Anti-procambarus clarkia white spot syndrome virus monoclonal antibody and application thereof

Abstract

The present application relates to a kind of anti-procambarus clarkii white spot syndrome virus monoclonal antibody and its application, the complementary determining region CDR1-3 sequence of the heavy chain variable region of the anti-procambarus clarkii white spot syndrome virus monoclonal antibody is sequentially shown as SEQ ID NO:3-5, the complementary determining region CDR1-3 sequence of the light chain variable region is sequentially shown as SEQ ID NO:6-8.The present application successfully obtains the high affinity of anti-VP28 monoclonal antibody, and it has excellent affinity to procambarus clarkii white spot syndrome virus.The monoclonal antibody can be used for specific detection to procambarus clarkii white spot syndrome virus, and has good virus neutralization activity.By adding the monoclonal antibody in water body, the resistance of procambarus clarkii to white spot syndrome virus can be greatly improved, easy to operate, without injection, suitable for large-scale breeding scene, provide a new technical means for the prevention and control of white spot syndrome virus disease in aquaculture.

Description

Technical Field

[0001] This invention belongs to the field of monoclonal antibodies and disease prevention and control in aquaculture, specifically relating to a monoclonal antibody against white spot virus in crayfish and its application. Background Technology

[0002] crayfish( Procambarus clarkii Crayfish is an important freshwater aquaculture species, and its farming scale has continued to expand in recent years. White Spot Syndrome (WSS), as one of the most serious viral diseases in crayfish farming, has caused huge economic losses to the industry.

[0003] White Spot Syndrome Virus (WSSV) is an enveloped double-stranded DNA virus belonging to the family Nimaviridae and the genus WSSV. Whispovirus It is one of the largest known animal viruses, with virus particles that are rod-shaped or elliptical, measuring approximately 80-120 × 250-380 nm. This virus has a wide host range, infecting a variety of aquatic animals, including crustaceans, and is highly contagious and deadly, with a mortality rate of 90%-100% within 3-10 days after infection.

[0004] Envelope proteins VP28, VP19, and VP26 are the main structural proteins of WSSV. Among them, VP28 is involved in the adsorption and membrane fusion of the virus with host cells and is an important antigen protein that induces the host to produce an immune response.

[0005] Developing efficient and specific prevention and control products against WSSV, especially immunoprophylaxis technologies based on WSSV-specific antibodies, is of great significance for protecting the crayfish farming industry. Summary of the Invention

[0006] We isolated a strain of white spot virus from the diseased crayfish. Cambarus clarkii The genome sequence and coding gene of whispovirus (WSSV-Cc) were analyzed, and its envelope protein VP28 was identified. A monoclonal antibody, VP28-2, was prepared using this protein. The monoclonal antibody prepared in this invention can specifically and sensitively recognize and bind to the VP28-2 envelope protein of whispovirus in crayfish.

[0007] Based on the above work, the present invention provides a monoclonal antibody against white spot virus of crayfish, wherein the CDR1-3 sequences of the heavy chain variable region are shown in SEQ ID NO:3-5, and the CDR1-3 sequences of the light chain variable region are shown in SEQ ID NO:6-8.

[0008] In one specific implementation, the heavy chain variable region sequence of the anti-crayfish white spot virus monoclonal antibody is shown in SEQ ID NO:1, and the light chain variable region sequence is shown in SEQ ID NO:2.

[0009] This invention also provides the application of the above-mentioned anti-crayfish white spot virus monoclonal antibody in detecting crayfish white spot virus in the environment.

[0010] This invention also provides the application of the above-mentioned anti-crayfish white spot virus monoclonal antibody in the preparation of a crayfish white spot virus infection detection agent.

[0011] The present invention also provides the application of the above-mentioned anti-crayfish white spot virus monoclonal antibody in the preparation of a formulation for preventing crayfish white spot virus infection.

[0012] Compared with the prior art, the present invention has the following advantages: (1) A high-affinity anti-VP28 monoclonal antibody was successfully obtained. This invention successfully prepared the hybridoma cell line VP28-2 using hybridoma technology and purified the monoclonal antibody VP28-2 from it. This antibody is an IgG1 subtype with an affinity constant Ka of 1.28 × 10⁻⁶. 6 (1 / MS), dissociation constant KD is 2.90 × 10⁻⁶. -10 M indicates that the monoclonal antibody VP28-2 has a very high affinity for the VP28 protein and can stably and specifically bind to the target antigen, laying a good foundation for subsequent detection and prevention applications.

