Shark nanobodies targeting cpv2 c- vp2 protein and uses thereof

Abstract

The application discloses a shark-derived nanobody targeting CPV2c-VP2 protein and application thereof. The amino acid sequence of the complementarity determining region 3 of the shark-derived nanobody is selected from the sequence shown in any one of SEQ ID NO. 17 to SEQ ID NO. 32, or a sequence homologous to the sequence shown in any one of SEQ ID NO. 17 to SEQ ID NO. 32; or the amino acid sequence of the shark-derived nanobody is selected from the sequence shown in any one of SEQ ID NO. 1 to SEQ ID NO. 16, or a sequence homologous to the sequence shown in any one of SEQ ID NO. 1 to SEQ ID NO. 16. The shark-derived VNAR nanobody has the advantages of small molecular weight, high affinity, high stability, easy genetic engineering, low production cost and the like.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to shark-derived VNAR nanobodies targeting the VP2 capsid protein of canine parvovirus (CPV2c) and their applications. Background Technology

[0002] Single-domain antibodies against the variable region (VNAR) of neoantigen receptor immunoglobulins in sharks can naturally recognize antigen molecules and are characterized by their small size, stability, ability to recognize hidden antigen sites, and ease of modification. Compared to camel-derived VHH nanobodies, shark-derived VNAR antibodies have smaller molecular weights and higher stability. However, due to factors such as the long immunization cycle in animals and the difficulty in preparation, their application and development in basic research, therapeutic drugs, and diagnostic reagents are insufficient.

[0003] Canine parvovirus (CPV) is generally believed to have evolved from feline panleukopenia virus (FPV), with the two sharing over 98% homology in their gene sequences. Canine parvovirus causes symptoms in infected dogs such as lethargy, loss of appetite, fever, vomiting, and severe diarrhea (usually bloody), and is highly contagious and fatal. Currently, routine treatment for canine parvovirus infection is generally limited to supportive care, including fluid replacement, antibiotics, antiemetics, and anti-inflammatory drugs. CPV is a non-enveloped, single-stranded DNA virus containing two open reading frames (ORFs). The first ORF encodes two non-structural proteins, NS1 and NS2, while the second ORF encodes two structural proteins: VP1 and VP2. VP1 and VP2 each encode portions of the viral capsid; VP2 is the major capsid protein, accounting for approximately 90% of the viral capsid and playing a crucial role in viral transmission and infection. VP2 is also a major antigenic protein, and its N-domain and ring domain contain several important B-cell epitopes that can induce effective neutralizing antibodies during viral infection.

[0004] Compared to feline panleukopenia virus (FPV), CPV mutates very rapidly. Discovered in canines in 1978 (the first known strain designated CPV2), CPV has undergone continuous genetic variation, and currently, CPV2a, CPV2b, and CPV2c are the three major antigenic variants of CPV. These variants are distinguished by amino acid substitutions on specific residues of the VP2 protein. From the original CPV2 to CPV2a, only a few amino acid changes occurred, including M87L, I101T, A300G, and D305Y. The CPV2b subtype arises from the N426D mutation of the CPV2a subtype, while the CPV2c subtype arises from the D426E mutation of the CPV2b subtype. According to recent molecular epidemiological studies, CPV2c has gradually replaced CPV2a as the new dominant variant in Asia, South America, North America, and Africa.

[0005] Neutralizing antibodies are an effective treatment for viral infections. They are typically produced after acute infection and reduce the virus's infectivity. Studies have shown that parvovirus neutralizing antibody therapy can significantly improve the survival and recovery rates of animals infected with parvovirus. In May 2023, the U.S. Department of Agriculture granted conditional approval to Elanco Animal Health's first canine parvovirus monoclonal antibody.

[0006] Currently, the neutralizing antibody formulations available for canine parvovirus infection in animals are traditional monoclonal antibodies, and there are no shark-derived nanobodies targeting CPV-related proteins. Summary of the Invention

[0007] To address the problems existing in the background art, this invention provides a shark-derived nanobody targeting the CPV2c-VP2 protein and its applications. The shark-derived heavy chain antibody variable region sequence (VNAR) provided by this invention, capable of binding to the CPV2c-VP2 capsid protein with high affinity, is also known as a nanobody or single-domain antibody. It can be used in the development of therapeutic neutralizing antibodies for canine parvovirus infection, and also in the development of diagnostic reagents for canine parvovirus infection.

[0008] The technical solution adopted in this invention is: I. A shark-derived nanobody targeting the CPV2c-VP2 protein The shark-derived nanobody includes a complementarity-determining region 3 (CDR3 region), the amino acid sequence of which is selected from any one of the sequences shown in SEQ ID NO.17 to SEQ ID NO.32, or a sequence that has at least 80% homology with the sequence shown in any one of SEQ ID NO.17 to SEQ ID NO.32 and retains antigen-binding function.

