Monoclonal antibody specifically combined with PPRV (Peste des petits ruminants virus) phosphoprotein

Abstract

The invention relates to the technical field of biology, in particular to a monoclonal antibody specifically combined with PPRV (Peste des petits ruminants virus) phosphoprotein. The invention discloses a monoclonal antibody 16E8A6 for resisting peste des petits ruminants virus phosphoprotein. The sequence of the monoclonal antibody 16E8A6 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 16E8A6 can be subjected to specific reaction with the peste des petits ruminants virus and the phosphoprotein thereof, has high affinity, can be used for detecting expression of the phosphoprotein of the peste des petits ruminants virus, and provides a new material for researching functions of the phosphoprotein of the peste des petits ruminants virus and explaining a pathogenic mechanism of the virus.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a monoclonal antibody specifically binding to phosphoprotein of peste des petits ruminants virus. Background Art

[0002] Peste des petits ruminants (PPR) is an acute, severe, contact-transmitted major infectious disease caused by the Peste des petits ruminants virus (PPRV). It can infect goats, sheep, gazelles and antelopes, and is mainly manifested by clinical symptoms such as fever, diarrhea, enteritis, and pneumonia, with high morbidity and mortality rates. In 2012, PPR was clearly listed as one of the 13 foreign animal diseases that require key prevention. Therefore, we must strengthen research on PPR.

[0003] The PPRV genome encodes a total of 6 structural proteins and 2 non-structural proteins (Mahapatra, et al, 2006). Among them, phosphoprotein (P) is a multifunctional protein that acts as a molecular chaperone to maintain the nucleoprotein (N) connected to the viral RNA in a soluble state, 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). In the past, due to people's misunderstanding of PPRV, there was little research on PPRV at home and abroad. There are more studies on the P proteins of other members of the measles virus genus, and it was found that the P protein is related to multiple functions such as 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 to induce the folding of the N protein, and the phosphorylation of the P protein is particularly important for RNA synthesis. Sai ki et al. (2008) found that the phosphorylation of the P protein plays an important regulatory role in the transcription and replication of the rinderpest virus genome. In addition, the P protein is the least conserved protein in the genus Morbillivirus, which also indicates that this protein plays an important role in PPR virus.

[0004] Studies have shown that PRRV P protein plays an important role in viral immune escape responses. Recent studies have found that PP RV P protein is a new antagonist that can inhibit the production of IFN in a variety of ways to counteract the host's antiviral innate immune response. P protein inhibits STAT1 phosphorylation by interacting with STAT1, thereby blocking JAK-STAT signaling, and exhibits a strong negative regulatory effect on the host's antiviral immune response (Li P et al., Viruses, 2019). PPRV P protein 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). PPRV P protein targets IFN-regulatory factor (IRF) 3 in human embryonic kidney 293T cells and primary goat fibroblasts, competes with TBK1 for binding to the IRF3 binding domain (IAD), blocks the interaction between TBK1 and IRF3, reduces the phosphorylation of IRF3, interferes with the dimerization of IRF3 and blocks the nuclear transport of IRF3, resulting in significant inhibition of RIG-I-like receptor pathway signaling, and impairs the expression of IFN-β and ISGs, thereby inhibiting the production of type I IFN and promoting viral replication (Li P et al., J Immunol, 2021). It can be seen that PPRV P protein plays an important role in viral pathogenesis and immune mechanisms.

[0005] Monoclonal antibodies are antibodies secreted by a single B lymphocyte clone and recognize only a single antigen epitope. They have high specificity, affinity and orientation and are widely used in disease diagnosis and treatment, biological research and industrial fields. The ability of an antibody 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). Summary of the invention

[0006] In view of the above technical problems, the present invention uses recombinant PPRV P protein to immunize animals, obtains a hybridoma cell line that stably secretes anti-PPRV P 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 research on the pathogenicity and immune mechanism of PPR virus. Specifically, it includes the following contents:

[0007] In a first aspect, the present invention provides a monoclonal antibody that specifically binds to the phosphoprotein 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 or a kit for detecting peste des petits ruminants virus.

[0014] In a fourth aspect, the present invention provides the use of the monoclonal antibody described in the first aspect in in vitro detection of PPR virus for non-disease diagnosis purposes or in research on the pathogenicity and immune mechanism of PPR virus.

[0015] In a fifth aspect, the present invention provides a fluorescently labeled monoclonal antibody, wherein the fluorescently labeled monoclonal antibody is the monoclonal antibody described in the first aspect above with a fluorescent marker.

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

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

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

[0019] Preferably, the reagent is used for detecting peste des petits ruminants virus by direct immunofluorescence method.

[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 the present invention are as follows: ① the present invention provides a monoclonal antibody that specifically binds to the phosphoprotein of PPR virus; ② the monoclonal antibody has high affinity and good reactivity to both the PPR virus and the PPR virus phosphoprotein; and can be used for in vitro detection of PPR virus for non-disease diagnosis purposes and research on the pathogenicity and immune mechanism of PPR virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is the subtype identification result of the monoclonal antibody of the present invention;

[0023] Figure 2 The reactogenicity identification result of the monoclonal antibody of the present invention;

[0024] Figure 3 is an abundance accumulation diagram of sequencing samples of the monoclonal antibody of the present invention;

[0025] Figure 4 This is the specific identification result after the monoclonal antibody of the present invention is labeled with FITC. DETAILED DESCRIPTION

[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 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.

[0027] The Nigeria 75 / 1 vaccine strain PPRV, PPRV positive serum and pCMV-Myc-His-PPRV-P recombinant plasmid 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.

[0028] 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.

