Preparation method of shrimp iridovirus div1 neutralizing antibody and application thereof

Abstract

The application provides a preparation method of a shrimp iridovirus DIV1 neutralizing antibody and application thereof. The gene sequence shown as SEQ ID NO. 1 and the amino acid sequence shown as SEQ ID NO. 2 are used to solve the problem that there is no neutralizing antibody for the shrimp iridovirus DIV1 at present. The antibody for the shrimp iridovirus DIV1 prepared by the application can specifically recognize the DIVI virus and has good virus neutralizing activity, can effectively reduce the death of shrimps caused by DIV1 virus infection, significantly improve the ability of shrimps to resist DIV1 virus infection, and effectively prevent the infection and transmission of the virus.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of molecular immunology and pathogenic biology, specifically relating to a method for preparing a neutralizing antibody against shrimp iridovirus DIV1 and its application. Background Technology

[0002] Shrimp iridovirus disease is an acute infectious disease caused by a new genus within the family Iridoviridae: Decapod iridescent virus 1 (DIV1). DIV1 is a cytoplasmic virus with linear double-stranded DNA. The virus exhibits an iridescent appearance under oblique light, hence its name. Shrimp infected with DIV1 exhibit symptoms such as a pale hepatopancreas, empty intestines and stomachs, and softened carapace, which can cause widespread mortality within a short period. Currently, shrimp farming primarily focuses on prevention of shrimp iridovirus, such as strict pond disinfection and timely waste collection and discharge. There are currently no effective control measures specifically for shrimp iridovirus disease.

[0003] Monoclonal antibody preparations possess excellent targeting properties. Current methods for preventing and treating shrimp diseases using monoclonal antibodies primarily involve developing neutralizing antibodies that target pathogens. These are immunoglobulins secreted by adaptive immune response cells that specifically bind to corresponding pathogens. The antibodies neutralize pathogens that invade cells or neutralize toxins produced by pathogens, thereby preventing viral infection. Recent studies have confirmed the effectiveness of monoclonal antibody preparations in preventing and treating various major shrimp farming diseases. For example, Chinese patent application CN 114702574 A discloses a method for preparing a single-chain antibody against the WSSV virus envelope protein VP28 and its application. This antibody can specifically recognize WSSV virus particles and effectively neutralize the virus, significantly reducing shrimp mortality caused by viral infection. Another example is Chinese patent application CN 110776565 A, which discloses an egg yolk antibody against Vibrio parahaemolyticus. This antibody can efficiently bind to pathogenic Vibrio, inhibit the growth of Vibrio parahaemolyticus, and has excellent preventive and therapeutic effects on premature mortality syndrome in shrimp caused by Vibrio parahaemolyticus infection. Currently, there is no existing technology for developing neutralizing antibodies specifically targeting DIV1 virus. Based on this, this invention is proposed. Summary of the Invention

[0004] This invention provides a method for preparing a neutralizing antibody against shrimp iridovirus DIV1 and its application, aiming to solve the problems mentioned in the background art.

[0005] This invention provides a method for preparing a neutralizing antibody against shrimp iridovirus DIV1 and its application. The technical solution is as follows:

[0006] A gene sequence of a neutralizing antibody against shrimp iridovirus DIV1 is shown in SEQ ID NO.1.

[0007] The amino acid sequence of a neutralizing antibody against shrimp iridovirus DIV1 is shown in SEQ ID NO.2.

[0008] A method for preparing a neutralizing antibody against shrimp iridovirus DIV1 includes the following steps:

[0009] S1: Using the striped bamboo shark as the immunization target, shrimp iridovirus DIV1 was used as the antigen for immunization. Total RNA was extracted from the peripheral blood lymphocytes of the immunized shark, reverse transcribed into cDNA, and the cDNA was used as a template to amplify the shark-derived single-domain antibody vNAR fragment of the striped bamboo shark.

[0010] S2: The amplified shark-derived single-domain antibody vNAR gene fragment was combined with a phage vector to construct a recombinant phage vector. The recombinant phage vector was transformed into competent cells and cultured to obtain the DIV1 phage antibody library.

