Nanobody against vp19 of white spot syndrome virus and application thereof

Abstract

This application relates to the technical field of disease prevention and control in aquaculture, specifically to a nanobody against white spot syndrome virus (WSSV) VP19 in shrimp and its application. The nanobody comprises CDR1, CDR2, and CDR3; wherein the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO. 1-3, respectively; or, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO. 8-10, respectively. The nanobody provided in this application can be administered orally by mixing with feed and exhibits high specificity and affinity. Furthermore, this nanobody can cross the tissue barrier and bind to WSSV epitope proteins, blocking the binding of WSSV to host cells, thereby achieving the purpose of preventing and treating WSSV.

Description

A nanobody against white spot syndrome virus VP19 in shrimp and its application Technical Field

[0001] This application relates to the technical field of disease prevention and control in aquaculture, and in particular to a nanobody against shrimp white spot syndrome virus VP19 and its application. Background Technology

[0002] Since 1992, White Spot Syndrome Virus (WSSV) has been prevalent in coastal aquaculture areas of my country, occurring almost universally in shrimp farming areas and causing significant damage, severely impacting shrimp farming. The incidence rate is significantly higher in marine aquaculture areas than in brackish water aquaculture areas. However, with the increasing use of freshwater aquaculture for Litopenaeus vannamei, the impact of WSSV on freshwater shrimp is gradually becoming apparent. In recent years, the incidence of WSSV in farmed Litopenaeus vannamei has shown an increasing trend.

[0003] WSSV primarily infects and damages the hematopoietic tissue, connective tissue, epithelium of the foregut and hindgut, blood cells, and gills of shrimp. Acute infection causes a sharp drop in shrimp's feed intake, and the carapace of the cephalothorax and abdominal segments is easily peeled off without adhering to the dermis (i.e., the shell is easily peeled off), with obvious white spots visible on the carapace. Most shrimp infected with WSSV show a pale red or reddish-brown body (especially evident in Litopenaeus vannamei). WSSV is highly virulent, with only 3-5 days, or even less, between the appearance of symptoms and death; the infection rate of WSSV is high, and within about 7 days, more than 90% of the shrimp in the pond can become diseased, or even die.

[0004] Currently, the prevention and control of WSSV in shrimp mainly faces the following technical challenges:

[0005] (1) To date, there is no specific treatment for WSSV. After shrimp are infected with WSSV, the main treatment is physical isolation, and the same treatment method used for bacterial white spots is applied, with oral administration of "Life-enhancing Agent + Florfenicol + Super Vitamin C". However, traditional antibiotics and chemical drugs are ineffective against WSSV and are prone to drug resistance and environmental pollution.

[0006] (2) As invertebrates, shrimp lack an acquired immune system and cannot produce WSSV-specific antibodies; only pathogen-specific "immunosensitization" may provide short-term protection. Inactivated virus, subunit antigen and DNA vaccines are still under development and there is a risk of variable efficacy. DNA vaccines are limited by the environment in which they are used due to stability issues.

[0007] (3) Vaccines based on the antiviral cell mechanism of RNA interference (RNAi) using double-stranded RNA (dsRNA) or small interfering RNA (siRNA) have the potential for application, but RNA has poor stability and high production cost.

[0008] (4) Conventional antibodies (such as polyclonal antibodies and monoclonal antibodies) have poor stability in the breeding environment and are difficult to tolerate harsh conditions such as high temperature during feed processing.

[0009] Based on the above, it is necessary to further explore the prevention and control methods of WSSV in shrimp. Summary of the Invention

[0010] This application provides a nanobody against white spot syndrome virus (WSSV) VP19 in shrimp and its application. This nanobody can be administered orally via feed mixing and exhibits high specificity and affinity. Furthermore, this nanobody can cross the tissue barrier and bind to WSSV epitope proteins, blocking the binding of WSSV to host cells, thereby achieving the purpose of preventing and treating WSSV.

