Shark nanobodies targeting fpv- vp2 protein and uses thereof

Abstract

The application discloses a shark-derived nanobody targeting FPV-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. 16 to SEQ ID NO. 30, or a sequence homologous to the sequence shown in any one of SEQ ID NO. 16 to SEQ ID NO. 30; 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. 15, or a sequence homologous to the sequence shown in any one of SEQ ID NO. 1 to SEQ ID NO. 15. 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 nanobodies of the VP2 capsid protein of feline panleukopenia virus (FPV) and their applications. Background Technology

[0002] Shark-derived VNAR nanobodies, containing the variable region of a neoantigen receptor immunoglobulin, 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 their preparation, their application and development in basic research, therapeutic drugs, and diagnostic reagents are insufficient.

[0003] Feline panleukopenia virus (FPV), also known as feline parvovirus, is a serious infectious disease in cats, often fatal to immunocompromised or unvaccinated animals such as kittens. Currently, routine treatment for FPV infection is generally limited to supportive care, including fluid replacement, antibiotics, antiemetics, and anti-inflammatory drugs. 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; however, there is currently no effective monoclonal antibody for feline panleukopenia virus available on the market.

[0004] The FPV-VP2 protein constitutes 90% of the viral capsid protein and can self-assemble into virus-like particles, playing a crucial role in viral transmission and infection. VP2 is also a major antigenic protein, with its N-domain and ring domain containing several important B-cell epitopes that can induce effective neutralizing antibodies during viral infection. Currently, there are no shark-derived nanobodies targeting FPV-related proteins. Summary of the Invention

[0005] To address the problems existing in the background art, this invention provides a shark-derived nanobody targeting the FPV-VP2 protein and its applications. The shark-derived heavy chain antibody variable region sequence (VNAR) provided by this invention, capable of binding to the FPV-VP2 capsid protein with high affinity, is also known as a nanobody or single-domain antibody. It can be used to prepare therapeutic neutralizing antibodies against FPV and also to prepare diagnostic reagents for FPV infection.

[0006] The technical solution adopted in this invention is:

[0007] I. A shark-derived nanobody targeting the FPV-VP2 protein

[0008] 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.16 to SEQ ID NO.30, or a sequence that has at least 80% homology with the sequence shown in any one of SEQ ID NO.16 to SEQ ID NO.30 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.15, 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.15 and retains antigen-binding function.

[0010] II. A biomaterial

[0011] The biomaterial includes at least one of the following biomaterials:

[0012] a) Nucleotides used to encode the shark-derived nanobodies as described above;

[0013] b) An expression vector containing the nucleotides;

[0014] c) A recombinant prokaryotic host containing the nucleotide or the expression vector.

[0015] III. Application of the above-mentioned biomaterials in the development of a shark-derived nanobody

[0016] The shark-derived nanobody and biomaterials can be used to prepare a detection reagent for feline panleukopenia virus.

[0017] 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.

[0018] Specifically, the detection target is the VP2 capsid protein of feline panleukopenia virus.

[0019] IV. Application of a shark-derived nanobody or biomaterial

[0020] The shark-derived nanobodies and biomaterials can be used to prepare drugs that can prevent and / or treat diseases in animals caused by or related to feline panleukopenia virus infection.

[0021] Specifically, the animal is a cat or another host animal of feline panleukopenia virus under natural conditions.

[0022] Specifically, the therapeutic target is the VP2 capsid protein of feline panleukopenia virus.

[0023] The beneficial effects of this invention are:

[0024] 1. The antibody of this invention is the first shark-derived VNAR nanobody targeting feline panleukopenia virus. Compared with traditional monoclonal antibodies, the shark-derived VNAR nanobody of this invention has the advantages of small molecular weight, high affinity, high stability, easy genetic engineering modification, and low production cost.

[0025] 2. Compared with camel-derived VHH nanobodies, the shark-derived VNAR antibody of this invention has advantages such as smaller molecular weight and higher stability, and has broad application prospects in the preparation of therapeutic antibody preparations for FPV-infected animals.

