Single-chain antibody capable of specifically recognizing artemisinin or antigen binding fragment of single-chain antibody and application of single-chain antibody

Abstract

The invention discloses a single-chain antibody for specifically recognizing artemisinin or an antigen binding fragment thereof and application thereof. The single-chain antibody for resisting artemisinin or the antigen binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises a CDR1 of which the amino acid sequence is shown as SEQ ID NO: 9, a CDR2 of which the amino acid sequence is shown as SEQ ID NO: 10 and a CDR3 of which the amino acid sequence is shown as SEQ ID NO: 11; the light chain variable region comprises a CDR1 of which the amino acid sequence is as shown in SEQ ID NO: 12, a CDR2 of which the amino acid sequence is as shown in SEQ ID NO: 13 and a CDR3 of which the amino acid sequence is as shown in SEQ ID NO: 14. The anti-artemisinin single-chain antibody provided by the scheme of the invention can specifically recognize artemisinin, can be directly used for artemisinin detection, and has high sensitivity.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a single-chain antibody specifically recognizing artemisinin or an antigen-binding fragment thereof and applications thereof. Background Art

[0002] Artemisinin, also known as artemisinin, is a sesquiterpene endoperoxide / δ-lactone drug used to treat malaria caused by the protozoan Plasmodium falciparum. According to the WHO report, artemisinin-based combination therapies (ACTs) remain the most effective treatment for falciparum malaria, and a total of 242 million doses of artemisinin drugs were distributed worldwide in 2021. Despite this, Africa remains the region with the heaviest malaria burden. As of now, Africa bears approximately 95% of malaria cases and 96% of malaria deaths worldwide. Due to the lack of efficient and rapid detection methods and conditions, many counterfeit and substandard artemisinin-based antimalarial drugs have entered the African market, aggravating the spread of malaria in Africa. Therefore, it is very necessary to establish an accurate, sensitive, rapid and simple analytical method for the detection of artemisinin-based antimalarial drugs.

[0003] Currently, the main methods reported for detecting artemisinin-based drugs include HPLC, TLC, LC-MS, infrared absorption spectroscopy, and immunoassay. Although chromatography and mass spectrometry have high accuracy and precision, they are not conducive to rapid and large-scale drug screening due to their disadvantages such as being cumbersome, time-consuming, and expensive. In contrast, immunoassays, such as enzyme-linked immunosorbent assay (ELISA), are simple and rapid, and are currently one of the main technologies for large-scale screening of small molecule drugs. As the core recognition element of immunoassays, the binding properties of antibodies directly determine the sensitivity, specificity, and scope of application of the detection method. Currently reported immunoassay methods for artemisinin detection are mostly broad-spectrum monoclonal antibodies with high cross-reaction rates and low specificity for artemisinin. Recombinant single-chain antibodies have the advantages of small molecular weight, large-scale production, and easy modification, and can meet the needs of high-sensitivity and high-throughput detection of artemisinin.

[0004] At present, there are no reports on single-chain antibodies that specifically recognize artemisinin at home or abroad. Summary of the Invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention provides an anti-artemisinin single-chain antibody or an antigen-binding fragment thereof.

[0006] The present invention also provides a recombinant protein.

[0007] The present invention also provides biological materials related to the antibody or antigen-binding fragment thereof described in the first aspect, or the recombinant protein described in the second aspect.

[0008] The present invention also provides a conjugate.

[0009] The invention also provides a product.

[0010] The present invention also provides uses of the antibody or antigen-binding fragment thereof described in the first aspect, the recombinant protein described in the second aspect, the biomaterial described in the third aspect, the conjugate described in the fourth aspect, or the product described in the fifth aspect.

[0011] The present invention also provides a method for detecting artemisinin or artemisinin content.

[0012] The present invention also provides a method for preparing the above-mentioned single-chain antibody or antigen-binding fragment thereof.

[0013] According to a first aspect of the present invention, an anti-artemisinin single-chain antibody or an antigen-binding fragment thereof is provided, wherein the anti-artemisinin single-chain antibody or the antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region;

[0014] The heavy chain variable region comprises a CDR1 with an amino acid sequence as shown in SEQ ID NO: 9, a CDR2 with an amino acid sequence as shown in SEQ ID NO: 10, and a CDR3 with an amino acid sequence as shown in SEQ ID NO: 11;

[0015] The light chain variable region comprises a CDR1 with an amino acid sequence as shown in SEQ ID NO: 12, a CDR2 with an amino acid sequence as shown in SEQ ID NO: 13, and a CDR3 with an amino acid sequence as shown in SEQ ID NO: 14.

[0016] According to some embodiments of the present invention, the amino acid sequence of the heavy chain variable region comprises:

[0017] a1) SEQ ID NO: 17; or

[0018] a2) an amino acid sequence obtained by substituting and / or deleting and / or adding one or more amino acids to SEQ ID NO: 17 and having the same function as the protein shown in SEQ ID NO: 1; or

[0019] a3a2) having an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, or 70% identical to SEQ ID NO: 17 and has the same function as the protein shown in SEQ ID NO: 17.

[0020] According to some embodiments of the present invention, the amino acid sequence of the light chain variable region comprises:

[0021] a1) SEQ ID NO: 18; or

[0022] a2) an amino acid sequence obtained by substituting and / or deleting and / or adding one or more amino acids to SEQ ID NO: 18 and having the same function as the protein shown in SEQ ID NO: 1; or

[0023] a3a2) having an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, or 70% identical to SEQ ID NO: 18 and has the same function as the protein shown in SEQ ID NO: 18.

[0024] According to some embodiments of the invention, the heavy chain variable region is connected to the light chain variable region with or without a connecting peptide.

[0025] According to some embodiments of the present invention, the connecting peptide is selected from a rigid connecting peptide or a flexible connecting peptide.

