A novel coronavirus neutralizing nanoantibody

Abstract

The present invention discloses a novel coronavirus neutralizing active nano antibody, which belongs to the field of biotechnology. The nano antibody of the present invention includes at least one of nano antibody B1-4, nano antibody C1-5 or nano antibody B-B2; the nucleotide sequence of the nano antibody B1-4 is shown in SEQ ID NO.1; the nucleotide sequence of the nano antibody C1-5 is shown in SEQ ID NO.2; the nucleotide sequence of the nano antibody B-B2 is shown in SEQ ID NO.3. The three nano antibodies B1-4, nano antibody C1-5 and nano antibody B-B2 of the present invention are specific to SARS-COV-2-RBD antigens, can be expressed in large quantities using a prokaryotic expression system, and have good solubility, low cost and good biological activity. The nano antibody targets a conserved epitope on SARS-COV-2-RBD, can neutralize various new coronavirus variants, and has the potential to be used in the treatment and prevention of new coronavirus infection.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a novel coronavirus neutralizing active nanoantibody. Background Art

[0002] The virus that causes COVID-19 is the novel coronavirus (SARS-CoV-2), an enveloped, positive-stranded RNA novel coronavirus that encodes the structural proteins spike (S), envelope (E), membrane (M), and nucleocapsid (N), 16 nonstructural proteins, and 5-8 accessory proteins. The S protein on the viral surface consists of 1273 amino acids and contains a furin cleavage site, which allows furin to cleave the S protein into two subunits, S1 and S2. The S1 subunit is highly immunogenic and contains two functional domains: the receptor binding domain (RBD) and the N-terminal domain (NTD). The S1 domain covers the upper portion of the S protein, with the RBD located at the tip. The RBD is a key site for viral binding to the target cell receptor ACE2. While primarily in a "downward" conformation, inaccessible to the receptor binding site, it can randomly flip, moving like a hinge, and temporarily shifting to an "upward" conformation to reveal the ACE2 receptor binding site. The RBD, as an antigen, elicits antibodies with strong neutralizing activity, and numerous therapeutic agents, therapies, and vaccines are currently under development targeting the RBD. However, while RBD is a hotspot for neutralizing antibodies, it also provides a lot of opportunities for viral mutations. Currently, a variety of variants carrying RBD mutations have emerged. On the Omicron strain (B.1.1.529), there are 15 mutation sites in the RBD. These variants increase the transmissibility of the new coronavirus or allow it to evade antibody neutralization, resulting in a decrease in the efficacy of antibodies elicited by most vaccines or a weakening of the effectiveness of protective neutralizing antibodies. Therefore, in order to combat the new coronavirus variants that have appeared or are about to appear, an antibody targeting a conserved epitope is urgently needed to resist the virus variants. It should be able to recognize the highly conserved neutralizing epitope on the S protein.

[0003] Heavy-chain antibodies exist in species of the Camelidae family. They are the variable region of the heavy chain of antibodies and lack the first constant region of the light chain and heavy chain that appear in conventional antibodies. Therefore, their size is usually only 12-14 kDa, and their size is in the nanometer range (about 2.5 nanometers in diameter and about 4 nanometers in height). They are also called nanobodies. Nanobodies are the smallest units that retain the same antigen affinity and specificity as conventional antibodies. They have a longer complementary determining region 3 (CDR3) than humans and can form finger-like structures to penetrate into the cavity on the surface of the antigen, allowing nanobodies to bind to antigenic epitopes that traditional antibodies cannot usually access. Nanobodies can also be mass-produced in microbial prokaryotic expression systems, have short operation cycles, and are generally very stable. They can be aerosolized for direct pulmonary delivery and are excellent candidates for the development of alternative routes of administration. Aquifex aeolicus lumazine synthase (AaLS) protein cage nanoparticles (PDB ID: 1HQK), isolated from a hyperthermophilic bacterium, are a promising protein scaffold for developing delivery and assembly systems. They can be used as a scaffold to display nanobodies, enhancing their affinity and neutralizing activity. AaLS is a hollow dodecahedron composed of 60 identical subunits, with an outer diameter of approximately 16 nanometers and an inner diameter of approximately 8 nanometers. It is uniform in size, structurally symmetrical, and thermally stable, with a Tm of approximately 120°C. Due to its hollow spherical structure, excellent encapsulation capabilities, and ligand-like properties, variants of AaLS can be used to encapsulate RNA and proteins, and small molecule drugs or antigens and antibodies can be bound to the outer surface of AaLS for delivery into cells. To attach antibodies to the AaLS surface, we engineered AaLS and nanobodies, exploiting the spontaneous formation of isopeptide bonds between the SpyCatcher003 protein and its peptide partner, SpyTag003, to link heterologously generated antibodies to the AaLS scaffold. Both the SpyTag and SpyCatcher can be located at different positions on the protein chain and react under a wide range of conditions (pH, buffer, and temperature).

