Anti-sars-cov-2 bispecific nanobody and preparation method and use thereof

Abstract

The application discloses an anti-novel coronavirus bispecific nanobody and a preparation method and application thereof, and belongs to the technical field of biological medicine. The application first prepares an anti-novel coronavirus parent nanobody with the SARS-CoV-2 Lambda strain S protein RBD domain as an antigen, and further constructs a bispecific nanobody capable of recognizing different antigen epitopes. The nanobody can be efficiently expressed in a prokaryotic system, is easy to produce industrially, is stable in physical and chemical properties, can be transported and stored at room temperature, is controllable in standard expression process, is low in production cost, is small in molecular weight, can pass through a physiological barrier, and recognizes a hidden epitope of an antigen. The bispecific anti-novel coronavirus nanobody provided by the application can effectively solve the problem of escape of novel coronavirus variants, has potential broad-spectrum neutralization capacity, and has important application value in prevention and treatment of novel coronavirus infection.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a bispecific nanobody against the novel coronavirus, its preparation method, and its uses. Background Technology

[0002] SARS-CoV-2, the novel coronavirus, is a single-stranded RNA virus that has mutated during transmission, resulting in various prevalent mutant strains. Examples include the Lambda, Delta, Omicron, XBB, and JN.1 mutant strains. The emergence of these mutants has led to immune escape from neutralizing antibodies, posing a potential risk to public health and property.

[0003] Currently, most approved monoclonal antibody drugs targeting the RBD domain, such as LY-CoV016+LY-CoV555 (bamlanivilab+etesevimab) and S309 (sotrovimab), have been evaded by variants, including AstraZeneca's Evusheld prophylactic antibody drug, which had previously entered the Chinese market. Due to the prevalence of these new mutant strains, the US FDA has also revoked Eli Lilly's authorization to use Bebtelovimab. Therefore, developing broadly neutralizing antibodies against SARS-CoV-2 mutants is an effective means of combating the spread of the virus.

[0004] Compared with monoclonal antibodies, nanobodies have many superior characteristics, such as small size (only 10-15kDa), low immunogenicity, high expression level, large-scale production capability, good stability, high temperature resistance, acid and alkali resistance, easy storage and transportation, and the ability to be designed into multivalent nanobodies to improve therapeutic effects.

[0005] In summary, the invention of a bispecific nanobody against SARS-CoV-2 with broad-spectrum neutralizing ability, low production cost, good heat resistance, and room temperature transport and storage shows promising clinical application prospects for the prevention and treatment of SARS-CoV-2. Summary of the Invention

[0006] In order to solve the above-mentioned problems in the existing technology, the purpose of this invention is to provide a bispecific nanobody against the novel coronavirus, its preparation method and uses.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides an anti-COVID-19 nanobody, wherein the complementarity-determining region of the anti-COVID-19 nanobody is composed of CDR1, CDR2, and CDR3. The amino acid sequence of CDR1 is shown in SEQ ID NO. 1, SEQ ID NO. 4, or SEQ ID NO. 7; the amino acid sequence of CDR2 is shown in SEQ ID NO. 2, SEQ ID NO. 5, or SEQ ID NO. 8; and the amino acid sequence of CDR3 is shown in SEQ ID NO. 3, SEQ ID NO. 6, or SEQ ID NO. 9.

[0008] Furthermore, the amino acid sequences of the anti-COVID-19 nanobody CDR1, CDR2 and CDR3 are shown in SEQ ID NO .1, SEQ ID NO .2 and SEQ ID NO .3, respectively.

[0009] Furthermore, the amino acid sequences of the anti-COVID-19 nanobody CDR1, CDR2 and CDR3 are shown in SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6, respectively.

[0010] Furthermore, the amino acid sequences of the anti-COVID-19 nanobody CDR1, CDR2 and CDR3 are shown in SEQ ID NO .7, SEQ ID NO .8 and SEQ ID NO .9, respectively.

[0011] Furthermore, the amino acid sequence of the anti-COVID-19 nanobody is shown in SEQ ID NO. 15, SEQ ID NO. 16 or SEQ ID NO. 17.