[0026] According to some embodiments of the present invention, the flexible connecting peptide is selected from (G3S) n 、(G4S) n or (G) n ; wherein n is an integer not less than 3. For example, n can be 3, 4, 5, 6, 7, 8, 9 or 10.

[0027] According to some embodiments of the present invention, the nucleotide sequence of the flexible connecting peptide is shown in SEQ ID NO:19.

[0028] According to some embodiments of the present invention, the amino acid sequence of the anti-artemisinin single-chain antibody or antigen-binding fragment thereof is any one of A1) to A3):

[0029] A1) the amino acid sequence shown in SEQ ID NO: 2;

[0030] A2) an amino acid sequence having the same function as the protein shown in SEQ ID NO: 2, wherein one or more amino acids are substituted and / or deleted and / or added;

[0031] A3) an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71% or 70% identical to the sequence of SEQ ID NO: 2, and has the same function as the protein of SEQ ID NO: 2.

[0032] According to some embodiments of the invention, the same function refers to specific binding to artemisinin.

[0033] The second aspect of the present invention provides a recombinant protein comprising the antibody or antigen-binding fragment thereof according to the first aspect of the present invention; and an optional tag.

[0034] According to some embodiments of the present invention, the tag may be attached to the N-terminus and / or C-terminus of the antibody or antigen-binding fragment thereof. The tag may be a tag that facilitates the solubilization and / or purification of the antibody or antigen-binding fragment thereof.

[0035] According to some embodiments of the present invention, the tag comprises at least one of a His tag, a FLAG tag, a Strep tag, a Nus tag, a maltose binding protein, and a GST tag. The recombinant protein of the present invention may comprise one or more tags; the multiple tags may comprise a combination of multiple identical tags or a combination of multiple different tags.

[0036] The third aspect of the present invention provides a biomaterial related to the antibody or antigen-binding fragment thereof described in the first aspect, or the recombinant protein described in the second aspect, wherein the biomaterial comprises at least one of B1) to B5);

[0037] B1) a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to the first aspect, or the recombinant protein according to the second aspect;

[0038] B2) an expression cassette containing the nucleic acid molecule described in B1);

[0039] B3) a recombinant vector containing the nucleic acid molecule described in B1) or the expression cassette described in B2);

[0040] B4) a recombinant microorganism containing the nucleic acid molecule described in B1), the expression cassette described in B2), or the recombinant vector described in B3);

[0041] B5) A transgenic cell line containing the nucleic acid molecule described in B1) or the expression cassette described in B2) or the recombinant vector described in B3).

[0042] According to some embodiments of the present invention, the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.

[0043] According to some embodiments of the present invention, the nucleic acid molecule is a DNA molecule encoding the antibody or antigen-binding fragment thereof, or the recombinant protein.

[0044] According to some embodiments of the present invention, the nucleic acid molecule in B1) includes any one of C1) to C2):

[0045] C1) the nucleotide sequence shown in SEQ ID NO: 1 or a degenerate sequence thereof;

[0046] C2) A DNA molecule that hybridizes with the DNA molecule defined in C1) under stringent conditions and encodes the antibody or antigen-binding fragment thereof or the recombinant protein.

[0047] According to some embodiments of the invention, the degenerate sequence has at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70% identity to the nucleotide sequence shown in SEQ ID NO:1.

[0048] According to some embodiments of the present invention, the expression cassette refers to a DNA capable of expressing the antibody or antigen-binding fragment thereof in a host cell, and includes at least one of a promoter, an enhancer sequence, an origin of replication, a terminator, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, and a reporter gene.

[0049] According to some embodiments of the present invention, the recombinant vector includes but is not limited to a plasmid vector, a cosmid vector and a viral vector (such as a phage vector, a lentiviral vector, an adenoviral vector, a retroviral vector and an adeno-associated viral vector). The recombinant vector is used to achieve the replication, integration, amplification and / or expression of the exogenous target gene in the recipient cell; it can be a cloning vector or an expression vector. For example, the recombinant vector can specifically be a vector that inserts the nucleic acid molecule into a pMD TM The recombinant vector obtained by 19-T vector.

[0050] According to some embodiments of the present invention, the recombinant microorganism may be a bacterium (such as Escherichia coli or Bacillus subtilis, etc.) or a fungus (such as yeast or Aspergillus, etc.).

[0051] According to some embodiments of the present invention, the transgenic cell line can be an insect cell (such as S2 Drosophila cells or Sf9 cells), an animal cell line (such as HEK 293T cells, 293F cells, CHO cells, COS cells, NSO cells, HeLa cells or BHK cells), or a plant cell (such as Arabidopsis cells or tobacco cells). The transgenic cell lines are all non-reproductive materials.

[0052] The fourth aspect of the present invention provides a conjugate comprising: the antibody or antigen-binding fragment thereof described in the first aspect, or the recombinant protein described in the second aspect;

[0053] and a coupling portion, wherein the coupling portion comprises at least one of a detectable label, a drug, a toxin, biotin, a spin label, an enzyme, a gold nanoparticle, and a nanomagnetic particle.

[0054] According to some embodiments of the present invention, the detectable marker is selected from at least one of a radioisotope, a fluorescent group, a chemiluminescent substance, a colored substance, an MRI (magnetic resonance imaging) contrast agent, and a CT (computer tomography) contrast agent.

[0055] According to some embodiments of the present invention, the radioactive isotopes include but are not limited to 3 H. 14 C. 15 N. 35 S. 90 Y. 99 Tc, 111 In, 125 I or 131 I.

[0056] According to some embodiments of the present invention, the fluorescent group includes but is not limited to FITC, rhodamine, and lanthanide phosphors.

[0057] According to some embodiments of the present invention, the chemiluminescent group includes but is not limited to luminol, isoluminol, luciferin or aequorin.

[0058] According to some embodiments of the present invention, the colored substance includes but is not limited to diaminobenzidine or 4-hydroxyazo-benzene-2-carboxylic acid.

