Shrimp tropomyosin specific nano antibody and application

Abstract

The invention provides a shrimp tropomyosin specific nano-antibody and application thereof. The specific nano-antibody is at least one of a nano-antibody Nb48, a nano-antibody Nb55, a nano-antibody Nb78, a nano-antibody Nb84, a nano-antibody Nb88, a nano-antibody Nb110, a nano-antibody Nb115 or a nano-antibody Nb141; the specific nano antibody comprises three complementary determining regions, namely a CDR1 (complementary determining region), a CDR2 (complementary determining region) and a CDR3 (complementary determining region); and for the nano antibody Nb48, the amino acid sequence of the CDR1 is as shown in SEQ ID NO.1, the amino acid sequence of the CDR2 is as shown in SEQ ID NO.2, and the amino acid sequence of the CDR3 is as shown in SEQ ID NO.3. The shrimp tropomyosin specific nano antibody disclosed by the invention is short in preparation period, low in cost and high in stability, and can realize economical, efficient, high-sensitivity and rapid detection of allergen shrimp tropomyosin in food.

Description

Technical Field

[0001] The invention belongs to the field of food, and in particular relates to a shrimp tropomyosin-specific nano antibody and an application thereof. Background Art

[0002] Food allergy is an immune-mediated adverse systemic immune response that has become a global health problem, and its frequency in the field of food safety is increasing. When a food allergy occurs in the human body, the immune system is stimulated, inducing an immunoglobulin E (IgE)-mediated hypersensitivity reaction, releasing allergic inflammatory mediators, and then triggering a severe acute hypersensitivity reaction. Common symptoms include rash, diarrhea, abdominal pain, and in severe cases, even anaphylactic shock, which directly threatens life. However, as of now, there is still a lack of completely effective clinical treatment for food allergies. For people who are prone to allergies, the only effective preventive measure is to avoid consuming foods containing allergens. In this context, the development of reliable and sensitive allergen detection technology to achieve accurate labeling and risk assessment of allergenic ingredients in food has become a top priority for ensuring food safety.

[0003] Over the past few decades, enzyme-linked immunosorbent assays (ELISA), polymerase chain reaction (PCR), and liquid chromatography-mass spectrometry (LC-MS) have been widely used in the labeling and management of food allergens. ELISA, with its high sensitivity and specificity, has been widely used in food allergen detection. The key to establishing ELISA methods lies in obtaining specific antibodies. In recent years, nanobodies, a new type of small-molecule antibody, have become a research hotspot. Nanobodies are the variable regions of heavy-chain antibodies (VHHs) naturally produced by camelids. They possess small molecular weight and strong penetrating power, enabling them to recognize structures and cryptic epitopes that conventional antibodies cannot. Compared to conventional monoclonal and polyclonal antibodies, nanobodies are also highly stable, resistant to high temperatures and acidic and alkaline environments, and easy to express. These unique advantages enable nanobodies to achieve analytical and diagnostic results that are difficult to achieve with conventional antibodies, making them an ideal choice for detecting illegal additives, pesticide residues, and allergenic ingredients in food.

[0004] Traditional monoclonal and polyclonal antibody preparation methods are complex, time-consuming, and costly. Nanobodies, on the other hand, are simple to prepare, require a short preparation time, are low-cost, and offer high stability. Despite advances in existing technologies, there is still room for improvement in food allergen detection, particularly in the highly sensitive and rapid detection of specific allergens. Summary of the Invention

[0005] In view of this, the present invention aims to overcome the defects in the prior art and proposes a shrimp tropomyosin-specific nanobody and its application.

[0006] To achieve the above object, the technical solution of the present invention is achieved as follows:

[0007] The present invention provides a shrimp tropomyosin-specific nanobody, wherein the specific nanobody is at least one of nanobody Nb48, nanobody Nb55, nanobody Nb78, nanobody Nb84, nanobody Nb88, nanobody Nb110, nanobody Nb115 or nanobody Nb141;

[0008] The specific nanobody includes three complementary determining regions CDR1, CDR2, and CDR3;

[0009] For Nanobody Nb48: the amino acid sequence of the CDR1 is shown in SEQ ID NO.1, the amino acid sequence of the CDR2 is shown in SEQ ID NO.2, and the amino acid sequence of the CDR3 is shown in SEQ ID NO.3;

[0010] For Nanobody Nb55: the amino acid sequence of the CDR1 is shown in SEQ ID NO.4, the amino acid sequence of the CDR2 is shown in SEQ ID NO.5, and the amino acid sequence of the CDR3 is shown in SEQ ID NO.6;

