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

Abstract

The invention 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. According to the present invention, paralichthys olivaceus rhabdovirus G protein gene is cloned, accurate screening is performed to obtain the B cell antigen epitope of the G protein, the hybridoma cell is prepared by using the epitope sequence as the antigen, and the specific single-chain antibody is efficiently produced by using the Escherichia coli expression system; the recombinant single-chain antibody can specifically recognize and combine with HIRRV, target a virus key functional region, effectively neutralize virus infection, reduce non-specific immunoreaction, and significantly improve the accuracy of rapid diagnosis, detection and treatment. The compound can be used as a paralichthys olivaceus rhabdovirus infection detection or diagnosis reagent and a paralichthys olivaceus rhabdovirus targeted blocking drug.

Description

TECHNICAL FIELD

[0001] The application relates to a recombinant antibody of hirame rhabdovirus and a preparation method and application thereof, in particular to a recombinant single-chain variable region antibody of hirame rhabdovirus and application thereof, and belongs to the field of molecular immunology. BACKGROUND

[0002] Hirame novirhabdovirus (HIRRV) is a highly contagious pathogen that can infect a variety of marine and freshwater fish, especially hirame, and cause severe hemorrhagic septicemia. HIRRV belongs to the Novirhabdovirus genus of the Rhabdoviridae family and is a single-stranded negative-sense RNA virus with a capsid. The virus particle is typically bullet-shaped. The genome of HIRRV is about 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). Among them, G protein is one of the important envelope proteins of HIRRV and has been shown to play a key role in the virus infection process. It is involved in the adsorption and fusion of the virus and host cells, is a key protein for viral invasion of host cells, and also participates in the assembly and release of virus particles.

[0003] As one of the important envelope proteins of HIRRV, G protein is a key target for virus detection and diagnosis, and is also a key neutralization target and potential vaccine candidate antigen for preventing and treating HIRRV infection. Previous studies have confirmed that G protein is an immunoprotective antigen of HIRRV virus, and the HIRRV nucleic acid and subunit vaccine constructed based on the protein can effectively induce the production of neutralizing antibodies in immunized fish, thereby exerting an immunoprotective effect. Therefore, anti-HIRRV-G protein monoclonal antibodies can specifically recognize and bind to the virus G protein and have the potential to neutralize viral virulence. However, the molecular weight of G protein is large, and it is extremely difficult to explore the neutralization site, which brings great challenges to the preparation of monoclonal antibodies with neutralizing activity. Traditional monoclonal antibody production has high cost, long time cycle and high operation requirement. At the same time, due to the large molecular weight and strong immunogenicity of the complete structure of the monoclonal antibody, it has the problems of poor tissue penetration and easy degradation in virus neutralization, targeted blocking and other clinical applications, which limits its large-scale application and promotion.

[0004] Recombinant single-chain variable fragment (scFv) has gradually become a research hotspot due to its small size, high specificity, easy genetic engineering operation, and convenient large-scale production. Recombinant antibodies are obtained by cloning the variable region genes of monoclonal antibodies and expressing them in bacteria, yeast or mammalian cells. scFv is formed by connecting the variable heavy chain (VH) and the variable light chain (VL) of the antibody through a flexible peptide, which contains six complementarity-determining regions (CDRs), which are the smallest functional units of antibody-antigen binding. Recombinant single-chain variable region antibodies not only maintain the antigen binding activity of the parent monoclonal antibody, but also have excellent tissue penetration and a molecular weight of only about 1 / 6 of the complete antibody, which has a broad application prospect in clinical diagnosis and treatment.

[0005] Chinese patent application with publication number CN112608383A discloses a single-chain antibody against Cobia rhabdovirus, which has a molecular weight of about 28kD and can specifically recognize Cobia rhabdovirus. It can be further used for the development of diagnostic and therapeutic preparations and the study of antigen epitopes. However, it has the problems of low antigen specificity, high production cost, complex process, low production efficiency, and potential immunogenicity risk. SUMMARY

[0006] To solve the above problems, the first object of the present application is to provide a Cobia rhabdovirus recombinant single-chain variable region antibody with small molecular weight, low immunogenicity, high specificity and virus neutralization activity, which can be used for the clinical prevention and treatment of Cobia rhabdovirus disease and the rapid and accurate detection of Cobia rhabdovirus.

[0007] The second object of the present application is to provide a preparation method of the above-mentioned Cobia rhabdovirus recombinant single-chain variable region antibody.

[0008] The application firstly uses bioinformatics software (BepiPred and IEDB software, etc.) to analyze and predict the B cell antigen epitope of the G protein of the Paralichthys olivaceus rhabdovirus, selects a peptide segment with an advantageous B cell antigen epitope; then constructs a G protein tertiary structure model of the Paralichthys olivaceus, selects a peptide segment located on the surface of the G protein molecule; finally, a plurality of peptide segments that meet the requirements of strong antigenicity and are located on the surface of the G protein molecule are spliced to form a Paralichthys olivaceus rhabdovirus epitope peptide, Balb / c mice are immunized, the lymph B cells of the mice are obtained, and the B cells are fused with myeloma cells to screen to obtain a monoclonal cell of the Paralichthys olivaceus rhabdovirus G protein, total RNA of the monoclonal cell is extracted, reverse transcribed into cDNA, amplified by using a degenerate primer of a mouse antibody conservative sequence, a sequence of a heavy chain variable region and a light chain variable region of a monoclonal antibody is obtained, a promoter, a linker and a His tag are added, and then the sequence is transferred into a pET-28a prokaryotic vector, DH5α competent cells are transformed, and after sequencing, expression is induced to obtain the Paralichthys olivaceus rhabdovirus recombinant single-chain variable region antibody.

[0009] The purpose of the application is realized by the following technical scheme: A Paralichthys olivaceus rhabdovirus recombinant single-chain variable region antibody comprises a heavy chain variable region and a light chain variable region, and 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.

