A single-chain antibody of a muscular stomach erosive factor, a kit and application and an immune detection method thereof

Abstract

The application belongs to the field of genetic engineering, and relates to a single-chain antibody of pepsinogen, a kit and application thereof and an immune detection method. The application constructs a single-chain antibody of pepsinogen and an expression vector thereof to obtain a single-chain antibody scFv 9-3 of pepsinogen with antigen binding activity, and the amino acid sequence of the single-chain antibody is shown as SEQ ID No. 7. 50 The IC50 value of the single-chain antibody is 87.41 ng / mL, the linear range is 27.02-282.77 ng / mL, and the detection limit is 13.60 ng / mL. The application also discloses a recognition mechanism of the antibody and pepsinogen through molecular docking technology, and provides a new genetic engineering antibody element for development of an immune detection method of pepsinogen.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering and relates to a single-chain antibody against gizzard erosion. Background Technology

[0002] Gizzard erosion amine is a biogenic amine produced during the heating and drying process of fishmeal, with the chemical formula C. 11 H 20 N4O2 has a molecular weight of 240. When poultry ingest feed containing gizzard erosion factor, the factor binds to histamine H2 receptors on gastric parietal cells, increasing intracellular cAMP concentration and promoting excessive gastric acid secretion, thus causing gizzard erosion and black vomiting disease in poultry. Specifically, gizzard erosion factor's ability to increase intracellular cAMP concentration, stimulate gastric acid secretion, and cause gizzard erosion is 1000 times, 10 times, and 300 times higher than histamine, respectively. When feed contains more than 12% spoiled fishmeal, poultry mortality exceeds 10%. Therefore, establishing a rapid detection method for gizzard erosion factor is crucial for feed safety and the health of farmed animals.

[0003] Immunoassays based on antigen-antibody reactions are widely used in fields such as feed safety, food safety, and disease control due to their advantages of rapid detection, low reaction cost, and simple experimental operation. Antibodies play a central role in immunoassays, and their performance directly affects the sensitivity and accuracy of the detection method. Traditional antibodies are obtained through animal immunization, but their instability and difficulty in mass production limit the further development of related immunoassay technologies. In contrast, genetically engineered antibodies, which are easy to express and modify, have stable and controllable production processes, smaller molecular weights, and stronger penetrating power, have become the rising stars in antibody development in recent years.

[0004] However, current research on immunoassay methods for gizzard erosion mainly focuses on traditional antibodies, primarily monoclonal and polyclonal antibodies. To date, no immunoassay method for gizzard erosion using genetically engineered antibodies as the main recognition element has been established. To meet practical testing needs, it is necessary to develop more antibodies for the detection of gizzard erosion, thereby enriching the application of related immunoassay methods in actual production. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention proposes a single-chain antibody against gizzard erosion, a reagent kit, its application, and an immunoassay method.

[0006] The technical solution of this invention is implemented as follows:

[0007] The first objective of this invention is to provide a single-chain antibody against gizzard erosion, wherein the heavy chain variable region of the single-chain antibody contains the VH amino acid sequence shown in SEQ ID No. 1-3.9-3 -CDR1-3; The light chain variable region of the antibody contains the VL of the amino acid sequence shown in SEQ ID No. 4-6. 9-3 -CDR1-3.

[0008] Furthermore, the amino acid sequence of the gizzard erosion single-chain antibody is shown in SEQ ID No. 7.

[0009] A second objective of this invention is to provide a nucleotide fragment encoding the above-mentioned single-chain antibody against gastric erosion; preferably, its nucleotide sequence is shown in SEQ ID No. 8.

[0010] A third objective of this invention is to provide a recombinant expression vector containing the aforementioned nucleotide fragments.

[0011] The fourth objective of this invention is to provide a recombinant engineered bacterium or recombinant expression cell comprising the above-described recombinant expression vector.

[0012] The fifth objective of this invention is to provide the application of the above-mentioned gizzard erosion single-chain antibody in the preparation of products for detecting gizzard erosion.

[0013] A sixth object of the present invention is to provide a kit for detecting gizzard erosion, wherein the kit comprises a gizzard erosion single-chain antibody.