[0011] For Nanobody Nb78: the amino acid sequence of the CDR1 is shown in SEQ ID NO.7, the amino acid sequence of the CDR2 is shown in SEQ ID NO.8, and the amino acid sequence of the CDR3 is shown in SEQ ID NO.9;

[0012] For Nanobody Nb84: the amino acid sequence of the CDR1 is shown in SEQ ID NO.10, the amino acid sequence of the CDR2 is shown in SEQ ID NO.11, and the amino acid sequence of the CDR3 is shown in SEQ ID NO.12;

[0013] For Nanobody Nb88: the amino acid sequence of the CDR1 is shown in SEQ ID NO.13, the amino acid sequence of the CDR2 is shown in SEQ ID NO.14, and the amino acid sequence of the CDR3 is shown in SEQ ID NO.15;

[0014] For Nanobody Nb110: the amino acid sequence of the CDR1 is shown in SEQ ID NO.16, the amino acid sequence of the CDR2 is shown in SEQ ID NO.17, and the amino acid sequence of the CDR3 is shown in SEQ ID NO.18;

[0015] For Nanobody Nb115: the amino acid sequence of the CDR1 is shown in SEQ ID NO.19, the amino acid sequence of the CDR2 is shown in SEQ ID NO.20, and the amino acid sequence of the CDR3 is shown in SEQ ID NO.21;

[0016] For Nanobody Nb141: the amino acid sequence of the CDR1 is shown in SEQ ID NO.22, the amino acid sequence of the CDR2 is shown in SEQ ID NO.23, and the amino acid sequence of the CDR3 is shown in SEQ ID NO.24.

[0017] Furthermore, the specific nanobody comprises four framework regions FR1, FR2, FR3, and FR4;

[0018] For Nanobody Nb48: the amino acid sequence of FR1 is shown in SEQ ID NO.25, the amino acid sequence of FR2 is shown in SEQ ID NO.26, the amino acid sequence of FR3 is shown in SEQ ID NO.27, and the amino acid sequence of FR4 is shown in SEQ ID NO.28;

[0019] For Nanobody Nb55: the amino acid sequence of FR1 is shown in SEQ ID NO.29, the amino acid sequence of FR2 is shown in SEQ ID NO.30, the amino acid sequence of FR3 is shown in SEQ ID NO.31, and the amino acid sequence of FR4 is shown in SEQ ID NO.32;

[0020] For Nanobody Nb78: the amino acid sequence of FR1 is shown in SEQ ID NO.33, the amino acid sequence of FR2 is shown in SEQ ID NO.34, the amino acid sequence of FR3 is shown in SEQ ID NO.35, and the amino acid sequence of FR4 is shown in SEQ ID NO.36;

[0021] For Nanobody Nb84: the amino acid sequence of FR1 is shown in SEQ ID NO.37, the amino acid sequence of FR2 is shown in SEQ ID NO.38, the amino acid sequence of FR3 is shown in SEQ ID NO.39, and the amino acid sequence of FR4 is shown in SEQ ID NO.40;

[0022] For Nanobody Nb88: the amino acid sequence of FR1 is shown in SEQ ID NO.41, the amino acid sequence of FR2 is shown in SEQ ID NO.42, the amino acid sequence of FR3 is shown in SEQ ID NO.43, and the amino acid sequence of FR4 is shown in SEQ ID NO.44;

[0023] For Nanobody Nb110: the amino acid sequence of FR1 is shown in SEQ ID NO.45, the amino acid sequence of FR2 is shown in SEQ ID NO.46, the amino acid sequence of FR3 is shown in SEQ ID NO.47, and the amino acid sequence of FR4 is shown in SEQ ID NO.48;

[0024] For Nanobody Nb115: the amino acid sequence of FR1 is shown in SEQ ID NO.49, the amino acid sequence of FR2 is shown in SEQ ID NO.50, the amino acid sequence of FR3 is shown in SEQ ID NO.51, and the amino acid sequence of FR4 is shown in SEQ ID NO.52;

[0025] For Nanobody Nb141: the amino acid sequence of FR1 is shown as SEQ ID NO.53, the amino acid sequence of FR2 is shown as SEQ ID NO.54, the amino acid sequence of FR3 is shown as SEQ ID NO.55, and the amino acid sequence of FR4 is shown as SEQ ID NO.56.