[0010] The complementarity determining region of the heavy chain variable region and the light chain variable region is composed of CDR1, CDR2 and CDR3; 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; 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; the coding nucleotide sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are shown as SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12 respectively; and the coding nucleotide sequences of CDR1, CDR2 and CDR3 of the light chain variable region are shown as SEQ ID NO: 14, SEQ ID NO: 16 and SEQ ID NO: 18 respectively.

[0011] A preparation method of a Paralichthys olivaceus rhabdovirus recombinant single-chain variable region antibody comprises the following steps: (1) using bioinformatics software to analyze and predict the B cell epitopes of the G protein of the Pufferfish Rhabdovirus, selecting the peptide segments with dominant B cell epitopes; then constructing a tertiary structure model of the G protein of the Pufferfish Rhabdovirus, selecting the peptide segments located on the surface of the G protein molecule, and finally splicing the peptide segments that meet the requirements of strong antigenicity and are located on the surface of the G protein molecule to prepare the Pufferfish Rhabdovirus epitope peptide, the sequence of which is shown as SEQ ID NO: 1; (2) immunizing Balb / c mice with the Pufferfish Rhabdovirus epitope peptide, obtaining the lymph B cells of the mice, and screening the anti-Pufferfish Rhabdovirus G protein monoclonal cells after fusion with myeloma cells; (3) extracting the total RNA of the monoclonal cells, reverse transcribing into cDNA, amplifying through the degenerate primers of the mouse antibody conservative sequence, obtaining the sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody, adding a promoter, a Linker and a His tag, and then transferring into a pET-28a prokaryotic vector, transforming BL21(DE3) competent cells, inducing expression after sequencing, and finally obtaining the Pufferfish Rhabdovirus recombinant single-chain variable region antibody.

[0012] The recombinant single-chain variable region antibody after purification was collected, and the results of immunofluorescence experiment showed that it could specifically bind to the natural G protein of HIRRV virus, and showed a fluorescent positive signal. The results of Western blotting experiment showed that the single-chain antibody against the Pufferfish Rhabdovirus G protein epitope peptide could recognize the natural Pufferfish Rhabdovirus G protein, and the supernatant of the myeloma cells as a control could not bind to the natural G protein of HIRRV virus. The results of virus neutralization experiment showed that the single-chain antibody against the Pufferfish Rhabdovirus G protein epitope peptide could effectively neutralize the Pufferfish Rhabdovirus and reduce the virulence of the virus.

[0013] The Pufferfish Rhabdovirus recombinant single-chain variable region antibody can specifically immunoreact with the G protein epitope peptide of 30.2 kDa of the Pufferfish Rhabdovirus, and show a positive reaction band.

[0014] The Pufferfish Rhabdovirus recombinant single-chain variable region antibody can specifically recognize the virus particles in the Epithelioma of Carassius auratus cells infected with the Pufferfish Rhabdovirus, and show green fluorescence.

[0015] The Pufferfish Rhabdovirus recombinant single-chain variable region antibody has significant neutralization activity on HIRRV through in vitro virus neutralization experiment, and can delay the pathological process of virus-infected cells. The Pufferfish Rhabdovirus recombinant single-chain variable region antibody can effectively neutralize virus infection and reduce the mortality rate of Pufferfish infected with the virus.

[0016] Therefore, the Pufferfish Rhabdovirus recombinant single-chain variable region antibody can be applied as a detection or diagnosis reagent for Pufferfish Rhabdovirus infection, and as a targeted blocking drug for Pufferfish Rhabdovirus.

[0017] The application has the advantages that by cloning the G protein gene of the Paralichthys olivaceus rhabdovirus, the B cell antigen epitope of the G protein is accurately screened, and a hybridoma cell is prepared by taking the epitope sequence as an antigen. E. coli Compared with the single-chain antibody produced by taking the purified virus as an antigen and by using the phage technology, the recombinant single-chain antibody has the significant advantages. The recombinant single-chain antibody developed by taking the G protein epitope peptide as an antigen can specifically recognize and combine with the HIRRV, targets the key functional region of the virus, effectively neutralizes the virus infection, reduces the non-specific immune response, and significantly improves the accuracy of rapid diagnosis, detection and treatment. E. coli The expression system is used, the production process is simple, the cycle is short, the yield is high, and the cost is much lower than that of the phage technology, so the expression system is suitable for large-scale industrial production, provides an efficient antibody preparation scheme for rapid response to the HIRRV epidemic, and the single-chain antibody expressed by the expression system has small molecular weight, low immunogenicity and excellent tissue permeability, so the single-chain antibody is suitable for development of in-vivo treatment antibody drugs and is convenient for subsequent function optimization. E. coli The recombinant single-chain antibody can effectively recognize and neutralize the activity of the HIRRV, and provides a key tool for rapid diagnosis, virus detection and prevention, and has important values for development of the HIRRV rapid detection diagnosis reagent and the HIRRV targeted blocking drug. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 It is an electrophoresis result diagram of the recombinant Paralichthys olivaceus rhabdovirus G protein epitope peptide.

[0019] In the drawings, lane M represents a molecular weight standard protein, lane 1 represents a strain that is not induced, lane 2 represents a strain that is induced by IPTG, and lane 3 represents the purified recombinant Paralichthys olivaceus rhabdovirus G protein epitope peptide protein.

[0020] Figure 2 It is a cell photo of EPC cells infected for 72 hours after the supernatant of each hybridoma cell strain 2B3, 6D9, 5E4 and 4H3 is incubated with the HIRRV at room temperature.

[0021] Figure 3 It is an SDS-PAGE result diagram of the recombinant single-chain variable region antibody.

[0022] In the drawings, lane M represents a molecular weight standard protein, lane 1 represents a strain that is not induced, lane 2 represents a strain that is induced by IPTG, and lane 3 represents the purified recombinant scFv.

[0023] Figure 4 It is a Western blotting result diagram of the recombinant single-chain variable region antibody and the recombinant G protein epitope peptide.

[0024] Lane M represents molecular weight marker; Lane 1 is the SDS-PAGE result of the genetically engineered strain expressing G protein epitope peptide; Lane 2 represents the immunoreaction result of the recombinant single-chain variable region antibody and the recombinant G protein epitope peptide; and Lane 3 is the negative control.