[0014] The seventh objective of this invention is to provide a method for detecting gizzard erosion in feed and food, comprising the following steps: using the above-mentioned gizzard erosion single-chain antibody as the antibody and gizzard erosion artificial antigen H1-OVA as the coating antigen, performing enzyme-linked immunosorbent assay (ELISA).

[0015] Preferably, the structural formula of the above-mentioned artificial antigen H1-OVA for gizzard erosion is as follows:

[0016] .

[0017] Preferably, the above-mentioned enzyme-linked immunosorbent assay (ELISA) is an indirect ELISA, specifically an indirect competitive enzyme-linked immunosorbent assay (icELISA).

[0018] As a more preferred embodiment, the present invention provides a specific method for detecting gizzard erosion factor:

[0019] S1. Prepare an ELISA plate coated with artificial antigen H1-OVA containing gizzard erosion liniment;

[0020] S2. Add the gizzard erosion pesticide standard or the sample to be tested into the microwell of the ELISA plate, and then add the single-chain antibody scFv 9-3;

[0021] S3. Add enzyme-labeled secondary antibody and incubate;

[0022] S4. Add colorimetric solution and incubate;

[0023] S5. Add the stop solution and measure;

[0024] S6. Using the logarithm of the drug's standard concentration. 10 The values ​​are plotted on the x-axis, and the ratio of the absorbance of each standard concentration to the absorbance of the zero standard well is plotted on the y-axis to establish a standard curve. The content of gizzard erosion pesticide in the test sample is then calculated based on the absorbance of the test sample.

[0025] The present invention has the following beneficial effects:

[0026] Based on the previously prepared anti-gizzard erosion monoclonal antibody 6F4, this invention utilizes genetic engineering technology to obtain a single-chain antibody scFv9-3 that specifically recognizes gizzard erosion, the amino acid sequence of which is shown in SEQ ID NO.7. This single-chain antibody can specifically recognize gizzard erosion pesticides, and the IC50 of the icELISA detection method... 50 The effective value was 87.41 ng / mL, the limit of detection was 13.60 ng / mL, and the linear range was 27.02-282.77 ng / mL. Further analysis using molecular docking technology revealed that the single-chain antibody recognizes gizzard erosion lin primarily through hydrogen bonding and hydrophobic interactions, forming a total of 7 hydrogen bonds and 2 hydrophobic interactions. The key amino acid sites for binding are Gln225, Ile134, Glu229, Asp133, and Trp47. The single-chain antibody provided by this invention features small molecular weight, ease of expression and modification, and offers a novel genetically engineered antibody element for the development of immunoassay methods for gizzard erosion lin. It can be used to establish enzyme-linked immunosorbent assay (ELISA) or immunochromatographic detection methods, and has broad application prospects in feed safety monitoring and on-site diagnosis of gizzard erosion. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 Screening of primers for VH gene PCR amplification.

[0029] Figure 2 Screening of primers for VL gene PCR amplification.

[0030] Figure 3Agar gel electrophoresis images for double enzyme digestion verification of VH and VL genes and vectors; where M and M1: DNA marker DL10000; M2: DNA marker DL2000; lane 1: pET-22b vector before enzyme digestion; lane 2: pET-22b vector after enzyme digestion; lanes 3, 4, 5, 6 and 7: target bands of VH7, VH5, VH9, VH12 and VH14 after enzyme digestion; lanes 8, 9 and 10: target bands of VL2, VL4 and VL6 after enzyme digestion.

[0031] Figure 4 Agarose gel electrophoresis image for bacterial culture PCR verification.

[0032] Figure 5 Amino acid sequence alignment for VH-positive monoclonal antibodies.

[0033] Figure 6 Amino acid sequence alignment for VL-positive monoclonal antibodies.

[0034] Figure 7 Agarose gel electrophoresis was used to identify the amplification of VH-linker, linker-VL, and VH-linker-VL.

[0035] Figure 8 Agarose gel electrophoresis image to verify the double digestion of VH9-3-Linker-VL4-1, VH7-1-Linker-VL4-1, and pET-22b.

[0036] Figure 9 SDS-PAGE electrophoresis images of purified scFv 7-1 and scFv 9-3.

[0037] Figure 10 icELISA standard curves for detecting erosives in scFv 7-1 and scFv 9-3.

