Shark-derived nanobodies targeting sars-cov-2 rbd protein and uses

Abstract

This invention discloses a shark-derived nanobody targeting the SARS-CoV-2 RBD protein and its applications. The nanobody's nucleotide sequence is shown in SEQ ID NO: 1, and its amino acid sequence is shown in SEQ ID NO: 5. Preparation involves immunizing a striped bamboo shark with the RBD protein, extracting total RNA from peripheral blood lymphocytes and reverse transcribing it into cDNA. The amplified VNAR fragment is then seamlessly ligated with amplified pR2 phage particles, transformed into TG1 competent cells, and a phage antibody library is constructed. SARS-CoV-2 RBD protein-specific nanobody sequences are screened from the library and recombinantly expressed to obtain the targeted shark-derived nanobody. The nanobody of this invention has a small molecular weight, allowing for the detection of the SARS-CoV-2 RBD antigen using paired nanobodies. It can be applied to immunoblotting, enzyme-linked immunosorbent assays (ELISA), and COVID-19 pharmaceutical applications related to the SARS-CoV-2 RBD protein.

Description

Shark-derived nanobodies targeting the SARS-CoV-2 RBD protein and their applications

[0001] This invention application is a divisional application with application number 2022101043013 and application date of 2022-01-28. Technical Field

[0002] This invention relates to a SARS-CoV-2 antibody and its preparation method in the field of biotechnology, specifically to a shark-derived nanobody targeting the SARS-CoV-2 RBD and its preparation method. Background Technology

[0003] SARS-CoV-2 is a coronavirus that enters cells and completes infection by binding to angiotensin-converting enzyme 2 (ACE2) on the surface of epithelial cells through the receptor-binding domain (RBD) of its spike protein.

[0004] Fully human antibodies isolated from recovered patients have been shown to have excellent antiviral activity. These traditional monoclonal antibodies consist of two heavy chains and two light chains, which have limitations such as large molecular weight, complex manufacturing processes, and difficulty in processing and modification. In contrast to traditional antibodies, camels or cartilaginous fish (such as sharks) possess antibodies composed of only two heavy chains, named heavy chain antibodies. Their variable region consists of only two identical heavy chain variable regions, and this region is called a single-domain antibody (sdAb). Single-domain antibody proteins are less than 10 nanometers in diameter, hence they are also called nanobodies. Alpaca and shark single-domain antibodies are called VHH and VNAR, respectively. Single-domain antibodies have many advantages over traditional scFv or Fab antibodies, including smaller molecular weight, stronger penetration, higher stability and solubility, and their function is independent of glycosylation modifications. Compared to traditional antibodies, single-domain antibodies typically have an extended CDR3, which can form a protruding surface structure to recognize antigenic epitopes, thus helping to identify hidden antigenic epitopes that are difficult for traditional antibodies to recognize. Currently, alpaca single-domain antibodies are the most advanced in research and development, having made significant progress in exploring the origin of antigen receptors and in developing vaccines, therapeutic drugs, diagnostic reagents, and biotechnology research tools. Similarly, shark-derived single-domain antibodies can be developed into diagnostic reagents and therapeutic antibody drugs. Currently, there are no patents for shark-derived nanobody sequences against SARS-CoV-2 RBD. Summary of the Invention

[0005] To address the problems existing in the background art, the present invention provides a shark-derived heavy chain antibody variable region sequence (VNAR) that can bind to the receptor-binding domain (RBD) of the SARS-CoV-2 virus with high affinity. This variable region sequence is also known as a nanobody, which can be used for the prevention, treatment and / or diagnosis of SARS-CoV-2 infection and can be used in related pharmaceutical applications.

[0006] The technical solution of the present invention is as follows:

[0007] I. A shark-derived nanobody targeting SARS-CoV-2 RBD:

[0008] The nucleotide sequence of the shark-derived nanobody is shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, or SEQ ID NO.4.