[0025] Figure 5 Figure 4 is a Western blotting detection result diagram of the recombinant single-chain variable region antibody and the HIRRV.

[0026] Lane M represents molecular weight marker; Lane 1 is the SDS-PAGE result of the HIRRV-infected EPC cell; Lane 2 represents the immunoreaction result of the recombinant single-chain variable region antibody (scFv) and the HIRRV G protein; Lane 3 is the positive control of the monoclonal antibody; and Lane 4 is the negative control.

[0027] Figure 6 Figure 6 is an indirect immunofluorescence experiment analysis result diagram of the specific reaction of the recombinant single-chain variable region antibody and the HIRRV in the EPC cell.

[0028] A1 represents the binding reaction of the recombinant single-chain variable region antibody and the virus in the HIRRV-infected EPC cell under the observation of a 40-fold objective lens; B1 is the fluorescence observation result of the myeloma cell supernatant instead of the recombinant scFv as a control; A2 and B2 are the fluorescence observation results of the DAPI-stained cell nucleus; and A3 and B3 are the superimposed diagrams of A1 and A2, and B1 and B2, respectively.

[0029] Figure 7 Figure 8 is an in-vitro virus neutralization experiment result diagram of the recombinant single-chain variable region antibody.

[0030] In experiment group 1, the recombinant scFv and the HIRRV are mixed and incubated, and then the EPC cell is infected, and the cytopathic effect is recorded at different time points; in negative control group 2, the same amount of bovine serum albumin is used to replace the recombinant scFv, and then the HIRRV is mixed and incubated, and then the EPC cell is infected.

[0031] Figure 8 Figure 10 is a cumulative mortality line graph of the in-vivo virus neutralization experiment of the recombinant single-chain variable region antibody. DETAILED DESCRIPTION

[0032] The present application will be further described below in conjunction with the accompanying drawings and through specific embodiments.

[0033] Example 1: Preparation of recombinant protein of the HIRRV envelope G protein epitope peptide and preparation of neutralizing monoclonal antibody thereof

[0034] 1. Preparation of recombinant protein of the HIRRV envelope G protein epitope peptide (1) According to the HIRRV genome sequence published on the NCBI website, the antigen sites of G protein were predicted by using the online tools of IEDB (http: / / tools.iedb.org / bcell / ) and BepiPred (BepiPred-3.0), and the B cell linear antigen site sequences of G 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.

[0035] (2) The final determination of the antigen peptide site was made by comprehensively analyzing the above prediction data. First, the antigen epitopes located in the signal peptide, transmembrane region, and intracellular region were excluded. Second, the antigen epitope parameters and the predicted potential B cell linear epitopes were analyzed. Finally, the antigen epitopes located in the random coil and beta-turn regions in the secondary structure were analyzed, and the specific sequence sites of the antigen peptide were screened and determined. The three-dimensional structure of the G protein epitope peptide molecule of Paralichthys olivaceus rhabdovirus was constructed, and a flexible linker GGGGS was used to separate the epitopes to avoid structural conflicts. The final G protein epitope peptide sequence of Paralichthys olivaceus rhabdovirus was: VYGQTIKPGVDSVSDQPTGGGGSSLSVGNNLGDIHTQGGGGSVSKDRRTYRAHQTSAFVAWGHPFGDEWGGGGSCPAAKLSKVSPSQLRCPRIFDDENRGLVAGGGGSVLRDPYTLGFLDSDFIEGKCSKSPCQTGGGGSIFVDKTSHHVVKATSYGHHPWGGGGGSLYNDTDTADITIRKIDSGGGGSRRPSMPAAPQEIPMYHLGGGGSTIEKALVEMKLAPREVGVYDTTTASAGGGGSDTTVGMRGSLDDFAGGGGSRSPHPGI (SEQ ID NO: 1), which was used for preparing G protein-specific monoclonal antibodies.

[0036] The above G protein coding sequence was synthesized and ligated into the pET-28a plasmid. After cloning and sequencing confirmation, it was transformed into BL21 (DE3) competent bacteria, induced and expressed, and the results are shown in Figure 1 , which shows that the recombinant G protein epitope peptide with a size of about 30.2 kDa is successfully induced and purified, which is consistent with the predicted protein size.

[0037] 2. Preparation of neutralizing monoclonal antibodies (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 two BALB / c mice were immunized intraperitoneally. Two weeks later, the same amount of recombinant G epitope peptide emulsified with Freund's incomplete adjuvant was injected intraperitoneally to enhance immunity. Then, the mice were injected with 100 μg of G epitope peptide through the tail vein every other week. Three days after the last injection, the mouse spleen and thymus cells were extracted under sterile conditions.

[0038] (2) The mouse was anesthetized with ether, and the spleen and thymus were removed under sterile conditions and passed through a 100-mesh screen to form a single-cell suspension with RPMI-1640 medium. The handling of experimental animals in this application complies with the requirements of animal welfare.

[0039] (3) The spleen cell suspension and thymus cell suspension were centrifuged at 1000 rpm for 3 min, and the supernatant was discarded. The spleen cell pellet was resuspended in RPMI-1640 medium, and the thymus cell pellet was resuspended in RPMI-1640 medium containing 1% HAT (10% fetal bovine serum).

[0040] (4) The spleen cell suspension and P3-X63-Ag8U1 myeloma cell suspension were mixed evenly, centrifuged at 1000 rpm for 3 min, and the supernatant was completely removed. The bottom of the centrifuge tube was tapped to mix the two cell pellets thoroughly into a paste. 1 ml of preheated polyethylene glycol solution at 37°C was drawn up with a pipette and added evenly to the centrifuge tube within 1 min, and then placed in a 37°C water bath for 5 min. Another 15 ml of preheated RPMI-1640 medium at 37°C was added to dilute the polyethylene glycol and lose its effect, and the RPMI-1640 medium was added to 40 ml, centrifuged at 1000 rpm for 3 min, and the supernatant was discarded.