[0009] Furthermore, the amino acid sequence of the shark-derived nanobody is selected from any one of the sequences shown in SEQ ID NO.1 to SEQ ID NO.16, or a sequence that has at least 85% homology with the sequence shown in any one of SEQ ID NO.1 to SEQ ID NO.16 and retains antigen-binding function.

[0010] II. A biomaterial The biomaterial includes at least one of the following: a) Nucleotides used to encode the shark-derived nanobody; b) An expression vector containing the nucleotides; c) A recombinant prokaryotic host containing the nucleotide or the expression vector.

[0011] III. An application of the shark-derived nanobody or the biomaterial described herein The shark-derived nanobody and the biomaterial can be used to prepare a detection reagent for canine parvovirus.

[0012] Specifically, the detection target is the VP2 capsid protein of canine parvovirus.

[0013] Specifically, the canine parvovirus is the CPV2c subtype.

[0014] Furthermore, the detection methods include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay, rapid diagnostic test strips, fluorescence immunoassay, and immunosensing assay.

[0015] IV. An application of the shark-derived nanobody or the biomaterial described herein The shark-derived nanobody and the biomaterial can be used to prepare drugs that can prevent and / or treat diseases in animals caused by or related to canine parvovirus infection.

[0016] Specifically, the animal is a dog, cat, or other animal that is a host for canine parvovirus under natural conditions.

[0017] Specifically, the therapeutic target is the VP2 capsid protein of canine parvovirus.

[0018] The beneficial effects of this invention are: 1. The shark-derived VNAR antibody provided by this invention is the first shark-derived VNAR nanobody targeting canine parvovirus. Compared with traditional monoclonal antibodies, the shark-derived VNAR nanobody of this invention has advantages such as small molecular weight, high affinity, high stability, ease of genetic engineering modification, and low production cost.

[0019] 2. Compared with camel-derived VHH nanobodies, the shark-derived VNAR antibody provided by this invention has advantages such as smaller molecular weight and higher stability.

[0020] 3. Compared to traditional monoclonal antibodies, the shark-derived VNAR antibody provided by this invention may enhance its neutralizing ability against viral infections due to its unique binding mode to the target protein. Furthermore, the ease of genetic modification of VNAR antibodies makes it easier to prepare multivalent antibodies, avoiding immune escape caused by viral mutations. Simultaneously, in the field of canine parvovirus infection diagnosis, the small size and ease of prokaryotic expression of VNAR nanobodies will effectively improve diagnostic sensitivity and reduce production costs. Attached Figure Description

[0021] Figure 1 The results of antibody ELISA screening for CPV2c-VP2 protein-binding phages were obtained.

[0022] Figure 2 Amino acid sequence analysis of CPV2c-VP2 binding shark-derived VNAR antibody.

[0023] Figure 3 Phylogenetic analysis of the amino acid sequence of CPV2c-VP2 binding shark-derived VNAR antibody.

[0024] Figure 4 The purification results are for five representative CPV2c-VP2 conjugated shark VNAR antibodies: FCB2, FCD1, FCE2, FCF6, and CC9.

[0025] Figure 5 The results show the SPR affinity assays of five representative CPV2c-VP2 binding shark VNAR antibodies FCB2, FCD1, FCE2, FCF6, and CC9 with the CPV2c-VP2 protein. Among them, (a) shows the affinity assay results for antibody FCB2, (b) shows the affinity assay results for antibody FCD1, (c) shows the affinity assay results for antibody FCE2, (d) shows the affinity assay results for antibody FCF6, and (e) shows the affinity assay results for antibody CC9. Detailed Implementation

[0026] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0027] This invention provides a shark-derived nanobody targeting the CPV2c-VP2 protein. The shark-derived nanobody includes a framework region (FR), complementarity-determining region 1 (CDR1), complementarity-determining region 3 (CDR3), and hypervariable regions 2 (HV2) and 4 (HV4).

[0028] Preferably, the amino acid sequence of the complementarity-determining region 3 of the shark-derived nanobody is selected from any one of the sequences shown in SEQ ID NO.17 to SEQ ID NO.32, or a sequence that has at least 80% homology with the sequence shown in any one of SEQ ID NO.17 to SEQ ID NO.32 and retains antigen-binding function.

[0029] Preferably, the amino acid sequence of the shark-derived nanobody is selected from any one of the sequences shown in SEQ ID NO.1 to SEQ ID NO.16, or a sequence that has at least 85% homology with the sequence shown in any one of SEQ ID NO.1 to SEQ ID NO.16 and retains antigen-binding function.

