Nanometer antibody combined with polyphenol oxidase and application thereof

Abstract

The invention discloses a nano antibody combined with polyphenol oxidase and application of the nano antibody. The nucleotide sequence of the nano antibody is as shown in SEQ ID NO.1, and the amino acid sequence corresponding to the nano antibody is as shown in SEQ ID NO.2. The nano antibody prepared by the invention has good stability, high specificity and high affinity, and can accurately inhibit the activity of polyphenol oxidase, thereby fundamentally preventing enzymatic browning; the prepared nano antibody is mainly used as a natural biological preservative and a processing aid, can be widely applied to quality maintenance of aquatic products and various easily browning foods, effectively blocks the enzymatic browning process by specifically inhibiting the activity of polyphenol oxidase, remarkably prolongs the shelf life and maintains the sensory quality of the product.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a nanobody bound to polyphenol oxidase and its application. Background Technology

[0002] Shrimp products are a major pillar of the global seafood market due to their rich nutrition and unique flavor. However, shrimp are highly susceptible to melanosis during harvesting, transportation, storage, and processing, primarily manifesting as persistent black or brown spots on the cephalothorax, arthropods, and tail. This phenomenon not only severely damages the product's appearance and reduces consumer acceptance but is also often mistaken for spoilage, leading to significant economic losses and resource waste. The essence of shrimp melanosis is enzymatic browning, with polyphenol oxidase as the core catalyst.

[0003] Polyphenol oxidase (PPO) exists primarily as prophenol oxidase (proPO) in the hemolymph of crustaceans and is a key component of the organism's innate immune system. When the organism is injured or stimulated by external factors, the proPO system is activated. PPO catalyzes the oxidation of substrates such as tyrosine to produce quinones, which then polymerize into melanin to encapsulate and eliminate pathogens. However, this defense mechanism becomes a major driver of quality deterioration in post-mortem shrimp. Therefore, effectively inhibiting PPO activity is a core strategy for controlling melanosis in shrimp and ensuring their commercial value.

[0004] Against this backdrop, the technology of precisely targeting and neutralizing PPO activity using highly specific biomolecules can meet the high-efficiency, safe, and environmentally friendly requirements of the aquatic product processing industry, and has enormous application potential. Antibody molecules, due to their excellent specificity and affinity, are considered ideal "molecular seals." However, traditional IgG antibodies have large molecular weights, limited stability, and high preparation costs, which restricts their application in complex food systems. The emergence of nanobodies (VHH) provides a revolutionary solution to this problem. As the smallest natural antigen-binding fragment composed solely of the heavy chain variable region, VHH possesses advantages such as small molecular weight (around 15 kDa), strong permeability, extremely high stability (resistant to high temperatures, acids and alkalis, and easily refoldable), and ease of genetic engineering modification. These characteristics allow them to more flexibly penetrate deep into tissues, approaching the active pockets or allosteric sites of PPO, achieving efficient and specific inhibition without chemical residue. Summary of the Invention

[0005] This study aims to develop specific nanobodies targeting shrimp polyphenol oxidase (PPO). An immune camel-derived VHH library was constructed and screened using phage display technology to obtain nanobodies capable of efficiently binding to and inhibiting PPO activity. This research lays a solid theoretical foundation for developing a new generation of biotechnology-based shrimp anti-melanosis preservatives and has significant scientific and application value for promoting the safer, higher-quality, and more sustainable development of the aquatic product processing industry.

[0006] The technical solution adopted in this invention is as follows: I. A nanobody bound to polyphenol oxidase The amino acid sequence of the nanobody is shown in SEQ ID NO.2. The nucleotide sequence corresponding to the encoding gene of the nanobody is shown in SEQ ID NO.1.

[0007] II. A method for preparing nanobodies conjugated with polyphenol oxidase, characterized in that, The method for preparing the nanobody bound to polyphenol oxidase includes: Step S1: After extracting total RNA from shrimp hemolymph, reverse transcription and cloning were performed sequentially to construct a recombinant expression plasmid containing the polyphenol oxidase gene. The recombinant expression plasmid was induced to express and purified to obtain recombinant polyphenol oxidase protein. The recombinant polyphenol oxidase protein was emulsified with different adjuvants and then used to immunize Bactrian camels multiple times to obtain cDNA from peripheral blood of Bactrian camels that had undergone the fourth immunization with polyphenol oxidase.

[0008] Step S2: Using cDNA from peripheral blood of Bactrian camels immunized with polyphenol oxidase for the fourth time as a template, specific amplification was performed to obtain nanobody nucleic acid sequences, and phage antibody immune libraries were constructed using the nanobody nucleic acid sequences.

[0009] Step S3: The PPO-5C sequence with polyphenol oxidase specificity is obtained by screening the phage antibody immune library, i.e., screening specific nanobodies.

