A monoclonal antibody against non-structural protein V of peste des petits ruminants virus

Abstract

This invention relates to the field of biotechnology, specifically to a monoclonal antibody against the non-structural protein V of peste des petits ruminants (PPR) virus and its gene sequence. The invention discloses a monoclonal antibody 3E10 against PPR non-structural protein V, wherein the sequence of monoclonal antibody 3E10 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 3E10 specifically reacts with PPR and exhibits high affinity, enabling its detection of PPR. This provides new material for functional studies of PPR V protein and the development of diagnostic reagents, and offers new technical support for the successful implementation of the global PPR elimination program.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a monoclonal antibody against the non-structural protein V of peste des petits ruminants virus. Background Technology

[0002] Peste des petits ruminants virus (PPRV) is the pathogen that causes peste des petits ruminants (PPR). This virus mainly infects ruminants such as goats, sheep, gazelles, and oryx, causing the animals to exhibit clinical symptoms such as fever, diarrhea, enteritis, and pneumonia.

[0003] The PPRV genome encodes six structural proteins and two non-structural proteins. The V protein is a non-structural protein resulting from a codon shift caused by the insertion of a G base at position 751 of the P gene during RNA editing. The length of the V protein varies among measlesviruses: PPRV is 298 aa, CDV and RPV are 299 aa, and MV and DMV are 300 and 303 aa, respectively. Theoretically, the V protein's molecular weight is estimated at 32.28 kDa, but its actual size is around 40 kDa. The phosphorylation of most (60%) of its Ser content is likely the main factor contributing to the increased molecular weight. The editing site (-5'-ttaaaagggcacag-3') in measlesviruses is conserved. In PPRV, this site is located at position 751 of the P gene, meaning the V gene shares the same N-terminus as the P gene. However, due to the codon shift caused by the insertion of the G base, the resulting V protein has a C-terminus rich in cysteine ​​(Cys). His and seven Cys are linked by two Zn... 2+ They combine to form a zinc finger structure, which plays an important role in antagonizing innate immunity.

[0004] Non-structural proteins are conserved not only in the genus Measlesvirus but also in the family Paramyxoviridae, indicating their crucial role in viral replication and pathogenicity within their respective species. The V protein of the Paramyxoviridae family can bind to the N and L proteins to regulate RNA synthesis and promote viral replication through transcription, but its specific mechanisms remain to be elucidated. The V protein of the Paramyxoviridae family plays a vital antagonistic role in viral evasion of the innate immune response, with two main antagonistic forms: (1) V protein inhibits upstream pathways of IFN production, primarily through inhibition of RIG-I-like receptor pathways or RLRs-like receptor pathways depending on the PAMPs; (2) V protein inhibits IFN-induced signal transduction. Studies have shown that the V protein of Paramyxoviridae viruses binds to MDA5 through its C-terminus containing a zinc finger structure, preventing the binding of dsRNA to MDA5 and MDA5 polymerization, thereby inhibiting IFN production. Different viral V proteins bind to MDA5 at specific sites. For example, the V proteins of PIV5 and NiV viruses contain a conserved Cys large loop, which is not essential for MDA5 binding. However, in the binding of MDA5 by the V proteins of MeV and MuV viruses, the zinc finger structure formed by Cys is crucial. Except for the V protein of rinderpest virus, the V proteins of different species of paramyxoviridae viruses, such as PIV5, hPIV2, MuV, MeV, HeV, NiV, and SeV, can bind to the helicase region (amino acid positions 701-816) of MDA5, hindering the steric hindrance of dsRNA-MDA5 binding. Crystal structure analysis shows that after the V protein binds to MDA5, its ATPase region unfolds, disrupting the hydrophobic sites in this region. This indicates that paramyxoviridae viral V proteins can bind to MDA5 using their C-terminus, but the molecular mechanism of binding is virus-specific. Recent studies have found that the PPRVV protein can bind to LGP2, thereby promoting its negative regulation of RIG-I, which explains the inhibitory mechanism of the V protein on RIG-I-dependent IFN production. Tyrosine residue 110 (Y110) of the V protein is a specific amino acid residue that interacts with STAT1; their binding can alter the subcellular distribution of STAT1. The V protein uses its C-terminal Cys and Trp residues to bind to STAT2, altering the cellular localization of STAT2 and inhibiting JAK-STAT signaling and IFN responses. Furthermore, recombinant viruses lacking the V protein or with a Cys-rich region exhibit weakened in vitro proliferation ability. Therefore, the V protein of paramyxoviridae viruses plays a crucial role in the viral life cycle and virus-induced innate immunity.