[0012] The present invention also provides a gene expressing an anti-SARS-CoV-2 nanobody, the nucleotide sequence of which is shown in SEQ ID NO. 10, SEQ ID NO. 11 or SEQ ID NO. 12.

[0013] The present invention also provides a bispecific nanobody against the novel coronavirus, the amino acid sequence of which is shown in SEQ ID NO. 18 or SEQ ID NO. 19.

[0014] The present invention also provides a gene expressing the above-mentioned anti-COVID-19 bispecific nanobody, the nucleotide sequence of which is shown in SEQ ID NO. 13 or SEQ ID NO. 14.

[0015] The present invention also provides a recombinant vector comprising the nucleotide sequence shown in SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13 or SEQ ID NO. 14.

[0016] The present invention also provides a recombinant cell comprising the recombinant vector described above.

[0017] The present invention has achieved the following beneficial effects: This invention first prepared a parental nanobody against SARS-CoV-2 using the RBD domain of the S protein of the SARS-CoV-2 Lambda strain as an antigen, and further constructed a bispecific nanobody capable of recognizing different antigenic epitopes. The nanobody of this invention can be efficiently expressed in prokaryotic systems, is easy to produce industrially, has stable physicochemical properties, good heat resistance, can be transported and stored at room temperature, has a standardized and controllable expression process, reduces production costs, has a small molecular weight, can cross physiological barriers, and recognize hidden antigenic epitopes. The bispecific anti-SARS-CoV-2 nanobody provided by this invention, capable of recognizing different antigenic epitopes, can effectively solve the problem of SARS-CoV-2 variant strain escape, possesses potentially broad-spectrum neutralizing ability, and has the potential to be developed into a nasal spray, showing significant application value in the prevention and treatment of SARS-CoV-2 infection.

[0018] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0019] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0020] Figure 1 These are DNA gel electrophoresis images of the seven anti-COVID-19 nanoantibodies screened by this invention.

[0021] Figure 2 These are the protein electrophoresis and gel filtration chromatography images of the seven anti-COVID-19 nanoantibodies screened by this invention after purification.

[0022] Figure 3 The binding activity of the anti-SARS-CoV-2 nanobody of this invention to the Spike protein and RBD protein of the SARS-CoV-2 Lambda mutant strain was detected by ELISA.

[0023] Figure 4The binding activity of the anti-SARS-CoV-2 nanobody of this invention to the RBD protein of SARS-CoV-2 Omicron BA.1 and BA.2 mutant strains was detected by ELISA.

[0024] Figure 5 The binding activity of the anti-SARS-CoV-2 nanobody of this invention to the S1 subunit of the spike protein of the SARS-CoV-2 Omicron BA.1 and BA.2 mutant strains was detected by ELISA.

[0025] Figure 6 The results of the neutralization experiment of the parental nanoantibodies against SARS-CoV-2 (L2, L25, L40) of this invention with SARS-CoV-2 Omicron BA.1 were used to test the results of the pseudovirus neutralization experiment.

[0026] Figure 7 The binding ability of parental nanobodies (L2, L25, L40) and bispecific nanobodies L2-L25 and L25-L40 to the SARS-CoV-2 Lambda rRBD domain was detected by MST assay. Detailed Implementation

[0027] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0028] The following experiments, where no temperature is specified, are reactions conducted under normal temperature conditions, which is room temperature, or 25±5℃.

[0029] Example 1: Construction of a single-chain antibody phage library The method for constructing a single-chain antibody phage library includes the following steps: (1) Mix 1 mg of protein (SARS-CoV-2 Lambda strain S protein RBD domain) with Freund's adjuvant in equal volume, emulsify it with an emulsifier and inject it into the neck lymph nodes of Bactrian camels. Immunize once every two weeks. After six to seven immunizations, collect blood, isolate peripheral blood lymphocytes after immunization in the laboratory, add lysis buffer and lyse.

