Antibody specifically binding to serpentine rhzomorph and application thereof

Abstract

The invention discloses an antibody specifically bound with serpentine rhzomorph and application of the antibody, and belongs to the technical field of detection. The invention provides an antibody which is simple to prepare and low in cost and detection limit. The antibody is a full-length antibody and comprises a heavy chain variable region VH and a light chain variable region VL, the amino acid sequence of the heavy chain variable region VH is as shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.2. When the antibody provided by the invention is used for detection, the detection limit is relatively low, and high-sensitivity detection of DAS can be realized. When the antibody provided by the invention is detected by a competitive ELISA (enzyme-linked immunosorbent assay) detection method, the IC50 is 7.61 ng / mL, and the detection limit of the antibody is 2.67-21.68 ng / mL, namely the scalar addition corresponding to the inhibition rate of 20-80%. And the crossover rate of the serpentine rhzomorph analogue is less than 1%.

Description

TECHNICAL FIELD

[0001] The present application relates to an antibody specifically binding to a serpentine and its application, and belongs to the technical field of detection. BACKGROUND

[0002] Serpentine, also known as diacetyl fusariene enol (DAS), is a highly toxic secondary metabolite produced by fusarium, which belongs to type A monodraconian enol family of toxins. It mainly pollutes grain crops and feed, and is more toxic than DON. It damages various tissues and organs of humans and animals, mainly manifested as anorexia, vomiting, growth inhibition, neurotoxicity, immunotoxicity, genetic toxicity and hepatotoxicity. It is urgent to develop a simple, rapid and quantitative detection method suitable for on-site detection.

[0003] Recognition is the basis of detection. The most common recognition element is antibody. Antibodies have the advantages of high affinity and specificity. In the prior art, the antigen-antibody detection mainly has the following schemes: Patent CN110724192A provides a hybridoma cell strain secreting a monoclonal antibody and its application. The detection sensitivity IC50 value of the monoclonal antibody secreted by the hybridoma cell strain is 5.97 ng / mL, which can be used to establish an immunological detection method for DAS, and detect DAS residues in food. Although the detection limit is relatively low, the preparation and separation method of the hybridoma cell strain is complex and the cost is high.

[0004] Patent CN111896738A provides a test strip for detection and its application, specifically a colloidal gold test strip for detecting DAS. The antibody is prepared by animal immunization and hybridoma cell preparation. In this scheme, when the DAS concentration added in the wheat and corn samples is 5 μg / kg, the analyzer shows negative; when the DAS concentration added is 10, 20 μg / kg, the analyzer shows positive.

[0005] In summary, in the existing schemes, the antibody is prepared by preparing hybridoma cells after animal immunization. On the one hand, this method is complicated and costly. On the other hand, the detection limit of the antibody provided by the prior art in DAS is still high, which limits the application scenarios in actual detection. Therefore, it is urgent to provide an antibody with simple preparation, low cost and low detection limit.

[0006] Genetically engineered antibodies are constructed by constructing the genes of antibodies into corresponding expression vectors, then introduced into suitable hosts for induced expression, and the target antibodies can be obtained after purification. At present, this technology has been widely used in the production of various mycotoxin single-chain antibodies, single-domain antibody fusion antibodies, etc. At present, the expression of genetically engineered antibodies is mainly based on the E. coli expression system, which has the characteristics of high expression yield and fast expression speed. However, since E. coli is a prokaryote, and antibodies are proteins with high-level structure, the prokaryote is difficult to correctly fold and modify the antibodies, which greatly reduces the quality of the antibodies, and has low affinity and specificity, thereby limiting the late application of the expressed antibodies. SUMMARY

[0007] The present application proposes a preparation method and use of a eukaryotic expression full-length antibody of DAS to solve the above problems in the prior art. The antibody obtained by eukaryotic expression can specifically bind to DAS and can be used for detection of DAS.

[0008] The present application provides an antibody with simple preparation, low cost and low detection limit. The antibody is a full-length antibody, which comprises a heavy chain variable region VH and a light chain variable region VL, the amino acid sequence of the heavy chain variable region VH is shown in SEQ ID NO. 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 2; the VH comprises HCDR1 HCDR3 with the amino acid sequences shown in SEQ ID NO. 3, SEQ ID NO. 4, and SEQ ID NO. 5, respectively; and the VL comprises LCDR1 LCDR3 with the amino acid sequences shown in SEQ ID NO. 6, LTS, and SEQ ID NO. 7, respectively. The present application provides an antibody or antigen-binding fragment thereof specifically binding to DAS, which comprises a light chain variable region VL and a heavy chain variable region VH; the light chain variable region VL comprises three complementarity determining regions LCDR1, LCDR2, and LCDR3, and the amino acid sequences thereof are shown in SEQ ID NO. 6, LTS, and SEQ ID NO. 7, respectively; The heavy chain variable region VH comprises three complementarity determining regions HCDR1, HCDR2, and HCDR3, and the amino acid sequences thereof are shown in SEQ ID NO. 3, SEQ ID NO. 4, and SEQ ID NO. 5, respectively.

