A monoclonal antibody specifically binding to the nucleocapsid protein of the red sea bream iridovirus and use thereof

Abstract

The application belongs to the technical field of biology and particularly relates to a monoclonal antibody specifically combined with the nucleocapsid protein of the red-winged virus and application thereof. The application firstly obtains a monoclonal antibody 6C5 against the nucleocapsid protein of the red-winged virus, wherein the monoclonal antibody 6C5 comprises a heavy chain variable region shown in SEQ ID NO. 6 and a light chain variable region shown in SEQ ID NO. 7, and the monoclonal antibody 6C5 can specifically react with the red-winged virus and the nucleocapsid protein thereof. Secondly, the monoclonal antibody 6C5 is labeled with FITC and can be used for directly detecting the red-winged virus by the immunofluorescence method, thereby providing new materials for studying the function of the red-winged virus and the nucleocapsid protein thereof and explaining the pathogenic mechanism of the virus.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a monoclonal antibody that specifically binds to the nucleocapsid protein of Akabane virus and its application. Background Technology

[0002] Akabane virus (AKAV) belongs to the genus Orthobunyavirus of the family Peribunyaviridae, and the Simbu serogroup. It is an enveloped, segmented, single-stranded, negative-sense RNA virus. The virus was first isolated from mosquitoes collected in Akabane Prefecture, Japan in 1959, hence its name (OYA et al., 1961). AKAV primarily infects ruminants such as cattle and sheep, causing reproductive disorders in females and leading to arthroryposis-hydranencephaly syndrome (AH) in fetuses or young animals, severely impacting ruminant reproduction and livestock production.

[0003] AKAV virus particles are typically spherical, with a diameter of approximately 70–130 nm, covered by an envelope and possessing spikes (TAKAHASHI et al., 1978). Its genome consists of three single-stranded negative-sense RNA segments: a large (L) segment, a medium (M) segment, and a small (S) segment. The L segment encodes the L protein; the M segment encodes a polyprotein, which is further processed into glycoproteins Gn and Gc, and the non-structural protein NSm; the S segment encodes the nucleocapsid protein (N protein) and the non-structural protein NSs (OKAJIMA et al., 2024). Compared to the more variable M segment, the L and S segments are relatively conserved. Therefore, the preparation of specific antibodies against the N protein encoded by the S segment is of great significance for AKAV antigen detection and research related to viral infection.

[0004] Monoclonal antibodies can specifically recognize corresponding antigenic epitopes and are characterized by high specificity, stability, and good batch-to-batch consistency. They have been widely used in pathogen detection, viral antigen localization, infection mechanism research, and diagnostic reagent development. The specific recognition of antigens by antibodies is mainly determined by the variable regions of the antibody heavy and light chains, among which the complementarity-determining region (CDR) is a key region involved in antigen binding. Obtaining monoclonal antibodies with clearly defined heavy and light chain variable regions and CDR sequences is beneficial for stable antibody preparation, sequence identification, and subsequent development and utilization.

[0005] Currently, there is still a need for further development of specific antibodies and matching detection reagents for AKAV research and detection. Therefore, the preparation of monoclonal antibodies that can specifically recognize AKAV nucleocapsid proteins and the identification of their heavy and light chain variable regions and CDR sequences are of great application value for the identification of AKAV-infected cells, the confirmation of viral isolates, the study of nucleocapsid protein expression and localization, and the development of related in vitro detection methods and reagents. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a monoclonal antibody that specifically binds to the nucleocapsid protein of Akabane virus, as well as the nucleic acid encoding the monoclonal antibody, the detection application of the monoclonal antibody, and a detection kit containing the monoclonal antibody, to meet the demand for specific antibodies in in vitro detection of Akabane virus, identification of nucleocapsid protein expression, and related research.

[0007] To achieve the above objectives, the present invention adopts the following technical solution.

[0008] In a first aspect, the present invention provides a monoclonal antibody that specifically binds to the nucleocapsid protein of Akabane virus, the monoclonal antibody comprising an antibody heavy chain and an antibody light chain; The variable region of the antibody heavy chain includes CDR1 with an amino acid sequence as shown in SEQ ID NO.1, CDR2 with an amino acid sequence as shown in SEQ ID NO.2, and CDR3 with an amino acid sequence as shown in SEQ ID NO.3; The variable region of the antibody light chain includes CDR1 with the amino acid sequence shown in SEQ ID NO.4, CDR2 with the amino acid sequence YTS, and CDR3 with the amino acid sequence shown in SEQ ID NO.5.