[0059] According to some embodiments of the present invention, the MRI contrast agent includes but is not limited to gadolinium (Gd), manganese (Mn) or iron (Fe). According to some embodiments of the present invention, the CT contrast agent includes but is not limited to barium (Ba) or cesium (Cs).

[0060] According to some embodiments of the invention, the toxin includes but is not limited to diphtheria toxin, ricin or cholera toxin.

[0061] According to some embodiments of the present invention, the enzyme includes but is not limited to β-galactosidase, peroxidase, alkaline phosphatase, horseradish peroxidase or acetylcholinesterase.

[0062] According to some embodiments of the invention, the spin label includes but is not limited to deuterium.

[0063] A fifth aspect of the present invention provides a product comprising at least one of D1) to D3);

[0064] D1) the antibody or antigen-binding fragment thereof according to the first aspect;

[0065] D2) the recombinant protein described in the second aspect;

[0066] D3) the recombinant protein conjugate described in the fourth aspect;

[0067] The product is selected from drugs, solid phase carriers, chips, reagents, test papers and test kits.

[0068] The sixth aspect of the present invention provides the use of the anti-artemisinin single-chain antibody or antigen-binding fragment thereof described in the first aspect, the recombinant protein described in the second aspect, the biomaterial described in the third aspect, the conjugate described in the fourth aspect, or the product of the fifth aspect in any one of E1) to E2);

[0069] E1) preparing products for detecting artemisinin;

[0070] E2) preparing artemisinin-binding products.

[0071] According to some embodiments of the present invention, the product is selected from a solid phase carrier, a chip, a reagent, a test paper and a kit.

[0072] According to some embodiments of the invention, the product is used to detect the presence or content of artemisinin.

[0073] According to some embodiments of the present invention, the product can be used for enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, immunofluorescence, immunoblotting, or immunoaffinity chromatography.

[0074] According to some embodiments of the present invention, the product further comprises one of an enzyme-linked immunosorbent assay (ELISA) detection reagent, an immunohistochemistry detection reagent, an immunofluorescence detection reagent, an immunoblotting detection reagent, and an immunoaffinity chromatography reagent. It will be understood that the product of the present invention is not limited to the reagents exemplified above. Other detection reagents common in the art may also be applied to the present invention.

[0075] According to the seventh aspect of the present invention, a method for detecting artemisinin or artemisinin content is proposed, comprising the following steps: using the above-mentioned anti-artemisinin single-chain antibody or its antigen-binding fragment, recombinant protein, biomaterial, conjugate or the above-mentioned product to detect the sample to be tested.

[0076] According to some embodiments of the present invention, the above-mentioned anti-artemisinin single-chain antibody or antigen-binding fragment thereof, recombinant protein, biological material, conjugate or the above-mentioned product is used to detect the sample by ELISA or BLEIA.

[0077] According to the eighth aspect of the present invention, a method for preparing the above-mentioned anti-artemisinin single-chain antibody or its antigen-binding fragment is proposed, comprising the following steps: obtaining it by culturing the recombinant microorganism or transgenic cell line described in the third aspect.

[0078] According to some embodiments of the present invention, at least the following beneficial effects are achieved: the anti-artemisinin single-chain antibody provided by the present invention can specifically recognize artemisinin and can be directly used for artemisinin detection with high sensitivity. At the same time, the artemisinin single-chain antibody is small in size and easy to modify, and can be directly used for the prediction of antigen epitopes and the quality detection of small molecule drugs containing artemisinin.

[0079] Other features and advantages of the present invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Figure 1 Electrophoresis diagram for constructing a specific artemisinin single-chain antibody expression vector; A is the nucleic acid electrophoresis diagram of the heavy and light chain fragments of the single-chain antibody, lanes 1 and 2 are the PCR products of the heavy and light chains, respectively, and lane M is the DL 2000 DNA marker; B is the nucleic acid electrophoresis diagram of pZOU-169C double-enzyme digestion, lane 1 is the double-enzyme digestion product, lane 2 is the original pZOU-169C plasmid, and lane M is the DL 15000 DNA marker;

[0081] Figure 2 Schematic diagram of the construction of a eukaryotic expression vector for a specific artemisinin single-chain antibody;

[0082] Figure 3 This is the fluorescence image of the specific artemisinin single-chain antibody stably transfected for 48 hours;

[0083] Figure 4 This is a fluorescence image of monoclonal cells of a specific artemisinin single-chain antibody;

[0084] Figure 5 This is the result of SDS-PAGE analysis of protein purification;

[0085] Figure 6 Figure 5 is the standard curve of artemisinin BLEIA and ELISA, where A is the BLEIA standard curve and B is the ELISA standard curve. DETAILED DESCRIPTION

[0086] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0087] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or those recommended by the manufacturer. All reagents or instruments used, if the manufacturer is not specified, are commercially available conventional products.

[0088] In the description of the present invention, the terms "comprises" and "having" and any variations thereof are intended to cover non-exclusive inclusions. For example, a process, method, system, product or apparatus that includes a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to the process, method, product or apparatus.