[0030] (2) Take the lysate of peripheral blood lymphocytes after immunization, extract total RNA using chloroform, and reverse transcribe cDNA using RT-PCR reaction program; (3) Using the above cDNA as a template, the first round of amplification was carried out using specific primers; Upstream primer: 5'-GTCCTGGCTGCTCTTCTACAAGG-3' (SEQ ID No: 15) Downstream primer: 5'-GGTACGTGCTGTTGAACTGTTCC-3' (SEQ ID No: 16) The PCR amplification procedure is as follows: ① Pre-denaturation at 94℃ for 4 min; ② 94℃ denaturation for 1 min, 58℃ annealing for 30 s, 72℃ extension for 80 s, 35 cycles; ③ Extend at 72℃ for 10 minutes.

[0031] (4) Separate the PCR products obtained in step (3) by 1% agarose gel electrophoresis to determine the target band (Heavy-Chain Antibodies, hcABs) of about 700bp. Cut the gel and recover the product to obtain the first-round amplification product. Then, use the first-round amplification product as a template for the second round of amplification. Upstream primer: 5'-CCGTGGCCCAGGCGGCCGAGCAGGTGCAGCTGCAGGAGTCTGGRGGAGG-3' (SEQ ID No: 17) Downstream primer: 5'-CTCCTGGCCGGCCTGGCCTGCGGCCGCTGAGGAGACGGTGACCTGGGT-3' (SEQ ID No: 18) The second round of PCR amplification procedure is as follows: ① Pre-denaturation at 95℃ for 7 minutes; ② 94℃ denaturation for 45s, 58℃ annealing for 45s, 72℃ extension for 45s, 20 cycles; ③ Extend at 72℃ for 10 minutes.

[0032] The recovered nanobody DNA fragments and pComb3 vector were simultaneously double-digested with SfiI enzyme and ligated for recombination. The resulting fragments were then electroporated into E. coli TG1 competent cells and plated. Serial dilutions were performed to calculate library capacity. Forty-eight single clones were randomly selected from the plates for colony PCR, and the results were verified by nucleic acid gel electrophoresis. The positive rate was over 95%. Positive clones were sequenced, and amino acid sequences were analyzed using MEGA11 software. The results showed virtually no repetitive sequences, indicating good sequence diversity.

[0033] Example 2: Screening and expression of nanobodies from SARS-CoV-2 Lambda mutant strains (1) Take 3 mL of the nanobody phage library prepared in Example 1 and add it to 300 mL of 2×YT-GA medium at a ratio of 1:100. Incubate at 37℃ and 230 rpm until the logarithmic growth phase. Add 20 times the number of helper phage M13KO7, mix well, and incubate at 37℃ for 30 minutes, then shake for 60 minutes. Centrifuge, discard the medium, add 2YT ampicillin-kanamycin medium, and incubate at 30℃ overnight with shaking. Centrifuge the next day, collect the supernatant, add 1 / 5 volume of PEG / NaCl, precipitate the recombinant phage at 4℃ for 1 hour, centrifuge to collect the phage pellet, and dissolve it in 200 µL of PBS to obtain the phage library. At the same time, take 10 μL for 20-fold serial dilution, infect the logarithmic growth phase TG1, incubate overnight, and calculate the titer.

[0034] (2) Dilute the Lambda RBD antigen to 10 μg / mL with PBS buffer, mix thoroughly, and coat it in a 96-well microplate at 100 μL / well. Coat overnight at 4°C, discard the supernatant, add 300 µL of blocking buffer (3% OVA-PBS), block at 37°C for 1 hour, wash 3 times with PBS, and set aside.

[0035] (3) Screening: One round of selection involves an investment of 4×10 11 Recombinant phages of CFU volume were added to 100 µL / well of phage library and incubated at 37 °C for 1 h. The cells were washed 6 times with 0.1% PBST for 2 min each time. Finally, the phages bound to the plate were eluted with 100 µL Gly-HCl elution buffer, and the elution buffer was immediately neutralized with 1M Tris-HCl (pH 7.4). The mixture was then mixed with logarithmic growth phase TG1, incubated statically for 30 min, shaken, and then plated and incubated upside down at 37 °C overnight.

