A monoclonal antibody that specifically binds to the phosphoprotein of peste des petits ruminants virus.

Abstract

This invention relates to the field of biotechnology, specifically to a monoclonal antibody that specifically binds to the phosphoprotein of peste des petits ruminants (PPR) virus. The invention discloses a monoclonal antibody 16E8A6 against PPR phosphoprotein. The sequence of monoclonal antibody 16E8A6 includes the heavy chain variable region shown in SEQ ID NO.7 and the light chain variable region shown in SEQ ID NO.8. Monoclonal antibody 16E8A6 specifically reacts with PPR and its phosphoprotein with high affinity, and can be used to detect the expression of PPR phosphoprotein, providing new materials for studying the function of PPR phosphoprotein and elucidating the viral pathogenic mechanism.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to a monoclonal antibody that specifically binds to the phosphoprotein of small ruminant virus. Background Technology

[0002] Peste des petits ruminants (PPR) is an acute, highly contagious, and deadly infectious disease caused by the peste des petits ruminants virus (PPRV). It can infect goats, sheep, elk, gazelles, and oryx, among others. The main clinical symptoms include fever, diarrhea, enteritis, and pneumonia, with high morbidity and mortality rates. In 2012, PPR was explicitly listed as one of the 13 key exotic animal diseases requiring prevention. Therefore, we must strengthen our research on PPR.

[0003] The PPRV genome encodes six structural proteins and two non-structural proteins (Mahapatra et al., 2006). Among these, the phosphoprotein (P) is a multifunctional protein that acts as a molecular chaperone to maintain the nucleoprotein (N) in a soluble state linked to viral RNA. It also binds to the N-terminus of the large protein (L) to form an active RNA polymerase and plays a cofactor role in the transcriptase complex (Chattopadhyay and Shaila, 2004; Cevik et al., 2004). Previously, due to misconceptions about PPRV, research on it was limited both domestically and internationally. More research has been conducted on P proteins from other members of the Measlesvirus genus, revealing that P proteins are involved in multiple functions, including regulating transcription, translation, and controlling the cell cycle in key biological processes. Fuentes et al. (2010) reported that the N-terminus of the measles virus P protein helps induce N protein folding, and P protein phosphorylation is particularly important for RNA synthesis. Saiki et al. (2008) found that P protein phosphorylation plays a crucial regulatory role in the transcription and replication of the rinderpest virus genome. Furthermore, the P protein is the least conserved protein in the measlesvirus genus, which suggests that it plays an important role in peste des petits ruminants virus.

[0004] Studies have shown that PRRV P proteins play important roles in viral immune evasion responses. Recent research has discovered that PRRV P proteins are a novel antagonist that can inhibit IFN production through multiple mechanisms, thereby counteracting the host's antiviral innate immune response. The P protein inhibits STAT1 phosphorylation by interacting with STAT1, thereby blocking JAK-STAT signaling and exhibiting a strong negative regulatory effect on the host's antiviral immune response (Li P et al., Viruses, 2019). PRRV P proteins can induce the expression of host cell growth arrest DNA damage protein (GADD34), inhibit eIF2α phosphorylation and SGs formation, thereby resisting the host immune response and promoting viral replication (Alfred N et al., Front Vet Sci, 2021). The PPRV P protein targets IFN-regulatory factor (IRF)3 in human embryonic kidney 293T cells and primary goat fibroblasts, competitively binding to the IRF3 binding domain (IAD) of TBK1, blocking the interaction between TBK1 and IRF3, reducing IRF3 phosphorylation, thereby interfering with IRF3 dimerization and blocking IRF3 nuclear transport. This results in significant inhibition of RIG-I-like receptor signaling pathways and impaired expression of IFN-β and ISGs, thus inhibiting type I IFN production and promoting viral replication (Li P et al., J Immunol, 2021). Therefore, the PPRV P protein plays a crucial role in viral pathogenesis and immune mechanisms.