[0011] Compared with traditional antibodies, nanobodies have the following significant advantages: Nanobodies have a molecular weight of only about 15 kDa, approximately one-tenth that of conventional antibodies, allowing them to penetrate tissue barriers; they are resistant to harsh conditions such as high temperatures, strong acids, and strong alkalis; they are highly soluble and do not easily aggregate; they exhibit strong specificity, high affinity, and low immunogenicity; and they can be recombinantly expressed in simple microorganisms, facilitating low-cost large-scale production and making them a substitute for traditional antibodies. Therefore, developing nanobodies targeting WSSV provides a new technological approach to solving the problem of WSSV prevention and control in shrimp.

[0012] The nanobody provided in this application is a specific antibody designed against the key WSSV protein VP19, and its stability is improved by binding the nanobody to the I53-50A protein. Furthermore, this application also constructs recombinant yeast for efficient extracellular expression of the nanobody.

[0013] In a first aspect, this application provides a nanobody against shrimp white spot syndrome virus VP19, employing the following technical solution:

[0014] A nanobody against white spot syndrome virus VP19 in shrimp, specifically including a nanobody named WSSV-1D or a nanobody named WSSV-1B.

[0015] The nanobody named WSSV-1D includes a complementarity-determining region (CDR), specifically CDR1, CDR2, and CDR3. The amino acid sequence of CDR1 is shown in SEQ ID NO. 1, the amino acid sequence of CDR2 is shown in SEQ ID NO. 2, and the amino acid sequence of CDR3 is shown in SEQ ID NO. 3.

[0016] The nanobody named WSSV-1B includes CDR1, CDR2, and CDR3. The amino acid sequence of CDR1 is shown in SEQ ID NO. 8, the amino acid sequence of CDR2 is shown in SEQ ID NO. 9, and the amino acid sequence of CDR3 is shown in SEQ ID NO. 10.

[0017] Furthermore, the aforementioned nanobody also includes a framework region (FR, i.e., the non-CDR portion), specifically including FR1, FR2, FR3, and FR4.

[0018] Optionally, CDR1, CDR2 and CDR3 are separated by FR1, FR2, FR3 and FR4.

[0019] Among them, the amino acid sequence of FR1 in the nanobody named WSSV-1D is shown in SEQ ID NO. 4, the amino acid sequence of FR2 is shown in SEQ ID NO. 5, the amino acid sequence of FR3 is shown in SEQ ID NO. 6, and the amino acid sequence of FR4 is shown in SEQ ID NO. 7.

[0020] The amino acid sequence of the above nanobody is shown in SEQ ID NO. 15.

[0021] Among them, the amino acid sequence of FR1 in the nanobody named WSSV-1B is shown in SEQ ID NO. 11, the amino acid sequence of FR2 is shown in SEQ ID NO. 12, the amino acid sequence of FR3 is shown in SEQ ID NO. 13, and the amino acid sequence of FR4 is shown in SEQ ID NO. 14.

[0022] The amino acid sequence of the above nanobody is shown in SEQ ID NO. 16.

[0023] Optionally, the CDR1 region contains an amino acid sequence that has at least 80% sequence similarity to the amino acid sequence shown in SEQ ID NO. 1 or SEQ ID NO. 8.

[0024] Optionally, the CDR1 region contains an amino acid sequence that has at least 90% sequence similarity to the amino acid sequence shown in SEQ ID NO. 1 or SEQ ID NO. 8.

[0025] Optionally, the CDR1 region contains an amino acid sequence that has at least 95% sequence similarity to the amino acid sequence shown in SEQ ID NO. 1 or SEQ ID NO. 8.

[0026] Optionally, the CDR1 region contains an amino acid sequence that has at least 99% sequence similarity to the amino acid sequence shown in SEQ ID NO. 1 or SEQ ID NO. 8.

[0027] Optionally, the CDR2 region contains an amino acid sequence that has at least 80% sequence similarity to the amino acid sequence shown in SEQ ID NO. 2 or SEQ ID NO. 9.

[0028] Optionally, the CDR2 region contains an amino acid sequence that has at least 90% sequence similarity to the amino acid sequence shown in SEQ ID NO. 2 or SEQ ID NO. 9.

[0029] Optionally, the CDR2 region contains an amino acid sequence that has at least 95% sequence similarity to the amino acid sequence shown in SEQ ID NO. 2 or SEQ ID NO. 9.