[0009] In an embodiment of the present application, the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO. 2, or an amino acid sequence with homology of at least 98% to SEQ ID NO. 2 and having the same function as the protein shown in SEQ ID NO. 2. The heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO. 1, or an amino acid sequence having at least 98% homology with SEQ ID NO. 1 and having the same function as the protein shown in SEQ ID NO. 1; In an embodiment of the present application, the antibody comprises an antibody heavy chain HC having the amino acid sequence shown in SEQ ID NO. 10 and an antibody light chain LC having the amino acid sequence shown in SEQ ID NO. 12.

[0010] The present application also provides a recombinant protein comprising the above-mentioned antibody or antigen-binding fragment thereof and a Pseudomonas exotoxin protein; and / or, an optional tag sequence for assisting expression and / or purification; the tag sequence is selected from at least one of the following group: His tag, GGGS sequence, FLAG tag. The present application also provides a recombinant vector containing a gene encoding the above-mentioned ophiosin antibody.

[0011] In an embodiment, the recombinant vector is a eukaryotic expression vector.

[0012] In an embodiment, the eukaryotic expression vector includes but is not limited to PCMV HA plasmid, PTT5 plasmid.

[0013] The present application also provides a cell carrying the above-mentioned recombinant vector.

[0014] In an embodiment, the cell is a mammalian cell.

[0015] In an embodiment, the cell is an Expi293F cell.

[0016] The present application also provides the above-mentioned ophiosin antibody or the above-mentioned recombinant vector or the above-mentioned cell.

[0017] In the application of detecting ophiosin, the application is not for the purpose of diagnosis and / or treatment of disease.

[0018] In an embodiment, the application is for preparing a kit or reagent for detecting ophiosin.

[0019] In an embodiment, the kit or reagent is used for ELISA detection, Western Blotting assay, immunohistochemistry or immunofluorescence.

[0020] In an embodiment, the kit further contains a standard of ophiosin.

[0021] The application also provides a nucleic acid molecule encoding the antibody or antigen binding fragment thereof; a nucleotide sequence encoding an antibody heavy chain HC is shown as SEQ ID NO. 11, and a nucleotide sequence encoding an antibody light chain LC is shown as SEQ ID NO. 13.

[0022] The application also provides a biological material comprising at least one of (a)-(g) below: (a) an expression cassette comprising the nucleic acid molecule described above; (b) a vector comprising the nucleic acid molecule described above; (c) a vector comprising the expression cassette of (a); (d) a transgenic cell line comprising the nucleic acid molecule described above; (e) a transgenic cell line comprising the expression cassette of (a); (f) a transgenic cell line comprising the vector of (b); (g) a transgenic cell line comprising the vector of (c).

[0023] In an embodiment, the cell line is selected from a microorganism or a mammalian cell.

[0024] The application also provides a kit containing the antibody or antigen binding fragment thereof described above or containing the nucleic acid molecule described above or containing the biological material described above.

[0025] The application also provides the use of the antibody or antigen binding fragment thereof described above or the biological material described above in the preparation of a reagent for detecting serpentine in a sample, which is not for the purpose of diagnosis and / or treatment of a disease.

[0026] The application also provides a method for preparing the antibody or antigen binding fragment thereof described above, which comprises culturing the transgenic cell line described above to obtain the antibody or antigen binding fragment thereof described above.

[0027] The application also provides a method for detecting whether serpentine exists in a sample or determining the content of serpentine in a sample, which comprises the step of contacting the antibody or antigen binding fragment thereof described above with the sample, which is not for the purpose of diagnosis and / or treatment of a disease.

[0028] Advantages (1) The antibody provided by the application has a low detection limit and can achieve high-sensitivity detection of DAS. The IC50 of the antibody provided by the application when detected by a competitive ELISA detection method is 7.61 ng / mL, and the detection limit, i.e., the amount of added standard corresponding to an inhibition rate of 20%-80%, is 2.67-21.68 ng / mL. The cross rate of serpentine analogues is less than 1%.

[0029] (2) The application realizes expression and purification of IgG by constructing pCMV HC, pCMV LC plasmids, co-transfecting Expi293F cells, inoculating the cells in a culture medium, culturing the cells at 30-40 DEG C under 5-10% CO2 for 6-7 days, collecting culture supernatant, and purifying by means of a Ni affinity column, so that expression and purification of IgG are realized.

[0030] (3) The antibody provided by the application does not need to be prepared by animal immunization, and the preparation method is simple.

[0031] (4) The recombinant full-length antibody expressed in the application can be genetically modified according to experimental purposes, and is widely applied to rapid preparation of antibodies of other targets. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 : Artificial antigen verification, wherein A: ultraviolet-visible spectrum of coated antigen DAS-BSA, B: SDS-PAGE gel electrophoresis diagram of coated antigen DAS-BSA, and C: ultraviolet-visible spectrum of immunized antigen DAS-HS-KLH.

[0033] Figure 2 : Detection results of serum titers of different mice.

[0034] Figure 3 : Agarose gel electrophoresis diagram of extracted total RNA.

[0035] Figure 4 : Agarose gel electrophoresis diagram of antibody variable region genes.

[0036] Figure 5 : Agarose gel electrophoresis diagram of assembled scFv.

