Monoclonal antibody for resisting non-structural protein V of peste des petits ruminants virus

Abstract

The invention relates to the technical field of biology, in particular to a monoclonal antibody for resisting peste des petits ruminants virus non-structural protein V and a gene sequence of the monoclonal antibody. The invention discloses a monoclonal antibody 3E10 for resisting a peste des petits ruminants virus non-structural protein V. The sequence of the monoclonal antibody 3E10 comprises a heavy chain variable region as shown in SEQ ID NO.7 and a light chain variable region as shown in SEQ ID NO.8; the monoclonal antibody 3E10 can be subjected to specific reaction with the peste des petits ruminants virus, has high affinity, can be used for detecting the peste des petits ruminants virus, provides a new material for functional research of peste des petits ruminants virus V protein and research and development of diagnostic reagents, and has broad application prospects. And a new technical support is provided for smooth implementation of a peste des petits ruminant global elimination plan.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a monoclonal antibody against nonstructural protein V of peste des petits ruminants virus. Background Art

[0002] Peste des petits ruminants virus (PPRV) is the pathogen that causes Peste des petits ruminants (PPR). The virus mainly infects ruminants such as goats, sheep, gazelles and antelopes, causing animals to show clinical symptoms such as fever, diarrhea, enteritis and pneumonia. PPR is a major cross-border animal infectious disease, listed as a legally reportable animal disease by OIE, and my country lists it as a Class I animal disease. In 2015, FAO and OIE organized a global ministerial meeting to adopt and launch the Global Strategy for the Control and Eradication of Peste des petits ruminants.

[0003] The PPRV genome encodes 6 structural proteins and 2 non-structural proteins. The V protein is a non-structural protein produced by the insertion of a base G at the 751th position of the P gene during RNA editing, resulting in a codon shift. The length of the V protein in the morbillivirus genus is different: PPRV is 298aa, CDV and RPV are 299aa, MV and DMV are 300 and 303aa, respectively. Theoretically, the molecular weight of the V protein is estimated to be 32.28kDa, but the actual size is around 40kD. Most of the Ser contained (60%) can be phosphorylated, which may be the main factor causing the increase in molecular weight. The editing site (-5'-ttaaaagggcacag-3') of the morbillivirus genus is conserved. In PPRV, this site is located at the 751bp position of the P gene, that is, the V gene and the P gene have the same N-terminus; but due to the codon shift after the insertion of the base G, the formed V protein has a cysteine ​​(Cys)-rich C-terminus. His and 7 Cys are bound by two Zn 2+ The binding forms a zinc finger structure that plays an important role in antagonizing innate immunity.

[0004] Nonstructural proteins are conserved not only in the Morbillivirus genus, but also in the Paramyxoviridae family, which indicates that they are critical for viral proliferation and pathogenicity in their respective species. The V protein of the Paramyxoviridae family can bind to the N protein and L protein to participate in the regulation of RNA synthesis, and also promote viral replication by participating in the transcription process, but its specific mechanism remains to be elucidated. The V protein of the Paramyxoviridae family plays a very important antagonistic role in the process of virus evasion of innate immune response. The antagonistic forms are mainly divided into two categories: (1) V protein inhibits the upstream pathway of IFN production, which is mainly divided into inhibition of RIG-I-like receptor pathway or RLRs-like receptor pathway according to different PAMPs; (2) V protein inhibits IFN-induced signal transduction. Studies have shown that the V protein of the Paramyxoviridae family virus binds to MDA5 through the C-terminus containing a zinc finger structure, preventing the binding of dsRNA to MDA5 and MDA5 polymerization, thereby inhibiting the production of IFN. The precise sites where different viral V proteins bind to MDA5 are specific. For example, the V proteins of PIV5 and NiV viruses contain a large loop of conserved Cys, which is not necessary in the process of binding to MDA5. However, the zinc finger structure formed by Cys is crucial in the process of binding to MDA5 by the V proteins of viruses such as MeV and MuV. In addition to the V protein of rinderpest virus, the V proteins of different species of Paramyxoviridae, such as PIV5, hPIV2, MuV, MeV, HeV, NiV, SeV, etc., can bind to the amino acid region 701-816 (helicase region) of MDA5, hindering the steric hindrance of dsRNA-MDA5 binding. The crystal structure shows that after the V protein binds to MDA5, its ATPase region changes from folding to stretching, destroying the hydrophobic site in this region. This shows that the V protein of Paramyxoviridae viruses can use its C-terminus to bind to MDA5, but the molecular mechanism of binding is virus-specific. Recent studies have found that PPRV V protein can bind to LGP2 and promote its negative regulation of RIG-I, which explains the inhibitory mechanism of V protein on RIG-I-dependent IFN production; the 110th tyrosine (Y110) of V protein is a specific amino acid residue that interacts with STAT1, and their binding can change the subcellular distribution of STAT1; V protein uses its C-terminal Cys residue and Trp residue to bind to STAT2, changing the cellular localization of STAT2 and inhibiting the signal transduction of the JAK-STAT pathway and inhibiting the IFN response. In addition, the recombinant virus lacking V protein or the Cys-rich region of V protein has a weaker ability to proliferate in vitro. It can be seen that the V protein of Paramyxoviridae virus plays a very critical role in the viral life cycle and virus-induced innate immunity.