[0030] Optionally, the CDR2 region contains an amino acid sequence that has at least 99% sequence similarity to the amino acid sequence shown in SEQ ID NO. 2 or SEQ ID NO. 9.

[0031] Optionally, the CDR3 region contains an amino acid sequence that has at least 80% sequence similarity to the amino acid sequence shown in SEQ ID NO. 3 or SEQ ID NO. 10.

[0032] Optionally, the CDR3 region contains an amino acid sequence that has at least 90% sequence similarity to the amino acid sequence shown in SEQ ID NO. 3 or SEQ ID NO. 10.

[0033] Optionally, the CDR3 region contains an amino acid sequence that has at least 95% sequence similarity to the amino acid sequence shown in SEQ ID NO. 3 or SEQ ID NO. 10.

[0034] Optionally, the CDR3 region contains an amino acid sequence that has at least 99% sequence similarity to the amino acid sequence shown in SEQ ID NO. 3 or SEQ ID NO. 10.

[0035] Optionally, any of the above amino acid sequences may further include a derived sequence that has been optionally added, deleted, modified and / or substituted with at least one (e.g., 1-3, preferably 1-2, more preferably 1) amino acid and can retain specific binding to VP19.

[0036] Optionally, all nanobodies are able to specifically bind to VP19.

[0037] Optionally, the nanobody can effectively block the interaction between VP19 and C-type lectin (LvCTL1).

[0038] Optionally, the nanobody includes humanized antibodies, camel-derived antibodies, and chimeric antibodies.

[0039] Optionally, the nanobody includes monomers, divalents, tetravalents, and / or multivalents.

[0040] Secondly, this application provides the use of the above-mentioned nanobody in the preparation of formulations for the prevention and / or treatment of diseases related to WSSV in shrimp.

[0041] Thirdly, this application provides a recombinant protein. The recombinant protein includes the nanobody of this application, a fusion protein following the linker peptide, an I53-50A sequence, and optionally a tag sequence to assist in expression and / or purification.

[0042] Optionally, the linker peptide is selected from the following sequence: (GaSb)n where a, b, n = 0 or 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 (preferably, a = 2, b = 1, n = 4).

[0043] Optionally, the linker peptide sequence is GGSGGSGGSGGS.

[0044] Optionally, the tag sequence includes an Fc tag, an HA tag, a cmyc tag, and a 6His tag.

[0045] Optionally, the recombinant protein specifically binds to the VP19 protein.

[0046] Fourthly, this application provides a pharmaceutical composition formulation. The pharmaceutical composition formulation includes the aforementioned nanobody.

[0047] Optionally, the preparation is a diagnostic reagent.

[0048] Optionally, the dosage form of the preparation is liquid or powder (such as aqueous solution, injection, lyophilized powder, spray-dried powder).

[0049] Optionally, the preparation is a shrimp feed additive.

[0050] Optionally, the preparation is a vaccine.

[0051] Optionally, the preparation is an antiviral antibody.

[0052] Optionally, the use is non-diagnostic and non-therapeutic.

[0053] Fifthly, this application provides a vaccine. The vaccine includes the aforementioned nanobody.

[0054] In summary, this application includes at least one of the following beneficial technical effects:

[0055] This application describes a method for obtaining nanobodies with good blocking activity through immunization and screening, which can effectively prevent, detect and treat WSSV in shrimp in actual aquaculture, especially shrimp farming, and for using Pichia pastoris to achieve efficient and inexpensive production of these nanobodies. Attached Figure Description

[0056] Figure 1 shows the SDS-PAGE detection results of the nanobody provided in this application expressed in Pichia pastoris (Lane 1: Protein Marker; Lane 2: WSSV-1D; Lane 3: WSSV-1B).

[0057] Figure 2 shows the experimental results of the nanobody provided in this application binding to shrimp white spot virus enzyme-linked immunosorbent assay (ELISA).

[0058] Figure 3 shows the results of the neutralization experiment of nanobodies in Japanese shrimp. Detailed Implementation

[0059] Before describing the embodiments of this application in detail, it should be understood that the terminology used herein is for the purpose of describing a particular embodiment only. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the term pertains.

[0060] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this application, unless otherwise stated, "multiple" means two or more.

