Paralichthys olivaceus rhabdovirus recombinant single-chain variable region antibody and application thereof

Abstract

The application discloses a Paralichthys olivaceus rhabdovirus recombinant single-chain variable region antibody and application thereof, and belongs to the field of molecular immunology. The recombinant single-chain variable region antibody comprises a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 4, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 5. The application is characterized in that the G protein B cell antigen epitope of the Paralichthys olivaceus rhabdovirus G protein gene is obtained through precise screening, the epitope sequence is used as an antigen to prepare a hybridoma cell, and the specific single-chain antibody is efficiently produced by using an E. coli expression system. The recombinant single-chain antibody can specifically recognize and combine with HIRRV, targets a key functional region of the virus, effectively neutralizes virus infection, reduces non-specific immune responses, and significantly improves the accuracy of rapid diagnosis, detection and treatment. The application can be used as a Paralichthys olivaceus rhabdovirus infection detection or diagnosis reagent and a targeted blocking drug for Paralichthys olivaceus rhabdovirus.

Description

Technical Field

[0001] This invention relates to a recombinant antibody against turbot rhabdovirus, its preparation method, and its application, specifically to a recombinant single-chain variable region antibody against turbot rhabdovirus and its application, belonging to the field of molecular immunology. Background Technology

[0002] Hirame novirhabdovirus (HIRRV) is a globally widespread infectious pathogen that infects various farmed fish species, particularly turbot, causing severe hemorrhagic septicemia. HIRRV belongs to the genus Novirhabdovirus in the family Rhabdoviridae. It is an enveloped, single-stranded, negative-sense RNA virus with a typically bullet-shaped viral particle. The HIRRV genome is approximately 11 kb in size and encodes five major structural proteins, including nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and RNA-dependent RNA polymerase (L). The G protein is one of the important envelope proteins of HIRRV and has been shown to play a crucial role in viral infection. It participates in the adsorption and fusion of the virus with host cells, making it a key protein for viral invasion of host cells. It also participates in the assembly and release of viral particles.

[0003] The G protein, as one of the important envelope proteins of HIRRV, is a key target for viral detection and diagnosis, as well as a crucial neutralizing target and potential vaccine candidate antigen for the prevention and treatment of HIRRV infection. Previous studies have confirmed that the G protein is an immunoprotective antigen of HIRRV, and HIRRV nucleic acid and subunit vaccines constructed based on this protein can effectively induce the production of neutralizing antibodies in immunized fish, exerting an immunoprotective effect. Therefore, anti-HIRRV-G protein monoclonal antibodies can specifically recognize and bind to the viral G protein and have the potential to neutralize viral virulence. However, the large molecular weight of the G protein and the extreme difficulty in identifying neutralizing sites pose a significant challenge to the preparation of monoclonal antibodies with neutralizing activity. Traditional large-scale production of monoclonal antibodies is costly, time-consuming, and requires sophisticated operations. Furthermore, due to the large molecular weight and strong immunogenicity of structurally intact monoclonal antibodies, their poor tissue penetration and susceptibility to degradation in clinical applications such as virus neutralization and targeted blocking limit their large-scale application and promotion.

[0004] Recombinant single-chain variable fragments (scFvs) have gradually become a research hotspot due to their advantages such as small size, high specificity, ease of genetic engineering, and suitability for large-scale production. Recombinant antibodies are obtained by cloning the variable region gene of monoclonal antibodies and expressing it in bacterial, yeast, or mammalian cells, ultimately yielding small molecule antibody fragments. scFvs are composed of the variable heavy chain (VH) and variable light chain (VL) of the antibody linked by a flexible peptide, and contain six complementarity-determining regions (CDRs), which are the smallest functional units for antibody-antigen binding. Recombinant single-chain variable fragment antibodies not only maintain the antigen-binding activity of the parent monoclonal antibody, but also have a molecular weight only about 1 / 6 that of the complete antibody, exhibiting excellent tissue permeability and showing broad application prospects in clinical diagnosis and treatment.

[0005] Chinese patent application CN112608383A discloses a single-chain antibody against turbot rhabdovirus. This single-chain antibody, ScFv-rHRV, has a molecular weight of approximately 28 kDa and can specifically recognize turbot rhabdovirus, potentially for use in the diagnosis, development of therapeutic agents, and epitope research of this virus. However, it suffers from problems such as low antigen specificity, high production cost, complex process, low production efficiency, and potential immunogenicity risks. Summary of the Invention

[0006] To address the aforementioned problems, the first objective of this invention is to provide a recombinant single-chain variable region antibody against turbot rhabdovirus with small molecular weight, low immunogenicity, high specificity, and virus-neutralizing activity, for the clinical prevention and treatment of turbot rhabdovirus disease, and for the rapid and accurate detection of turbot rhabdovirus.

[0007] A second objective of this invention is to provide a method for preparing the above-mentioned recombinant single-chain variable region antibody against turbot rhabdovirus.

[0008] This invention first uses bioinformatics software (BepiPred and IEDB software, etc.) to analyze and predict B cell antigenic epitopes of turbot rhabdovirus G protein, and selects peptides with dominant B cell antigenic epitopes. Next, a tertiary structure model of turbot G protein is constructed, and peptides located on the surface of the G protein molecule are selected. Finally, multiple peptides with strong antigenicity and located on the surface of the G protein molecule that meet the requirements are spliced ​​together to form turbot rhabdovirus epitope peptides. These peptides are then used to immunize Balb / c mice to obtain mouse B lymphocytes, which are fused with myeloma cells and screened to obtain anti-turbo rhabdovirus G protein monoclonal cells. Total RNA is extracted from the monoclonal cells, reverse transcribed into cDNA, and amplified using degenerate primers with conserved mouse antibody sequences to obtain the heavy chain variable region and light chain variable region sequences of the monoclonal antibody. After adding a promoter, linker, and His tag, the antibody is transformed into a pET-28a prokaryotic vector, transformed into DH5α competent cells, sequenced, and induced to express, yielding the recombinant single-chain variable region antibody against turbot rhabdovirus.

[0009] The objective of this invention is achieved through the following technical solution:

[0010] A recombinant single-chain variable region antibody against turbot rhabdovirus, comprising a heavy chain variable region and a light chain variable region, characterized in that: the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 4, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 5. The coding nucleotide sequence of the heavy chain variable region of the recombinant antibody is shown in SEQ ID NO: 2, and the coding nucleotide sequence of the light chain variable region is shown in SEQ ID NO: 3.