[0037] Figure 6 : Agarose gel electrophoresis diagrams before and after pCANTAB-5E enzyme digestion, wherein lane 1 is before enzyme digestion, and lane 2 is after enzyme digestion.

[0038] Figure 7 : Colony PCR identification of pCANTAB5E-scFv recombinant plasmid.

[0039] Figure 8 : Phage antibody library titer determination plate.

[0040] Figure 9 : Phage-ELISA verification results.

[0041] Figure 10A : Mammalian cell expression plasmid pCMV-HC; Figure 10B : Mammalian cell expression plasmid pCMV-LC.

[0042] Figure 11 SDS-PAGE gel electrophoresis of DAS-scFv, wherein lanes 1-4 are cell supernatant, cell precipitate, permeate, and wash liquid, respectively; and lanes 5 and 6 are eluate.

[0043] Figure 12 SDS-PAGE gel electrophoresis of DAS-IgG, wherein A: non-reducing condition loading; and B: reducing condition loading.

[0044] Figure 13 Ic-ELISA absorbance value concentration curve of DAS-IgG antibody. DETAILED DESCRIPTION

[0045] The technical solutions of the present application will be further described below in conjunction with the accompanying drawings and through specific embodiments. Those skilled in the art should understand that the embodiments are only to help understand the present application and should not be regarded as specific limitations of the present application.

[0046] In the present disclosure, unless otherwise specified, the scientific and technical terms used herein have meanings commonly understood by those skilled in the art. Also, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology related terms and laboratory operation steps used herein are the terms and conventional steps widely used in the corresponding fields. At the same time, in order to better understand the present disclosure, the definitions and explanations of related terms are provided as follows.

[0047] In the present application, the amino acids at the corresponding positions are represented by the recognized IUPAC single-letter abbreviations, wherein each amino acid and its abbreviation are as follows: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine (Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V).

[0048] In the present application, the term "about" or "approximately" should be understood to include all numerical values within the permissible range of measurement error.

[0049] In the present invention, the terms "nucleic acid", "nucleic acid molecule", "nucleic acid sequence", "nucleotide sequence", and "polynucleotide" can be used interchangeably and refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double- or triple-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising, consisting of, or consisting essentially of, purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0050] In the present invention, the term "nucleic acid construct" refers to a nucleotide sequence comprising an entire expression cassette capable of expressing a gene of interest or a protein of interest. In addition to a coding sequence encoding a gene of interest or a protein of interest, the nucleic acid construct can further comprise genetic elements such as a promoter, an enhancer, a terminator, and / or a polyadenylation signal for regulating gene expression.

[0051] In the present invention, the term "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host or a nucleic acid molecule capable of inserting a gene fragment of interest into a host genome, which transfers the gene fragment of interest carried thereby to a host cell and / or between host cells. The vector can include a vector mainly used for inserting DNA or RNA into a cell, a vector mainly used for replicating DNA or RNA, and a vector mainly used for expression of transcription and / or translation of DNA or RNA. The vector further includes a vector having a plurality of the above-described functions. The vector can be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Generally, the vector can produce a desired expression product by culturing a suitable host cell containing the vector.

[0052] In the present invention, a "light chain variable region" (VL) or a "heavy chain variable region" (VH) consists of "framework" regions separated by three "complementarity determining regions" or "CDRs". The framework regions serve to align the CDRs that specifically bind to an epitope of an antigen. The CDRs include amino acid residues in an antibody that are primarily responsible for antigen binding. Both the VL domain and the VH domain comprise the following framework regions (FRs) and CDR regions from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The CDR1, CDR2, and CDR3 of the VL domain are also referred to herein as LCDR1, LCDR2, and LCDR3, respectively; and the CDR1, CDR2, and CDR3 of the VH domain are also referred to herein as HCDR1, HCDR2, and HCDR3, respectively.

[0053] The following example section contains the following components and sources: LB plate medium (Amp+): Yeast extract 5.0 g / L, Tryptone 10.0 g / L, NaCl 10.0 g / L, Agar 15 g / L, Ampicillin 100 μg / L; LB liquid medium: Yeast extract 5.0 g / L, Tryptone 10.0 g / L, NaCl 10.0 g / L; Expi293F cells involved in the following examples were purchased from Thermo Fisher Scientific, USA.

[0054] Relevant reagents used in the following examples: cationic transfection reagent, transfection enhancer I, enhancer II from Thermo Fisher Scientific, USA; TMB color developing solution A, TMB color developing solution B from Yikesheng Biotechnology (Shanghai) Co., Ltd.

[0055] The CAS number of DAS involved in the following examples is 2270-40-8, the CAS number of the catalyst DMAP involved is 1122-58-3, the CAS number of EDC involved is 25952-53-8, the CAS number of KLH involved is 9013-72-3, the CAS number of CDI involved is 530-62-1, and the CAS number of BSA involved is 9048-46-8.