[0061] The endpoints and any values ​​of the ranges disclosed in this application are not limited to the precise ranges or values, and such ranges or values ​​should be understood to include values ​​close to such ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0062] In this application, the terms "comprising" or "including" are open-ended expressions, meaning they include the content specified in this application but do not exclude other aspects.

[0063] To make the objectives, technical solutions, and advantages of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0064] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0065] This application selects VP19, a key protein of WSSV, as the target for designing specific nanobodies. VP19 is the second most abundant protein in the envelope of WSSV after VP28, making it a key target for designing immunoprophylactic drugs. The anti-VP19 nanobodies obtained in this application can specifically bind to VP19 and inhibit viral assembly.

[0066] The present application will be further described in detail below with reference to the embodiments and test results.

[0067] Example 1

[0068] This embodiment provides the design, expression, immunization, and screening of anti-WSSV-VP19 nanobodies.

[0069] Specifically, the following steps are included:

[0070] (1) Expression design of antigen VP19

[0071] Targeting the conserved epitope of the key WSSV protein VP19, the amino acid sequence (as shown in SEQ ID NO. 17) and nucleotide sequence (as shown in SEQ ID NO. 18) encoding the target epitope were synthesized via gene synthesis and cloned into an E. coli vector (such as pET28a). Purification was performed using an immobilized metal ion affinity chromatography column such as Ni-NTA.

[0072] (2) Immunity

[0073] Antigen preparation: The amino acid sequence of the antigen is shown in SEQ ID NO. 17. Emulsify the antigen and adjuvant (Frederick adjuvant, Thermo) 1:1 to form a homogeneous antigen mixture and store at 4°C. The total antigen amount for each immunization is 2 mg, and the volume of each immunization is less than 2 mL.

[0074] Shark Immunization: Select suitable sharks and ensure they are fit. Record the ear tag and begin the immunization experiment. Inject 0.4 mL of antigen mixture into two injection points on each side near the neck lymph nodes. Observe the shark for half an hour after immunization to confirm it is in good condition and shows no signs of discomfort. Immunize every two weeks for a total of seven immunizations.

[0075] Blood collection: Blood is collected 5-7 days after the 6th and 7th immunizations. The blood is taken from the shark's neck vein, and 25-30 mL of blood is collected each time, divided into 3 blood collection tubes.

[0076] Serum separation: Blood was collected for immune evaluation before the 4th, 5th, and 6th immunizations. 5 mL of blood was collected from the shark's jugular vein each time. The blood was centrifuged at 400 x g for 30 min on the same day using a pre-cooled 25°C centrifuge, and the supernatant serum was separated and preserved.

[0077] Lymphocyte separation: Add 3 mL of cell separation medium to a 15 mL centrifuge tube, then slowly add 3 mL of blood. Add the blood carefully and slowly to prevent mixing with the separation medium. After centrifuging at 25°C for 30 min at 400 x g, observe the blood separation in the centrifuge tube. Carefully aspirate the middle cotton-like upper layer of immune cells using a 200 μL pipette into a new 15 mL centrifuge tube. Store the upper serum layer in a new centrifuge tube at -80°C.

[0078] Add 10 mL of room temperature PBS buffer to each tube and centrifuge at 25°C, 400 x g for 20 min. Remove the supernatant, add 5 mL of room temperature PBS buffer to each tube, and centrifuge at 25°C, 400 x g for 20 min. Count the cells using a hemocytometer. Remove the supernatant, and lyse the isolated lymphocytes using RNAiso Plus to obtain 10-1 cells. 7 / mL of solution, store at -80℃.

[0079] (3) Library construction and phage screening

[0080] RNA extraction: Transfer peripheral blood lymphocytes preserved in Trizol to a 1.5 mL centrifuge tube, add 1 / 5 volume of chloroform and mix well; let stand at room temperature for 5 min, then centrifuge at 4°C and 12000g for 15 min; carefully transfer the supernatant after centrifugation to a new centrifuge tube; add 0.5-1 volume of isopropanol to the new centrifuge tube; let stand at room temperature for 10 min, then centrifuge at 4°C and 12000g for 10 min; wash the precipitate with an equal volume of 75% ethanol to the peripheral blood lymphocytes preserved in Trizol, centrifuge at 4°C and 7500g for 5 min, and then dissolve in an appropriate amount of RNase-free water.

