A shark-derived single-domain antibody binding to polyphenol oxidase and its application

Abstract

The present invention discloses a shark-derived single-domain antibody that binds to polyphenol oxidase and its application. The nucleotide sequence of the single-domain antibody is annotated as SEQ ID NO.1, and the amino acid sequence corresponding to the single-domain antibody is recorded as SEQ ID NO.2. The shark-derived single-domain antibody is used to detect the concentration of polyphenol oxidase. The single-domain antibody preparation method is to use polyphenol oxidase as the target protein to perform biopanning on the shark-derived single-domain antibody library, and finally screen out a single-domain antibody sequence with high specificity for polyphenol oxidase. This single-domain antibody sequence is constructed into a eukaryotic expression vector, and the shark-derived single-domain antibody Fc fusion protein is expressed and purified using HEK 293F cells. The single-domain antibody prepared by the present invention exhibits good stability, high specificity and high affinity. The present invention provides a theoretical basis for the inhibition of shrimp melanization and the quality and safety monitoring of aquatic products with polyphenol oxidase as the target. The single-domain antibody can stably detect the concentration of polyphenol oxidase.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a shark-derived single-domain antibody that binds to polyphenol oxidase and an application thereof. Background Art

[0002] Melanosis is the phenomenon of black spots forming at the junction of the trunk and injured parts of dead crustaceans. Caused by a series of auto-oxidation and polymerization reactions, melanosis is a typical characteristic of crustacean quality decline. Polyphenol oxidase (PPO) is a key enzyme responsible for melanosis. PPO is a copper-containing metalloenzyme that activates molecular oxidation and catalyzes the hydroxylation of phenolic compounds. It is widely found in animals, plants, microorganisms, and fungi. PPO is associated with enzymatic browning reactions and causes melanin formation in animals. Therefore, the development of a nanobody that specifically binds to PPO provides a theoretical basis for the development of PPO-specific inhibitors, delaying melanosis in shrimp, and increasing the commercial value of shrimp. In 1993, biologists discovered heavy chain antibodies naturally lacking light chains in camel serum. These antibodies are homodimers composed of two heavy chains. After years of research, Flajinik et al. also discovered natural heavy-chain antibodies in cartilaginous fish. This type of heavy-chain antibody was named the immunoglobulin new antigen receptor (IgNAR). The antigen-binding fragment of IgNAR is called the variable domain of immunoglobulin new antigen receptor (VNAR), which is located at the end of a single heavy chain. The molecular weight of VNAR is approximately 12kDa, only one-tenth the size of a traditional monoclonal antibody. It is the smallest antigen-binding unit known to date and is also called a single-domain antibody. Because of its small molecular weight, single-domain antibodies are also called nanobodies and are the smallest known natural antibody fragments. Single-domain antibodies have high stability, a relatively simple structure, and a small size. They generally require less metabolic energy for synthesis and folding, making them easy to express in large quantities in a variety of microorganisms, such as Escherichia coli and yeast systems. This unique advantage makes them more suitable for industrial application. In addition, single-domain antibodies can quickly penetrate tissue barriers while maintaining their high specificity and affinity, which can effectively overcome the limitations of traditional antibodies, making them an effective supplement to antibody therapy. Therefore, single-domain antibodies have great development potential in the fields of drug development, disease diagnosis and treatment. Currently, there are no shark-derived single-domain antibodies that bind to polyphenol oxidase. Summary of the Invention

[0003] To address the problems presented in the prior art, the present invention provides a shark-derived single-domain antibody that binds to polyphenol oxidase and its applications. This invention provides a shark-derived heavy chain antibody variable region sequence that binds to polyphenol oxidase with high affinity. This is crucial for detecting polyphenol oxidase and inhibiting melanization in aquatic products such as shrimp, thereby improving the quality and safety of these products.

[0004] The technical solutions adopted in the present invention are as follows:

[0005] 1. A shark-derived single-domain antibody that binds to polyphenol oxidase:

[0006] The amino acid sequence of the shark-derived single-domain antibody is shown in SEQ ID NO.2.