[0009] Preferably, the amino acid sequence of the variable region of the antibody heavy chain is shown in SEQ ID NO.6, and the amino acid sequence of the variable region of the antibody light chain is shown in SEQ ID NO.7.

[0010] Preferably, the heavy chain of the monoclonal antibody is of type IgG1, and the light chain is of type κ.

[0011] In a second aspect, the present invention provides a nucleic acid that encodes the antibody heavy chain and antibody light chain of the monoclonal antibody described in the first aspect above.

[0012] Preferably, the nucleotide sequence encoding the variable region of the antibody heavy chain is shown in SEQ ID NO.8, and the nucleotide sequence encoding the variable region of the antibody light chain is shown in SEQ ID NO.9.

[0013] Thirdly, the present invention provides the application of the monoclonal antibody described in the first aspect above in the preparation of reagents for detecting Akabane disease virus.

[0014] Fourthly, the present invention provides the application of the monoclonal antibody described in the first aspect above in the in vitro detection of Akabane disease virus for non-disease diagnosis purposes.

[0015] Fifthly, the present invention provides the application of the monoclonal antibody described in the first aspect above in the study of the pathogenesis and immune mechanism of Akabane virus.

[0016] In a sixth aspect, the present invention provides a fluorescently labeled monoclonal antibody, wherein the fluorescently labeled monoclonal antibody is the monoclonal antibody described in the first aspect above labeled with a fluorescent marker.

[0017] Preferably, the fluorescent marker is a detectable small molecule fluorescent marker.

[0018] Preferably, the fluorescent marker is the small molecule fluorescent organic dye FITC.

[0019] In a seventh aspect, the present invention provides the application of the fluorescently labeled monoclonal antibody described in the sixth aspect above in the preparation of a reagent for detecting Akabane virus.

[0020] Preferably, the reagent is used for the detection of Akabane disease virus by direct immunofluorescence assay.

[0021] Eighthly, the present invention provides a kit for detecting Akabane virus, the kit comprising the monoclonal antibody described in the first aspect above, or the fluorescently labeled monoclonal antibody described in the sixth aspect above.

[0022] The beneficial effects of this invention are as follows: Compared with the prior art, this invention firstly provides a monoclonal antibody that can specifically bind to the nucleocapsid protein of Akabane virus, and clarifies the heavy chain and light chain complementarity-determining region sequences and the variable region nucleic acid sequence of the monoclonal antibody; the monoclonal antibody has a high antibody titer of 1:128000 in indirect ELISA detection; the monoclonal antibody has good reactivity with both Akabane virus and its nucleocapsid protein, and can identify Akabane virus-infected cells by direct immunofluorescence; it can be used for in vitro detection of Akabane virus and research on the pathogenic and immune mechanisms of Akabane virus. Attached Figure Description

[0023] Figure 1 The results are SDS-PAGE analysis of samples before and after purification of the monoclonal antibody described in this invention.

[0024] Figure 2 This is the result of the indirect ELISA titer determination of the monoclonal antibody described in this invention.

[0025] Figure 3 This is the result of the monoclonal antibody subtype identification described in this invention.

[0026] Figure 4 This is for the verification of the reactivity of the monoclonal antibody described in this invention.

[0027] Figure 5 This is a stacked map of the abundance of sequencing samples of the monoclonal antibody described in this invention.

[0028] Figure 6 This is the specificity identification result of the monoclonal antibody labeled with FITC as described in this invention. Detailed Implementation

[0029] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0030] The Akabane virus (AKAV), AKAV positive serum, and pET30a-His-AKAV N recombinant plasmid used in the following examples were preserved by the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Lanzhou Branch of the China Animal Health and Epidemiology Center). The experimental animals used for antigen immunization were clean-grade 6-8 week old female BALB / c mice, provided by the Laboratory Animal Center of the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Lanzhou Branch of the China Animal Health and Epidemiology Center).

[0031] In addition, unless otherwise specified, all reagents used in the following examples are commercially available or can be synthesized according to the methods described in the text or known to the art. For reaction conditions not listed, they are also readily available to those skilled in the art. Example 1: Preparation and identification of monoclonal antibodies against the nucleocapsid protein of Akabane virus.