[0041] (5) 1 ml of the cell suspension was added to the prepared thymus cell suspension, mixed evenly, and added to a 96-well culture plate. The culture plate was placed in a 37°C, 4.5% CO2 incubator for culture, and the cell growth was observed under an inverted microscope. After about two weeks, the hybridoma cell culture supernatant was collected for detection.

[0042] (6) After fusion, when the hybridoma cell population grew to about 1 / 3 of the area of the 96-well culture plate, positive hybridoma cells were screened using indirect enzyme-linked immunoassay, including the following steps: ① Antigen coating: HIRRV was separated and purified using differential centrifugation, and the HIRRV virus suspension was diluted with carbonate coating solution (pH 9.6) to a concentration of 50 μg / ml, added to a 96-well enzyme-labeled plate (100 μl / well), and coated at 4°C overnight. ② Aspiration of the coating liquid, washing with phosphate buffer solution (PBST) containing 0.05% Tween-20, 5 minutes each time, three times; ③ Add 200µl of 3% bovine serum albumin blocking solution (3% BSA blocking solution) per well, block at 37°C for 1h; ④ Three times of washing as in ②; ⑤ Add the hybridoma cell culture supernatant as the first antibody to the enzyme-labeled plate at 50µl per well, incubate in a 37°C incubator for 1h; ⑥ Three times of washing as in ②; ⑦ Add the alkaline phosphatase-labeled anti-His tag antibody (1:4000 dilution) as the second antibody to the enzyme-labeled plate at 50µl per well, incubate in a 37°C incubator for 1h; ⑧ Three times of washing as in ②; then add 100µl of 4-nitrophenyl phosphate (pNPP) application solution per well, react in the dark for 5-20min, add 50µl of 2M NaOH solution per well, and stabilize for 3-5min, then measure the OD value at 405nm working wavelength.

[0043] Take the bone marrow tumor cell supernatant as the negative control, and take the light absorption value of each well at 405nm wavelength as the calculation basis, calculate the ratio (P / N) of the light absorption value of each experimental well to that of the negative control, and when P / N≥2.1, the well is positive.

[0044] (7) Cloning: the positive hybridoma cells detected are cloned by limited dilution method, and the steps are as follows: ① After the mouse is anesthetized with ether, the thymus is taken out under sterile conditions, ground on a 100-mesh screen, and blown to form a single-cell suspension with RPMI-1640 medium; ② Centrifuge the thymus cell suspension at 1000rpm for 3min, discard the supernatant, and resuspend the thymus cell precipitate with 10ml of RPMI-1640 cell culture medium containing 10% fetal bovine serum; ③ Count the cells in the positive cell well to be cloned with a hemocytometer, then dilute them with the culture medium in a 10-fold gradient, take out 100 hybridoma cells, and put them into the thymus cell suspension; ④ Drop the cell suspension evenly into a 96-well culture plate with a dropper, 100µl per well, and each well contains an average of one hybridoma cell; ⑤ Place it in a CO2 incubator for culture; ⑥ After two weeks, detect the hybridoma cell culture supernatant of each well by indirect enzyme-linked immunoassay, and clone the hybridoma cells in the positive clone well again according to the above method to ensure the formation of a single clone.

[0045] (8) Take the growing, good hybridoma cells, make cell suspension, 1000g centrifugal 3min, discard the supernatant, add freezing solution (9 parts RPMI-1640 medium + 1 part dimethyl sulfoxide), the final cell density is 5x10 6 6D9, 5E4, 4H3.

[0046] Example 2: Screening of HIRRV envelope G protein epitope peptide monoclonal antibody with virus neutralization ability

[0047] In order to screen out monoclonal antibody strains with significant virus neutralization ability, the culture supernatant of 2B3, 6D9, 5E4, 4H3 hybridoma cells prepared in Example 1 was mixed with an equal volume of HIRRV suspension, incubated at room temperature for 1 hour, then infected EPC cells, and the virus neutralization activity of different strains of monoclonal antibody was evaluated by observing the CPE difference of each experimental group.

[0048] (1) The frozen EPC cell strain was taken out from the -80°C refrigerator and immediately placed in a preheated 37°C water bath to melt, and after complete melting, 1000 rpm centrifugal 3min at room temperature, carefully pour off the supernatant in the clean bench, add 1ml M199 cell culture medium to suspend the cells, transfer to 24-well cell culture plate, and place in 4.5% CO2 cell incubator for culture.

[0049] (2) Mix the hybridoma cell culture supernatant with an equal volume of HIRRV diluent, incubate at room temperature for 1 hour, and prepare for infection of EPC cells.

[0050] (3) After infection, continuously track and monitor the CPE changes of different experimental groups of cells, and record the different CPE of the cells in different monoclonal antibody neutralization groups.

[0051] Results: After 72 hours of infection, all treatment groups showed different degrees of CPE, but the EPC cells added with 4H3 hybridoma cell culture supernatant showed a significant delay in the occurrence of CPE, and the number of cells with pathological changes was significantly less than the control group and other 3 strains of monoclonal antibodies (such as Figure 2 ). It is shown that the monoclonal antibody produced by hybridoma 4H3 strain has significant neutralization effect on HIRRV infection.

[0052] Example 3: Recombinant single-chain variable region antibody gene cloning of HIRRV envelope G protein epitope peptide neutralizing monoclonal antibody 4H3

[0053] 1. Resuscitation of hybridoma cell line, extraction of total RNA and synthesis of cDNA template (1) Take the frozen anti-HIRRV-G neutralizing monoclonal antibody 4H3 hybridoma cell line from the -80°C refrigerator, immediately put it into the preheated 37°C water bath to melt, after complete melting, centrifuge at 1000 rpm at room temperature for 3 minutes, carefully pour off the supernatant in the clean bench, add 1 ml of 37°C preheated GIT cell culture medium to suspend the cells gently, transfer to a 24-well cell culture plate, and place in a 4.5% CO2 cell incubator for culture.