[0007] The nucleotide sequence of the shark-derived single-domain antibody encoding gene is shown in SEQ ID NO.1.

[0008] The shark-derived single-domain antibody is used in the preparation of a polyphenol oxidase concentration detection reagent.

[0009] The application of the shark-derived single-domain antibody in the preparation of a shrimp melanosis inhibitory reagent.

[0010] 2. A method for detecting the polyphenol oxidase concentration of a shark-derived single-domain antibody, comprising the following steps:

[0011] Step S1: First, setting a standard group, a sample group, and a blank group, wherein the standard group adds polyphenol oxidase standards of different concentrations to an immunowell plate, the sample group adds a polyphenol oxidase sample to be tested to the immunowell plate, and the blank group does not add any sample to the immunowell plate;

[0012] Polyphenol oxidase standards generally refer to polyphenol oxidase kits with known concentrations.

[0013] Step S2: add shark-derived single domain antibody to the standard group, sample group and blank group, and measure the absorbance OD of the standard group and sample group after adding shark-derived single domain antibody. 450 value;

[0014] Step S3: Based on the absorbance OD of the standard group and the sample group 450 The polyphenol oxidase concentration of the sample group to be tested is obtained.

[0015] The step S2 is specifically as follows: the standard group, sample group and blank group are blocked with skim milk, and then shark-derived single domain antibodies are added to the immune well plates of the standard group, sample group and blank group. After incubation, the immune well plates are washed with PBST, and goat anti-human IgG Fc-HRP antibody is added to each well of the immune well plate. After incubation, the immune well plate is washed again with PBST, and then TMB is added to each well for color development. After color development, sulfuric acid H2SO4 is added to terminate the reaction, and the absorbance OD of the standard group, sample group and blank group is measured respectively. 450 Then, the absorbance OD values ​​of the standard group and the sample group were calculated. 450 The absorbance OD of the blank group was subtracted from the value 450 The corrected absorbance OD values ​​of the standard group and sample group were obtained. 450 value.

[0016] The step S3 is specifically as follows: according to the concentration of the polyphenol oxidase standard in the standard group and the corrected absorbance OD 450 Value, plot absorbance OD 450 The standard curve of the absorbance OD 450 The standard curve of the values ​​was used to obtain the OD value after correction of the sample group. 450 The concentration corresponding to the value is the polyphenol oxidase concentration of the sample group.

[0017] Specifically, the concentration of polyphenol oxidase standard in the standard group is used as the horizontal axis, and the corresponding corrected absorbance OD 450 The value was used as the vertical axis and linear regression analysis was performed to obtain the absorbance OD 450 The standard curve of the values.

[0018] 3. A method for preparing a shark-derived single-domain antibody that binds to polyphenol oxidase, comprising the following steps:

[0019] Step S1: Screening specific single domain antibodies;

[0020] Step S2: Recombinant expression of single domain antibodies to produce shark-derived single domain antibodies.

[0021] The step S1 is specifically as follows:

[0022] S11. First, the existing shark-derived single-domain antibody synthetic library is inoculated into a liquid culture medium. Helper phage is then added to the liquid culture medium to promote phage proliferation. After static culture and centrifugation to collect phage, the phage is shaken and cultured overnight to obtain more phage. The phage is then enriched using PEG precipitation to determine the titer of the enriched phage.

[0023] S12, dissolving polyphenol oxidase in a protein-free solution GFBE to prepare a polyphenol oxidase solution, then coating 0.1 mg / mL of the polyphenol oxidase solution in an immunoplate as a polyphenol oxidase group, and using an immunoplate not coated with the polyphenol oxidase solution as a negative control group, respectively adding phage enriched in S11 to the immunoplates of the polyphenol oxidase group and the negative control group so that the phage binds to the polyphenol oxidase in the polyphenol oxidase group, then washing the immunoplate with PBST (phosphate buffered saline), and after washing, obtaining phage detached from the polyphenol oxidase group and the negative control group, respectively;