[0004] Although nanobodies have opened up important possibilities in biomedical research, to date, most nanobodies have been obtained by immunizing camels, and the research and development process is long and expensive, which is a huge barrier to entry for most laboratories. Natural nanoantibody libraries do not require animal immunization, saving time and cost. Antibody sequences can be obtained in 2-3 weeks. They have the following advantages when screening nanoantibodies: (1) Diverse antibody monoclonals can be isolated; (2) Antibodies against weak antigens, self-antigens, and toxic antigens that do not cause immune responses in the body can be isolated; (3) Multiple antigens can be screened to obtain multiple antibodies; (4) High-affinity antibodies can be obtained when the library capacity is large enough. Summary of the Invention

[0005] The purpose of the present invention is to overcome the shortcomings of the existing technology and provide a novel coronavirus neutralizing active nano antibody. The nano antibody of the present invention has the characteristics of small molecular weight, low immunogenicity and stability. It can overcome the disadvantages of traditional monoclonal antibodies that cannot be expressed in prokaryotic systems, have a long research and development cycle, and high production costs. The nano antibody has good development prospects in the treatment and prevention of novel coronavirus infection.

[0006] To achieve the above-mentioned purpose, the technical solution adopted by the present invention is: a novel coronavirus neutralizing active nanobody, the nanobody comprising at least one of nanobody B1-4, nanobody C1-5 or nanobody B-B2; the nucleotide sequence of the nanobody B1-4 is shown in SEQ ID NO.1; the nucleotide sequence of the nanobody C1-5 is shown in SEQ ID NO.2; the nucleotide sequence of the nanobody B-B2 is shown in SEQ ID NO.3.

[0007] The natural nanoantibody library used in the present invention collected blood samples from 103 healthy adult alpacas, with a blood volume of 2660 ml and the number of isolated PBMC cells was about 1.06×10 10 Using nested PCR, the complete set of antibody heavy chain variable region genes were amplified from isolated PBMCs. These genes were then cloned into the phage vector pADL-10b using phage display technology, with the VHH expressed as a fusion protein on the surface of the phage shell. Using the phage display library as the mobile phase and the SARS-CoV-2 receptor binding domain protein RBD as the target antigen, the researchers utilized antigen-antibody specific binding. After incubation, unbound free phage were washed away, and phage bound to the target molecule were eluted. The eluted phage were then infected with SS320 competent cells, propagated, and amplified before undergoing another round of elution. After 2-3 cycles of "adsorption-elution-amplification," nanobodies with affinity for the target protein SARS-CoV-2-RBD were obtained. After initial screening, the isolated antibody monoclonals were further specifically screened using phage ELISA to obtain the target monoclonal clones. After sequencing and comparison, they were named "B1-4", "C1-5" and "B-B2". The above-mentioned nanoantibodies have the characteristics of small molecular weight, low immunogenicity and stability. They can overcome the disadvantages of traditional monoclonal antibodies that cannot be expressed in the prokaryotic system, have a long research and development cycle and high production costs. This nanoantibody has good development prospects in the treatment and prevention of new coronavirus infections.

