An anti-HBsAg shark single-domain antibody or its antigen-binding fragment and its application

Abstract

The present invention discloses an anti-HBsAg shark single domain antibody or an antigen binding fragment thereof and an application thereof, relates to an anti-HBsAg shark single domain antibody and an amino acid and nucleotide coding sequence thereof, and also relates to a recombinant plasmid and a recombinant expression bacteria comprising the nucleotide coding sequence and an application thereof. The antibody has higher affinity and other pharmacological properties, is helpful to improve the medical level, is suitable for the preparation of drugs, and has broad market prospects.

Description

Technical Field

[0001] The present invention relates to anti-HBsAg shark single domain antibodies, more specifically, to anti-HBsAg shark single domain antibodies and nucleotides thereof. The present invention also relates to recombinant plasmids and recombinant expression bacteria comprising the nucleotide coding sequence, and belongs to the field of single domain antibody technology development. Background Art

[0002] Immunoglobulin New Antigen Receptor IgNAR is another known heavy chain antibody besides camel heavy chain antibodies. It exists in cartilaginous fish and was first isolated in the nurse shark (Ginglymostomacirratum). It was later found in the striped bamboo shark (Chiloscylliumplagiosum), a small shallow-bottom-dwelling shark.

[0003] The variable domain of IgNAR (VNAR) is the smallest antigen-binding region known to date. VNAR has some of the same desirable properties as VHH and has been identified as a possible biotherapeutic agent based on the following reasons: (1) Compared with IgG, the small size and penetrating ability of VNAR make it possible to enter dense tissues that IgG cannot enter; (2) the high urea content in shark serum makes VNAR have excellent stability and solubility; (3) VNAR has a tendency to bind to antigenic clefts and block the active site of enzymes, which is conducive to its more stable existence in vivo; (4) VNAR has high (potentially sub-nanomolecule) binding affinity for the active sites of multiple antigen-cleaving enzymes. The application development of shark-derived VNAR fragments is still in the preclinical stage. However, recent studies have shown that the variable domain of IgNAR has versatility in biomedical applications. Antigen-specific VNAR molecules have been isolated to combat a wide range of disease-related targets, including proteins, cytokines, toxins, and viral targets associated with cancer and arthritis.

[0004] Hepatitis B (HBV) is a disease caused by the infection of the hepatitis B virus (HBV) in living organisms. There are two main types of hepatitis B: acute hepatitis B and chronic hepatitis B. After being infected with HBV, the human body can easily develop from acute hepatitis B to chronic hepatitis B, or even to cirrhosis and hepatocellular carcinoma. According to the WHO's guidelines for the prevention and treatment of chronic hepatitis B, the goal of treating chronic hepatitis B is to eliminate HBV in the patient's body through various means, reduce inflammatory necrosis of liver cells and liver fibrosis, delay the deterioration of the patient's condition, and thereby reduce the incidence of adverse complications such as cirrhosis, liver failure, and liver cancer, improve the patient's quality of life, and prolong the patient's survival time. At present, the main treatment strategy for chronic hepatitis B is antiviral treatment. Drugs are used to inhibit various steps in the viral growth cycle, thereby inhibiting viral amplification and delaying the deterioration of the disease. In recent years, research on the treatment of chronic hepatitis B has focused more on various therapeutic vaccines, including multi-epitope vaccines and DNA vaccines.

[0005] However, there are relatively few studies on IgNAR in striped bamboo sharks, and there are no reports on VNAR against hepatitis B surface antigen (HBsAg). Using the immune characteristics of sharks to explore the preparation of single-domain antibodies for HBsAg has a positive clinical application prospect. It is also necessary and of great medical value to develop single-domain antibodies with higher affinity and other pharmacological properties for HBsAg. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a single-domain antibody against HBsAg, and at the same time provide the coding sequence of the single-domain antibody, a plasmid containing the coding sequence, and a recombinant IgNAR protein having binding activity with human or mouse homologous HBsAg protein.

[0007] In order to achieve the above object, the present invention is implemented by the following technical solutions:

[0008] The present invention discloses an anti-HBsAg shark single-domain antibody or an antigen-binding fragment thereof, wherein the anti-HBsAg shark single-domain antibody or the antigen-binding fragment thereof comprises a variable region VNAR of an IgNAR protein, and the amino acid sequence of the variable region VNAR comprises PZ-cluster 1329; the sequence of PZ-cluster 1329 is:

[0009] NVFTQRVEQTPTTTTKEAGESLTINCVLKGSRYALCKTYWYFTKKGATKKESLTNGGRYSVTMNKASKSFLLGISDLRVEDSGTYNCKAYSSGYTVHCDTGGQYYEGGGTILTVKPGKQPSPPIISLHYSAT.