[0081] Reverse transcription of cDNA: Following the instructions of the reverse transcription kit, reverse transcribe the RNA obtained in the previous step into cDNA.

[0082] Amplifying antibody fragments: Specific antibody fragments are amplified from reverse-transcribed cNDA using PCR amplification with Taq DNA Polymerase Hot Start enzyme.

[0083] Cloning to the phage plasmid: Digest the antibody gene sequence and phage vector obtained in the previous step using BglI. The digestion system is as follows: 12 μg of amplified gene or 3 μg of vector, 3 μL of 10X BglI Buffer, 4.5 μL of BglI, and add water to a final volume of 30 μL. Incubate the digestion at 37°C for 3-4 hours.

[0084] After enzyme digestion, the vector and amplified gene were recovered using a DNA purification and recovery kit according to the manufacturer's instructions, followed by ligation. The ligation reaction mixture consisted of: 200 ng of vector, 80 ng of amplified gene, 2 μL of T4 ligase, 5 μL of 10X ligation buffer, and water added to a final volume of 50 μL. The mixture was incubated overnight at 4°C. The ligation product was then recovered using a DNA purification and recovery kit according to the manufacturer's instructions and dissolved in ultrapure water.

[0085] Transformation of SS320: Pre-cool the electroporation cuvette on ice. After the SS320 competent cells thaw, add 1 μL of the recovered ligation product. Transfer the mixed competent cells and ligation product to the pre-cooled electroporation cuvette and electroporate using the Bacteria transformation program preset on the electroporator. Immediately after electroporation, add 1 mL of SOC medium to the electroporation cuvette. After thawing the cells at 37°C for 60 min, plate them on LB culture plates containing tetracycline and ampicillin resistance for overnight growth.

[0086] After overnight growth, the cells on the culture plate were rinsed and scraped off with LB medium and a spreader, and then stored at -80°C after adding 20% ​​glycerol.

[0087] Amplification and purification of the phage library: Approximately 10^9 cells scraped from the previous step were transferred to 100 mL of 2X YT medium pre-added with tetracycline and ampicillin antibiotics, and incubated at 37°C and 220 rpm until the OD600 reached 0.5. Helper phage was added at a ratio of 20:1 to bacterial cells, and the culture was continued at 37°C for 30 min. Kanamycin and 0.2 mM IPTG were added to a final concentration, and the culture was incubated overnight at 30°C.

[0088] Centrifuge overnight cultured cells at 4°C and 13,000 rpm for 5 min. Transfer the supernatant to a new centrifuge tube and add 1 / 4 volume of pre-chilled 5X PEG8000 / NaCl. Incubate on ice for 30 min. Centrifuge at 4°C and 13,000 rpm for 10 min to remove the supernatant. Add 1 mL of PBS buffer to dissolve the precipitate. Add 250 μL of 5X PEG8000 / NaCl again and incubate on ice for 10 min. Centrifuge at 4°C and 16,000 g for 15 min, remove the supernatant, and dissolve the precipitate in 1 mL of PBS to obtain the phage library.

[0089] (4) Antibody screening process

[0090] 1) Coating antigen: Dilute the antigen protein to 2.5-10 μg / ml with coating buffer (pH 9.6 50mM Na2CO3•NaHCO3), and coat each well with 100-200 μl of diluted antigen onto an ELISA plate and incubate overnight at 4°C.

[0091] 2) Washing: Discard the coating solution and wash 3 times with PBS, then pat dry.

[0092] 3) Blocking: Add 5% skim milk powder, fill all wells and block at 37℃ for 2 hours; supernatant: PBSM=3:2 and block for 30-60 minutes.

[0093] 4) Washing: Discard the blocking solution and wash 3 times with PBS, then pat dry.

[0094] 5) Add supernatant: Add 200 μl of the pre-blocked supernatant to each well of the microplate and react at 37°C for 1 h.

[0095] 6) Washing: First round: 5 washes with PBST, 5 washes with PBS; Second round: 10 washes with PBST, 10 washes with PBS; Third round: 20 washes with PBST, 20 washes with PBS.