[0011] S3: Screen for positive clone D70 from the DIV1 phage antibody library and perform sequencing analysis on the gene sequence of positive clone D70;

[0012] S4: Amplify the vNAR gene fragment contained in D70, clone the amplified product into the expression vector to obtain the recombinant expression vector, transform the recombinant expression vector into competent cells, screen out positive strains, expand the culture, add an inducer to induce expression, collect and purify the antibody protein, that is, the neutralizing antibody against shrimp iridovirus DIV1.

[0013] The beneficial effects of the embodiments of the present invention are as follows:

[0014] 1. This invention provides a neutralizing antibody against shrimp iridovirus DIV1, employing the nucleotide sequence shown in SEQ ID NO.1 and the amino acid sequence shown in SEQ ID NO.2, thus addressing the current lack of neutralizing antibodies targeting shrimp iridovirus DIV1. The neutralizing antibody against shrimp iridovirus DIV1 prepared by this invention can specifically recognize DIV1 virus and exhibits good virus neutralizing activity, effectively reducing shrimp mortality caused by DIV1 virus infection, significantly improving shrimp's resistance to DIV1 virus infection, and effectively preventing virus infection and transmission.

[0015] 2. The present invention provides the application of a neutralizing antibody against shrimp iridovirus DIV1 in the preparation of feed for the prevention and treatment of shrimp iridovirus DIV1. Experiments show that using the neutralizing antibody against shrimp iridovirus DIV1 as a feed additive and orally feeding shrimp feed containing the neutralizing antibody can reduce the mortality rate of shrimp infected with iridovirus. Attached Figure Description

[0016] Figure 1 A schematic diagram of the SDS-PAGE gel electrophoresis results of the neutralizing antibody against DIV1 prepared according to the present invention;

[0017] Figure 2 A schematic diagram of the results of an ELISA assay to detect the recognition of natural DIV1 virus by neutralizing antibodies prepared for use;

[0018] Figure 3 This is a schematic diagram showing the results of Western Blot detection of the specific recognition of the prepared neutralizing antibody against the DIV1 virus.

[0019] Figure 4 The results of immunogenicity testing of the prepared neutralizing antibodies;

[0020] Figure 5 The results show the effectiveness of the prepared neutralizing antibody in neutralizing DIV1 virus.

[0021] Figure 6 The results show the mortality rate of shrimp infected with DIV1 virus via oral administration.

[0022] Figure 7 The results show the therapeutic effect of oral feeding DIV1 neutralizing antibodies on shrimp iridovirus disease. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with specific embodiments of the present application and with reference to the accompanying drawings.

[0024] Example 1:

[0025] Single-domain antibodies (SdAbs) contain only a single variable region (VH / VL). Like conventional antibodies, they can selectively bind to specific antigens. Single-domain antibodies have a molecular weight of only 12-15 kDa, much smaller than ordinary antibodies (150-160 kDa) which consist of two heavy chains and two light chains.

[0026] Shark-derived single-domain antibodies, also known as shark-derived variable domains of immunoglobulin new antigen receptors (vNARs), are antigen-binding domains obtained through genetic engineering based on naturally occurring immunoglobulin new antigen receptors (IgNARs) in sharks. With a molecular weight of only 12 kDa, they are the smallest known antigen-binding domains in vertebrates. Shark-derived single-domain antibodies possess unique advantages such as small molecular weight, high stability, ease of genetic engineering modification, and suitability for large-scale production, making them excellent raw materials for developing molecular probes, disease diagnostic kits, and therapeutic drugs.

[0027] A method for preparing a neutralizing antibody against shrimp iridovirus DIV1 includes the following steps:

[0028] 1. Construction of phage antibody library:

[0029] (1) Striped bamboo sharks were immunized with purified shrimp iridovirus DIVI virus. The specific method is as follows: 5x10 6 A dose of DIV1 virus (copies / kg) was administered to striped bamboo sharks via subcutaneous injection at multiple sites, which was considered one immunization. A total of six immunizations were administered, with a 14-day interval between adjacent immunizations.