[0010] Step S4: The PPO-5C sequence is recombinantly expressed to obtain polyphenol oxidase nanobody, i.e. eukaryotic expression camel-derived nanobody.

[0011] In step S1, more specifically, total shrimp hemolymph RNA is extracted from shrimp hemolymph and reverse transcribed into shrimp hemolymph cDNA. The shrimp hemolymph cDNA is then cloned to obtain the polyphenol oxidase gene. A recombinant expression plasmid containing the polyphenol oxidase gene is constructed using this gene. The polyphenol oxidase recombinant expression plasmid is then transformed into *E. coli*. BL In 21 cells, the target protein was expressed by IPTG and the recombinant polyphenol oxidase was obtained by purification using a Ni-affinity chromatography column.

[0012] Recombinant polyphenol oxidase was mixed with Freund's complete adjuvant (volumes equal), and thoroughly emulsified to obtain a mixed emulsion. This emulsion was then administered subcutaneously to Bactrian camels for primary immunization. Following primary immunization, booster immunizations were administered monthly by subcutaneous injection of an emulsion formed from a mixture of recombinant polyphenol oxidase protein and Freund's incomplete adjuvant (volumes equal). On day 15 after the fourth immunization, venous blood was collected from Bactrian camels, and total RNA was extracted and reverse transcribed into cDNA.

[0013] In step S1, primers 1-F and 1-R are used for polyphenol oxidase gene sequence amplification. The forward F sequence of primer 1 is shown in SEQ ID NO.3, and its reverse R sequence is shown in SEQ ID NO.4: 5'-GCTCCGTCGACAAGCTTGCGGCCGCATGGACAAGAGTCGGAAGAA- 3', SEQ ID NO.3; 5'-GGTGGTGGTGCTCGAGTGCGGCCGCTCAGTCTCGGTTCAGCCTCTC- 3', SEQ ID NO. 4.

[0014] In step S2, primers 2-F and 2-R are used for amplifying the nanobody nucleic acid sequence. The forward F sequence of primer 2-R is shown in SEQ ID NO.5, and its reverse R sequence is shown in SEQ ID NO.6. 5'-GCTGCACAGCCTGCTATGGCACAGKTGCAGCTCGTGGAGTCTGGGGG-3'; SEQ ID NO.5; 5'-GAGTTTTTGTTCGGCTGCTGCTGAGGAGACGGTGACCTGGGTCCCC-3'; SEQ ID NO. 6.

[0015] In step S2, primers 3-F and 3-R are used for pR2 phage amplification. The forward F sequence of primer 3-R is shown in SEQ ID NO.7, and its reverse R sequence is shown in SEQ ID NO.8. 5'-AGCAGCCGAACAAAAACTCATCTCAGAAGAG-3'; SEQ ID NO.7; 5'-CCATAGCAGGCTGTGCAGCATAGAAAGGTACCACTAAAGGAATTGC-3'; SEQ ID NO. 8.

[0016] Step S2 specifically involves: S21: The specific amplification in step S2 is as follows: the VHH fragment is obtained by amplifying the cDNA of peripheral blood from Bactrian camels using a high-fidelity enzyme; at the same time, the pR2 phage is amplified using a high-fidelity enzyme using pR2 phage as a template, and the amplified pR2 phage is digested with the restriction enzyme Dpn I. The VHH fragment is then ligated to the digested and amplified pR2 phage through seamless cloning to obtain recombinant pR2 phage.

[0017] S22: In step S2, the specific steps for constructing the phage antibody immune library using the nanobody nucleic acid sequence are: activating TG1 Escherichia coli to OD200. 600 Activated TG1 Escherichia coli were obtained at a concentration of approximately 0.5. The activated TG1 Escherichia coli were then subjected to ice bath and repeated resuspending in 10% glycerol and centrifugation to prepare TG1 competent bacteria. Recombinant pR2 phage particles were electroporated into TG1 competent bacteria to obtain bacteria carrying phage-camel-derived immune antibodies. The bacteria carrying phage-camel-derived immune antibodies were diluted and coated onto bacterial scraping plates to obtain bacterial scraping plates. The bacterial scraping plates were then cryopreserved to obtain a phage-camel-derived immune antibody library.

[0018] In practice, the size of the phage antibody library can be counted. If the library size is less than a preset threshold, the phage antibody library is discarded and a new antibody library is prepared.

[0019] In step S3, the screening using the phage antibody immune library specifically involves: S31: First, the phage nanobody immunotherapy library was activated, and helper phages were added to promote phage proliferation, resulting in a phage sample. After static incubation, the phage sample was centrifuged to collect phage particles, and then incubated overnight with shaking to achieve phage amplification, yielding an amplified phage sample. The phages in the amplified sample were collected using PEG precipitation to obtain an enriched phage solution, and the phage titer in the enriched solution was determined.