[0011] The complementary regions of the determinant clusters in both the heavy chain variable region and the light chain variable region are composed of CDR1, CDR2, and CDR3. The amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region are shown in SEQ ID NO: 9, SEQ ID NO: 11, and SEQ ID NO: 13, respectively. The amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region are shown in SEQ ID NO: 15, SEQ ID NO: 17, and SEQ ID NO: 19, respectively. The encoding nucleotide sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region are shown in SEQ ID NO: 8, SEQ ID NO: 10, and SEQ ID NO: 12, respectively. The encoding nucleotide sequences of CDR1, CDR2, and CDR3 in the light chain variable region are shown in SEQ ID NO: 14, SEQ ID NO: 16, and SEQ ID NO: 18, respectively.

[0012] A method for preparing a recombinant single-chain variable region antibody against turbot rhabdovirus includes the following steps:

[0013] (1) Bioinformatics software was used to analyze and predict the B cell antigenic epitopes of the turbot rhabdovirus G protein, and peptides with dominant B cell antigenic epitopes were selected. Then, a tertiary structure model of the turbot G protein was constructed, and peptides located on the surface of the G protein molecule were selected. Finally, multiple peptides with strong antigenicity and located on the surface of the G protein molecule that meet the requirements were spliced ​​to form turbot rhabdovirus epitope peptides, the sequence of which is shown in SEQ ID NO:1. (2) Balb / c mice were immunized with the above turbot rhabdovirus epitope peptides to obtain mouse lymphocytes B cells. After fusion with myeloma cells, anti-turbo rhabdovirus G protein monoclonal cells were obtained. (3) Total RNA was extracted from the monoclonal cells, reverse transcribed into cDNA, and amplified using degenerate primers with conserved mouse antibody sequences to obtain the heavy chain variable region and light chain variable region sequences of the monoclonal antibody. After adding promoter, linker and His tag, the cDNA was transformed into pET-28a prokaryotic vector and transformed into BL21(DE3). Competent cells were sequenced and induced to express the recombinant single-chain variable region antibody of turbot rhabdovirus.

[0014] Purified recombinant single-chain variable region antibodies were collected and immunofluorescence assays showed that they specifically bound to the native HIRRV virus G protein, exhibiting a positive fluorescence signal. Western blotting results showed that the single-chain antibody against the epitope peptide of the turbot rhabdovirus G protein could recognize the native turbot rhabdovirus G protein, while the supernatant of myeloma cells (as a control) could not bind to the native HIRRV virus G protein. Virus neutralization assays showed that the single-chain antibody against the epitope peptide of the turbot rhabdovirus G protein could effectively neutralize turbot rhabdovirus and reduce viral virulence.

[0015] The recombinant single-chain variable region antibody against turbot rhabdovirus can specifically react with the 30.2 kDa G protein epitope peptide of turbot rhabdovirus, showing a positive reaction band.

[0016] The recombinant single-chain variable region antibody against turbot rhabdovirus can specifically recognize viral particles in carp epithelioma cells infected with turbot rhabdovirus, exhibiting green fluorescence.

[0017] The recombinant single-chain variable region antibody against turbot rhabdovirus was confirmed by in vitro virus neutralization experiments to have significant neutralizing activity against HIRRV and to delay the pathological progression of virus-infected cells. This antibody effectively neutralizes viral infection and reduces the mortality rate of virus-infected turbot.

[0018] Therefore, the recombinant single-chain variable region antibody against turbot rhabdovirus can be used as a detection or diagnostic reagent for turbot rhabdovirus infection, as well as as a targeted blocking drug for turbot rhabdovirus.

[0019] The advantage of this invention lies in the precise screening of B-cell antigenic epitopes of the G protein obtained by cloning the turbot rhabdovirus G protein gene, and the preparation of hybridoma cells using this epitope sequence as an antigen, employing *Escherichia coli* (E. coli). E. coli This invention utilizes a G protein epitope peptide as an antigen to efficiently produce specific single-chain antibodies (scFv), offering significant advantages over single-chain antibodies produced using purified virus as an antigen via phage technology. The recombinant single-chain antibody developed using this invention specifically recognizes and binds to HIRRV, targeting key functional regions of the virus, effectively neutralizing viral infection, reducing non-specific immune responses, and significantly improving the accuracy of rapid diagnosis, detection, and treatment. E. coli The expression system boasts a simple, short-cycle, and high-yield production process, with costs significantly lower than phage technology, making it suitable for large-scale industrial production and providing an efficient antibody preparation solution for rapid response to the HIRRV epidemic; through E. coli The expressed single-chain antibody has a small molecular weight, low immunogenicity, and excellent tissue permeability, making it suitable for developing in vivo therapeutic antibody drugs and facilitating subsequent functional optimization. Immunological testing and in vitro / in vivo experiments have validated that this recombinant single-chain antibody can effectively recognize and neutralize HIRRV activity, providing a key tool for rapid diagnosis, virus detection, and prevention. It is of significant value for the development of rapid HIRRV diagnostic reagents and HIRRV targeted blocking drugs. Attached Figure Description

[0020] Figure 1 Electrophoresis results of the G protein epitope peptide of recombinant turbot rhabdovirus.

[0021] In this study, lane M represents the molecular weight standard protein; lane 1 is the uninduced strain; lane 2 is the IPTG-induced strain; and lane 3 is the purified recombinant turbot rhabdovirus G protein epitope peptide.

[0022] Figure 2 Cell photographs of EPC cells 72 hours after incubation of supernatants from hybridoma cell lines 2B3, 6D9, 5E4, and 4H3 with HIRRV at room temperature.

[0023] Figure 3 This is an SDS-PAGE result of the recombinant single-chain variable region antibody.

[0024] In this study, lane M represents the molecular weight standard protein; lane 1 represents the uninduced strain; lane 2 represents the IPTG-induced strain; and lane 3 represents the purified recombinant scFv.

[0025] Figure 4 This is a Western blotting result of recombinant single-chain variable region antibody and recombinant G protein epitope peptide.

[0026] In this study, lane M represents the molecular weight standard protein; lane 1 represents the SDS-PAGE results of the genetically engineered strain expressing G protein epitope peptides; lane 2 represents the results of the immune response between the recombinant single-chain variable region antibody and the recombinant G protein epitope peptides; and lane 3 represents the negative control.

[0027] Figure 5 The image shows the results of Western blotting detection of recombinant single-chain variable region antibody and turbot rhabdovirus.