[0005] Monoclonal antibodies are antibodies secreted by a single B lymphocyte clone that recognize only a single antigenic epitope. They possess high specificity, affinity, and directionality, and are widely used in disease diagnosis and treatment, biological research, and industry. The binding ability of an antibody to a specific antigen depends on the variable region of each light / heavy chain pair, and is mainly determined by the complementarity-determining region (CDR). Summary of the Invention

[0006] To address the aforementioned technical problems, this invention utilizes recombinant PPRV P protein to immunize animals, obtaining hybridoma cell lines that stably secrete monoclonal antibodies against PPRV P protein. Sequencing yielded the complementarity-determining region and variable region sequences of this monoclonal antibody, which can be used for in vitro detection of peste des petits ruminants (PPR) virus or for research on the pathogenic and immune mechanisms of PPR virus. Specifically, it includes the following:

[0007] In a first aspect, the present invention provides a monoclonal antibody that specifically binds to the phosphoprotein of peste des petits ruminants virus, the monoclonal antibody comprising an antibody heavy chain and an antibody light chain;

[0008] The variable region CDR 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;

[0009] The variable region CDR of the antibody light chain includes CDR1 with an amino acid sequence as shown in SEQ ID NO.4, CDR2 with an amino acid sequence as shown in SEQ ID NO.5, and CDR3 with an amino acid sequence as shown in SEQ ID NO.6.

[0010] Preferably, the amino acid sequence of the variable region of the antibody heavy chain is shown in SEQ ID NO.7, and the amino acid sequence of the variable region of the antibody light chain is shown in SEQ ID NO.8.

[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.9, and the nucleotide sequence encoding the variable region of the antibody light chain is shown in SEQ ID NO.10.

[0013] Thirdly, the present invention provides the application of the monoclonal antibody described in the first aspect above in the preparation of reagents or kits for detecting peste des petits ruminants 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 peste des petits ruminants virus for non-disease diagnosis purposes or in the study of the pathogenesis and immune mechanism of peste des petits ruminants virus.

[0015] Fifthly, 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.

[0016] Preferably, the fluorescent marker is a detectable small molecule fluorescent marker.

[0017] Preferably, the fluorescent marker is the small molecule fluorescent organic dye FITC.

[0018] In a sixth aspect, the present invention provides the application of the fluorescently labeled monoclonal antibody described in the fifth aspect above in the preparation of a reagent for detecting peste des petits ruminants virus.

[0019] Preferably, the reagent is used for the detection of peste des petits ruminants virus by direct immunofluorescence assay.

[0020] In a seventh aspect, the present invention provides a direct immunofluorescence kit for detecting peste des petits ruminants virus, the kit comprising the fluorescently labeled monoclonal antibody described in the fifth aspect above.

[0021] The beneficial effects of this invention are: ① This invention provides a monoclonal antibody that specifically binds to the phosphoprotein of peste des petits ruminants (PPR); ② The monoclonal antibody has high affinity and good reactivity with both PPR and its phosphoprotein; it can be used for in vitro detection of PPR for non-disease diagnosis purposes and for research on the pathogenesis and immune mechanisms of PPR. Attached Figure Description

[0022] Figure 1 The results of the subtype identification of the monoclonal antibody described in this invention;

[0023] Figure 2 The results of the reactivity identification of the monoclonal antibody described in this invention;

[0024] Figure 3 This is a stacked map of the abundance of sequencing samples of the monoclonal antibody described in this invention;

[0025] Figure 4 This is the specificity identification result of the monoclonal antibody labeled with FITC as described in this invention. Detailed Implementation

[0026] The embodiments of the present invention are described in detail below. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting 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.

[0027] The Nigeria 75 / 1 vaccine strain PPRV, PPRV positive serum, and pCMV-Myc-His-PPRV-P recombinant plasmid used in the following examples were all preserved by the Lanzhou Veterinary Research Institute and were available to those skilled in the art or prepared using conventional methods; the experimental animals were clean-grade 8-10 week old female BALB / c mice provided by the Experimental Animal Center of the Lanzhou Veterinary Research Institute.