[0020] S32: Prepare a 50 μg / mL polyphenol oxidase solution and coat it into immunoassay plates as the polyphenol oxidase experimental group. Use uncoated polyphenol oxidase plates as the negative control group. Add the S31 enriched phage solution to both groups of plates, and then wash both groups of plates with phosphate buffer containing Tween-20. After multiple washes, obtain washed polyphenol oxidase experimental plates and washed negative control plates. Add trypsin to the washed polyphenol oxidase experimental plates and washed negative control plates, respectively, to elute and obtain phages that specifically bind polyphenol oxidase and control phages.

[0021] S33: TG1 bacteria were infected with phages specifically binding to polyphenol oxidase and control phages, respectively. The two phage groups were then diluted and plated, and cultured overnight to obtain single colonies in both the polyphenol oxidase group and the negative control group. The number of single colonies in both groups was counted. If the number of single colonies in the polyphenol oxidase group was less than 10 times that in the control group, the process returned to S32 to reduce the polyphenol oxidase concentration in the immunopores and new phages were prepared until the number of single colonies in the polyphenol oxidase group met the requirements.

[0022] S34: Select single colonies of the polyphenol oxidase group into a 96-well cell culture plate, add helper phages, and amplify to obtain monoclonal phages.

[0023] S35: 0.5 μg / mL polyphenol oxidase solution and protein-free solution were coated onto 96 immunoassay plates, serving as the second polyphenol oxidase experimental group and control group, respectively. Monoclonal phages were added to both groups to bind to proteins within the immunoassay plate. Horseradish peroxidase-labeled phage-specific antibodies were then added to each group to obtain the treated second polyphenol oxidase experimental group and the treated control group. The phage-specific antibodies in the treated second polyphenol oxidase experimental group and the treated control group were then developed using 3,3',5,5'-tetramethylbenzidine chromogenic solution. The absorbance of the solutions was measured after development. The absorbance OD of the treated polyphenol oxidase group was then calculated. 450 The absorbance OD of the treated control group 450 Monoclonal phages with a ratio greater than 20 were considered positive phages. Sequencing was performed using primer 5'-CAGGAAACAGCTATGAC-3', and repetitive sequences were removed. Sequencing and identification confirmed that the nucleotide sequence encoded a nanobody targeting polyphenol oxidase.

[0024] Step S4 specifically involves: The PPO-5C sequence obtained in step S3 was constructed into the mammalian expression vector pTT5-TEV-Fc. The mammalian expression vector pTT5-TEV-Fc carrying the nanobody sequence was then transfected into HEK 293 cells. The nanobody fused with human IgG1 Fc for expression. The nanobody Fc fusion protein was then purified by agarose gel rProtein A affinity chromatography to obtain the polyphenol oxidase nanobody.

[0025] The nanobody was then characterized, including the following steps: a) Coat 1 μg / mL polyphenol oxidase solution onto a 96 immunoassay plate overnight. A protein-free solution is used as the negative control. After washing with phosphate buffer, the plate is blocked with skim milk. The fusion protein is then serially diluted and added to the immunoassay plate for incubation. The plate is then washed multiple times with phosphate buffer containing 0.1% Tween-20, and HRP anti-IgG1 Fc secondary antibody is added. The plate is then developed with 3,3',5,5'-tetramethylbenzidine chromogenic solution. The chromogenic reaction is terminated with acidic solution, and the absorbance (OD) of the solution is measured. 450 The half-effect concentration EC was fitted using graphing software. 50 Value, EC 50 It is a key indicator for evaluating the sensitivity of enzyme-linked immunosorbent assay (ELISA).

[0026] b) First, polyphenol oxidase was biotinylated. The streptavidin (SA) biosensor was pre-wetted with 0.02% Tween-20 phosphate-buffered saline (PBST) for 10 min. When the biotinylated polyphenol oxidase antigen was immersed in the SA sensor at a concentration of 250 nM, the SA captured the antigen, causing a signal increase. Unbound antigen was washed away with PBST buffer to stabilize the signal. Then, single-domain fusion antibodies of different concentrations were immersed in the sensor. The antigen and nanobody bound, causing a signal increase. Finally, the SA sensor was immersed in PBST to dissociate the antibody, resulting in a signal decrease. The signal changes at each step constitute a complete binding-dissociation sensing map. From the sensing map, three key kinetic parameters can be extracted by the software: the association rate constant (ka), the dissociation rate constant (kd), and the affinity constant (K). D ), K D The smaller the value, the higher the affinity.

[0027] The phage antibody immune library in step S2 includes the nucleic acid sequence SEQ ID NO.1 as described in claim 2.

[0028] III. Applications of Nanobodies Conjugated with Polyphenol Oxidase Application in products for detecting the effect / activity of polyphenol oxidase inhibition. The product for detecting the effect / activity of polyphenol oxidase inhibition is one or more combinations of a polyphenol oxidase neutralizing antibody inhibition effect assay kit and a polyphenol oxidase activity inhibition assay reagent.