[0013] (2) Specific detection of white spot virus in crayfish was achieved. The monoclonal antibody VP28-2 described in this invention was verified by Western blot analysis. It showed a clear, specific band at approximately 28 kDa in the total protein of the gill tissue of crayfish infected with white spot disease virus (WSV), while no band was detected in the uninfected control group. These results indicate that the monoclonal antibody can be used for the specific immunodetection of WSV in crayfish, exhibiting good detection sensitivity and specificity.

[0014] (3) It has good virus neutralizing activity. The monoclonal antibody VP28-2 described in this invention was verified through neutralization experiments to effectively block viral infection after pre-incubation with the virus. No infection events occurred in the experimental group of crayfish, while the infection rate in the control group was approximately 70%. This demonstrates that the monoclonal antibody has good neutralizing ability against white spot virus in crayfish and can be used for passive immunization.

[0015] (4) It has a significant preventive and therapeutic effect on white spot virus infection in crayfish. This invention, through a crayfish immersion challenge experiment, demonstrated that after adding the aforementioned monoclonal antibody to the culture water, the mortality rate of crayfish in the experimental group after 14 days did not exceed 35%, and only some showed white spot symptoms; while the mortality rate in the control group exceeded 80%, and the vast majority showed white spot symptoms. This indicates that adding this monoclonal antibody to the water can significantly improve the resistance of crayfish to white spot virus, significantly reduce the infection rate and mortality rate of crayfish white spot virus, and has a good preventive and therapeutic effect on white spot virus disease.

[0016] (5) A novel strategy for the prevention and control of white spot virus in crayfish is provided. This invention is the first to apply anti-VP28 monoclonal antibody to the prevention and control of white spot virus in crayfish. Passive immune protection of crayfish can be achieved by immersion in water. The operation is simple, requires no injection, and is suitable for large-scale aquaculture. It provides a new technical means for the prevention and control of white spot virus disease in aquaculture. Attached Figure Description

[0017] Figure 1 Western blot images showing the detection of white spot virus in crayfish using the monoclonal antibody VP28-2. Lane 1 shows proteins prepared from crayfish tissue infected with the virus, lane 2 shows total proteins from gill tissue of uninfected crayfish, and lane M is a protein marker.

[0018] Figure 2 This study investigated the infection of crayfish with white spot virus after incubation with monoclonal antibody VP28-2.

[0019] Figure 3 A graph showing the mortality rate of patients who underwent immersion challenge experiments. Detailed Implementation

[0020] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0021] 1. Obtaining hybridoma cells The gene encoding amino acids 28-204 of the VP28 protein was cloned into the pGEX-4T-1 vector, transformed into E. coli (DE3), and positive clones were selected for prokaryotic expression and protein purification.

[0022] Balb / c mice were immunized with purified protein. After the third immunization, serum was collected for ELISA testing. Mice with a titer of 1:64000 or higher were used for shock immunization.

[0023] One week after the initial immunization, mouse spleen cells were fused with SP2 / 0 myeloma cells. The fusion supernatant was analyzed using ELISA with purified VP28 protein. Cells from positive wells were used for subclonal selection. The OD of the positive control was [not specified]. 450 The value is 1.21, so OD is selected. 450 Hybridoma cells with values ​​close to or exceeding those of the positive control were selected as candidate hybridoma cells.

[0024] Hybridoma cell cloning was performed using a limiting dilution method: hybridoma cells with higher measured values ​​were diluted to approximately 1 cell / 0.1 ml, and 100 μl was seeded into each well of a 96-well plate. ELISA was performed after the cell supernatant turned yellow. The resulting hybridoma cell line VP28-2 exhibited good growth characteristics and stable antigen-binding ability. It was then expanded and cryopreserved for subsequent experiments.

[0025] 2. Purification, identification, and sequencing of monoclonal antibody VP28-2 Hybridoma cells that tested positive for digestion were prepared into a cell suspension and injected intraperitoneally into mice (approximately 1 × 10⁻⁶ cells). 6 (Cells / mouse). After the mice's abdomens swelled, ascites fluid was extracted, and the antibodies were purified by affinity chromatography.

[0026] Analysis revealed that the antibody type was IgG1. Its affinity constant was determined, and the affinity analysis results are as follows: Ka = 1.28 × 10⁻⁶. 6 (1 / MS), Kd=3.72×10 -4 (1 / S), KD=2.90×10 -10 (M). It can be seen that the monoclonal antibody VP28-2 has a high affinity for the antigen VP28 protein.