[0026] 3. The high-affinity VNAR antibody of this invention possesses a unique binding mode to the target protein, which can lead to enhanced neutralization ability against viral infections. Furthermore, the ease of genetic modification of VNAR antibodies makes them easier to prepare multivalent antibodies, avoiding immune escape due to viral mutations. Simultaneously, in the field of FPV infection diagnosis, the small size and prokaryotic expression capability of VNAR nanobodies will effectively improve diagnostic sensitivity and reduce production costs. Attached Figure Description

[0027] Figure 1 The results of antibody ELISA screening for FPV-VP2 protein-binding phages were obtained.

[0028] Figure 2 Amino acid sequence analysis of FPV-VP2-binding shark-derived VNAR antibody.

[0029] Figure 3 Phylogenetic analysis of the amino acid sequence of FPV-VP2-binding shark-derived VNAR antibody.

[0030] Figure 4 The purification results of four representative FPV-VP2 bound shark-derived VNAR antibodies, FCB2, FCD1, FCE2 and FCF6, are shown.

[0031] Figure 5 The results show the affinity assays of four representative FPV-VP2 binding shark VNAR antibodies FCB2, FCD1, FCE2, and FCF6 with the FPV-VP2 protein SPR. 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, and (d) shows the affinity assay results for antibody FCF6. Detailed Implementation

[0032] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0033] This invention provides a shark-derived nanobody targeting the FPV-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).

[0034] 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.16 to SEQ ID NO.30, or a sequence that has at least 80% homology with the sequence shown in any one of SEQ ID NO.16 to SEQ ID NO.30 and retains antigen-binding function.

[0035] 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.15, 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.15 and retains antigen-binding function.

[0036] The present invention also provides a biomaterial.

[0037] Specifically, the biomaterial includes at least one of the following:

[0038] a) Nucleotides used to encode the aforementioned shark-derived nanobodies;

[0039] b) An expression vector containing the above nucleotides;

[0040] c) A recombinant prokaryotic host containing the above-mentioned nucleotides or the above-mentioned expression vectors.

[0041] Preferably, the expression vector includes, but is not limited to, plasmids, viral vectors, and phage display vectors.

[0042] Furthermore, viral vectors include, but are not limited to, lentiviruses and adenoviruses.

[0043] Preferably, the recombinant prokaryotic host is the BL21(DE3) strain.

[0044] Furthermore, the recombinant host can also be a eukaryotic host cell.

[0045] The present invention also provides the application of the above-mentioned shark-derived nanobody or the above-mentioned biomaterial in the preparation of a detection reagent for feline panleukopenia virus.

[0046] Specifically, the detection target is the VP2 capsid protein of feline panleukopenia virus.

[0047] 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.

[0048] The present invention also provides the use of the above-mentioned shark-derived nanobody or the above-mentioned biomaterial in the preparation of a medicament capable of preventing and / or treating diseases caused by or related to feline panleukopenia virus infection in animals.

[0049] Specifically, the therapeutic target is the VP2 capsid protein of feline panleukopenia virus.

[0050] Specifically, the GenBank accession number for the DNA sequence encoding the VP2 capsid protein of feline panleukopenia virus is MT270585.1.

[0051] Specific embodiments of the present invention are as follows:

[0052] Example

[0053] In this embodiment, full-length FPV-VP2 protein was selected as the antigen, and multiple high-affinity VNAR nanobodies targeting FPV-VP2 were prepared by immunizing striped bamboo sharks. These nanobodies can serve as excellent candidate molecules for neutralizing antibodies required for the treatment of feline parvovirus infection.

[0054] The preparation process is as follows:

[0055] 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.

[0056] 2) Preparation of FPV-VP2 protein antigen: The FPV-VP2 protein DNA sequence (GenBank: MT270585.1) was synthesized as a whole gene 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 FPV-VP2 protein exceeded 85% and was used for immunization of striped bamboo shark.