[0005] Monoclonal antibodies are antibodies made by highly consistent immune cells that originate from a single parent cell. Monoclonal antibodies recognize a specific antigen epitope and have monovalent affinity. Their ability to bind 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). Therefore, monoclonal antibodies can be used to detect or purify substances containing specific antigen epitopes and have become an important tool in the fields of biochemistry, molecular biology, and medical research. Summary of the invention

[0006] In view of the above technical problems, the present invention uses recombinant PPRV V protein to immunize animals, obtains a hybridoma cell line that stably secretes anti-PPRV V protein monoclonal antibodies, and sequences the complementary determining region and variable region sequences of the monoclonal antibody, which can be used for in vitro detection of PPR virus or functional research of PPR virus V protein. Specifically, it includes the following contents:

[0007] In a first aspect, the present invention provides a monoclonal antibody against nonstructural protein V of peste des petits ruminants virus, wherein the monoclonal antibody comprises 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 as SEQ ID NO.7, and the amino acid sequence of the variable region of the antibody light chain is shown as SEQ ID NO.8.

[0011] In a second aspect, the present invention provides a nucleic acid encoding 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 as SEQ ID NO.9, and the nucleotide sequence encoding the variable region of the antibody light chain is shown as SEQ ID NO.10.

[0013] In a third aspect, the present invention provides use of the monoclonal antibody described in the first aspect in the preparation of a reagent for detecting antibodies against non-structural proteins of peste des petits ruminants virus.

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

[0015] In a fourth aspect, 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 to the monoclonal antibody in (i).

[0018] In a fifth aspect, the present invention provides an ELISA kit for detecting antibodies to 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 comprises an ELISA plate coated with peste des petits ruminants virus antigen, a control serum, a blocking solution, a diluent, the enzyme conjugate described in the fourth aspect, a washing solution, a color developer, and a 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 use 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 PPRV V protein to 1 μg / mL with coating solution (0.05 mol / L sodium carbonate-sodium bicarbonate buffer, pH 9.6), 50 μL / well, seal the plate with sealing film, and coat overnight at 4°C;

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

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

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

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

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

[0029] (7) Detect the OD of each well using an 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阴性对照 )×100%, the result judgment criteria are: S / Nc%>50%, antibody negative; S / Nc%≤50%, antibody positive.

[0031] The beneficial effects of the present invention are: ① the present invention provides a monoclonal antibody against non-structural protein V of peste des petits ruminants virus; ② the monoclonal antibody has high affinity and good reactivity to peste des petits ruminants virus; and can be used for detection for the purpose of diagnosing naturally infectious peste des petits ruminants and studying the function of V protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 is the subtype identification result of the monoclonal antibody of the present invention;

[0033] Figure 2 The reactogenicity identification result of the monoclonal antibody of the present invention;

[0034] Figure 3 This is an abundance accumulation diagram of sequencing samples of the monoclonal antibody described in the present invention. DETAILED DESCRIPTION

[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 cannot be understood as limiting the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all 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 are all preserved by the Lanzhou Veterinary Research Institute and can be obtained by those skilled in the art, or prepared by conventional methods; the experimental animals are 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 explicitly stated, all reagents used in the following examples are commercially available or can be synthesized according to text or known methods, and reaction conditions not listed are also easily obtained by 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] The recombinant protein pET-B2m-PPRV V was prepared using a prokaryotic expression system, and BALB / c mice were immunized. The monoclonal antibody that specifically binds to the PPRV V protein was screened using B cell cloning technology. First, clones that bind to the PPRV V protein were selected using ELISA; then clones that bind to PPRV were screened. Seven positive hybridoma cells were screened, subcloned three times, expanded, and finally preserved at OD 450 The hybridoma cell line with the highest value was 3E10. The above immunization and screening processes were entrusted to Wuhan Jinkairui Bioengineering Co., Ltd.