[0008] As a preferred embodiment of the novel coronavirus neutralizing active Nanobody of the present invention, the amino acid public sequence of the Nanobody B1-4 is shown as SEQ ID NO.4; the amino acid public sequence of the Nanobody C1-5 is shown as SEQ ID NO.5; the amino acid public sequence of the Nanobody B-B2 is shown as SEQ ID NO.6.

[0009] As a preferred embodiment of the novel coronavirus neutralizing active Nanobody of the present invention, the Nanobody comprises a complementary determining region; the complementary determining region consists of CDR1, CDR2 and CDR3.

[0010] As a preferred embodiment of the novel coronavirus neutralizing active Nanobodies of the present invention, the CDR1, CDR2 and CDR3 amino acid sequences of the Nanobody B1-4 are shown in SEQ ID NO.4 from the N-terminal positions 30-39, 51-63 and 101-121, respectively; the CDR1, CDR2 and CDR3 amino acid sequences of the Nanobody C1-5 are shown in SEQ ID NO.5 from the N-terminal positions 30-39, 51-63 and 101-121, respectively; the CDR1, CDR2 and CDR3 amino acid sequences of the Nanobody B-B2 are shown in SEQ ID NO.6 from the N-terminal positions 30-39, 51-63 and 101-121, respectively.

[0011] As a preferred embodiment of the novel coronavirus-neutralizing nanobodies described herein, the nanobodies are capable of recognizing an antigenic epitope with an amino acid sequence as shown in SEQ ID NO. 7. After coupling the three nanobodies provided herein to the AaLS nanoscaffold, SDS-PAGE and negative staining electron microscopy confirmed their display on the surface of the self-assembled AaLS protein cage structure. Furthermore, all three nanobodies, after being attached to the nanoscaffold, can recognize the SARS-CoV-2-RBD antigen-binding epitope.

[0012] The present invention also provides a nucleotide molecule, which encodes the nanobody.

[0013] The present invention also provides a recombinant vector plasmid containing the nucleotide molecule. The nanobody gene sequence is inserted between the NdeI and XhoI restriction sites of the pET-28a(+) expression vector. Both the N-terminus and C-terminus of the antibody sequence can be fused with the 6×His tag on the pET-28a(+) vector for expression, facilitating purification and obtaining a soluble nanobody fragment with a purity of >90%.

[0014] The present invention also provides a host cell containing the recombinant vector plasmid.

[0015] The present invention also provides the use of the nanobody, the nucleotide molecule, the recombinant vector plasmid or the host cell in the preparation of a drug for preventing or treating novel coronavirus infection.

[0016] The present invention also provides the use of the nanoantibody, the nucleotide molecule, the recombinant vector plasmid or the host cell in the preparation of a kit for detecting novel coronavirus infection.

[0017] The beneficial effects of the present invention include providing novel coronavirus-neutralizing nanobodies. The three nanobodies, B1-4, C1-5, and B-B2, specifically target the SARS-CoV-2-RBD antigen and can be expressed in large quantities using a prokaryotic expression system. These nanobodies exhibit good solubility, low cost, and excellent biological activity. These nanobodies target a conserved epitope on the SARS-CoV-2-RBD and can neutralize various novel coronavirus variants, demonstrating their potential for use in the treatment and prevention of novel coronavirus infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the map of the pET-28a(+)-target gene expression plasmid vector.

[0019] Figure 2 This is the SDS-PAGE diagram of nanobody purification.

[0020] Figure 3 To identify the activity of nanoantibodies binding to antigens.

[0021] Figure 4 SPR was used to detect the binding kinetics of nanobodies.

[0022] Figure 5 The neutralization activity of nanobodies was detected by alternative neutralization assay.

[0023] Figure 6 The neutralization activity of nanobodies was tested for pseudovirus neutralization assay.

[0024] Figure 7 This is the SDS-PAGE diagram of the coupling of nanoantibodies and AaLS scaffolds.

[0025] Figure 8 This is a negative staining electron micrograph of the nanoantibody coupled to the AaLS scaffold.