[0009] The amino acid sequences of the shark-derived nanoantibodies are shown in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, or SEQ ID NO.8, and the antibodies are named sh-aRBD-2, sh-aRBD-5, sh-aRBD-17, and sh-aRBD-18, respectively.

[0010] II. A method for preparing shark-derived nanobodies targeting SARS-CoV-2 RBD:

[0011] The preparation method of the shark-derived nanobody is carried out according to the following steps:

[0012] Sharks were immunized with in vitro recombinant SARS-CoV-2 RBD protein as an antigen. Peripheral blood was collected after immunization. Lymphocytes were obtained from the peripheral blood by density gradient centrifugation using a 30% sucrose solution. Total RNA was extracted from the lymphocyte solution and then reverse transcribed into cDNA.

[0013] Using cDNA as a template, the VNAR gene sequence was amplified with specific primers. The VNAR gene sequence was then inserted into the pR2 phage amplification product, electroporated into TG1 competent bacteria, cultured on a plate, and colonies were scraped from the plate to obtain a phage display antibody library. After multiple screenings, recombinant expression, and purification, the SARS-CoV-2RBD shark-derived nanobody was obtained.

[0014] This method characterizes the affinity of nanobodies for SARS-CoV-2RBD, yielding high-affinity shark-derived nanobodies targeting the SARS-CoV-2RBD protein; and the paired nanobodies are then used to detect the SARS-CoV-2RBD antigen.

[0015] The multiple screening, recombinant expression, and purification process involves the following steps: the RBD phage antibody library is first panned and screened by phage ELISA to obtain positive clones, and the antibody sequences are identified by sequencing. The antibody sequences are then constructed into a recombinant expression vector, induced to express, and purified to obtain SARS-CoV-2 RBD shark-derived nanobodies.

[0016] The specific protocol for immunizing sharks is as follows: a total of five immunizations are administered, with the first three being subcutaneous injections and the last two being tail vein injections; the interval between subcutaneous injections is 10 days, and the interval between tail vein injections is 30 days; the tail vein injection is administered 30 days after the third subcutaneous injection; and peripheral blood of the shark is collected via the tail vein 15 days after the last tail vein injection.

[0017] The forward primer used for amplifying the VNAR gene sequence is SEQ ID NO.11, namely 5'-GCTGCA CAGCCTGCTATGGCAACTCAACGGGTTGAACAAACACCGA-3', and the reverse primer is SEQ ID NO.12, namely 5'-GAGTTTTTGTTCGGCTGCTGCTGGTTTTACAGTCAGA ATGGTGCCGC-3'.

[0018] The pR2 phage amplification product was obtained by amplifying pR2 phage particles. The forward primer used was SEQ ID NO.13, i.e., 5'-AGCAGCCGAACAAAAACTCATCTCAGAAGAG-3'; the reverse primer was SEQ ID NO.14, i.e., 5'-CCATAGCAGGCTGTGCAGCATAGAAAGGTACCA CTAAAGGAATTGC-3'.

[0019] III. A biepisode dimerizing antibody:

[0020] A dual-epitope dimerized antibody was constructed using SARS-CoV-2RBD shark-derived nanobodies, by linking two SARS-CoV-2RBD shark-derived nanobodies with different sequences using a flexible polypeptide chain. The two SARS-CoV-2RBD shark-derived nanobodies have different sequences.

[0021] The biepithelialized antibody refers to an antibody that can bind to the two epitopes of SARS-CoV-2RBD by connecting two nanoantibodies that are respectively bound to two independent epitopes on SARS-CoV-2RBD with a flexible polypeptide chain.

[0022] The nucleotide sequence of the flexible polypeptide chain is shown in SEQ ID NO.9, and the amino acid sequence is shown in SEQ ID NO.10.

[0023] The dual-epitope dimerized antibody exhibits superior performance compared to the SARS-CoV-2 RBD shark-derived nanobody before being linked with a flexible polypeptide chain.