[0030] (2) After completing the immunization in step (1), peripheral blood mononuclear cells of the immunized shark were collected. Total RNA was extracted from these immunized shark peripheral blood mononuclear cells using the QIAGEN RNA extraction kit, and then analyzed using the PrimeScript assay from TAKARA. TM The II 1st Strand cDNASynthesis Kit reverse transcribed the total RNA from the immune shark peripheral blood mononuclear cells into cDNA.

[0031] (3) Using the striped bamboo shark vNAR gene amplification specific primers we previously designed, among which;

[0032] Upstream primer: CGTGGCCCAGGCGGCCGGGCCCCCTGGTTACCAAATGT;

[0033] Downstream primer: CGTGGCCCAGGCGGCCGGGCCCTTTGCCAGGTTTCACAGTCAG; Using the cDNA obtained in step (2) as a template, the vNAR gene fragment was obtained by PCR amplification. The amplified and purified vNAR gene fragment and the phage vector pComb3XSS were digested with Sfi I restriction endonuclease and then ligated to construct a recombinant phage vector.

[0034] (4) The recombinant phage vector constructed in step (3) is transferred into XL1-Blue competent cells by electroporation. XL1-Blue cells can ensure the stable replication of high-copy plasmids, thereby obtaining a primary antibody library.

[0035] (5) Take an appropriate amount of the primary antibody library obtained in step (4) and inoculate it into 100 mL of 2xYTG medium (containing 2% glucose, 100 μg / mL ampicillin and 50 μg / mL tetracycline), and culture at 37℃ and 250 rpm with shaking until the bacterial density OD600 reaches 0.5.

[0036] Add 10 12 PFU VCSM13 helper phage was incubated at room temperature for 15 min, then cultured at 37°C with shaking at 150 rpm for 1 h; the cells were collected by centrifugation at 4000 rpm for 20 min.

[0037] Use 200 mL of 2xYTG amp+tet+kana The culture medium (containing 2% glucose, 100 μg / mL, 50 μg / mL tetracycline and 50 μg / mL kanamycin) was resuspended and cultured at 37°C with shaking at 250 rpm for 16 h.

[0038] Transfer the cultured bacterial culture to a sterile centrifuge tube and centrifuge at 10,000 rpm and 4°C for 20 min. Collect the supernatant and add polyethylene glycol / sodium chloride (PEG / NaCl) solution to the supernatant. The volume ratio of the added PEG / NaCl solution to the supernatant is 1:5. Incubate at 4°C on ice for 1 h to precipitate the phage. Centrifuge at 10,000 rpm and 4°C for 20 min and collect the precipitate. Dissolve the precipitate in 10 mL of phosphate buffer. This is the DIV1 phage antibody library.

[0039] 2. Sieving:

[0040] The DIV1 phage antibody library underwent three rounds of screening and enrichment. The first round of screening and enrichment methods are as follows:

[0041] (1) Set the copy number to 1x10 7 Dissolve copies of DIV1 virus particles in 2 mL of phosphate buffer, transfer to an immunoassay tube, pour in coating solution, and incubate overnight at 4°C.

[0042] (2) Discard the coating solution, wash 3 times with phosphate buffer, add 3 mL of 2% bovine serum albumin (BSA) solution, and slowly shake at room temperature for 2 hours to block.

[0043] (3) Wash the immunotubes three times with phosphate buffer, add 1 mL of the phage antibody obtained in step 1, and incubate at room temperature with gentle shaking for 2 h;

[0044] (4) After washing the immunoturbine tubes 10 times with phosphate Tween buffer solution (PBST), add 2 mL of L-lycine-HCl glycine hydrochloride (pH=2.0) to the immunoturbine tubes, shake slowly at room temperature for 15 minutes, add 0.2 mL of Tris-HCl (pH=9.0) to adjust the pH to about 7.5, then add 5 mL of XL1-Blue bacterial culture (OD600=0.6), transfer to a 50 mL centrifuge tube, and incubate at 37 °C with shaking for 1 h;