[0029] Applications in the preparation of natural biological preservatives.

[0030] IV. A kit for detecting the inhibitory effect of polyphenol oxidase neutralizing antibody This includes the aforementioned nanobodies that are bound to polyphenol oxidase.

[0031] This invention discloses a nanobody conjugated with polyphenol oxidase and its applications. The nucleotide sequence of the nanobody is labeled SEQ ID NO.1, and the corresponding amino acid sequence is recorded as SEQ ID NO.2. This invention successfully obtained a high-affinity nanobody that efficiently targets shrimp polyphenol oxidase by immunizing camels and constructing a nanobody phage display library, followed by multiple rounds of biological screening. This nanobody exhibits good stability, high specificity, and high affinity, and can precisely inhibit polyphenol oxidase activity, thereby fundamentally preventing enzymatic browning. This product is mainly used as a natural biological preservative and processing aid, and can be widely applied to the quality maintenance of aquatic products and various browning-prone foods. By specifically inhibiting polyphenol oxidase activity, it effectively blocks the enzymatic browning process, significantly extends shelf life, and maintains the sensory quality of the product.

[0032] This invention utilizes cDNA prepared from camel peripheral blood that has undergone multiple polyphenol oxidase immunizations as a template to amplify the VHH fragment, which is then ligated with phage particles to construct a camel-derived immune library. A specific antibody sequence against polyphenol oxidase, named PPO-5C, is obtained through phage display technology.

[0033] A pTT5-TEV-Fc eukaryotic recombinant plasmid containing the PPO-5C nanobody sequence was successfully constructed through seamless cloning. After secretory expression in HEK 293F cells and purification using rProtein A affinity chromatography, a PPO-5C-Fc fusion protein yield of over 1.78 mg was obtained. Enzyme-linked immunosorbent assay (ELISA) and biomembrane interference assays showed that the PPO-5C-Fc fusion antibody had a half-maximal effective concentration (EC50) of 0.09 nM and an affinity constant of 1.49 nM, successfully yielding a highly sensitive and high-affinity nanobody targeting polyphenol oxidase.

[0034] The beneficial effects of this invention are: 1. The camel-derived nanobody of the present invention has an extremely small molecular weight, only about 15 kDa, and a simple structure consisting of a single monomeric domain. This characteristic gives it excellent penetration and diffusion capabilities in complex food matrices (such as shrimp shells and shrimp meat tissues), enabling it to efficiently reach the microscopic region where polyphenol oxidase (PPO) is located, thereby achieving efficient and precise inhibition of enzymatic browning reactions from the source and improving the blackening control effect.

[0035] 2. The camel-derived nanobody molecules of this invention exhibit exceptional physicochemical stability, including strong tolerance to high temperatures, extreme pH values, and organic solvents. Under food processing or harsh storage conditions, the VHH inhibitor can maintain its biological activity and binding capacity for extended periods without easily becoming inactive. This stability holds promise for developing a natural biological preservative suitable for a variety of diverse industrial environments.

[0036] 3. The nanobody structure of the present invention is simple and easy to further optimize through genetic engineering, such as constructing multivalent and multispecific antibodies or fusing them with other functional modules, thereby developing a multifunctional platform that can be used not only for inhibition but also for highly sensitive detection of PPO activity, thus expanding its technological scope and application value. Attached Figure Description