[0032] 1. Immunized animals Immunogens were prepared by transforming *E. coli* BL21(DE3) with the pET30a-His-AKAV N recombinant plasmid. The purified immunogen—recombinant protein His-AKAV N—was diluted to 200 μg / mL with PBS and thoroughly emulsified with an equal volume of Freund's complete adjuvant to prepare a stable water-in-oil emulsion. Six- to eight-week-old female BALB / c mice were selected, and each mouse received 100 μg of the emulsion via multiple subcutaneous injections on its back for the first immunization. On days 15 and 30 after the first immunization, the same dose of recombinant protein His-AKAV N was emulsified with an equal volume of Freund's incomplete adjuvant, and booster immunizations were performed in the same manner. Seven days after the third immunization, blood was collected via the tail vein, and serum was separated. The titer of anti-AKAVN protein antibodies in mouse serum was detected using an indirect ELISA method. Mice with a strong immune response were selected based on the test results. Three days before cell fusion, the selected mice were intraperitoneally injected with 50 μg of adjuvant-free recombinant protein His-AKAV N for shock immunization.

[0033] 2. Cell fusion Mice were euthanized by dislocation, and spleens were removed under aseptic conditions to prepare spleen cell suspensions. Sp2 / 0 myeloma cells were mixed with spleen cells at a ratio of 1:8, centrifuged, and the supernatant was discarded. Polyethylene glycol 2000 was added to initiate cell fusion. Subsequently, incomplete RPMI-1640 medium was gradually added to terminate the fusion. The fused cells were resuspended in HAT selective medium, seeded in 96-well cell culture plates, and cultured at 37 °C and 5% CO2.

[0034] 3. Screening and subcloning of positive hybridoma cells After cell fusion, half-volume medium replacement was performed every 2–4 days. After 7–8 days of culture, the growth of cell clones in each well was observed. When the hybridoma cell growth area reached approximately one-tenth of the well bottom area, the culture supernatant was collected, and positive cell wells secreting anti-AKAV N protein antibodies were screened using an indirect ELISA method. Positive hybridoma cells selected using the limiting dilution method were subjected to three rounds of subcloning until a monoclonal hybridoma cell line stably secreting specific antibodies was obtained. The culture was then expanded, and finally, OD (endogenous oxidative stress) was preserved. 450 The hybridoma cell with the highest value was 6C5 in the cell pore.

[0035] 4. Preparation of monoclonal antibodies Female BALB / c mice aged 8–10 weeks were selected. Each mouse was pretreated with 0.5 mL of Freund's incomplete adjuvant via intraperitoneal injection. Seven days later, 1 × 10⁻⁶ mg / L of the adjuvant was injected intraperitoneally. 66C5 hybridoma cells in good growth condition were collected. Ascites fluid was collected 7–10 days post-inoculation when the mice exhibited significant abdominal distension and difficulty moving. The ascites fluid-derived monoclonal antibody was initially purified using saturated ammonium sulfate precipitation, followed by further purification using Protein G affinity chromatography.

[0036] SDS-PAGE electrophoresis results are as follows: Figure 1 As shown, the purified monoclonal antibody sample showed distinct bands at approximately 55 kDa and 25 kDa, corresponding to the heavy and light chains of the mouse IgG antibody, respectively, thus obtaining a high-purity anti-AKAV N protein monoclonal antibody 6C5.

[0037] 5. Monoclonal antibody titer determination The titer of monoclonal antibodies was detected by indirect ELISA using recombinant protein His-AKAV N coated at 0.1 μg / well on ELISA plates. Normal mouse ascites fluid served as a negative control, and AKAV-positive serum served as a positive control. Serially diluted monoclonal antibody 6C5 was used as the test sample. The ELISA titer of 6C5 was determined by the highest dilution with a p / n ratio > 2.1.

[0038] The results are as follows Figure 2 As shown, the indirect ELISA titer of the obtained monoclonal antibody 6C5 was 1:128000, indicating that the monoclonal antibody 6C5 described in this application can specifically react with the recombinant protein His-AKAV N.

[0039] 6. Identification of monoclonal antibody 6C5 subtypes The heavy and light chain isotypes of monoclonal antibody 6C5 were identified using a commercially available mouse monoclonal antibody isotype identification kit, and the specific procedures were performed in accordance with the kit instructions.

[0040] The results are as follows Figure 3 As shown, the heavy chain subtype of the monoclonal antibody 6C5 described in this application is IgG1, and the light chain type is κ.

[0041] 7. Reactivity identification of monoclonal antibody 6C5 Recombinant AKAV N protein, AKAV-infected MDBK cell samples, and uninfected MDBK cell samples were prepared separately. The reactivity of the monoclonal antibody 6C5 was detected by Western blot using the monoclonal antibody as the primary antibody.

