Shark source single-domain antibody combined with polyphenol oxidase and application of shark source single-domain antibody

Abstract

The invention discloses a shark source single-domain antibody combined with polyphenol oxidase and application of the shark source single-domain antibody. The nucleotide sequence of the single-domain antibody is labeled as SEQ ID NO.1, the amino acid sequence corresponding to the single-domain antibody is recorded as SEQ ID NO.2, the shark-source single-domain antibody is used for detecting the concentration of polyphenol oxidase, and the preparation method of the single-domain antibody comprises the following steps: carrying out biological elutriation on a shark-source single-domain antibody library by taking polyphenol oxidase as target protein; and finally, screening to obtain a single-domain antibody sequence with high specificity to polyphenol oxidase, constructing the single-domain antibody sequence into an eukaryotic expression vector, and expressing and purifying shark source single-domain antibody Fc fusion protein by using HEK 293F cells. The prepared single-domain antibody shows good stability, high specificity and high affinity, a theoretical basis is provided for prawn blackening inhibition with polyphenol oxidase as a target spot and aquatic product quality safety monitoring, and 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 specifically relates to a shark-derived single-domain antibody combined with polyphenol oxidase and an application thereof. Background Art

[0002] Melanosis is the phenomenon of black spots at the junction of the trunk and injured parts of crustaceans after death. It is caused by a series of auto-oxidation and polymerization reactions. Melanosis is a typical feature of the decline in the quality of crustaceans. Polyphenol oxidase (PPO) is the key enzyme that causes melanosis. Polyphenol oxidase is a metalloenzyme containing copper ions that can activate molecular oxidation and catalyze the hydroxylation of phenolic compounds. It is widely present in animals, plants, microorganisms and fungi. PPO is related to enzymatic browning reactions and causes the formation of melanin in animals. Therefore, the development of a nano-antibody that specifically binds to polyphenol oxidase provides a theoretical reference for the development of PPO-specific inhibitors, delaying the melanosis of shrimp, and improving the commercial value of shrimp. In 1993, biologists discovered heavy chain antibodies that naturally lack light chains from camel serum, which are homodimers composed of two heavy chains. After years of research, Flajinik et al. also found natural heavy chain antibodies in cartilaginous fish. Such heavy chain antibodies were named 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 about 12kDa, which is only one-tenth the size of traditional monoclonal antibodies. It is the smallest antigen binding unit known so far and is also called a single domain antibody. Because of its small molecular weight, single domain antibodies are also called nanobodies, which are the smallest natural antibody fragments known so far. Single domain antibodies have high stability, simple structure, and small size. They usually require less metabolic energy to synthesize and fold, so they are easy to express in large quantities in a variety of microorganisms, such as Escherichia coli, yeast and other systems. This unique advantage makes it easier for industrial applications. In addition, single domain antibodies can quickly penetrate tissue barriers and maintain 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 is no shark-derived single domain antibody that binds to polyphenol oxidase. Summary of the invention

[0003] In order to solve the problems existing in the background technology, the purpose of the present invention is to provide a shark-derived single domain antibody that binds to polyphenol oxidase and its application. The present invention provides a shark-derived heavy chain antibody variable region sequence that can bind to polyphenol oxidase with high affinity, which is very important for the detection of polyphenol oxidase and inhibiting the blackening of aquatic products such as shrimp to improve the quality and safety of aquatic products.

[0004] The technical solution adopted by the present invention is 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 shark-derived single-domain antibody is used in the preparation of a shrimp melanosis inhibition 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 into the immunoplate, the sample group adds the polyphenol oxidase sample to be tested into the immunoplate, and the blank group does not add any sample into the immunoplate;

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

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

[0014] Step S3: according to 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, the sample group and the blank group are blocked with skim milk, and then the shark-derived single domain antibody is added to the immune well plates of the standard group, the sample group and the 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, the sample group and the blank group is measured respectively. 450 Then, the absorbance OD of the standard group and the sample group were 450 The absorbance OD of the blank group was subtracted from the value 450 The corrected absorbance OD of the standard group and sample group was 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 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 the polyphenol oxidase standard in the standard group is used as the horizontal axis, and the corresponding corrected absorbance OD 450 The absorbance OD was obtained by linear regression analysis. 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 obtain 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, and then helper phage is added to the liquid culture medium to promote the proliferation of phage. After static culture and centrifugal collection of phage, the phage is shaken and cultured overnight to obtain more phage, and then the phage is enriched by PEG precipitation method to obtain 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 S11-enriched phages to the immunoplates of the polyphenol oxidase group and the negative control group so that the phages bind to the polyphenol oxidase in the polyphenol oxidase group, and then washing the immunoplate with PBST (phosphate buffer), and after washing, obtaining the detached phages in the polyphenol oxidase group and the negative control group;