[0036] (4) Repeat the screening 2-3 times. The specific steps are as described in step (3). Theoretically, the titer of the selected product increases in each round.

[0037] (5) Phage ELISA verification: Select a single clone, culture at 37°C with shaking for 8 hours, add 20 times the amount of helper phage, incubate at 37°C for 15 minutes, then culture with shaking for 45 minutes. Replace the culture medium with 2YT ampicillin-kanamycin medium and culture at 30°C with shaking overnight. Take 100 μL of supernatant coated with 2 µg / mL Lambda-RBD antigen and add it to the enzyme-labeled wells at a rate of 100 µL / well. Coat overnight at 4°C. Wash the plate 4 times with 0.1% PBST, add 100 μL of HRP-labeled anti-M13 antibody to each well, incubate at 37°C for 1 hour, wash the plate 5 times with 0.1% PBST, add 100 μL of TMB chromogenic substrate to each well, incubate at 37°C in the dark for 15 minutes, add 50 μL of 1M phosphate to stop the reaction, detect the absorbance at 450 nm, and send 100 µL of the positive clone culture for sequencing.

[0038] (6) Expression of BL21 in *E. coli*: Positive clones verified by phage ELISA were sequenced to obtain antibodies with different sequences. Different antibody sequences were amplified by PCR and constructed into the pet20b vector, such as... Figure 1 As shown in the figure. The plasmid with the correct sequence was transformed into BL21 E. coli for expression. After collecting the bacteria, they were lysed under high pressure and purified by affinity chromatography, finally obtaining the desired result. Figure 2 The seven purified anti-SARS-CoV-2 nanobodies shown are L2, L6, L12, L15, L23, L25, and L40. (R5 is a commercially available anti-SARS-CoV-2 wild-type RBD nanobodies, used as a positive control.) Example 3: ELISA method for detecting the binding ability of the nanobody of the present invention (1) Dilute the mutant RBD protein (Lambda-RBD, Omicron-RBD) or the full-length S protein of the OmicronBA.1 mutant to 2 μg / mL with PBS, add 100 µL / well to the microplate, and coat overnight at 4℃; remove the microplate and wash it twice with 0.05% PBST, pat dry the liquid in the microplate, add blocking buffer (PBST containing 5% skim milk, 5% PBSTM), 100 µL / well, and incubate at 37℃ for 1 h.

[0039] (2) Wash the plate twice with 0.05% PBST and blot dry the liquid inside the plate. Add 100 µL of the purified nanobody obtained in Example 2 (serially diluted twofold) to each well and incubate at 37 °C for 1 h. Wash the plate four times with 0.05% PBST and blot dry. Add 100 µL of the secondary antibody HRP_x0002_Anti-DYKDDDDK (1:30000) diluted with 5% skim milk to each well and incubate at 37 °C for 1 h. Wash the plate five times with 0.05% PBST and blot dry. Add 100 µL of TMB single-component chromogenic solution to each well and incubate at 37 °C for 8 min. Add 50 µL of phosphate stop solution to each well and gently shake to mix. Read the absorbance value at 450 nM. The results are as follows. Figure 3-5 As shown, L2, L25, L40, and L23 can bind not only the Lambda RBD domain but also the RBD domain or the S1 subunit of the spike protein in OmicronBA.1 and OmicronBA.2 mutant strains. ELISA assays showed that L2, L25, L40, and L23 bind to the EC1 subunit of the full-length S protein of OmicronBA.1. 50 The values ​​were 9.53 nM, 7.142 nM, 15.39 nM, and 109.8 nM, respectively.

[0040] The CDR1 of nanobody L2 is the amino acid sequence shown in SEQ ID NO. 1, the CDR2 is the amino acid sequence shown in SEQ ID NO. 2, and the CDR3 is the amino acid sequence shown in SEQ ID NO. 3; the CDR1 of nanobody L25 is the amino acid sequence shown in SEQ ID NO. 4, the CDR2 is the amino acid sequence shown in SEQ ID NO. 5, and the CDR3 is the amino acid sequence shown in SEQ ID NO. 6; the CDR1 of nanobody L40 is the amino acid sequence shown in SEQ ID NO. 7, the CDR2 is the amino acid sequence shown in SEQ ID NO. 8, and the CDR3 is the amino acid sequence shown in SEQ ID NO. 9.