[0005] Monoclonal antibodies are antibodies made from highly homogeneous immune cells derived from a single parent cell. They recognize a specific antigenic epitope, exhibiting monovalent affinity. Their binding ability to a particular antigen depends on the variable regions of each light / heavy chain pair, primarily determined by the complementarity-determining regions (CDRs). Therefore, monoclonal antibodies can be used to detect or purify substances containing specific antigenic epitopes and have become important tools in biochemistry, molecular biology, and medical research. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention utilizes recombinant PPRV V protein to immunize animals, obtaining hybridoma cell lines that stably secrete monoclonal antibodies against PPRV V 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 virus (PPR) or functional studies of PPR V protein. Specifically, it includes the following:

[0007] In a first aspect, the present invention provides a monoclonal antibody against the non-structural protein V 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 for detecting antibodies against non-structural proteins of peste des petits ruminants virus.

[0014] Preferably, the reagent is a blocking ELISA kit.

[0015] Fourthly, the present invention provides an enzyme conjugate, the enzyme conjugate comprising:

[0016] (i) the monoclonal antibody described in the first aspect above;

[0017] (ii) and horseradish peroxidase conjugated with the monoclonal antibody in (i).

[0018] Fifthly, the present invention provides an ELISA kit for detecting antibodies against the nonstructural protein V of peste des petits ruminants virus, the kit comprising the enzyme conjugate described in the fourth aspect above.

[0019] Preferably, the kit includes an enzyme-labeled plate coated with peste des petits ruminants virus antigen, control serum, blocking solution, diluent, the enzyme conjugate described in the fourth aspect above, washing solution, chromogenic agent, and stop solution.

[0020] Preferably, the coating antigen is peste des petits ruminants virus V protein.

[0021] In a sixth aspect, the present invention provides the application of the monoclonal antibody described in the first aspect, or the enzyme conjugate described in the fourth aspect, or the ELISA kit described in the fifth aspect, in the detection of natural infectivity of peste des petits ruminants virus for non-disease diagnosis purposes or in the study of the function of peste des petits ruminants virus V protein.

[0022] In a seventh aspect, the present invention provides a blocking ELISA method for detecting peste des petits ruminants virus, wherein the method comprises the following steps:

[0023] (1) Dilute the purified recombinant PPRVV protein to 1 μg / mL with coating buffer (0.05 mol / L sodium carbonate-sodium bicarbonate buffer, pH 9.6), 50 μL / well, seal with sealing membrane, and coat overnight at 4°C;

[0024] (2) Wash 3 times with 1×PBST, add 1% BSA blocking solution, 100μL / well, seal the plate with sealing membrane, and incubate at 37℃ for 1h;

[0025] (3) Wash 3 times with 1×PBST, add positive serum, negative serum and test serum respectively, 50μL / well, seal with sealing film, and incubate at 37℃ for 45min;

[0026] (4) Wash 3 times with 1×PBST, then add HRP-3E10 diluted with PBST (1:8×10⁻⁶). 3 ), 50 μL / well, seal with sealing film, incubate at 37°C for 30 min;

[0027] (5) Add substrate TMB, 50 μL / well, seal the plate with sealing membrane, and react at 37°C for 15 min;

[0028] (6) Add stop solution (1M sulfuric acid), 50 μL / well;

[0029] (7) Detect the OD of each well using an enzyme-linked immunosorbent assay (ELISA) reader. 450 value;

[0030] (8) Result determination: Calculate the inhibition rate (S / Nc%) of each sample according to the formula, S / Nc% = (OD 450待检样品 / OD 450 (Negative control) × 100%, the result interpretation criteria are: S / Nc% > 50%, antibody negative; S / Nc% ≤ 50%, antibody positive.