[0057] 3) The striped bamboo shark underwent a total of 7 immunizations: The first immunization was performed by emulsifying 45 μg of FPV-VP2 protein antigen and 45 μg of CPV2c-VP2 protein antigen (VP2 protein of CPV2c type canine parvovirus) with an equal volume of Freund's complete adjuvant, and then injecting it subcutaneously at multiple points in the abdomen and pectoral fins of the anesthetized shark; the remaining immunizations were performed by emulsifying 45 μg of FPV-VP2 protein antigen and 45 μg of CPV2c-VP2 protein antigen with an equal volume of Freund's incomplete adjuvant, and then injecting it subcutaneously at multiple points in the abdomen and pectoral fins of the anesthetized shark.

[0058] 4) Obtaining and cloning the VNAR gene fragment: After immunization, shark venous blood and spleen tissue were collected. Lymphocytes were isolated from the venous blood and spleen tissue. 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 gene fragment was ligated to the pADL22c digested vector at a 3:1 molar ratio. The ligation product was purified using a PCR product recovery kit.

[0059] 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 volume of the constructed immunoglobulin was approximately 2.25 × 10⁻⁶ cells / mL. 8 .

[0060] 6) Pulverizing FPV-VP2 specific binding phages: Before each round of phage pulverization, M13KO7 helper phages were added to rescue and amplify phage particles displaying nanobodies, and the number of rescued phages used for pulverization was estimated by preparing phage gradient dilutions and infecting TG1 cells in the growth phase of the infection index.

[0061] The first round of FPV-VP2 specific binding phage panning was performed in MaxiSorp 96-well plates, with 1 μg of FPV-VP2 protein incubated in each well, and PBS negative control wells included; the plates were blocked with 5% skim milk; 10 μg of FPV-VP2 protein was added to each panning well. 11 The rescued and amplified phages were incubated on a shaker at room temperature to allow specific phages to bind to the target protein; the bound phages were eluted with 0.05% trypsin; 10 μl of the eluted phages were titrated by serial dilution to estimate the number of infectious recombinant phages recovered; the remaining eluted phages were used to infect TG1 cells and cultured overnight in LB medium (100 µg / ml ampicillin + 2% glucose) to recover the bound phages; the next day, an equal volume of 40% glycerol was added to the bacterial culture and aliquoted for cryopreservation for phage amplification and subsequent panning.

[0062] The second and third rounds of panning were similar to the first round, but the concentration of the coating antigen protein in the wells was halved each time to enrich high-affinity target protein-binding phages. Panning was stopped when the phage titers eluted from the antigen-coated wells differed by two orders of magnitude from those from the negative control wells.

[0063] 7) Screening for high-affinity antibody-displaying phages for FPV-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 VNAR nanobody and FPV-VP2 protein ELISA analysis. 0.2 µg of FPV-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; 5% skim milk was used for blocking; 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 after stopping the reaction with ELISA stop solution, absorbance was measured at 450 nm (see attached image). Figure 1 ).

[0064] Figure 1 This study presents the ELISA screening results of 188 potential TG1 monoclonal periplasmic space-expressed nanobodies that bind to the FPV-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 monoclonal clones selected from the sub-liquidity of the phage panned sub-liquidity library spread on solid plates. In the figure, darker blue indicates absorbance (OD) at 450 nm. 450 The higher the OD, 450 Experimental wells with a reading ratio greater than 4 to negative control wells H11 and H12 were selected as positive nanobody clones.

[0065] 8) Nanobody sequence analysis: OD of experimental wells and random nanobody sequences. 450 Clones with a read ratio greater than 4 were selected as positive clones. Multiple positive clones underwent DNA sequencing and were translated into amino acid sequences to obtain nanobody sequences (see attached). Figure 2 ).

[0066] Figure 2 The amino acid sequence alignment results of 30 FPV-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.

[0067] 9) Nanobody Grouping: Based on sequence affinity analysis, FPV-VP2-bound VNAR antibodies can be divided into 4 groups with CDR3 region lengths of 15, 16, 18, and 17 amino acids, respectively (see appendix).Figure 3 ).

[0068] like Figure 3 As shown, the 30 FPV-VP2 binding VNAR antibody amino acid sequences can be divided into 4 groups according to their phylogenetic relationship, and are distinguished by different colors.