[0024] S13. The phages from the polyphenol oxidase group and the negative control group were infected with TG1 bacteria (Escherichia coli). The phages infected with TG1 bacteria were then spread on a plate and cultured for 12 hours to obtain single colonies. The number of single colonies in the polyphenol oxidase group and the negative control group was counted and compared:

[0025] If the number of single colonies in the polyphenol oxidase group is 10 times greater than that in the negative control group, it indicates that the number of single colonies in the polyphenol oxidase group meets the requirement, and the process proceeds to S14;

[0026] Otherwise, return to S12 and prepare new phages until the number of single colonies of the polyphenol oxidase group meets the requirement;

[0027] S14, selecting a single colony from the polyphenol oxidase group and placing it in an immune well plate, adding a helper phage, and amplifying to obtain a monoclonal phage;

[0028] S15. Use polyphenol oxidase solution to coat the immunoplate where the monoclonal phage is located, then add horseradish peroxidase-labeled phage-specific antibody to the immunoplate, and then add TMB (3,3',5,5'-tetramethylbenzidine) color development solution to develop the monoclonal phage. The absorbance OD 450 The monoclonal phage of S1 was sequenced and duplicate sequences were removed using sequence alignment software. The nucleotide sequence obtained after sequencing and removal of duplicate sequences was used as the nucleotide sequence of the gene encoding the shark-derived single domain antibody. Among them, the nucleotide sequence obtained from S1 was specific for polyphenol oxidase.

[0029] The S2 is specifically:

[0030] The gene encoding the shark-derived single-domain antibody was constructed into the eukaryotic expression vector pTT5-TEV-Fc, and then expressed in HEK293F (human embryonic kidney) cells. The shark-derived single-domain antibody (i.e., a single-domain antibody-Fc fusion protein specific for polyphenol oxidase) was obtained by purification via an agarose gel rProtein A affinity chromatography column.

[0031] 4. Characterization of shark-derived single-domain antibodies includes the following steps:

[0032] S31, polyphenol oxidase antigen was coated onto the immunoplate at a concentration of 1 μg / mL, and a protein-free solution was used as the negative control group. The plates were coated overnight at 4°C. The next day, the liquid was removed, the plates were washed with phosphate buffered saline (PBS), and blocked with MPBS (phosphate buffered saline plus 5% milk). Subsequently, the expressed antibodies were diluted to multiple concentrations and added to the wells for incubation. After incubation, the wells were washed with PBST (phosphate buffered saline plus 0.1% Tween-20), incubated with goat anti-human IgG Fc-HRP antibody, and then washed again. TMB was used for color development and H2SO4 was used for termination. The OD was measured. 450 The half-maximal effective concentration (EC) of the antibody was obtained by fitting 50 value.

[0033] S32. Before the experiment, the sensor was pre-wetted with 200 μL of phosphate-buffered saline (PBST) for 10 minutes. The experimental procedure was as follows: the antigen polyphenol oxidase was first biotinylated and loaded onto the SA biosensor at 500 nM, with a fixed value of 1.0 nm. The sensor was then incubated with a serial dilution of 16-11G-Fc at room temperature for 240 seconds and allowed to dissociate for 240 seconds.

[0034] The present invention utilizes the constructed shark-derived single-domain antibody synthetic library and biopanning with polyphenol oxidase to ultimately isolate a high-affinity single-domain antibody, which is named 16-11G. The nucleotide and amino acid sequences are as follows:

[0035] Nucleotide sequence:

[0036] GCACAACGGGTTGAAAAACACCGACAACGACAACAAAGGAGGCAGGCGAATCACTGACCATCAATTGCGTCCTAAAAGGTTCCAGCTATGCATTGGGTAGCACGTACTGGTATTTCACAAAAAAGGGCGCAACAAAGAAGGCGAGCTTATCAACTGGCG GACGATACTCGGACACAAAGAATACGGCATCAAAGTCCTTTTCCTTGCGAATTAGTGACCTAAGAGTTGAAGACAGTGGTACATATCACTGTAAAGCGTATTTCCATACTCCTCATTACGCTCATGTCCCTACTTGGGAGCGGGCGGAGGAAGGAGGCGG CACCATTCTGACTGTAAAAGCA