[0089] Example 1 Anti-artemisinin single-chain antibody and its preparation method

[0090] This example provides an anti-artemisinin single-chain antibody, the sequence of which is as follows:

[0091] Nucleotide sequence encoding artemisinin single-chain antibody:

[0092] GAGGTTCAGCTCCAGCAGTCTGGGACTGTGCTGGCAAGGCCTGGGGCTTCCGTGAAGATGTCCTGCAAGGCCTCTGGCTACAGCTTTACCAGCTACTGGATGCTCTGGATAAAACAGAGGCCTGGACAGGGTCTAGAATGGATTGCAGGTTTTTATCCTGGAAATAGTGATACTTGGTACAACCAGAAGTTCACGGGCAAGGCCAAACTGACTGCAGTCACATCCGCCAGCACTGCCTACATGGAGCTCAGCAGCCTGACAAATGAGGACTCTGCGGTCTATTACTGTACAAGAGGGGATAGTAACTCCTTTGCTATGGACTACTGGGGTCAGGGAACCGCAGTCACCGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGCTCCGGCGGTGGCGGTTCCCAGGCTGTTGTGACTCAGGAATCTGCACTCACCACATCACCTGGTGAAACAGTCACACTCACTTGTCGCTCAAGTACTGGGGCTGTTACAACTAGTAACTATGCCAACTGGGTCCAAGAAAAACCAGATCATTTATTCACTGGTCTAATAGGTGGTACCAACAACCGAGCTCCAGGTGTTCCTGCCAGATTCTCAGGCTCCCTGATTGGAGACAAGGCTGCCCTCACCATCACAGGGGCACAGACTGAGGATGAGGCAATATATTTCTGTGCTCTATGGTACAGCAACCATTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTA(SEQ ID NO:1).

[0093] Amino acid sequence of the single-chain antibody against artemisinin:

[0094] EVQLQQSGTVLARPGASVKMSCKASGYSFTSYWMLWIKQRPGQGLEWIAGFYPGNSDTWYNQKFTGKAKLTAVTSASTAYMELSSLTNEDSAVYYCTRGDSNSFAMDYWGQGTAVTVSSGGGGSGGGGSGGGGSQAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTKLTVL(SEQ ID NO:2).

[0095] Nucleotide sequence of the heavy chain variable region of the single-chain antibody:

[0096] GAGGTTCAGCTCCAGCAGTCTGGGACTGTGCTGGCAAGGCCTGGGGCTTCCGTGAAGATGTCCTGCAAGGCCTCTGGCTACAGCTTTACCAGCTACTGGATGCTCTGGATAAAACAGAGGCCTGGACAGGGTCTAGAATGGATTGCAGGTTTTTATCCTGGAAATAGTGATACTTGGTACAACCAGAAGTTCACGGGCAAGGCCAAACTGACTGCAGTCACATCCGCCAGCACTGCCTACATGGAGCTCAGCAGCCTGACAAATGAGGACTCTGCGGTCTATTACTGTACAAGAGGGGATAGTAACTCCTTTGCTATGGACTACTGGGGTCAGGGAACCGCAGTCACCGTCTCCTCA(SEQ ID NO:15).

[0097] Nucleotide sequence of the light chain variable region of the single-chain antibody:

[0098] CAGGCTGTTGTGACTCAGGAATCTGCACTCACCACATCACCTGGTGAAACAGTCACACTCACTTGTCGCTCAAGTACTGGGGCTGTTACAACTAGTAACTATGCCAACTGGGTCCAAGAAAAACCAGATCATTTATTCACTGGTCTAATAGGTGGTACCAACAAC CGAGCTCCAGGTGTTCCTGCCAGATTCTCAGGCTCCCTGATTGGAGACAAGGCTGCCCTCACCATCACAGGGGCACAGACTGAGGATGAGGCAATATATTTCTGTGCTCTATGGTACAGCAACCATTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTA(SEQ ID NO:16).

[0099] Amino acid sequence of the variable region of the heavy chain of the single-chain antibody:

[0100] EVQLQQSGTVLARPGASVKMSCKASGYSFTSYWMLWIKQRPGQGLEWIAGFYPGNS DTWYNQKFTGKAKLTAVTSASTAYMELSSLTNEDSAVYYCTRGDSNSFAMDYWGQGTAVT VSS (SEQ ID NO: 17).

[0101] Amino acid sequence of the light chain variable region of the single-chain antibody:

[0102] QAVVTQESALTTSPGETVTTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRAP GVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTKLTVL (SEQ ID NO: 18).

[0103] Single-chain antibody CDR region nucleotide sequence:

[0104] Nucleotide sequence encoding CDR1 of the heavy chain variable region: AGCTACTGGATGCTC (SEQ ID NO: 3);

[0105] The nucleotide sequence encoding CDR2 of the heavy chain variable region: GGTTTTTATCCTGGAAATAGTGATACTTGGTACAACCAGAAGTTCACGGGC (SEQ ID NO: 4);

[0106] Nucleotide sequence encoding CDR3 of the heavy chain variable region: GGGGATAGTAACTCCTTTGCTATGGACTAC (SEQ ID NO: 5);

[0107] The nucleotide sequence encoding CDR1 of the light chain variable region: CGCTCAAGTACTGGGGCTGTTACAACTAGTAACTATGCCAAC (SEQ ID NO: 6);

[0108] Nucleotide sequence encoding light chain variable region CDR2: GGTACCAACAACCGAGCTCCA (SEQ ID NO: 7);

[0109] Nucleotide sequence encoding light chain variable region CDR3: GCTCTATGGTACAGCAACCATTGGGTG (SEQ ID NO: 8);

[0110] amino acid sequence of CDR1 of the heavy chain variable region: SYWML (SEQ ID NO: 9);

[0111] The amino acid sequence of CDR2 of the heavy chain variable region is: GFYPGNSDTWYNQKFTG (SEQ ID NO: 10);

[0112] amino acid sequence of CDR3 of the heavy chain variable region: GDSNSFAMDY (SEQ ID NO: 11);

[0113] amino acid sequence of CDR1 of the light chain variable region: RSSTGAVTTSNYAN (SEQ ID NO: 12);

[0114] amino acid sequence of light chain variable region CDR2: GTNNRAP (SEQ ID NO: 13);

[0115] The amino acid sequence of the light chain variable region CDR3 is: ALWYSNHWV (SEQ ID NO: 14).