[0041] SEQ ID NO. 1: NYYMG.

[0042] SEQ ID NO.2:GIEADGTTSYADSVKG.

[0043] SEQ ID NO.3: DEATWVASIRTLTWNY.

[0044] SEQ ID NO. 4: RYCMG.

[0045] SEQ ID NO.5: TIDKNGLTTYTESVKG.

[0046] SEQ ID NO.6: DPPSIETMTCDVLAFWVNH.

[0047] SEQ ID NO. 7: AYCMN.

[0048] SEQ ID NO.8:AIDSDGNTRYAESVKG.

[0049] SEQ ID NO.9: ARGSGGACYGYDFNYYGMDY.

[0050] The nucleotide sequence of nanobody L2 is shown in SEQ ID NO. 10; the nucleotide sequence of nanobody L25 is shown in SEQ ID NO. 11; and the nucleotide sequence of nanobody L40 is shown in SEQ ID NO. 12.

[0051] The amino acid sequence of nanobody L2 is shown in SEQ ID NO. 15; the amino acid sequence of nanobody L25 is shown in SEQ ID NO. 16; and the amino acid sequence of nanobody L40 is shown in SEQ ID NO. 17.

[0052] SEQ ID NO. 10: ATGCAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTCGTGCAGGCTGGAGGGTCTCTGAGACTCACCTGTGCAGCCTCTGGATCCATCGACAGTAACTACTACATGGGCTGGTTCCGCCAGGCTCCAGGAAAGGAGCGCGAGGGACTCGCAGGTATTGAAGCTGATGGAACCACGTCCTACGCAGACTCCGTGA AGGGCCGATTCACCATCTCCAAAGACAACGCCCAGAACACCGTGTATCTACAAATGAACAGCCTCAAACCTGAGGACTCTGGCATGTACTACTGTGCGGCAGACGAGGCGACGTGGGTCTCGATACGGACTTTGACCTGGAACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCGGCGGCCGCACTCGAG SEQ ID NO. 11: ATGCAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGGCTCTCCTGTTTAGTCTCCGGAGACACTTCCAGCCGCTACTGCATGGGCTGGTTCCGCCAGGCTCCGTGGAAGGAGCGCGAAGCGGTCGCGACTATTGATAAGAATGGTTTAACAACGTACACAGAATCCGTGAAGGGCCGATTCACCATCTCCAAAGACAACGCCAAGAACACTCTCAATCTACAAATGAACAGCCTGAAACCCGAGGACAGTGCCATGTACTACTGTGCGGCAGATCCACCCAGTATAGAGACTATGACGTGTGATGTCTTGGCGTTTTGGGTTAATCACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCGGCGGCCGCACTCGAG SEQ ID NO .12: ATGCAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATACAGCTTTGGTGCATACTGCATGAACTGGTTCCGCCAGACTCCAGGGAAGGAGCGCGAGGCGGTCGCGGCTATTGATAGTGATGGTAACACAAGGTACGCAGAGTCCGTGAAGGGCCGATTCACCATCTCCAAAGACAACGCCAAGAACACTCTGTATCTGCAAATGGACAGCCTGAAACCTGAAGACACTGCCACGTACTACTGTGCGGCCGCCCGCGGTAGTGGTGGTGCCTGCTACGGGTACGACTTTAATTACTACGGCATGGACTACTGGGGCAAAGGAACCCAGGTCACCGTCTCCTCGGCGGCCGCACTCGAG SEQ ID NO .15: MQVQLQESGGGSVQAGGSLRLTCAASGSIDSNYYMGWFRQAPGKEREGLAGIEADGTTSYADSVKGRFTISKDNAQNTVYLQMNSLKPEDSGMYYCADEATWVSIRTLTWNYWGQGTQVTVSSAAALE SEQ ID NO. 16: MQVQLQESGGGSVQAGGSLRLSCLVSGDTSSRYCMGWFRQAPWKEREAVATIDKNGLTTYTESVKGRFTISKDNAKNTLNLQMNSLKPEDSAMYYCAADPPSIETMTCDVLAFWVNHWGQGTQVTVSSAAALE SEQ ID NO. 17: MQVQLQESGGGSVQAGGSLRLSCAASGYSFGAYCMNWFRQTPGKEREAVAAIDSDGNTRYAESVKGRFTISKDNAKNTLYLQMDSLKPEDTATYYCAAARGSGGACYGYDFNYYGMDYWGKGTQVTVSSAAALE Example 4: Detection of the neutralizing ability of the nanobody of the present invention against OmicronBA.1 using a pseudovirus method (1) 5 µg of SARS-CoV-2-S-pcDNA3.1 plasmid and 5 µg of pNL4-3.Luc.RE plasmid were co-transfected into 293T cells with 30 µg PEI (polyethyleneimine). After changing the medium, the supernatant was collected after 48 h of culture and centrifuged at 800 rpm for 3 min to remove cell debris. The pseudovirus was aliquoted into 1 mL tubes and stored at -80℃ for later use. The pseudovirus was serially diluted with DMEM complete medium and then used to infect 293T cells transfected with hACE2 receptor. After 48 h, the 96-well cell culture plate was removed, the liquid in the wells was discarded, and 65 µL of substrate was added. After reacting in the dark at room temperature for 5 min, the reaction was performed using the Luciferase preset program in the luminescence detector. The pseudovirus titer was calculated based on the fluorescence value (relative light unit, RLU).