[0037] Amino acid sequence:

[0038] AQRVEQTPTTTTKEAGESLTINCVLKGSSYALGSTYWYFTKKGATKKASLSTGGRYSDTKNTASKSFSLRISDLRVEDSGTYHCKAYFHTPHYAHVPTWERAEEGGGTILTVK

[0039] The single-domain antibody includes two complementary determining regions (CDR1 and CDR3), namely the first complementary determining region CDR1 and the second complementary determining region CDR3. The sequences of the two complementary determining regions are as follows:

[0040] CDR1: KGSSYALGSTYW

[0041] CDR3: FHTPHYAHVPTWERAE

[0042] The single-domain antibody 16-11G was fused with the human IgG1 Fc segment and cloned into the pTT5 vector. Secretory expression was performed in mammalian 293F cells. After 3 days of expression, the 16-11G-Fc fusion protein in the culture supernatant was purified using an rProtein A affinity chromatography column, and the antibody yield was found to be above 12 mg / L.

[0043] The 16-11G antibody can bind to polyphenol oxidase with high affinity. ELISA assays showed that the affinity of the 16-11G-Fc antibody disclosed herein for binding to polyphenol oxidase was 6.48 nM. Biolayer interferometry (BLI) assays showed that the equilibrium dissociation constant (K) between polyphenol oxidase and the 16-11G-Fc antibody was 2.68 nM. D ) is 2885nM.

[0044] Compared with the prior art, the present invention has the following advantages:

[0045] 1. Since the single-domain antibody of the present invention is derived from shark heavy chain antibodies, it has the characteristics of high stability, high expression level and high affinity. The shark-derived single-domain antibody can stably detect the concentration of polyphenol oxidase.

[0046] 2. The shark-derived single-domain antibody of the present invention has a small molecular weight, high solubility, easy recombinant expression, good thermal stability, strong folding ability, good tissue permeability, low aggregation tendency and strong target binding ability.

[0047] 3. The single-domain antibodies screened by the present invention exhibit good stability, high specificity and high affinity, providing a theoretical basis for the subsequent detection of polyphenol oxidase and the inhibition of melanization of aquatic products such as shrimp to improve the quality and safety of aquatic products. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 This is a graph of the monoclonal phage ELISA results;

[0049] Figure 2 This is the SDS-PAGE gel electrophoresis result of 16-11G-Fc antibody;

[0050] Figure 3 This is a graph showing the binding affinity of 16-11G-Fc antibody to polyphenol oxidase measured by enzyme-linked immunosorbent assay (ELISA);

[0051] Figure 4 This is a graph showing the affinity between a single domain antibody and polyphenol oxidase detected by BLI. DETAILED DESCRIPTION

[0052] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

[0053] Example 1. Screening and characterization of shark-derived single-domain antibodies that bind to polyphenol oxidase

[0054] 1. Screening of single-domain antibodies targeting polyphenol oxidase

[0055] Step S1: Inoculate the shark-derived single domain antibody synthesis library into 2×TY liquid culture medium containing 100 μg / mL ampicillin (Amp) and 2% glucose (G) (volume 100 mL) to make the initial OD 600 The culture was shaken at 37°C and 200 rpm until the OD 600 About 0.5~0.6(OD 600 The value refers to the absorbance value of the solution at a wavelength of 600 nm). Then 1 μL 10 13A volume of pfu of KM13 helper phage was incubated at 37°C for 45 minutes. After incubation, the culture was centrifuged for 10 minutes (37°C, 3500g), and the supernatant was removed. The pellet was resuspended in 200 mL of 2TY / Amp / kanamycin sulfate / 0.1% G medium and incubated at 25°C, 200 rpm, and shaken for 16 hours. After incubation, the entire cell suspension was centrifuged at 4°C, 3500g, for 30 minutes. The entire supernatant was added to 20% PEG / NaCl (polyethylene glycol / sodium chloride) in a 4:1 ratio. Mixed, the suspension was incubated on ice for 1 hour, and then centrifuged for 30 minutes (4°C, 3500g). The supernatant was discarded, and the pellet was resuspended in autoclaved PBS (phosphate-buffered saline). The pellet was vortexed, transferred to a centrifuge tube, and centrifuged for 10 minutes (4°C, 12,000 rpm). Transfer the supernatant to a new centrifuge tube and measure the OD 260 value.