[0028] In this study, lane M represents the molecular weight standard protein; lane 1 represents the SDS-PAGE results of carp epithelioma cells infected with HIRRV; lane 2 represents the immune response results of recombinant single-chain variable region antibody (scFv) and turbot rhabdovirus G protein; lane 3 is the monoclonal antibody positive control; and lane 4 is the negative control.

[0029] Figure 6 The figure shows the results of an indirect immunofluorescence assay analyzing the specific reaction between the recombinant single-chain variable region antibody and HIRRV in carp epithelioma cells.

[0030] In this diagram, A1 represents the binding reaction of recombinant single-chain variable region antibody to HIRRV-infected carp epithelioma (EPC) cells observed under a 40x objective lens; B1 is the fluorescence observation result of myeloma cell supernatant as a control instead of recombinant scFv; A2 and B2 are the fluorescence observation results of DAPI-stained cell nuclei; and A3 and B3 are overlays of A1 and A2, and B1 and B2, respectively.

[0031] Figure 7 The figure shows the results of an in vitro virus neutralization experiment using recombinant single-chain variable region antibodies.

[0032] In the experimental group 1, EPC cells were infected with a mixture of recombinant scFv and HIRRV, and the cytopathic effects were recorded at different time points. In the negative control group 2, EPC cells were infected with a mixture of recombinant scFv and HIRRV, with an equal amount of bovine serum albumin instead of scFv.

[0033] Figure 8 Line graph showing the cumulative mortality rate in in vivo virus neutralization experiments using recombinant single-chain variable region antibodies. Detailed Implementation

[0034] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0035] Example 1: Preparation of recombinant protein from epitope peptides of G protein in the envelope of turbot rhabdovirus and preparation of neutralizing monoclonal antibody

[0036] 1. Preparation of recombinant protein containing G protein epitope peptides from turbot rhabdovirus envelope

[0037] (1) Based on the HIRRV genome sequence published on the NCBI website, the antigenic sites of G protein were predicted using the online tool IEDB (http: / / tools.iedb.org / bcell / ) and BepiPred (BepiPred-3.0). The linear antigenic site sequences of G on B cells were found to be 20-37, 47-75, 82-95, 117-142, 151-153, 171-197, 208-214, 223-244, 274-276, 285-298, 311-313, 325-326, 335-341, 365-391, 404-408, 410-412, 419-428, 436-452, and 488-504.

[0038] (2) Based on the above prediction data, the site of the antigenic peptide was finally determined. First, antigenic epitopes located in signal peptides, transmembrane regions, and intracellular regions were excluded; second, antigenic epitope parameters and predicted potential linear B-cell antigenic epitopes were analyzed; finally, antigenic epitopes located in random coil and β-turn regions of secondary structures were analyzed to screen and determine the specific sequence site of the antigenic peptide. The three-dimensional structure of the turbot rhabdovirus G protein epitope peptide molecule was constructed. The flexible linker GGGGS was used to separate the epitopes to avoid structural conflicts. The final turbot rhabdovirus G protein epitope peptide sequence is as follows:

[0039] VYGQTIKPGVDSVSDQPTGGGGSSLSVGNNLGDIHTQGGGGSVSKDRRTYRAHQTSAFVAWGHPFGDEWGGGGSCPAAKLSKVSPSQLRCPRIFDDENRGLVAGGGGSVLRDPYTLGFLDSDFIEGKCSKSPCQTGGGGSIFVDKTSHHVVKATSYGHHPWGGGGGSLYNDTDTADITIRKIDSGGGGSRRPSMPAAPQEIPMYHLGGGGSTIEKALVEMKLAPREVGVYDTTTASAGGGGSDTTVGMRGSLDDFAGGGGSRSPHPGI (SEQ ID NO:1) is used to prepare G protein-specific monoclonal antibodies.

[0040] The G protein coding sequence described above was synthesized and ligated into the pET-28a plasmid. After confirmation by cloning and sequencing, it was transformed into BL21(DE3) competent bacteria for induced expression. The results are as follows: Figure 1 As shown, the recombinant G protein epitope peptide of approximately 30.2 kDa was successfully induced and purified, which is consistent with the predicted protein size.

[0041] 2. Preparation of neutralizing monoclonal antibodies

[0042] (1) 100 µg of purified recombinant G protein epitope peptide was mixed with an equal volume of Freund's complete adjuvant and emulsified, and then administered intraperitoneally to two BALB / c mice. Two weeks later, the same amount of recombinant G epitope peptide was emulsified with Freund's incomplete adjuvant and administered intraperitoneally to boost the immunization. Then, 100 µg of G epitope peptide was injected into the mice via the tail vein every week. Three days after the last injection, the spleen and thymocytes of the mice were extracted under sterile conditions.

[0043] (2) Mice were anesthetized with ether. Under aseptic conditions, the spleen and thymus were removed, passed through a 100-mesh sieve, and then pipetted with RPMI-1640 medium to form a single-cell suspension. The handling of experimental animals in this invention complies with animal welfare requirements.

[0044] (3) Centrifuge the spleen cell suspension and thymocyte suspension at 1000 rpm for 3 min respectively, discard the supernatant, resuspend the spleen cell pellet in RPMI-1640 medium, and resuspend the thymocyte pellet in RPMI-1640 selective cell culture medium containing 1% HAT (containing 10% fetal bovine serum).

[0045] (4) Mix the spleen cell suspension with the P3-X63-Ag8U1 myeloma cell suspension evenly, centrifuge at 1000 rpm for 3 min, completely remove the supernatant, gently tap the bottom of the centrifuge tube to mix the two cell precipitates thoroughly into a paste; use a pipette to draw 1 ml of polyethylene glycol solution preheated to 37℃ and add it dropwise into the centrifuge tube at a uniform rate within 1 min, and then let it stand in a 37℃ water bath for 5 min; continue to add 15 ml of RPMI-1640 medium preheated to 37℃ to dilute the polyethylene glycol and make it ineffective, add RPMI-1640 medium to 40 ml, centrifuge at 1000 rpm for 3 min, and discard the supernatant.

[0046] (5) Add 1 ml of cell suspension to the prepared thymocyte suspension, mix well and drop into a 96-well culture plate; place the culture plate in an incubator at 37°C and CO2 concentration of 4.5% and observe cell growth under an inverted microscope. After about two weeks, take the hybridoma cell culture supernatant for testing.