[0038] Figure 11 The amino acid sequence is scFv 9-3.

[0039] Figure 12 The results are the predictions for the secondary structure of scFv 9-3.

[0040] Figure 13 The results are the predictions for the tertiary structure of scFv 9-3.

[0041] Figure 14 Two-dimensional molecular docking diagram of scFv 9-3 and gizzard erosionin.

[0042] Figure 15 A flowchart outlining the overall technical process for the preparation, application, and immunoassay of gizzard erosion single-chain antibodies. Detailed Implementation

[0043] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0044] Unless otherwise specified, the experimental methods used in the following experimental examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0045] Example 1: Cloning of the single-chain antibody gene for gizzard erosion.

[0046] 1. Total RNA extraction and cDNA synthesis from hybridoma cells

[0047] The 6F4 hybridoma cell line containing the anti-gastric erosion monoclonal antibody was revived and cultured until the cell number was sufficient and the cells were in good condition, at which point total RNA was extracted. Lyse cells with 2 mL of TRNSOL lysis buffer and transfer evenly to two 1.5 mL centrifuge tubes. Add 200 μL of chloroform to each tube, shake vigorously for 15 s, and incubate on ice for 5 min. Centrifuge at 12000 r / min for 10 min at 4 ℃. Transfer the supernatant to a new centrifuge tube, add 0.7 volumes of anhydrous ethanol, vortex to mix, and then transfer to a GBC adsorption column. Centrifuge at 12000 r / min for 1 min at 4 ℃. Add 500 μL of Wash Buffer I to the GBC adsorption column, centrifuge at 12000 r / min for 1 min after washing, and discard the waste liquid at the bottom. Place the GBC adsorption column back into the collection tube. Add 600 μL of Wash Buffer II to wash, centrifuge at 12000 r / min for 1 min after washing, and discard the waste liquid at the bottom. Place the GBC adsorption column in a fume hood for 5-10 min to thoroughly dry the residual ethanol in the adsorption material. Finally, rinse with 30-100 μL of RNase-free... RNA was eluted with water. The obtained RNA was identified by agarose gel electrophoresis, and its concentration and purity were determined using a Nanodrop 2000c micro UV spectrophotometer. It was then stored at -80 ℃ for later use.

[0048] Use 2 μg of the above RNA as a template, prepare reverse transcription reaction system one according to Table 1, react at 65 ℃ for 5 min in a PCR instrument, remove and place on an ice box to cool; then prepare reverse transcription reaction system two according to Table 2, react at 42 ℃ for 60 min in a PCR instrument for reverse transcription, stop the reaction at 70 ℃ for 5 min, and freeze the obtained cDNA at -80 ℃ for later use.

[0049] Table 1 Reverse transcription solution system 1

[0050]

[0051] Table 2 Reverse Transcription Solution System II

[0052]

[0053] 2. PCR amplification of VH and VL genes

[0054] To amplify the antibody heavy chain variable region (VH) gene, 15 upstream primers and 4 downstream primers were paired and combined. The VH upstream primer sequences are shown in Table 3, and the VH downstream primer sequences are shown in Table 4. The PCR amplification reaction system was prepared according to Table 5, and the PCR amplification program was set according to Table 6.

[0055] Table 3. Upstream primer sequences for VH

[0056]

[0057] Table 4. VH downstream primer sequences

[0058]

[0059] To amplify the antibody light chain variable region (VL) gene, eight upstream primers and three downstream primers were paired and combined. The sequences of the upstream VL primers are shown in Table 5, and the sequences of the downstream VL primers are shown in Table 6. The PCR amplification reaction system and procedure were the same as those in Table 5 and Table 6, respectively.

[0060] Table 5. Upstream primer sequences for VL

[0061]

[0062] Table 6. VL downstream primer sequences

[0063]

[0064] Table 7 PCR amplification system

[0065]

[0066] Table 8 PCR Amplification Procedure

[0067]

[0068] After the reaction, the PCR products were analyzed by 1% agarose gel electrophoresis. Primer combinations capable of amplifying the target bands were selected based on the size (approximately 300-500 bp for VH and VL genes) and brightness. Figure 1As shown, five primer combinations were successfully cloned from the VH gene: VH5 / JH2, VH7 / JH2, VH9 / JH2, VH12 / JH2, and VH14 / JH2; Figure 2 As shown, three primer combinations were successfully cloned from the VL gene: VK4 / JK1 / 2, VK6 / JK1 / 2, and VK2 / JK4. The target bands were excised and recovered using a DNA purification kit for later use.