[0024] S13. The phages of the polyphenol oxidase group and the negative control group were used to infect TG1 bacteria (Escherichia coli), and then the phages infected with TG1 bacteria were spread on a plate. After culturing for 12 hours, single colonies were obtained. The number of single colonies of 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 greater than 10 times that of the negative control group, it means that the number of single colonies in the polyphenol oxidase group meets the requirement, and the process goes 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 in 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) colorimetric solution to develop the monoclonal phage. Measure the absorbance OD 450 The monoclonal phage with a size of >1 is sequenced and the repeated sequences are removed using sequence alignment software, and the nucleotide sequence obtained after sequencing and removing the repeated sequences is used as the nucleotide sequence of the coding gene of the shark-derived single domain antibody. Among them, the nucleotide sequence obtained from S1 has polyphenol oxidase specificity.

[0029] The S2 is specifically:

[0030] The coding gene of 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) cells. The shark-derived single-domain antibody (i.e., a single-domain antibody-Fc fusion protein specific for polyphenol oxidase) was obtained by purification through 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 on the immunoplate at a concentration of 1 μg / mL. The negative control group was a protein-free solution. The plate was coated overnight at 4°C. The next day, the liquid was removed, the plate was washed with phosphate buffer PBS, and blocked with MPBS (phosphate buffer plus 5% milk). Subsequently, the expressed antibody was diluted to multiple concentrations and added to the wells for incubation. After incubation, the wells were washed with PBST (phosphate buffer plus 0.1% Tween-20), incubated with goat anti-human IgG Fc-HRP antibody, washed again, developed with TMB, terminated with sulfuric acid H2SO4, and the OD was measured. 450 The half 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 min. The experimental process was as follows: the antigen polyphenol oxidase was first biotinylated and loaded into the SA biosensor at 500 nM with a solidification value of 1.0 nm, and then incubated with gradient dilutions of 16-11G-Fc at room temperature for 240 s and dissociated for 240 s.

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

[0035] Nucleotide sequence:

[0036] GCACAACGGGTTGAAAAACACCGACAACGACAACAAAGGAGGCAGGCGAATCACTGACCATCAATTGCGTCCTAAAAGGTTCCAGCTATGCATTGGGTAGCACGTACTGGTATTTCACAAAAAAGGGCGCAACAAAGAAGGCGAGCTTATCAACTGGCG GACGATACTCGGACACAAAGAATACGGCATCAAAGTCCTTTTCCTTGCGAATTAGTGACCTAAGAGTTGAAGACAGTGGTACATATCACTGTAAAGCGTATTTCCATACTCCTCATTACGCTCATGTCCCTACTTGGGAGCGGGCGGAGGAAGGAGGCGG CACCATTCTGACTGTAAAAGCA

[0037] Amino Acid Sequence:

[0038] AQRVEQTPTTTTKEAGESLTINCVLKGSSYALGSTYWYFTKKGATKKASLSTGGRYSDTKNTASKSFSLRISDLRVEDSGTYHCKAYFHTPHYAHVPTWERAEEGGGTILTVK

[0039] The single domain antibody comprises two complementary antigen determining regions (CDR1 and CDR3), namely, a first complementary antigen determining region CDR1 and a second complementary antigen determining region CDR3. The sequences of the two complementary antigen 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. The mammalian cell 293F was used for secretory expression. After 3 days of expression, the 16-11G-Fc fusion protein in the culture supernatant was purified using an rProtein A affinity chromatography column. The antibody yield was found to be above 12 mg / L.

[0043] The 16-11G antibody can bind to polyphenol oxidase with high affinity. The ELISA test showed that the affinity of the 16-11G-Fc antibody of the present disclosure for binding to polyphenol oxidase was 6.48 nM. The biolayer interferometry (BLI) test showed that the equilibrium dissociation constant (K) of polyphenol oxidase and the 16-11G-Fc antibody was 2.64 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 targeted binding ability.

[0047] 3. The single-domain antibodies screened by the present invention exhibit good stability, high specificity and high affinity, which provide a theoretical basis for the subsequent detection of polyphenol oxidase and the inhibition of blackening 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 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 the binding affinity diagram of 16-11G-Fc antibody and polyphenol oxidase measured by ELISA;