[0042] The results are as follows Figure 4 As shown, the monoclonal antibody 6C5 described in this application can specifically recognize recombinant proteins His-AKAVN and AKAV-infected MDBK cell samples, showing a specific band at approximately 28 kDa; while it does not react with normal MDBK cell samples, demonstrating good specificity. Example 2: Determination of the variable region sequences of the 6C5 heavy and light chains of monoclonal antibody.

[0043] 1. Obtaining the variable region gene of the heavy and light chains of monoclonal antibodies Based on the preliminary screening, monoclonal antibody 6C5 was selected, and its hybridoma cells were cultured on a large scale. Once the cells reached the logarithmic growth phase, the hybridoma cells were counted and collected, reaching a density of 5 × 10⁶ cells / year. 6 Total RNA was extracted using the HiPure RNA Mini Columns (Magen) kit, following the instructions in the product manual. RNA was dissolved in RNase-free water, and the concentration and integrity of total RNA were assessed using NanoDrop and nucleic acid electrophoresis. Reverse transcription was performed using SMART Scribe Reverse Transcriptase (Takara) and its oligo-dT and template switch oligo (TSO), following the instructions in the product manual. The obtained double-stranded cDNA was used as a template for amplification, with the upstream primer anchored to the TSO and the downstream primer binding to the constant region of either the heavy or light chain. The 5' ends of the upstream and downstream primers were labeled with P5 and P7 adapters, respectively. Heavy and light chain fragments were amplified independently in the first round of PCR. The first-round PCR product was purified using magnetic beads, and a second round of PCR was performed using the purified product as a template. In this stage, index primers were ligated to both ends of the first-round PCR product to form a TruSeq dual-index library. The library was purified using magnetic beads, quantified using Qubit, and then sequenced using an Illumina MiSeq PE300.

[0044] The 10 sequences with the highest abundance were selected for abundance analysis, and the abundance packing plot is shown below. Figure 5 As shown, the sequence with the most productive gene and the highest ranking was selected as the target gene sequence.

[0045] 2. Analysis of gene sequencing results of the variable domains of the heavy and light chains of monoclonal antibodies First, the original FASTQ files were quality-assessed. Cutadapt (v1.9.1) was used to remove adapters and bases with low quality scores (Q value < 20) to generate pruned data; FLASH (v2.2.00) was used to assemble paired-end sequencing reads. Based on the sequencing results, the gene sequence of the 6C5 heavy-light chain variable domain of the monoclonal antibody was analyzed using NCBI-IgBLAST (v1.17.0) and the IMGT database (http: / / www.imgt.org / ).

[0046] The results showed that the heavy chain variable region VH gene of the monoclonal antibody 6C5 described in this application is 369 bp in length, encoding 123 amino acid residues, and includes the highly variable regions CDR1-H, CDR2-H, and CDR3-H; the light chain variable region VL gene is 321 bp in length, encoding 107 amino acid residues, and includes the highly variable regions CDR1-L, CDR2-L, and CDR3-L. Details are shown below: CDR1-H: GYTFTNYW (shown in SEQ ID NO.1); CDR2-H: IVPSDSYT (shown as SEQ ID NO.2); CDR3-H: ARSFFYGTTYGFLFDY (shown as SEQ ID NO.3); CDR1-L: QDINNY (shown as SEQ ID NO.4); CDR2-L: YTS; CDR3-L: QQYSQLPWT (shown as SEQ ID NO.5); Heavy chain variable region: QVPLQQPGAALVEPGASVELSCKASGYTFTNYWMHWVKQRPGRGLEWIGEIVPSDSYTNYNQVFKGKATLTVDKSSTTAYLQLSSLTSEDSAVYFCARSFFYGTTYGFLFDYWGQGTTLTVSS (shown in SEQ IDNO.6); Heavy chain variable region gene sequence: CAGGTCCCACTGCAGCAGCCTGGGGCTGCGCTTGTGGAGCCTGGGGCTTCAGTGGAGCTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAACTGGATGCACTGGGTTAAACAGAGGCCTGGACGTGGCCTTGAATGGATCGGAGAGATTGTTCCTTCTGATAGTTATACTAACTATA ATCAAGTTTTCAAGGGCAAGGCCACATTGACTGTCGACAAGTCCTCCACCACAGCCTACTTGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTTCTGTGCAAGATCTTTTTTTCTACGGTACTACCTACGGATTTCTCTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO.8 shown); Light chain variable region: DIQMTQTTSSLSASLGDRVTISCSASQDINNYLNWYQQKPDGTVKVLIYYTSSSHSGVPSRFSGSGSGTEYSLTISNLEPEDIATYYCQQYSQLPWTFGGGTKLEIK (shown as SEQ ID NO.7); Light chain variable region gene sequence: GATATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGTGCAAGTCAGGACATTAACAATTATTTAAACTGGTATCAGCAGAAACCAGATGGAACTGTTAAAGTCCTGATCTATTACACATCAA GTTCACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGGACAGAATATTCTCTCACCATCAGCAACCTGGAACCTGAAGATATTGCCACTTACTATTGTCAGCAATATAGTCAACTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA (SEQ ID NO.9 shown). Example 3: Application of monoclonal antibody 6C5 in IFA detection