[0024] When the shark-derived nanobodies of the present invention were paired, the RBD antigen protein was detected by colloidal gold method. Among them, the detection limit of the paired sh-aRBD-2 and sh-aRBD-18 antibodies was the lowest, which was 0.390 μg / mL.

[0025] Subsequently, this invention discovered that the four nanobodies prepared can recognize three epitopes of SARS-CoV-2RBD, of which sh-aRBD-5 and sh-aRBD-17 bind to the same epitope of SARS-CoV-2RBD, while sh-aRBD-2 and sh-aRBD-18 bind to two other different epitopes, respectively. The detection limit for sh-aRBD-2 and sh-aRBD-18 is even lower, at 0.390 μg / mL.

[0026] Therefore, biepisode dimerizing antibodies sh-aRBD-2-5, sh-aRBD-2-17, sh-aRBD-2-18, sh-aRBD-5-18, and sh-aRBD-17-18 were constructed using these combinations.

[0027] Applications of the shark-derived nanobody in Western blot analysis, enzyme-linked immunosorbent assay (ELISA), and COVID-19 pharmaceutical manufacturing of SARS-CoV-2 RBD protein.

[0028] The SARS-CoV-2RBD shark-derived nanobodies were used to detect SARS-CoV-2RBD antigen proteins and to screen for paired antibodies with low detection limits.

[0029] The beneficial effects of this invention are:

[0030] The shark nanobody of this invention has a small molecular weight, simple structure, and good stability. The shark-derived nanobody of this invention has a molecular weight of only 12kDa, which is about 1 / 10 of that of conventional antibodies and about 20% smaller than alpaca nanobody. It is the smallest known natural single-domain antibody in vertebrates. It can be expressed in vitro using prokaryotic or eukaryotic expression systems, is easy to prepare, has high expression levels, and is low in cost.

[0031] The shark-derived nanobody described in this invention can be applied to Western blot analysis of SARS-CoV-2 RBD protein, enzyme-linked immunosorbent assay (ELISA), and pharmaceutical applications for the prevention, treatment, and diagnosis of COVID-19. Attached Figure Description

[0032] Figure 1 shows the SARS-CoV-2 RBD protein immunization protocol for sharks.

[0033] Figure 2 shows the results of monoclonal phage ELISA.

[0034] Figure 3 shows the amino acid sequence alignment results of the nanobody.

[0035] Figure 4 shows the SDS-PAGE gel electrophoresis results of the nanobody Fc fusion protein (A) and nanobody (B). Lane M represents the standard protein.

[0036] Figure 5 shows the results of ELISA characterization of the binding between the nanobody Fc fusion protein and SARS-CoV-2 RBD.

[0037] Figure 6 shows the results of gel filtration chromatography characterizing the binding between the nanobody Fc fusion protein and SARS-CoV-2 RBD.

[0038] Figure 7 shows the affinity between the nanobody Fc fusion protein (A) and nanobody (B) and SARS-CoV-2 RBD, characterized using BLI. The solid lines represent the real-time monitored kinetic curves, and the dashed lines represent the software-fitted curves. The kinetic curves for different antibody concentration gradients correspond from top to bottom to the concentrations indicated on the right, from top to bottom.

[0039] Figure 8 shows the results of the paired detection of RBD antigen protein by the nanobodies. The concentration of the detected RBD antigen is indicated above the test strip, decreasing by a factor of two from 50 μg / mL to 0.097 μg / mL. Test strips recorded as 1-2 are those with the test line coated with sh-aRBD-2 and those with colloidal gold-labeled sh-aRBD-18. The remaining test strips are all colloidal gold-labeled with sh-aRBD-2, and the antibody information at the test line is shown above the test strip.

[0040] Figure 9 shows the affinity between the dimerized nanobody and SARS-CoV-2 RBD characterized using BLI. Detailed Implementation

[0041] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. The following embodiments are explanations of the present invention, but the present invention is not limited to the following embodiments.