[0026] Figure 9 It is the potential antigenic epitope for the binding of nanoantibodies to SARS-COV-2-RBD antigen. DETAILED DESCRIPTION

[0027] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0028] Example 1 Screening and preparation of nanoantibodies targeting SARS-COV-2-RBD protein

[0029] 1. Construction of Nanobody Natural Phage Display Library

[0030] The natural nanoantibody library used in this example was provided by Shenzhen Kangti Life Science Technology Co., Ltd. The natural nanoantibody library was prepared by collecting peripheral blood cells (PBMC) from 103 healthy adult alpacas, extracting total RNA, and reverse transcribing it into cDNA. The heavy chain antibody VHH gene fragment was amplified using specific primers and ligated into the phage plasmid pADL-10b to construct a phage plasmid library containing the amplified nanofragments. The fragments were electroporated into SS320 competent cells multiple times, and the VHH region fragments were displayed on the phage surface under the amplification and rescue of the helper phage M13K07 to obtain the alpaca natural phage library. The antibody library had a capacity of 2×109 cfu and a titer of 1.28×10 13 pfu / mL. Nanosequences that can specifically bind to SARS-COV-2-RBD antigen were screened based on the constructed natural library.

[0031] 2. Use phage display technology to select and enrich SARS-COV-2-RBD nanobodies

[0032] Immunofluorescence tubes were coated with 50 μg of SARS-CoV-2-RBD (Arg319-Phe541) antigen (derived from a wild-type COVID-19 strain, NCBI reference number YP_009724390.1 (provided by Beijing Sino Biological Science and Technology Co., Ltd.)) using CBS coating buffer (pH 9.6) overnight at 4°C. The next day, unbound antigen was discarded from the tubes and the tubes were washed three times with 2 ml of PBS buffer at room temperature for 5 minutes each. The tubes were then blocked with 3% BSA blocking buffer at room temperature for 2 hours, the blocking buffer discarded, and the tubes were washed three times with 2 ml of PBS buffer at room temperature for 5 minutes each. 100 μl of phage antibody library was added to each well and incubated at room temperature for 1 hour. Unbound phage were discarded from the tubes and washed 20 times with PBS containing 0.1% Tween-20 for 5 minutes each. Elution was performed with 1 ml of 0.25 mg / mL Trypsin solution at room temperature for 30 minutes. Add 10 μl of 10% AEBSF to stop the eluate, collect the solution in the immunotube, which is the first round of phage screening eluate. Immediately add the eluted phage to 5 ml of SS320 bacterial solution with an OD600 of 0.5-0.6, culture at 37°C for 30 minutes, spread on 2YT-GA (containing a final concentration of 100 μg / ml AMP, 2% glucose) plates, and culture at 37°C overnight. The next day, use a spreading rod to scrape all the colonies from the overnight culture, and take about 400 μl of the scraped bacteria and transfer them to 100 ml of 2×YT liquid medium (containing 10 μg / ml Tet and 100 μg / ml AMP), with an initial OD600 of 0.1. Cultivate at 37°C and 250 rpm until the bacterial solution grows to an OD600 of 0.5-0.6, and add helper phage M13K07 (titer of 10 13 20 μl of 5% PEG / ml (10 μg / ml) was added and incubated at 220 rpm for 30 min. KAN and 0.2 mM IPTG were added to a final concentration of 50 μg / ml, respectively, and the culture was continued overnight at 30°C and 250 rpm. The next day, after centrifugation, the supernatant was collected and 20% PEG / 2.5M NaCl was added to precipitate the phage. The first-round phage sublibrary was collected and used for the next round of screening. This panning process was repeated three times to enrich for single clones of phage expressing nanobodies on their surfaces. The results of these three rounds of screening are shown in Table 1.