[0037] Figure 1 This is a comparison diagram of the amino acid sequences of polyphenol oxidase and EF565469.1; Figure 2 This is a graph showing the absorbance ratio between the monoclonal phage polyphenol oxidase group and the control group; Figure 3 This is the SDS-PAGE gel electrophoresis result of the PPO-5C-Fc antibody. Lane M is the standard protein. Figure 4 It is an enzyme-linked immunosorbent assay (ELISA) image; Figure 5 This is a binding-dissociation sensing diagram of PPO-5C-Fc and polyphenol oxidase using biomembrane interferometry. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0039] Example 1: Screening and characterization of nanobodies bound to polyphenol oxidase I. Immunity against polyphenol oxidase in Bactrian camels: Hemolymph was extracted from shrimp, and an equal proportion of anticoagulant was added. Total RNA was extracted using the Total RNA Kit and reverse transcribed into cDNA using the Evo M-MLV RT kit. Specific primers 1-F and 1-R were designed based on the polyphenol oxidase gene sequence (PPO-2, Gene Bank number: EF565469.1) and the pET-30a vector. The polyphenol oxidase gene sequence was amplified using the high-fidelity enzyme PrimeSTAR Max DNA Polymerase, primers 1-F and 1-R. The PCR program was: 94 ℃, 1 min; 98 ℃, 15 s; 66 ℃, 20 s; 72 ℃, 3 min, for a total of 35 cycles. The peT-30a plasmid was digested with the restriction endonuclease NotI and recovered. The amplified polyphenol oxidase gene sequence was ligated to the digested peT-30a vector using T7 ligase. The ligation product was added to... transT1 competent cells were cultured at 37 ℃ and 220 rpm for 1 h, then plated onto LB / Kana plates. Colony PCR was performed for verification, and sequencing was conducted. The sequencing results are as follows: Figure 1 As shown. A recombinant polyphenol oxidase plasmid was obtained using a plasmid miniprep kit, and the recombinant polyphenol oxidase plasmid was added to... BL In 21 competent cells, cultured at 37 ℃ and 220 rpm for a period of time, when the OD of the bacterial culture... 600 When the concentration reached approximately 0.6, IPTG was added to a final concentration of 0.5 mmol / L. Expression was induced at 16 °C for 18 h, followed by centrifugation at 9000 g for 20 min at 4 °C. The precipitate was reconstituted with sonication buffer (20 mmol / L Tris-HCl, 0.2 mmol / L NaCl, pH=8.0), sonicated (5 s on, 5 s off, 40%), and centrifuged (4 °C, 11000 g, 20 min). The supernatant was filtered through a 0.22 μm syringe and purified using a Ni-affinity chromatography column. Gradient elution was performed with 0-250 mmol / L sonication buffer. Proteins with single, biologically active bands were collected and concentrated to obtain recombinant polyphenol oxidase protein. The recombinant polyphenol oxidase protein was mixed with an equal volume of Freund's complete adjuvant, thoroughly emulsified, and subcutaneously injected into Bactrian camels. After the initial immunization, booster immunizations were performed monthly using an emulsion of recombinant polyphenol oxidase protein mixed with an equal volume of Freund's incomplete adjuvant. A single immunization dose consisted of 500 μg of recombinant polyphenol oxidase injected into one Bactrian camel. Immunization of the Bactrian camels was completed after four immunizations. Venous blood was collected from the Bactrian camels 15 days after the fourth immunization, and total RNA was extracted and reverse-engineered into cDNA.

[0040] II: Construction of a Nanobody Library Targeting Polyphenol Oxidase Step S1: Using cDNA prepared from camel peripheral blood immunized with polyphenol oxidase for the fourth time as a template, the VHH sequence with the pR2 homologous arm was amplified. PCR amplification was performed using a program of 94 °C pre-denaturation for 5 min, 98 °C denaturation for 10 s, 57 °C annealing for 15 s, and 72 °C extension for 25 s, for a total of 35 cycles, followed by a final extension at 72 °C for 1 min. The VHH amplification product was recovered using a kit. Simultaneously, pR2 was also amplified and recovered using a kit. The amplification program was 98 °C denaturation for 10 s, 55 °C annealing for 15 s, and 68 °C extension for 4 min 45 s, for a total of 30 cycles. The amplified pR2 was digested with restriction endonuclease Dpn I and recovered. The recovered VHH sequence and digested pR2 were cloned seamlessly at 50 °C for 1 h using 2×Uniclone Seamless Cloning Mix at a ratio of 4:1. Finally, the recombinant plasmid was recovered using a recovery kit.

[0041] Step S2: Streak the frozen TG1 bacteria on a 2×TY plate and incubate overnight. Pick a single bacterium and transfer it to 20 mL of 2×TY. Incubate at 37°C and 200 rpm for 6 h using a shaker. Then, transfer the bacterial culture at a 1:100 ratio to 300 mL of 2×TY and incubate until OD reaches the target value. 600 ≈0.5, immediately centrifuge at 4 ℃, 5000 g for 15 min, collect the precipitate, resuspend the precipitate in 300 mL of 10% glycerol, centrifuge at 4 ℃, 5000 g for 15 min, repeat the operation 3 times, then resuspend the precipitate in 50 mL of 10% glycerol, centrifuge at 4 ℃, 3300 g for 10 min. Finally, resuspend the bacterial cells in 1 mL of 10% glycerol. The recombinant plasmid product after seamless cloning is mixed with 500 μL of TG1 competent cells, electroporated at 2500 V for 5 ms, and the electroporation product is transferred to 20 mL of 2×TY and cultured at 37 ℃, 220 rpm for 1 h with shaking. The cultured bacterial solution is serially diluted to 90 mm solid plates, cultured overnight, and the number of colonies is counted and the size of the constructed antibody library is calculated. The remaining bacterial solution is plated to 150 mm solid plates, cultured overnight, and the bacterial growth is scraped off and frozen at -80 ℃. This is the constructed nanobody immunotherapy library.