[0051] Figure 4 This is a graph of the affinity between a single domain antibody and polyphenol oxidase detected by BLI using antibody affinity determination. 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 synthetic 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 of the solution at 600nm wavelength). Then add 1μL 10 13pfu of KM13 helper phage, and let it stand at 37°C for 45 minutes. After the standing, centrifuge the bacterial solution for 10 minutes (37°C, 3500g), and remove the supernatant. Resuspend the precipitated bacteria in 200mL of 2TY / Amp / Kanamycin sulfate (Kana) / 0.1% G medium, and shake and culture at 25°C and 200rpm for 16 hours. After the end, centrifuge all the bacterial solution at 4°C and 3500g for 30 minutes. Take all the supernatant, add 20% PEG / NaCl (polyethylene glycol / sodium chloride) in a ratio of 4:1, mix well, let it stand in an ice bath for 1 hour, and centrifuge for 30 minutes (4°C, 3500g). Discard the supernatant, resuspend the precipitate with PBS (phosphate buffered saline) sterilized by high temperature and high pressure, vortex the precipitate and put it into a centrifuge tube, and centrifuge it for 10 minutes (4°C, 12000rpm). Transfer the supernatant to a new centrifuge tube and measure the OD 260 value.

[0056] Step S2, panning. Coat the polyphenol oxidase antigen at a concentration of 0.1 mg / mL in the immunowell plate, and set up a negative control of protein-free solution GFBE. After standing overnight at 4°C, discard the coating solution and wash three times with 280 μL of PBS. Then add 280 μL of MPBS (phosphate buffer solution) and block the reaction at room temperature for 2 hours. Then wash twice with 280 μL of PBST (phosphate buffer), add 1×10 11 pfu The phage solution prepared above was incubated at 80 rpm for 1 h, the waste liquid was discarded, and 280 μL of PBST was used for 20 washes. 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 taken to infect 900 μL OD 600 About 0.5% TG1 bacterial solution was incubated in a 37°C water bath for 45 min. 50 μL and 5 μL of the infected bacterial solution were spread on LB / Amp plates respectively and cultured overnight.

[0057] Step S3, screening of single domain antibody monoclonal against polyphenol oxidase by phage ELISA:

[0058] Step S31, preparation of monoclonal phage: 95 monoclones were picked from the overnight culture plate and inoculated into 100 μL 2×TY / Amp / 2% G respectively. Another well was used as a negative control and cultured in a 96-well culture plate with shaking for 6-8 hours (37°C, 250 rpm). Subsequently, 3 μL of bacterial solution was taken from each well and re-inoculated into 100 μL 2×TY / Amp / 2% G / KM13 culture medium (3 μL KM13 was added to 12.5 mL 2×TY / Amp / 2% G), and cultured again with shaking for 1.5 hours (37°C, 250 rpm), and then allowed to stand at 37°C for 45 minutes. An equal amount of 30% glycerol was added to the remaining 97 μL bacterial solution wells, and the mixed solution was stored in a -80°C refrigerator. After standing, the bacterial solution was thoroughly mixed and 50 mL was discarded, centrifuged at 3200g 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, the plate was centrifuged for 40 min (4°C, 3200 g), and 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 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 OD 450 Value, the result is Figure 1 shown.

[0060] Step S33: All OD 450 The 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 out 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-terminal of the gene sequence of the nanobody to guide secretory expression, and the human IgG1 Fc was fused to the C-terminal of the gene sequence of the nanobody. 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 cells HEK 293F with 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 2. Figure 2 As shown, highly pure 16-11G-Fc antibody was purified.

[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, all at 4°C overnight. The next day, the protein coating solution and the liquid in the protein-free control wells were removed, and the wells were washed twice with PBS and 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, OD was measured. 450 The result is Figure 3 As shown in Figure 2, 16-11G-Fc antibody has a good affinity for polyphenol oxidase, with a half effective concentration (EC 50 The value is 6.84nM.

[0065] Step S2: using biomembrane interference technology (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 of 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 measured using SA sensor, where the solid line is the real-time monitoring 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 description is only a specific 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.

[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 single-domain antibody from shark

[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. A 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. 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 shrimp melanosis inhibition reagent.

5. A method for detecting polyphenol oxidase concentration of 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 into the immunoplate, the sample group adds the polyphenol oxidase sample to be tested into the immunoplate, and the blank group does not add any sample into the immunoplate; Step S2: adding shark-derived single domain antibodies to the standard group, sample group and blank group, and measuring the absorbance OD of the standard group and sample group after adding shark-derived single domain antibodies 450 value; Step S3: according to 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.

6. The method for detecting polyphenol oxidase concentration of shark-derived single domain antibodies according to claim 5, characterized in that: The step S2 is specifically as follows: the standard group, the sample group and the blank group are blocked with skim milk, and then the shark-derived single domain antibody is added to the immune well plates of the standard group, the sample group and the 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, the sample group and the blank group is measured respectively. 450 Then, the absorbance OD of the standard group and the sample group were 450 The absorbance OD of the blank group was subtracted from the value 450 The corrected absorbance OD of the standard group and sample group was obtained. 450 value.

7. The method for detecting polyphenol oxidase concentration of shark-derived single domain antibodies according to claim 5, 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 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.

Images