[0033] Table 1 Phage enrichment ratios after three rounds of panning for SARS-COV-2-RBD

[0034]

[0035] 3. SARS-COV-2-RBD Phage-ELISA detection of positive clones

[0036] The ELISA plate was coated with 1 ng / μl SARS-COV-2-RBD antigen using CBS (pH 9.6) and BSA as a control at the same time, and the coating was incubated at 4°C overnight. 192 individual colonies were picked and transferred to a sterile 96-well plate. 200 μl of 2×YT liquid medium (containing 100 μg / ml AMP and 10 μg / ml Tet) was added to each well and cultured overnight at 37°C. The next day, 2 μl of the overnight culture was transferred to a fresh 96-well plate containing 200 μl of 2×YT liquid medium (containing 100 μg / ml AMP and 10 μg / ml Tet). The plates were incubated at 37°C for 3-5 hours. Helper phage M13K07 was added to achieve a phage:bacteria ratio of 20:1. The plates were incubated at 37°C for 30 minutes. Finally, 50 μg / ml KAN and 0.2 mM IPTG were added, respectively, and the cultures were incubated overnight at 30°C, 250 rpm. The cultures were centrifuged the next day, and the supernatant was collected for analysis. Take the coated ELISA plate and block each well with 3% BSA at room temperature for 1 hour. Wash the plate three times at room temperature. Add an equal amount of monoclonal phage supernatant to each well of the experimental and control groups and incubate at room temperature for 2 hours. Wash each well three times with 200 μl of PBST. Add M13 Bacteriophage Antibody (HRP) and incubate at room temperature for 1 hour. Wash five times with PBST. Add TMB colorimetric solution to each well and develop in the dark for 2-3 minutes. Add 1M HCl stop solution and read the OD450 value using a microplate reader. A positive clone is identified when the ratio of the experimental well to the BSA control well is greater than 10 and the absorbance value of the experimental well is ≥1.

[0037] 4. Positive clone gene sequence analysis

[0038] Three Nanobodies against SARS-COV-2-RBD were identified by phage-ELISA, and the complementarity-determining regions (CDRs) of the heavy chain antibodies were determined by DNA sequencing analysis. They were named "B1-4," "C1-5," and "B-B2," respectively. The heavy chain nucleotide sequences of the Nanobodies B1-4, C1-5, and B-B2 are shown in SEQ ID NOs. 1 to 3, respectively, and the VHH region amino acid sequences are shown in SEQ ID NOs. 4 to 6, respectively. The amino acid sequences at positions 30-39 are CDR1, the amino acid sequences at positions 51-63 are CDR2, and the amino acid sequences at positions 101-121 are CDR3.

[0039] Example 2 Soluble prokaryotic expression and purification of SARS-COV-2-RBD nanobody

[0040] 1. Construction of pET-28a(+) prokaryotic expression vector plasmid

[0041] The nanobody gene sequence was inserted into the NdeI and XhoI restriction sites of the pET-28a(+) expression vector. The N-terminus and C-terminus of the nanobody sequence were fused with the 6×His tag on the expression vector, which can be used for the purification of the fusion protein. The vector map of pET-28a(+)-target gene is shown in Figure 1 , and commissioned Shanghai Jierui Bioengineering Co., Ltd. to synthesize the plasmid.

[0042] 2. Soluble prokaryotic expression of nanobodies

[0043] Evenly spread the recombinant expression plasmid-pierced bacteria onto a plate containing LB solids (final KAN concentration of 50 μg / mL) and incubate at 37°C for 12-14 hours. Pick a single colony and expand it into a sterile test tube containing 5 mL of LB liquid medium. Shake the culture at 250 rpm at 37°C for 12-14 hours to obtain a seed culture. Transfer the seed culture to an initial OD600 of 0.1 and incubate at 37°C with shaking at 220 rpm. When the cells grow to an OD600 of 0.6-0.8, induce them with IPTG at a final concentration of 0.5 mmol / L and incubate at 18°C ​​for 12-14 hours. Centrifuge at 12,000 rpm at 4°C for 8 minutes, discard the supernatant, resuspend the cells in pre-chilled sterile PBS, lyse them using an ultrasonic cell disruptor, and centrifuge at 13,000 rpm at 4°C for 5 minutes. Collect the supernatant and pellet separately.