[0026] Furthermore, the amino acid sequence of the Nanobody Nb48 is shown as SEQ ID NO.57; the amino acid sequence of the Nanobody Nb55 is shown as SEQ ID NO.58; the amino acid sequence of the Nanobody Nb78 is shown as SEQ ID NO.59; the amino acid sequence of the Nanobody Nb84 is shown as SEQ ID NO.60; the amino acid sequence of the Nanobody Nb88 is shown as SEQ ID NO.61; the amino acid sequence of the Nanobody Nb110 is shown as SEQ ID NO.62; the amino acid sequence of the Nanobody Nb115 is shown as SEQ ID NO.63; and the amino acid sequence of the Nanobody Nb141 is shown as SEQ ID NO.64.

[0027] The present invention also provides an application of a shrimp tropomyosin-specific nanobody, and the application of the specific nanobody in shrimp tropomyosin immunoassay for purposes other than disease diagnosis.

[0028] The present invention also provides an application of a shrimp tropomyosin-specific nanobody, and the application of the specific nanobody in epitope identification of shrimp tropomyosin.

[0029] The present invention also provides an application of a shrimp tropomyosin-specific nanobody, and the application of the specific nanobody in the purification and tracing of shrimp tropomyosin that is not an antibody based on the purpose of disease diagnosis.

[0030] Compared with the prior art, the present invention has the following advantages:

[0031] The shrimp tropomyosin-specific nanoantibody of the present invention has a short preparation cycle, low cost, and high stability, and can realize economical, efficient, highly sensitive, and rapid detection of the allergen shrimp tropomyosin in food.

[0032] The present invention aims to utilize the characteristics of nanoantibodies to construct a system for detecting shrimp tropomyosin in food, providing a new solution for the highly sensitive and rapid detection of trace allergens in food. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Identification of shrimp tropomyosin as described in the examples of the present invention;

[0034] Figure 2 This is the screening result of the shrimp tropomyosin-specific nanobody described in the examples of the present invention;

[0035] Figure 3 This is the electrophoresis diagram of the specific nanobody protein containing HA- and His-tags against shrimp tropomyosin described in the examples of the present invention;

[0036] Figure 4 This is an immunoblot image of the specific nanobody against shrimp tropomyosin containing HA- and His-tags described in the examples of the present invention;

[0037] Figure 5 The specific nanobody binding activity assay of the shrimp tropomyosin containing a His-tag as described in the examples of the present invention;

[0038] Figure 6 This is the electrophoresis diagram of the specific nanobody protein containing a His-tag against shrimp tropomyosin described in an embodiment of the present invention;

[0039] Figure 7 This is an immunoblot image of the specific nanobody containing a His-tag against shrimp tropomyosin described in an embodiment of the present invention;

[0040] Figure 8 This is a diagram of the immunoprecipitation of shrimp tropomyosin and specific nanoantibodies described in an embodiment of the present invention;

[0041] Figure 9 Affinity determination of the shrimp tropomyosin nanobody described in the embodiments of the present invention;

[0042] Figure 10 Screening for shrimp tropomyosin-specific nanoantibodies as described in the embodiments of the present invention;

[0043] Figure 11 Optimization of the concentration of nanoantibodies for shrimp tropomyosin detection according to the embodiments of the present invention;

[0044] Figure 12 The invention relates to the construction of the shrimp tropomyosin immunoassay method described in the embodiments of the present invention. DETAILED DESCRIPTION

[0045] Unless otherwise defined, the technical terms used in the following examples have the same meanings as commonly understood by those skilled in the art to which this invention belongs. The experimental reagents used in the following examples, unless otherwise specified, are conventional biochemical reagents; the experimental methods described, unless otherwise specified, are conventional methods.

[0046] The present invention will be described in detail below with reference to the embodiments.

[0047] Example 1 Extraction of shrimp tropomyosin

[0048] Remove the shrimp heads and tails, grind the meat in PBS at low temperature to obtain a homogenate, and add an equal volume of -20°C pre-chilled acetone. Stir at 4°C for 20 minutes, then centrifuge at 3500 rpm for 15 minutes, and discard the supernatant. Collect the precipitate and mix it with the same volume of -20°C pre-chilled acetone. Repeat the centrifugation and stirring three times. The final precipitate is collected and dried to obtain shrimp fibrin powder. Prepare an extraction buffer consisting of 20 mM Tris, 1 M KCl, and 0.1 mM DTT (pH 7.4). The powder was extracted overnight with a 1:7 (w / v) extraction buffer, and the supernatant was salted out with 20% (NH4)2SO4 (pH = 7.5) by mass. After stirring for 30 minutes at 4°C, the supernatant was discarded after centrifugation at 10000 rpm for 30 minutes. The precipitate was dissolved with PBS and the pH of the solution was adjusted to 4.6. After stirring for 30 minutes at 4°C, the precipitate was centrifuged at 10000 rpm for 30 minutes. The precipitate was dissolved with PBS, dialyzed, and freeze-dried to obtain shrimp tropomyosin. Figure 1 shown.