[0045] (5) Add a copy number of 10 12 Copies of VCSM13 helper phage were incubated at room temperature for 30 minutes, then cultured with shaking at 150 rpm and 37°C for 1 hour. The precipitate was then centrifuged at 4000 rpm for 20 minutes, collected, and resuspended in 100 mL of 2×YTG. amp+tet+kana Culture medium, shaken at 37°C for 16 h at 250 rpm;

[0046] (6) Transfer the cultured bacterial solution to a sterile centrifuge tube, centrifuge at 10,000 rpm and 4°C for 20 min, collect the supernatant, add PEG / NaCl solution to the supernatant with a volume ratio of 1:5, and incubate at 4°C on ice for 1 h to precipitate the phage; centrifuge at 10,000 rpm and 4°C to collect the precipitate, and then dissolve the phage with 5 mL of PBS.

[0047] The subsequent second and third rounds of screening and enrichment steps are the same as the first round.

[0048] 3. Screening for positive recombinant antibodies:

[0049] Phage ELISA, a qualitative and quantitative method for detecting immune responses based on the specific binding of antigens and antibodies, is used to screen for antigen-positive recombinant antibodies. The specific method is as follows:

[0050] (1) Take a 72-well culture plate and add 0.4 mL of 2×YTG to each well. amp+tet+kana Culture medium;

[0051] (2) Randomly select antibody single colonies from the three-level antibody library (i.e., the DIV1 antibody library obtained after three rounds of screening), inoculate them into the above culture plate, label it as Master Plate, and incubate with shaking at 250 rpm and 37°C overnight.

[0052] (3) Take another 72-well culture plate and take 0.4 mL of a solution containing 1 x 10⁻⁶ ppm. 10 2xYTG of pfu VCSM13 helper phage amp +tet+kanaAdd culture medium to each well; take 50 μL of culture medium from each well of the Master Plate in step (2) and add it to the corresponding well, label it P1 plate, and incubate with shaking at 150 rpm and 37℃ for 2 h; centrifuge at 4000 rpm for 20 min, discard the supernatant, and add 0.4 mL of 2×YT to each well. amp+tet+kana Culture medium, incubate overnight at 37℃ and 250 rpm with shaking; after centrifugation at 4000 rpm for 20 min, collect the supernatant and store at 4℃ for later use. This completes the preparation of phage recombinant antibody.

[0053] (4) The number of copies is 5x10 5 Copies of DIV1 virus were added to a 96-well microplate, coating solution was poured in, and the plate was incubated overnight at 4°C. The coating solution was then removed, and the plate was washed three times with PBS. 2% bovine serum albumin (BSA) solution was added, and the plate was slowly shaken at room temperature for 2 hours to block the virus. The phage recombinant antibody prepared in step (3) was added accordingly, and the plate was slowly shaken at room temperature for 2 hours to incubate. The plate was washed six times with PBST, and the enzyme-labeled secondary antibody Anti-M13-HRP (diluted with PBST at a ratio of 1:5000) was added. The plate was slowly shaken at room temperature for 1 hour to incubate. The plate was washed six times with PBST, and 100 μL of TBM colorimetric solution was added to each well. After incubation in the dark for 10 minutes, 100 μL of 2M H2SO4 was added to each well to stop the colorimetric reaction. The plate was read at OD450 nm using a microplate reader. Positive clones were selected based on the OD450 value of the experimental group being more than three times higher than that of the negative control group. The positive clones were sequenced and analyzed. The positive clones were named D70.