[0028] 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.

[0029] Example 1: Preparation of Monoclonal Antibodies

[0030] 1. Construction of hybridoma cell lines that stably secrete monoclonal antibodies

[0031] Immunogens were prepared by transfecting 293T cells with the pCMV-Myc-His-PPRV P recombinant plasmid. BALB / c mice were immunized, and monoclonal antibodies specifically binding to the PPRV P protein were screened using B-cell cloning techniques. Clones binding to the PPRV P protein were first selected using ELISA, followed by screening for clones binding to PPRV. The five selected positive hybridoma cell lines underwent three subclonal assays, were expanded in culture, and finally, OD cells were preserved. 450 The hybridoma cells with the highest concentration were 16E8A6. The above immunization and screening processes were outsourced to Anhui Global Gene Technology Co., Ltd.

[0032] 2. Preparation of ascites-type monoclonal antibodies

[0033] Each 8–10 week old female BALB / c mouse was intraperitoneally injected with 0.2 mL of liquid paraffin, followed by an intraperitoneal injection of 1 × 10⁻⁶ mol / L paraffin 7 days later. 6 Five well-growing hybridoma cells (16E8A6) were collected. The mice were observed daily for 5 days after inoculation. When the mice's abdomens became significantly distended and they had difficulty moving, they were euthanized by dislocation. The dark red liquid was aspirated from the mice's abdomens using a sterile syringe, centrifuged at 3000 rpm for 20 min, and the clear supernatant was aliquoted, labeled, and stored at -20℃ for later use.

[0034] 3. Determination of ascites titer

[0035] The ascites titer was determined by indirect ELISA using the Nigeria 75 / 1 vaccine strain PPRV (1:10) as the coating antigen. Sp2 / 0 cell supernatant (0.222) and normal mouse ascites (0.217) were used as negative controls, and PPRV-positive serum (0.737) was used as a positive control. The specific criterion was that the maximum dilution of ascites with a P / N ratio > 2.0 was taken as the ELISA titer. The results are shown in Table 1. Table 1 shows that the ascites titer of the hybridoma cell line 16E8A6 monoclonal antibody was 1:128 × 10⁻⁶. 3 .

[0036] Table 1. Results of ascites titer detection for hybridoma cell lines

[0037]

[0038] Example 2: Identification of monoclonal antibody 16E8A6 against peste des petits ruminants virus P protein

[0039] 1. Determination of monoclonal antibody subtypes

[0040] Take 200 μL of hybridoma cell culture supernatant and use the Isostrip Mouse Monoclonal Antibody Isotyping Kit to identify the monoclonal antibody subtype: Take 20 μL of hybridoma cell supernatant and add 180 μL of pH 7.2 PBS to dilute 1:20; take 150 μL of this dilution and add it to the test tube containing blue powder, gently shake to mix until the blue powder is completely dissolved; insert the Isotrip colloidal gold test strip into the bottom of the tube and observe the results within 5-10 minutes.

[0041] After detection using the Mouse Monoclonal Antibody Isotyping Kit, the monoclonal antibody subtype secreted by hybridoma cell line 16E8A6 was identified as IgG1, with a light chain type of κ. The results are as follows: Figure 1 As shown.

[0042] 2. Monoclonal antibody relative affinity test

[0043] PPRV (1:10) and recombinant P protein (1 μg / mL) were used as coating antigens (100 μL / well), respectively. Monoclonal antibody was added at 5-fold serial dilutions, and the relative affinity of monoclonal antibody 16E8A6 was determined using an indirect ELISA method. The results are shown in Table 2. Monoclonal antibody 16E8A6 showed high affinity for both antigens PPRV and recombinant P protein, and the affinity was stable.

[0044] Table 2. Relative affinity of monoclonal antibodies

[0045]

[0046] 3. Indirect immunofluorescence assay for the reactivity of monoclonal antibodies.

