Hemoglobin shark nanobody and preparation method and application thereof

Abstract

The application discloses a hemoglobin shark-derived nanobody and a preparation method and application thereof. The nucleotide sequence of the nanobody is as shown in SEQ ID NO:1-3, and the amino acid sequence is as shown in SEQ ID NO:4-6. The preparation method comprises the following steps: immunizing a striped spiny dogfish with a hemoglobin antigen, extracting total RNA of peripheral blood lymphocytes and reverse transcribing the total RNA into cDNA; amplifying a VNAR fragment, connecting the VNAR fragment with a phagemid, and constructing a phage antibody library; screening a hemoglobin-specific nanobody sequence from the library and recombinantly expressing the hemoglobin-specific nanobody sequence, so as to obtain a shark-derived nanobody targeting hemoglobin. The nanobody disclosed by the application is derived from the striped spiny dogfish, has small molecular weight, high stability, strong tissue penetration ability and can recognize hidden antigen epitopes, and can be used in the fields of affinity purification of hemoglobin antibodies, immunological detection, cell imaging, disease diagnosis and the like, and can avoid problems such as instability of existing glycated hemoglobin or hemoglobin detection kits.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a hemoglobin shark-derived nanobody, its preparation method, and its application. Background Technology

[0002] Hemoglobin (Hb) is an iron-containing metalloprotein in the blood used to transport oxygenated red blood cells and plays a vital role in the body's metabolism. Hemoglobin is the most abundant protein in human serum, and its level is crucial to human health. Abnormal Hb levels in human serum can lead to certain health problems, such as anemia and polycythemia. Hemoglobin is of great significance in disease diagnosis. Anti-human hemoglobin antibodies specifically bind to human hemoglobin without reacting with other proteins, such as myoglobin and animal hemoglobin, and are not affected by various factors that could lead to false negatives or false positives. Furthermore, this method requires no special equipment, is simple to operate, and reacts rapidly, making it highly valuable for clinical applications.

[0003] Traditional monoclonal antibodies are often produced using mammalian cell lines to ensure correct structural folding and post-translational modifications such as glycosylation during manufacturing. Furthermore, the antigen-binding surfaces of traditional antibody Fab fragments and single-chain antibody scFvs often form concave topologies, typically only recognizing sites located on the antigen surface, thus limiting their widespread use and therapeutic efficacy. Using traditional antibodies to detect hemoglobin (Hb) may be affected by Hb variants. For example, common Hb variants like HbF and HbS can introduce errors in Hb measurement results, leading to low specificity or inaccurate quantification.

[0004] In the 1990s, Hamers Casterman and Muyldermans of the Free University of Brussels, Belgium, discovered a naturally occurring heavy chain antibody in the serum of camels, such as dromedary camels, Bactrian camels, and llamas. This antibody lacked the light chain portion and contained only the heavy chain portion. Subsequently, heavy chain antibodies were also detected in cartilaginous fish such as sharks. These antibodies had a variable region consisting of only one heavy chain variable region, which was called a single-domain antibody (sdAb). Single-domain antibody proteins are less than 10 nanometers in diameter and are therefore 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 penetrability, 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. Therefore, they can be used to help identify hidden antigenic epitopes that are difficult for traditional antibodies to recognize. It has potential applications in molecular imaging, disease diagnosis, immune detection, and environmental monitoring. Currently, there are no patents for shark-derived nanobody sequences that target hemoglobin. Summary of the Invention

[0005] To address the problems existing in the background art, the present invention aims to provide a hemoglobin-derived shark nanobody, its preparation method, and its applications. The present invention provides a shark heavy chain antibody variable region sequence (VNAR) capable of binding to hemoglobin with high affinity. This variable region sequence, also known as a nanobody, can be used in fields such as affinity purification of hemoglobin, immunological detection, cell imaging, disease diagnosis, and pharmaceutical manufacturing.

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

[0007] I. A type of hemoglobin shark-derived nanobody:

[0008] The nucleotide sequence of the shark-derived nanobody is SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3; the amino acid sequence of the shark-derived nanobody is SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO.6.

[0009] II. Application of a hemoglobin-derived shark nanobody:

[0010] Applications of the shark-derived nanobodies in the fields of affinity purification of hemoglobin, immunological detection, cell imaging, and disease diagnosis.