[0049] Example 2 Alpaca immunization and construction of nanobody library based on shrimp tropomyosin

[0050] Alpacas were immunized with 0.1 mg of extracted shrimp tropomyosin at weekly intervals for a total of six immunizations. Three days after the sixth immunization, blood was collected from the alpacas' jugular vein. Lymphocytes were extracted from the blood using density gradient centrifugation. Total RNA was then extracted from the lymphocytes using TRIzol™ reagent (Invitrogen), and the precipitated RNA was isolated and precipitated. Reverse transcription was performed using the extracted RNA as a template to obtain cDNA. Nested PCR was then performed using the cDNA as a template to amplify the VHH fragment. The first round of PCR was performed using primers CALL001 and CALL002. The amplified product primarily consisted of two fragments of varying molecular weights. These fragments were separated by 1% agarose gel electrophoresis and the approximately 700 bp fragment was excised and purified using the QIAquick Gel Extraction Kit (QIAGEN). A second PCR amplification of the first-round PCR product was performed using primers PMCF and A6E. These primers contain NotI and PstI restriction enzyme sites, providing enzyme digestion and ligation sites. The amplified product was verified by 1% agarose gel electrophoresis and purified using a PCR purification kit (QIAGEN). The purified PCR product was double-digested with the pMECS plasmid vector using NotI and PstI restriction endonucleases (NEB). The digested product was purified using a DNA recovery kit (Tiangen) and ligated with T4 DNA ligase (Thermo Scientific) to form a recombinant plasmid. The recombinant plasmid was purified using phenol:chloroform:isoamyl alcohol (25:24:1) and transformed into Escherichia coli TG1 competent cells (Lucigen) using a GenePulser Xcell™ electroporator (BIO-RAD). The transformed cells were plated on ampicillin-resistant LB plates and cultured overnight at 37°C. The next day, the grown strain was harvested using a cell scraper and resuspended in LB liquid medium supplemented with 100% glycerol for long-term storage at -80°C. At the same time, 48 random colonies were selected for colony PCR, and the correct insertion rate of the VHH fragment was calculated by 1% agarose gel electrophoresis. The library capacity and diversity were calculated by serial dilution of the colony concentration and plate counting.

[0051] Example 3 Screening and identification of specific nanobodies

[0052] The purified phages were subjected to three rounds of panning. That is, by coating the antigen overnight in a 96-well plate, TG1 cells with high affinity for the antigen were selected. First, 1 ml of TG1 cells containing the nanobody library was inoculated into 300 ml of 2×TY medium (containing 100 μg / ml ampicillin and 1% glucose), and cultured at 37°C in a shaker at 220 rpm for 2 hours. When the cells reached the logarithmic growth phase, ~10 12VCSM13 assisted phage infection of TG1 cells for 30 minutes. After the infection, the cells were divided into 50 ml centrifuge tubes and centrifuged at 4 ° C to collect the bacteria. Take 1 ml of 2×TY medium (containing 100 μg / ml ampicillin and 100 μg / ml kanamycin) to resuspend the bacteria and inoculate them into 300 ml of 2×TY medium (containing 100 μg / ml ampicillin and 100 μg / ml kanamycin) and culture them overnight at 37 ° C. The next day, the supernatant was collected by centrifugation at 4 ° C and mixed with pre-cooled PEG / NaCl to precipitate the phage in the supernatant. After centrifugation, the precipitate was resuspended in 1 ml of sterile PBS and its concentration was determined to be 10 12 pfu / ml. 11 Phages were added to the wells coated with antigen overnight ("+") and the negative control wells coated with PBS overnight ("-"). After incubation at room temperature for 1 hour, the wells were washed multiple times with PBST to remove phages that did not specifically bind to the antigen. The bound phages were eluted with 100 μl triethylamine (100 mM TEA, pH 11.0), neutralized with 100 μl Tris-HCl (1.0 M, pH 7.4), and transferred to a sterile centrifuge tube. At this point, 200 μl of specific phages were obtained, of which 180 μl was used for re-expansion culture, 10 μl was used for enrichment, and 10 μl was used for backup. During enrichment, 10 μl of phages were taken and diluted tenfold from top to bottom in a 96-well plate with 90 μl PBS added to each well until 10 was reached. -7 10 μl of each phage dilution was added to 90 μl of TG1 cells in the logarithmic growth phase and infected for 30 minutes. Then, 10 μl of the corresponding TG1 cell suspension was plated onto LB agar plates and incubated overnight at 37°C. 180 μl of phage was re-cultured for the next round of panning, and this was continued until three rounds of panning were completed.