[0010] The present invention discloses an anti-HBsAg shark single-domain antibody or an antigen-binding fragment thereof, characterized in that the anti-HBsAg shark single-domain antibody or the antigen-binding fragment thereof comprises a variable region VNAR of an IgNAR protein, and the amino acid sequence of the variable region VNAR comprises PZ-cluster 4364; the PZ-cluster4364 sequence is:

[0011] NVFTQWVEQTPTTTTTEAGESLTINCVLRGSSYALCNTYWYFTKKGATKKEILSNGGRYAATVNKASKSFSLRISDLRVEDSGTYHCVTTGCDGIAYYYEGGGTILTVKPGKQPSPPIISLHYSAT.

[0012] The present invention provides a nucleotide sequence encoding the anti-HBsAg shark single-domain antibody or its antigen-binding fragment, characterized in that it comprises nucleotides encoding a polypeptide, wherein the polypeptide comprises the shark single-domain antibody variable region VNAR; the variable region VNAR comprises PZ-cluster 1329, and the variable region VNAR can bind to human HBsAg.

[0013] The present invention provides a nucleotide sequence encoding the anti-HBsAg shark single-domain antibody or its antigen-binding fragment, characterized in that it comprises nucleotides encoding a polypeptide, wherein the polypeptide comprises the shark single-domain antibody variable region VNAR; the variable region VNAR comprises PZ-cluster4364, and the variable region VNAR can bind to human HBsAg.

[0014] The present invention provides a nucleotide sequence, the nucleotide sequence is PZ-cluster 1329', which belongs to the optimized VNAR protein nucleotide sequence, and the PZ-cluster 1329' is

[0015] AACGTGTTTACCCAGCGTGTGGAACAGACCCCGACCACCACCACCAAAGAAGCCGGTGAAAGTCTGACCATTAATTGCGTGCTGAAAGGTAGTCGCTATGCACTGTGCAAAACCTATTGGTATTTTACCAAAAAGGGTGCCACCAAAAAAGAAAGCCTGACCAATGGCGGTCGCTATAGCGTGACCATGAATAAGGCA AGTAAAAGTTTTCTGCTGGGCATTAGCGATCTGCGCGTTGAAGATAGTGGTACCTATAATTGCAAAGCATATAGTAGCGGCTATACCGTTCATTGCGATACCGGCGGTCAGTATTATGAAGGCGGTGGCACCATTCTGACCGTTAAACCGGGCAAACAGCCGAGTCCGCCGATTATTAGCCTGCATTATAGTGCCACC.

[0016] The nucleotide sequence of claim 4 of the present invention is an optimized VNAR protein nucleotide sequence, and the nucleotide sequence is PZ-cluster4364';

[0017] The PZ-cluster 4364' is:

[0018] AACGTGTTTACCCAGTGGGTGGAACAGACCCCGACCACCACCACAGAAGCAGGCGAAAGTCTGACCATTAATTGCGTGCTGCGCGGTAGCAGTTATGCCCTGTGTAATAACCTATTGGTATTTTACCAAAAAGGGTGCCACCAAAAAAGAAATTCTGAGCAATGGTGGTCGCTATGCCGCCACCGTG AATAAGGCAAGCAAAAGTTTTTAGCCTGCGTATTAGTGATCTGCGCGTTGAAGATAGTGGTACCTATCATTGTGTTACCACCGGCTGCGATGGTATTGCATATTATTATGAAGGTGGTGGTACCATTCTGACCGTGAAACCGGGTAAACAGCCGAGTCCGCCGATTATTAGTCTGCATTATAGCGCAACC.

[0019] The invention discloses a recombinant plasmid containing a nucleotide sequence.

[0020] The invention discloses a recombinant expression bacterium containing a recombinant plasmid.

[0021] The invention discloses an application of an anti-HBsAg shark single-domain antibody in the preparation of a hepatitis B therapeutic antibody drug.

[0022] The invention discloses an application of a nucleotide sequence in preparing a therapeutic antibody drug for hepatitis B surface antigen HBsAg.