[0042] Example 1: Immunizing sharks with SARS-CoV-2 RBD protein

[0043] Sharks were immunized using recombinantly expressed SARS-CoV-2 RBD protein, as shown in Figure 1. A total of five immunizations were administered: the first three were subcutaneous injections, and the last two were tail vein injections. The interval between subcutaneous injections was 10 days, and the interval between tail vein injections was 30 days. The tail vein injection was performed 30 days after the third subcutaneous immunization. Peripheral blood was collected from the sharks via the tail vein 15 days after the tail vein injection. Three striped bamboo sharks were used in this invention, with each shark receiving 250 μg of RBD protein per immunization.

[0044] Example 2: Screening of SARS-CoV-2 RBD nanobodies

[0045] 1) Slowly add the extracted shark blood to the supernatant of an equal volume of 30% (m / v) sucrose solution, centrifuge at 500g for 20 min, collect the intermediate lymphocyte layer, wash with PBS, and centrifuge to collect the cell pellet. Extract total RNA using an RNA extraction kit and reverse transcribe it into cDNA.

[0046] 2) Using cDNA as a template, the VNAR sequence was amplified with specific primers. The forward primer was 5'-GCTGCACAGCCTGCTATGGCAACTCAACGGGTTGAACAAACACCGA-3', and the reverse primer was 5'-GAGTTTTTGTTCGGCTGCTGCTGGTTTTACAGTCAGAATG GTGCCGC-3'. A high-fidelity DNA polymerase was used for amplification, and the amplification program was: 98℃, 10s; 57℃, 15s; 72℃, 25s, for 30 cycles. The amplified VNAR fragment was recovered using a kit. Using pR2 phage as a template, the pR2 phage was amplified with specific primers. The forward primer was 5'-AGCAGCCGAACAAAAACTCATCTCAGAAGAG-3', and the reverse primer was 5'-CCATAGCAGGCTGTGCAGCATAGAAAGGTACCACTAAAGGAATTGC-3'. The DNA polymerase used for amplification was a high-fidelity enzyme, and the amplification program was: 98℃, 10s; 53℃, 15s; 68℃, 4min, 15s, for 30 cycles. The template pR2 phage was digested in the amplification product using Nco I and Not I, and then the amplified pR2 phage was recovered using a kit. The pR2 phage and VNAR fragment were ligated using a seamless clone at a molar ratio of 1:4. The ligation product was electroporated into TG1 competent cells and cultured at 37℃, 200rpm for 1h. 0.2μL and 0.02μL (dilution method) of the bacterial culture were then spread onto 10cm solid plates and cultured for 13h. The colony count was then performed, and the size of the constructed antibody library was calculated. The remaining bacterial culture was centrifuged and spread onto 150mm solid plates, cultured at 37℃ for 13h, and then the bacterial colony was scraped off, flash-frozen in liquid nitrogen, and stored at -80℃. This constitutes the nanobody library.

[0047] 3) Activate the frozen antibody library bacteria and add KM13 helper phage to assist the growth and reproduction of M13 phage. Take the bacterial culture supernatant and measure the phage titer; this is the amplified phage. Coat the SARS-CoV-2 RBD antigen onto the immunoassay plate at a concentration of 0.1 mg / mL and add 1 x 10⁻⁶ ppm. 11 Phages amplified with PFU or higher were incubated at room temperature for 1 hour. Phages specifically bound to the SARS-CoV-2 RBD antigen were eluted with trypsin and then used to infect TG1 bacteria. 50 μL and 5 μL of the infecting bacterial suspension were plated on solid plates, and the total colony count was recorded.