[0031] The beneficial effects of this invention are: ① This invention provides a monoclonal antibody against the non-structural protein V of peste des petits ruminants virus; ② The monoclonal antibody has high affinity and good reactivity with peste des petits ruminants virus; it can be used for the detection of naturally occurring peste des petits ruminants and for the study of V protein function. Attached Figure Description

[0032] Figure 1 The results of the subtype identification of the monoclonal antibody described in this invention;

[0033] Figure 2 The results of the reactivity identification of the monoclonal antibody described in this invention;

[0034] Figure 3 This is a stacked map of the abundance of sequencing samples of the monoclonal antibody described in this invention. Detailed Implementation

[0035] 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.

[0036] The Nigeria 75 / 1 vaccine strain PPRV, PPRV positive serum, recombinant PPRV V protein and recombinant plasmid pCMV-3×Flag-PPRV-V 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.

[0037] 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.

[0038] Example 1: Preparation of Monoclonal Antibodies

[0039] 1.1 Construction of hybridoma cell lines that stably secrete monoclonal antibodies

[0040] Recombinant protein pET-B2m-PPRV V was prepared using a prokaryotic expression system and immunized BALB / c mice. Monoclonal antibodies specifically binding to PPRV V protein were screened using B-cell cloning techniques. First, clones binding to PPRV V protein were selected using ELISA; then, clones binding to PPRV were further screened. Seven positive hybridoma cell lines were identified, and three subclonal assays were performed. The cells were then expanded and OD cells were finally preserved. 450 The hybridoma cell line with the highest value was 3E10. The above immunization and screening processes were outsourced to Wuhan Jinkairui Biotechnology Co., Ltd.

[0041] 1.2 Preparation of Ascites-type Monoclonal Antibodies

[0042] 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 (3E10) 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 cervical 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.

[0043] 1.3 Determination of ascites titer

[0044] The ascites titer was determined by indirect ELISA using the Nigeria 75 / 1 vaccine strain PPRV (1:50) 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 highest dilution of ascites with a P / N ratio > 2.0 was taken as the ELISA titer. Table 1 shows that the ascites titer of the hybridoma cell line 3E10 monoclonal antibody was 1:1.6 × 10⁻⁶. 4 .

[0045] Table 1. Results of ascites titer detection

[0046]

[0047] Example 2: Identification of monoclonal antibody 3E10 against peste des petits ruminants virus V protein

[0048] 2.1 Determination of Monoclonal Antibody Subtypes

[0049] 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 1:10; 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.

[0050] After testing with the Mouse Monoclonal Antibody Isotyping Kit, the monoclonal antibody subtype secreted by hybridoma cell line 3E10 was identified as IgG1, with a light chain type of κ. The results are as follows: Figure 1 As shown.

[0051] 2.2 Monoclonal antibody relative affinity test

[0052] PPRV (1:10) and recombinant PPRV V protein (1 μg / mL) were used as coating antigens (100 μL / well), with 100 μL coated per well. The relative affinity of the virus to the 5-fold serially diluted monoclonal antibody 3E10 was determined by indirect ELISA. The results are shown in Table 2, indicating that monoclonal antibody 3E10 has a high affinity for PPRV.