[0069] Example 2: Construction of a single-chain antibody expression vector

[0070] 1. Enzyme digestion of VH and VL gene fragments

[0071] Since the primers for amplifying the VL gene contain NotⅠ and SalⅠ restriction sites, and the primers for amplifying the VH gene contain NcoⅠ and XhoⅠ restriction sites, and the pET-22b vector carries all four restriction sites, NcoⅠ and XhoⅠ enzymes can be used to double-digest the VH gene fragment and the pET-22b vector, respectively. The digestion reaction system was prepared according to Table 9, and digestion was carried out at 37 ℃ for 3 h. The digestion products were identified by 1% agarose gel electrophoresis, and the results are shown in the table below. Figure 3 Lanes a and 3b, where M is the DNA marker DL10000, lane 1 is the pET-22b vector before digestion, lane 2 is the pET-22b vector after digestion, and lanes 3, 4, 5, 6, and 7 are the target bands VH7, VH5, VH9, VH12, and VH14 after digestion, respectively. The VL gene fragment and pET-22b vector were also double-digested using NotI and SalI enzymes, respectively. The results are shown in [Figure 1]. Figure 3 c, where M1 is DNA marker DL10000, M2 is DNA marker DL2000, and lanes 8, 9, and 10 represent the target bands of VL2, VL4, and VL6 after enzyme digestion. The VH and VL gene fragments were cleaned and recovered. The 5400 bp band of the double-digested pET-22b vector was recovered by gel extraction. The concentration was determined using Nanodrop 2000c and stored at -20℃ for later use.

[0072] Table 9 Enzyme digestion reaction system

[0073]

[0074] 2. Construction of pET-22b-VL and pET-22b-VH vectors

[0075] The VL fragment recovered from double enzyme digestion was ligated with the linearized pET-22b vector according to the enzyme ligation reaction system in Table 10, and ligated at 16 ℃ for 16 h. 2 μL of the ligation product was transformed into 100 μL of E. coli DH5α competent cells, incubated on ice for 30 min, heat-shocked at 42 ℃ for 90 s, incubated on ice for 5 min, and then 500 μL of LB liquid medium was added. The cells were cultured at 37 ℃ with shaking at 250 r / min for 1 h, and then plated onto LB solid medium containing 100 μg / mL ampicillin and incubated upside down at 37 ℃ overnight.

[0076] Table 10 Enzyme-linked reaction system

[0077]

[0078] The following day, single colonies were randomly selected from the plate for colony PCR verification. Some identification results are shown below. Figure 4 As shown, positive clones were selected and sent to a sequencing company for sequencing. The sequencing results showed that the pET-22b-VL vector was successfully constructed.

[0079] Using the same method, the VH fragment recovered from double enzyme digestion was ligated into the linearized pET-22b vector, transformed, and identified to obtain the pET-22b-VH vector. Sequence alignment of the VH gene was performed (see results below). Figure 5 It was found that, except for VH9-3, which consists of 351 bp (117 amino acids), the other four sequences all consist of 330 bp (110 amino acids). These sequences are identical except for the FR1 region. Further analysis of the FR1 region revealed four different sequences. Comparison with the starting amino acid sequence of the universal single-chain antibody VH sequence showed that VH9-3 was the most similar. The other three sequences were likely due to different primers used for amplification. Therefore, after comprehensive consideration, VH9-3 and VH7-1 were selected for subsequent experiments. Sequence alignment of the VL gene (results see...) Figure 6 All six sequences consist of 333 bp (111 amino acids). Among these six VL gene sequences, only the FR1 region of VL4-2 differs by one amino acid sequence, so they can be considered as the same VL gene sequence. After comprehensive consideration, VL4-1 was selected for subsequent experiments.

[0080] 3. Assembly of VH-Linker-VL single-chain antibody gene

[0081] Nested PCR was used to ligate VH, VL, and the linker into a single-chain antibody gene in the form of VH-Linker-VL. First, primers were designed to introduce the linker sequence downstream of VH and upstream of VL. Primer sequences are shown in Table 11.