[0048] 4) Randomly select 95 single colonies from the above plates and activate them overnight. Add KM13 helper phage, centrifuge, and collect the supernatant after lysis; this is the monoclonal phage. Coat SARS-CoV-2RBD antigen at a concentration of 1 μg / mL onto a 96-well immunoassay plate, adding the prepared monoclonal phage solution to each well, and incubate at room temperature for 1 h. Capture the phages bound to the SARS-CoV-2RBD antigen using HRP-anti-M13 antibody, and develop the color with TMB substrate for 5 min. Terminate the reaction with 1M H2SO4 solution. Record the OD using a microplate reader. 450 The values ​​were then compiled into a bar chart as shown in Figure 2.

[0049] 5) Select OD 450 Wells with a value greater than 1 were used for sequencing analysis. The sequencing primers were: 5'-CCCTCATAGTTAGCGTAACGA-3'.

[0050] 6) By comparing and analyzing the obtained antibody sequences and excluding duplicate clones, four different nanobody sequences were obtained (Figure 3). SEQ ID NO.1-4 shows the nanobody nucleotide sequences, from which the nanobody amino acid sequences shown in SEQ ID NO.5-8 can be obtained.

[0051] Example 3: Expression and purification of SARS-CoV-2 RBD nanobody and its Fc fusion protein

[0052] The nanobody sequence of this invention was constructed into the mammalian expression vector pTT5 with a signal peptide, and then fused with human IgG1 Fc for expression. The Fc fragment was expressed at the C-terminus and could be digested with TEV enzyme to obtain the nanobody. The recombinant plasmid was transfected into HEK 293 cells, and the culture supernatant was collected and purified by rProtein A affinity chromatography. As shown in Figure 4A, we obtained high-purity SARS-CoV-2RBD nanobody Fc fusion protein. After TEV digestion, high-purity SARS-CoV-2RBD nanobody protein was obtained (Figure 4B).

[0053] Example 4 characterizes the SARS-CoV-2 RBD nanobody.

[0054] 1) The binding of the nanobody Fc fusion protein to SARS-CoV-2 RBD was characterized by gel filtration chromatography. The nanobody Fc fusion protein and SARS-CoV-2 RBD protein were mixed at a molar ratio of 2:1, incubated on ice for 1 h, and then loaded onto a Superdex 200 gel filtration chromatography column. The absorbance change at 280 nm was recorded, and a chromatographic pattern was plotted. Simultaneously, nanobody Fc fusion protein and SARS-CoV-2 RBD protein controls were set up. As shown in Figure 5, sh-aRBD-2-Fc, sh-aRBD-5-Fc, and sh-aRBD-18-Fc could all bind to SARS-CoV-2 RBD, while sh-aRBD-17-Fc showed a weaker binding affinity to SARS-CoV-2 RBD.

[0055] 2) The binding of the nanobody Fc fusion protein to SARS-CoV-2 RBD was characterized using a non-competitive ELISA: SARS-CoV-2 RBD protein at a concentration of 10 μg / mL was coated onto an immunoassay plate, and serially diluted 1:5 solutions of the nanobody Fc fusion protein and ACE2-Fc protein were added sequentially. The plate was incubated at room temperature for 1 h. Then, HRP anti-IgG1 Fc antibody was added to detect the bound VNAR-Fc and ACE2-Fc. The results are shown in Figure 6. The binding affinity of the four nanobodies to SARS-CoV-2 RBD was in the following order: sh-aRBD-5-Fc > sh-aRBD-18-Fc > sh-aRBD-2-Fc > sh-aRBD-17-Fc. 50 The values ​​were 0.037, 2.155, 145.819, and 510.841 nM, respectively. Among them, sh-aRBD-5-Fc and sh-aRBD-18-Fc had higher affinity than ACE2-Fc (4.119 nM).