[0069] The sequences of representative antibodies (those capable of binding to FPV-VP2 protein and whose CDR3 region amino acid sequence differs from other binding sequences) in each group are as follows:

[0070] ①The CDR3 region is 15 amino acids in length:

[0071] 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.16;

[0072] Shark-derived nanobody FCB2-B3: The amino acid sequence is shown in SEQ ID NO.2, and positions 88-102 are the CDR3 region, the amino acid sequence of which is shown in SEQ ID NO.17;

[0073] Shark-derived nanobody FCB2-C2: 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.18;

[0074] Shark-derived nanobody FCB2-D9: The amino acid sequence is shown in SEQ ID NO.4, and positions 88-102 are the CDR3 region, the amino acid sequence of which is shown in SEQ ID NO.19;

[0075] Shark-derived nanobody FCB2-E10: The amino acid sequence is shown in SEQ ID NO.5, and positions 88-102 are the CDR3 region, the amino acid sequence of which is shown in SEQ ID NO.20;

[0076] Shark-derived nanobody FCB2-E12: 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.21;

[0077] Shark-derived nanobody FCB2-F10: 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.22;

[0078] Shark-derived nanobody FCB2-F11: 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.23;

[0079] 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.24;

[0080] ②The CDR3 region is 17 amino acids in length:

[0081] 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.25;

[0082] 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.26;

[0083] ③The CDR3 region is 16 amino acids in length:

[0084] 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.27;

[0085] Shark-derived nanobody FCE2-C11: 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.28;

[0086] ④ The CDR3 region is 18 amino acids in length:

[0087] 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.29;

[0088] Shark-derived nanobody FCF6-A8: The amino acid sequence is shown in SEQ ID NO.15, and positions 88-105 are the CDR3 region, the amino acid sequence of which is shown in SEQ ID NO.30.

[0089] 10) Nanobody Expression: Four representative VNAR antibodies (named FCB2, FCD1, FCE2, and FCF6, with amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.10, SEQ ID NO.12, and SEQ ID NO.14, respectively) capable of binding to the FPV-VP2 protein were expressed in the BL21(DE3) prokaryotic system using the pCZN1 vector and purified using His-tag affinity. The purity of all bands was greater than 90%, and the molecular weight of the bands was approximately 14 kDa, meeting expectations. (See attached image) Figure 4 ).

[0090] 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. First, FPV-VP2 protein was immobilized on a CM5 chip as a ligand. Each nanobody was diluted to several concentrations in a 96-well plate, and the nanobodies were coupled to the target protein through the chip from low to high concentrations. After each concentration point was run through, 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 ).

[0091] SPR measurements showed that four representative shark-derived nanobodies exhibited very high affinity for the FPV-VP2 protein: the equilibrium dissociation constants of FCB2, FCD1, FCE2, and FCF6 with the FPV-VP2 protein were 2.04 × 10⁻⁶. -10 M, 1.92×10 -11 M, 8.83×10 -8 M, 1.73×10 -9 M.

[0092] It is evident that the equilibrium dissociation constants of shark-derived nanobodies FCB2 and FCD1 with FPV-VP2 protein can reach the picomolar level.

[0093] In summary, the high-affinity VNAR antibody targeting the FPV capsid protein involved in this invention can be used for the immunological diagnostic detection of feline panleukopenia virus infection. Furthermore, based on the excellent properties of VNAR antibodies, nanobody immunological detection reagents have advantages over traditional monoclonal antibody-based detection reagents, such as high stability and low production cost.

[0094] 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.

[0095] 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.

[0096] The amino acid sequence involved in this invention is as follows:

[0097] SEQ ID NO.1:

[0098] Name: Amino acid sequence of shark-derived nanobody FCB2 targeting FPV-VP2

[0099] Type: AA

[0100] Biological source: Striped bamboo shark ( Cihiloscyllium plagiasum )

[0101] MAARLEQTPTTTTKEAGESLTINCVLKGSSCALGRTYWYFTKKGATKKASLSTGGRYSDTKNTASKSFSLRISDLRVEDSGTYHCKAYREILGCQELGMIRYEGGGTILTVK

[0102] SEQ ID NO.2:

[0103] Name: Amino acid sequence of shark-derived nanobody FCB2-B3 targeting FPV-VP2