[0047] (6) After fusion, when the hybridoma cell population grows to occupy about 1 / 3 of the bottom area of ​​the 96-well culture plate, detection begins. Indirect enzyme-linked immunosorbent assay (ELISA) is used to screen for positive hybridoma cells, including the following steps:

[0048] ① Coating antigen: HIRRV was isolated and purified by differential centrifugation. The HIRRV virus suspension was diluted to a concentration of 50 µg / ml with carbonate coating buffer (pH 9.6) and added to a 96-well microplate (100 µl / well). The plate was coated overnight at 4°C.

[0049] ② Aspirate the coating solution and wash with phosphate-buffered saline (PBST) containing 0.05% Tween-20 for 5 minutes each time, for a total of three washes;

[0050] ③ Add 200µl of 3% bovine serum albumin blocking solution (3% BSA blocking solution) to each well and block at 37℃ for 1 hour;

[0051] ④ Wash three times as described in ②;

[0052] ⑤ Use the hybridoma cell culture supernatant as the primary antibody, add 50 µl to each well of the ELISA plate, and incubate at 37°C for 1 h;

[0053] ⑥ Wash three times as described in ②;

[0054] ⑦ Add 50 µl of alkaline phosphatase-labeled anti-His tag antibody (1:4000 dilution) to each well of the microplate as the second antibody and incubate at 37°C for 1 h.

[0055] ⑧ Wash three times as in ②; then add 100µl of 4-nitrophenol phosphate (pNPP) working solution to each well, react in the dark for 5~20min, add 50µl of 2M NaOH solution to each well, stabilize for 3~5min, and then measure the OD value using a working wavelength of 405nm.

[0056] Using myeloma cell supernatant as a negative control, the absorbance of each well at a wavelength of 405 nm was used to calculate the ratio (P / N) between the absorbance of each experimental well and the negative control. A well was considered positive when P / N ≥ 2.1.

[0057] (7) Cloning: The detected positive hybridoma cells were cloned using the limiting dilution method, and the steps are as follows:

[0058] ① After anesthetizing mice with ether, the thymus was removed under sterile conditions, ground on a 100-mesh sieve, and pipetted with RPMI-1640 medium to form a single-cell suspension;

[0059] ② Centrifuge the thymocyte suspension at 1000 rpm for 3 min, discard the supernatant, and resuspend the thymocyte pellet in 10 ml of RPMI-1640 cell culture medium (containing 10% fetal bovine serum);

[0060] ③ Count the cells in the positive cell wells to be cloned using a hemocytometer, then dilute them 10-fold with culture medium, take out 100 hybridoma cells, and put them into thymocyte suspension;

[0061] ④ Mix the cell suspension evenly with a dropper and add it to a 96-well culture plate, 100 µl per well, with an average of one hybridoma cell per well;

[0062] ⑤ Place in a CO2 incubator for incubation;

[0063] ⑥ Two weeks later, the culture supernatant of hybridoma cells in each well was tested by indirect enzyme-linked immunosorbent assay (ELISA). The hybridoma cells from the positive clone wells were cloned again using the above method to ensure the formation of monoclonal cells.

[0064] (8) Take vigorous, well-morphologically healthy hybridoma cells, prepare a cell suspension, centrifuge at 1000g for 3 minutes, discard the supernatant, and add cryopreservation solution (9 parts RPMI-1640 medium + 1 part dimethyl sulfoxide) to make the final cell density 5×10⁻⁶. 6 Cells / ml: 1 ml of cell suspension was placed into a 2 ml cryovial, the cap was tightened, and then the cryovial was placed in a small box containing cotton. The cryovial was placed in an ultra-low temperature freezer at -80℃ overnight (8~12h), and then immersed in liquid nitrogen for long-term preservation. A total of four hybridoma cell lines were obtained and named 2B3, 6D9, 5E4 and 4H3, respectively.

[0065] Example 2: Screening for HIRRV envelope G protein epitope monoclonal antibodies with virus-neutralizing ability

[0066] To screen for monoclonal antibody strains with significant virus neutralizing ability, the culture supernatant of four hybridoma cell strains (2B3, 6D9, 5E4, and 4H3) prepared in Example 1 was mixed with an equal volume of HIRRV suspension and incubated at room temperature for 1 hour to infect EPC cells. The virus neutralizing activity of different monoclonal antibodies was evaluated by observing the differences in CPE of cells in each experimental group.

[0067] (1) Take the frozen EPC cell line out of the -80°C freezer and immediately put it into a preheated 37°C water bath to thaw. After it is completely thawed, centrifuge at 1000 rpm at room temperature for 3 minutes. Carefully discard the supernatant in a clean bench, add 1 ml of M199 cell culture medium to gently suspend the cells, transfer them to a 24-well cell culture plate, and incubate in a 4.5% CO2 cell culture incubator.

[0068] (2) Mix the hybridoma cell culture supernatant with an equal volume of HIRRV dilution solution and incubate at room temperature for 1 hour to prepare for EPC cell infection.

[0069] (3) After infection, the changes in CPE of cells in different experimental groups were continuously monitored, and the differential cytopathic effects of infected cells in different monoclonal antibody neutralization groups were recorded.

[0070] Results: At 72 hours post-infection, all treatment groups showed varying degrees of CPE. However, the EPC cells treated with 4H3 hybridoma cell culture supernatant showed a significantly delayed onset of CPE, and the number of diseased cells was significantly lower than that of the control group and the other three monoclonal antibodies (e.g., ...). Figure 2 This indicates that the monoclonal antibody produced by hybridoma strain 4H3 has a significant neutralizing effect on HIRRV infection.

[0071] Example 3: Cloning of the recombinant single-chain variable region antibody gene of HIRRV envelope G protein epitope peptide neutralizing monoclonal antibody 4H3

[0072] 1. Resuscitation of hybridoma cell lines, extraction of total RNA, and synthesis of cDNA templates.

[0073] (1) Take the frozen anti-HIRRV-G neutralizing monoclonal antibody 4H3 hybridoma cell line out of the -80°C freezer and immediately put it into a preheated 37°C water bath to thaw. After complete thawing, centrifuge at 1000 rpm at room temperature for 3 minutes. Carefully discard the supernatant in a clean bench, add 1 ml of preheated 37°C GIT cell culture medium to gently suspend the cells, transfer them to a 24-well cell culture plate, and incubate in a 4.5% CO2 cell culture incubator.