[0054] The nucleotide sequence of D70 (SEQ ID NO.1) is as follows:

[0055] ATGAATATTTTCTGTTTTCGTGCCTTGTAGCCTGGTTACCAAATGTCTTCACTGCACGGGTTGAACAAACCACCGACAACGACAACAAAGGAGGCAGGCGAATCACTGACCATCAATTGCGTCCTAAGAGATTCCAACTGTGCGGTGGATAGCACGAACTGGTATTTCACAAAAAAGGGCGCAACAAAGAAG GAGAGCTTTTCAAATGGCGGACGATACGCGGAAACAGTGAACAAGGCATCAAAGTCCTTTTCTTTGCGAATTAGTGACCTAAGAGTTGAAGACAGTGGTACATATCACTGTAAAGCGTATAGTTGCCGCGGACTGCTTACTGGGGGCGATTATGAAGGAGGCGGCACCATTCTGACTGTGAAACCTGGCAAA

[0056] The amino acid sequence of D70 (SEQ ID NO.2) is as follows:

[0057] MNIFLFSCLVAWLPNVFTARVEQTPTTTTKEAGESLTINCVLRDSNCAVDSTNWYFTKKGATKKESFSNGGRYAETVNKASKSFSLRISDLRVEDSGTYHCKAYSCRGLLTGGDYEGGGTILTVKPGK

[0058] 4. Expression of DIV1 neutralizing antibody

[0059] Based on the sequencing results, primers were designed to amplify the vNAR gene fragment of the neutralizing antibody D70, and the fragment was cloned into the Pet30a expression vector using homologous recombination. The recombinant expression vector was transformed into E. coli Shuffle T7 competent cells. After confirming the positive strain, the cells were inoculated into LB medium containing kanamycin resistance and cultured at 200 rpm until the OD600 reached 0.6-1.0. IPTG solution with a final concentration of 0.5 mmol / L was added, and expression was induced at 200 rpm and 18℃ for 18 h. After induction, the bacterial cells were collected by centrifugation, and the cells were disrupted by sonication. The supernatant was collected after centrifugation, and the antibody protein was purified using conventional His-Tag affinity chromatography. The purified recombinant antibody protein of D70 had a purity >90%. The SDS-PAGE electrophoresis results are shown below. Figure 1 As shown.

[0060] Example 2:

[0061] Antibody performance testing

[0062] 1. ELISA detection of the recognition of naturally occurring DIV1 virus by neutralizing antibodies prepared in this study.

[0063] Indirect enzyme-linked immunosorbent assay (ELISA) is a qualitative and quantitative detection method for antigen-antibody specific binding and immune reactions. DIV1 virus is serially diluted to a titer of 10. 8 copies / mL, 10 7 copies / mL, 10 6 copies / mL, 10 5 copies / mL, 10 4 copies / mL, 10 3 copies / mL, 10 2 DIV1 virus at different titers (copies / mL and 10 copies / mL) was added to each well of an ELISA plate, followed by coating buffer. The plate was incubated overnight at 4°C. The next day, the coating buffer was discarded, and the plate was washed with PBS. 2% BSA was added, and the plate was blocked at room temperature for 2 hours. After washing three times with PBS, affinity-purified D70 antibody (0.2 μg / mL) was added, and the plate was incubated at room temperature with shaking for 2 hours. After washing six times with PBST, HRP-conjugated mouse anti-striped bamboo shark IgNAR secondary antibody was added, and the plate was incubated at room temperature with shaking for 1.5 hours. After washing with PBST, 100 μL of TBM substrate was added to each well, and the plate was incubated in the dark for 10 minutes. 100 μL of 2M H2SO4 was added to each well to stop the color development. The OD450 value was read at 450 nm using an ELISA reader. The binding was assessed when the OD450 value of the experimental group was more than three times higher than that of the negative control group. The experimental results are as follows: Figure 2 As shown, the experimental results indicate that the D70 antibody can specifically bind to the natural DIV1 virus, and the detection sensitivity is 10. 3 copies / mL.