[0056] Step S2, panning. The polyphenol oxidase antigen was coated in the immunoplate at a concentration of 0.1 mg / mL, and a protein-free solution GFBE negative control was set. After standing overnight at 4°C, the coating solution was discarded and washed three times with 280 μL of PBS. Then 280 μL of MPBS (phosphate buffer solution) was added and the reaction was blocked at room temperature for 2 hours. Subsequently, 280 μL of PBST (phosphate buffer solution) was used for two washes, and 1×10 11 The phage solution prepared above was incubated at 80 rpm for 1 h, the waste liquid was discarded, and 20 washes were performed with 280 μL of PBST. 100 μL of trypsin (0.5 mg / mL) was added to the polyphenol oxidase group Hb and negative control wells, and the phages were eluted at 80 rpm for 1 h. Then 100 μL of the eluted phage was used to infect 900 μL of OD 600 Incubate the TG1 bacterial solution at a concentration of approximately 0.5% in a 37°C water bath for 45 minutes. Spread 50 μL and 5 μL of the infected bacterial solution onto LB / Amp plates, respectively, and incubate overnight.

[0057] Step S3: Phage ELISA screening of anti-polyphenol oxidase single domain antibody monoclonals:

[0058] Step S31, Preparation of Monoclonal Phage: 95 single clones were picked from the overnight culture plate and inoculated into 100 μL of 2×TY / Amp / 2% G. Another well served as a negative control and was shaken in a 96-well culture plate for 6-8 hours (37°C, 250 rpm). Subsequently, 3 μL of bacterial solution was aspirated from each well and re-inoculated into 100 μL of 2×TY / Amp / 2% G / KM13 medium (3 μL of KM13 was added to 12.5 mL of 2×TY / Amp / 2% G). The culture was shaken again for 1.5 hours (37°C, 250 rpm) and allowed to stand at 37°C for 45 minutes. An equal amount of 30% glycerol was added to each well containing the remaining 97 μL of bacterial solution, and the mixture was stored in a -80°C freezer. After standing, the bacterial solution was thoroughly mixed and 50 mL was discarded. The solution was centrifuged at 3200 g for 20 minutes, and the supernatant was completely removed. The pellet was resuspended in 200 μL 2TY / Amp / Kana / 0.1% G medium and cultured for 20 h (25°C, 250 rpm). After the culture was completed, the plate was centrifuged for 40 min (4°C, 3200 g). The monoclonal phage supernatant was transferred to a new 96-well plate and stored at 4°C.

[0059] Step S32, monoclonal phage ELISA detection: dilute polyphenol oxidase to 10 μg / mL with GFBE, take 100 μL to coat the 96-well immunoplate, and set up protein-free solution GFBE as a negative control, and coat overnight at 4°C. Wash 3 times with PBS, add 300 μL MPBS to each well, and block at room temperature for 2 hours. Add 100 μL of the phage MPBS mixture prepared above to each well and incubate at room temperature for 1 hour. Wash the plate 4 times with PBST. Use MPBS to moderately dilute the HRP-anti M13 antibody (diluted in MPBS at a ratio of 1:8000), add 100 μL to each well of the above immunoplate, and incubate at room temperature for 1 hour. Wash the plate 4 times with PBST. Add 100 μL TMB colorimetric substrate to each well and react at room temperature for 5 minutes in the dark. Add 50 μL of 1M H2SO4 to each well to terminate the reaction and measure the OD 450 Value, the result is Figure 1 shown.