[0104] Type: AA

[0105] Biological source: Striped bamboo shark ( Cihiloscyllium plagiasum )

[0106] MAARLEQTPTTTTKEAGESLTINCVLKGSSCALGSTYWYFTKKGATKKASLSTGGRYAETVNKASKSFSLRISDLRVEDSGTYTCEAYRGILGCQELGAIRYEGGGTLVTVN

[0107] SEQ ID NO.3:

[0108] Name: Amino acid sequence of shark-derived nanobody FCB2-C2 targeting FPV-VP2

[0109] Type: AA

[0110] Biological source: Striped bamboo shark ( Cihiloscyllium plagiasum )

[0111] MAQRLEQTPTTTTKEAGESLTINCVLKGSSCALGSTFWYFTKKGATKKASLATGGRYSDTKNTASKSFSLRISDLRVEDSGTYHCEAYRRLLGCQELGAIRFEGSGTLLTVK

[0112] SEQ ID NO.4:

[0113] Name: Amino acid sequence of shark-derived nanobody FCB2-D9 targeting FPV-VP2

[0114] Type: AA

[0115] Biological source: Striped bamboo shark ( Cihiloscyllium plagiasum )

[0116] MAARVEQTPTTTTKEAGESLTINCVLKGSSCALGRTYWYFTKKGATQKASLSTGGRYSDTKNTASKSFSLRISDLRVEDSGTYHCEAYRSLLGCQELGLIRFEGGGTTLTVK

[0117] SEQ ID NO.5:

[0118] Name: Amino acid sequence of shark-derived nanobody FCB2-E10 targeting FPV-VP2

[0119] Type: AA

[0120] Biological source: Striped bamboo shark ( Cihiloscyllium plagiasum )

[0121] MAARVEQTPTTITKESGESLTINCVLKGSSCALGNTYWYFTKKGATKKASLSTGGRYSDTKNTASKSFSLRISDLRVEDSGTYHCEAYRGILGCQELGLIRFEGGGTIVTVN

[0122] SEQ ID NO.6:

[0123] Name: Amino acid sequence of shark-derived nanobody FCB2-E12 targeting FPV-VP2

[0124] Type: AA

[0125] Biological source: Striped bamboo shark ( Cihiloscyllium plagiasum )

[0126] MAARVEQTPTTTTKEAGESLTINCVLKGSNCALGNTNWYFTKKGATQKASLSTGGRYSDTKNTASKSFSLRISDLRVEDSGTYHCEAYRRLLGCQELGSIRFEGGGTLLTVK

[0127] SEQ ID NO.7:

[0128] Name: Amino acid sequence of shark-derived nanobody FCB2-F10 targeting FPV-VP2

[0129] Type: AA

[0130] Biological source: Striped bamboo shark ( Cihiloscyllium plagiasum )

[0131] MAARLEQTPTTTTKKAGESLTINCVLKGSTCALGSTLWYFTKKGATKKARLSTGGRYSDTKNTASKSFSLRISDLRVEDSGTYHCEAYRGILGCQELGAIRFEGGTTVTVK

[0132] SEQ ID NO.8:

[0133] Name: Amino acid sequence of shark-derived nanobody FCB2-F11 targeting FPV-VP2

[0134] Type: AA

[0135] Biological source: Striped bamboo shark ( Cihiloscyllium plagiasum )

[0136] MAARLEQTPTTTTKEAGESLTINCVLKGSSCALGSTFWYFTKKGATKKASLSTGGRYSDTKNTASKSFSLRISDLRVEDSGTYHCEAYRGILGCQELGLIRYEGGGTILTVN

[0137] SEQ ID NO.9:

[0138] Name: Amino acid sequence of shark-derived nanobody FCB2-F12 targeting FPV-VP2

[0139] Type: AA

[0140] Biological source: Striped bamboo shark ( Cihiloscyllium plagiasum )

[0141] MAPRVEQTPTTTTKEAGESLTINCVLKGSSCALGRTYWYFTKKGATQKASLLTGGRYSETKNTASKSFSLRISDLRVEDSGTYHCEAYRSVLGCQELGLIRFEGGGTILTVN

[0142] SEQ ID NO.10:

[0143] Name: Amino acid sequence of shark-derived nanobody FCD1 targeting FPV-VP2

[0144] Type: AA

[0145] Biological source: Striped bamboo shark ( Cihiloscyllium plagiasum )

[0146] MAPRVEQTPTTTTKEAGESLTINCVLKGTSYTLCNTYWYFTKKDATKKESLSNGGRYAETVHKASKSFSLRISDLRVEDSGTYHCKTYTRHPSIPTASRCILYYEGGGTLVTVK

[0147] SEQ ID NO.11:

[0148] Name: Amino acid sequence of shark-derived nanobody FCD1-A5 targeting FPV-VP2

[0149] Type: AA

[0150] Biological source: Striped bamboo shark ( Cihiloscyllium plagiasum )

[0151] MAPRLEQTPTTTTKEAGESLTINCVLKGTSYTLCNTYWYFTKKDATKKESLSNGGRYAETVHKASKSFSLRISDLRVEDSGTYHCKTYTRHPSLPTASRCILYYEGGGTIVTVK

[0152] SEQ ID NO.12:

[0153] Name: Amino acid sequence of shark-derived nanobody FCE2 targeting FPV-VP2

[0154] Type: AA

[0155] Biological source: Striped bamboo shark ( Cihiloscyllium plagiasum )

[0156] MAERVEQTPTTTTKEAGESLTINCVLKGSNCAAGTTYWYFTKKGATKKARLSTGGRYSDTKNTASKSFSLRISDLRVEDSGTYHCETYPITGAGCLWIAPYSYEGGGTILTVK

[0157] SEQ ID NO.13:

[0158] Name: Amino acid sequence of shark-derived nanobody FCE2-C11 targeting FPV-VP2

[0159] Type: AA

[0160] Biological source: Striped bamboo shark ( Cihiloscyllium plagiasum )

[0161] MAPRVEQTPTTTTKEAGESLTINCVLKGFSCALGNTYWYFTKKGATKKASLSTGGRYSDTKNTASNTFSLRISDLRVEDSGTYHCEIYPTTGAGCLWIAPYSYEGGGTTVTVK

[0162] SEQ ID NO.14:

[0163] Name: Amino acid sequence of shark-derived nanobody FCF6 targeting FPV-VP2

[0164] Type: AA

[0165] Biological source: Striped bamboo shark ( Cihiloscyllium plagiasum )

[0166] MAQRLEQTPTTTTKEAGESLTINCVLKGSSCSLGSTSWYFTKKGATKKASLSTGGRYSDTKNTASKSFSLRISDLRVEDSGTYYCQAFIVGLGCGSTPGSHHRYIEGGGTTVTVK

[0167] SEQ ID NO.15:

[0168] Name: Amino acid sequence of shark-derived nanobody FCF6-A8 targeting FPV-VP2

[0169] Type: AA

[0170] Biological source: Striped bamboo shark ( Cihiloscyllium plagiasum )

[0171] MAPRVEQTPTTTTKEAGESLTINCVLKGSSCSLGSTFWYFTKKGATKKASLSTGGRYSDTKNTASKSFSLRISDLRVEDSGTYHCQAFVLGLGCGTTPGSHHRYIEGGGTIVTVK

[0172] SEQ ID NO.16:

[0173] Name: Amino acid sequence of the CDR3 region of shark-derived nanobody FCB2 targeting FPV-VP2

[0174] Type: AA

[0175] Biological source: Striped bamboo shark ( Cihiloscyllium plagiasum )

[0176] YREILGCQELGMIRY

[0177] SEQ ID NO.17:

[0178] Name: Amino acid sequence of the CDR3 region of shark-derived nanobody FCB2-B3 targeting FPV-VP2

[0179] Type: AA

[0180] Biological source: Striped bamboo shark ( Cihiloscyllium plagiasum )

[0181] YRGILGCQELGAIRY

[0182] SEQ ID NO.18:

[0183] Name: Amino acid sequence of the CDR3 region of shark-derived nanobody FCB2-C2 targeting FPV-VP2

[0184] Type: AA

[0185] Biological source: Striped bamboo shark ( Cihiloscyllium plagiasum )