[0030] The present invention also provides at least one of the following biomaterials: a) Nucleotides used to encode the aforementioned shark-derived nanobody; b) An expression vector containing the above nucleotides; c) A recombinant prokaryotic host containing the above-mentioned nucleotides or the above-mentioned expression vectors.

[0031] Preferably, the expression vector includes, but is not limited to, plasmids, viral vectors, and phage display vectors.

[0032] Furthermore, viral vectors include, but are not limited to, lentiviruses and adenoviruses.

[0033] Preferably, the recombinant prokaryotic host is the BL21(DE3) strain.

[0034] Furthermore, the recombinant host can also be a eukaryotic host cell.

[0035] The present invention also provides the application of the above-mentioned shark-derived nanobody or biomaterial in the preparation of a detection reagent for canine parvovirus.

[0036] Specifically, the tests include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay, rapid diagnostic test strips, fluorescence immunoassay, and immunosensing assay.

[0037] Preferably, the detection target is the VP2 capsid protein of canine parvovirus.

[0038] More preferably, the canine parvovirus is the CPV2c subtype.

[0039] The present invention also provides the use of the above-mentioned shark-derived nanobody or biomaterial in the preparation of a medicament capable of preventing and / or treating diseases in animals caused by or related to canine parvovirus infection.

[0040] Preferably, the animal is a dog, cat, or other animal that is a host for canine parvovirus under natural conditions.

[0041] Preferably, the therapeutic target is the VP2 capsid protein of canine parvovirus.

[0042] More preferably, the canine parvovirus is the CPV2c subtype.

[0043] Specific embodiments of the present invention are as follows: Example In this embodiment, the main capsid protein VP2 of CPV2c virus was used as the immunogenic antigen for the preparation of CPV neutralizing antibodies. Multiple high-affinity VNAR nanobodies targeting CPV2c-VP2 were prepared by immunizing striped bamboo sharks. These nanobodies can serve as excellent candidate molecules for neutralizing antibodies required for the treatment of canine parvovirus infection.

[0044] Example 1) Keeping striped bamboo sharks: Striped bamboo sharks are kept in a 25°C constant temperature seawater aquarium with a filtration system (salinity of about 3%). The seawater is kept circulating and changed regularly. Fresh shrimp are fed to them weekly and their growth is observed.

[0045] 2) Preparation of CPV2c-VP2 protein antigen: The DNA sequence of the antigen CPV2c-VP2 protein (GenBank: MH476592.1) was synthesized in its entirety and inserted into the pET28a prokaryotic expression vector. It was expressed by BL21(DE3) Escherichia coli and purified by Ni-IDA affinity. The purity of the purified CPV2c-VP2 protein exceeded 85%, which was used for immunization of striped bamboo shark.

[0046] 3) The striped bamboo shark underwent a total of 7 immunizations: The first immunization was performed by emulsifying 45 μg of CPV2c-VP2 protein antigen and 45 μg of FPV-VP2 protein antigen with an equal volume of Freund's complete adjuvant, and then injecting the mixture subcutaneously at multiple points in the abdomen and pectoral fins of the anesthetized shark; the remaining immunizations were performed by emulsifying 45 μg of CPV2c-VP2 protein antigen and 45 μg of FPV-VP2 protein antigen with an equal volume of Freund's incomplete adjuvant, and then injecting the mixture subcutaneously at multiple points in the abdomen and pectoral fins of the anesthetized shark.

[0047] 4) Obtaining and cloning the VNAR gene fragment: After immunization, shark venous blood and spleen tissue were collected. Lymphocytes from the venous blood and spleen were isolated, and total RNA was extracted from the lymphocytes and whole spleen tissue and reverse transcribed into single-stranded cDNA. After one round of PCR amplification, the VNAR gene set of the striped bamboo shark was obtained. The PCR product and pADL22c phage vector were digested with SfiI and BglI restriction enzymes, respectively, and then recovered. The VNAR fragment was ligated to the pADL22c digested vector at a 3:1 molar ratio, and the ligation product was purified using a PCR product recovery kit.

[0048] 5) Phage library construction: The ligation product was electroporated into TG1 competent cells. The transformed cells were evenly spread on four 245 mm square LB agar plates (100 µg / ml ampicillin and 2% (wt / vol) glucose) as phage libraries. Simultaneously, the electroporated TG1 cells were serially diluted and plated. The constructed immunoglobulin library volume was measured to be approximately 2.25 × 10⁸.

[0049] 6) Selection of CPV2c-VP2 protein-specific phages: Before each round of phage selection, M13KO7 helper phages were added to rescue and amplify phage particles displaying nanobodies, and the number of rescued phages used for selection was estimated by preparing phage gradient dilutions and infecting TG1 cells in the growth phase of the infection index.