[0053] (2) 293T cells expressing hACE2 were evenly seeded into 96-well plates and incubated at 27 ℃ with 5% CO2 for 18-24 h. The antibody was serially diluted with sterile PBS, and 10 µL of each antibody concentration was added to 90 µL of Omicron mutant pseudovirus. After mixing, the mixture was incubated at 37 ℃ for 1 h. The culture plate was placed in a 37 ℃ incubator and incubated for 48 h. The supernatant was discarded, and 65 µL of substrate was added to each well. The reaction was carried out at room temperature in the dark for 5 min. The fluorescence value was then detected and the half-maximal inhibitory concentration (IC50) of the antibody was calculated. 50 )like Figure 6 As shown, L25, L2, and L40 all exhibit strong neutralizing activity, effectively inhibiting the invasion of Omicron pseudoviruses into host cells. Furthermore, the inhibition rate increases with increasing antibody concentration. The half-maximal inhibitory concentration (IC50) of L25, L2, and L40 is [not specified in the original text]. 50 The values ​​are 3.729 nM, 34.67 nM, and 0.7 nM, respectively.

[0054] Example 5: MST method for detecting the binding ability of the nanobodies L2-L25 and L25-L40 of the present invention to Lambda RBD Methods for preparing bispecific nanobodies: A gene with the nucleotide sequence shown in SEQ ID NO. 13 was constructed and expressed and purified using the vector pcomb3 to obtain the fusion protein, namely the bispecific nanobody L2-L25. The amino acid sequence of L2-L25 is the amino acid sequence of the two nanobodies L2 and L25 linked by (GGGGS)3, as shown in SEQ ID NO. 18.

[0055] A gene with the nucleotide sequence shown in SEQ ID NO. 14 was constructed and expressed and purified using the vector pcomb3 to obtain the fusion protein, namely the bispecific nanobody L25-L40. The amino acid sequence of L25-L40 is the amino acid sequence of the two nanobodies L25 and L40 linked by (GGGGS)3, as shown in SEQ ID NO. 19.

[0056] The nucleotide sequences of bispecific nanobodies L2-L25 are shown in SEQ ID NO. 13; the nucleotide sequences of bispecific nanobodies L25-L40 are shown in SEQ ID NO. 14.