[0116] The single-chain antibody against artemisinin can be synthesized artificially or prepared by the following preparation method:

[0117] 1. Cloning of variable region genes

[0118] Total RNA was extracted from an artemisinin-positive hybridoma cell line (screened by the research group of Guo Suqin at Wuyi University) using an RNA extraction kit (purchased from Invitrogen, following the manufacturer's instructions). cDNA was synthesized using RNA as a template and specific primers using a reverse transcription kit K1621 (purchased from Thermofisher, following the manufacturer's instructions). The cDNA was sent to GeneWeizhi Biotechnology Co., Ltd. for sequencing. The nucleotide sequences of the VH and VL genes were obtained by sequencing. After sequence analysis, the heavy and light chain variable region genes were amplified by PCR.

[0119] 2. Variable region gene amplification

[0120] The plasmid returned by Jinweizhi Biotechnology Co., Ltd. was used as a template. The heavy chain variable region gene was amplified by PCR with primers QHS VHF-BamHI and QHS VHR-linker; the light chain variable region gene was amplified with primers HJM K Linker-VLF and HJM K VLR-HindⅢ (primer sequences are shown in Table 5). The results of agarose gel electrophoresis were as follows: Figure 1 The PCR amplification system is shown in Figure A.

[0121] Table 1 Heavy chain variable region gene amplification system

[0122]

[0123]

[0124] PCR reaction conditions were: 95°C for 3 min; entering the cycling phase: 95°C for 30 sec, 55°C for 30 sec, 72°C for 1 min, 35 cycles; and finally 72°C for 5 min.

[0125] Table 2 Light chain variable region gene amplification system

[0126]

[0127] The PCR reaction conditions were: 95°C for 3 min; entering the cycling phase: 95°C for 30 sec, 55°C for 30 sec, 72°C for 1 min, 35 cycles; and finally 72°C for 5 min.

[0128] 3. Assembly of specific artemisinin single-chain antibody

[0129] Using the gene sequence encoding the flexible peptide ((Gly4Ser)3) (GGTGGCGGTGGCTCGGGCGGTGGTGGCTCCGGCGGTGGCGGTTCC (SEQ ID NO: 19)) as a linker, the VL and VH genes were assembled into full-length scFv genes containing restriction enzyme cleavage sites by overlap extension PCR technology. The first PCR reaction system is as follows: first, in a conventional PCR reaction system (50 μL), equimolar 2, variable region gene amplification obtained by VL and VH genes were added, and HJM K Linker-VLF and QHS VHR-linker were used as primers (sequences shown in Table 5) for PCR amplification. The amplification system is shown in Table 3 below.

[0130] Table 3 SOE-PCR reaction system

[0131]

[0132]

[0133] The first PCR reaction procedure was as follows: pre-denaturation at 98°C for 30 seconds; entering the cycling stage: 98°C for 15 seconds, 58°C for 15 seconds, 72°C for 1 minute, for a total of 10 cycles, and extension at 72°C for 5 minutes.

[0134] The PCR product obtained in the first PCR reaction was used as a template and the primers QHS VHF-BamHI and HJMK VLR-HindIII (sequences shown in Table 5) were used for the second PCR. The reaction system was shown in Table 4 below.

[0135] Table 4 Second PCR amplification system

[0136]

[0137] The second PCR reaction program was as follows: pre-denaturation at 98°C for 30 seconds; entering the cycling stage: 98°C for 15 seconds, 58°C for 15 seconds, 72°C for 1 minute, for a total of 30 cycles, and extension at 72°C for 5 minutes.

[0138] After the reaction product is identified by agarose gel electrophoresis, the target fragment is recovered to obtain a specific artemisinin single-chain antibody.

[0139] Table 5 Primer sequences

[0140]

[0141]

[0142] Example 2 Construction of a Stable Expression Vector for a Specific Anti-artemisinin Single-chain Antibody

[0143] 1. Amplification of specific artemisinin single-chain antibody

[0144] Using a specific artemisinin single-chain antibody as a template and primers QHS S1 and HJMK S2 (sequences shown in Table 2), the reaction system was 50 μL. The reaction product was identified by agarose gel electrophoresis, and the target fragment was recovered. The PCR system is shown in Table 6 below:

[0145] Table 6 PCR amplification system

[0146]

[0147] The PCR program was as follows: pre-denaturation at 98°C for 30 seconds; cycling: 98°C for 15 seconds, 58°C for 15 seconds, 72°C for 1 minute, for a total of 30 cycles, and extension at 72°C for 5 minutes.

[0148] 2. Enzyme digestion of stable expression vector

[0149] The pZOU-169C stable expression vector (constructed by Guo Suqin's research group at Wuyi University) was digested with EcoRI and BamHI restriction endonucleases. The total digestion system was 50 μL. The reaction was incubated at 37°C for 3 hours. The reaction product was identified by agarose gel electrophoresis and the target fragment was recovered. The identification results of the recovered target fragment are as follows: Figure 1 As shown in Figure B.

[0150] 3. Homologous recombination and transformation

[0151] The recovered scFv fragment and the linear expression vector after enzyme digestion were recombined using a seamless cloning kit (White Shark Easy Biotechnology Co., Ltd.) (incubated at 56°C for 30 minutes). 50 μL of E. coli Top 10 competent cells were thawed on ice, and the recombinant solution was added. The cells were placed on ice for 30 minutes, heat-shocked at 42°C for 50 seconds, and then placed on ice for 3 minutes. The recombinant solution was completely aspirated and spread on LB solid culture medium containing 100 μg / mL ampicillin. After inverted overnight culture at 37°C for 12 hours, single clones were picked the next day and cultured in 1 mL of LB liquid culture medium for 4-6 hours. 500 μL of bacterial solution was taken and sent to Guangzhou Qingke Biotechnology Co., Ltd. for sequencing. After sequence determination and comparison, the strain with correct sequence was selected for expansion culture. The plasmid was extracted using an endotoxin-free plasmid medium extraction kit (Tiangen Biotechnology Co., Ltd.), and the plasmid was named pZOU-169C-ART scFv-Nluc. The schematic diagram of the stable expression vector is shown in the figure below. Figure 2 shown.