[0041] 1.2 Preparation of ascites monoclonal antibodies

[0042] Each female BALB / c mouse aged 8 to 10 weeks was intraperitoneally injected with 0.2 mL of liquid paraffin. Seven days later, 1 × 10 6 The mouse abdomen was observed every day for 5 days after inoculation. When the mouse abdomen was obviously swollen and had difficulty in moving, the mouse was killed by cervical dislocation. The dark red liquid was sucked out from the mouse abdomen with a sterile syringe, and centrifuged at 3000r / min for 20min. The clear supernatant was aliquoted, labeled, and stored at -20℃ for later use.

[0043] 1.3 Determination of ascites titer

[0044] Nigeria 75 / 1 vaccine strain PPRV (1:50) was used as the coating antigen, and the ascites titer was detected by indirect ELISA. 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 positive control. The specific judgment standard was: the maximum dilution of ascites when P / N>2.0 was the ELISA titer of ascites. From Table 1, it can be seen that the ascites titer of the hybridoma cell 3E10 strain monoclonal antibody is 1:1.6×10 4 .

[0045] Table 1 Ascites titer test results

[0046]

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

[0048] 2.1 Determination of monoclonal antibody subtypes

[0049] Take 200 μL of hybridoma cell culture supernatant and use Isostrip Mouse Monoclonal Antibody Isotyping Kit to identify the subtype of the monoclonal antibody: take 20 μL of hybridoma cell supernatant, add 180 μL of PBS with pH 7.2 to make a 1:10 dilution; take 150 μL of the dilution and add it to the experimental tube containing blue powder, gently shake and mix until the blue powder is completely melted; insert the Isotrip colloidal gold test strip into the bottom of the tube and observe the results within 5 to 10 minutes.

[0050] After testing with the Mouse Monoclonal Antibody Isotyping Kit, the monoclonal antibody secreted by the hybridoma cell line 3E10 was identified as IgG1 and the light chain type was κ. Figure 1 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), 100 μL was coated in each well, and the relative affinity between the virus and the 5-fold dilution of the monoclonal antibody 3E10 was determined by indirect ELISA method. The results are shown in Table 2, and the monoclonal antibody 3E10 has a high affinity with 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 inoculated and replaced with fresh DMEM culture medium containing 10% fetal bovine serum after 2 hours of adsorption. At 24hpi, the culture medium was discarded, the cells were washed with PBS, and 4% paraformaldehyde was added to fix the cells at room temperature for 30 minutes. Indirect immunofluorescence test was performed using monoclonal antibody 3E10 and fluorescently labeled secondary antibody. The results are shown in Figure 2 As shown, monoclonal antibody 3E10 can react with PPRV. This indicates that monoclonal antibody 3E10 has good reactivity and can be used to study the functional mechanism of PPRV V protein and develop diagnostic preparations.

[0057] Example 3 Determination of the heavy and light chain variable region sequences of the anti-peste des petits ruminants virus V protein monoclonal antibody 3E10

[0058] According to the previous screening, monoclonal antibody 3E10 was selected and its hybridoma cells were expanded and cultured. When the cells grew to the logarithmic phase, the hybridoma cells were counted and collected, and the number of cells reached 5×10 6 Total RNA was extracted using HiPure RNAMini Columns (Magen) kit according to the product instructions. RNase-free water was added to dissolve RNA, and the concentration and integrity of total RNA were detected by NanoDrop and nucleic acid electrophoresis. Reverse transcription was performed using SMART Scribe Reverse Transcriptase (Takara) and its oligo-dT and template switch oligo (TSO). For specific operations, refer to 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 was bound to the constant region of the heavy chain or light chain. The 5' ends of the upstream and downstream primers were labeled with P5 and P7 adapters, respectively. The heavy chain and light chain fragments were amplified independently in the first round of PCR. The first round PCR products were purified using magnetic beads, and the purified products were used as templates for the second round of PCR. At this stage, index primers were connected to both ends of the first round PCR products to form a TruSeq dual-index library. The library was purified using magnetic beads, quantified by Qubit, and sequenced using Illumina MiSeq PE300.