[0057] SEQ ID NO. 13: ATGCAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGGCTCTCCTGTTTAGTCTCCGGAGACACTTCCAGCCGCTACTGCATGGGCTGGTTCCGCCAGGCTCCGTGGAAGGAGCGCGAAGCGGTCGCGACTATTGATAAGAATGGTTTAACAACGTACACAGAATCCGTGAAGGGCCGATTCACCATCTCCAAAGACAACGCCAAGAACACTCTCAATCTACAAATGAACAGCCTGAAACCCGAGGACAGTGCCATGTACTACTGTGCGGCAGATCCACCCAGTATAGAGACTATGACGTGTGATGTCTTGGCGTTTTGGGTTAATCACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGGAGGTGGAGGTAGTGGAGGTGGAGGTTCTGGTGGCGGAGGGAGCCAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCACCTGTGCAGCCTCTGGATCCATCGACAGTAACTACTACATGGGCTGGTTCCGCCAGGCTCCAGGAAAGGAGCGCGAGGGACTCGCAGGTATTGAAGCTGATGGAACCACGTCCTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAAAGACAACGCCCAGAACACCGTGTATCTACAAATGAACAGCCTCAAACCTGAGGACTCTGGCATGTACTACTGTGCGGCAGACGAGGCGACGTGGGTCTCGATACGGACTTTGACCTGGAACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCGGCGGCCGCACTCGAG SEQ ID NO .14: ATGCAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGGCTCTCCTGTTTAGTCTCCGGAGACACTTCCAGCCGCTACTGCATGGGCTGGTTCCGCCAGGCTCCGTGGAAGGAGCGCGAAGCGGTCGCGACTATTGATAAGAATGGTTTAACAACGTACACAGAATCCGTGAAGGGCCGATTCACCATCTCCAAAGACAACGCCAAGAACACTCTCAATCTACAAATGAACAGCCTGAAACCCGAGGACAGTGCCATGTACTACTGTGCGGCAGATCCACCCAGTATAGAGACTATGACGTGTGATGTCTTGGCGTTTTGGGTTAATCACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGGAGGTGGAGGTAGTGGAGGTGGAGGTTCTGGTGGCGGAGGGAGCCAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATACAGCTTTGGTGCATACTGCATGAACTGGTTCCGCCAGACTCCAGGGAAGGAGCGCGAGGCGGTCGCGGCTATTGATAGTGATGGTAACACAAGGTACGCAGAGTCCGTGAAGGGCCGATTCACCATCTCCAAAGACAACGCCAAGAACACTCTGTATCTGCAAATGGACAGCCTGAAACCTGAAGACACTGCCACGTACTACTGTGCGGCCGCCCGCGGTAGTGGTGGTGCCTGCTACGGGTACGACTTTAATTACTACGGCATGGACTACTGGGGCAAAGGAACCCAGGTCACCGTCTCCTCGGCGGCCGCACTCGAG SEQ ID NO .18: MQVQLQESGGGSVQAGGSLRLSCLVSGDTSSRYCMGWFRQAPWKEREAVATIDKNGLTTYTESVKGRFTISKDNAKNTLNLQMNSLKPEDSAMYYCAADPPSIETMTCDVLAFWVNHWGQGTQVTVSSGGGGSGG GGSGGGGSQVQLQESGGGSVQAGGSLRLTCAASGSIDSNYYMGWFRQAPGKEREGLAGIEADGTTSYADSVKGRFTISKDNAQNTVYLQMNSLKPEDSGMYYCAADEATWVSIRTLTWNYWGQGTQVTVSSAAALE SEQ ID NO. 19: MQVQLQESGGGSVQAGGSLRLSCLVSGDTSSRYCMGWFRQAPWKEREAVATIDKNGLTTYTESVKGRFTISKDNAKNTLNLQMNSLKPEDSAMYYCAADPPSIETMTCDVLAFWVNHWGQGTQVTVSSGGGGSGGGGS GGGGSQVQLQESGGGSVQAGGSLRLSCAASGYSFGAYCMNWFRQTPGKEREAVAAIDSDGNTRYAESVKGRFTISKDNAKNTLYLQMDSLKPEDTATYYCAAARGSGGACYGYDFNYYGMDYWGKGTQVTVSSAAALE (1) Dilute the fluorescently labeled RBD to 100 nM with 0.05% PBST and mix well for later use; dilute the nanobody to be tested with 0.05% PBST by two-fold and incubate it with the fluorescently labeled RBD protein at room temperature for 30 min.