[0152] 4. Expression and purification of specific anti-artemisinin single-chain antibody

[0153] (1) pZOU-169C-ART scFv-Nluc plasmid transfection into HEK 293T cells

[0154] HEK 293T cells were cultured in a 24-well plate to a cell density of 60%-70% per well. Fresh 10% complete medium (10% FBS, 1% PS, 81% DMEM) was replaced. 500 ng of pZOU-169C-ART scFv-Nluc plasmid and 500 ng of transposase were co-transfected into HEK 293T cells using a Lipo 8000 transfection kit (Biyuntian Biotechnology Co., Ltd.). After culturing at 37°C for 24 h, positive cells with green fluorescence were observed. The results are shown in Figure 2. Figure 3 shown.

[0155] (2) Picking positive monoclonal cells

[0156] After the positive cells were replaced with a culture medium containing 1 μg / mL puro and cultured for 24 hours, the supernatant was removed and the cells were washed three times with PBS. After adding 0.05% trypsin for 1 minute, the trypsin digestion was terminated with 10% complete culture medium. The cell mixture was transferred to a 1.5 mL sterile centrifuge tube and centrifuged at 1000 rpm for 5 minutes. The supernatant was removed and the cells were resuspended with 10% complete culture medium. 1 / 10 of the cell suspension was transferred to a 10 cm cell culture dish and cultured for about 10 days. During this period, fresh complete culture medium was replaced four times. When the single cells grew into a cluster, the monoclonal cells were picked and cultured in a 24-well plate. The results are as follows: Figure 4 shown.

[0157] (3) Purification of anti-artemisinin single-chain antibody

[0158] Monoclonal cells were passaged into 10 cm cell culture plates. Once confluent, mitomycin was added at a final concentration of 10 μg / mL to inhibit cell growth. Cell supernatants were collected every three days and frozen at -20°C until needed. The collected cell supernatants were centrifuged at 5000 rpm for 10 minutes. The supernatant was added to an equal volume of PBS, filtered through a 0.45 μm aqueous filter, and purified using Protein GAgarose medium (Biyuntian Biotechnology Co., Ltd.) using the following purification procedure:

[0159] 1) Fill the gravity column and drain the protective liquid;

[0160] 2) Wash the purification column with 10 column volumes of ultrapure water;

[0161] 3) Equilibrate the purification column with 20 column volumes of PBS (pH 7.4);

[0162] 4) Incubate the filler and cell supernatant at 4°C for 1 hour;

[0163] 5) Pass the mixed solution through the column together, and repeat the flow-through through the column three times;

[0164] 6) Wash three times with 10 column volumes of PBS (pH 7.4) to remove nonspecifically bound proteins;

[0165] 7) After washing, elute with 100 mM glycine-HCl buffer (pH 3) for 3 tubes, 1.5 mL / tube. Continue eluting with 100 mM glycine-HCl buffer (pH 2) for 7 tubes, 1.5 mL / tube. Add 1 / 10 volume of Tris-HCl (pH 8.8) buffer to each tube of eluate. Collect the eluted protein and freeze it at -20°C until use.

[0166] 8) The purified scFv protein was subjected to SDS-PAGE gel electrophoresis analysis. The results were as follows: Figure 5 As shown, there are many impurity bands in the cell supernatant before purification, the target band in the flow-through after binding to the filler is lighter, there is no target band in the wash solution, and the molecular weight of the target protein in the eluate is consistent with the expected size of 74kDa;

[0167] 9) The eluates containing the target protein were mixed, concentrated using ultrafiltration tubes (Amicon Ultra-15 Centrifug Filter Units, 30 kD), snap-frozen in liquid nitrogen, and stored at -80°C.

[0168] Example 3 Application of single-chain antibodies in the detection of artemisinin

[0169] 1. Establishment of artemisinin standard curve by BLEIA method

[0170] (1) Dilute the artemisinin-coated antigen to 0.25 μg / mL with coating buffer (pH 9.6, 0.05 M carbonate buffer) and add it to a 96-well transparent ELISA plate at 100 μL / well. Coat at 37°C for 3 h. Wash three times with washing buffer (pH 7.4, 0.01 M PBS buffer containing 0.1% Tween 20) and spin dry.

[0171] (2) Accurately weigh 1 mg of artemisinin standard and dissolve it in 1 mL of methanol to obtain a 1 mg / mL artemisinin standard solution. The artemisinin standard solution was diluted with sample diluent (PBS buffer, pH 7.4, 0.01 M, containing 0.1% Tween 20 and 0.1 g / mL gelatin) to the following concentrations: 8, 4, 2, 1, 0.5, 0.25, 0.13, and 0 ng / mL;

[0172] (3) Add different concentrations of artemisinin standard dilution to the ELISA plate coated in step (1) as experimental wells, 50 μL / well; replace the artemisinin standard dilution with 50 μL sample dilution as the control well;

[0173] (4) The artemisinin single-chain antibody prepared in Example 1 was diluted to 0.10 μg / mL with sample diluent and added to the experimental wells and control wells at a volume of 50 μL per well. The wells were incubated at 37°C for 30 min. The cells were washed three times with washing solution and dried.

[0174] (5) Dilute the luminescent substrate according to the instructions of the Nano-Light Luciferase Reporter Gene Assay Kit, add 80-100 μL of luminescent substrate to each well, and immediately measure the luminescence value of each well in a microplate reader, recording the luminescence value every 5 minutes for 15-20 minutes;

[0175] The standard curve was drawn with the concentration of artemisinin standard solution (ng / mL) as the X-axis and the ratio of luminescence values (L / L0, where L is the average luminescence value of the experimental well and L0 is the average luminescence value of the control well) as the Y-axis. Figure 6 As shown in Figure A.