[0023] The present invention provides a method for preparing an anti-HBsAg shark single domain antibody, characterized in that the preparation method comprises the following steps:

[0024] Step 1: preparing an expression vector containing a nucleotide sequence for expressing the anti-HBsAg shark single domain antibody or an antigen-binding fragment thereof;

[0025] Step 2: construct a recombinant expression bacterium containing the expression vector of step 1;

[0026] Step 3: Cultivate the recombinant expression bacteria constructed in step 2;

[0027] Step 4: Separate and purify to obtain the anti-HBsAg shark single domain antibody or its antigen-binding fragment.

[0028] Beneficial effects: It has the characteristics of high stability and good solubility, and can pass through the blood-brain barrier, breaking the previous disadvantage that the antibody cannot effectively reach the lesion due to its large molecular weight; VNAR can be used for disease diagnosis and research and development of drugs for the treatment of acute and chronic diseases; according to epidemiological surveys, nearly 80% of the total number of viral hepatitis cases is caused by infection with HBV. Chronic hepatitis B is an infectious disease that is prevalent worldwide. At present, antiviral drugs are mainly used, and there are few studies on the direct use of antibodies as therapeutic agents. The main limiting reasons are that ordinary double-chain antibodies have a large molecular weight, strong immunogenicity, low antibody preparation volume, and complex processes. The present invention uses the striped bamboo shark IgNAR as a research object. The VNAR in IgNAR has a small molecular weight and strong binding activity, which is more conducive to use as a therapeutic antibody. It has a simple structure and only requires a disulfide bond to form an effective antibody structure to meet the binding requirements. It can be expressed in a prokaryotic system, greatly reducing production costs, increasing production volume, and providing the possibility for mass preparation. This study immunized the striped bamboo shark with HBsAg and screened specific antibodies, providing new ideas for the treatment of chronic hepatitis B and a reference for the screening of specific antibodies for other antigens. It is suitable for the preparation of drugs and has broad market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 SDS-PAGE electrophoresis of whole bacteria before and after protein 1329 induction, supernatant and precipitate after disruption.

[0030] Figure 2 SDS-PAGE electrophoresis of whole bacteria, supernatant and precipitate after protein 4364 induction.

[0031] Figure 3 SDS-PAGE electrophoresis of protein 1329 purified by nickel column.

[0032] Figure 4 SDS-PAGE electrophoresis of protein 4364 purified by nickel column.

[0033] Figure 5 SDS-PAGE electrophoresis of the purified protein samples.

[0034] Figure 6 Double antibody sandwich Elisa method to detect protein in vitro activity. DETAILED DESCRIPTION

[0035] The effective amount described in the present invention refers to the amount or dosage of the product described in the present invention (preferably anti-HBsAg antibody or antigen-binding fragment thereof) that provides the desired treatment after being administered to a patient, organ or individual in a single or multiple doses.

[0036] The "include" or "comprising" described in the present invention is an open description, which contains the specified components or steps described, as well as other specified components or steps that do not substantially affect the technical effect. When it is used in this application to describe the sequence of a protein or nucleic acid, the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the activity described in the present invention.

[0037] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0038] The present invention is described in detail below in conjunction with specific embodiments, but the present invention is not limited to these implementations, and any improvement or substitution based on the basic spirit of the present embodiment still falls within the scope of protection required by the claims of the present invention.

[0039] Example 1

[0040] Acquisition of HBsAg single domain antibody sequence

[0041] Sequence screening:

[0042] Sharks were immunized with commercial HBsAg-ad antigen, and the spleen and lymphocytes of the immunized sharks were taken for transcriptome sequencing, the serum of the immunized sharks was taken for mass spectrometry detection, and the RNA of the spleen and lymphocytes of the immunized sharks was extracted for RT-PCR to obtain a high-throughput database containing only the shark VNAR region. The obtained transcriptome high-throughput sequencing results were compared with the high-throughput database of the VNAR region, and sequences that were contained in both databases and had a high content were screened; on the other hand, the mass spectrometry results were compared with the high-throughput database of the shark VNAR region, and sequences that were contained in both databases and had a high content were screened. The VNAR variable region sequences PZ-cluster 1329 and PZ-cluster 4364 were obtained by combining the two screening results; IgNAR protein is a dimer, and each chain contains 1 variable region (VNAR) and 5 constant regions (CNAR);