[0096] 7) Add 100 μl of TAE to each well and elute for 15 min, then pipette every 5 min; immediately after elution, add an equal volume of Tris-HCl to neutralize.

[0097] 8) Add 750 μl of elution buffer to 5 ml of XL1-BLUE (OD=0.5), incubate at 37℃ for 30 min, and then incubate at 37℃ and 200 rpm for 30 min.

[0098] 9) Centrifuge at 3800 rpm for 10 min, resuspend in 1 ml of 2YT medium, take 10 μl to determine the concentration, and spread the remainder onto two 150 mm² YT-ATG plates and incubate at 37°C overnight.

[0099] 10) Use 25ml of 2YT medium to wash off all the bacteria from the two 150mm 2YT-ATG plates for the next round of washing, and freeze the remaining bacteria in 15% glycerol.

[0100] 11) Select 96 single colonies and transfer them to a 96-well cell culture plate containing 2X YT medium with tetracycline and ampicillin. Incubate at 37°C for 3-4 hours, then add kanamycin and helper phage at a ratio of 20:1 to the wells and incubate overnight at 30°C. The next day, centrifuge the overnight cell culture to obtain the supernatant.

[0101] (5) Phage ELISA

[0102] 1) Coating: Dilute the antigen protein to 2.5-10 μg / ml with coating buffer (pH 9.6 50mM Na2CO3•NaHCO3), and coat each well with 100-200 μl of diluted antigen and incubate overnight at 4°C.

[0103] 2) Washing: Discard the coating solution and wash 3 times with PBS, then pat dry;

[0104] 3) Blocking: Add 5% skim milk powder, fill all wells and block at 37℃ for 2 hours; Recombinant antibody: PBSM=3:2 blocking for 30-60 minutes;

[0105] 4) Washing: Discard the blocking solution and wash 3 times with PBS, then pat dry;

[0106] 5) Add recombinant antibody: Add the pre-blocked supernatant to the ELISA plate, 200 μl per well for the experimental group (containing the supernatant of the phage library) and the control group (PBS), and react at 37°C for 1 h;

[0107] 6) Washing: Discard the recombinant antibody solution, wash 3 times with PBST, and then wash 3 times with PBS;

[0108] 7) Add enzyme-labeled secondary antibody: Dilute the enzyme-labeled secondary antibody with 5% PBSM at a ratio of 1:10000, add 100 μl to each well, and react for 1 h;

[0109] 8) Washing: Discard the enzyme-labeled antibody solution, wash 3 times with PBST, and then wash 3 times with PBS;

[0110] 9) Color development: Add 100 μl of TMB color development solution to each well and react at room temperature for 5-10 min;

[0111] 10) Termination: Add 50 μl of stop solution to each well and measure the absorbance at 450 nm. Take a picture of the result for record-keeping.

[0112] Add the phage supernatant obtained in the previous step to the overnight-coated antigen and 3% BSA-blocked 96-well ELISA plate, and incubate at room temperature for 1 hour. After washing three times with PBS containing 0.05% Tween, use the phage antibody as the primary antibody and the corresponding secondary antibody TMB for color development. Read the absorbance of each well at a wavelength of 450 nm. Select the SS320 colony with the highest absorbance reading for sequencing to obtain the antibody gene sequence.

[0113] Sequencing yielded the gene and amino acid sequences of the anti-WSSV-VP 19 nanobody.

[0114] Two nanobody sequences were obtained.

[0115] The first one is named WSSV-1D, and its amino acid sequence is shown in SEQ ID NO. 15.

[0116] The nanobody comprises CDR1, CDR2, and CDR3. The amino acid sequence of CDR1 is shown in SEQ ID NO. 1, the amino acid sequence of CDR2 is shown in SEQ ID NO. 2, and the amino acid sequence of CDR3 is shown in SEQ ID NO. 3.

[0117] The nanobody also includes FR1, FR2, FR3, and FR4. The amino acid sequence of FR1 is shown in SEQ ID NO. 4, the amino acid sequence of FR2 is shown in SEQ ID NO. 5, the amino acid sequence of FR3 is shown in SEQ ID NO. 6, and the amino acid sequence of FR4 is shown in SEQ ID NO. 7.