[0082] Table 11 Nested PCR Primer Sequences

[0083]

[0084] First round of PCR: Using pET-22b-VH9-3 as a template, primers F9 and R9 were used to amplify the VH9-3-Linker fragment; using pET-22b-VH7-1 as a template, primers F7 and R7 were used to amplify the VH7-1-Linker fragment; using pET-22b-VL4-1 as a template, primers F4 and R4 were used to amplify the Linker-VL4-1 fragment. The PCR reaction system was the same as in Table 7, and the amplification program was: 94 ℃ for 5 min; 94 ℃ for 30 s, 55 ℃ for 30 s, 72 ℃ for 1 min, 30 cycles; 72 ℃ for 10 min. The amplified products were detected by 1% agarose gel electrophoresis, and the results are shown in the table below. Figure 7 a, M is DNA marker DL2000, lanes 1, 2 and 3 are VH7-1-Linker, VH9-3-Linker and linker-VL4-1 respectively, and the target fragment is recovered by gel cutting.

[0085] Second round PCR: The VH9-3-Linker and Linker-VL4-1 fragments recovered in the first round were used as templates and primers (50 ng each) for nested PCR amplification to obtain the full-length VH9-3-Linker-VL4-1 fragment. The VH7-1-Linker-VL4-1 fragment was obtained using the same method. The PCR reaction system was: 25 μL of 2×Taq Master Mix, 50 ng of each template, and water added to a final volume of 50 μL. The amplification program was: 94 ℃ for 5 min; 94 ℃ for 30 s, 58 ℃ for 30 s, 72 ℃ for 1 min 30 s, 30 cycles; 72 ℃ for 10 min. The amplification products were detected by 1% agarose gel electrophoresis. The results are shown in [Figure number missing]. Figure 7 b, M is DNA marker DL2000, lanes 4 and 5 are VH9-3-Linker-VL4-1 and VH7-1-Linker-VL4-1, respectively, and the target band (approximately 750 bp) is recovered by gel cutting.

[0086] 4. Construction of the pET-22b-scFv expression vector

[0087] The recovered VH9-3-Linker-VL4-1 and VH7-1-Linker-VL4-1 fragments were double-digested with the pET-22b empty vector using NcoI and XhoI, respectively, according to the digestion system shown in Table 9. After electrophoresis identification, the digestion products were recovered from the gel and ligated according to the enzyme ligation reaction system shown in Table 10. The ligation products were then transformed into E. coli DH5α competent cells, plated, and single clones were picked for colony PCR verification and sequencing. The successfully sequenced recombinant plasmids were named pET-22b-scFv9-3 and pET-22b-scFv7-1, respectively.

[0088] Example 3: Expression and purification of gizzard erosion single-chain antibody

[0089] 1. Induced expression of single-chain antibodies

[0090] The correctly sequenced recombinant plasmids pET-22b-scFv9-3 and pET-22b-scFv7-1 were transformed into E. coli BL21(DE3) competent cells, yielding single colonies of positive engineered bacteria. These colonies were inoculated into LB broth and cultured overnight at 37 °C with shaking at 250 rpm. The following day, a 1% inoculum was added to 1 L of LB broth and cultured at 37 °C with shaking at 250 rpm until the logarithmic growth phase. IPTG was then added to a final concentration of 1 mmol / L, and the cells were induced to grow at 22 °C with shaking at 200 rpm for 16 h. After centrifugation at 12000 rpm for 20 min, the supernatant was discarded, and the bacterial cells were collected.

[0091] 2. Extraction of soluble proteins from the periplasmic cavity

[0092] Soluble proteins in the periplasmic cavity were extracted using the sucrose osmotic pressure method. 4 mL of TES solution (0.2 mol / L Tris-HCl pH 8.0, 0.5 mmol / L EDTA, 20% sucrose) was added to the collected bacterial cells to fully resuspend the cells, and the cells were frozen at -80 °C for at least 3 h. After thawing at room temperature, 12 mL of a 5-fold diluted TES solution (TES:water = 1:4) was added, and the mixture was vortexed and incubated at 16 °C and 180 r / min for 30 min. The mixture was then centrifuged at 4 °C and 12000 r / min for 20 min, and the supernatant was collected as the periplasmic cavity soluble protein extract.