[0043] Gene sequence synthesis:

[0044] The amino acid fragments screened from the amino acid sequence of the variable region VNAR of the IgNAR protein were selected, the codons of the encoded nucleotide sequence were optimized for E. coli expression, and restriction sites (preferably BamH and XhoI) were added to both ends of the optimized gene for artificial synthesis. The synthetic nucleotide sequence was PZ-cluster1329':

[0045] AACGTGTTTACCCAGCGTGTGGAACAGACCCCGACCACCACCACCAAAGAAGCCGGTGAAAGTCTGACCATTAATTGCGTGCTGAAAGGTAGTCGCTATGCACTGTGCAAAACCTATTGGTATTTTACCAAAAAGGGTGCCACCAAAAAAGAAAGCCTGACCAATGGCGGTCGCTATAGCGTGACCATGAATAAGGCA AGTAAAAGTTTTCTGCTGGGCATTAGCGATCTGCGCGTTGAAGATAGTGGTACCTATAATTGCAAAGCATATAGTAGCGGCTATACCGTTCATTGCGATACCGGCGGTCAGTATTATGAAGGCGGTGGCACCATTCTGACCGTTAAACCGGGCAAACAGCCGAGTCCGCCGATTATTAGCCTGCATTATAGTGCCACC

[0046] PZ-cluster4364':

[0047] AACGTGTTTACCCAGTGGGTGGAACAGACCCCGACCACCACCACAGAAGCAGGCGAAAGTCTGACCATTAATTGCGTGCTGCGCGGTAGCAGTTATGCCCTGTGTAATAACCTATTGGTATTTTACCAAAAAGGGTGCCACCAAAAAAGAAATTCTGAGCAATGGTGGTCGCTATGCCGCCACCGTG AATAAGGCAAGCAAAAGTTTTTAGCCTGCGTATTAGTGATCTGCGCGTTGAAGATAGTGGTACCTATCATTGTGTTACCACCGGCTGCGATGGTATTGCATATTATTATGAAGGTGGTGGTACCATTCTGACCGTGAAACCGGGTAAACAGCCGAGTCCGCCGATTATTAGTCTGCATTATAGCGCAACC

[0048] The nucleotide sequence of recombinant IgNAR protein is artificially synthesized, the sequence is accurate, it can be obtained in large quantities through fermentation, the production cost is low, it is environmentally friendly, and can be prepared in large quantities; the recombinant IgNAR protein can effectively bind to antigen proteins and successfully replace or partially replace immunoglobulin IgG; the recombinant IgNAR protein has a small molecular weight, and the V region of the recombinant single-domain antibody has a molecular weight of only about 15kDa compared to traditional antibodies. This feature enables it to carry drugs into cells or penetrate the blood-brain barrier.

[0049] Implementation 2

[0050] Construction of recombinant expression plasmid:

[0051] The gene sequence was inserted into the expression vector pET-22b by double enzyme digestion to obtain the pET-22b-IgNAR recombinant plasmid.

[0052] Example 3

[0053] Construction of recombinant expression bacteria:

[0054] 1) Prepare competent E. coli cells. The strain may be E. coli BL21 (DE3), Rosetta (DE3) or other expression bacteria, preferably E. coli BL21 (DE3).

[0055] 2) Slowly add 2 μL of recombinant plasmid pET-22b-IgNAR (no more than 50 ng) into the clean bench, and slowly stir with the tip of a pipette to mix evenly, and place on ice for 30 minutes.

[0056] 3) Heat shock in a 42°C constant temperature water bath for 90 seconds. Do not touch the competent cells during the heat shock process. After the heat shock, quickly transfer to ice and incubate for 5 minutes.

[0057] 4) Add 1 mL of LB liquid culture medium preheated at 37°C to the clean bench, place in a 37°C shaker, and shake and culture at 220 rpm for 1 hour to revive the competent cells.

[0058] 5) After recovery, centrifuge at room temperature at 10,000 rpm for 5 minutes.

[0059] 6) Discard 1 mL of supernatant in a clean bench, mix the remaining supernatant and precipitate, and aspirate 50 μL to spread on an ampicillin-resistant plate.

[0060] 7) Place the plate in a 37°C biochemical incubator and incubate upside down for about 12 hours.