[0054] (2) Take the cells in the logarithmic growth phase for total RNA extraction. The cells in the logarithmic growth phase are transparent, regular in shape, and round and transparent. Collect about 1 x 10 7 hybridoma cells, centrifuge at 1000 rpm at room temperature for 3 minutes, discard the supernatant, add 1 ml of Trizol, and repeatedly blow and lyse the cells to a uniform state. Then add 200 μl of chloroform, shake vigorously, then 12000g, 4°C for 3 minutes, 4°C centrifuge for 15 minutes. Take the upper aqueous phase, add an equal volume of isopropanol, mix well, then 4°C for 10 minutes to precipitate DNA, then 4°C, 12000g centrifuge for 15 minutes, the obtained precipitate is total RNA.

[0055] (3) After obtaining the total RNA of the hybridoma cells, directly synthesize the corresponding cDNA by reverse transcription PCR for amplification of the target sequence.

[0056] 2. Amplification of monoclonal antibody heavy and light chain variable region genes The obtained cDNA was used as a template to amplify the heavy and light chain variable region sequences (V H , V L ) of the 4H3 antibody using PCR primers specific for amplifying murine antibody variable region sequences. V H was amplified using commercial Heavy Primers 1 and 2, and V L was amplified using commercial Light Primers Mix. After detecting the PCR amplification results by agarose gel electrophoresis and confirming the expected target band, the PCR product was recovered using a PCR product recovery kit, and the DNA fragment concentration was determined using a Nano Drop 8000 microvolume nucleic acid analyzer. The 3' end of the DNA fragment amplified by Taq DNA polymerase was added with an "A" characteristic, the target fragment was ligated to PMD-19T vector for T-A cloning, and the target gene was sequenced by screening positive strains to obtain its cDNA sequence.

[0057] The nucleotide sequence of the variable region of the heavy chain of the 4H3 antibody is as follows: CAGGTTACTCTGAAAGAGTCTGGCCCTGGGATATTGCAGCCCTCCCAGACCCTCAGTCTGACTTGTTCTTTCTCTGGGTTTTCACTGAGCACTTCTGGTATGAGTGTGGGCTGGATTCGTCAGCCTTTAGGGAAGGGTCTGGAGTGGCTGGCACACATTTTGTGGAATGATGATAAATTCTATAATACAGCCCTGAAAAGCCGGCTCACAATCTCCAAGGATACTTCCAACAATCAGGTATTCCTCAAGATCGCCAGTGTGGTCAATGCAGATACTGCCACATATTACTGTGCTCGAGCCTTCTACTATGGGGGCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA (SEQ ID NO: 2).

[0058] The nucleotide sequence of the variable region of the light chain of the 4H3 antibody is as follows: GACATTGTGATGTCACAGTCTCCATCCTCCCTAGCTGTGTCAGTTGGAGAGAAGGTTACTATGAGCTGCAAGTCCAGTCAGAGCCTTTTATATAGTAAGAATCAAAAGAAATACTTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATTTTCTGGGCATCCACTAGGGAGTCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGAAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAATATTATACCTATCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA (SEQ ID NO: 3).

[0059] The amino acid sequence of the variable region of the heavy chain of the 4H3 antibody is as follows: QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMSVGWIRQPLGKGLEWLAHILWNDDKFYNTALKSRLTISKDTSNNQVFLKIASVVNADTATYYCARAFYYGGWFAYWGQGTLVTVSA (SEQ ID NO: 4).

[0060] The amino acid sequence of the light chain variable region of the 4H3 antibody is as follows: DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSKNQKKYLAWYQQKPGQSPKLLIFWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYTYPWTFGGGTKLEIK (SEQ ID NO: 5).

[0061] 3. Construction of single-chain antibody gene by SOE-PCR The recombinant single-chain variable region antibody gene prepared by the present application adopts the order of V L -Linker-V H -Linker-His-tag, so when amplifying the V L and V H sequences for SOE-PCR, part of the Linker sequence needs to be added at the 3' end of the original V L sequence and the 3' end and 5' end of the original V H sequence, respectively, to facilitate subsequent overlap extension; in addition, restriction enzyme cutting sites and corresponding protection bases are introduced for the convenience of subsequent cloning and expression of the recombinant scFv gene. The primers designed for amplifying the V L and V H sequences of the single antibody containing the Linker sequence are arranged in Table 1. The V H and V L sequences of the single antibody 4H3 containing the linker peptide gene are amplified by PCR, and after recovery and sequencing, the full-length extension amplification is performed by overlap extension PCR. The overlap extension PCR product is used to amplify the scFv-4H3 gene by using the primers scFv-4H3V H F and scFv-4H3V L R in Table 2.

[0062] Table 1 Primers for amplifying the heavy and light chain variable region sequences of the 4H3 antibody

[0063] Table 2 Primers for amplifying the heavy and light chain variable region sequences of the 4H3 antibody by SOE-PCR

[0064] The nucleotide sequence of the recombinant single-chain variable region antibody of the 4H3 antibody was finally determined as follows: GACATTGTTATGAGCCAGAGCCCGAGTAGTCTGGCGGTTAGCGTTGGCGAAAAAGTTACCATGAGCTGTAAAAGCAGCCAAAGCCTGCTGTACAGCAAAAACCAGAAAAAATACCTGGCGTGGTATCAGCAAAAACCGGGTCAATCTCCGAAACTGCTGATCTTTTGGGCAAGTACCCGCGAATCTGGCGTTCCGGATCGTTTTACCGGTTCTGGTTCTGGTACCGATTTTACCCTGACCATCAGCAGCGTTAAAGCGGAAGATCTGGCCGTGTATTACTGCCAGCAGTATTACACCTATCCGTGGACCTTTGGCGGCGGTACCAAACTGGAAATTAAAGGCGGCGGCGGTTCTGGCGGCGGTGGTTCCGGCGGCGGTGGTAGTCAAGTTACCCTGAAAGAAAGCGGTCCGGGTATTCTGCAACCGAGTCAAACCCTGAGTCTGACCTGTAGCTTTAGCGGCTTTAGCCTGTCTACCTCTGGTATGAGCGTTGGCTGGATTCGTCAACCGCTGGGTAAAGGTCTGGAGTGGCTGGCTCATATCCTGTGGAACGACGACAAATTCTACAACACCGCGCTGAAAAGTCGTCTGACCATTAGCAAAGACACCAGCAACAACCAGGTCTTCCTGAAAATCGCGAGCGTTGTTAACGCGGATACCGCAACCTATTATTGCGCACGCGCATTTTATTACGGCGGCTGGTTTGCATATTGGGGTCAAGGTACGCTGGTTACCGTTTCTGCAGGTGGCGGCGGTAGTCATCATCATCATCATCAT(SEQ ID NO:6)