[0118] The second name is WSSV-1B, and its amino acid sequence is shown in SEQ ID NO. 16.

[0119] The nanobody comprises CDR1, CDR2, and CDR3. The amino acid sequence of CDR1 is shown in SEQ ID NO. 8, the amino acid sequence of CDR2 is shown in SEQ ID NO. 9, and the amino acid sequence of CDR3 is shown in SEQ ID NO. 10.

[0120] The nanobody also includes FR1, FR2, FR3, and FR4. The amino acid sequence of FR1 is shown in SEQ ID NO. 11, the amino acid sequence of FR2 is shown in SEQ ID NO. 12, the amino acid sequence of FR3 is shown in SEQ ID NO. 13, and the amino acid sequence of FR4 is shown in SEQ ID NO. 14.

[0121] Example 2

[0122] This embodiment uses Pichia pastoris to express the nanobody prepared in Example 1.

[0123] Specifically, the following steps are included:

[0124] (1) The nanobody sequence obtained in Example 1 was constructed into the pPICZαA vector.

[0125] (2) Linearized with BglII restriction endonuclease and then electroporated into X-33 competent cells.

[0126] (3) Spread the electroporated samples onto YPDS plates containing different concentrations of bleomycin-resistant medium and incubate them upside down in a 30°C incubator for 3-4 days.

[0127] (4) After single clones grow on the plate culture medium, pick single clones from plates of different concentrations and place them in BMGY medium. When the OD value of BMGY culture medium reaches about 20, collect the cells and replace them in BMMY medium. Culture at 28℃ and 250rpm.

[0128] (5) Samples were taken every 24 hours thereafter, and methanol with a final volume concentration of 1% was added and sampled again; the samples were centrifuged at 12000 rpm for 5 min, the supernatant was collected and stored at -20℃; the induction was continued for 5 days and the culture was ended.

[0129] (6) Perform SDS-PAGE analysis on the obtained supernatant sample.

[0130] The results are shown in Figure 1.

[0131] As shown in Figure 1, the nanobody provided in this application was successfully expressed in Pichia pastoris.

[0132] Example 3

[0133] In this embodiment, ELISA was used to detect the binding activity of the nanobody prepared in Example 1 with the virus.

[0134] Specifically, the following steps are included:

[0135] The shrimp white spot virus was diluted to a concentration of 1×10⁻⁶ with 50mM sodium carbonate-carbonate buffer at pH 9.6. 9The concentration of cfu / mL was then aliquoted into 100 μL per well of a 96-well plate and incubated overnight at 4°C. After washing five times with PBST, 300 μL of 1% BSA blocking buffer was added, and the plate was incubated at 37°C for 1 h. After washing five times with PBST, 100 μL of serially diluted nanobody sample was added, and the plate was incubated at 37°C for 1 h. After washing five times with PBST, 100 μL of anti-His tag-HRP (1:4000 dilution) was added, and the plate was incubated at 37°C for 1 h. After washing five times with PBST, 100 μL of TMB chromogenic solution was added, and the plate was incubated at 37°C for 10 min. The reaction was terminated by adding 2M H2SO4 to 50 μL of each well, and the absorbance was measured at 450 nm using a microplate reader.

[0136] The results are shown in Table 1 and Figure 2.

[0137] Table 1. Results of enzyme-linked immunosorbent assay (ELISA) for the binding of nanobodies to shrimp white spot virus.

[0138]

[0139] As shown in Table 1 and Figure 2, the nanobodies of this application exhibit good blocking activity against the VP19 antigen, especially the WSSV-1D nanobodies.

[0140] Example 4

[0141] This embodiment uses the nanobody prepared in Example 1 to conduct animal experiments.

[0142] (1) Experimental shrimp larvae

[0143] The shrimp used in the experiment were juvenile Litopenaeus vannamei (SPF) shrimp from the Qisha breeding base of the Guangxi Fisheries Research Institute. The average body length was 4.5 ± 0.55 cm, and the average weight was 0.69 ± 0.31 g. After being temporarily raised for 7 days, shrimp of similar size were selected for the experiment.