[0074] (2) Cells in the logarithmic growth phase were used for total RNA extraction. Cells in the logarithmic growth phase are transparent, regularly shaped, and round. Approximately 1×10⁶ cells were collected. 7 Centrifuge hybridoma cells at 1000 rpm for 3 minutes at room temperature, discard the supernatant, add 1 ml of Trizol, and repeatedly pipette to lyse the cells until homogeneous. Then add 200 μl of chloroform, vortex vigorously, and incubate at 12000g and 4°C for 3 minutes, followed by centrifugation at 4°C for 15 minutes. Collect the upper aqueous phase, add an equal volume of isopropanol, mix well, and incubate at 4°C for 10 minutes to precipitate DNA. Then centrifuge at 12000g for 15 minutes at 4°C. The resulting precipitate is the total RNA.

[0075] (3) After obtaining the total RNA of hybridoma cells, the corresponding cDNA is directly synthesized by reverse transcription PCR for the amplification of the target sequence.

[0076] 2. Amplification of the variable region gene of the heavy and light chains of monoclonal antibodies

[0077] Using the obtained cDNA as a template, PCR primers for specifically amplifying the variable region sequence of the mouse antibody were used to amplify the heavy and light chain variable region sequence (V) of the 4H3 antibody. H V L ), to commercially available Heavy Primers 1 and 2 to expand V H Expanding V with commercially available Light Primers Mix LAfter confirming the PCR amplification results by agarose gel electrophoresis and obtaining the expected target band, the PCR product was recovered using a PCR product recovery kit, and the DNA fragment concentration was measured using a Nano Drop 8000 micro-volume nucleic acid analyzer. Taking advantage of the characteristic that the DNA fragment amplified by Taq DNA polymerase has an "A" added to the 3' end, the target fragment was ligated into the PMD-19T vector for TA cloning. The target gene was then sequenced using a method of screening for positive strains to obtain its cDNA sequence.

[0078] The final nucleotide sequence of the heavy chain variable region of the 4H3 antibody was determined as follows:

[0079] CAGGTTACTCTGAAAGAGTCTGGCCCTGGGATATTGCAGCCCTCCCAGACCCTCAGTCTGACTTGTTCTTTCTCTGGGTTTTCACTGAGCACTTCTGGTATGAGTGTGGGCTGGATTCGTCAGCCTTTAGGGAAGGGTCTGGAGTGGCTGGCACACATTTTGTGGAATGATGATAAATTCTA TAATACAGCCCTGAAAAGCCGGCTCACAATCTCCAAGGATACTTCCAACAATCAGGTATTCCTCAAGATCGCCAGTGTGGTCAATGCAGATACTGCCACATATTACTGTGCTCGAGCCTTCTACTATGGGGGCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA (SEQ ID NO:2).

[0080] The nucleotide sequence of the light chain variable region of the 4H3 antibody is as follows:

[0081] GACATTTGTGATGTCACAGTCTCCATCCTCCCTAGCTGTGTCAGTTGGAGAGAAGGTTACTATGAGCTGCAAGTCCAGTCAGAGCCTTTTATAGTAAGAATCAAAAGAAATACTTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATTTTCTGGGCA TCCACTAGGGAGTCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGAAGGCTGAAGACCTGGCAGTTTTATTACTGTCAGCAATATTATACCTATCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA (SEQ ID NO:3).

[0082] The amino acid sequence of the heavy chain variable region of the 4H3 antibody has been determined as follows:

[0083] QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMSVGWIRQPLGKGLEWLAHILWNDDKFYNTALKSRLTISKDTSNNQVFLKIASVVNADTATYYCARAFYYGGWFAYWGQGTLVTVSA (SEQ ID NO: 4).

[0084] The amino acid sequence of the light chain variable region of the 4H3 antibody has been determined as follows:

[0085] DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSKNQKKYLAWYQQKPGQSPKLLIFWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYTYPWTFGGGTKLEIK (SEQ ID NO: 5).

[0086] 3. SOE-PCR construction of single-chain antibody genes

[0087] The recombinant single-chain variable region antibody gene prepared in this invention uses V L -Linker-V H The -Linker-His-tag sequence, so in the amplification of V for SOE-PCR L and V H When performing a sequence, it is necessary to use the existing V. L The 3' end of the sequence and the original V HPartial linker sequences were added to the 3' and 5' ends of the sequence to facilitate subsequent overlap extension; additionally, restriction enzyme sites and corresponding protective bases were introduced to facilitate the subsequent cloning and expression of the recombinant scFv gene. A V-type linker sequence containing the designed linker sequence was used for amplifying the monoclonal antibody. L and V H Primers for the sequences are listed in Table 1. PCR was used to amplify the V gene containing the linker peptide of monoclonal antibody 4H3. H and V L After recovery and sequencing, full-length extension amplification was performed using overlap extension PCR. The overlap extension PCR product was then amplified using the primers scFv-4H3V listed in Table 2. H F and scFv-4H3V L R is used to amplify the scFv-4H3 gene.

[0088] Table 1. Primers used to amplify the heavy and light chain variable region sequences of the 4H3 antibody.

[0089]

[0090] Table 2 Primers used for SOE-PCR amplification of the heavy and light chain variable region sequences of 4H3 antibody

[0091]

[0092] The final nucleotide sequence of the recombinant single-chain variable region antibody of the 4H3 antibody was determined as follows:

[0093] GACATTGTTATGAGCCAGAGCCCGAGTAGTCTGGCGGTTAGCGTTGGCGAAAAAGTTACCATGAGCTGTAAAAGCAGCCAAAGCCTGCTGTACAGCAAAAACCAGAAAAAATACCTGGCGTGGTATCAGCAAAAACCGGGTCAATCTCCGAAACTGCTGATCTTTTGGGCAAGTACCCGCGAATCTGGCGTTCCGGATCGTTTTACCGGTTCTGGTTCTGGTACCGATTTTACCCTGACCATCAGCAGCGTTAAAGCGGAAGATCTGGCCGTGTATTACTGCCAGCAGTATTACACCTATCCGTGGACCTTTGGCGGCGGTACCAAACTGGAAATTAAAGGCGGCGGCGGTTCTGGCGGCGGTGGTTCCGGCGGCGGTGGTAGTCAAGTTACCCTGAAAGAAAGCGGTCCGGGTATTCTGCAACCGAGTCAAACCCTGAGTCTGACCTGTAGCTTTAGCGGCTTTAGCCTGTCTACCTCTGGTATGAGCGTTGGCTGGATTCGTCAACCGCTGGGTAAAGGTCTGGAGTGGCTGGCTCATATCCTGTGGAACGACGACAAATTCTACAACACCGCGCTGAAAAGTCGTCTGACCATTAGCAAAGACACCAGCAACAACCAGGTCTTCCTGAAAATCGCGAGCGTTGTTAACGCGGATACCGCAACCTATTATTGCGCACGCGCATTTTATTACGGCGGCTGGTTTGCATATTGGGGTCAAGGTACGCTGGTTACCGTTTCTGCAGGTGGCGGCGGTAGTCATCATCATCATCATCAT (SEQ ID NO:6).