[0053] Table 2. Relative affinity of monoclonal antibodies

[0054]

[0055] 2.3 Indirect Immunofluorescence Assay

[0056] When the density of Vero cells in the confocal culture dish reached 80%, PPRV (MOI = 0.01) was seeded. After 2 hours of adsorption, the medium was replaced with fresh DMEM containing 10% fetal bovine serum. The medium was aspirated after 24 hours per index (hpi), the cells were washed with PBS, and fixed with 4% paraformaldehyde at room temperature for 30 minutes. Indirect immunofluorescence assay was performed using monoclonal antibody 3E10 and fluorescently labeled secondary antibody. The results are as follows: Figure 2 As shown, monoclonal antibody 3E10 can react with PPRV. This indicates that monoclonal antibody 3E10 has good reactivity and can be used for research on the functional mechanism of PPRV V protein and the development of diagnostic agents.

[0057] Example 3: Determination of the heavy and light chain variable region sequence of the monoclonal antibody 3E10 against peste des petits ruminants virus V protein.

[0058] Based on the preliminary screening, monoclonal antibody 3E10 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 total of 5 × 10⁶ cells / year. 6 Total RNA was extracted using the HiPure RNA Mini Columns (Magen) kit, following the product instructions. 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 product instructions. The obtained double-stranded cDNA was used as a template for amplification. The upstream primer was anchored to TSO, and the downstream primer bound 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 and quantified using Qubit. Sequencing was performed using an Illumina MiSeq PE300.

[0059] The 10 sequences with the highest abundance were selected for abundance analysis. The abundance packing diagram of the samples is shown below. Figure 3 Select the most relevant and highest-ranking sequence as the target gene sequence.

[0060] 3.2 Analysis of gene sequencing results of the variable domains of the heavy and light chains of monoclonal antibodies

[0061] Based on the sequencing results, the gene sequences of the heavy and light chain variable domains of the monoclonal antibody 3E10 were analyzed using NCBI-IgBLAST (v1.17.0) and the IMGT database (http: / / www.imgt.org / ). The analysis showed that the VH gene of the heavy chain variable region of the hybridoma cell line 3E10 antibody is 342 bp in length, encoding 114 amino acid residues, and includes the highly variable regions CDR1-H, CDR2-H, and CDR3-H; the VL gene of the light chain variable region is 336 bp in length, encoding 112 amino acid residues, and includes the highly variable regions CDR1-L, CDR2-L, and CDR3-L. Details are shown below:

[0062] CDR1-H: GFTFSNYR (shown in SEQ ID NO.1);

[0063] CDR2-H: IPGKSDNFGT (shown in SEQ ID NO.2);

[0064] CDR3-H: SRGAY (shown in SEQ ID NO.3);

[0065] CDR1-L: QSLLDSDGKTY (shown in SEQ ID NO.4);

[0066] CDR2-L: LVS (shown in SEQ ID NO.5);

[0067] CDR3-L: WQGTHFPQT (shown in SEQ ID NO.6);

[0068] VH: QVQLVEAGGGLVRPGNSLKLSCVTSGFTF SNYRMYWLRQPPGRRLEWIAVIPGK SDNFGTNYAESVKGRFIISRDDSKSSVYLQMNSLREEDTATYYCSRGAYWGQGTLVTVSA (shown in SEQ ID NO.7); Gene sequence: CAGGTGCAGCTTGTAGAGGCCGGGGGAGGCTTGGT GAGGCCTGGAAATTCTCTGAAACTCTCCTGTGTTACCTCGGGATTCACTTTCAGTAACTACCGGATGTACTGGCTCCGCCAGCCTCCAGGGAGGAGGCTGGAGTGGATTGCTGTAATTCCAGGCAAATCTGATAATTTTGGAACAAATTATGCAGAGTCTGTGAAAGGCAGATTCATTATTTCAAGAGATGATTCAAAAAGCAGTGTCTACCTGCAGATGAACAGCTTAAGAGAGGAAGACACTGCCACTTATTATTGTAGTAGAGGGGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA (shown in SEQ ID NO.9);

[0069] VL: DVVMTQTPLTLSVTIGQPASMSCKSSQSL LDSDGKTYLNWLLQRPGQSPKRLIYL VSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPQTFGGGTKLEIK (shown in SE Q ID NO.8); gene sequence: GATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTTA CCATTGGACAACCAGCCTCCATGTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTACCTGGTGTCTAAACTGGACTCTGGAGTCC CTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGCAGGGGACACATTTTCCTCAGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA(SEQ ID NO.10 shown).