[0042] III. Screening of Nanobodies Targeting Polyphenol Oxidase Step S1: Activate the nanobody immune library to achieve initial OD 600 ≈0.1, incubated in a shaker at 37 ℃ until OD 600 Add ≈0.5 after the final concentration is 1×10. 11 PFU KM13 helper phage was incubated at 37 °C for 45 min, then centrifuged for 10 min (3500 g). The supernatant was discarded, and the precipitate was resuspended in 200 mL of 2×TY (containing 100 µg / mL ampicillin, 100 µg / mL kanamycin sulfate, and 0.1% glucose). The phage was cultured at 25 °C and 200 rpm for 16 h. After incubation, the culture was centrifuged at 4 °C and 3500 g for 30 min, and the supernatant was collected. 50 mL of 20% polyethylene glycol / sodium chloride mixture was added, mixed well, and incubated on ice for 1 h to precipitate the phage. The precipitate was then centrifuged at 4 °C and 3500 g for 30 min, the supernatant was discarded, and the precipitate was collected. The precipitate was resuspended in 500 µL of phosphate buffer, centrifuged at 4 °C and 12000 g for 10 min, and the phage supernatant was collected and the titer calculated.

[0043] Step S2: Dissolve polyphenol oxidase to a final concentration of 50 μg / mL (concentrations for the second and third rounds are 10 μg / mL and 2 μg / mL, respectively), add 100 µL to a 96-well immunoassay plate, and prepare a protein-free solution as a control. Incubate all coating solutions overnight at 4°C. Wash three times with 280 µL of phosphate buffer, then block with 280 µL of skim milk for 2 h. After blocking, add 100 µL of 1×10⁻⁶ phosphate buffer. 11 PFU phages were incubated at 80 rpm for 1 h on a shaker. After incubation, the phages were washed 20 times with 280 µL phosphate-buffered saline (containing 0.1% Tween-20) to remove non-specific proteins. Phages specifically bound to polyphenol oxidase were eluted with 0.5 mg / mL trypsin solution and then used to infect TG1 bacteria. 50 µL and 5 μL of the infection solution were plated onto LB agar plates (containing ampicillin), incubated overnight, and colony counts were recorded.

[0044] Step S3: Phage ELISA screening for monoclonal phages resistant to polyphenol oxidase. Step S31: Randomly pick 95 single bacteria from the positive counting plate and transfer them to a 96-well culture plate. Mix well by pipetting, leaving one well as a negative control. Incubate at 37 ℃ and 250 rpm for 6-8 h. Transfer 3 µL of bacterial culture to 100 µL of a 96-well culture plate containing KM13 helper phage. Incubate at 37 ℃ and 250 rpm for 1.5 h. Preserve the remaining bacterial culture with 100 µL of 30% glycerol. After incubation, incubate at 37 ℃ for 45 min, then centrifuge at 3500 g for 20 min at room temperature. After centrifugation, aspirate the supernatant and resuspend the bacteria in each well of the 96-well plate with 200 µL of 2×TY (containing 100 µg / mL ampicillin, 100 µg / mL kanamycin sulfate, and 0.1% glucose). Incubate at 25 ℃ and 250 rpm for 20 h. After the culture was completed, the phage supernatant was collected after centrifugation at 3200 g for 40 min at 4 ℃.

[0045] Step S32: Coat 0.5 µg / mL polyphenol oxidase solution onto a 96-well plate, with a 96-well protein-free solution serving as a control group. Coat overnight at 4 °C. Wash twice with 280 µL phosphate buffer, then block with 280 µL skim milk for 2 h. After blocking, wash twice more with 280 µL phosphate buffer. After washing, add 100 µL of the prepared monoclonal phage to each well and incubate at 80 rpm for 1 h. After incubation, each well was washed four times with 280 µL of phosphate buffer containing 0.1% Tween-20, followed by the addition of 100 µL of diluted HRP-anti M13 antibody and incubation at 80 rpm for 1 h. After incubation, each well was washed three times with phosphate buffer containing 0.1% Tween-20, and then incubated for 5 min in 100 µL of 3,3',5,5'-tetramethylbenzidine chromogenic solution in the dark. Finally, the chromogenic reaction was terminated with 50 µL of 1M H2SO4, and the OD450 of the chromogenic solution was measured. The ratio (P / N) of the absorbance value (OD450) of the polyphenol oxidase experimental group at 450 nm to that of the corresponding control group was plotted on the ordinate, with the well number of the 96-well plate on the x-axis. Figure 2 The scatter plot is shown. All positive clones with a P / N value greater than 20 were selected and sent to the company for sequencing using primer 5'-CAGGAAACAGCTATGAC-3'. Finally, a nanobody sequence was obtained and named PPO-5C.

[0046] Step S33: The camel-derived nanobody sequence PPO-5C was constructed into the mammalian expression vector pTT5-TEV-Fc, transfected into HEK 293 cells, and cultured for 3 days. The cells were then centrifuged at 5000 g for 15 min at 4°C. The supernatant protein was collected and eluted using a lipoglycogel rProtein A affinity chromatography column with a linear gradient elution of 0.1 M acetic acid solution (0-100%, 20 min). The eluent was collected and concentrated, and the fusion protein was verified by SDS-PAGE. Figure 3 As shown, a high-purity polyphenol oxidase nanobody Fc fusion protein was obtained, with a yield of over 1.78 mg.