[0011] The application of shark-derived nanobodies in affinity purification of hemoglobin specifically refers to the enrichment and purification of hemoglobin from blood or tissue samples using shark-derived nanobodies.

[0012] The application of shark-derived nanobodies in the field of disease diagnosis includes their use in the preparation of drugs for diagnosing polycythemia / anemia.

[0013] The immunological tests include enzyme-linked immunosorbent assay (ELISA) for hemoglobin, chemiluminescent immunoassay, fluorescence immunoassay, colloidal gold immunoassay, and immunosensing assay.

[0014] The cell imaging includes immunofluorescence imaging and in vivo localization imaging.

[0015] III. A method for preparing a hemoglobin shark-derived nanobody, comprising the following steps:

[0016] S1: Preparation of hemoglobin solution;

[0017] S2: Immunize against the striped bamboo shark;

[0018] S3: Construct a phage antibody library;

[0019] S4: Screening for specific nanobodies;

[0020] S5: Nanobody Recombinant Expression: The antibody sequence is constructed into a prokaryotic or eukaryotic expression vector, and the recombinant protein is induced to be expressed. The antibody is then purified by affinity chromatography.

[0021] Specifically, S1 involves: firstly, isolating and purifying hemoglobin antigen from human blood cells, and then dissolving the hemoglobin antigen in a specific solvent / water to obtain a hemoglobin solution.

[0022] S2 specifically involves mixing hemoglobin solution with Freund's complete adjuvant at a volume ratio of 1:1. After complete emulsification, the mixture is subcutaneously injected into the lateral fin of the striped bamboo shark, and this subcutaneous injection is considered as one immunization. The striped bamboo sharks are immunized a total of four times, each time by subcutaneous injection. The interval between subcutaneous injections is 20 days, and the single immunization dose is 100 μg per striped bamboo shark.

[0023] Specifically, the hemoglobin solution prepared by S1 is first mixed with Freund's complete adjuvant at a volume ratio of 1:1. After complete emulsification, the mixture is subcutaneously injected into the lateral fin of the striped bamboo shark; this is the first immunization. Then, booster immunizations are administered to the striped bamboo shark every 20 days. Each booster immunization involves mixing the hemoglobin solution with Freund's incomplete adjuvant at a volume ratio of 1:1, and after complete emulsification, subcutaneously injecting the mixture into the lateral fin of the striped bamboo shark. This process is repeated four times to complete the immunization of the striped bamboo shark.

[0024] Specifically, S3 is:

[0025] S31. Ten days after the last immunization, the snout and gills of the striped bamboo shark were sprayed with MS-222 anesthetic with a mass fraction of 0.1% to anesthetize the striped bamboo shark. Then, peripheral blood of the striped bamboo shark was collected through the tail vein. The peripheral blood was centrifuged to collect lymphocyte solution. Total RNA was then extracted from the lymphocyte solution and reverse transcribed into cDNA.

[0026] S32. Using cDNA as a template, the VNAR fragment was amplified using a high-fidelity enzyme.

[0027] S33. Using pR2 phage particles as templates, amplify the pR2 phage particles with a high-fidelity enzyme, and digest the pR2 phage particle template with an endonuclease; the endonuclease is specifically Dpn I.

[0028] S34. The VNAR fragment and the enzyme-digested pR2 phage particles were ligated seamlessly, electrotransformed into competent bacteria, diluted and spread on the bacteria, and the bacteria were scraped and frozen to obtain a phage antibody library.

[0029] In practice, the size of the phage antibody library can be counted. If the library size is less than a preset threshold, the phage antibody library is discarded and step S3 is repeated to reprocess and prepare the antibody library.

[0030] Specifically, S4 is:

[0031] S41, the strains of the activated phage antibody library, and helper phages were added. After static culture and centrifugation to collect the bacterial cells, the bacterial cells were cultured overnight with shaking.

[0032] S42. Enrich phages using the PEG precipitation method and calculate the titer of the enriched phages.