[0059] Select the 10 sequences with the highest abundance ratio for abundance analysis. The abundance accumulation diagram of the sample is as follows: Figure 3 The productive and top-ranked sequences were selected as the target gene sequences.

[0060] 3.2 Analysis of gene sequencing results of monoclonal antibody heavy and light chain variable domains

[0061] According to the sequencing results, the gene sequences of the heavy and light chain variable domains of the monoclonal antibody 3E10 were analyzed by NCBI-IgBLAST (v1.17.0) and the IMGT database (http: / / www.imgt.org / ). The analysis results showed that the VH gene of the heavy chain variable region of the hybridoma cell line 3E10 antibody was 342 bp in length, encoding 114 amino acid residues, and including the hypervariable 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 including the hypervariable regions CDR1-L, CDR2-L and CDR3-L. The details are as follows:

[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 anti-peste des petits ruminants virus V protein monoclonal antibody 3E10

[0071] 4.1 Labeling of monoclonal antibody 3E10

[0072] The monoclonal antibody 3E10 (HRP-3E10) prepared in Example 1 was labeled using Peroxidase Labeling Kit-NH2 (Dojindo), and the specific operation was performed according to the instruction manual.

[0073] 4.2PPRV 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 the serum to be tested to the remaining wells;

[0075] (2) Incubation: 37°C for 45 min;

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

[0077] (4) Enzyme conjugate: Add buffer 4 to each well to dilute to 1× enzyme conjugate 100 μL, 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 incubate at room temperature in the dark for 15 minutes;

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

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

[0082] (9) Result determination: According to the formula: S / N% = (sample OD 450 / Negative control OD 450 )×100%, calculate the S / N% of each serum sample; S / N%>60% is judged as negative for PPR virus antibody, S / N%≤50% is judged as positive for PPR virus antibody, 50%<S / N%≤60% is judged as suspicious and the test is repeated.

[0083] According to the test results of the ID.vet kit on the serum samples stored in this laboratory, combined with the competitive ELISA judgment standard (PI = 100-(OD T ÷OD C )×100, PI>80 was determined as strongly positive, 50<PI≤80 was determined as weakly positive, and PI≤50 was determined as negative), and 3 strongly positive samples, 3 weakly positive samples, and 3 negative samples of PPR virus were selected for the establishment of the subsequent blocking ELISA detection method.

[0084] 4.3 The monoclonal antibody of the present invention is used for blocking ELISA detection

[0085] The recombinant PPRV V protein was used as the coating antigen, and the monoclonal antibody HRP-3E10 prepared in 4.1 of Example 4 of the present invention was used as the detection antibody to detect strong positive, weak positive and negative sera of peste des petits ruminants virus, as well as FMDV-O, BTV and LSDV positive sera, and analyze the reaction characteristics of the monoclonal antibody prepared in Example 1 of the present invention.

[0086] 4.3.1 Optimal antigen-antibody reaction concentration

[0087] (1) The ELISA plate was coated with different concentrations (2, 1, 0.5, and 0.25 μg / mL) of recombinant PPRV V protein, 50 μL / well, at 4°C overnight;

[0088] (2) Wash with PBST three times and add 1:1×10 3 , 1:2×10 3 , 1:4×10 3 、1∶8×10 3 , 1:1.6×10 4 50 μL / well of the 10-fold diluted monoclonal antibody HRP-3E10 working solution, seal the plate, and react at 37°C for 1 h;

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

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

[0091] (5) Measure OD using an ELISA reader 450nm Value, according to OD 450nm The optimal reaction concentration was screened out.

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

[0093] Table 3 Optimal reaction concentration test results

[0094]

[0095] 4.3.2 Optimal sample addition amount

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

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

[0098] (3) Wash 3 times with PBST and add 1:8×10 3 Diluted monoclonal antibody HRP-3E10 working solution, 50 μL / well, seal the plate, and react at 37°C for 1 h;

[0099] (4) Wash three times with PBST, add TMB solution (50 μL / well), seal the plate, and react at 37°C in the dark for 15 min;

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

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

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

[0103] Table 4 Optimal sample addition test results

[0104]

[0105] 4.3.3 Optimal blocking solution

[0106] The operation method is the same as 4.3.2, and the blocking solutions are PBST, 1% BSA solution, and 2% calf serum solution. OD was measured by microplate reader 450 The S / Nc% was calculated and the best blocking solution was screened out.