[0065] The amino acid sequence of the recombinant single-chain variable region antibody of the 4H3 antibody was determined as follows: DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSKNQKKYLAWYQQKPGQSPKLLIFWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYTYPWTFGGGTKLEIKGGGGSGGGGSGGGGSQVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMSVGWIRQPLGKGLEWLAHILWNDDKFYNTALKSRLTISKDTSNNQVFLKIASVVNADTATYYCARAFYYGGWFAYWGQGTLVTVSAGGGGSHHHHHH (SEQ ID NO: 7).

[0066] 4. Analysis of antibody variable region sequences The obtained antibody variable region genes were analyzed in IMGT / V-Quest in IMGT (http: / / www.ebi.ac.uk / imgt). The cloned genes were in line with the characteristics of mouse immunoglobulin variable region genes, and the light and heavy chain variable region genes each had three CDRs, and had characteristic cysteine residues necessary for maintaining the structure of the antibody variable region and correct positions.

[0067] (1) Heavy chain variable region sequence information: ① Nucleotide sequence of CDR1 of heavy chain variable region: ACCTCTGGTATGAGCGTTGGC (SEQ ID NO: 8); amino acid sequence: TSGMSVG (SEQ ID NO: 9).

[0068] ② Nucleotide sequence of CDR2 of heavy chain variable region: CATATCCTGTGGAACGACGACAAATTCTACAACACCGCGCTGAAAAGT (SEQ ID NO: 10); amino acid sequence: HILWNDDKFYNTALKS (SEQ ID NO: 11).

[0069] ③ Nucleotide sequence of CDR3 of heavy chain variable region: GCATTTTATTACGGCGGCTGGTTTGCATAT (SEQ ID NO: 12); amino acid sequence: AFYYGGWFAY (SEQ ID NO: 13).

[0070] (2) Light chain variable region sequence information: The nucleotide sequence of CDR1 of the light chain variable region: AAAAGCAGCCAAAGCCTGCTGTACAGCAAAAACCAGAAAAAATACCTGGCG (SEQ ID NO: 14); the amino acid sequence: KSSQSLLYSKNQKKYLA (SEQ ID NO: 15).

[0071] The nucleotide sequence of CDR2 of the light chain variable region: TGGGCAAGTACCCGCGAATCT (SEQ ID NO: 16); the amino acid sequence: WASTRES (SEQ ID NO: 17).

[0072] The nucleotide sequence of CDR3 of the light chain variable region: CAGCAGTATTACACCTATCCGTGGACC (SEQ ID NO: 18); the amino acid sequence: QQYYTYPWT (SEQ ID NO: 19).

[0073] Example 4: Recombinant expression of the single-chain variable region recombinant antibody of the application

[0074] (1) The primer scFv-4H3V F and scFv-4H3V R were used to amplify the scFv-4H3 gene, and the target gene was recovered by PCR product recovery kit and prepared for use. L F and scFv-4H3V H R were used to amplify the scFv-4H3 gene, and the target gene was recovered by PCR product recovery kit and prepared for use.

[0075] (2) The DNA fragment obtained in the previous step and the pET-28a vector were subjected to double enzyme digestion.

[0076] (3) The T4 DNA ligase ligation system was prepared according to the molar ratio of the vector to the target gene of 1:10 (0.03 pmol:0.3 pmol), and the reaction was carried out at 16°C overnight.

[0077] (4) All the ligation systems were added to the DH5a competent cells for transformation operation, and the positive clones were screened by colony PCR and sent to the company for sequencing.

[0078] (5) The correct strain on the plate was picked up and cultured in liquid LB medium containing kanamycin overnight, and the plasmid was extracted and transformed into BL21 (DE3) competent cells. The positive clones were identified by colony PCR, and the induction expression of recombinant protein was prepared.

[0079] (6) The positive clone strain obtained in the previous step was picked up and cultured in fresh liquid LB medium containing kanamycin to OD 600=0.8, IPTG was added to a final concentration of 1 mM, and the culture was continued for 4-5 hours. The bacteria were harvested by centrifugation at 8000 rpm for 5 minutes, resuspended in 0.1 M phosphate buffer saline (PBS), and disrupted by ultrasonic wave for denaturing polyacrylamide gel electrophoresis analysis.

[0080] (8) The bacteria after ultrasonic disruption were added with an equal volume of 2x loading buffer and boiled in boiling water for 5 minutes. The sample was carefully added to the gel hole by a pipette, and electrophoresis was performed at a constant current of 30 mA until the bromophenol blue indicator reached the bottom of the gel. The gel was dyed and decolored, and the expression of the target protein was analyzed by gel electrophoresis system.

[0081] (9) The recombinant protein was purified by Ni-NTA (GE Healthcare, Chicago) affinity chromatography, and the purified recombinant scFv recombinant protein was renatured by step-by-step dialysis. Finally, the protein was freeze-dried after dialysis with ultrapure water, and the purity and protein concentration were detected by SDS-PAGE after adjusting the concentration.

[0082] Results: SDS-PAGE showed that the protein band of the strain induced by IPTG was obviously thickened, and the recombinant scFv recombinant protein band after purification and renaturation was single (as shown in Figure 3 ), indicating that the recombinant scFv of monoclonal antibody 4H3 was successfully expressed and could be used for subsequent experiments.

[0083] Example 5: Identification of the single-chain variable region recombinant antibody of the application binding to the G protein epitope peptide by immunoblotting experiment

[0084] (1) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis: ① The recombinantly expressed G protein epitope peptide was added to a sample buffer containing an equal proportion of sodium dodecyl sulfate and boiled in boiling water for 5 min.