[0056] 3) The affinity of nanobodies and their Fc fusion proteins to SARS-CoV-2 RBD was characterized using BLI. To characterize the affinity of the nanobodies' Fc fusion proteins to RBD, RBD was first biotinylated to obtain biotinylated RBD protein (biotin-RBD). Then, it was immobilized on an SA biosensor, and different concentration gradients of the nanobodies' Fc fusion proteins were set to detect the affinity between biotin-RBD and the nanobodies' Fc fusion proteins. The results are shown in Figure 7A. The binding forces of the four nanobodies' Fc fusion proteins to SARS-CoV-2 RBD were in the following order: sh-aRBD-5-Fc > sh-aRBD-18-Fc > sh-aRBD-2-Fc > sh-aRBD-17-Fc, with an affinity constant K. DThe values ​​were 3.88, 9.20, 28.3, and 211 nM, respectively. To characterize the affinity between the nanobody and RBD, the nanobody Fc fusion protein was first immobilized on a Protein A biosensor. Different SARS-CoV-2 RBD concentration gradients were set, and the affinity between RBD and the nanobody was detected. The results are shown in Figure 7B. The binding forces of the four nanobodies to SARS-CoV-2 RBD were in the following order: sh-aRBD-5 > sh-aRBD-18 > sh-aRBD-2 > sh-aRBD-17, and their affinity constants K0 were also determined. D The values ​​are 38.5, 60.3, 429, and 2720 nM, respectively.

[0057] Example 5 describes the pairing of nanobodies for detecting RBD antigen proteins.

[0058] sh-aRBD-2, sh-aRBD-5, sh-aRBD-17, and sh-aRBD-18 were paired and coated onto the detection line or labeled with colloidal gold to prepare RBD antigen protein detection strips. The test strip width was 3.5 mm, the antibody concentration used for coating was 1.5 mg / mL, and the antibody concentration used for colloidal gold labeling was 20 μg / mL. The RBD antigen concentration was decreased by a factor of two from 50 μg / mL to 0.097 μg / mL, and the RBD protein loading volume was 40 μL for each step. The results are shown in Figure 8. The detection limit was lowest for the paired antibodies sh-aRBD-2 and sh-aRBD-18, at 0.390 μg / mL.

[0059] Example 6 characterizes the dimerized nanobody.

[0060] The four nanobodies described in this invention can recognize three epitopes of SARS-CoV-2 RBD. sh-aRBD-5 and sh-aRBD-17 bind to the same epitope of SARS-CoV-2 RBD, while sh-aRBD-2 and sh-aRBD-18 bind to two other different epitopes, respectively. Therefore, biepithelial dimerized antibodies sh-aRBD-2-5, sh-aRBD-2-17, sh-aRBD-2-18, sh-aRBD-5-18, and sh-aRBD-17-18 were constructed using these combinations. Recombinant expression of sh-aRBD-2-5 and sh-aRBD-2-17 antibodies was performed, and their affinity constant K with SARS-CoV-2 RBD was detected. D Value. As shown in Figure 9, the affinity of the dimerized nanobody is higher than that of the nanobody monomer, sh-aRBD-2-5 and sh-aRBD-2-17, with the affinity constant K. D The values ​​are 6.39 and 32.1 nM, respectively.

[0061] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0062] The sequence involved in this invention is as follows:

[0063] SEQ ID NO.1:

[0064] Name: SARS-CoV-2 RBD shark-derived nanobody sh-aRBD-2. Nucleotide sequence source: Striped bamboo shark (Cihiloscyllium plagiasum).

[0065] ACTCAACGGGTTGAACAAACACCGACAACGACAACAAAGGAGGCAGGCGAATCACTGACCATCAATTGCGTCTTAAAAGGTTCCAGCTGTGCATTGGGTAGCACGTACTGGTATTTCACAAAAAAGGGCGCAACAAAGAAGGCGAGCTTATCAACTGGCGGACGATACTCG GACACAAAGAATACGGCATCAAAGTCCTTTTCCTTGCGAATTAGTGACCTAAGAGTTGAAGACAGTGGTACATATCACTGTGAAGCGTATGAAACAGCTGGGCCGGACTGTTCCTATAGCTGGGGATATAGCTATATTGAAGGAGGCGGCACCATTCTGACTGTAAAACCT

[0066] SEQ ID NO.2:

[0067] Name: Nucleotide sequence of SARS-CoV-2 RBD shark-derived nanobody sh-aRBD-5. Source: Striped bamboo shark (Cihiloscyllium plagiasum).