[0094] The amino acid sequence of the recombinant single-chain variable region antibody of the 4H3 antibody is as follows:

[0095] DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSKNQKKYLAWYQQKPGQSPKLLIFWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYYTYPWTFGGGTKLEIKGGGGSGGGGSGGGGSQVT LKESGPGILQPSQTLSLTCSFSGFSLSTSGMSVGWIRQPLGKGLEWLAHILWNDDKFYNTALKSRLTISKDTSNNQVFLKIASVVNADTATYYCARAFYYGGWFAYWGQGTLVTVSAGGGGSHHHHHH (SEQ ID NO: 7).

[0096] 4. Antibody variable region sequence analysis

[0097] The obtained antibody variable region gene was analyzed in IMGT / V-Quest at IMGT (http: / / www.ebi.ac.uk / imgt). The cloned gene conforms to the characteristics of mouse immunoglobulin variable region genes. Both the light and heavy chain variable region genes have 3 CDRs and contain the characteristic cysteine ​​residues necessary to maintain the structure of the antibody variable region, and their positions are correct.

[0098] (1) Heavy chain variable region sequence information:

[0099] ① Nucleotide sequence of heavy chain variable region CDR1: ACCTCTGGTATGAGCGTTGGC (SEQ ID NO: 8); Amino acid sequence: TSGMSVG (SEQ ID NO: 9).

[0100] ② The nucleotide sequence of the heavy chain variable region CDR2: CATATCCTGTGGAACGACGACAAATTCTACAACACCGCGCTGAAAAGT (SEQ ID NO:10); amino acid sequence: HILWNDDKFYNTALKS (SEQ ID NO:11).

[0101] ③ Nucleotide sequence of heavy chain variable region CDR3: GCATTTTATTACGGCGGCTGGTTTGCATAT (SEQ ID NO:12); Amino acid sequence: AFYYGGWFAY (SEQ ID NO:13).

[0102] (2) Light chain variable region sequence information:

[0103] ① Nucleotide sequence of the light chain variable region CDR1: AAAAGCAGCCAAAGCCTGCTGTACAGCAAAAACCAGAAAAAATACCTGGCG (SEQ ID NO:14); Amino acid sequence: KSSQSLLYSKNQKKYLA (SEQ ID NO:15).

[0104] ② The nucleotide sequence of the light chain variable region CDR2: TGGGCAAGTACCCGCGAATCT (SEQ ID NO:16); amino acid sequence: WASTRES (SEQ ID NO:17).

[0105] ③ Nucleotide sequence of the light chain variable region CDR3: CAGCAGTATTACACCTATCCGTGGACC (SEQ ID NO:18); Amino acid sequence: QQYYTYPWT (SEQ ID NO:19).

[0106] Example 4: Recombinant expression of the single-chain variable region recombinant antibody of the present invention

[0107] (1) Using primers scFv-4H3V L F and scFv-4H3V H R was used to amplify the scFv-4H3 gene, and the target gene was recovered using a PCR product recovery kit for later use.

[0108] (2) Take the DNA fragments and pET-28a vector obtained in the previous step and perform double enzyme digestion experiments respectively.

[0109] (3) Prepare the T4 DNA ligase ligation system according to the molar ratio of vector to target gene of 1:10 (0.03pmol: 0.3pmol) and react overnight at 16°C.

[0110] (4) DH5α competent cells were added to the entire ligation system for transformation. Positive clones were screened by colony PCR and sent to the company for sequencing.

[0111] (5) Select the correctly sequenced strains from the plate, culture them overnight in liquid LB medium containing kanamycin, extract the plasmids, and transform them into BL21 (DE3) competent cells. Similarly, identify positive clones by colony PCR and prepare for the induction expression of recombinant proteins.

[0112] (6) Select the positive clones obtained in the previous step and culture them in fresh liquid LB medium containing kanamycin until OD. 600=0.8, add IPTG to a final concentration of 1mM, continue culturing for 4-5 hours, centrifuge at 8000 rpm for 5 minutes to harvest the bacterial cells, resuspend the bacterial cells in 0.1M phosphate buffer (PBS), and after sonication, use them for denaturing polyacrylamide gel electrophoresis analysis.

[0113] (8) After ultrasonic disruption, the bacterial cells were added to an equal volume of 2× loading buffer and boiled in boiling water for 5 minutes. The sample was carefully added to the gel wells by pipette. The electrophoresis tank was kept at a constant current of 30 mA until bromophenol blue ran out of the bottom of the gel. After staining and destaining, the gel was analyzed for the expression of the target protein by gel electrophoresis system.

[0114] (9) The recombinant protein was purified by Ni-NTA (GE Healthcare, Chicago) affinity chromatography, and the purified recombinant scFv recombinant protein was refolded by stepwise dialysis. Finally, after dialysis with ultrapure water, it was lyophilized, the concentration was adjusted, and the purity and protein concentration were detected by SDS-PAGE.

[0115] Results: SDS-PAGE showed a significant increase in protein bands in the IPTG-induced strains, while the purified and refolded recombinant scFv protein bands were uniform (e.g., Figure 3 As shown in the figure, this indicates that the recombinant scFv of monoclonal antibody 4H3 was successfully expressed and can be used for subsequent experiments.

[0116] Example 5: Immunoblotting assay to identify the binding of the single-chain variable region recombinant antibody of the present invention to G protein epitope peptide.

[0117] (1) Sodium dodecyl sulfonate-polyacrylamide gel electrophoresis:

[0118] ① Add the recombinant G protein epitope peptide to a sample buffer containing sodium dodecyl sulfate in equal proportion and boil in boiling water for 5 minutes.

[0119] ② Add the sample treated in ① to the sample wells, 10 µl of sample to each well, and perform electrophoresis under constant current conditions; keep the current constant at 30 mA until the bromophenol blue indicator reaches the bottom edge, then stop electrophoresis and remove the gel; part of the gel is used for membrane transfer, and part is placed in Coomassie brilliant blue staining solution for 1 h.

[0120] ③ Cut a piece of nitrocellulose membrane (0.22µm pore size) the same size as the electrophoresis gel, wet it in Western blot transfer buffer, and place it on the electrophoresed gel. Cut off a corner of the nitrocellulose membrane to mark the starting end of the sample sequence. Support it with wetted filter paper, and attach a second wetted filter paper to the other side of the gel sheet; follow the above placement order to form a "gel sandwich" with the gel sheet, nitrocellulose membrane, and filter paper.