[0050] The first round of CPV2c-VP2 specific binding phage panning was performed in MaxiSorp 96-well plates. 1 μg of CPV2c-VP2 protein was incubated in each well, with a PBS negative control well included. The plates were blocked with 5% skim milk. 10¹¹ rescued and amplified phages were added to each panning well and incubated at room temperature with a shaker to allow the specific phages to bind to the target protein. The bound phages were eluted with 0.05% trypsin. 10 μl of the eluted phage was serially diluted to estimate the number of infectious recombinant phages recovered. The remaining eluted phages were used to infect TG1 cells and incubated overnight in LB medium (100 µg / ml ampicillin + 2% glucose) to recover the bound phages. The following day, an equal volume of 40% glycerol was added to the bacterial culture, and the cells were aliquoted and frozen for phage amplification and subsequent panning. The second and third rounds of panning followed similar procedures, but the concentration of the coating antigen protein in each well was halved to enrich high-affinity target protein-binding phages. Panning was stopped when the phage titers eluted from the antigen-coated wells and the negative control wells differed by two orders of magnitude.

[0051] 7) Screening for high-affinity antibody-displaying phages for CPV2c-VP2 protein: 188 TG1 monoclonal antibodies were picked from the panned phage sub-library plate and used to prepare E. coli periplasmic space extract for ELISA analysis of VNAR nanobodies and CPV2c-VP2 protein. 0.2 µg of CPV2c-VP2 protein was added to each well of a 96-well Maxisorp plate and incubated with PBS solution, leaving a control well without the target protein in PBS. The plates were blocked with 5% skim milk. 50 µl of monoclonal TG1 periplasmic protein extract, 10 µl of blocking solution, and 40 µl of PBS solution were added to each protein-coated well. One protein-coated well contained unpanned random TG1 periplasmic protein extract as a negative control. The primary antibody used was rabbit anti-HA tag antibody (1:3000), and the secondary antibody used was HRP-conjugated anti-rabbit IgG antibody (1:5000); TMB chromogenic substrate was used, and the reaction was terminated by adding ELISA stop solution. The absorbance was then measured at 450 nm. (See attached image) Figure 1 ).

[0052] Figure 1 This study presents the ELISA screening results of 188 potential TG1 monoclonal periplasmic space-expressing nanobodies that bind to the CPV2c-VP2 protein. H11 represents randomly selected TG1 monoclonal control wells before panning, H12 represents uninoculated E. coli PBS control wells, and the remaining wells contain the 188 TG1 monoclonals selected from the phage panning sub-liquidity library diluted and plated on solid plates. In the figure, darker blue indicates a higher absorbance (OD450) at 450 nm. Wells with an OD450 ratio greater than 4 compared to the negative control wells H11 and H12 were selected as positive nanobodies.

[0053] 8) Nanobody Sequence Analysis: Nanobodies with an OD450 reading ratio greater than 4 were selected as positive clones. Multiple positive clones were subjected to DNA sequencing and translated into amino acid sequences to obtain the nanobody sequences (see attached). Figure 2 ).

[0054] Figure 2 The results of amino acid sequence alignment for 30 CPV2c-VP2 binding VNAR antibodies are presented. Amino acid residues with a match of less than 20% with highly conserved consensus sequences are highlighted in blue. The amino acid sequences of the complementarity-determining regions CDR1 / 3 and the hypervariable regions HV2 / 4 are labeled above the aligned sequences. Antibody sequences with 100% similarity were excluded during the alignment process.

[0055] 9) Nanobody Grouping: Based on sequence affinity analysis, CPV2c-VP2 binding VNAR antibodies can be divided into 5 groups, with CDR3 region lengths of 15, 18, 16, 13, and 17 amino acids respectively (see appendix). Figure 3 ).