[0093] 3. Ni-NTA affinity chromatography purification

[0094] The extract was filtered through a 0.22 μm microporous membrane and mixed with 1 mL of Ni-NTA agarose gel packing. The mixture was incubated overnight at 4°C with shaking. The next day, the mixture was loaded into a gravity column. The column was first equilibrated with 0.01 mol / L PBS (pH 7.4), and then eluted sequentially with PBS buffers containing 10 mmol / L, 20 mmol / L, 40 mmol / L, and 200 mmol / L imidazole. Each eluted fraction was collected.

[0095] Each eluted fraction was subjected to SDS-PAGE protein electrophoresis for identification. Results are shown below. Figure 9 As shown, M is a 180 kDa marker. Lanes 1 and 5 are purified scFv 7-1 and scFv 9-3, respectively, with the target protein band appearing at approximately 34 kDa, consistent with the expected size. Lanes 2, 3, and 4 contain 500 mmol / L, 20 mmol / L, and 40 mmol / L imidazole eluents, respectively, with the 200 mmol / L imidazole eluent showing higher purity of the target protein. The eluent containing the target protein was concentrated using an ultrafiltration tube (10 kDa molecular weight cutoff) and dialyzed against 0.01 mol / L PBS to remove salt. Protein concentration was determined using a Nanodrop 2000c, and the eluent was aliquoted and stored at -20 °C for later use.

[0096] Example 4: Identification of Single-Chain Antibody Activity

[0097] 1. Establishment of an indirect competitive ELISA detection method

[0098] Using the artificial antigen H1-OVA of gizzard erosion as the coating agent, the antigen-binding activity of the purified single-chain antibody was detected by icELISA. The specific operational steps are as follows:

[0099] (1) Coating: Dilute the original H1-OVA to 2 μg / mL with carbonate buffer (CB, pH 9.6), add 100 μL to each well of a 96-well microplate, and coat overnight at 4 ℃.

[0100] (2) Washing: Discard the liquid in the well, add 300 μL PBST (PBS containing 0.05% Tween-20) to each well and wash twice, then pat dry.

[0101] (3) Sealing: Add 120 μL of sealing solution (5% skim milk powder) to each well, seal at 37 ℃ for 3 h, discard the sealing solution, pat dry, and then dry in an oven at 37 ℃ upside down for 1 h.

[0102] (4) Sample addition: The gizzard erosion standard was serially diluted to a series of concentrations with PBS. 50 μL of gizzard erosion standard of different concentrations and 50 μL of single-chain antibody diluted to an appropriate concentration were added to each well (50 μL of PBS and 50 μL of single-chain antibody were added to each titer well). The wells were incubated at 37 °C for 40 min. The wells were washed 5 times with PBST and patted dry.

[0103] (5) Add secondary antibody: Add 100 μL of mouse anti-His tag monoclonal antibody (1:5000 dilution) to each well and incubate at 37 ℃ for 30 min. Wash 5 times with PBST and pat dry.

[0104] (6) Add enzyme-labeled secondary antibody: Add 100 μL of HRP-labeled goat anti-mouse IgG (1:5000 dilution) to each well and incubate at 37 ℃ for 30 min. Wash 5 times with PBST and pat dry.

[0105] (7) Color development: Add 100 μL of TMB color development solution to each well and incubate at 37 °C in the dark for 10 min.

[0106] (8) Termination: Add 50 μL of stop solution (10% H2SO4) to each well and measure the absorbance at 450 nm using a microplate reader (A). 450 ).

[0107] 2. Establishment of the standard curve

[0108] Using the logarithm of the concentration of the gizzard erosion standard as the x-axis and the ratio of the absorbance at each concentration point to the absorbance of the zero standard well (B / B0) as the y-axis, a four-parameter fitting was performed using Origin 2021 software to establish the icELISA standard curve.

[0109] Figure 10 a displays the IC of scFv7-1 50 The value was 110.04 ng / mL, the limit of detection was 9.47 ng / mL, and the linear detection range was 23.43-516.85 ng / mL. Figure 10 b shows the IC of scFv 9-3 50 The effective value was 87.41 ng / mL, the limit of detection was 13.60 ng / mL, and the linear detection range was 27.02–282.77 ng / mL. Both single-chain antibodies exhibited good antigen-binding activity.

