at -80°C.

[0145] 2.9 Silver staining to identify the purification effect of antibody supernatant

[0146] Mix the antibody supernatant with 5× protein loading buffer and boil in a metal bath at 99°C for 10 minutes to denature the protein. Prepare a 12% lower gel. Spot the protein marker and sample. Electrophorese the upper gel at 80V for 15 minutes and the lower gel at 120V for 50 minutes. Place the gel in a fixative (50% ethanol, 10% acetic acid and 40% pure water) and keep at 4°C overnight. Add 30% ethanol and shake on a shaker at room temperature for 10 minutes. Add pure water and shake on a shaker at room temperature for 10 minutes. Add silver staining sensitizer and shake on a shaker at room temperature for 2 minutes. Add pure water and wash twice, each time on a shaker at room temperature for 1 minute. Add silver solution and shake on a shaker at room temperature for 10 minutes. Add pure water and wash once, and shake on a shaker at room temperature for 1 minute. Add silver staining developer and shake on a shaker at room temperature for 3 minutes until the expected ideal protein bands appear. Add silver staining stop solution and shake on a shaker at room temperature for 5 minutes. The results are shown in the attached figure. Figure 11 As shown in the figure, the impurity proteins of both antibodies were reduced after purification, and the protein of about 70KD was BSA, which could not be removed by nickel column purification.

[0147] 2.10 Desalting and replacing eluent

[0148] Break off the tail of the desalting column and loosen the cap. Place the column in a 50 mL centrifuge tube. Centrifuge at 1000 × g for 2 minutes to remove the storage buffer. Add 5 mL of PBS buffer to the column and centrifuge at 1000 × g for 2 minutes to remove the buffer. Repeat this step twice. Place the column in a new 50 mL centrifuge tube, remove the cap, and slowly add the sample to the center of the column. Centrifuge at 1000 × g for 2 minutes to collect the sample and store at -80°C.

[0149] 2.11 Silver staining to identify the desalting effect of antibody supernatant

[0150] Mix the antibody supernatant with 5× protein loading buffer and boil in a metal bath at 99°C for 10 minutes to denature the protein. Prepare a 12% lower gel. Spot the protein marker and sample. Electrophorese the upper gel at 80V for 15 minutes and the lower gel at 120V for 50 minutes. Place the gel in the fixative and keep it at 4°C overnight. Add 30% ethanol and shake it at room temperature for 10 minutes. Add pure water and shake it at room temperature for 10 minutes. Add silver staining sensitizer and shake it at room temperature for 2 minutes. Add pure water and wash twice, each time on a shaker at room temperature for 1 minute. Add silver solution and shake it at room temperature for 10 minutes. Add pure water and wash once, and shake it at room temperature for 1 minute. Add silver staining developer and shake it at room temperature for 3 minutes until the expected ideal protein bands appear. Add silver staining stop solution and shake it at room temperature for 5 minutes. The results are as follows. Figure 12 As shown in the figure, after the two antibodies replaced the eluent, the impurity proteins were also reduced to a certain extent, but the antibody concentration was reduced.

[0151] 2.12 Antibody Neutralization Activity Detection

[0152] Thaw the SARS-CoV-2-Fluc pseudovirus from -80°C on ice and dilute it to 1×10 using DME medium containing 10% FBS. 4 TCID50 / mL. Dilute the antibody in a 3-fold gradient. Add 90 μL of the diluted antibody solution to each well of a 96-well cell culture plate. Add 90 μL of the diluted pseudovirus solution to each well. Set up a cell control group and a virus control group. Place the 96-well cell culture plate in a 37°C incubator and incubate for 1 hour. Transfer the mixed solution in the cell culture plate to a 96-well white plate and set up 3 replicates at 50 μL / well. Dilute 293T-ACE2 cells to 4×10 6 Cells were cultured at a concentration of 50 μL / well of the cell suspension in a 96-well plate and incubated in a 37°C incubator. After 24 hours of incubation, 25 μL of 37°C pre-warmed DME medium containing 10% FBS was added to each well. Culture was continued for 48 hours. The 96-well plate was then removed and 5 μL of luciferase assay substrate was added to each well. The mixture was mixed and the chemiluminescence value was measured using a microplate reader.

[0153] Antibody inhibition virus curve Figure 13 and Figure 14 As shown, the virus inhibition rate of the original concentration S-pro-ab-750 antibody reached 84%, and 25μL of the antibody could neutralize 205TCID50 new coronavirus pseudoviruses; the virus inhibition rate of the original concentration S-pro-ab-753 antibody reached 55%, and 25μL of the antibody could neutralize 135TCID50 new coronavirus pseudoviruses.

[0154] The above is only a preferred specific implementation method of the present application, but the scope of protection of the present application is not limited thereto. Any changes or replacements that can be easily thought of by any technician familiar with this technical field within the technical scope disclosed in this application should be covered by the scope of protection of the present application.

Claims

1. Antibodies or antigen-binding fragments that specifically bind to the novel coronavirus spike protein, wherein: The antibody or antigen-binding fragment comprises: (1) The hypervariable region CDR of the heavy chain variable region; comprising HCDR1, HCDR2, and HCDR3, wherein HCDR1 has the amino acid sequence shown in SEQ ID NO. 2; HCDR2 has the amino acid sequence shown in SEQ ID NO. 4; and HCDR3 has the amino acid sequence shown in SEQ ID NO. 6; (2) The hypervariable region CDR of the light chain variable region; it comprises LCDR1, LCDR2 and LCDR3, wherein LCDR1 is the amino acid sequence shown in SEQ ID NO.9; LCDR2 is the amino acid sequence shown in SEQ ID NO.11; and LCDR3 is the amino acid sequence shown in SEQ ID NO.

13.

2. The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment comprises: (1) The framework region FR of the heavy chain variable region; comprising HFR1, HFR2, HFR3, and HFR4, wherein HFR1 has the amino acid sequence shown in SEQ ID NO. 1; HFR2 has the amino acid sequence shown in SEQ ID NO. 3; HFR3 has the amino acid sequence shown in SEQ ID NO. 5; and HFR4 has the amino acid sequence shown in SEQ ID NO. 7; (2) The framework region FR of the light chain variable region comprises LFR1, LFR2, LFR3 and LFR4, wherein LFR1 is the amino acid sequence shown in SEQ ID NO.8; LFR2 is the amino acid sequence shown in SEQ ID NO.10; LFR3 is the amino acid sequence shown in SEQ ID NO.12; and LFR4 is the amino acid sequence shown in SEQ ID NO.

14.

3. The antibody or antigen-binding fragment of any one of claims 1 or 2, comprising: (1) a heavy chain variable region; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 29; and (2) Light chain variable region; the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

30.

4. A nucleic acid encoding the antibody or antigen-binding fragment of any one of claims 1 or 2.

5. A vector comprising the nucleic acid of claim 4. A host cell comprising the vector of claim 5 .

7. Use of the antibody or antigen-binding fragment of claim 3, the nucleic acid of claim 4, or the vector of claim 5 in the preparation of a reagent for detecting and / or treating novel coronavirus infection.

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