[0070] Example 4: Application of monoclonal antibody 3E10 against peste des petits ruminants virus V protein

[0071] 4.1 Labeling of monoclonal antibody 3E10

[0072] The monoclonal antibody 3E10 (HRP-3E10) prepared in Example 1 was labeled using the Peroxidase Labeling Kit-NH2 (Dojindo), following the instructions in the manufacturer's manual.

[0073] 4.2 PPRV Competitive ELISA Antibody Detection Kit (ID.vet)

[0074] (1) Sample addition: Add 25 μL of buffer 13 to each well, then add 25 μL of positive control to wells A1 and B1, 25 μL of negative control to wells C1 and D1, and 25 μL of serum to be tested to the remaining wells.

[0075] (2) Incubation: React at 37℃ for 45 min;

[0076] (3) Washing: Discard the solution in the wells, add 300 μL of washing solution to each well, and wash 3 times;

[0077] (4) Enzyme conjugate: Add buffer 4 to each well to dilute to 100 μL of 1× enzyme conjugate, and react at room temperature for 30 min.

[0078] (5) Washing: Wash 3 times as above;

[0079] (6) Color development: Add 100 μL of substrate solution to each well and let it stand at room temperature in the dark for 15 minutes.

[0080] (7) Termination: Add 100 μL of termination solution to each well;

[0081] (8) Detection: Measure the absorbance of each well at a wavelength of 450 nm using an ELISA reader;

[0082] (9) Result determination: According to the formula: S / N%=(sample OD 450 / Negative control OD 450 Calculate the S / N% of each serum sample by multiplying the value by 100%. If S / N% > 60%, the sample is considered negative for peste des petits ruminants (PPR) antibodies; if S / N% ≤ 50%, the sample is considered positive for PPR antibodies; and if 50% < S / N% ≤ 60%, the sample is considered suspicious and requires repeated testing.

[0083] Based on the test results of serum samples stored in our laboratory using the ID.vet kit, and in accordance with the competitive ELISA criteria in the national standard (GB / T27982-2011) for the diagnosis of peste des petits ruminants (PI = 100 - (OD)), T ÷OD C (PI>80 is considered strong positive, 50<PI≤80 is considered weak positive, and PI≤50 is considered negative). Three samples of strong positive, three samples of weak positive, and three samples of negative PPR virus were selected for subsequent establishment of the blocking ELISA detection method.

[0084] 4.3 The monoclonal antibody described in this invention is used to block ELISA detection.

[0085] Using recombinant PPRVV protein as the coating antigen, the monoclonal antibody HRP-3E10 prepared in Example 4.1 of this invention was used as the detection antibody to detect strongly positive, weakly positive, and negative serum of peste des petits ruminants virus, as well as positive serum of FMDV-O, BTV, and LSDV, and to analyze the reaction characteristics of the monoclonal antibody prepared in Example 1 of this invention.

[0086] 4.3.1 Optimal antigen-antibody reaction concentration

[0087] (1) The microplates were coated with recombinant PPRVV protein at different concentrations (2, 1, 0.5 and 0.25 μg / mL), 50 μL / well, and coated overnight at 4℃;

[0088] (2) Wash 3 times with PBST, and add 1:1×10 each time. 3 1:2×10 3 1:4×10 3 1:8×10 3 1:1.6×10 4 The working solution of monoclonal antibody HRP-3E10, diluted 5 times, was used in a 50 μL / well plate. The plate was then sealed and reacted at 37°C for 1 h.

[0089] (3) Wash 3 times with PBST, add 50 μL of substrate TMB solution per well, seal the plate, and react at 37°C in the dark for 15 min.