[0056] Example 1: Screening of murine single-chain antibody (scFv) sequences I. Experimental methods 1. Synthesis of hapten, immunogen and coating antigen (1) Preparation of hapten DAS-HS: DAS (5 mg) was weighed and dissolved in 1500 μL of pyridine, and succinic anhydride (36 mg) and catalyst DMAP (17 mg) were added. Under light shielding conditions, magnetic stirring was carried out at 50°C for 5 hours. 250 μL of water was added to quench the reaction and dried in a drying machine. The residue was extracted with water and chloroform (1:1) three times. The organic phase (lower layer) was combined and dried to obtain DAS-HS hapten, which was a brown oil.

[0057] (2) Preparation of immunogen DAS-HS-KLH: The prepared hapten DAS-HS was dissolved in 1.2 mL DMF, and NHS (6 mg) and EDC (10.2 mg) were added, and stirred at room temperature for 6-8 hours. 400 μL of the reaction solution was added dropwise to KLH (10 mg) dissolved in 4 mL PBS solution, and stirred at 4°C in the dark overnight. The reaction solution was dialyzed against PBS at 4°C for 3 days to obtain the immunogen DAS-HS-KLH, which was stored in a -20°C refrigerator for later use.

[0058] (3) Preparation of coated antigen DAS-BSA: DAS (5 mg) was dissolved in 1000 μL DMF, and CDI (32 mg) was added, and stirred at 37°C for 2 h. The reaction solution was added dropwise to BSA (8 mg) dissolved in 4 mL PBS solution, and stirred at 4°C in the dark overnight. The reaction solution was dialyzed against PBS at 4°C for 3 days to obtain the coated antigen DAS-BSA, which was stored in a -20°C refrigerator for later use. The synthesized antigen was verified by ultraviolet spectrum and SDS-PAGE.

[0059] 2. Mouse immunization Mouse immunization was performed with the immunogen, and the mouse serum titer was detected.

[0060] Mouse immunization: 6-8 week old Balb / c mice were used, and the prepared immunogen DAS-HS-KLH with a concentration of 1 mg / mL was mixed with an equal amount of Freund's adjuvant, and completely emulsified. Each mouse was injected with 100 μL, and the spleen B lymphocytes were gradually stimulated to produce DAS antibody genes. The first immunization used Freund's complete adjuvant (FCA), and the subsequent booster immunization used Freund's incomplete adjuvant (FICA). Immunization was performed every 2 weeks, and a total of 4 booster immunizations were performed.

[0061] Detection of mouse serum titer: 100-200 μL of tail tip blood was taken from each immunized mouse after each immunization for 1 week, and centrifuged at 3000 r / min for 15 min to obtain the upper serum. After gradient dilution, the titer was detected by ELISA. The OD value of the mouse serum to be tested was P, the OD value of the healthy mouse serum was N, and the maximum dilution factor of the serum was selected when P / N>2.1 was the antibody titer. 450 nm 450 nm

[0062] 3. Construction of recombinant vector ​​The spleen cells of the immunized mice were taken for total RNA extraction, the integrity of the extracted total RNA was verified by agarose gel electrophoresis, and the first strand cDNA was obtained by reverse transcription. The obtained cDNA was used as a template, and the antibody VH and VL fragments were amplified by using a mouse antibody universal primer. The VH and VL were assembled into scFv by using a nest PCR amplification technology, and Not I and Sfi I enzyme cutting sites were introduced at both ends of the scFv for the construction of a recombinant plasmid. The pCANTAB-5E vector was double-cut by using Not I and Sfi I endonucleases, and the scFv was connected to the pCANTAB-5E vector by means of T4 ligase to form a pCANTAB5E-scFv recombinant plasmid.

[0063] 4. Antibody screening The recombinant plasmid pCANTAB5E-scFv was electroporated into the E. coli TG1 competent cells, and after infection and amplification by the helper phage M13K07, the DAS phage antibody library was obtained, and affinity panning was performed. The wells with a sample absorbance value / negative control group absorbance value (P / N) > 2.1 were selected as positive single colony phages, and the positive clones were obtained, and the plasmid was extracted, the bacteria were preserved, and were sent to Suzhou Jinweizhi Company for sequencing.

[0064] II. Experimental results: The identification of the coated antigen DAS-BSA and the immunizing antigen DAS-HS-KLH is shown in Figure 1 . The successful synthesis of the DAS coated antigen DAS-BSA was verified by ultraviolet-visible spectroscopy ( Figure 1 ), and SDS-PAGE gel electrophoresis ( Figure 1 ). The absorption peak of DAS-BSA was blue-shifted, and the SDS-PAGE gel electrophoresis showed that the molecular weight was slightly larger than that of BSA. These results indicated that the coupling reaction between DAS and BSA was successful. It can be seen from Figure 1 B that the ultraviolet absorption spectrum of the immunizing antigen DAS-HS-KLH product is between the hapten DAS-HS and KLH, and is relatively close to the ultraviolet absorption curve of KLH, which may be caused by the excess of KLH molecules and the low concentration of DAS-KLH.

[0065] The results of the detection of the titer of the mouse serum are shown in Table 1, Figure 2 The immunization effect of the mouse numbered 1 was the best, and the titer (maximum dilution multiple) of the antiserum reached 256,000.