[0144] (2) Experimental grouping and conditions

[0145] The shrimp were randomly divided into 5 groups, and the experiment was conducted in a 500L round plastic tank. The effective water volume during the experiment was 450L, and the temperature was room temperature. To prevent the shrimp from jumping out, the experimental tank was covered with a light-blocking net.

[0146] (3) Statistical indicators

[0147] After 30 days of rearing, the growth statistics are as follows:

[0148] Survival rate = Number of harvested animals / Number of released animals × 100%.

[0149] (4) WSSV prevention and control experiment

[0150] 1) Shrimp feed formulation:

[0151] Shrimp feed was divided into three groups based on the content of nanoantibodies, as follows:

[0152] Feed A – Basic shrimp feed (New Hope Group tiger prawn compound feed), made into pellets with a particle size of 1.5mm.

[0153] Feed B – Add 0.1% by weight of nano-antibody WSSV-1D fermentation supernatant spray-dried powder to the shrimp basic feed to make granules with a particle size of 1.5 mm.

[0154] Feed C – Add 0.1% by weight of nano-antibody WSSV-1B fermentation supernatant spray-dried powder to the shrimp base feed to make granules with a particle size of 1.5 mm.

[0155] 2) The shrimp were divided into groups, including a negative control group, a positive control group, experimental group 1 (Ab WSSV-1D), and experimental group 1 (Ab WSSV-1B), as detailed below:

[0156] Negative control group: No virus was introduced, and shrimp basal feed (feed A) was used.

[0157] Positive control group: continuously inoculated with the virus for 3 days and fed with shrimp basic feed (feed A);

[0158] Experimental group 1 (Ab WSSV-1D): The shrimp were first continuously inoculated with the virus for 3 days, and then treated with nano-antibody fermentation supernatant sprayed dry powder (feed B) with 0.1% by weight added to the shrimp basic feed.

[0159] Experimental group 2 (Ab WSSV-1B): The shrimp were first continuously inoculated with the virus for 3 days, and then treated with nano-antibody fermentation supernatant sprayed dry powder (feed C) with 0.1% by weight added to the shrimp basic feed.

[0160] Infection method: Each group was fed with leukoderma syndrome virus material (TCIDs0=10) and challenged with a dose of 10 TCIDs0. Observation was conducted for 7 days, and the cumulative mortality rate was recorded.

[0161] The results are shown in Table 2 and Figure 3.

[0162] Table 2. Results of shrimp challenge experiment: Shrimp mortality rate (%) and challenge time statistics.

[0163]

[0164] As shown in Table 2 and Figure 3, the nanobodies of this application can effectively prevent and control WSSV in shrimp in actual aquaculture, especially shrimp farming, and reduce shrimp mortality, especially the WSSV-1D nanobodies.

[0165] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0166] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this application.

Claims

1. A nanobody against white spot syndrome virus VP19 in shrimp, characterized in that, The nanobody comprises CDR1, CDR2, and CDR3; wherein the amino acid sequence of CDR1 is shown in SEQ ID NO. 1, the amino acid sequence of CDR2 is shown in SEQ ID NO. 2, and the amino acid sequence of CDR3 is shown in SEQ ID NO. 3; or, the amino acid sequence of CDR1 is shown in SEQ ID NO. 8, the amino acid sequence of CDR2 is shown in SEQ ID NO. 9, and the amino acid sequence of CDR3 is shown in SEQ ID NO.

10.

2. The nanobody according to claim 1, characterized in that, The amino acid sequence of the nanobody is shown in SEQ ID NO.

15.

3. The nanobody according to claim 1, characterized in that, The amino acid sequence of the nanobody is shown in SEQ ID NO.

16.

4. The use of any one of the nanobody claims 1-3 in the preparation of an agent for the prevention and / or treatment of white spot syndrome in shrimp.

5. A recombinant protein, characterized in that, The recombinant protein comprises the following components: a nanobody having any one of claims 1-3, and a tag sequence to assist in expression and / or purification.

6. A pharmaceutical combination formulation, characterized in that, The drug combination formulation includes nanobodies having any one of claims 1-3.

7. A vaccine, characterized in that, The vaccine includes nanobodies having any one of claims 1-3.

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