[0047] Vero cells were seeded in confocal culture dishes. When the cell density reached 80%, PPRV cells were seeded at an MOI of 0.01. After 2 hours of adsorption, the medium was replaced with fresh DMEM containing 10% fetal bovine serum. After 24 hours, the culture medium was discarded, the cells were washed with PBS, and fixed with 4% paraformaldehyde at room temperature for 30 minutes. Indirect immunofluorescence assays were performed using monoclonal antibody 16E8A6 and fluorescently labeled secondary antibody. The results are as follows: Figure 2 As shown, monoclonal antibody 16E8A6 exhibits good reactivity with PPRV. This indicates that monoclonal antibody 16E8A6 can be used for research on the function and mechanism of action of PPRV P protein.

[0048] Example 3: Determination of the heavy / light chain variable region sequence of the monoclonal antibody 16E8A6 against the P protein of small ruminant virus. 1. Obtaining the gene of the heavy / light chain variable region of the monoclonal antibody.

[0049] Based on the preliminary screening, monoclonal antibody 16E8A6 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 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.

[0050] The 10 sequences with the highest abundance were selected for abundance analysis, and the abundance packing plot is shown below. Figure 3 Select the most productive gene sequence as the target gene sequence.

[0051] 2. Analysis of gene sequencing results of the variable domains of the heavy and light chains of monoclonal antibodies

[0052] First, the original fastq files were quality-assessed. Cutadapt (v1.9.1) was used to remove linkers and bases with low quality scores (Q value < 20) to generate pruned data; Pandaseq (v2.10) was used to merge peer reads.

[0053] Based on sequencing results, the genes of the heavy and light chain variable domains of the anti-PPRV P protein monoclonal antibody were analyzed using NCBI-IgBLAST (v1.17.0) and the IMGT database (http: / / www.imgt.org / ). The results showed that the VH gene of the heavy chain variable region of the 16E8A6 antibody in the hybridoma cell line was 342 bp in length, encoding 114 amino acid residues, and included the highly variable regions CDR1-H, CDR2-H, and CDR3-H; the VL gene of the light chain variable region was 336 bp in length, encoding 112 amino acid residues, and included the highly variable regions CDR1-L, CDR2-L, and CDR3-L. Details are shown below:

[0054] CDR1-H: GFTFSTYT (shown in SEQ ID NO.1);

[0055] CDR2-H: ISSGGST (shown in SEQ ID NO.2);

[0056] CDR3-H: ARGGHMDY (shown in SEQ ID NO.3);

[0057] CDR1-L: QSLLNNDGKTY (shown in SEQ ID NO.4);

[0058] CDR2-L: LVS (shown in SEQ ID NO.5);

[0059] CDR3-L: WQGTHFPQT (shown in SEQ ID NO.6);

[0060] VH: EVKLVESGGGLVKPGGSLKLSCAASGFTFSTYTISWVRQTPEKRLEWVASISSGGS TYYPDSVKGRFTISRDNARNILYLQMSSLRSEDTAMYYCARGGHMDYWGQGTSVTVSS (shown in SEQ ID NO.7); Gene sequence: GAAGTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGA AGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTACCTATACCATATCTTGGGTTCGCCAGACTCCAGAGAAGAGGCTGGAGTGGGTCGCATCCATTAGTAGTGGTGGTAGCACCTACTATCCAGACAGTGTGAAGGGCCGATTCACCATCTCCAGAGATAATGCCAGGAACATCCTGTACCTGCAAATGAGCAGTCTGAGGTCTGAGGACACGGCCATGTATTACTGTGCAAGAGGCGGTCATATGGATTATTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA (shown in SEQ ID NO.9);

[0061] VL: DVVMTQTPLTLSVTIGQPASISCKSSQSLLNNDGKTYLNWLLQRPGQSPKRLIYLV SKLDSGVPDRFTGSGSGTDFTLKISRVEADDLGVYYCWQGTHFPQTFGGGTKLEIK (shown in SEQ ID NO.8); gene sequence: GATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACC ATTGGACAGCCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAAATAATGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCC CTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGACGATTTGGGAGTTTATTATTGCTGGCAAGGTACACATTTTCCTCAGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA(SE Q ID NO.10 shown).