[0068] ACTCAACGGGTTGAACAAACACCGACAACGACAACAAAGGAGGCAGGCGAATCACTGACCATCAATTGCGTCCTAAGAGATTCCAGCTGTGCATTGGATAGCACGTACTGGTATTTCACAAAAAAGGGCGCAACAAAGAAGGAGAGCTTATCAAATGGCGGACGATACGCGG AAACAGTGAACAAGGCATCAAAGTCCTTTTCTTTGCGAATTAGTGACCTAAGAGTTGAAGACAGTGGTACATATCACTGTGAAGCGTATAAGCCCCCTCTACAGCTGGGATCTCGGGCGTTATAACCTTAGCTGGAATTGCGAGGGAGGCGGCACCATTCTGACTGTAAAACCT

[0069] SEQ ID NO.3:

[0070] Name: Nucleotide sequence of SARS-CoV-2 RBD shark-derived nanobody sh-aRBD-17. Source: Striped bamboo shark (Cihiloscyllium plagiasum).

[0071] ACTCAACGGGTTGAACAAACACCGACAACGACAACAAAGGAGGCAGGCGAATCACTGACCATCAATTGCGTCCTAAGAGATTCCAGCTGTGCATTGGATAGCACGTACTGGTATTTCACAAAAAAGGGCGCAACAAAGAAGGAGAGCTTATCAAATGGCGGACGATAC GCGGAAACAGTGAACAAGGCATCAAAGTCCTTTTCTTTCGAATTAGTGACCTAAGAGTTGAAGACAGTGGTACATATCACTGTAAAGGACAGCTGAATGAGGGCTGTTATGGGAGCTGGAATCGCAACTATTATGAAGGAGGCGGCACCATTCTGACTGTAAAACCT

[0072] SEQ ID NO.4:

[0073] Name: Nucleotide sequence of SARS-CoV-2 RBD shark-derived nanobody sh-aRBD-18. Source: Striped bamboo shark (Cihiloscyllium plagiasum).

[0074] ACTCAACGGGTTGAACAAACACCGACAACGACAACAAAGGAGGCAGGCGAATCACTGACCATCAATTGCGTCCTAAGAGATTCCAGTTGTACATTGACTAGCACGCACTGGTATTTCACAAAAAAGGGCGCAACAAAGAAGGAGAGCTTATCAAATGGCGGACGA TACGCGGAAACAGTGAACAAGGCATCAAAGTCCTTTTCTTTGCGAATTAGTGACCTAAGAGTTGAAGACAGTGGTACATATCACTGTGAGGCTTACAGCTGGATGTGAAGGGCGATACTATAACTGGGATGGAGGAGGCGGCACCATTCTGACTGTAAAACCT

[0075] SEQ ID NO.5:

[0076] Name: SARS-CoV-2 RBD Shark-Derived Nanobody sh-aRBD-2 Amino acid sequence source: Striped bamboo shark (Cihiloscyllium plagiasum)

[0077] TQRVEQTPTTTTKEAGESLTINCVLKGSSCALGSTYWYFTKKGATKKASLSTGGRYSDTKNTASKSFSLRISDLRVEDSGTYHCEAYETAGPDCSYSWGYSYIEGGGTILTVKP

[0078] SEQ ID NO.6:

[0079] Name: Amino acid sequence of SARS-CoV-2 RBD shark-derived nanobody sh-aRBD-5. Source: Striped bamboo shark (Cihiloscyllium plagiasum).