[0053] After the third round of panning, single colonies were selected from the plates obtained in each round of panning, a total of 200 colonies were selected, and they were inoculated into 2×TY medium supplemented with 10% (w / v) glycerol, 2% (w / v) glucose and 100μg / ml ampicillin, and cultured at 37°C overnight. Inoculate 1ml of 2×TY culture medium containing 0.1% (w / v) glucose and 100μg / ml ampicillin into a sterile 2ml deep-well plate (Axygen), transfer 10μl of the culture solution of each single colony into it and mark it. The deep-well plate was shaken and cultured at 37°C for about 4 hours until the OD 600When it reaches 1, add IPTG to each well at a final concentration of 1mM and continue shaking culture at 37°C to induce the expression of nanoantibodies. After induction, the bacteria are collected by centrifugation and frozen at -80°C overnight. The next day, the bacteria are taken out and resuspended in PBS to obtain periplasmic lysate by freeze-thaw method. The periplasmic lysate is used for ELISA verification to select specific strains. That is, 20μl periplasmic lysate and 80μl PBS are added to the positive wells coated with 1.5μg shrimp tropomyosin overnight and the negative wells coated with PBS, and incubated at room temperature for 1 hour. After blocking with 5% skim milk powder for 2 hours, 100μl of Mouse anti-His MAb (Invitrogen) diluted at 1:5000 as the primary antibody and 1:5000 diluted Goat anti-Mouse MAb labeled with alkaline phosphatase (Invitrogen) are incubated as the secondary antibody. After adding the colorimetric agent, the OD is measured every 15 minutes. 450 Measure 4 times in total. Select the colonies whose OD value in the positive well is at least 2 times that of the negative well (positive colonies). Extract plasmids from the positive strains and sequence them, and select specific strains with different sequences. Figure 2-4 shown.

[0054] Example 4 Expression and purification of specific nanobodies

[0055] Extract plasmids from specific strains with different sequences to transform E. coli WK6 competent cells. Add 1 μl of plasmid to 50 μl of competent cells and incubate on ice for 30 minutes. After incubation in a 42°C water bath for 45 seconds, quickly place on ice for 5 minutes to obtain competent cells that have also been transformed with the plasmid. Add 1 ml of LB liquid culture medium and culture in an incubator at 200 rpm and 37°C for 1 hour to revive the competent cells. Spread the above liquid on an LB agar plate and culture at 37°C overnight. Pick a single colony and inoculate it in 10 ml of LB liquid culture medium containing 100 μg / ml ampicillin for overnight culture. Inoculate 5 ml of bacterial liquid into 500 ml of TB culture medium containing 2.5 ml of 20% (w / v) glucose, 100 μg / ml ampicillin and 500 μl of 2M MgCl2, and culture in a shaker at 37°C and 220 rpm until OD 600 When the concentration of the cell reached about 0.6, 500 μl of IPTG with a final concentration of 1 mM was added and cultured overnight in a shaker at 28°C to induce the expression of nanobodies. The next day, the cells were collected by centrifugation and the periplasmic extract containing specific nanobodies was obtained by osmotic shock method. The periplasmic extract was mixed with HisPur TMAfter binding to Ni-NTA resin (Thermo-Scientific) for 1 hour, the antibody was passed through a PD-10 column (GE Healthcare) and washed three times with PBS to remove non-specific components. The nanobody was eluted with 500 mM imidazole. The purified nanobody was collected, further purified by size exclusion chromatography (SEC), and identified by SDS-PAGE and immunoblotting analysis. Figure 5-7 shown.