[0044] 3. Nanobody Ni-NTA affinity chromatography column purification

[0045] The affinity tag of the fusion protein is 6×His, and the nanoantibody is purified using Ni-NTA affinity chromatography resin from Shanghai Bioengineering. Take the Ni column precipitation resin, wash it 2-3 times with sterile ddH2O, equilibrate the Ni column with 10mM imidazole buffer, add the prepared ultrasonic lysate supernatant to the Ni column, incubate at 4°C for 1 hour, use 2 times the column volume of eluent (containing 20mM imidazole) to wash away the impurities, and finally elute the target protein with an equal volume of eluent (containing 250mM imidazole), and collect the eluent; use ultrafiltration concentrator tubes to concentrate the sample. 15% SDS-PAGE was used to detect the protein purification, and the results are as follows Figure 2 As shown. Figure 2 It can be seen that the nanobody provided by the present invention has good solubility, is easy to express and purify in a prokaryotic system, has a purity of >90%, and has low preparation cost.

[0046] Example 3 Western Blot Identification of Antigenicity of Nanobodies Binding to RBD

[0047] In this example, the purified nanobody was validated by Western blot using the SARS-COV-2-RBD antigen. 0.1 μg of SARS-COV-2-RBD antigen was loaded, subjected to 15% SDS-PAGE, and transferred to the membrane. Blocked with 5% skim milk blocking buffer at room temperature for 1 hour, the purified nanobody was used as the primary antibody, diluted to 1:100 with blocking buffer, incubated at room temperature for 1 hour, and then transferred to 4°C for overnight incubation. Anti-His-HRP antibody diluted 1:2000 was used as the secondary antibody and incubated at room temperature for 1 hour. The membrane was washed, developed with ECL, and exposed to light. The results are shown in Figure 2. Figure 3 As shown. Figure 3 It can be seen that the nanoantibodies provided by the present invention can all recognize SARS-COV-2-RBD antigens, and the bands are luminescent strongly, indicating that the concentration and purity of the purified samples are relatively high.

[0048] Example 4 SPR detection of the kinetic properties of nanobody binding to RBD protein

[0049] In this embodiment, the COOH chip was installed according to the standard operating procedure of the OpenSPRTM instrument. Start running at the maximum flow rate (150 μL / min), the detection buffer was PBS (PH7.4), and after reaching the signal baseline, 200 μL of isopropanol was loaded, and the bubbles were discharged after running for 10 seconds. After reaching the baseline, the sample loop was rinsed with buffer and emptied with air. After the signal reached the baseline, the buffer flow rate was adjusted to 20 μL / min. The chip was activated by loading EDC / NHS (1:1) solution. 200 μL of SARS-COV-2-RBD diluted with activation buffer was loaded and run for 4 minutes, the sample loop was rinsed with PBS, and emptied with air. 200 μL of Blocking solution was loaded, the sample loop was rinsed with PBS, and emptied with air. Observe the baseline for 5 minutes to ensure stability. The nanoantibody was diluted with buffer, and the selected nanoantibody was loaded at 20 μL / min. The protein and ligand binding time was 240 seconds, and the natural dissociation time was 360 seconds. TraceDrawer software was used to calculate and analyze the kinetic parameters of the binding reaction, using the One To One analysis model. The results were as follows: Figure 4 The results showed that the affinity of nanobody B1-4 binding was 1.61×10 -8 M, the binding constant is 1.11×10 5 1 / Ms, and the dissociation constant is 1.79×10 -3 1 / s; the affinity of C1-5 binding is 3.17×10 -7 M, the binding constant is 1.83×10 4 1 / Ms, and the dissociation constant is 5.80×10 -3 1 / s, and the affinity of B-B2 binding was 3.79×10 -7 M, the binding constant is 7.15×103 1 / Ms, and the dissociation constant is 2.71×10 -3 1 / s, it can be seen that the nanobody provided by the present invention can bind to the antigen with moderate affinity.