[0085] ② The sample treated in ① was added to the loading hole, and 10 μl of sample was added to each hole. Electrophoresis was performed under constant current conditions; the electrophoresis was stopped when the bromophenol blue indicator reached the bottom edge, and the gel was removed. Part of the gel was used for membrane transfer, and part was placed in the coomassie brilliant blue staining solution for 1 h.

[0086] ③ A piece of nitrocellulose membrane (pore size 0.22 μm) with the same size as the electrophoresis gel was placed in the western blot rapid membrane transfer liquid, and then placed on the electrophoresed gel. An angle was cut on the nitrocellulose membrane to mark the starting end of the sample order. A piece of wet filter paper was placed on the other side of the gel piece; the gel piece, nitrocellulose membrane, and filter paper formed a set of sandwiched "gel sandwich" according to the above placement order. (4) Put 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 perform electrophoresis at a constant current of 400 mA for 20 min; (5) After the transfer is completed, remove the nitrocellulose membrane.

[0087] 2) Immunoblotting: (1) Wash the nitrocellulose membrane with PBS for 10 min, and then place it in a protein-free blocking solution and block at 37°C for 1 h; (2) Wash with PBST for 3 times, each for 5 min; (3) Place the nitrocellulose membrane in a recombinant scFv recombinant protein solution (100 μg / ml) and slowly shake at 37°C for 1 h, with PBS as a negative control; (4) Wash as in (2) for 3 times; (5) Place the nitrocellulose membrane in an anti-His tag mouse antibody (1:3000) and slowly shake at 37°C for 45 min; (6) Wash as in (2) for 3 times; (7) Place the nitrocellulose membrane in a peroxidase-labeled goat anti-mouse antibody (1:3000) and slowly shake at 37°C for 45 min; (8) Wash as in (2) for 3 times; (9) Place the nitrocellulose membrane in an enhanced chemiluminescence reagent (ECL color developing solution) and develop color until the color is clear; (10) At the same time, cook the gel placed in Coomassie brilliant blue in boiling water until the bands are clear.

[0088] Results: The single-chain variable region recombinant antibody of the application specifically reacts with the G protein epitope peptide 30.2 kDa protein, showing a black band, which is the same as the positive control band position, and no band is shown for the negative control (as shown in Figure 4 ).

[0089] Example 6: Immunoblotting experiment for identifying the binding of the single-chain variable region recombinant antibody of the application to the natural G protein

[0090] (1) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis: (1) Centrifuge the HIRRV-infected EPC cells at 12000 g for 10 min, discard the supernatant, resuspend in 1 ml of PBS, and add an equal proportion of sample buffer containing sodium dodecyl sulfate, and cook in boiling water for 5 min. At the same time, set up a sample of healthy EPC cells not infected with HIRRV as a control.

[0091] ② The sample treated in ① was added to the loading well, 10 μl of sample was added to each well, and electrophoresis was performed under constant current conditions; the electrophoresis was stopped when the bromophenol blue indicator reached the bottom edge, the gel was removed, and the gel was used for membrane transfer and part of the gel was placed in the coomassie brilliant blue dye solution for 1 h of staining.

[0092] ③ A piece of nitrocellulose membrane (pore size 0.22 μm) with the same size as the electrophoresis gel was cut, wetted in the Western blot rapid membrane transfer solution, and placed on the electrophoresed gel. A corner was cut off the nitrocellulose membrane to mark the starting end of the sample order. A second piece of wet filter paper was placed on the other side of the gel piece; the gel piece, nitrocellulose membrane and filter paper formed a sandwiched "gel sandwich" in the above order of placement; ④ The "gel sandwich" was placed in the electrophoresis tank containing the transfer buffer, the nitrocellulose membrane was facing the anode, the gel was facing the cathode, and electrophoresis was performed under constant current conditions of 400 mA for 30 min; ⑤ After transfer, the nitrocellulose membrane was removed.

[0093] (2) Immunoblotting: ① The nitrocellulose membrane was washed with PBS for 10 min, then placed in a protein-free blocking solution, and blocked at 37°C for 1 h; ② Washed with PBST for 3 times, 5 min each time; ③ The nitrocellulose membrane was placed in a recombinant scFv protein solution (100 μg / ml) and slowly shaken at 37°C for 1 h, with PBS as a negative control; ④ Washed three times as in ②; ⑤ The nitrocellulose membrane was placed in an anti-His tag mouse antibody (1:3000) and slowly shaken at 37°C for 45 min; ⑥ Washed three times as in ②; ⑦ The nitrocellulose membrane was placed in a horseradish peroxidase-labeled goat anti-mouse antibody (1:3000) and slowly shaken at 37°C for 45 min.

[0094] ⑧ Washed three times as in ②; ⑨ The nitrocellulose membrane was placed in an enhanced chemiluminescence reagent (ECL color developing solution) for color development until the color was clear; ⑩ At the same time, the gel placed in the coomassie brilliant blue was boiled in boiling water until the bands were clear.

[0095] Results: The recombinant scFv of the present application specifically reacts with the 60 kDa protein in the EPC cells infected with HIRRV, showing a black band, which has the same molecular weight as the theoretical molecular weight of HIRRV G protein and has the same position as the positive control band, while the recombinant scFv does not react with any protein in the healthy EPC cells in the control group, and no positive reaction band is shown (as shown in Figure 5

[0096] Example 7: Immunofluorescence experiment for identifying the single-chain variable region recombinant antibody of the present application

[0097] ① The trypsin-digested EPC cells are added to the 24-well plate with cell climbing sheets, and after the cells are fixed, the culture medium is aspirated, and the HIRRV with MOI = 0.1 is added, and after incubation at room temperature for two hours, the virus liquid is aspirated, and 1 ml of M199 culture medium containing 2% fetal bovine serum is added, and incubation is carried out at 20°C until the CPE phenomenon appears.

[0098] ② The culture liquid is aspirated, and the cell climbing sheets are washed with PBST for three times, and 1 ml of -20°C pre-cooled methanol is added, and fixed at -20°C for 10 min.