[0060] Step S33: All OD 450 Positive clones with values ​​greater than 1 were sent to the company for sequencing, and the sequencing results were analyzed and compared. Finally, a single-domain antibody sequence was screened and named as the above shark-derived single-domain antibody 16-11G.

[0061] 2. Expression and purification of single-domain antibodies and their Fc fusion proteins

[0062] Primers were designed to fuse the IFNα protein signal peptide to the N-terminus of the nanobody gene sequence to guide secretory expression, and the human IgG1 Fc to the C-terminus of the nanobody gene sequence. A TEV restriction site was introduced between them and then cloned into the mammalian expression vector pTT5. The constructed recombinant vector was transiently transfected into mammalian HEK 293F cells using PEI. After 3 days of culture, the supernatant was collected and the 16-11G-Fc fusion protein in the supernatant was purified using an rProtein A affinity chromatography column and analyzed by SDS-PAGE electrophoresis. The results are shown in Figure 5. Figure 2 As shown, highly pure 16-11G-Fc antibody was obtained by purification.

[0063] 3. Characterization of Single Domain Antibodies

[0064] Step S1: On the immunoplate, polyphenol oxidase antigen was coated at a concentration of 5 μg / mL, and protein-free solution GFBE was used as a negative control. Both were coated overnight at 4°C. The next day, the protein coating solution and the liquid in the protein-free control wells were removed, the wells were washed twice with PBS, and then blocked with MPBS for 2 hours. The purified 16-11G-Fc antibody was added at an initial concentration of 10 3 nM as the starting point, and serial dilution to 10 -4 nM, a total of 16 gradient concentrations were set. All dilution steps were performed in MPBS. After blocking, each concentration gradient antibody protein solution was added to the pre-coated wells (100 μL per well) and incubated at 80 rpm for 1 hour. Only an equal amount of MPBS was added to the control wells. To ensure data reliability, three parallel wells were set for each concentration. After incubation, the wells were washed 4 times with PBST. 100 μL of anti-IgG Fc-HRP antibody (diluted in MPBS at a ratio of 1:10000) was added to each well, incubated at 80 rpm for 1 hour, and washed again with PBST 3 times. After TMB color development and termination with 1moL / L sulfuric acid H2SO4 solution, the OD was measured. 450 The result is as follows Figure 3 As shown in Figure 2, 16-11G-Fc antibody has a good affinity for polyphenol oxidase, with a half effective concentration (EC) of 50 The value is 6.84nM.

[0065] Step S2: using biofilm interferometry (BLI) to characterize the affinity between the single domain antibody and polyphenol oxidase.

[0066] Streptavidin affinity (SA) biosensor was used in the experiment. SA biosensor provides support for kinetic and quantitative experiments based on its high sensitivity and temperature baseline. Before the experiment, the sensor needs to be pre-wetted with 200μL of phosphate buffered saline (PBST) for 10 minutes. The experimental process is as follows: the antigen polyphenol oxidase is first biotinylated and loaded into the SA biosensor at 500nM with a solidification value of 1.0nm, and then incubated with gradient diluted 16-11G-Fc at room temperature for 240s and dissociated for 240s. According to Figure 4 It can be seen that the equilibrium dissociation constant K between 16-11G-Fc and polyphenol oxidase is D The value is 2885nM. Figure 4 As shown, the interaction between 16-11G-Fc and polyphenol oxidase was determined using SA sensor, where the solid line is the real-time kinetic curve, using BLItz Pro TM The software performs fitting analysis based on the 1:1 binding model to obtain the association constant (ka), dissociation constant (kd) and calculate the equilibrium dissociation constant (K D ), the kinetic curves of different antibody concentration gradients correspond to the concentrations marked on the right from top to bottom.

[0067] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above are only specific embodiments of the present invention and are 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.