[0047] IV. Characterizing Nanobodies Step S1, Enzyme-Linked Immunosorbent Assay: 1 μg / mL polyphenol oxidase solution was coated onto a 96 immunoassay plate overnight. A protein-free solution was used as the negative control. After washing twice with phosphate buffer, the plate was blocked with skim milk for 2 h. Additionally, the purified single-domain fusion antibody PPO-5C-Fc was serially diluted with phosphate buffer (containing 0.1% Tween-20), resulting in 15 dilution gradients. Each gradient was repeated three times, increasing the concentration of the nanobody from 10... 3 nM diluted to 1×10 -4nM was added to the immunoassay plate and incubated on a shaker at 80 rpm for 1 h. The remaining steps were similar to step S32. The secondary antibody used was HRP anti-IgG1 Fc. The logarithm of the nanobody fusion protein concentration was plotted on the x-axis, and the corresponding OD... 450 The absorbance value was plotted on the ordinate, and the data was fitted to obtain a typical S-shaped curve. For example... Figure 4 As shown, the half-maximal effect concentration (EC50) is 0.09 nM, and the sensitivity reaches the nanomolar level.

[0048] Step S2: Detection of single-domain fusion antibody affinity using biolayer interferometry: First, polyphenol oxidase is biotinylated. After the polyphenol oxidase antigen and biotinylation reagent are mixed evenly, the reaction is carried out at room temperature in the dark for 60 min. Then, desalting is performed using a desalting column, and the eluent is collected and its protein concentration is measured. The SA probe is pre-soaked in phosphate buffer (0.02% Tween-20, pH=7.4) for 10 min, and then the SA probe is mounted onto the instrument. The biotinylated polyphenol oxidase and PPO-5C-Fc single-domain fusion antibody are subsequently diluted with the above phosphate buffer for experiments. After the antigen solidification height reaches more than 1 nm, the baseline is solidified. Subsequent binding and dissociation with different concentrations of PPO-5C-Fc antibody are then analyzed using BLItz Pro. TM The software analyzes and fits the data to obtain the affinity constant (K). D ), the binding constant (ka) and the dissociation constant (kd). According to Figure 5 It can be seen that the affinity constant K between PPO-5C-Fc and polyphenol oxidase is... D The value is 1.49 nM, and the affinity reaches the nanomolar level.

[0049] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely 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 within the protection scope of the present invention.

[0050] The amino acid sequence involved in this invention is as follows: SEQ ID NO.1: Name: Nucleotide sequence of nanobody DNA type: other DNA Biological origin: Bactrian Camel CAGGTGCAGCTCGTGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTTTAGTCTCTGGGGTCACTTTTGATGACATGGGCTGGTACCGCCAGGCTCCAGGGAATGAGTGCGAGTTCGTCGCGCTTATTAGTAGTGATGGTAATACATACTATGAAGACT CCGTGAAGGGCCGATTCACCATCTCCCAAGACACACCCAGAACGCGATATATCTGCAAATGAACAGCCTGAAACCTGAAGACACGGGCCGTGTATTACTGTGCGTCAGCTGGTCCCCTGACTATGGGTTTTAGTCGTTGCGGTTACTGGGGCCAGGGGGACCCAGGTCACCGTCTCCTCA SEQ ID NO.2: Name: Amino acid sequence of nanobody Sequence type: AA Biological origin: Bactrian Camel QVQLVESGGGSVQAGGSLRLSCLVSGVTFDDMGWYRQAPGNECEFVALISSDGNTYYEDSVKGRFTISQDNTQNAIYLQMNSLKPEDTAVYYCASAGPLTMGFSRCGYWGQGTQVTVSS.

[0051] SEQ ID NO.3: Name: 1-F primer gene sequence Sequence type: other DNA Biological origin: synthetic construct 5'-GCTCCGTCGACAAGCTTGCGGCCGCATGGACAAGAGTCGGAAGAA- 3'.

[0052] SEQ ID NO.4: Name: 1-R primer gene sequence Sequence type: other DNA Biological origin: synthetic construct 5'-GGTGGTGGTGCTCGAGTGCGGCCGCTCAGTCTCGGTTCAGCCTCTC- 3'.

[0053] SEQ ID NO.5: Name: 2-F primer gene sequence Sequence type: other DNA Biological origin: synthetic construct 5'-GCTGCACAGCCTGCTATGGCACAGKTGCAGCTCGTGGAGTCTGGGGG-3'.

[0054] SEQ ID NO.6: Name: 2-R primer gene sequence Sequence type: other DNA Biological origin: synthetic construct 5'-GAGTTTTTGTTCGGCTGCTGCTGAGGAGACGGTGACCTGGGTCCCC-3'.

[0055] SEQ ID NO.7: Name: 3-F primer gene sequence Sequence type: other DNA Biological origin: synthetic construct 5'-AGCAGCCGAACAAAAACTCATCTCAGAAGAG-3'.