[0066] Table 1: ELISA detection of the titer of the tail blood of the immunized mice

[0067] The agarose gel electrophoresis diagram of the total RNA of the spleen of the immunized mice is shown in Figure 3Clear 28S and 18S bands are visible, indicating good integrity of the total RNA, which can be used as a template for reverse transcription into cDNA. Electrophoresis results of antibody VH and VL fragment amplification are as follows... Figure 4 The VL band, approximately 300 bp, and the VH band, approximately 350 bp, are consistent with the expected single-chain antibody gene fragment sizes, indicating successful amplification of the light and heavy chain fragments, which can be used to assemble scFv. The scFv was identified by agarose gel electrophoresis, and the approximately 750 bp band was recovered and purified using a gel extraction kit. Verification results are shown in [link to verification results]. Figure 5 As can be seen from the figure, the amplified scFv bands are clear and the size is as expected, indicating that the scFv fragment was successfully assembled and can be used to construct recombinant plasmids.

[0068] The agarose gel electrophoresis results of the pCANTAB-5E vector plasmid and its Not I and Sfi I double digestion are as follows: Figure 6 As shown in the figure, the band changes of the vector before and after enzyme digestion are obvious, and it becomes a digested vector with a slightly smaller molecular weight, indicating that the enzyme digestion was successful and it can be used for ligation and transformation.

[0069] The constructed recombinant plasmid pCANTAB5E-scFv was electroporated into TG1 competent cells, and the cells grew well on 2×YT-Amp plates, indicating successful transformation. Twenty-four transformant single colonies were randomly selected for colony PCR identification, and the results are as follows: Figure 7 As shown in the figure, all 24 colonies amplified the target fragment, with a positive insertion rate of 100%. The 24 monoclonal bacterial cultures containing the correct-sized target fragment were sent to Sangon Biotech in Shanghai for sequencing. Sequencing analysis revealed that all colonies successfully inserted different target gene sequences of approximately 750 bp between the Not I and Sfi I restriction sites.

[0070] The constructed DAS single-chain antibody library had a volume greater than 10, as determined by titer analysis. 9 pfu / mL. After amplification by helper phage M13K07, as... Figure 8 As shown, the titer of the DAS primary phage antibody library obtained was approximately 4 × 10⁻⁶. 14 The pfu / mL concentration can be used for subsequent screening. After each round of screening, the titers of the elution and amplification products were determined, and the number of phages enriched in each round was calculated. Specific titer results are shown in Table 2.

[0071] Table 2: DAS-scFv titer determination during screening process

[0072] As can be seen from Table 2, with the increase of the number of panning, the phage was obviously enriched, and each round was obviously improved than the last round, indicating that the screening method was feasible. 24 single colonies were randomly selected, expanded, infected with M13K07, and monoclonal phage was prepared for phage-ELISA identification. The clones with P / N value greater than 2.1 were selected as positive clones. The verification results are shown in Figure 8 As can be seen from the figure, the P / N value of No. 11 clone is obviously better than that of other single colonies, so No. 11 clone (scFv11) is determined as a positive clone, and plasmid extraction, bacteria preservation and sequencing are carried out by Shanghai Sangon Biotech Company. After analysis, the full length of scFv11 is 744 bp, in which the size of VH is 348 bp, the size of VL is 318 bp, and the linker is GGGGSGGGGSGGGGS. The ScFv11 is annotated using IMGT coding rules, and the specific annotation results are as follows: Heavy chain variable region VH amino acid sequence (DAS-ScFv11_VH, heavy chain CDR region is bold and underlined) : SEQ ID NO. 1: QVQLQQSGAELMKPGASVKISCKAT GYTFSSYWI EWVKQRPGHGLEWIGE ILPGSGST NYNEKFKGKATFTADTSSNTAYMQLSSLTSEDSAVYYC ARRAVWFAY WGQGTTVTVSS; Among them, the heavy chain CDR region (HCDR) is as follows: Heavy chain CDR1 region, SEQ ID NO. 3: GYTFSSYW; Heavy chain CDR2 region, SEQ ID NO. 4: ILPGSGST; Heavy chain CDR3 region, SEQ ID NO. 5: ARRAVWFAY.

[0073] Light chain variable region VL amino acid sequence (DAS-ScFv11_VL, light chain CDR region is bold and underlined) : SEQ ID NO. 2: DIELTQSPAIMSASPGEKVTITCSAS SSVSY MHWFQLKPGTSPKPWIY LTS NLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYC QQWSSNPPT FGGGTKLDLK; Among them, the light chain CDR region (LCDR) is as follows: Light chain CDR1 region, SEQ ID NO. 6: SSVSY; Light chain CDR2 region amino acid sequence: LTS; Light chain CDR3 region, SEQ ID NO. 7: QQWSSNPPT.

[0074] Example 2: Construction and biological expression of recombinant full-length antibody IgG plasmid of Ophiocordin I. Experimental method 1. The sequence of Ophiocordin ScFv11 obtained by screening in Example 1: The nucleotide sequence of the DAS-ScFv11_VH coding gene is shown as SEQ ID NO. 8, and the nucleotide sequence of the DAS-ScFv11_VL coding gene is shown as SEQ ID NO. 9.