[0064] 2. Western blot analysis of antibody specificity for DIV1 virus recognition

[0065] Western blotting is an immunoassay for specific proteins, with a copy number of 1 x 10⁻⁶. 6The DIV1 virus particles from copies were used as the sample. After SDS-PAGE electrophoresis, the protein was transferred to a PVDF membrane using a semi-dry transfer method. The membrane was blocked with 5% skim milk powder and incubated at room temperature with shaking for 2 hours. Affinity-purified D70 antibody (0.2 μg / mL) was added, and the membrane was incubated at room temperature with shaking for 2 hours. After washing 6 times with TBST, HRP-conjugated mouse anti-striped bamboo shark IgNAR secondary antibody was added, and the membrane was incubated at room temperature with shaking for 1.5 hours. After washing 5 times with TBST, equal volumes of ECL chemiluminescence solutions A and B were mixed and poured onto the membrane. The membrane was then exposed and developed using a chemiluminescent gel imaging system (BIORAD ChemiDoc XRS). The experimental results are as follows: Figure 3 As shown, there is a distinct specific band between 70-100 kDa, indicating that the prepared D70 neutralizing antibody can specifically recognize DIV1 virus.

[0066] 3. Mass spectrometry analysis was used to identify the structural proteins of the DIV1 virus bound by the prepared antibodies.

[0067] With a copy number of 1x10 6 Copies of DIV1 viral particles were used as the sample. After SDS-PAGE electrophoresis, the electrophoretic gel was stained with Coomassie Brilliant Blue. A single electrophoretic band with a molecular weight of 70kDa–100kDa was cut from the gel with a clean scalpel and placed in an EP tube for mass spectrometry identification by Shanghai Apticon Biotechnology Co., Ltd. The mass spectrometry results showed that the D70 neutralizing antibody specifically recognizes the DIV1 viral structural protein Swarming motility protein YbiA.

[0068] 4. Immunogenicity test of the prepared neutralizing antibodies

[0069] Experimental and control groups were set up. The experimental group was injected with D70 neutralizing antibody at a concentration of 200 μg / mL, while the control group was injected with BSA solution of the same concentration, with an injection dose of 20 μL for both groups. The shrimp used in the experiment were purchased from a shrimp farm in Zhangzhou, Fujian Province, and all shrimp were 10 cm in size and tested negative for DIV1 before the experiment. After being temporarily held for 96 hours, the immunogenicity of D70 neutralizing antibody was tested by subcutaneous injection in the abdominal segments, with 30 shrimp in each group. The survival rate of the shrimp was observed and recorded every 24 hours after injection, and the survival rate curve was calculated and plotted. The experimental results are as follows: Figure 4 As shown, 14 days after injection, the survival rate of shrimp in the experimental group was 83.3% (25 / 30), and the survival rate of shrimp in the control group was 86.7% (26 / 30). The deaths of shrimp in both the experimental and control groups were due to natural attrition, indicating that the D70 neutralizing antibody has low immunogenicity when injected into live shrimp and will not cause physiological toxicity to shrimp.

[0070] 5. Detection of the neutralizing effect of the prepared neutralizing antibody on DIV1 virus

[0071] Ninety healthy and vigorous shrimp were selected and divided into three groups: an experimental group, a positive control group, and a negative control group, with 30 shrimp in each group. All shrimp tested negative for DIV1 before the experiment. Specific treatment methods are as follows:

[0072] Experimental group:

[0073] 20 μL 1x10 8 A DIV1 virus solution of copies / μL and 20μL of antibody solution were mixed (antibody concentration was 200μg / mL). After incubation in a metal bath at 200rpm and 30℃ for 2h, the mixture was injected subcutaneously into the abdominal segments.

[0074] Positive control group:

[0075] 20 μL 1x10 8 A solution of DIV1 virus (copies / μL) and 20 μL of BSA solution (concentration of 100 μg / mL) were mixed and incubated in a metal bath at 200 rpm and 30°C for 2 h before being injected subcutaneously into the abdominal segments.

[0076] Negative control group:

[0077] Take 40 μL of BSA (concentration of 100 μg / mL), incubate it in a metal bath at 200 rpm and 30℃ for 2 h, and then inject it subcutaneously into the abdominal segment;

[0078] After injection, shrimp survival was observed and recorded every 24 hours for 14 consecutive days. Survival rate curves were calculated and plotted. Experimental results are as follows: Figure 5 As shown, the mortality rate in the positive control group reached 50% on day 4 after injection and 100% on day 8; in the experimental group, 1 animal died on day 2, 2 on day 5, 4 on day 7, and 2 on day 8, for a total of 9 deaths, with a survival rate of 70%; in the negative control group, 3 animals died during the observation period, which was considered natural attrition, with a survival rate of 90%. These results indicate that the prepared neutralizing antibody has a good neutralizing effect on DIV1 virus.