[0047] 1. Labeling of monoclonal antibodies The monoclonal antibody 6C5 (FITC-6C5) prepared in Example 1 was labeled using the FITC fluorescence conjugation kit (abcam), following the instructions in the manufacturer's manual.

[0048] 2. Specificity of FITC-labeled monoclonal antibodies detected by direct immunofluorescence assay MDBK cells were seeded in confocal microscopy dishes. When the cell density reached 80%, AKAV cells were seeded at an MOI of 0.1. After 2 hours of adsorption, the medium was replaced with fresh DMEM containing 10% fetal bovine serum. The cells were incubated at 37°C with 5% CO2 for 32 hours, then the medium was discarded. The cells were washed with PBS and fixed with 4% paraformaldehyde at room temperature for 15 minutes. After washing three times with PBS, 0.1% Triton X-100 was added, and the cells were permeabilized at room temperature for 15 minutes. After washing again, the cells were blocked with 5% BSA at 37°C for 60 minutes, and the blocking solution was discarded. After washing again, FITC-6C5 was added, and the cells were incubated at 37°C in the dark for 1 hour. After washing again, DAPI was added, and the cells were incubated at room temperature in the dark for 10 minutes. The cells were observed and photographed using a laser confocal microscope. Normal MDBK cells not infected with AKAV were treated in the same way as a negative control.

[0049] The results are as follows Figure 6 As shown, FITC-6C5 reacts only with AKAV-infected MDBK cells and not with normal MDBK cells. This indicates that the monoclonal antibody 6C5 described in this application specifically reacts only with the AKAV N protein.

[0050] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A monoclonal antibody that specifically binds to the nucleocapsid protein of Akabane virus, characterized in that, The monoclonal antibody comprises an antibody heavy chain and an antibody light chain; The variable region of the antibody heavy chain includes CDR1 with an amino acid sequence as shown in SEQ ID NO.1, CDR2 with an amino acid sequence as shown in SEQ ID NO.2, and CDR3 with an amino acid sequence as shown in SEQ ID NO.3; The variable region of the antibody light chain includes CDR1 with the amino acid sequence shown in SEQ ID NO.4, CDR2 with the amino acid sequence YTS, and CDR3 with the amino acid sequence shown in SEQ ID NO.

5.

2. The monoclonal antibody as described in claim 1, characterized in that, The amino acid sequence of the variable region of the antibody heavy chain is shown in SEQ ID NO.6, and the amino acid sequence of the variable region of the antibody light chain is shown in SEQ ID NO.

7.

3. A nucleic acid, characterized in that, The nucleic acid encodes the antibody heavy chain and antibody light chain of the monoclonal antibody of claim 1 or 2.

4. The nucleic acid as described in claim 3, characterized in that, The nucleotide sequence encoding the variable region of the antibody heavy chain is shown in SEQ ID NO.8, and the nucleotide sequence encoding the variable region of the antibody light chain is shown in SEQ ID NO.

9.

5. The use of the monoclonal antibody as described in claim 1 or 2 in the preparation of reagents for detecting Akabane virus, or in the in vitro detection of Akabane virus for non-disease diagnosis purposes, or in the study of the pathogenesis and immune mechanism of Akabane virus.

6. A fluorescently labeled monoclonal antibody, characterized in that, The fluorescently labeled monoclonal antibody is the monoclonal antibody described in claim 1 or 2 that is labeled with a fluorescent marker.

7. The fluorescently labeled monoclonal antibody as described in claim 6, characterized in that, The fluorescent label is a detectable small molecule fluorescent label.

8. The use of the fluorescently labeled monoclonal antibody as described in claim 6 or 7 in the preparation of reagents for detecting Akabane virus.

9. The application as described in claim 8, characterized in that, The reagent is used for the direct immunofluorescence assay to detect Akabane disease virus.

10. A reagent kit for detecting Akabane virus, characterized in that, The kit includes the monoclonal antibody as described in claim 1 or 2, or the fluorescently labeled monoclonal antibody as described in claim 6 or 7.

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