[0061] 8) Use an inoculation loop to pick up a single colony and inoculate it into 5 mL of LB liquid culture medium. Then add 5 μL Amp and place it in a shaker at 37°C and 220 rpm for 12 h.

[0062] 9) Sequencing confirmed that the transformation was successful and the recombinant expression bacteria E. coli BL21-pET-22b-IgNAR were obtained.

[0063] Example 4

[0064] Inducible expression of recombinant proteins:

[0065] 1) The recombinant expression bacteria were inoculated into LB medium containing Amp resistance at a ratio of 1:100 and cultured at 37°C and 220 rpm / min for 4 h.

[0066] 2) Take 1 mL of bacterial solution as the pre-induction sample.

[0067] 3) Add IPTG to make the final concentration of IPTG 1-0.5 mM, preferably 0.5 mM, and continue culturing at 6°C for 12-16 h (preferably 12 h).

[0068] Example 5

[0069] 1) After induction, take 1 mL of bacterial solution as the post-induction sample, centrifuge the sample at 12000 rpm and 4°C for 10 min, discard the supernatant, add 1 mL of PBS to wash once, add 100 μL of PBS to resuspend, add 25 μL of Loading Buffer to mix, and place in a metal bath at 100°C for 10 min for protein gel electrophoresis.

[0070] 2) After the remaining cells were centrifuged, an appropriate amount of PBS was added, mixed and then ultrasonically disrupted on ice for 3 seconds and then 3 seconds, until the cells became transparent, and then centrifuged at 12000rpm and 4℃ for 20min. 100μL of the supernatant and precipitate were taken for protein gel electrophoresis. Figure 1 As shown, the results of 4364 protein induction are as follows Figure 2 shown.

[0071] 3) Recombinant IgNAR was expressed in the supernatant at 16°C, and the supernatant was purified according to the nickel column purification instructions. The His buffer containing 25, 50, 100, 200, 250, 300, 400, and 500 mM imidazole was used for elution to explore the optimal elution concentration. The results are as follows: Figure 3 As shown, the results of 4364 proteins are as follows Figure 4 As shown, the elution effect of the eluent containing 250 mM imidazole is better. The eluent containing 250 mM imidazole is collected, and the impurity proteins are washed away with the eluent containing 50 or 100 mM imidazole.

[0072] 4) The collected eluate containing the target protein was placed in a dialysis bag (dialysis conditions: molecular weight of the dialysis bag was 3 kDa, 4°C, and the liquid was changed every 6 hours) using PBS as the buffer solution overnight.

[0073] 5) The dialyzed protein is concentrated to 1-2 mL using a 3 kDa ultrafiltration tube at 5000 rpm and 4°C to obtain the purified protein.

[0074] 6) Identification: The purified recombinant VNAR was subjected to SDS-PAGE detection to identify the purification results, such as Figure 5 shown.

[0075] Example 6

[0076] In vitro activity detection (double antibody sandwich Elisa):

[0077] 1) Dilute the recombinant IgNAR protein to different concentrations with sodium carbonate-sodium bicarbonate buffer (CBS, pH 9.6), and plate 100 μL per well. The control is sodium carbonate-sodium bicarbonate buffer. Plate at 4°C overnight.

[0078] 2) The next day, the cells were washed three times with PBST for 5 min each time, incubated with 5% skim milk powder at 37°C for 2 h, and then washed three times with PBST for 5 min each time.

[0079] 3) Incubate with primary antibody. Dilute HBsAg protein to 2 μg / mL with 1% BSA, 2% skim milk powder solution, add 100uL to each well, incubate at 37°C for 2h, wash 4 times with PBST, 5min each time, and pat dry the plate.

[0080] 4) Mouse anti-HBsAg HRP-tagged antibody was used as the secondary antibody, diluted 1:10,000, incubated at 37°C for 2 h, washed 4 times with PBST, 5 min each time, and the plate was patted dry.

[0081] 5) Add 100 μL of TMB colorimetric solution to each well and react in the dark at 37°C for 15 min. After the reaction, add 50 μL of 2M H2SO4 stop solution to each well and measure the absorbance at 450 nm with a microplate reader.

[0082] Under different concentrations of single-domain antibody IgNAR, when the antigen HBsAg protein concentration was 2μg / mL, the results were as follows Figure 6 As shown in the figure, when the absorbance of the experimental group (OD450) / the absorbance of the blank group (OD450)>2.1, the result is positive. The experimental results show that the detection difference is significant, indicating that the single-domain antibody IgNAR has good binding properties with the antigen HBsAg protein.