[0186] YRRLLGCQELGAIRF

[0187] SEQ ID NO.19:

[0188] Name: Amino acid sequence of the CDR3 region of shark-derived nanobody FCB2-D9 targeting FPV-VP2

[0189] Type: AA

[0190] Biological source: Striped bamboo shark ( Cihiloscyllium plagiasum )

[0191] YRSLLGCQELGLIRF

[0192] SEQ ID NO.20:

[0193] Name: Amino acid sequence of the CDR3 region of shark-derived nanobody FCB2-E10 targeting FPV-VP2

[0194] Type: AA

[0195] Biological source: Striped bamboo shark ( Cihiloscyllium plagiasum )

[0196] YRGILGCQELGLIRF

[0197] SEQ ID NO.21:

[0198] Name: Amino acid sequence of the CDR3 region of shark-derived nanobody FCB2-E12 targeting FPV-VP2

[0199] Type: AA

[0200] Biological source: Striped bamboo shark ( Cihiloscyllium plagiasum )

[0201] YRRLLGCQELGSIRF

[0202] SEQ ID NO.22:

[0203] Name: Amino acid sequence of the CDR3 region of shark-derived nanobody FCB2-F10 targeting FPV-VP2

[0204] Type: AA

[0205] Biological source: Striped bamboo shark ( Cihiloscyllium plagiasum )

[0206] YRGILGCQELGAIRF

[0207] SEQ ID NO.23:

[0208] Name: Amino acid sequence of the CDR3 region of shark-derived nanobody FCB2-F11 targeting FPV-VP2

[0209] Type: AA

[0210] Biological source: Striped bamboo shark ( Cihiloscyllium plagiasum )

[0211] YRGILGCQELGLIRY

[0212] SEQ ID NO.24:

[0213] Name: Amino acid sequence of the CDR3 region of shark-derived nanobody FCB2-F12 targeting FPV-VP2

[0214] Type: AA

[0215] Biological source: Striped bamboo shark ( Cihiloscyllium plagiasum )

[0216] YRSVLGCQELGLIRF

[0217] SEQ ID NO.25:

[0218] Name: Amino acid sequence of the CDR3 region of shark-derived nanobody FCD1 targeting FPV-VP2

[0219] Type: AA

[0220] Biological source: Striped bamboo shark ( Cihiloscyllium plagiasum )

[0221] YTRHPSIPTASRCILYY

[0222] SEQ ID NO.26:

[0223] Name: Amino acid sequence of the CDR3 region of shark-derived nanobody FCD1-A5 targeting FPV-VP2

[0224] Type: AA

[0225] Biological source: Striped bamboo shark ( Cihiloscyllium plagiasum )

[0226] YTRHPSLPTASRCILYY

[0227] SEQ ID NO.27:

[0228] Name: Amino acid sequence of the CDR3 region of shark-derived nanobody FCE2 targeting FPV-VP2

[0229] Type: AA

[0230] Biological source: Striped bamboo shark ( Cihiloscyllium plagiasum )

[0231] YPITGAGCLWIAPYSY

[0232] SEQ ID NO.28:

[0233] Name: Amino acid sequence of the CDR3 region of shark-derived nanobody FCE2-C11 targeting FPV-VP2

[0234] Type: AA

[0235] Biological source: Striped bamboo shark ( Cihiloscyllium plagiasum )

[0236] YPTTGAGCLWIAPYSY

[0237] SEQ ID NO.29:

[0238] Name: Amino acid sequence of the CDR3 region of shark-derived nanobody FCF6 targeting FPV-VP2

[0239] Type: AA

[0240] Biological source: Striped bamboo shark ( Cihiloscyllium plagiasum )

[0241] FIVGLGCGSTPGSHHRYI

[0242] SEQ ID NO.30:

[0243] Name: Amino acid sequence of the CDR3 region of shark-derived nanobody FCF6-A8 targeting FPV-VP2

[0244] Type: AA

[0245] Biological source: Striped bamboo shark ( Cihiloscyllium plagiasum )

[0246] FVLGLGCGTTPGSHHRYI.

Claims

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

10.

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

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.

Images