[0075] SEQ ID NO. 8: caggtccaactgcaacagtcaggagctgagctgatgaagcctggggcctcagtgaagatatcctgcaaggctactggctacacattcagtagctactggatagagtgggtaaagcagaggcctggacatggccttgagtggattggagagattttacctggaagtggtagtactaactacaatgagaagttcaagggcaaggccacattcactgcagatacatcctccaacacagcctacatgcaactcagcagcctgacatctgaggactctgccgtctattactgtgcaagaagggcggtttggtttgcttactggggccaagggaccacggtcaccgtctcctca SEQ ID NO. 9: gacattgagctcacccagtctccagcaatcatgtctgcatctccaggggagaaggtcaccataacctgcagtgccagctcaagtgtcagttacatgcactggttccagctgaagccaggcacttctcccaaaccctggatttatctcacatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagcatggaggctgaagatgctgccacttattactgccagcagtggagtagtaacccacccacgttcggaggggggaccaagctggacctgaaa In the recombinant IgG full-length antibody, the full-length antibody heavy chain HC amino acid sequence is shown as SEQ ID NO. 10: SEQ ID NO. 10: mgwsciilflvatatgvhsqvqlqqsgaelmkpgasvkisckatgytfssywiewvkqrpghglewigeilpgsgstnynekfkgkatftadtssntaymqlssltsedsavyycarravwfaywgqgttvtvssakttppsvyplapgsaaqtnsmvtlgclvkgyfpepvtvswntgslssgvhtfpavlqsdlytlsssvtvpsstwpsetvtcnvahpasstkvdkkivprdcgckpcictvpevssvfifppkpkdvltitltpkvtcvvvdiskddpevqfswfvddvevhtaqtqpreeqfnstfrsvselpimhqdwlngkefkcrvnsaafpapiektisktkgrpkapqvytipppkeqmakdkvsltcmitdffpeditvewqwngqpaenykntqpimntngsyfvysklnvqksnweagntftcsvlheglhnhhtekslsh The full-length antibody heavy chain HC encoding gene nucleotide sequence is shown as SEQ ID NO. 11; SEQ ID NO. 11: The full-length antibody light chain LC amino acid sequence is shown in SEQ ID NO.12: SEQ ID NO.12: mgwsciilflvatatgvhsdieltqspaimsaspgekvtitcsasssvsymhwfqlkpgtspkpwiyltsnlasgvparfsgsgsgtsysltissmeaedaatyycqqwssnppt fgggtkldlkgqpksspsvtlfppsseeletnkatlvctitdfypgvvtvdwkvdgtpvtqgmettqpskqsnnkymassyltltarawerhssyscqvtheghtvekslspaecl The nucleotide sequence of the full-length antibody light chain LC encoding gene is shown in SEQ ID NO.13.

[0076] SEQ ID NO.13: atgggttggagttgcatcatcttgtttctggtcgccacagccaccggtgtacattccgacattgagctcacccagtctccagcaatcatgtctgcatctccaggggagaaggtcaccataacctgcagtgccagctcaagtgtcagttacatgcactggttccagctgaagccaggcacttctcccaaaccctggatttatctcacatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagcatggaggctgaagatgctgccacttattactgccagcagtggagtagtaacccacccacgttcggaggggggaccaagctggacctgaaagggcagcccaagagcagccctagcgtgaccctgttcccccctagcagcgaggagctggagaccaacaaggccaccctggtgtgcaccatcaccgacttctaccccggcgtggtgaccgtggactggaaggtggacggcacccccgtgacccaaggcatggagaccacacagcctagcaagcagagcaacaacaagtacatggctagcagctacctgaccctgaccgctagagcctgggagagacacagcagctacagctgccaagtgacccacgagggccacaccgtggagaagagcctgagccccgccgagtgcctg 2. The two enzyme cutting sites of Ncol and Xhol between DAS-scFv were introduced into pET-22b expression vector by seamless cloning, and the plasmid pET-22b-DAS-scFv was constructed (DAS-scFv on the vector: VH with nucleotide sequence as shown in SEQ ID NO. 8 and VL with nucleotide sequence as shown in SEQ ID NO. 9 were connected by Linker: GGGGSGGGGSGGGGS), and the expression of recombinant single-chain antibody (DAS-scFv) was carried out in BL21; E. coli BL21; Using the same method, the target genes (full-length antibody light chain LC and full-length antibody heavy chain HC) were cloned into the pCMV vector. A start codon was added to the 5' end of the gene, and a stop codon was added to the 3' end to ensure successful gene expression. As shown in Figure 10, plasmids pCMV-HC and pCMV-LC, containing full-length antibody light chain LC (SEQ ID NO.13) and full-length antibody heavy chain HC (SEQ ID NO.11), were constructed respectively. Expression of the full-length recombinant antibody (pCMV-HC:pCMV-LC = 1:1.5) was performed in mammalian Expi293F cells. The specific steps are as follows: (1) Prokaryotic expression of single-chain antibody DAS-scFv: Recombinant bacteria containing the pET-22b-DAS-scFv plasmid were streaked and incubated overnight at 37°C. Single colonies were picked and inoculated into LB broth and incubated overnight. The next day, the plasmid was extracted and quantified using Nanodrop. 2 μg of plasmid was added to BL21(DE3) competent cells and cultured with shaking at 37°C and 220 r / min for 40 min, followed by plating and incubation overnight. Single colonies were picked and inoculated into LB / Amp broth and incubated until OD500. 600 >1. Add the bacterial culture to 100 mL of LB / Amp liquid medium and incubate until OD reaches 1. 600 Induced overnight at 16℃ and 220 r / min with 0.6-0.8 mM IPTG. The next day, the cells were centrifuged at 12000 r / min and 4℃ for 5 min, and the bacterial pellet and supernatant were identified by SDS-PAGE gel electrophoresis.