[0056] Example 5 Confirmation of specific nanoantibodies targeting allergens

[0057] Co-immunoprecipitation (Co-IP) and high-resolution liquid chromatography-mass spectrometry (LC-MS / MS) were used to confirm the target protein of the specific nano-antibody. HisPurTM Ni-NTA magnetic beads (Thermo-Scientific) were used as magnetic beads. Three groups were set up. After adding 15μl of magnetic beads to each group, both sides were washed with 300μl PBS. The washing solution and magnetic beads were separated by a magnetic stand to remove the ethanol in the magnetic beads. The beads were resuspended again with 300μl PBS. 15μg of specific nano-antibody was added to two of the groups, and the other group was added with an equal volume of PBS as the first two groups as a blank control. 100μL of PBST was added to the three groups and mixed for 1h. 10μg of shrimp tropomyosin (TM) was added to the group with specific nano-antibody as the experimental group, and the other group was the control group. The above three groups were mixed at room temperature for 2-3h. The above liquid was washed with washing buffer (PBST containing 50mM imidazole; pH 8.0) to remove non-specific components adsorbed on the magnetic beads. Wash three times, add 30μL of elution buffer (PBS containing 250mM imidazole; pH 8.0) to rinse the magnetic bead separation, and use a metal bath for 15 minutes, vortex every 3 minutes during the period, and incubate for 15 minutes by shaking. The solution is collected by a magnetic stand. The solution contains nanoantibodies and their target proteins. The nanoantibodies and target proteins are separated by SDS-PAGE, and the target protein bands are cut out and digested with trypsin. The peptides after vacuum drying are desalted using Sep-Pak@VAc 1cc (50mg) C18Cartridges and the final sample of station-controlled drying is subjected to LC-MS / MS analysis. The analysis results are compared with the TM-Pen sequence in Uniprot to confirm that the target protein of the obtained nanoantibody is shrimp tropomyosin. The results are as follows Figure 8 shown.

[0058] The affinity (K) of the nanobody was evaluated by coating the CM5 chip with shrimp tropomyosin antigen at a concentration of 10 μg / mL and then performing a series of gradient dilutions (1000, 500, 250, 125, 62.5, 31.25 and 15.625 nM) on the nanobody. The binding kinetics of the antibody and antigen were measured based on surface plasmon resonance technology.D ), the smaller the KD value, the higher the affinity of the nanobody. As shown in the figure, the selected nanobodies all have good affinity. In the double antibody sandwich detection system, the selection of antibody pairs depends on the high affinity of the antibody to target the antigen, such as Figure 9 shown.

[0059] Example 6 Pairing screening of nanobodies

[0060] The purified pHEN6c plasmid was recombined using enzyme digestion and ligation. The vector selected was pHEN6c, and the VHH fragment from pMECS was selected as the template. After double digestion with the restriction enzymes PstI-HF and BESTEII-HF, ligation was performed using T4 ligase at 16°C for 12-16 hours. After ligation, the T4 ligase was inactivated at 65°C for 15 minutes. A plasmid containing only the His-tag was obtained and purified. The plasmid was transformed into Escherichia coli WK6 and plated on solid culture medium to obtain single colonies. Successful ligation was verified by PCR amplification and agarose gel electrophoresis, with a size of approximately 700 bp considered successful. Successfully ligated E. coli WK6 cells were amplified and the above-described third round of panning procedures were repeated to induce expression of the nanobody, yielding a specific nanobody containing only the His-tag. All antibodies were then paired to screen for suitable antibody pairs for a double-antibody sandwich ELISA, using the nanobody containing only the His-tag as the capture antibody and the nanobody containing both the HA and His-tags as the detection antibodies. First, different capture antibodies were coated at 10 μg / ml overnight at 4°C. The cells were washed three times with PBST, incubated with 5% skim milk at room temperature for 2 hours, and then washed three times with PBST. The experimental group was incubated with shrimp tropomyosin at a concentration of 5 μg / ml for 1 hour at room temperature, while the control group was incubated with the same volume of PBS and washed five times with PBST. Different HA- and His-tagged nanobodies were then incubated at a concentration of 10 μg / ml for 1 hour at room temperature and washed five times with PBST. A 1:5000 diluted HA Tag Monoclonal Antibody was used as the primary antibody and incubated for 1 hour at room temperature, followed by five washes with PBST. HRP-conjugated goat anti-mouse mAb was used as the secondary antibody and incubated for 1 hour at room temperature, followed by five washes with PBST. Each well was developed with 100 μl of TMB as a substrate for 10 minutes, stopped with 50 μl of sulfuric acid, and the absorbance at 450 nm was measured. Select appropriate antibody pairs for subsequent method establishment, and the results are as follows Figure 10 shown.