[0027] Total RNA was extracted from the hybridoma cell line VP28-2 and reverse transcribed to obtain cDNA. PCR amplification was performed using IgG1 subtype-specific primers, and the product was ligated into the pMD18-T vector, transformed into *E. coli*, and positive clones were selected for sequencing analysis. The results showed that the heavy chain variable region sequence of the monoclonal antibody VP28-2 is shown in SEQ ID NO:1, and the corresponding three complementarity-determining regions (CDRs) 1-3 are shown in SEQ ID NO:3-5, respectively; the light chain variable region sequence is shown in SEQ ID NO:2, and the corresponding three complementarity-determining regions (CDRs) 1-3 are shown in SEQ ID NO:6-8, respectively.

[0028] 3. Western blot detection of white spot virus in crayfish Total protein was extracted from the gill tissue of crayfish infected with white spot virus and prepared as an electrophoresis sample. The sample was separated by 5-20% continuous SDS-PAGE gel electrophoresis. After electrophoresis, the gel was equilibrated in transfer buffer (25 mM Tris pH 8.3; 192 mM glycine; 20% methanol) for 10 min, and then transferred to a PVDF membrane at 80 V for 40 min. The membrane was blocked with 5% skim milk powder (dissolved in TBST) at room temperature for 1 h. A monoclonal antibody (1:4000 dilution) was added as the primary antibody and incubated at room temperature for 2 h. The membrane was washed three times with TBST and incubated with horseradish peroxidase-labeled goat anti-mouse IgG as the secondary antibody for 2 h. After washing, the membrane was developed using an ECL chemiluminescence immunoassay kit. Total protein from the gill tissue of uninfected crayfish was used as a control.

[0029] The results are as follows Figure 1 As shown, no bands were detected in the control group (lane 2), while a clear specific band was detected at approximately 28 kDa in the protein prepared from crayfish tissue infected with the virus, indicating that the monoclonal antibody VP28-2 can be used to specifically detect white spot virus in crayfish.

[0030] 4. Neutralization Experiment The white spot disease virus of crayfish was incubated with monoclonal antibody VP28-2, and then crayfish were infected. The virus without monoclonal antibody was used as the control group.

[0031] The results are as follows Figure 2 As shown, the infection rate of crayfish in the control group was approximately 70%, and no infection events occurred in the experimental group. This demonstrates that the monoclonal antibody VP28-2 has good neutralizing ability against crayfish white spot virus.

[0032] 5. Crayfish soaking challenge test The monoclonal antibody was added to the crayfish culture water, and the crayfish were soaked in it for 4 hours. Then, white spot virus dilution was added to the crayfish culture water to make the virus concentration 1×10⁻⁶. 6 Copies / mL, continue soaking for 24 h, with no monoclonal antibody added as a control.

[0033] After soaking, remove the shrimp, rinse them quickly with disinfectant water, and transfer them to a normal breeding tank; replace the water with fresh breeding water and begin normal breeding.

[0034] Observe the crayfish farming situation, and count the number of deaths after 14 days. The results are as follows: Figure 3 As shown, after 14 days, the mortality rate in the control group exceeded 85%, and the vast majority of crayfish exhibited white spot symptoms, while the mortality rate in the experimental group was less than 35%, with only some crayfish showing white spot symptoms. This indicates that adding monoclonal antibodies to the culture water can prevent and treat white spot virus infection.

[0035] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A monoclonal antibody against the envelope protein VP28 of crayfish white spot virus, characterized in that, It includes a heavy chain and a light chain. The CDR1-3 sequences of the variable region of the heavy chain are shown in SEQ ID NO:3-5, and the CDR1-3 sequences of the variable region of the light chain are shown in SEQ ID NO:6-8.

2. The monoclonal antibody against crayfish white spot virus envelope protein VP28 according to claim 1, characterized in that, The heavy chain variable region sequence is shown in SEQ ID NO:1, and the light chain variable region sequence is shown in SEQ ID NO:

2.

3. The application of the monoclonal antibody against the VP28 envelope protein of crayfish white spot virus as described in claim 1 in the preparation of a reagent for detecting crayfish white spot virus infection.

4. The use of the monoclonal antibody against the VP28 envelope protein of crayfish white spot virus as described in claim 1 in the preparation of a formulation for preventing crayfish white spot virus infection.

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