[0079] Example 3:

[0080] Detection of the therapeutic effect of the prepared neutralizing antibody on shrimp iridovirus disease

[0081] 1. Experiment on oral infection of shrimp with DIV1 virus

[0082] Sixty healthy and vigorous shrimp were selected and divided into two groups: an experimental group and a control group, with 30 shrimp in each group. All shrimp tested negative for DIV1 before the experiment. The experimental group was fed 12g of DIV1-infected shrimp per meal, while the control group was fed the same amount of healthy shrimp meat twice daily. Feeding was stopped after 5 days, and shrimp survival was observed and recorded every 24 hours for 14 days. The mortality rate curve was calculated and plotted. The experimental results are as follows: Figure 6 As shown, the experimental group began to die on the 3rd day after feeding was stopped, the mortality rate exceeded 50% on the 8th day and exceeded 90% on the 9th day; the control group had a total of 5 deaths during the observation period, with a mortality rate of 16.7%, which was attributed to natural attrition.

[0083] 2. Detection of the therapeutic effect of neutralizing antibodies prepared by oral feeding on shrimp iridovirus disease.

[0084] The prepared anti-DIV1 virus neutralizing antibody was used as a feed additive to determine its therapeutic effect against shrimp iridovirus disease; the specific treatment method is as follows:

[0085] Weigh out 100g of ordinary shrimp feed per group, and spray a small amount of distilled water onto the feed to slightly moisten it; dilute 10mg of the prepared neutralizing antibody with distilled water to 10mL; mix the diluted neutralizing antibody solution with the feed (mixing amount is 1‰), dry it, add 5g of seaweed powder as a binder, mix it again, dry it, and store it at -80℃. Before feeding, raise it to room temperature.

[0086] Sixty healthy and vigorous shrimp were selected and divided into two groups: an experimental group and a control group, with 30 shrimp in each group. All shrimp tested negative for DIV1 before the experiment. The experimental group was fed shrimp feed containing DIV1 neutralizing antibodies, while the control group was fed normal shrimp feed. Feeding was done twice daily, with uneaten feed removed one hour after feeding, for seven consecutive days. Starting on the eighth day, shrimp infected with DIV1 were fed for infection for five consecutive days (during infection, the experimental group was fed two meals of infected shrimp, followed by one meal of shrimp feed containing DIV1 neutralizing antibodies, and the control group was fed one meal of normal shrimp feed). After infection, the shrimp were fed shrimp feed containing DIV1 neutralizing antibodies for another three days, then the normal shrimp feed was resumed. Shrimp survival was observed and recorded every 24 hours after infection began, for 14 consecutive days. The survival rate curve was calculated and plotted. The experimental results are as follows: Figure 7 As shown, mortality began in the experimental group on the 4th day after infection, with a total of 12 deaths and 18 survivors, resulting in a survival rate of 60%. Mortality also began in the control group on the 4th day after infection, with a mortality rate exceeding 50% on the 9th day and reaching 90% on the 10th day. These results indicate that the neutralizing antibodies prepared by oral feeding have a good effect on the treatment of shrimp iridovirus disease.

[0087] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention specification, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

[0088]

[0089]

Claims

1. A shrimp iridescent virus DIV1 neutralizing antibody, characterized in that, The gene sequence of the neutralizing antibody against the shrimp iridescent virus DIV1 is shown as SEQ ID NO. 1, and the amino acid sequence is shown as SEQ ID NO.

2.

2. Use of the neutralizing antibody against the shrimp iridescent virus DIV1 according to claim 1 in the preparation of a feed additive for preventing and treating the shrimp iridescent virus DIV1.

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