[0077] (2) Purification of single-chain antibody DAS-scFv: Purify the liquid containing the target protein using a His-tagged protein purification kit: Filter through a 0.22 μm filter membrane, load 1 mL of His-tagged purification resin into an empty affinity chromatography column, and equilibrate 2-3 times with 0.5 column volumes of non-denaturing lysis buffer. Add the liquid to be purified to the affinity chromatography column, collect the flow-through, and cycle 2-3 times. Elute with a gradient concentration of imidazole solution and collect the target protein. Verify the purified sample by 12.5% ​​SDS-PAGE gel electrophoresis. Dialyze with 0.01 M, pH 7.4 PBS at 4°C for 3 days, changing the dialysis buffer 2-3 times daily.

[0078] (3) Expression and purification of recombinant full-length antibody DAS-IgG: Expi293F cells were pre-resuscitated and passaged to ensure cell viability. On the day of transfection, the total cell count reached 7.5 × 10⁻⁶ cells / year. 7Cells were cultured and their volume adjusted using fresh Expi293 serum-free medium. 30 μg of plasmid (pCMV-HC:pCMV-LC = 1:1.5) was added to 1.5 mL of Opti-MEMⅠ serum-free medium and mixed thoroughly. 81 μL of cationic transfection reagent was added to 1.5 mL of Opti-MEMⅠ serum-free medium and mixed thoroughly, then incubated at room temperature for 5 min. The above suspension was mixed and incubated at room temperature for 20 min. 3 mL of this mixture was then added to the cells and cultured in a shaker at 37°C, 8% CO2, and 125 r / min. 12 h after transfection, 150 μL of transfection enhancer I and 1.5 mL of enhancer II were added, mixed thoroughly, and cultured for another 6-7 days. The target protein was then collected. Purification of the full-length antibody DAS-IgG was performed in the same manner as in step (2).

[0079] (4) Evaluation of single-chain antibody DAS-scFv and full-length antibody DAS-IgG: Add coating antigens DAS-BSA and BSA to 96-well plates, respectively, and incubate at 37°C for 2 h. Wash three times with 0.05% PBST and blot dry. Add 350 μL of 3% skim milk powder to each well and block at 37°C for 2 h. Wash three times and blot dry. Add 100 μL of antibody DAS-scFv or DAS-IgG to each well and incubate at 37°C for 1 h. Wash three times and blot dry. Add 100 μL of anti-His-HRP secondary antibody and incubate at 37°C for 30 min. Wash three times and blot dry. Add 50 μL each of TMB chromogenic solutions A and B, react at 37°C for 15 min, add stop solution, and measure OD. 450nm Absorbance values. 0.125, 0.25, 1, and 2 μg / mL DAS-BSA were coated into the wells of an ELISA plate, respectively. After blocking as described above, different concentrations of DAS-scFv or DAS-IgG antibodies were added, and subsequent steps were the same as above.

[0080] II. Experimental Results (1) Prokaryotic expression of single-chain antibody DAS-scFv: The SDS-PAGE gel electrophoresis results of the single-chain antibody DAS-scFv expressed and purified in E. coli are as follows: Figure 11 As shown in the diagram, lane 1 contains the fermentation supernatant, lane 2 contains the bacterial precipitate, and lanes 5-6 represent the target proteins eluted sequentially. The results indicate that the single-chain antibody DAS-scFv expressed by *E. coli* is present in the bacterial precipitate. The theoretical molecular weight of DAS-scFv is approximately 30 kDa, which matches the bands in lanes 5-6.

[0081] (2) Cellular expression of recombinant full-length antibody DAS-IgG: SDS-PAGE results are as follows Figure 12As shown, Figure 12 As shown in the left side of FIG. 1, under non-reducing conditions, the molecular weight of the recombinant full-length antibody is slightly greater than 180 kd, which is likely due to the glycosylation modification of eukaryotic cells. Figure 12 As shown in the right side of FIG. 1, under reducing conditions, two separate bands appear on the gel because the disulfide bond connecting the heavy chain and the light chain is destroyed. After purification, a band with a molecular weight of about 25 kDa is the light chain, and a band with a molecular weight of about 55 kDa is the heavy chain.

[0082] (3) Evaluation of single-chain antibody DAS-scFv and recombinant full-length antibody DAS-IgG: After dialysis of the purified antibody protein in PBS, the same concentration of DAS-BSA and BSA was used to coat the 96-well plate, and the antibody activity was verified by non-competitive ELISA.

[0083] Single-chain antibody DAS-scFv: When the same concentration of antigen and BSA was added, the absorbance values of the antigen group and the BSA group at 450 nm were both about 0.1, and the P / N value was less than 2.1, indicating that DAS-scFv had almost no activity.