[0050] Example 5 Alternative Neutralization Experiment Based on SARS-COV-2-RBD

[0051] This example uses KingScript's SARS-CoV-2 (new coronavirus) alternative virus neutralization test (sVNT) kit to identify the neutralizing activity of nanobodies. According to the instructions, the gradient diluted nanobody was mixed with an equal volume of RBD pre-coupled with HRP. Three RBD proteins were used in the present invention, namely wild strain, Delta strain (B.1.617.2) and Omicron strain (B.1.1.529). The EP tube containing the mixture was incubated at 37°C for 30 minutes. Then 100 μL of the mixture was added to a microtiter plate pre-coated with ACE2 and incubated at 37°C for 15 minutes. A competitive inhibition reaction occurred between the HRP-coupled RBD protein and the SARS-CoV-2 neutralizing nanobody. After thorough washing, TMB substrate solution was added to the wells and incubated for 15 minutes, and then an equal volume of stop solution was added to stop the reaction. The color intensity was measured spectrophotometrically at 450nm. The results are shown in Figure 2. Figure 5 As shown. It can be seen that the nanoantibodies provided by the present invention have a certain blocking and inhibitory effect on the RBD of the three SARS-CoV-2.

[0052] Example 6 Pseudovirus Neutralization Test

[0053] The SARS-CoV-2 pseudovirus neutralization test in this example used 293T cells overexpressing ACE2 (293T-hACE2). The nanobody to be tested was diluted to 100 μg / ml with complete culture medium. 100 μl of nanobody was mixed with 50 μl of 1.3×10 4 TCID 50 / mL SARS-CoV-2 pseudovirus mixture. Two pseudoviruses were used in this invention: the wild-type strain and the Omicron strain (B.1.1.529). The negative control group consisted of cells alone. The mixture was incubated in a 37°C, 5% CO2 incubator for 1 hour. After incubation, 100 μL of freshly trypsinized 293T-hACE2 cells were added to a 96-well plate. The plate was incubated in a 37°C, 5% CO2 incubator for 24 hours. After 24 hours, the supernatant from the cell culture plate was gently aspirated, leaving 100 μL per well. 100 μL of luciferase substrate was then added to each well and lysed using a pipette 6-8 times. After incubation at room temperature for 2 minutes, 150 μL of the lysate was transferred to a white, opaque 96-well plate and luminescence was measured using a multi-function microplate reader. The value for the positive control should be a relative luminescence unit value ten times higher than the cell background. The inhibition rate of the antibody was calculated according to the following formula: Inhibition rate = (1-(average RLU of the sample-average RLU of the cell control group) / (average RLU of the virus control group-average RLU of the cell control group))*100%.

[0054] The results are as follows Figure 6 As shown by Figure 6 It can be seen that the nanoantibodies provided by the present invention have an inhibitory effect on the SARS-COV-2 wild strain and Omicron (B.1.1.529) variant pseudovirus at a concentration of 100 μg / ml, and there is no statistical difference in the inhibition rate of the wild strain and the Omicron (B.1.1.529) variant.

[0055] Example 7 Antibodies coupled to AaLS nanoscaffold proteins to form multimeric nanobodies

[0056] The C-terminus of the nanobody sequence was added with Spytag003 (GenBank: MT945421.1) connected with a flexible short peptide (G4S)3, and the C-terminus of the AaLS scaffold sequence was added with SpyCatcher003 (GenBank: QGX07219.1) connected with a flexible short peptide (GGS)4. The two sequences were inserted into the pET-28a (+) expression vector, and the plasmid was synthesized by Shanghai Jierui Bioengineering Co., Ltd. The two proteins were expressed and purified in prokaryotes according to the method of Example 2, and coupled with the nanobody: AaLS scaffold at a molar ratio of 2:1. The antibody and scaffold were diluted with 1M TBS buffer at pH 8.5, mixed in molar ratio, and incubated at 4°C overnight. The coupling efficiency was analyzed by 12% SDS-PAGE and observed using negative staining electron microscopy. The results are shown as follows: Figure 7 、 Figure 8 shown.

[0057] The SDS-PAGE image shows that the nanoantibody provided by the present invention can be coupled to the AaLS nanoscaffold, the protein band size is consistent, and negative staining electron microscopy observes that the nanoantibody can be coupled to the AaLS nanoscaffold and maintain the spherical structure of the nanoscaffold.