[0121] ④ Place the “gel sandwich” into an electrophoresis tank containing transfer buffer, with the nitrocellulose membrane facing the anode and the gel facing the cathode, and electrophore for 20 minutes under a constant current of 400mA.

[0122] ⑤ After the transfer is complete, remove the nitrocellulose membrane.

[0123] 2) Immunoblotting:

[0124] ① Wash the nitrocellulose membrane with PBS for 10 min, then place it in protein-free blocking buffer and block at 37°C for 1 h;

[0125] ② Wash three times with PBST, 5 minutes each time;

[0126] ③ Place the nitrocellulose membrane in a recombinant scFv recombinant protein solution (100 μg / ml) and shake slowly at 37°C for 1 h. Use PBS as a negative control.

[0127] ④ Same as ②, wash three times;

[0128] ⑤ Place the nitrocellulose membrane in anti-His tag mouse antibody (1:3000) and shake slowly at 37°C for 45 minutes;

[0129] ⑥ Same as ②, wash three times;

[0130] ⑦ Place the nitrocellulose membrane in peroxidase-labeled goat anti-mouse antibody (1:3000) and shake slowly at 37°C for 45 min;

[0131] ⑧ Same as ② Wash three times;

[0132] ⑨ Immerse the nitrocellulose membrane in an enhanced chemiluminescence reagent (ECL color-developing solution) until the color becomes clear;

[0133] ⑩ At the same time, boil the gel placed in Coomassie Brilliant Blue in boiling water until the bands are clear.

[0134] Results: The single-chain variable region recombinant antibody of this invention specifically reacted with the 30.2 kDa G protein epitope peptide, showing a black band at the same position as the positive control band, while the negative control showed no band (e.g., Figure 4 (As shown).

[0135] Example 6: Immunoblotting assay to identify the binding of the single-chain variable region recombinant antibody of the present invention to the native G protein.

[0136] (1) Sodium dodecyl sulfonate-polyacrylamide gel electrophoresis:

[0137] ① After centrifuging HIRRV-infected EPC cells at 12000g for 10 min, discard the supernatant, add 1 ml of PBS to resuspend the cells, add an equal proportion of sample buffer containing sodium dodecyl sulfate, and boil in water for 5 min. Simultaneously, set up a control group of healthy EPC cells not infected with HIRRV.

[0138] ② Add the sample treated in ① to the sample wells, 10 µl of sample to each well, and perform electrophoresis under constant current conditions; keep the current constant at 30 mA until the bromophenol blue indicator reaches the bottom edge, then stop electrophoresis and take out the gel; part of the gel is used for membrane transfer, and part is placed in Coomassie brilliant blue staining solution for 1 h.

[0139] ③ Cut a piece of nitrocellulose membrane (0.22µm pore size) the same size as the electrophoresis gel, wet it in Western blot transfer buffer, and place it on the electrophoresed gel. Cut off a corner of the nitrocellulose membrane to mark the starting end of the sample sequence. Support it with wetted filter paper, and attach a second wetted filter paper to the other side of the gel sheet; follow the above placement order to form a "gel sandwich" with the gel sheet, nitrocellulose membrane, and filter paper.

[0140] ④ Place the “gel sandwich” into the electrophoresis tank containing the transfer buffer, with the nitrocellulose membrane facing the anode and the gel facing the cathode, and electrophore for 30 minutes under a constant current of 400mA.

[0141] ⑤ After the transfer is complete, remove the nitrocellulose membrane.

[0142] (2) Immunoblotting:

[0143] ① Wash the nitrocellulose membrane with PBS for 10 min, then place it in protein-free blocking buffer and block at 37°C for 1 h;

[0144] ② Wash three times with PBST, 5 minutes each time;

[0145] ③ Place the nitrocellulose membrane in a recombinant scFv protein solution (100 μg / ml) and shake slowly at 37°C for 1 h. Use PBS as a negative control.

[0146] ④ Same as ②, wash three times;

[0147] ⑤ Place the nitrocellulose membrane in anti-His tag mouse antibody (1:3000) and shake slowly at 37°C for 45 minutes;

[0148] ⑥ Same as ②, wash three times;

[0149] ⑦ Place the nitrocellulose membrane in horseradish peroxidase-labeled goat anti-mouse antibody (1:3000) and shake slowly at 37°C for 45 minutes.

[0150] ⑧ Same as ② Wash three times;

[0151] ⑨ Immerse the nitrocellulose membrane in an enhanced chemiluminescence reagent (ECL color-developing solution) until the color becomes clear;

[0152] ⑩ At the same time, boil the gel placed in Coomassie Brilliant Blue in boiling water until the bands are clear.

[0153] Results: The recombinant scFv of this invention specifically reacted with the 60kDa protein in HIRRV-infected EPC cells, showing a black band. This molecular weight is consistent with the theoretical molecular weight of HIRRV G protein and is located in the same position as the positive control band. In contrast, the recombinant scFv in the control group did not react with any protein in healthy EPC cells, and no positive reaction band was shown (e.g., Figure 5 (As shown).

[0154] Example 7: Immunofluorescence assay for the recombinant antibody with the single-chain variable region of the present invention

[0155] ① Add EPC cells digested with trypsin to a 24-well plate containing cell crawling smears. After the cells are fixed, aspirate the culture medium, add turbot rhabdovirus with an MOI of 0.1, incubate at room temperature for two hours, aspirate the virus solution, add 1 ml of M199 medium containing 2% fetal bovine serum, and culture at 20°C until CPE occurs.

[0156] ② Aspirate the culture medium, wash the cell slides three times with PBST, add pre-cooled methanol at -20℃, and fix at -20℃ for 10 min.

[0157] ③ Remove methanol, wash the cell slides three times with PBST, add 1 ml of 5% BSA, and incubate at room temperature for 1 h.

[0158] ④ After aspirating BSA, add 0.1 mg / ml recombinant scFv as the primary antibody, and use myeloma cell supernatant as a negative control.

[0159] ⑤ Incubate in a humidified chamber at 37°C for 1.5 hours. Wash thoroughly with PBST by shaking three times for 5 minutes each time to remove unbound primary antibody.

[0160] ⑥ Remove PBST, add anti-His-tag mouse antibody (1:3000 dilution) as the secondary antibody, incubate in a humidified chamber at 37°C for 45 min, then remove the slide and wash three times with PBST for 5 min each time.