[0033] S43. Coat the wells of a 96-well immunoplate with 0.1 mg / mL hemoglobin solution. The wells coated with protein are designated as the hemoglobin group, and the wells not coated with protein are designated as the negative control group. Enriched bacteriophages are added to both groups of wells to allow the bacteriophages to bind to the protein on the immunoplate. After multiple washes, bacteriophages that specifically bind to hemoglobin are obtained by elution with trypsin.

[0034] Specifically, hemoglobin solution and negative control were coated into the wells of an immunoassay plate, and then the enriched phages were added to the hemoglobin solution experimental group and the negative control group.

[0035] S44. Infect TG1 bacteria with bacteriophages that specifically bind to hemoglobin, dilute and spread on plates, culture to obtain single colonies, and count the number of colonies in the hemoglobin group and the negative control group, ensuring that the number of colonies in the hemoglobin group is greater than that in the negative control group.

[0036] S45. Pick single colonies of the hemoglobin group into a 96-well immunoassay plate, add helper phage, and amplify to obtain monoclonal phage;

[0037] S46. Coat the 96-well immunoplate again with 1 μg / mL hemoglobin solution, and use the wells coated with protein as the hemoglobin group and the wells not coated with protein as the negative control group. Add monoclonal phage to both groups to allow the monoclonal phage to bind to the protein on the immunoplate. Add horseradish peroxidase-labeled phage-specific antibody, use TMB chromogenic solution for color development, measure the absorbance of the solution after color development, screen out positive colonies, and thus obtain the hemoglobin-specific antibody sequence.

[0038] The positive result here refers to OD. 450 The value is greater than 1.0. Negative colonies are those on the plate where bacteriophages have not bound to hemoglobin, while positive colonies are those on the plate where bacteriophages have bound to hemoglobin. Specifically, sequencing primers are used to sequence positive colonies, multiple sequence alignment is performed, and repetitive sequences are removed to determine the hemoglobin-specific antibody sequence, as shown in SEQ ID NO.1 to SEQ ID NO.3.

[0039] The sequencing primers are specifically: 5'-CAGGAAACAGCTATGAC-3'.

[0040] The seamless cloning ligation in S34 is specifically as follows: the ligation system consists of 100 μL of seamless cloning ligase, 0.5 pmol pR2 phage, and 2 pmol VNAR fragment, with the total volume of pR2 phage and VNAR fragment being 50 μL; the ligation reaction conditions are a 50℃ water bath for 1 h.

[0041] The nucleotide sequences of the nanobodies of this invention are as shown in SEQ ID NO: 1-3, and the amino acid sequences are as shown in SEQ ID NO: 4-6. The preparation involves immunizing striped bamboo sharks with hemoglobin antigen, extracting total RNA from peripheral blood lymphocytes and reverse transcribing it into cDNA; amplifying the VNAR fragment and ligating it with phage particles to construct a phage antibody library; screening hemoglobin-specific nanobodies from the library and recombinantly expressing them to obtain shark-derived nanobodies targeting hemoglobin.

[0042] Compared with the prior art, the present invention has the following advantages:

[0043] 1. The nanobody of the present invention is derived from the striped bamboo shark. It has the advantages of small molecular weight, easy modification and expression, low immunogenicity, high antigen binding, and strong tissue penetration. It can be used in the fields of affinity purification of hemoglobin, immunological detection, in vivo localization and imaging, disease diagnosis and protein structure research.

[0044] 2. The screening of shark-derived nanobodies requires only two sharks. These nanobodies possess structural stability and antigen-binding activity comparable to the original heavy chain antibodies, and are the smallest known units capable of binding target antigens. They exhibit high specificity, good affinity, and high stability, and are widely used in biochemical mechanism research, structural biology, and the diagnosis and treatment of diseases such as tumors.

[0045] 3. Nanobodies exhibit high solubility and strong refolding ability, and can be obtained in large quantities through recombinant expression systems. Furthermore, nanobodies have already achieved intracellular drug delivery via intracellular antibody delivery and are being applied in clinical practice. Currently, there is considerable research on alpaca-derived single-domain antibodies, but less research on shark-derived single-domain antibodies. Therefore, this invention addresses the current situation of limited research on shark-derived single-domain antibodies, optimizing the shortcomings of traditional human monoclonal antibodies such as large molecular weight, weak affinity, and complex operation. Attached Figure Description

[0046] Figure 1 This is a schematic diagram of a shark immunization program using hemoglobin.