[0068] SEQ ID NO.1:

[0069] Name: Nucleotide sequence of shark-derived single-domain antibody

[0070] DNA type: other DNA

[0071] Organism source: Chiloscyllium plagiosum

[0072] GCACAACGGGTTGAACAAACACCGACAACGACAACAAAGGAGGCAGGCGAATCACTGACCATCAATTGCGTCCTAAAAGGTTCCAGCTATGCATTGGGTAGCACGTACTGGTATTTCACAAAAAAGGGCGCAACAAAGAAGGCGAGCTTATCAACTGGCGGACGATACTCGGACACAAAGAATACGGCATCAAAGTCCTTTTCCTTGCGAATTAGTGACCTAAGAGTTGAAGACAGTGGTACATATCACTGTAAAGCGTATTTCCATACTCCTCATTACGCTCATGTCCCTACTTGGGAGCGGGCGGAGGAAGGAGGCGGCACCATTCTGACTGTAAAAGCASEQ ID NO.2:

[0073] Title: Amino Acid Sequence of Shark-derived Single Domain Antibody

[0074] Sequence Type: AA

[0075] Organism Source: Chiloscyllium plagiosum

[0076] AQRVEQTPTTTTKEAGESLTINCVLKGSSYALGSTYWYFTKKGATKKAS LSTGGRYSDTKNTASKSFSLRISDLRVEDSGTYHCKAYFHTPHYAHVPTWER AEEGGGTILTVK。

Claims

1. A shark-derived single-domain antibody that binds to polyphenol oxidase, characterized in that: The amino acid sequence of the shark-derived single-domain antibody is shown in SEQ ID NO.

2.

2. The shark-derived single-domain antibody that binds to polyphenol oxidase according to claim 1, characterized in that: The nucleotide sequence of the shark-derived single-domain antibody encoding gene is shown in SEQ ID NO.

1.

3. The use of a shark-derived single domain antibody that binds to polyphenol oxidase according to any one of claims 1 to 2, characterized in that: The shark-derived single-domain antibody is used in the preparation of a polyphenol oxidase concentration detection reagent.

4. A method for detecting polyphenol oxidase concentration using a shark-derived single domain antibody according to any one of claims 1 to 2, characterized in that: The following steps are involved: Step S1: First, setting a standard group, a sample group, and a blank group, wherein the standard group adds polyphenol oxidase standards of different concentrations to an immunowell plate, the sample group adds a polyphenol oxidase sample to be tested to the immunowell plate, and the blank group does not add any sample to the immunowell plate; Step S2: add shark-derived single domain antibody to the standard group, sample group and blank group, and measure the absorbance OD of the standard group and sample group after adding shark-derived single domain antibody. 450 value; Step S3: Based on the absorbance OD of the standard group and the sample group 450 The polyphenol oxidase concentration of the sample group to be tested is obtained.

5. The method for detecting polyphenol oxidase concentration of a shark-derived single-domain antibody according to claim 4, characterized in that: The step S2 is specifically as follows: the standard group, sample group and blank group are blocked with skim milk, and then shark-derived single domain antibodies are added to the immune well plates of the standard group, sample group and blank group. After incubation, the immune well plates are washed with PBST, and goat anti-human IgG Fc-HRP antibody is added to each well of the immune well plate. After incubation, the immune well plate is washed again with PBST, and then TMB is added to each well for color development. After color development, sulfuric acid H2SO4 is added to terminate the reaction, and the absorbance OD of the standard group, sample group and blank group is measured respectively. 450 Then, the absorbance OD values ​​of the standard group and the sample group were calculated. 450 The absorbance OD of the blank group was subtracted from the value 450 The corrected absorbance OD values ​​of the standard group and sample group were obtained. 450 value.

6. The method for detecting polyphenol oxidase concentration of a shark-derived single-domain antibody according to claim 4, characterized in that: The step S3 is specifically as follows: according to the concentration of the polyphenol oxidase standard in the standard group and the corrected absorbance OD 450 Value, plot absorbance OD 450 The standard curve of the absorbance OD 450 The standard curve of the values ​​was used to obtain the OD value after correction of the sample group. 450 The concentration corresponding to the value is the polyphenol oxidase concentration of the sample group.

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