[0110] Example 5: Molecular docking analysis of antigen-antibody recognition mechanism

[0111] 1. Bioinformatics analysis of single-chain antibodies

[0112] Bioinformatics analysis was performed using scFv 9-3, which has high sensitivity. Its amino acid sequence SEQ ID No. 7 is shown below. Figure 11 The nucleotide sequence is SEQ ID No. 8. The secondary structure of the protein was predicted using the SOPMA online website. Figure 12 Its secondary structure is mainly composed of random coils, extended strands, beta turns, and alpha helixes.

[0113] Using the SWISS-MODEL online website for homologous modeling, a 3D structural model of scFv 9-3 was obtained. Figure 13 a). The model was evaluated using the PROCHECK procedure. The Ramachandran plot showed that 90.5% of the amino acid residues were located in the optimal region, and 8.5% were located in other allowed regions, indicating that the model conformation was reasonable. Figure 13 b). The overall quality coefficient evaluated by the ERRAT procedure was 94.79, indicating that the model has high reliability. Figure 13 c).

[0114] 2. Molecular docking analysis

[0115] The molecular docking of scFv 9-3 with gizzard erosion was performed using Lead IT 2.1.8 software, and the docking conformation with the lowest binding free energy was selected for analysis.

[0116] Figure 14 Molecular docking results of scFv 9-3 and gizzard erosion showed that scFv 9-3 and gizzard erosion mainly recognize each other through hydrogen bonding and hydrophobic interaction, forming a total of 7 hydrogen bonds and 2 hydrophobic interactions. The key amino acid sites for binding were Gln225, Ile134, Glu229, Asp133 and Trp47.

[0117] Figure 15 A flowchart summarizing the overall technical process for the preparation, application, and immunoassay of gizzard erosion single-chain antibodies is presented.

[0118] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0119] Statement regarding funding

[0120] This research was supported by the National Natural Science Foundation of China (No. 32502384 and No. 62501228), the Key Research and Development and Extension Project of Henan Province (Science and Technology Breakthrough) (No. 252102311167 and No. 262102211018), the Doctoral Research Start-up Fund Project of Henan Academy of Agricultural Sciences (No. 2026BX76), and the Excellent Young Scientists Fund of Henan Academy of Agricultural Sciences (No. 2026YQ12).

Claims

1. A single-chain antibody against gizzard erosion, characterized in that: The heavy chain variable region of the gizzard erosion single-chain antibody contains the VH amino acid sequence shown in SEQ ID No. 1-3. 9-3 -CDR1-3; The light chain variable region of the antibody contains the VL of the amino acid sequence shown in SEQ ID No. 4-6. 9-3 -CDR1-3.

2. The single-chain antibody against gizzard erosion as described in claim 1, characterized in that: The amino acid sequence of the gizzard erosion single-chain antibody is shown in SEQ ID No.

7.

3. A nucleotide fragment encoding the gizzard erosion single-chain antibody as described in claim 1 or 2.

4. The nucleotide fragment according to claim 3, wherein the nucleotide sequence is shown in SEQ ID No.

8.

5. A recombinant expression vector comprising the nucleotide fragment of claim 4.

6. Recombinant engineered bacteria or recombinant expression cells containing the recombinant expression vector of claim 5.

7. The use of the gizzard erosion single-chain antibody according to claim 1 or 2 in the preparation of products for detecting gizzard erosion.

8. A kit for detecting erosive substances in the gizzard, characterized in that: It contains the single-chain antibody containing gizzard erosion as described in claim 1 or 2.

9. A method for detecting gizzard erosion factor in feed or food, characterized in that, The steps are as follows: using the single-chain antibody of gizzard erosion as described in claim 1 or 2 as the antibody, and the artificial antigen of gizzard erosion, H1-OVA, as the coating antigen, enzyme-linked immunosorbent assay (ELISA) is performed.

10. The method for detecting gizzard erosion factor in feed or food according to claim 9, characterized in that, The structural formula of the artificial antigen H1-OVA for gastric erosion is as follows: 。

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