[0047] Figure 2 This is a diagram of the results of a monoclonal phage ELISA test.

[0048] Figure 3 This is a diagram showing the amino acid sequence alignment results of the nanobody.

[0049] Figure 4 The results of SDS-PAGE gel electrophoresis of the nanobody Fc fusion protein and lane M are the standard protein.

[0050] Figure 5 This is a graph showing the results of ELISA characterization of the binding between the nanobody Fc fusion protein and hemoglobin.

[0051] Figure 6 This is a schematic diagram illustrating the affinity between the nanobody and hemoglobin using BLI (Body Liposome Therapy). The solid line represents the real-time monitored kinetic curve, and the dashed line represents the software-fitted curve. The kinetic curves for different hemoglobin concentration gradients correspond sequentially from top to bottom to the concentrations indicated on the right, from top to bottom. Detailed Implementation

[0052] 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.

[0053] The embodiments of the present invention are as follows:

[0054] Example 1: Hemoglobin Immunization of Sharks

[0055] Sharks were immunized using isolated and purified hemoglobin, with the immunization protocol as follows: Figure 1As shown. This invention involves four immunizations, all administered subcutaneously. The immunization interval is 20 days. Ten days after the fourth immunization, peripheral blood was collected from the shark via the tail vein. This invention uses two striped bamboo sharks, with each shark receiving a hemoglobin dose of 100 μg per immunization.

[0056] Example 2: Screening of Hemoglobin Nanobodies

[0057] 1) Slowly add an equal volume of 30% (m / v) sucrose solution to the upper layer of the extracted shark peripheral blood, centrifuge at 300g for 30 min, collect the intermediate lymphocyte layer, wash twice with PBS, and centrifuge to collect the cell pellet. Extract total RNA using an RNA extraction kit and reverse transcribe it into cDNA.

[0058] 2) Using cDNA as a template, amplify the VNAR sequence with specific primers. The forward primers are:

[0059] The amplified VNAR fragment was amplified using the following primers: 5'-GCTGCACAGCCTGCTATGGCAACTCAACGGGTTGAA CAAACACCGAC-3', and the reverse primer was 5'-GAGTTTTTGTTCGGCTGCT GCTGGTTTTACAGTCAGAATGGTGCCGC-3'. The high-fidelity DNA polymerase used was PrimeSTAR Max DNA Polymerase, 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, specific primers were used to amplify the pR2 phage. The forward primer was 5'-AGCAGCC.

[0060] The reverse primer was 5'-CCATA GCAGGCTGTGCAGCATAGAAAGGTACCACTAAAGGAATTGC-3'. The DNA polymerase used for amplification was the high-fidelity enzyme PrimeSTAR GXL DNAPolymerase. The amplification program was: 98℃, 10s; 53℃, 15s; 68℃, 4min, 15s, for 30 cycles. The template pR2 phage was digested with Dpn I, and then the amplified pR2 phage was recovered using a kit. The pR2 phage and VNAR fragment were ligated using a seamless cloning method at a molar ratio of 1:4. The ligation product was recovered using a kit, and sterile water was used for elution. The ligation product was electroporated into TG1 competent cells and cultured at 37°C and 200 rpm for 1 h. 0.2 μL and 0.02 μL (dilution method) of the bacterial culture were then plated onto 90 mm agar plates and cultured for 13 h. The number of colonies was counted, and the size of the constructed antibody library was calculated. The remaining bacterial culture was centrifuged and plated onto five 150 mm agar plates, cultured at 37°C for 13 h, and then the bacterial colony was scraped off, flash-frozen in liquid nitrogen, and stored at -80°C. This constitutes the phage antibody library.

[0061] 3) Activate the frozen antibody library bacteria and add KM13 helper phage. Take the bacterial culture supernatant and measure the phage titer; this is the amplified phage. Coat the immunoassay plate with hemoglobin at a concentration of 0.1 mg / mL and add 1×10⁻⁶ ppm. 11 Phages amplified with PFU or higher were incubated at room temperature for 1 hour. Phages specifically bound to hemoglobin were eluted with trypsin solution, 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.