[0090] (4) Add stop solution (1M sulfuric acid), 50 μL / well;

[0091] (5) OD was measured using an enzyme-linked immunosorbent assay (ELISA) reader. 450nm Value, based on OD 450nm The optimal reaction concentration was determined by screening.

[0092] The results are shown in Table 3. The optimal antigen coating amount was 0.05 μg / well, while the optimal dilution of the monoclonal antibody HRP-3E10 was 1:8 × 10⁻⁶. 3 .

[0093] Table 3 Results of Optimal Reaction Concentration Detection

[0094]

[0095] 4.3.2 Optimal Sample Addition Amount

[0096] (1) Dilute the recombinant PPRVV protein to 1 μg / mL, 50 μL / well, and coat overnight at 4℃;

[0097] (2) Add PPRV negative and positive serum stock solution and serum solution diluted with PBS at a ratio of 1:1, 50 μL / well, and incubate at 37°C for 1 h;

[0098] (3) Wash 3 times with PBST, add 1:8×10 3 Diluted monoclonal antibody HRP-3E10 working solution, 50 μL / well, sealed plate, reacted at 37℃ for 1 h;

[0099] (4) Wash 3 times with PBST, add 50 μL of substrate TMB solution per well, seal the plate, and react at 37°C in the dark for 15 min.

[0100] (5) Add stop solution (1M sulfuric acid), 50 μL / well;

[0101] (6) Determination of OD 450The ratio (S / Nc%) of the OD value of the test sample to the OD value of the negative control is calculated to screen out the optimal sample amount.

[0102] The S / Nc% is shown in Table 4. Compared with the results of a sample volume of 25 μL, when the sample volume is 50 μL, the S / Nc% of the positive sample is smaller and the S / Nc% of the negative sample is larger, and the judgment results are consistent.

[0103] Table 4 Results of Optimal Sample Dosage Testing

[0104]

[0105] 4.3.3 Optimal Sealing Fluid

[0106] The procedure is the same as in 4.3.2, using PBST, 1% BSA solution, and 2% fetal bovine serum solution as blocking solutions. OD is measured using a microplate reader. 450 The value is used to calculate S / Nc%, and the optimal blocking solution is selected.

[0107] The test results are shown in Table 5. When using 1% BSA solution as the blocking solution, OD 450 The values ​​and S / Nc% were relatively stable, and the results of the serum test were consistent with the results of the neutralization test.

[0108] Table 5 Detection results of the best sealing fluid

[0109]

[0110] 4.3.4 Optimal Response Time

[0111] The procedure is the same as in 4.3.2, with serum reaction times of 45 and 60 min, and HRP-3E10 reaction times of 30 and 45 min, based on the measured OD values. 450 Calculate the S / Nc% value to screen for the optimal reaction time.

[0112] The test results are shown in Table 6. It was found that different reaction time combinations yielded consistent results and did not affect the interpretation of the results. Considering the difference in S / Nc% values ​​of positive samples and the need for rapid measurement, reaction times of 45 min for serum and 30 min for HRP-3E10 were selected.

[0113] Table 6 Results of Optimal Reaction Time Detection

[0114]

[0115] 4.3.5 Determination of Standard Operating Procedures for Blocking ELISA Detection Methods

[0116] Based on the above optimized conditions, the established blocking ELISA procedure was determined as follows:

[0117] (1) Dilute the purified recombinant PPRVV protein to 1 μg / mL with coating buffer (0.05 mol / L sodium carbonate-sodium bicarbonate buffer, pH 9.6), 50 μL / well, seal with sealing membrane, and coat overnight at 4°C;

[0118] (2) Wash 3 times with 1×PBST, add 1% BSA blocking solution, 100μL / well, seal the plate with sealing membrane, and incubate at 37℃ for 1h;

[0119] (3) Wash 3 times with 1×PBST, add positive serum, negative serum and test serum respectively, 50μL / well, seal with sealing film, and incubate at 37℃ for 45min;

[0120] (4) Wash 3 times with 1×PBST, then add HRP-3E10 diluted with PBST (1:8×10⁻⁶). 3 ), 50 μL / well, seal with sealing film, incubate at 37°C for 30 min;

[0121] (5) Add substrate TMB, 50 μL / well, seal the plate with sealing membrane, and react at 37°C for 15 min;

[0122] (6) Add stop solution (1M sulfuric acid), 50 μL / well;

[0123] (7) Detect the OD of each well using an enzyme-linked immunosorbent assay (ELISA) reader. 450 value.