[0099] ③ The methanol is aspirated, and the cell climbing sheets are washed with PBST for three times, and 1 ml of 5% BSA is added, and incubated at room temperature for 1 h.

[0100] ④ After the BSA is aspirated, 0.1 mg / ml of the recombinant scFv is added as the first antibody, and the myeloma cell supernatant is used as the negative control.

[0101] ⑤ It is placed in a wet box and incubated at 37°C for 1.5 h. It is washed with PBST for three times, 5 min each time, and the unbound first antibody is washed away.

[0102] ⑥ The PBST is aspirated, the anti-His-tag mouse antibody (1:3000 dilution) is used as the second antibody, and after incubation at 37°C in the wet box for 45 min, the glass slide is taken out and washed with PBST for three times, 5 min each time ⑦ The goat anti-mouse antibody labeled with horseradish peroxidase (1:3000) is incubated at room temperature for 45 min and washed as above.

[0103] ⑧ The glass slide is taken out and washed with PBST for three times, 5 min each time. DAPI (1:2000 dilution) is added dropwise, incubated at room temperature for 15 min and washed as above.

[0104] ⑨ After spinning dry, the anti-fluorescence quenching mounting medium is added dropwise in the dark, covered with a cover glass, and stored at 4°C for standby.

[0105] ⑩ It is observed under a fluorescence microscope.

[0106] ​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).

[0107] Example 8: Detection of the in vitro neutralization effect of the single-chain variable region recombinant antibody of the present invention on HIRRV

[0108] 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.

[0109] 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.

[0110] 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.

[0111] ③ Take pictures of EPC cells every 24 hours after infection until obvious CPE phenomenon appears.

[0112] The results are as follows Figure 7 The 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.

[0113] Example 9: Detection of the effect of the recombinant scFv turbot on neutralizing HIRRV in vivo according to the present invention

[0114] To explore whether the recombinant scFv protein can neutralize the infection of HIRRV in the body of Paralichthys olivaceus, 100 μg / ml of the recombinant protein is mixed with an equal volume of HIRRV suspension, and then the mixed solution is incubated at room temperature, and then the infected Paralichthys olivaceus is injected, and the difference in the death of fish in each experimental group is observed and recorded after infection, and the in-vivo virus neutralization activity of the recombinant scFv is evaluated based on the cumulative mortality data of each group.

[0115] ①Paralichthys olivaceus is purchased from a certain breeding farm in Qingdao Rizhao, about 10-12 cm, and is bred in a water tank with continuous aeration, and is fed daily; qCPR method detection is HIRRV negative; and after temporary breeding for one week, it is used for virus neutralization experiment of HIRRV.

[0116] ②In the neutralization experiment group, 100 μg / ml of the recombinant scFv is mixed with an equal volume of HIRRV virus suspension (2.5 × 10 6.5 TCID 50 / 100 μL) to prepare a mixed solution, and then the mixed solution is incubated at room temperature for 1 hour, and then the infected Paralichthys olivaceus is injected, and each fish is injected with 50 μl.

[0117] ③In the infection control group, the Paralichthys olivaceus is injected with the same concentration of bovine serum albumin (BSA) suspension instead of the recombinant scFv suspension, and then the mixed solution of the BSA suspension and the HIRRV virus suspension is incubated under the same conditions, and then the infected Paralichthys olivaceus is injected. The negative control group of Paralichthys olivaceus is injected with 50 μl of PBS per fish.

[0118] ④After infection, the death of Paralichthys olivaceus is observed and recorded, and a cumulative mortality curve is prepared, and the results show (as shown in Figure 8 ): the Paralichthys olivaceus in the HIRRV and BSA mixed infection group starts to die on the second day after infection, and the rapid death period is from the third day to the eighth day, and the cumulative mortality rates are 93.33%, respectively, while the Paralichthys olivaceus in the recombinant scFv neutralization group starts to die on the third day after infection, and the cumulative mortality rate reaches 55.00% on the eleventh day after infection, and the relative immune protection rate is 41.07%. The Paralichthys olivaceus in the negative control group survives throughout the experiment. The experimental results show that the recombinant scFv of 100 μg / ml has a significant neutralization effect on HIRRV infection.

[0119] Those skilled in the art will understand that modifications, additions and substitutions are possible within the scope of the present application, and all of them are possible without exceeding the scope of the present application.

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 encoding nucleotide sequence of the heavy chain variable region of the recombinant antibody is shown as SEQ ID NO: 2, and the encoding 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. A method for preparing the recombinant single-chain variable region antibody of Paralichthys olivaceus rhabdovirus according to claim 1, characterized in that, The method comprises the following steps: (1) using bioinformatics software to analyze and predict the B cell antigen epitopes of the G protein of the Cobia rhabdovirus, selecting a peptide segment with dominant B cell antigen epitopes; then constructing a Cobia G protein tertiary structure model, selecting a peptide segment located on the surface of the G protein molecule, and finally splicing multiple peptide segments that meet the requirements of strong antigenicity and are located on the surface of the G protein molecule to prepare the Cobia rhabdovirus epitope peptide, the sequence of which is shown as SEQ ID NO: 1; (2) immunizing Balb / c mice with the Cobia rhabdovirus epitope peptide, obtaining the lymph B cells of the mice, fusing the lymph B cells with myeloma cells, and screening to obtain the anti-Cobia rhabdovirus G protein monoclonal cells; (3) extracting the total RNA of the monoclonal cells, reverse transcribing into cDNA, amplifying through the degenerate primers of the mouse antibody conservative sequence, obtaining the sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody, adding a promoter, a Linker and a Histag, and then transferring into a pET-28a prokaryotic vector, transforming BL21(DE3) competent cells, inducing expression after sequencing, and finally obtaining the Cobia rhabdovirus recombinant single-chain variable region antibody.

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

7. Use of the Cobia rhabdovirus recombinant single-chain variable region antibody according to any one of claims 1-4 in the preparation of a Cobia rhabdovirus targeted blocking drug.

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