[0062] 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 hemoglobin with 1 μg / mL of the prepared monoclonal phage solution into each well of a 96-well immunoplate, and incubate at room temperature for 1 h. Capture the phage bound to hemoglobin 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 Numerical values, and organize them as follows Figure 2 The bar chart shown.

[0063] 5) Select OD 450 Wells with a value greater than 1 were used for sequencing analysis. The sequencing primer was 5'-CAGGAAACAGCTATGAC-3'.

[0064] 6) The measured antibody sequences were subjected to comparative analysis, and after excluding duplicate clones, a total of three distinct nanobody sequences were obtained( Figure 3 ). The nanobody nucleotide sequences are shown in SEQ ID NO.1-3, from which the nanobody amino acid sequences shown in SEQ ID NO.4-6 can be derived.

[0065] Example 3 Expression and purification of the obtained hemoglobin nanobody Fc fusion protein

[0066] The nanobody sequences of the present invention were constructed into the mammalian expression vector pTT5 carrying a signal peptide, to achieve fusion expression with human IgG1 Fc, wherein the Fc fragment was expressed at the C-terminus, and the nanobody could be obtained by TEV enzyme digestion. The recombinant plasmid was transfected into HEK 293 cells, the culture supernatant was collected, and the nanobody Fc fusion protein was purified via rProtein A affinity chromatography column. As shown Figure 4 , we obtained the high-purity hemoglobin nanobody Fc fusion protein.

[0067] Example 4 Characterization of the hemoglobin nanobody

[0068] 1) Non-competitive ELISA was used to characterize the binding of the nanobody Fc fusion protein to hemoglobin: hemoglobin at a concentration of 10 μg / mL was coated on an immunoplate, and the nanobody Fc fusion protein subjected to 1:5 gradient dilution was added, followed by incubation at room temperature for 1 h. Afterwards, HRP anti-IgG1 Fc antibody was added to detect the bound VNAR-Fc. The results are shown in Figure 5 , the binding force of the two nanobodies to hemoglobin follows the order: 7-11A-Fc>5-10C, and their EC 50 values are 8.01 nM and 531.04 nM respectively.

[0069] 2) BLI was used to characterize the affinity between the nanobody and hemoglobin. To characterize the affinity between the nanobody and hemoglobin, hemoglobin was first labeled with biotin to obtain biotinylated hemoglobin (biotin-Hb). Afterwards, it was immobilized on an SA biosensor, different nanobody concentration gradients were set, and the affinity of different nanobody Fc fusion protein concentration gradients to hemoglobin was detected. The results are shown in Figure 6 , the binding force of the three nanobodies to hemoglobin follows the order: 5-10C-Fc<7-11A-Fc<T-12-4D-Fc, and their affinity constant K D values are 1.08×10 -6 M, 5.627×10 -7 M and 5.96×10 -9 M respectively.

[0070] 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.

[0071] The sequence involved in this invention is as follows:

[0072] SEQ ID NO.1:

[0073] Name: Nucleotide sequence of 5-10C of hemoglobin shark-derived nanobody

[0074] DNA type: other DNA

[0075] Source: Striped bamboo shark (Cihiloscyllium plagiasum)

[0076] ACTCAACGGGTTGAACAAACACCGACAACGACAACAAAGGAGGCAGGCGAATCACTGACCATCAATTGCGTCCTAAGAGATTCCAGCTGTGCATTGGATAGCACGTACTGGTATTTCACAAAAAAGGGCGCAACAAAGAAGGAGAGCTTATCAAATGGCGGACGAT ACGCGGAAACAGTGAACAAGGCATCAAAGTCCTTTTCTTTGCGAATTAGTGACCTAAGAGTTGAAGACAGTGGTACATATCACTGTAAAGCGTATAGTACCTTCCCTTTCCCTACGCGGTTTCGGGTGTGGGGCGAAGGAGGCGGCACCATTCTGACTGTAAAACCT

[0077] SEQ ID NO.2:

[0078] Name: Nucleotide sequence of hemoglobin shark-derived nanobody 7-11A

[0079] DNA type: other DNA

[0080] Source: Striped bamboo shark (Cihiloscyllium plagiasum)