[0061] Example 7 Optimization of Double Antibody Sandwich ELISA Method Conditions

[0062] Based on the captured and detected nanoantibody pairs screened, the concentration of the nanoantibody was optimized. First, different concentrations of the capture antibody screened in the previous step were coated overnight at 4°C and washed 3 times with PBST. Then, the cells were incubated with 5% skim milk at room temperature for 2 hours and washed 3 times with PBST. The experimental group was incubated with shrimp tropomyosin at a concentration of 5μg / ml for 1 hour at room temperature, and the control group was added with the same volume of PBS and washed 5 times with PBST. Then, different concentrations of the HA- and His-tagged nanoantibodies screened in the previous step were incubated at room temperature for 1 hour and washed 5 times with PBST. HA Tag Monoclonal Antibody diluted 1:5000 was used as the primary antibody for incubation at room temperature for 1 hour and washed 5 times with PBST. HRP-labeled Goat anti-Mouse Mab diluted 1:5000 was used as the secondary antibody for incubation at room temperature for 1 hour and washed 5 times with PBST. Each well was developed with 100 μl TMB as substrate for 10 min and then stopped with 50 μl sulfuric acid and the absorbance at 450 nm was measured. The concentrations of capture antibody and detection antibody were determined. The results were as follows: Figure 11 shown.

[0063] Example 8 Establishment of standard curve for double antibody sandwich ELISA method

[0064] According to the optimized conditions, a nano-antibody double-antibody sandwich ELISA detection method was constructed to determine the standard curve of shrimp tropomyosin. Shrimp tropomyosin was diluted according to a certain concentration ratio, and the logarithm of the antigen concentration was used as the horizontal axis, corresponding to OD 450 The value is plotted as the vertical axis, and the results are shown in the figure. The best linear range is selected based on the plotted curve. The detection limit (LOD) is the concentration value on the curve corresponding to the blank well average plus three times the standard deviation (SD), and the quantification limit (LOQ) is the concentration value on the curve corresponding to the blank well average plus ten times the standard deviation (SD). The results are shown in the figure. Figure 12 shown.

[0065] 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 in the scope of protection of the present invention.

Claims

1. A shrimp tropomyosin-specific nanobody, characterized in that: The specific nanobody is at least one of nanobody Nb48, nanobody Nb55, nanobody Nb78, nanobody Nb84, nanobody Nb88, nanobody Nb110, nanobody Nb115 or nanobody Nb141; The specific nanobody includes three complementary determining regions CDR1, CDR2, and CDR3; For Nanobody Nb48: the amino acid sequence of the CDR1 is shown in SEQ ID NO.1, the amino acid sequence of the CDR2 is shown in SEQ ID NO.2, and the amino acid sequence of the CDR3 is shown in SEQ ID NO.3; For Nanobody Nb55: the amino acid sequence of the CDR1 is shown in SEQ ID NO.4, the amino acid sequence of the CDR2 is shown in SEQ ID NO.5, and the amino acid sequence of the CDR3 is shown in SEQ ID NO.6; For Nanobody Nb78: the amino acid sequence of the CDR1 is shown in SEQ ID NO.7, the amino acid sequence of the CDR2 is shown in SEQ ID NO.8, and the amino acid sequence of the CDR3 is shown in SEQ ID NO.9; For Nanobody Nb84: the amino acid sequence of the CDR1 is shown in SEQ ID NO.10, the amino acid sequence of the CDR2 is shown in SEQ ID NO.11, and the amino acid sequence of the CDR3 is shown in SEQ ID NO.12; For Nanobody Nb88: the amino acid sequence of the CDR1 is shown in SEQ ID NO.13, the amino acid sequence of the CDR2 is shown in SEQ ID NO.14, and the amino acid sequence of the CDR3 is shown in SEQ ID NO.15; For Nanobody Nb110: the amino acid sequence of the CDR1 is shown in SEQ ID NO.16, the amino acid sequence of the CDR2 is shown in SEQ ID NO.17, and the amino acid sequence of the CDR3 is shown in SEQ ID NO.18; For Nanobody Nb115: the amino acid sequence of the CDR1 is shown in SEQ ID NO.19, the amino acid sequence of the CDR2 is shown in SEQ ID NO.20, and the amino acid sequence of the CDR3 is shown in SEQ ID NO.21; For Nanobody Nb141: the amino acid sequence of the CDR1 is shown in SEQ ID NO.22, the amino acid sequence of the CDR2 is shown in SEQ ID NO.23, and the amino acid sequence of the CDR3 is shown in SEQ ID NO.