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

[0108] Table 5 Optimal blocking solution test results

[0109]

[0110] 4.3.4 Optimal response time

[0111] The operation method is the same as 4.3.2. The serum reaction time is 45 and 60 minutes respectively, and the HRP-3E10 reaction time is 30 and 45 minutes. 450 Calculate the S / Nc% value and screen out the optimal reaction time.

[0112] The test results are shown in Table 6. It is found that the results of different reaction time combinations are consistent and do not affect the result determination. From the perspective of the difference in S / Nc% values ​​of positive samples and rapid determination, the reaction times of serum and HRP-3E10 were selected to be 45 minutes and 30 minutes respectively.

[0113] Table 6 Best reaction time test results

[0114]

[0115] 4.3.5 Determination of standard operating procedures for blocking ELISA detection method

[0116] Determine the established blocking ELISA operation steps based on the above optimized conditions:

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

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

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

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

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

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

[0123] (7) Detect the OD of each well using an 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阴性对照 )×100%, the result judgment criteria are: S / Nc%>50%, antibody negative; S / Nc%≤50%, antibody positive.

[0125] 4.3.6 Specificity test

[0126] The established blocking ELISA method was used to detect FMDV-O, BTV, LSDV and GPV positive sera. 450 The S / Nc% value is calculated.

[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%, that is, the monoclonal antibodies prepared in Example 1 or Example 4.1 have good specificity.

[0128] Table 7 Specificity test results

[0129]

[0130] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A monoclonal antibody against nonstructural 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 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 according to 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 according to claim 1 or 2.

4. The nucleic acid according to 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. Use of the monoclonal antibody according to claim 1 or 2 in the preparation of a reagent for detecting antibodies against nonstructural proteins of PPR virus, or in the detection of natural infectivity of PPR virus for purposes other than disease diagnosis, or in the study of the function of PPR virus V protein.

6. The use according to claim 5, characterized in that The reagent is a blocking ELISA kit.

7. An enzyme conjugate, characterized in that The enzyme conjugate comprises: (i) the monoclonal antibody according to claim 1 or 2; (ii) and horseradish peroxidase conjugated to the monoclonal antibody in (i).

8. An ELISA kit for detecting antibodies to nonstructural protein V of peste des petits ruminants virus, characterized in that: The kit comprises the enzyme conjugate according to claim 7.

9. The ELISA kit according to claim 8, wherein The kit comprises an ELISA plate coated with peste des petits ruminants virus antigen, a control serum, a blocking solution, a diluent, the enzyme conjugate according to claim 7, a washing solution, a color developer, and a stop solution.

10. A blocking ELISA method for detecting peste des petits ruminants virus, characterized in that: The method comprises the following steps: (1) Diluting the purified recombinant PPRV V protein to 1 μg / mL with coating solution, 50 μL / well, sealing the plate with a sealing film, and coating at 4°C overnight; the coating solution is 0.05 mol / L sodium carbonate-sodium bicarbonate buffer, pH 9.6; (2) Wash three times with 1× PBST, add 1% BSA blocking solution, 100 μL / well, seal the plate with sealing film, and incubate at 37°C for 1 h; (3) Wash three times with 1× PBST, add positive serum, negative serum and test serum, 50 μL / well, seal the plate with sealing film, and incubate at 37°C for 45 min; (4) Wash 3 times with 1×PBST and add 1:8×10 3 The enzyme conjugate HRP-3E10 according to claim 7 was diluted with PBST, 50 μL / well, the plate was sealed with a sealing film, and incubated at 37°C for 30 min; (5) Add TMB substrate, 50 μL / well, seal the plate with sealing film, and react at 37°C for 15 min; (6) Add 1 M sulfuric acid, 50 μL / well; (7) Detect the OD of each well using an ELISA reader 450 value; (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阴性对照 )×100%, the result judgment criteria are: S / Nc%>50%, antibody negative; S / Nc%≤50%, antibody positive.

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