[0081] ACTCAACGGGTTGAAAAACACCGACAACGACAAGAAAGGAGACAG GCGAATCACTGACCATCAATTGCGTCCTAAAAGGTTCCAGATATGCATTGTGTAATACGTTTTGGGTATTTCACAAAAAAGGGCGCTACGAAGAAGGAGAACTTATCAACGGGCGGACGATACGCGGAAACAGTGAACAAGGCATC AAGGACCTTTTCTTTGCGAATTAGTGACCTAAGAGTTGAAGACAGTGGTACATATCACTGTAAAGTGTATATCCCCTCAGCTTGGGGAGGCTGTACTTGGGGGTACTATTATGAAGGAGGCGGCACCATTCTGACTGTAAAACCA

[0082] SEQ ID NO.3:

[0083] Name: Nucleotide sequence of hemoglobin shark-derived nanobody T-12-4D

[0084] DNA type: other DNA

[0085] Source: Striped bamboo shark (Cihiloscyllium plagiasum)

[0086] ACTCAACGGGTTGAACAAACACCGACAACGACAACAAAGGAGGCAGGCGAATCACTGACCATCAATTGCGTCCTAAAAGGTTCCAGTTATGCATTGTGTGACACGTACTGGTATTTCACAAAAAAGGGCGCAACAAAGAAGGAGAGCTTATCAAATGGCGGACGATAC GCGGAAACAGTGAACGAGGCATCAAAGTCCTTTTCTTTCGAATTAGTGACCTAAGAATTGAAGACAGTGGTACATATCACTGCAAAGCGTTATACACTCGGGAAGACCATTACTGTAACTGGAATGCTCCTTATGAAGGAGGCGGCACCATTCTGACTGTAAAACCT

[0087] SEQ ID NO.4:

[0088] Name: Amino acid sequence of 5-10C of hemoglobin shark-derived nanobody

[0089] Source: Striped bamboo shark (Cihiloscyllium plagiasum)

[0090] TQRVEQTPTTTTKEAGESLTINCVLRDSSCALDSTYWYFTKKGATKKESLSNGGRYAETVNKASKSFSLRISDLRVEDSGTYHCKAYSTFPFPTRFRVWGEGGGTILTVKP

[0091] SEQ ID NO.5:

[0092] Name: Amino acid sequence of hemoglobin shark-derived nanobody 7-11A

[0093] Source: Striped bamboo shark (Cihiloscyllium plagiasum)

[0094] TQRVEQTPTTTRKETGESLTINCVLKGSRYALCNTFWYFTKKGATKKENLSTGGRYAETVNKASRTFSLRISDLRVEDSGTYHCKVYIPSAWGGCTWGYYYEGGGTILTVKP

[0095] SEQ ID NO.6:

[0096] Name: Amino acid sequence of hemoglobin shark-derived nanobody T-12-4D

[0097] Source: Striped bamboo shark (Cihiloscyllium plagiasum)

[0098] TQRVEQTPTTTTKEAGESLTINCVLKGSSYALCDTYWYFTKKGATKKES

[0099] LSNGGRYAETVNEASKSFSLRISDLRIEDSGTYHCKALYTREDHYCNWNAPY

[0100] EGGGTILTVKP.

Claims

1. A hemoglobin shark-derived nanobody, characterized in that: The nucleotide sequence of the shark-derived nanobody is SEQ ID NO.1, SEQ ID NO.2, or SEQ ID NO.

3.

2. The application of the hemoglobin shark-derived nanobody as described in claim 1, characterized in that: Applications of the shark-derived nanobody in the fields of affinity purification of hemoglobin, immunological detection, cell imaging, and disease diagnosis and pharmaceuticals.

3. The application of the hemoglobin shark-derived nanobody according to claim 2, characterized in that: Application of the shark-derived nanobody in the preparation of drugs for diagnosing polycythemia and anemia.

4. The application of the hemoglobin shark-derived nanobody according to claim 2, characterized in that: The immunological tests include enzyme-linked immunosorbent assay (ELISA) for hemoglobin, chemiluminescent immunoassay, fluorescence immunoassay, colloidal gold immunoassay, and immunosensing assay.

5. The application of the hemoglobin shark-derived nanobody according to claim 2, characterized in that: The cell imaging includes immunofluorescence imaging and in vivo localization imaging.

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