24.

2. The shrimp tropomyosin-specific nanobody according to claim 1, characterized in that: The specific nanobody comprises four framework regions FR1, FR2, FR3, and FR4; For Nanobody Nb48: the amino acid sequence of FR1 is shown in SEQ ID NO.25, the amino acid sequence of FR2 is shown in SEQ ID NO.26, the amino acid sequence of FR3 is shown in SEQ ID NO.27, and the amino acid sequence of FR4 is shown in SEQ ID NO.28; For Nanobody Nb55: the amino acid sequence of FR1 is shown in SEQ ID NO.29, the amino acid sequence of FR2 is shown in SEQ ID NO.30, the amino acid sequence of FR3 is shown in SEQ ID NO.31, and the amino acid sequence of FR4 is shown in SEQ ID NO.32; For Nanobody Nb78: the amino acid sequence of FR1 is shown in SEQ ID NO.33, the amino acid sequence of FR2 is shown in SEQ ID NO.34, the amino acid sequence of FR3 is shown in SEQ ID NO.35, and the amino acid sequence of FR4 is shown in SEQ ID NO.36; For Nanobody Nb84: the amino acid sequence of FR1 is shown in SEQ ID NO.37, the amino acid sequence of FR2 is shown in SEQ ID NO.38, the amino acid sequence of FR3 is shown in SEQ ID NO.39, and the amino acid sequence of FR4 is shown in SEQ ID NO.40; For Nanobody Nb88: the amino acid sequence of FR1 is shown in SEQ ID NO.41, the amino acid sequence of FR2 is shown in SEQ ID NO.42, the amino acid sequence of FR3 is shown in SEQ ID NO.43, and the amino acid sequence of FR4 is shown in SEQ ID NO.44; For Nanobody Nb110: the amino acid sequence of FR1 is shown in SEQ ID NO.45, the amino acid sequence of FR2 is shown in SEQ ID NO.46, the amino acid sequence of FR3 is shown in SEQ ID NO.47, and the amino acid sequence of FR4 is shown in SEQ ID NO.48; For Nanobody Nb115: the amino acid sequence of FR1 is shown in SEQ ID NO.49, the amino acid sequence of FR2 is shown in SEQ ID NO.50, the amino acid sequence of FR3 is shown in SEQ ID NO.51, and the amino acid sequence of FR4 is shown in SEQ ID NO.52; For Nanobody Nb141: the amino acid sequence of FR1 is shown as SEQ ID NO.53, the amino acid sequence of FR2 is shown as SEQ ID NO.54, the amino acid sequence of FR3 is shown as SEQ ID NO.55, and the amino acid sequence of FR4 is shown as SEQ ID NO.

56.

3. The shrimp tropomyosin-specific nanobody according to claim 2, characterized in that: The amino acid sequence of the nanobody Nb48 is shown in SEQ ID NO.57; The amino acid sequence of the nanobody Nb55 is shown in SEQ ID NO.58; The amino acid sequence of the nanobody Nb78 is shown in SEQ ID NO.59; The amino acid sequence of the Nanobody Nb84 is shown in SEQ ID NO.60; The amino acid sequence of the nanobody Nb88 is shown in SEQ ID NO.61; The amino acid sequence of the nanobody Nb110 is shown in SEQ ID NO.62; The amino acid sequence of the nanobody Nb115 is shown in SEQ ID NO.63; The amino acid sequence of the nanobody Nb141 is shown in SEQ ID NO.

64.

4. The use of the shrimp tropomyosin-specific nanobody according to any one of claims 1 to 3, characterized in that: The specific nanoantibody is used in shrimp tropomyosin immunoassay for purposes other than disease diagnosis.

5. The use of the shrimp tropomyosin-specific nanobody according to any one of claims 1 to 3, characterized in that: The specific nanobody is used in the epitope identification of shrimp tropomyosin.

6. The use of the shrimp tropomyosin-specific nanobody according to any one of claims 1 to 3, characterized in that: The specific nano-antibody is used in the purification and tracing of shrimp tropomyosin for purposes other than disease diagnosis.

7. The use of the shrimp tropomyosin-specific nanobody according to any one of claims 1 to 3, characterized in that: The specific nanobody is used in shielding shrimp tropomyosin-sensitized epitopes.

8. The use of the shrimp tropomyosin-specific nanobody according to any one of claims 1 to 3, characterized in that: The application of the specific nanobody in shrimp tropomyosin binding co-crystallization.

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