[0062] Example 4: Application of monoclonal antibody 16E8A6 against peste des petits ruminants virus P protein

[0063] 1. Labeling of monoclonal antibody 16E8A6

[0064] The monoclonal antibody 16E8A6 (FITC-16E8A6) prepared in Example 1 and the monoclonal antibody 13-3 against PPRV N protein (obtained or prepared according to patent ZL201210278970.9) (FITC-13-3) were labeled using the FITC fluorescence conjugation kit (abcam). The specific operation was performed according to the instructions.

[0065] 2. Specificity of FITC-labeled monoclonal antibodies detected by direct immunofluorescence assay

[0066] Vero cells were seeded in confocal culture dishes. When the cell density reached 80%, PPRV was seeded at an MOI of 0.01. After adsorption for 2 hours, the medium was replaced with fresh DMEM containing 10% fetal bovine serum. The cells were incubated at 37°C with 5% CO2 for 36 hours, then the medium was discarded. The cells were washed with PBS and fixed with 4% paraformaldehyde at room temperature for 30 minutes. Simultaneously, pCMV-myc-PPRVP, pCMV-myc-PPRV N plasmids, and JET Prime transfection reagent were mixed at a ratio of 1 μg:2 μL and incubated for 15 minutes. When the cell density in the confocal culture dish reached approximately 80%, the medium was replaced with fresh medium. The transfection mixture was added to the cells, gently shaken to mix, and incubated at 37°C with 5% CO2 for 36 hours. The medium was then discarded, and the cells were washed and fixed as before.

[0067] Direct immunofluorescence detection of fixed samples was performed using FITC-labeled monoclonal antibodies: (1) The fixative was discarded, washed with PBS, and permeabilized with Triton X-100 at room temperature for 5 min; (2) Washed as above, and blocked with 5% BSA at 37℃ for 1 h; (3) Washed as above, and added the corresponding FITC-labeled monoclonal antibodies respectively, and incubated at 37℃ in the dark for 1 h; (4) Washed as above, and added DAPI and incubated at room temperature in the dark for 10 min; (5) Observed and photographed using a laser confocal microscope.

[0068] The results are as follows Figure 4 As shown, FITC-13-3 reacts with PPRV N protein, while FITC-16E8A6 reacts with PPRV and its P protein, but not with PPRV N protein. This indicates that the monoclonal antibody 16E8A6 described in this application reacts only with PPR VP protein and has good specificity.

[0069] 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 phosphoprotein of peste des petits ruminants virus, characterized in that, The monoclonal antibody comprises an antibody heavy chain and an antibody light chain; The variable region CDR 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 CDR of the antibody light chain includes CDR1 with an amino acid sequence as shown in SEQ ID NO.4, CDR2 with an amino acid sequence as shown in SEQ ID NO.5, and CDR3 with an amino acid sequence as shown in SEQ ID NO.

6.

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.7, and the amino acid sequence of the variable region of the antibody light chain is shown in SEQ ID NO.

8.

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.9, and the nucleotide sequence encoding the variable region of the antibody light chain is shown in SEQ ID NO.

10.

5. The use of the monoclonal antibody as described in claim 1 or 2 in the preparation of reagents for detecting peste des petits ruminants virus.

6. A fluorescently labeled monoclonal antibody, characterized in that, The fluorescently labeled monoclonal antibody is the monoclonal antibody as described in claim 1 or 2.

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 a reagent for detecting peste des petits ruminants virus.

9. The application as described in claim 8, characterized in that, The reagent is used for the detection of peste des petits ruminants virus by direct immunofluorescence assay.

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