[0080] TQRVEQTPTTTTKEAGESLTINCVLRDSSCALDSTYWYFTKKGATKKESLSNGGRYAETVNKASKSFSLRISDLRVEDSGTYHCEAYKPPLQLGSRALYLSWNCEGGGTILTVKP

[0081] SEQ ID NO.7:

[0082] Name: Amino acid sequence of SARS-CoV-2 RBD shark-derived nanobody sh-aRBD-17. Source: Striped bamboo shark (Cihiloscyllium plagiasum).

[0083] TQRVEQTPTTTTKEAGESLTINCVLRDSSCALDSTYWYFTKKGATKKESLSNGGRYAETVNKASKSFSLRISDLRVEDSGTYHCKGQLNEGCYGSWNRNYYEGGGTILTVKP

[0084] SEQ ID NO.8:

[0085] Name: Amino acid sequence of SARS-CoV-2 RBD shark-derived nanobody sh-aRBD-18. Source: Striped bamboo shark (Cihiloscyllium plagiasum).

[0086] TQRVEQTPTTTTKEAGESLTINCVLRDSSCTLTSTHWYFTKKGATKKESLSNGGRYAETVNKASKSFSLRISDLRVEDSGTYHCEAYTAGCEGRYYNWDGGGGTILTVKP

[0087] SEQ ID NO.9:

[0088] Name: Nucleotide sequence of a flexible polypeptide chain

[0089] Source: Artificial Sequence

[0090] GGTGGCGGAGGGTCTGGTGGCGGAGGGTCTGGTGGCGGAGGGTCT

[0091] SEQ ID NO.10:

[0092] Name: Amino acid sequence of a flexible polypeptide chain

[0093] Source: Artificial Sequence

[0094] GGGGSGGGGSGGGGS

[0095] SEQ ID NO.11

[0096] Name: Forward primers for VNAR gene sequence amplification

[0097] Source: Artificial Sequence

[0098] GCTGCACAGCCTGCTATGGCAACTCAACGGGTTGAACAAACACCGASEQ ID NO.12

[0099] Name: Reverse primers for VNAR gene sequence amplification

[0100] Source: Artificial Sequence

[0101] GAGTTTTTGTTCGGCTGCTGCTGGTTTTACAGTCAGAATGGTGCCGC

[0102] SEQ ID NO.13

[0103] Name: Forward primers for pR2 phage amplification

[0104] Source: Artificial Sequence

[0105] AGCAGCCGAACAAAAACTCATCTCAGAAGAG

[0106] SEQ ID NO.14

[0107] Name: Reverse primers for pR2 phage amplification

[0108] Source: Artificial Sequence

[0109] CCATAGCAGGCTGTGCAGCATAGAAAGGTACCACTAAAGGAATTGC.

Claims

1. A shark-derived nanobody targeting SARS-CoV-2 RBD, characterized in that: The nucleotide sequence of the shark-derived nanobody is SEQ ID NO.

1.

2. A biepisotope dimerizing antibody, characterized in that: A dual-epitope dimerized antibody was constructed using the SARS-CoV-2 RBD shark-derived nanobody described in claim 1, which consists of two SARS-CoV-2 RBD shark-derived nanobodies with different sequences linked together by a flexible polypeptide chain; the nucleotide sequence of the other SARS-CoV-2 RBD shark-derived nanobody with a different sequence is SEQ ID NO.2 or SEQ ID NO.

3.

3. The biepisode dimerizing antibody according to claim 2, characterized in that: The nucleotide sequence of the flexible polypeptide chain is shown in SEQ ID NO.9, and the amino acid sequence is shown in SEQ ID NO.

10.

4. The application of the shark-derived nanobody targeting SARS-CoV-2 RBD as described in claim 1 or the biepisode dimerizing antibody as described in claim 3, characterized in that: Application of the shark-derived nanobody in the immunoblotting kit and enzyme-linked immunosorbent assay kit for the preparation of SARS-CoV-2 RBD protein.

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