[0084] Recombinant full-length antibody DAS-IgG: When the same concentration of antigen and BSA was added, the absorbance value of the antigen group at 450 nm was significantly higher than that of BSA, and the P / N value was significantly greater than 2.1, indicating that DAS-IgG had good specificity.

[0085] Therefore, DAS-IgG can be selected for the establishment of an enzyme-linked immunoassay method in the future.

[0086] Example 3: ELISA kit for serpulin 1. Establishment of competitive ELISA detection method: (1) Chessboard titration method to determine optimal DAS BSA coating concentration and optimal antibody concentration: Serpulin antigen and purified antibody DAS-IgG obtained in Example 2 were diluted to 0.125 μg / mL, 0.25 μg / mL, 0.5 μg / mL, 1 μg / mL, 1.5 μg / mL, and 2 μg / mL, respectively, and the subsequent operation was the same as in Example 2 (4) Evaluation of single-chain antibody DAS-scFv and full-length antibody DAS-IgG. OD 450 nm The antigen-antibody concentration corresponding to a value close to 1 is the optimal concentration.

[0087] (2) 100 μL of optimal concentration of DAS After BSA coating and blocking, 50 μL of different concentrations of serpentine toxin (0.125-100 ng / mL) standard and 50 μL of twice the optimal concentration of DAS were added IgG, and the subsequent operation is the same as (4) in Example 2. The standard curve was established with the DAS standard concentration as the abscissa and the inhibition rate B / B0 as the ordinate. Among them, B is the absorbance value corresponding to different concentrations of serpentine toxin standard; B0 is the absorbance value corresponding to the concentration of serpentine toxin standard being 0.

[0088] 2. Experimental results: According to the checkerboard titration method, the optimal DAS The BSA coating concentration was 1 μg / mL, and the optimal DAS The IgG concentration was 1 μg / mL. The competition standard curve of DAS concentration in the range of 0.125-100 ng / mL is shown in Figure 13 The detection limit, i.e. the amount corresponding to the inhibition rate of 20%-80%, was 2.67-21.68 ng / mL, and the IC50 value was 7.61 ng / mL. The cross rate of serpentine toxin analogues was less than 1%.

[0089] Although the present application has been disclosed with the above preferred embodiments, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application. Therefore, the protection scope of the present application should be defined by the claims.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to anguimycin, characterized in that, The antibody or antigenic fragment thereof comprises a light chain variable region VL and a heavy chain variable region VH; the light chain variable region VL comprises LCDR1, LCDR2, LCDR3, the amino acid sequences of which are respectively shown as SEQ ID NO. 6, LTS, SEQ ID NO. 7; The heavy chain variable region VH comprises HCDR1, HCDR2, HCDR3, the amino acid sequences of which are respectively shown as SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO.

5.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein, The light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO. 2, or an amino acid sequence with homology of at least 98% to SEQ ID NO. 2 and having the same function as the protein shown in SEQ ID NO. 2; The heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO. 1, or an amino acid sequence with homology of at least 98% to SEQ ID NO. 1 and having the same function as the protein shown in SEQ ID NO.

1.

3. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein, The antibody comprises an antibody heavy chain HC having the amino acid sequence shown in SEQ ID NO. 10, and an antibody light chain LC having the amino acid sequence shown in SEQ ID NO.

12.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the antibody or antigenic fragment thereof of any one of claims 1-3; the nucleotide sequence encoding the antibody heavy chain HC is shown as SEQ ID NO. 11, and the nucleotide sequence encoding the antibody light chain LC is shown as SEQ ID NO.

13.

5. A biomaterial, characterized by, The biological material comprises at least one of the following (a)-(g): (a) an expression cassette comprising the nucleic acid molecule of claim 4; (b) a vector comprising the nucleic acid molecule of claim 4; (c) a vector comprising the expression cassette of (a); (d) a transgenic cell line comprising the nucleic acid molecule of claim 4; (e) a transgenic cell line comprising the expression cassette of (a); (f) a transgenic cell line comprising the vector of (b); (g) a transgenic cell line comprising the vector of (c).

6. The biomaterial of claim 5, wherein, The cell line is selected from a microorganism or a mammalian cell.

7. A kit, characterized in that, The kit contains the antibody or antigenic fragment thereof of any one of claims 1-3, or contains the nucleic acid molecule of claim 4, or contains the biological material of claim 5.

8. Use of the antibody or antigenic fragment thereof of any one of claims 1-3, the nucleic acid molecule of claim 4, or the biological material of claim 5 or 6 in the preparation of a reagent for detecting opheliin in a sample.

9. A method of producing an antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that, The method comprises culturing the transgenic cell line of claim 5 or 6 to prepare the antibody or antigenic fragment thereof of any one of claims 1-3.

10. A method for detecting the presence or absence of a serpentine in a sample or for determining the amount of a serpentine in a sample, characterized in that, The method comprises the step of contacting the antibody or antigenic fragment thereof of any one of claims 1-3 with a sample, and the method is not for the purpose of diagnosis and / or treatment of a disease.

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