[0083] Example 7

[0084] The present invention provides an application of an anti-HBsAg single domain antibody or an antigen binding fragment thereof in antibody-drug conjugates and pharmaceutical carriers.

[0085] Example 8

[0086] The present invention provides an application of an anti-HBsAg single domain antibody or an antigen binding piece thereof in a pharmaceutical composition.

[0087] Example 9

[0088] The invention discloses an application of an anti-HBsAg single domain antibody or an antigen binding fragment thereof in preparing a product for treating hepatitis B-related diseases.

[0089] Finally, it should be noted that the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by ordinary technicians in this field should be considered as the protection scope of the present invention. Invention Name: An anti-HBsAg shark single-domain antibody or its antigen-binding fragment and its application First applicant: Zhejiang Sci-Tech University The sequence of PZ-cluster 1329 is: NVFTQRVEQTPTTTTKEAGESLTINCVLKGSRYALCKTYWYFTKKGATKKESLTNGGRYSVTMNKASKSFLLGISDLRVEDSGTYNCKAYSSGYTVHCDTGGQYYEGGGTILTVKPGKQPSPPIISLHYSAT. The sequence of PZ-cluster4364 is: NVFTQWVEQTPTTTTTEAGESLTINCVLRGSSYALCNTYWYFTKKGATKKEILSNGGRYAATVNKASKSFSLRISDLRVEDSGTYHCVTTGCDGIAYYYEGGGTILTVKPGKQPSPPIISLHYSAT. The sequence of PZ-cluster 1329’ is: AACGTGTTTACCCAGCGTGTGGAACAGACCCCGACCACCACCACCAAAGAAGCCGGTGAAAGTCTGACCATTAATTGCGTGCTGAAAGGTAGTCGCTATGCACTGTGCAAAACCTATTGGTATTTTACCAAAAAGGGTGCCACCAAAAAAGAAAGCCTGACCAATGGCGGTCGCTATAGCGTGACCATGAATAAGGCAAGTAAAAGTTTTCTGCTGGGCATTAGCGATCTGCGCGTTGAAGATAGTGGTACCTATAATTGCAAAGCATATAGTAGCGGCTATACCGTTCATTGCGATACCGGCGGTCAGTATTATGAAGGCGGTGGCACCATTCTGACCGTTAAACCGGGCAAACAGCCGAGTCCGCCGATTATTAGCCTGCATTATAGTGCCACC. The sequence of PZ-cluster 4364’ is: AACGTGTTTACCCAGTGGGTGGAACAGACCCCGACCACCACCACCACAGAAGCAGGCGAAAGTCTGACCATTAATTGCGTGCTGCGCGGTAGCAGTTATGCCCTGTGTAATACCTATTGGTATTTTACCAAAAAGGGTGCCACCAAAAAAGAAATTCTGAGCAATGGTGGTCGCTATGCCGCCACCGTGAATAAGGCAAGCAAAAGTTTTAGCCTGCGTATTAGTGATCTGCGCGTTGAAGATAGTGGTACCTATCATTGTGTTACCACCGGCTGCGATGGTATTGCATATTATTATGAAGGTGGTGGTACCATTCTGACCGTGAAACCGGGTAAACAGCCGAGTCCGCCGATTATTAGTCTGCATTATAGCGCAACC。

Claims

1. An anti-HBsAg shark single domain antibody, characterized in that: The anti-HBsAg shark single domain antibody comprises the variable region VNAR of the IgNAR protein, and the amino acid sequence of the variable region VNAR is shown in the sequence of PZ-cluster 1329 or PZ-cluster 4364; the sequence of PZ-cluster 1329 is: NVFTQRVEQTPTTTTKEAGESLTINCVLKGSRYALCKTYWYFTKKGATKKESLTNGGRYSVTMNKASKSFLLGISDLRVEDSGTYNCKAYSSGYTVHCDTGGQYYEGGGTILTVKPGKQPSPPIISLHYSAT; The PZ-cluster4364 sequence is: NVFTQWVEQTPTTTTTEAGESLTINCVLRGSSYALCNTYWYFTKKGATKKEILSNGGRYAATVNKASKSFSLRISDLRVEDSGTYHCVTTGCDGIAYYYEGGGTILTVKPGKQPSPPIISLHYSAT.

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