[0058] Example 8 Peptide ELISA to identify potential antigenic epitopes binding to nanoantibodies and SARS-COV-2-RBD

[0059] Seventeen short peptides covering the SARS-COV-2-RBD (320-553) protein, with a length of 9-30 amino acids, were artificially synthesized with a purity of more than 90%. The above 17 peptides were dissolved in ddH2O or DMSO, and then diluted to 1μg / ml with coating solution to coat the ELISA plate as a capture antigen. The ZIKA EDIII peptide was used as a negative control and incubated at 4°C overnight. The next day, PBST blocking solution containing 3% BSA was used and incubated at 37°C for 1h. After washing with PBST 3 times for 5 minutes each time, 100μg / ml of poly-nanoantibodies were added to each well. After incubation at 37°C for 1h, the plate was washed 3 times with PBST, and a 1:2000 diluted anti-His-HRP antibody was added to each well. The plate was incubated at 37°C for 1 hour and washed 3 times. TMB was reacted at room temperature in the dark for 10min, and the reaction was terminated with 1M HCl. The OD450 value was read by a microplate reader to detect the bound potential epitope. The results are as follows Figure 9 It can be seen that the three nanobodies provided by the present invention can react with peptide 16, and the difference is statistically significant compared with the control group. Peptide 16 may be a potential epitope of the nanobodies provided by the present invention.

[0060] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A novel coronavirus neutralizing active nanobody, characterized in that: The nanobody is nanobody B1-4, nanobody C1-5 or nanobody B-B2; the nucleotide sequence of the nanobody B1-4 is shown in SEQ ID NO.1; the nucleotide sequence of the nanobody C1-5 is shown in SEQ ID NO.2; the nucleotide sequence of the nanobody B-B2 is shown in SEQ ID NO.

3.

2. The novel coronavirus neutralizing active nanobody according to claim 1, characterized in that The amino acid public sequence of the Nanobody B1-4 is shown in SEQ ID NO.4; the amino acid public sequence of the Nanobody C1-5 is shown in SEQ ID NO.5; the amino acid public sequence of the Nanobody B-B2 is shown in SEQ ID NO.

6.

3. The novel coronavirus neutralizing active nanobody according to claim 1, characterized in that The Nanobody comprises a complementarity determining region; the complementarity determining region consists of CDR1, CDR2 and CDR3.

4. The novel coronavirus neutralizing active nanobody according to claim 1, characterized in that The CDR1, CDR2 and CDR3 amino acid sequences of the Nanobody B1-4 are shown in SEQ ID NO.4 from the 30th to 39th, 51st to 63rd and 101st to 121st positions from the N-terminus, respectively; the CDR1, CDR2 and CDR3 amino acid sequences of the Nanobody C1-5 are shown in SEQ ID NO.5 from the 30th to 39th, 51st to 63rd and 101st to 121st positions from the N-terminus, respectively; the CDR1, CDR2 and CDR3 amino acid sequences of the Nanobody B-B2 are shown in SEQ ID NO.6 from the 30th to 39th, 51st to 63rd and 101st to 121st positions from the N-terminus, respectively.

5. The novel coronavirus neutralizing active nanobody according to claim 1, characterized in that The nanobody can recognize the antigenic epitope whose amino acid sequence is shown in SEQ ID NO.

7.

6. A nucleotide molecule, characterized in that The nucleotide molecule encodes the Nanobody according to any one of claims 1 to 5.

7. A recombinant vector plasmid containing the nucleotide molecule according to claim 6. A host cell containing the recombinant vector plasmid according to claim 7 .

9. Use of the nanobody according to any one of claims 1 to 5, the nucleotide molecule according to claim 6, the recombinant vector plasmid according to claim 7, or the host cell according to claim 8 in the preparation of a drug for preventing or treating novel coronavirus infection.

10. Use of the nanobody according to any one of claims 1 to 5, the nucleotide molecule according to claim 6, the recombinant vector plasmid according to claim 7, or the host cell according to claim 8 in the preparation of a kit for detecting novel coronavirus infection.

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