[0056] The sequences of representative antibodies (those capable of binding to CPV2c-VP2 protein and whose CDR3 region amino acid sequence differs from other binding sequences) in each group are as follows: ①The CDR3 region is 15 amino acids in length: Shark-derived nanobody FCB2: The amino acid sequence is shown in SEQ ID NO.1, with positions 88-102 being the CDR3 region, and the amino acid sequence is shown in SEQ ID NO.17; Shark-derived nanobody FCB2-A1: The amino acid sequence is shown in SEQ ID NO.2, with positions 88-102 being the CDR3 region, and the amino acid sequence is shown in SEQ ID NO.18; Shark-derived nanobody FCB2-A2: The amino acid sequence is shown in SEQ ID NO.3, with positions 88-102 being the CDR3 region, and the amino acid sequence is shown in SEQ ID NO.19; Shark-derived nanobody FCB2-B3: The amino acid sequence is shown in SEQ ID NO.4, with positions 88-102 being the CDR3 region, and the amino acid sequence is shown in SEQ ID NO.20; Shark-derived nanobody FCB2-C2: The amino acid sequence is shown in SEQ ID NO.5, with positions 88-102 being the CDR3 region, and the amino acid sequence is shown in SEQ ID NO.21; Shark-derived nanobody FCB2-D9: The amino acid sequence is shown in SEQ ID NO.6, with positions 88-102 being the CDR3 region, and the amino acid sequence is shown in SEQ ID NO.22; Shark-derived nanobody FCB2-E10: The amino acid sequence is shown in SEQ ID NO.7, with positions 88-102 being the CDR3 region, and the amino acid sequence is shown in SEQ ID NO.23; Shark-derived nanobody FCB2-F10: The amino acid sequence is shown in SEQ ID NO.8, with positions 88-102 being the CDR3 region, and the amino acid sequence is shown in SEQ ID NO.24; Shark-derived nanobody FCB2-F12: The amino acid sequence is shown in SEQ ID NO.9, with positions 88-102 being the CDR3 region, and the amino acid sequence is shown in SEQ ID NO.25; ②The CDR3 region is 17 amino acids in length: Shark-derived nanobody FCD1: The amino acid sequence is shown in SEQ ID NO.10, with positions 88-104 being the CDR3 region, and the amino acid sequence is shown in SEQ ID NO.26; Shark-derived nanobody FCD1-A5: The amino acid sequence is shown in SEQ ID NO.11, with positions 88-104 being the CDR3 region, and the amino acid sequence is shown in SEQ ID NO.27; ③The CDR3 region is 16 amino acids in length: Shark-derived nanobody FCE2: The amino acid sequence is shown in SEQ ID NO.12, with positions 88-103 being the CDR3 region, and the amino acid sequence is shown in SEQ ID NO.28; Shark-derived nanobody FCE2-C6: The amino acid sequence is shown in SEQ ID NO.13, with positions 88-103 being the CDR3 region, and the amino acid sequence is shown in SEQ ID NO.29; ④ The CDR3 region is 18 amino acids in length: Shark-derived nanobody FCF6: The amino acid sequence is shown in SEQ ID NO.14, with positions 88-105 being the CDR3 region, and the amino acid sequence is shown in SEQ ID NO.30; Shark-derived nanobody FCF6-F1: The amino acid sequence is shown in SEQ ID NO.15, with positions 88-105 being the CDR3 region, and the amino acid sequence is shown in SEQ ID NO.31; ⑤ The CDR3 region is 13 amino acids in length: Shark-derived nanobody CC9: The amino acid sequence is shown in SEQ ID NO.16, with positions 88-100 being the CDR3 region, and the amino acid sequence is shown in SEQ ID NO.32.

[0057] like Figure 3As shown, the 30 CPV2c-VP2 binding VNAR antibody amino acid sequences can be divided into 5 groups according to their phylogenetic relationship, and are distinguished by different colors.

[0058] 10) Nanobody Expression: Five representative VNAR antibodies binding to CPV2c-VP2 protein (named FCB2, FCD1, FCE2, FCF6, and CC9; amino acid sequences as shown in SEQ ID NO.1, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, and SEQ ID NO.16, respectively) were expressed using the pCZN1 vector in the BL21(DE3) prokaryotic system and purified using His-tag affinity. The purity of all bands was greater than 90%, and the molecular weight of each band was approximately 14 kDa, meeting expectations. (See attached image) Figure 4 ).

[0059] 11) Affinity Detection of Nanobodies: Surface plasmon resonance (SPR) technology was used to quantitatively detect the affinity and binding kinetics between VNAR antibodies and target proteins. CPV2c-VP2 protein was immobilized on a CM5 chip as a ligand protein; each nanobody was diluted to several concentrations in a 96-well plate, and coupled to the target protein through the chip from low to high concentrations; after each concentration point, the chip was regenerated using glycine hydrochloride solution (pH 2.0), and this process was repeated until all corresponding antibody concentrations were obtained; the data were globally fitted to a 1:1 Langmuir binding model using Biacore Insight evaluation software to obtain the binding and dissociation constants (see attached). Figure 5 ).

[0060] SPR measurements showed that five representative shark-derived nanobodies all exhibited very high affinity for the CPV2c-VP2 protein: the equilibrium dissociation constants of FCB2, FCD1, FCE2, FCF6, and CC9 with the CPV2c-VP2 protein were 6.28 × 10⁻¹⁰ M, 2.17 × 10⁻¹¹ M, 5.38 × 10⁻⁸ M, 3.1 × 10⁻⁹ M, and 3.88 × 10⁻⁸ M, respectively.