[0058] (2) Samples were aspirated and tested on the instrument, with LED / excitation power set to 40% and medium MST power. The results of repeated measurements were analyzed using the MST affinity analysis software MO. Affinity Analysis to determine the dissociation equilibrium constant (KD value). The results are as follows: Figure 7 As shown, compared with the parental nanobody, the bispecific nanobodies L2-L25 and L25-L4 of this invention significantly improve the binding ability with Lambda RBD.

[0059] In summary, this invention first prepared a parental nanobody against SARS-CoV-2 using the RBD domain of the S protein of the SARS-CoV-2 Lambda strain as an antigen, and further constructed a bispecific nanobody capable of recognizing different antigenic epitopes. The nanobody of this invention can be efficiently expressed in prokaryotic systems, is easy to produce industrially, has stable physicochemical properties, can be transported and stored at room temperature, has a standardized and controllable expression process, low production cost, small molecular weight, and can cross physiological barriers to recognize hidden antigenic epitopes. The bispecific anti-SARS-CoV-2 nanobody provided by this invention, capable of recognizing different antigenic epitopes, can effectively solve the problem of SARS-CoV-2 variant strain escape, possesses potentially broad-spectrum neutralizing ability, and has the potential to be developed into a nasal spray, thus having important application value in the prevention and treatment of SARS-CoV-2 infection.

Claims

1. A nanobody against the novel coronavirus, characterized in that: The complementarity-determining region of the anti-SARS-CoV-2 nanobody is composed of CDR1, CDR2, and CDR3. The amino acid sequence of CDR1 is shown in SEQ ID NO. 1, SEQ ID NO. 4, or SEQ ID NO. 7, the amino acid sequence of CDR2 is shown in SEQ ID NO. 2, SEQ ID NO. 5, or SEQ ID NO. 8, and the amino acid sequence of CDR3 is shown in SEQ ID NO. 3, SEQ ID NO. 6, or SEQ ID NO.

9.

2. The anti-COVID-19 nanobody according to claim 1, characterized in that: The amino acid sequences of the anti-COVID-19 nanoantibody CDR1, CDR2 and CDR3 are shown in SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3, respectively.

3. The anti-COVID-19 nanobody according to claim 1, characterized in that: The amino acid sequences of the anti-COVID-19 nanobody CDR1, CDR2 and CDR3 are shown in SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6, respectively.

4. The anti-COVID-19 nanobody according to claim 1, characterized in that: The amino acid sequences of the anti-COVID-19 nanobody CDR1, CDR2 and CDR3 are shown in SEQ ID NO. 7, SEQ ID NO. 8 and SEQ ID NO. 9, respectively.

5. The anti-COVID-19 nanobody according to claim 1, characterized in that: The amino acid sequence of the anti-COVID-19 nanobody is shown in SEQ ID NO. 15, SEQ ID NO. 16 or SEQ ID NO.

17.

6. A gene expressing an anti-SARS-CoV-2 nanobody, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO. 10, SEQ ID NO. 11 or SEQ ID NO.

12.

7. A bispecific nanobody against the novel coronavirus, characterized in that: The amino acid sequence of the anti-COVID-19 bispecific nanobody is shown in SEQ ID NO. 18 or SEQ ID NO.

19.

8. A gene expressing the anti-SARS-CoV-2 bispecific nanobody of claim 7, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO. 13 or SEQ ID NO.

14.

9. A recombinant vector, characterized in that: The recombinant vector comprises the nucleotide sequence shown in SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13 or SEQ ID NO.

14.

10. A recombinant cell, characterized in that: The recombinant cells include the recombinant vector as described in claim 9.

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