[0161] ⑦ Incubate with horseradish peroxidase-labeled goat anti-mouse antibody (1:3000) at room temperature for 45 min and wash as above.

[0162] ⑧ Remove the slide, wash three times with PBST for 5 minutes each time. Add DAPI (1:2000 dilution), incubate at room temperature for 15 minutes, and wash again as above.

[0163] ⑨ After drying, add anti-fluorescence quenching mounting medium in the dark, cover with a coverslip, and store at 4°C for later use.

[0164] ⑩ Observe under a fluorescence microscope.

[0165] Results: The recombinant scFv of this invention showed a positive binding reaction with HIRRV-infected EPC cells, exhibiting a significant fluorescent signal, while the negative control group showed no positive reaction. This indicates that the recombinant scFv protein can bind to native HIRRV in cells (e.g., Figure 6 (As shown).

[0166] Example 8: Detection of the in vitro neutralization effect of the single-chain variable region recombinant antibody of the present invention on HIRRV

[0167] To verify whether the recombinant scFv expressing the prokaryotic anti-HIRRV-G neutralizing monoclonal antibody 4H3 possesses neutralizing activity against HIRRV, the recombinant scFv was diluted to 50 μg / mL and mixed with HIRRV suspension (2 × 10⁻⁶). 3 TCID 50 The mixture was prepared in equal volumes ( / ml) and incubated at 20°C for 45 minutes before being inoculated into infected EPC cells. A negative control was prepared by replacing recombinant scFv with His-tagged protein of equal concentration. CPE of cells from different experimental groups was continuously observed, and the differences in disease progression and severity were recorded to assess the neutralizing activity of scFv against HIRRV.

[0168] ① Take the frozen EPC cell line out of the -80°C freezer and immediately place it in a preheated 37°C water bath to thaw. After complete thawing, centrifuge at 1000 rpm at room temperature for 3 minutes. Carefully discard the supernatant in a clean bench, add 1 ml of M199 cell culture medium to gently suspend the cells, transfer them to a 24-well cell culture plate, and incubate in a 4.5% CO2 cell culture incubator.

[0169] ② Mix recombinant scFv with an equal volume of HIRRV dilution solution and incubate at room temperature for 1 hour to prepare for infecting EPC cells.

[0170] ③ Take pictures of EPC cells every 24 hours after infection until obvious CPE phenomenon appears.

[0171] The results are as follows Figure 7The results showed that after prokaryotic expression of recombinant scFv and HIRRV were incubated with EPC cells, CPE (cytopathic effect) was observed in both experimental group 1 and control group 2. However, in the experimental group, the onset of CPE in EPC cells was significantly delayed, and the number of diseased cells was lower. These results indicate that recombinant scFv has a significant neutralizing effect on HIRRV infection.

[0172] Example 9: Detection of the effect of the recombinant scFv turbot on neutralizing HIRRV in vivo according to the present invention

[0173] To investigate whether the recombinant scFv protein of this invention can neutralize HIRRV infection in turbot, 100 µg / ml of recombinant protein was mixed with an equal volume of HIRRV suspension, incubated at room temperature, and then injected into turbot to infect them. The differential mortality of fish in each experimental group was continuously observed and recorded after infection, and the in vivo virus neutralizing activity of recombinant scFv was evaluated based on the cumulative mortality data of each group.

[0174] ① The turbot were purchased from a fish farm in Rizhao, Qingdao. They were about 10-12cm long and were raised in a water tank with continuous aeration. They were fed daily. The qCPR method test was negative for HIRRV. After being temporarily raised for one week, they were used for HIRRV virus neutralization experiments.

[0175] ② The neutralization experimental group was prepared with 100 μg / ml recombinant scFv and an equal volume of HIRRV viral suspension (2.5 × 10⁻⁶). 6.5 TCID 50 Mix well with 100 μL of the mixture and incubate at room temperature for 1 hour. Inject 50 μL of the infected turbot into the peritoneum.

[0176] ③ In the infected control group, turbot were injected with bovine serum albumin (BSA) suspension instead of recombinant scFv suspension, mixed with HIRRV virus suspension under the same conditions. In the negative control group, each turbot was injected with 50 μl PBS.

[0177] ④ After infection, continuously observe and record the mortality of turbot, construct a cumulative mortality curve, and show the results (e.g.) Figure 8 In the HIRRV and BSA co-infection group, turbot began to die on day 2 post-infection, with a rapid mortality period from day 3 to day 8, and a cumulative mortality rate of 93.33%. In contrast, turbot in the recombinant scFv neutralization group began to die on day 3 post-infection, with a cumulative mortality rate reaching 55.00% by day 11 post-infection, and a relative immune protection rate of 41.07%. All turbot in the negative control group survived throughout the experiment. The experimental results indicate that 100 μg / ml of recombinant scFv exhibits a significant neutralizing effect against HIRRV infection.

[0178] Those skilled in the art will understand that modifications, additions, and substitutions to the above embodiments are possible within the scope of protection of this invention, and none of them exceed the scope of protection claimed by this invention.

Claims

1. A recombinant single-chain variable region antibody of a Paralichthys olivaceus rhabdovirus, comprising a heavy chain variable region and a light chain variable region, characterized in that: The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 4, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO:

5.

2. The recombinant single-chain variable region antibody of Paralichthys olivaceus rhabdovirus according to claim 1, wherein: The coding nucleotide sequence of the heavy chain variable region of the recombinant antibody is shown as SEQ ID NO: 2, and the coding nucleotide sequence of the light chain variable region is shown as SEQ ID NO:

3.

3. The recombinant single-chain variable region antibody of the Paralichthys olivaceus rhabdovirus according to claim 1, wherein the amino acid sequence of the VL region is SEQ ID NO: 1, and the amino acid sequence of the VH region is SEQ ID NO:

2. The amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are shown as SEQ ID NO: 9, SEQ ID NO: 11 and SEQ ID NO: 13, respectively.

4. The turbot rhabdovirus recombinant single-chain variable region antibody as described in claim 1, characterized in that: The amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region are shown as SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 19, respectively.

5. Use of the Paralichthys olivaceus rhabdovirus recombinant single-chain variable region antibody according to any one of claims 1-4 in the preparation of a Paralichthys olivaceus rhabdovirus infection detection or diagnosis reagent.

6. Use of the Paralichthys olivaceus rhabdovirus recombinant single-chain variable region antibody according to any one of claims 1-4 in the preparation of a Paralichthys olivaceus rhabdovirus target blocking drug.

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