[0061] It is evident that the equilibrium dissociation constants of FCB2, FCD1, and CPV2c-VP2 proteins even reach the picomolar level.

[0062] In summary, the high-affinity VNAR antibody against CPV capsid protein involved in this invention can also be used for the immunological detection of canine parvovirus infection, such as virus test strips. Based on the excellent properties of VNAR antibodies, nanobody immunological detection reagents have advantages such as high stability and low production cost compared with traditional monoclonal antibody-based detection reagents.

[0063] The above specific embodiments are used to explain and illustrate the present invention, but not to limit the present invention. Any modifications and changes made to the present invention within the spirit and scope of the claims shall fall within the protection scope of the present invention.

[0064] The above description is only a preferred embodiment of the present invention. Therefore, all equivalent changes or modifications made to the structure, features and principles described in the claims of this patent application are included in the scope of this patent application.

[0065] The amino acid sequence involved in this invention is as follows: SEQ ID NO.1: Name: Amino acid sequence of shark-derived nanobody FCB2 targeting CPV2c-VP2 amino acid sequence Type: AA Biological source: Striped bamboo shark (Cihiloscyllium plagiasum) MAARLEQTPTTTTKEAGESLTINCVLKGSSCALGRTYWYFTKKGATKKASLSTGGRYSDTKNTASKSFSLRISDLRVEDSGTYHCKAYREILGCQELGMIRYEGGGTILTVK SEQ ID NO.2: Name: Amino acid sequence of shark-derived nanobody FCB2-A1 targeting CPV2c-VP2 Type: AA Biological source: Striped bamboo shark (Cihiloscyllium plagiasum) MAPRVEQTPTTTTKEAGESLTINCVLKGSSCALGRTNWYFTKKGATKKASLSTGGRYSDTKNTASKSFSLRISDLRVEDSGTYYCEAYRGILGCQELGLIRYEGGGTTVTVN SEQ ID NO.3: Name: Amino acid sequence of shark-derived nanobody FCB2-A2 targeting CPV2c-VP2 Type: AA Biological source: Striped bamboo shark (Cihiloscyllium plagiasum) MAARVEQTPTTTTKEAGESLTINCVLKGSTCALSKTYWYFTKKGASKKASLSTGGRYSATKNTASKSFSLRISDLRVEDSGTYHCEAYRRLLGCQELGSIRFEGGGTTLTVK SEQ ID NO.4: Name: Amino acid sequence of shark-derived nanobody FCB2-B3 targeting CPV2c-VP2 Type: AA Biological source: Striped bamboo shark (Cihiloscyllium plagiasum) MAARLEQTPTTTTKEAGESLTINCVLKGSSCALGSTYWYFTKKGATKKASLSTGGRYAETVNKASKSFSLRISDLRVEDSGTYTCEAYRGILGCQELGAIRYEGGGTLVTVN SEQ ID NO.5: Name: Amino acid sequence of shark-derived nanobody FCB2-C2 targeting CPV2c-VP2 Type: AA Biological source: Striped bamboo shark (Cihiloscyllium plagiasum) MAQRLEQTPTTTTKEAGESLTINCVLKGSSCALGSTFWYFTKKGATKKASLATGGRYSDTKNTASKSFSLRISDLRVEDSGTYHCEAYRRLLGCQELGAIRFEGSGTLLTVK SEQ ID NO.6: Name: Amino acid sequence of shark-derived nanobody FCB2-D9 targeting CPV2c-VP2 Type: AA Biological source: Striped bamboo shark (Cihiloscyllium plagiasum) MAARVEQTPTTTTKEAGESLTINCVLKGSSCALGRTYWYFTKKGATQKASLSTGGRYSDTKNTASKSFSLRISDLRVEDSGTYHCEAYRSLLGCQELGLIRFEGGGTTLTVK SEQ ID NO.7: Name: Amino acid sequence of shark-derived nanobody FCB2-E10 targeting CPV2c-VP2 Type: AA Biological source: Striped bamboo shark (Cihiloscyllium plagiasum) MAARVEQTPTTITKESGESLTINCVLKGSSCALGNTYWYFTKKGATKKASLSTGGRYSDTKNTASKSFSLRISDLRVEDSGTYHCEAYRGILGCQELGLIRFEGGGTIVTVN SEQ ID NO.8: Name: Amino acid sequence of shark-derived nanobody FCB2-F10 targeting CPV2c-VP2 Type: AA Biological source: Striped bamboo shark (Cihiloscyllium