[0124] (8) Result determination: Calculate the inhibition rate (S / Nc%) of each sample according to the formula, S / Nc% = (OD 450待检样品 / OD 450 (Negative control) × 100%, the result interpretation criteria are: S / Nc% > 50%, antibody negative; S / Nc% ≤ 50%, antibody positive.

[0125] 4.3.6 Specificity test

[0126] FMDV-O, BTV, LSDV, and GPV-positive serum were detected using the established blocking ELISA method, and the results were analyzed based on the measured OD values. 450 Calculate its S / Nc% value.

[0127] The test results are shown in Table 7. The S / Nc% values ​​of FMDV-O, BTV, LSDV and GPV positive sera are all greater than 80%, which means that the monoclonal antibodies prepared in Example 1 or Example 4.1 have good specificity.

[0128] Table 7 Results of Specificity Tests

[0129]

[0130] 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 against the non-structural protein V 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 of LVS, 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 (PPR) V protein, or in the detection of natural infectivity of PPR for non-disease diagnosis purposes.

6. The application as described in claim 5, characterized in that, The reagent is a blocking ELISA kit.

7. An enzyme conjugate, characterized in that, The enzyme conjugate includes: (i) the monoclonal antibody as described in claim 1 or 2; (ii) and horseradish peroxidase conjugated with the monoclonal antibody in (i).

8. An ELISA kit for detecting antibodies against the non-structural protein V of peste des petits ruminants virus, characterized in that, The kit comprises the enzyme conjugate of claim 7.

9. The ELISA kit as described in claim 8, characterized in that, The kit includes an enzyme-labeled plate coated with peste des petits ruminants virus antigen, control serum, blocking buffer, diluent, the enzyme conjugate as described in claim 7, washing buffer, chromogenic agent, and stop solution.

10. A blocking ELISA method for detecting peste des petits ruminants virus for non-disease diagnosis purposes, characterized in that, The method includes the following steps: (1) Dilute the purified recombinant PPRVV protein to 1 μg / mL with coating buffer, 50 μL / well, seal with sealing membrane, and coat overnight at 4°C; the coating buffer is 0.05 mol / L sodium carbonate-sodium bicarbonate buffer, pH 9.6; (2) Wash 3 times with 1×PBST, add 1% BSA blocking solution, 100μL / well, seal the plate with sealing membrane, and incubate at 37℃ for 1h; (3) Wash 3 times with 1×PBST, add positive serum, negative serum and test serum respectively, 50μL / well, seal with sealing film, and incubate at 37℃ for 45min; (4) Wash 3 times with 1×PBST, then add 1:8×10 3 The enzyme conjugate of claim 7 diluted with PBST, 50 μL / well, sealed with sealing film, and incubated at 37°C for 30 min; (5) Add substrate TMB, 50 μL / well, seal the plate with sealing membrane, and react at 37°C for 15 min; (6) Add 1M sulfuric acid, 50 μL / well; (7) Detect the OD of each well using an enzyme-linked immunosorbent assay (ELISA) reader. 450 value; (8) Result determination: Calculate the inhibition rate S / Nc% for each sample according to the formula, S / Nc% = (OD 450待检样品 / OD 450阴性对 The result is calculated as follows: (S / Nc%) × 100%, and the criteria for judging the result are: S / Nc% > 50%, antibody negative; S / Nc% ≤ 50%, antibody positive.

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