[0176] 2. Establishment of artemisinin standard curve by ELISA

[0177] (1) Dilute the artemisinin coating antigen to 0.5 μg / mL with coating buffer. The coating steps are the same as those of the BLEIA method.

[0178] (2) Dilute the artemisinin standard solution with sample diluent to the following concentrations: 50, 25, 12.5, 6.25, 3.13, 1.6, 0.78, and 0 ng / mL;

[0179] (3) Add different concentrations of artemisinin standard dilution to the ELISA plate coated in step (1) as experimental wells, 50 μL / well; replace the artemisinin standard dilution with 50 μL sample dilution as the control well;

[0180] (4) The artemisinin single-chain antibody prepared in Example 1 was diluted to 2.5 μg / mL with sample diluent and added to the experimental wells and control wells at a volume of 50 μL per well. The wells were incubated at 37°C for 30 min. The cells were washed three times with washing solution and dried.

[0181] (5) Add 100 μL of horseradish peroxidase-labeled rabbit anti-human Fc antibody (SE134, Lambda) to each well and incubate at 37°C for 30 min; wash three times with washing buffer and spin dry;

[0182] (6) Add 100 μL of TMB colorimetric solution to each well and develop the color for 10 min in the dark;

[0183] (7) Add 50 μL of stop solution to each well and measure the OD of each well at 450 nm using a microplate reader. 450 value.

[0184] The standard curve is drawn with the concentration of artemisinin standard solution (ng / mL) as the X-axis and the absorbance ratio (B / B0, where B is the average absorbance value of the experimental well and B0 is the average absorbance value of the control well) as the Y-axis. Figure 6 As shown in Figure B.

[0185] 3. Comparison of BLEIA and ELISA methods for detecting artemisinin using artemisinin single-chain antibody

[0186] IC of artemisinin single-chain antibody for artemisinin detection determined by BLEIA 50 (The corresponding artemisinin concentration when L / L0 is 50%) is 0.78±0.22ng mL -1 , the detection range is 0.11-4.02ng mL -1 The LOD value (artemisinin concentration at 10% inhibition) was 0.03 ng mL -1 IC of artemisinin single-chain antibody for artemisinin detection determined by ELISA 50 (The corresponding artemisinin concentration when B / B0 is 50%) is 7.44±0.88ng mL -1 , the detection range is 1.83–40.38 ng mL -1 , the LOD value was 0.84 ng mL -1 The IC values of parental monoclonal antibodies for artemisinin detection were determined by ELISA. 50 2.6 ng mL -1 By comparison, it can be found that the IC values of artemisinin single-chain antibody and parental monoclonal antibody determined by conventional ELISA method are 50 Both values are higher than those measured by BLEIA, indicating that the ELISA method is less sensitive than the BLEIA method. Furthermore, the ELISA test process requires the addition of additional detection antibodies, increasing testing time and cost. In comparison, the BLEIA method is less time-consuming and less expensive than conventional ELISA, making it a promising immunoassay method.

[0187] 4. Specific detection of artemisinin single-chain antibody:

[0188] Referring to the methods described in 1. Establishing a Standard Curve for Artemisinin by BLEIA and ELISA, three structural analogs, artesunate (China Food and Drug Inspection Institutes, Catalog No. 100201), dihydroartemisinin (China Food and Drug Inspection Institutes, Catalog No. 100202), and artemether (China Food and Drug Inspection Institutes, Catalog No. 101323), were used in place of artemisinin. The cross-reactivity rate of artemisinin structural analogs was analyzed to investigate whether artesunate, dihydroartemisinin, and artemether interfered with the assay. The test results are shown in Table 4.

[0189] Cross-reaction rate (%) = (IC 50 IC of artemisinin analogs 50 )×100.

[0190] Table 4

[0191]

[0192] The test results are shown in Table 4. It can be seen from the table that the artemisinin single-chain antibody prepared in the embodiment of the present invention was assayed by BLEIA. The reaction rate of the artemisinin single-chain antibody to dihydroartemisinin and artesunate was less than 0.01%, and there was no cross-reactivity to artemether; the single-chain antibody assayed by ELISA had no cross-reactivity to artesunate, dihydroartemisinin, and artemether.

[0193] As the core recognition element of immunoassays, antibodies' binding properties directly determine the sensitivity and applicability of detection methods. Specific single-chain antibodies (scFvs) can specifically recognize a single target molecule, ensuring the accuracy of test results. The scFv constructed in this invention is a specific antibody that specifically recognizes artemisinin, with cross-reactivity with its derivatives less than 0.01%. Experimental results demonstrate that both BLEIA and ELISA assays have high specificity, but BLEIA is approximately nine times more sensitive than ELISA.

[0194] Currently reported immunoassays for artemisinin mostly use monoclonal antibodies, polyclonal antibodies, and other recombinant antibodies, including the anti-artemisinin Fab recombinant antibody developed by Paudel et al., which has cross-reactivity rates of 100% for artemisinin and 71.82% for artesunate; the monoclonal antibody (MAb) 1C1 constructed by Tanaka et al. has clear specificity for artemisinin, but cross-reactivity rates with artesunate and dihydroartemisinin are 630% and 29.9%, respectively; the anti-artemisinin polyclonal antibody constructed by Ferreira et al. has a 100% cross-reactivity rate for artesunate and a cross-reactivity rate of less than 1% for deoxyartemisinin; and the anti-artemisinic acid single-chain antibody F170-10 constructed by Eggelte et al. has a cross-reactivity rate of 3-5% for artemisinin and artemether. By comparison, it was found that the specificity and sensitivity of the above immunoassay methods are lower than those of the BLEIA method and ELISA method developed by the present invention. The BLEIA method also offers a shorter detection time (results can be obtained in approximately 40 minutes), significantly improving detection efficiency and reducing testing costs, making it more suitable for constructing immunoassays for artemisinin-based drugs. The specific single-chain antibody constructed in this study can recognize a single target molecule and has extremely low cross-reactivity with artemisinin structural analogs. Its high specificity, high sensitivity, and low molecular weight offer significant advantages in pharmacokinetic analysis, making it a promising immunoassay for artemisinin pharmacokinetics.