[0056] SEQ ID NO.8: Name: 3-R primer gene sequence Sequence type: other DNA Biological origin: synthetic construct 5'-CCATAGCAGGCTGTGCAGCATAGAAAGGTACCACTAAAGGAATTGC-3'.

Claims

1. A nanobody conjugated with polyphenol oxidase, characterized in that, The amino acid sequence of the nanobody is shown in SEQ ID NO.

2.

2. The nanobody conjugated with polyphenol oxidase according to claim 1, characterized in that, The nucleotide sequence of the nanobody is shown in SEQ ID NO.

1.

3. A method for preparing a nanobody conjugated with polyphenol oxidase as described in any one of claims 1-2, characterized in that, The preparation method includes: Step S1: Total RNA was extracted from shrimp hemolymph and then reverse transcribed, cloned, and a recombinant expression plasmid containing the polyphenol oxidase gene was constructed. The recombinant expression plasmid was induced to express and purified to obtain recombinant polyphenol oxidase protein. The recombinant polyphenol oxidase protein was emulsified with different adjuvants and then used to immunize Bactrian camels multiple times to obtain cDNA from peripheral blood of Bactrian camels that had undergone the fourth immunization with polyphenol oxidase. Step S2: Using cDNA from peripheral blood of Bactrian camels immunized with polyphenol oxidase for the fourth time as a template, specific amplification was performed to obtain nanobody nucleic acid sequences, and phage antibody immune libraries were constructed using the nanobody nucleic acid sequences. Step S3: The PPO-5C sequence with polyphenol oxidase specificity is obtained by screening the phage antibody immune library. Step S4: Recombinantly express the PPO-5C sequence to obtain polyphenol oxidase nanobody.

4. The method for preparing nanobodies conjugated with polyphenol oxidase according to claim 3, characterized in that, Step S2 specifically involves: S21: The specific amplification in step S2 is as follows: the VHH fragment is obtained by amplifying the cDNA of peripheral blood from Bactrian camels using a high-fidelity enzyme; at the same time, the pR2 phage is amplified using a high-fidelity enzyme using pR2 phage as a template, and the amplified pR2 phage is digested with the restriction enzyme Dpn I. The VHH fragment is then ligated to the digested and amplified pR2 phage through seamless cloning to obtain recombinant pR2 phage. S22: In step S2, the specific steps for constructing the phage antibody immune library using the nanobody nucleic acid sequence are: activating TG1 Escherichia coli to OD200. 600 Activated TG1 Escherichia coli were obtained at a concentration of approximately 0.

5. The activated TG1 Escherichia coli were then subjected to ice bath and repeated resuspending in 10% glycerol and centrifugation to prepare TG1 competent bacteria. Recombinant pR2 phage particles were electroporated into TG1 competent bacteria to obtain bacteria carrying phage-camel-derived immune antibodies. The bacteria carrying phage-camel-derived immune antibodies were diluted and coated onto bacterial scraping plates to obtain bacterial scraping plates. The bacterial scraping plates were then cryopreserved to obtain a phage-camel-derived immune antibody library.

5. The method for preparing nanobodies conjugated with polyphenol oxidase according to claim 3, characterized in that, Step S4 specifically involves: constructing the PPO-5C sequence obtained in step S3 into the mammalian expression vector pTT5-TEV-Fc, then transfecting the mammalian expression vector pTT5-TEV-Fc carrying the nanobody sequence into HEK 293 cells, and then purifying the nanobody Fc fusion protein by agarose gel rProtein A affinity chromatography column to obtain the polyphenol oxidase nanobody.

6. The method for preparing nanobodies conjugated with polyphenol oxidase according to claim 3, characterized in that, The phage antibody immune library in step S2 includes the nucleic acid sequence SEQ ID NO.1 as described in claim 2.

7. The application of a polyphenol oxidase-bound nanobody according to any one of claims 1-2 or a polyphenol oxidase-bound nanobody prepared by any one of claims 3-6, characterized in that, Application in detecting products that inhibit polyphenol oxidase.

8. The application of the nanobody bound to polyphenol oxidase according to claim 7, characterized in that, The product used to detect polyphenol oxidase inhibition is one or more of the following: a polyphenol oxidase neutralizing antibody inhibition effect test kit and a test reagent for inhibiting polyphenol oxidase activity.

9. The application of an antibody conjugated with polyphenol oxidase nanoparticles as described in any one of claims 1-2, or a polyphenol oxidase nanobody prepared by any one of claims 3-6, characterized in that, Applications in the preparation of natural biological preservatives.

10. A kit for detecting the inhibitory effect of polyphenol oxidase neutralizing antibody, characterized in that, This includes the polyphenol oxidase-bound nanobody as described in any one of claims 1-2 or the polyphenol oxidase-bound nanobody prepared by any one of claims 3-6.

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