plagiasum) MAARLEQTPTTTTKKAGESLTINCVLKGSTCALGSTLWYFTKKGATKKARLSTGGRYSDTKNTASKSFSLRISDLRVEDSGTYHCEAYRGILGCQELGAIRFEGGTTVTVK SEQ ID NO.9: Name: Amino acid sequence of shark-derived nanobody FCB2-F12 targeting CPV2c-VP2 Type: AA Biological source: Striped bamboo shark (Cihiloscyllium plagiasum) MAPRVEQTPTTTTKEAGESLTINCVLKGSSCALGRTYWYFTKKGATQKASLLTGGRYSETKNTASKSFSLRISDLRVEDSGTYHCEAYRSVLGCQELGLIRFEGGGTILTVN SEQ ID NO.10: Name: Amino acid sequence of shark-derived nanobody FCD1 targeting CPV2c-VP2 Type: AA Biological source: Striped bamboo shark (Cihiloscyllium plagiasum) MAPRVEQTPTTTTKEAGESLTINCVLKGTSYTLCNTYWYFTKKDATKKESLSNGGRYAETVHKASKSFSLRISDLRVEDSGTYHCKTYTRHPSIPTASRCILYYEGGGTLVTVK SEQ ID NO.11: Name: Amino acid sequence of shark-derived nanobody FCD1-A5 targeting CPV2c-VP2 Type: AA Biological source: Striped bamboo shark (Cihiloscyllium plagiasum) MAPRLEQTPTTTTKEAGESLTINCVLKGTSYTLCNTYWYFTKKDATKKESLSNGGRYAETVHKASKSFSLRISDLRVEDSGTYHCKTYTRHPSLPTASRCILYYEGGGTIVTVK SEQ ID NO.12: Name: Amino acid sequence of shark-derived nanobody FCE2 targeting CPV2c-VP2 Type: AA Biological source: Striped bamboo shark (Cihiloscyllium plagiasum) MAERVEQTPTTTTKEAGESLTINCVLKGSNCAAGTTYWYFTKKGATKKARLSTGGRYSDTKNTASKSFSLRISDLRVEDSGTYHCETYPITGAGCLWIAPYSYEGGGTILTVK SEQ ID NO.13: Name: Amino acid sequence of shark-derived nanobody FCE2-C6 targeting CPV2c-VP2 Type: AA Biological source: Striped bamboo shark (Cihiloscyllium plagiasum) MAPRVEQTPTTTTKEAGESLTINCVLKGSSCALGRTYWYFTKKGATQKASLSTGGRYSDTKNTASNTFSLRISDLRVEDSGTYHCEIYPTTGAGCLWIAPYSYEGGGTLVTVK SEQ ID NO.14: Name: Amino acid sequence of shark-derived nanobody FCF6 targeting CPV2c-VP2 Type: AA Biological source: Striped bamboo shark (Cihiloscyllium plagiasum) MAQRLEQTPTTTTKEAGESLTINCVLKGSSCSLGSTSWYFTKKGATKKASLSTGGRYSDTKNTASKSFSLRISDLRVEDSGTYYCQAFIVGLGCGSTPGSHHRYIEGGGTTVTVK SEQ ID NO.15: Name: Amino acid sequence of shark-derived nanobody FCF6-F1 targeting CPV2c-VP2 Type: AA Biological source: Striped bamboo shark (Cihiloscyllium plagiasum) MAPRVEQTPTTTTKEAGESLTINCVLKGSSCSLGSTFWYFTKKGATKKASLSTGGRYSDTKNTASKSFSLRISDLRVEDSGTYHCQAFVLGLGCGTTPGSHHRYIEGGGTTLTVK SEQ ID NO.16: Name: Amino acid sequence of CC9, a shark-derived nanobody targeting CPV2c-VP2 Type: AA Biological source: Striped bamboo shark (Cihiloscyllium plagiasum) MAARVEQTPTTTTKEAGESLTINCVLKGSSCAVGSTFWYFTKKGATKKASLSTGGRYSDTKNTASKSFSLRISDLRVEDSGTYHCKAYVLGLLCRGGVYIEGGGTTVTVK SEQ ID NO.17: Name: Amino acid sequence of the CDR3 region of shark-derived nanobody FCB2 targeting CPV2c-VP2 Type: AA Biological source: Striped bamboo shark (Cihiloscyllium plagiasum) YREILGCQELGMIRY SEQ ID NO.18: Name: Amino acid sequence of the CDR3 region of shark-derived nanobody FCB2-A1 targeting CPV2c-VP2 Type: AA Biological source: Striped bamboo shark (Cihiloscyllium plagiasum) YRGILGCQELGLIRY SEQ ID NO.19: Name: Amino acid sequence of the CDR3 region of shark-derived nanobody FCB2-A2 targeting CPV2c-VP2 Type: AA Biological source: Striped bamboo shark (Cihiloscyllium plagiasum) YRRLLGCQELGSIRF SEQ ID NO.20: Name: Amino acid sequence of the CDR3 region of shark-derived nanobody FCB2-B3 targeting CPV2c-VP2 Type: AA Biological source: Striped bamboo shark (Cihiloscyllium plagiasum) YRGILGCQELGAIRY SEQ ID NO.21: Name: Amino acid sequence of the CDR3 region of shark-derived nanobody FCB2-C2 targeting CPV2c-VP2 Type: AA Biological source: Striped bamboo shark (Cihiloscyllium plagiasum) YRRLLGCQELGAIRF SEQ ID NO.22: Name: Amino acid sequence of the CDR3 region of shark-derived nanobody FCB2-D9 targeting CPV2c-VP2 Type: AA Biological source: Striped bamboo shark (Cihiloscyllium plagiasum) YRSLLGCQELGLIRF SEQ ID NO.23: Name: Amino acid sequence of the CDR3 region of shark-derived nanobody FCB2-E10 targeting CPV2c-VP2 Type: AA Biological source: Striped bamboo shark (Cihiloscyllium plagiasum) YRGILGCQELGLIRF SEQ ID NO.24: Name: Amino acid sequence of the CDR3 region of shark-derived nanobody FCB2-F10 targeting CPV2c-VP2 Type: AA Biological source: Striped bamboo shark (Cihiloscyllium plagiasum) YRGILGCQELGAIRF SEQ ID NO.25: Name: Amino acid sequence of the CDR3 region of shark-derived nanobody FCB2-F12 targeting CPV2c-VP2 Type: AA Biological source: Striped bamboo shark (Cihiloscyllium plagiasum) YRSVLGCQELGLIRF SEQ ID NO.26: Name: Amino acid sequence of the CDR3 region of shark-derived nanobody FCD1 targeting CPV2c-VP2 Type: AA Biological source: Striped bamboo shark (Cihiloscyllium plagiasum) YTRHPSIPTASRCILYY SEQ ID NO.27: Name: Amino acid sequence of the CDR3 region of shark-derived nanobody FCD1-A5 targeting CPV2c-VP2 Type: AA Biological source: Striped bamboo shark (Cihiloscyllium plagiasum) YTRHPSLPTASRCILYY SEQ ID NO.28: Name: Amino acid sequence of the CDR3 region of shark-derived nanobody FCE2 targeting CPV2c-VP2 Type: AA Biological source: Striped bamboo shark (Cihiloscyllium plagiasum) YPITGAGCLWIAPYSY SEQ ID NO.29: Name: Amino acid sequence of the CDR3 region of shark-derived nanobody FCE2-C6 targeting CPV2c-VP2 Type: AA Biological source: Striped bamboo shark (Cihiloscyllium plagiasum) YPTTGAGCLWIAPYSY SEQ ID NO.30: Name: Amino acid sequence of the CDR3 region of shark-derived nanobody FCF6 targeting CPV2c-VP2 Type: AA Biological source: Striped bamboo shark (Cihiloscyllium plagiasum) FIVGLGCGSTPGSHHRYI SEQ ID NO.31: Name: Amino acid sequence of the CDR3 region of shark-derived nanobody FCF6-F1 targeting CPV2c-VP2 Type: AA Biological source: Striped bamboo shark (Cihiloscyllium plagiasum) FVLGLGCGTTPGSHHRYI SEQ ID NO.32: Name: Amino acid sequence of the CDR3 region of CC9, a shark-derived nanobody targeting CPV2c-VP2 Type: AA Biological source: Striped bamboo shark (Cihiloscyllium plagiasum) YVLGLLCRGGVYI.

Claims

1. A shark-derived nanobody targeting CPV2 c-VP2 protein, characterized in that: The amino acid sequence of the shark-derived nanobody is shown in SEQ ID NO.

1.

2. A biomaterial, characterized by, Includes at least one of the following biological materials: a) Nucleic acid for encoding the shark-derived nanobody as described in claim 1; b) An expression vector containing the nucleic acid; c) A recombinant prokaryotic host containing the nucleic acid or the expression vector.

3. The application of a shark-derived nanobody as described in claim 1 or a biomaterial as described in claim 2, characterized in that: This reagent is used to prepare a detection reagent for canine parvovirus, wherein the canine parvovirus is the CPV2c subtype.

4. The application according to claim 3, characterized in that: The detection methods include enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay, rapid diagnostic test strips, fluorescence immunoassay, and immunosensing assay.

5. The application according to claim 3, characterized in that: The detection target is the VP2 capsid protein of canine parvovirus.

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