[0195] The embodiments of the present invention are described in detail above in conjunction with the embodiments, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge scope of ordinary technicians in the relevant technical field without departing from the purpose of the present invention.

Claims

1. An anti-artemisinin single-chain antibody or an antigen-binding fragment thereof, characterized in that: including heavy chain variable regions and light chain variable regions; The heavy chain variable region comprises a CDR1 with an amino acid sequence as shown in SEQ ID NO: 9, a CDR2 with an amino acid sequence as shown in SEQ ID NO: 10, and a CDR3 with an amino acid sequence as shown in SEQ ID NO: 11; The light chain variable region comprises a CDR1 with an amino acid sequence as shown in SEQ ID NO: 12, a CDR2 with an amino acid sequence as shown in SEQ ID NO: 13, and a CDR3 with an amino acid sequence as shown in SEQ ID NO:

14.

2. The anti-artemisinin single-chain antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region comprises: a1) SEQ ID NO: 17; or a2) an amino acid sequence obtained by substituting and / or deleting and / or adding one or more amino acids in SEQ ID NO: 17 and having the same function as the protein shown in SEQ ID NO: 1; or a3a2) an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70% identical to SEQ ID NO: 17 and has the same function as the protein set forth in SEQ ID NO: 17; And / or, the amino acid sequence of the light chain variable region comprises: a1) SEQ ID NO: 18; or a2) an amino acid sequence obtained by substituting and / or deleting and / or adding one or more amino acids in SEQ ID NO: 18 and having the same function as the protein shown in SEQ ID NO: 1; or a3a2) an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70% identical to SEQ ID NO: 18 and has the same function as the protein set forth in SEQ ID NO: 18; and / or, the heavy chain variable region is connected to the light chain variable region via or without a connecting peptide; Preferably, the connecting peptide is selected from a rigid connecting peptide or a flexible connecting peptide; More preferably, the flexible connecting peptide is selected from (G3S) n 、(G4S) n or (G) n ; wherein n is an integer not less than 3.

3. The anti-artemisinin single-chain antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The amino acid sequence of the anti-artemisinin single-chain antibody or antigen-binding fragment thereof is any one of A1) to A3): A1) the amino acid sequence shown in SEQ ID NO: 2; A2) an amino acid sequence having the same function as the protein shown in SEQ ID NO: 2, wherein one or more amino acids are substituted and / or deleted and / or added; A3) an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71% or 70% identical to the sequence of SEQ ID NO: 2, and has the same function as the protein of SEQ ID NO:

2.

4. A recombinant protein, characterized in that A method comprising the anti-artemisinin single-chain antibody or antigen-binding fragment thereof according to any one of claims 1 to 3; and an optional tag; Preferably, the tag can be attached to the N-terminus and / or C-terminus of the antibody or antigen-binding fragment thereof; Preferably, the tag comprises at least one of a His tag, a FLAG tag, a strep tag, a nus tag, a maltose binding protein, and a GST tag.

5. A biomaterial related to the anti-artemisinin single-chain antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, or the recombinant protein according to claim 4, characterized in that: The biomaterial comprises at least one of B1) to B5); B1) a nucleic acid molecule encoding the anti-artemisinin single-chain antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, or the recombinant protein according to claim 4; B2) an expression cassette containing the nucleic acid molecule described in B1); B3) a recombinant vector containing the nucleic acid molecule described in B1) or the expression cassette described in B2); B4) a recombinant microorganism containing the nucleic acid molecule described in B1), the expression cassette described in B2), or the recombinant vector described in B3); B5) a transgenic cell line containing the nucleic acid molecule described in B1), the expression cassette described in B2), or the recombinant vector described in B3); Preferably, the nucleic acid molecule B1) includes any one of C1) to C2): C1) the nucleotide sequence shown in SEQ ID NO: 1 or a degenerate sequence thereof; C2) A DNA molecule that hybridizes with the DNA molecule defined in C1) under stringent conditions and encodes the antibody or antigen-binding fragment thereof or the recombinant protein.

6. A conjugate, characterized in that Comprising: the anti-artemisinin single-chain antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, or the recombinant protein according to claim 4; and a coupling portion, wherein the coupling portion comprises at least one of a detectable label, a drug, a toxin, biotin, a spin label, an enzyme, a gold nanoparticle, and a nanomagnetic particle.

7. A product, characterized in that including at least one of D1) to D3); D1) the anti-artemisinin single-chain antibody or antigen-binding fragment thereof according to any one of claims 1 to 3; D2) the recombinant protein according to claim 4; D3) the conjugate according to claim 6; The product is selected from drugs, solid phase carriers, chips, reagents, test papers and test kits.

8. Use of the anti-artemisinin single-chain antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the recombinant protein according to claim 4, the biomaterial according to claim 5, the conjugate according to claim 6, or the product according to claim 7 in any one of E1) to E2); E1) preparing products for detecting artemisinin; E2) preparing products combining artemisinin; Preferably, the product is selected from a solid phase carrier, a chip, a reagent, a test paper and a kit.

9. A method for detecting artemisinin or artemisinin content, characterized in that: The following steps are involved: The sample to be tested is detected using the anti-artemisinin single-chain antibody or its antigen-binding fragment according to any one of claims 1 to 3, the recombinant protein according to claim 4, the biomaterial according to claim 5, the conjugate according to claim 6 or the product according to claim 7.

10. A method for preparing the above-mentioned anti-artemisinin single-chain antibody or antigen-binding fragment thereof, comprising the following steps: The method is obtained by culturing the recombinant microorganism or transgenic cell line according to claim 5.

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