[0029] Example 1 Preparation of monoclonal antibodies

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

[0031] The pCMV-Myc-His-PPRV P recombinant plasmid was used to transfect 293T cells to prepare immune antigens. BALB / c mice were immunized and monoclonal antibodies that specifically bind to PPRV P protein were screened using B cell cloning technology. ELISA was used to first select clones that bind to PPRV P protein, and then clones that bind to PPRV were screened. The five positive hybridoma cells screened were subcloned three times, expanded and cultured, and finally OD was preserved. 450 The hybridoma cell 16E8A6 in the well with the highest value. The above immunization and screening process was entrusted to Anhui Global Gene Technology Co., Ltd.

[0032] 2. Preparation of Ascites Monoclonal Antibodies

[0033] 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 abdomen of the mouse was obviously swollen and the mouse had difficulty in moving, it was killed by dislocation. The dark red liquid was sucked out from the abdomen of the mouse with a sterile syringe, and centrifuged at 3000r / min for 20min. The clear supernatant was taken and packaged, labeled, and stored at -20℃ for later use.

[0034] 3. Determination of Ascites Titer

[0035] Nigeria 75 / 1 vaccine strain PPRV (1:10) 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. The results are shown in Table 1. From Table 1, it can be seen that the ascites titer of the hybridoma cell line 16E8A6 monoclonal antibody was 1:128×10 3 .

[0036] Table 1 Detection results of ascites titer of hybridoma cell lines

[0037]

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

[0039] 1. Determination of monoclonal antibody subtypes

[0040] 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 1:20 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.

[0041] After detection by Mouse Monoclonal Antibody Isotyping Kit, the monoclonal antibody secreted by hybridoma cell line 16E8A6 was identified as IgG1 and the light chain type was κ. Figure 1 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), and 5-fold diluted monoclonal antibodies were added, and the relative affinity of monoclonal antibody 16E8A6 was determined by indirect ELISA. The results are shown in Table 2. Monoclonal antibody 16E8A6 has high affinity with both antigen PPRV and recombinant P protein, and the affinity is stable.

[0044] Table 2 Relative affinity of monoclonal antibodies

[0045]

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

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

[0048] Example 3 Determination of the heavy and light chain variable region sequences of the monoclonal antibody 16E8A6 against PPR virus P protein 1. Acquisition of the heavy and light chain variable region genes of the monoclonal antibody

[0049] According to the previous screening, monoclonal antibody 16E8A6 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 to reach 5×10 6 Total RNA was extracted using HiPure RNAMini Columns (Magen) kit. For the corresponding operation, please refer to the product manual. 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, please refer to the product manual. 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 and quantified by Qubit, and then sequenced using Illumina MiSeq PE300.

[0050] Select the 10 sequences with the highest abundance ratio for abundance analysis. The abundance accumulation diagram is as follows: Figure 3 Select the gene sequence with the most productive and top ranking as the target gene sequence.

[0051] 2. Analysis of gene sequencing results of monoclonal antibody heavy and light chain variable domains

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

[0053] According to the sequencing results, the genes of the heavy and light chain variable domains of the anti-PPRV P protein monoclonal antibody were analyzed by NCBI-IgBLAST (v1.17.0) and IMGT database (http: / / www.imgt.org / ). The results showed that the VH gene of the heavy chain variable region of the hybridoma cell line 16E8A6 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:

[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 CTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGACGATTTGGGAGTTTTATTGCTGGCAAGGTACACATTTTCCTCAGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA(SE Q ID NO.10 shown).

[0062] Example 4 Application of monoclonal antibody 16E8A6 against PPR 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 respectively using a FITC fluorescence coupling kit (abcam), and the specific operations were performed according to the instructions.

[0065] 2. Detection of the specificity of FITC-labeled monoclonal antibodies by direct immunofluorescence

[0066] Vero cells were plated in confocal culture dishes. When the cell density reached 80%, PPRV was inoculated at MOI = 0.01. After adsorption for 2 hours, fresh DMEM culture medium containing 10% fetal bovine serum was replaced. After 36 hours of constant temperature culture at 5% CO2 and 37°C, the culture medium was discarded, washed with PBS, and fixed at room temperature for 30 minutes with 4% paraformaldehyde. At the same time, pCMV-myc-PPRVP, pCMV-myc-PPRV N plasmids and transfection reagent JET Prime were mixed at a ratio of 1μg:2μL, and allowed to stand for 15 minutes; fresh culture medium was replaced for Vero cells in the confocal culture dish with a cell density of about 80%, and the transfection mixture was added to the cells, gently shaken to mix, and after 36 hours of constant temperature culture at 5% CO2 and 37°C, the culture medium was discarded, and washed and fixed as above.

[0067] Direct immunofluorescence detection of fixed samples was performed using FITC-labeled monoclonal antibodies: (1) discard the fixative, wash with PBS, and add TritonX-100 for permeabilization at room temperature for 5 min; (2) wash as above, add 5% BSA for blocking at 37°C for 1 h; (3) wash as above, add the corresponding FITC-labeled monoclonal antibodies, and incubate at 37°C in the dark for 1 h; (4) wash as above, add DAPI, and incubate at room temperature in the dark for 10 min; (5) observe with a laser confocal microscope and take pictures.

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

[0069] 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 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 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 peste des petits ruminants virus.

6. Use of the monoclonal antibody according to claim 1 or 2 in in vitro detection of PPR virus for non-disease diagnosis purposes or in research on the pathogenicity and immune mechanism of PPR virus.

7. A fluorescently labeled monoclonal antibody, characterized in that: The fluorescently labeled monoclonal antibody is the monoclonal antibody according to claim 1 or 2.

8. The fluorescently labeled monoclonal antibody according to claim 7, characterized in that The fluorescent marker is a detectable small molecule fluorescent marker.

9. Use of the fluorescently labeled monoclonal antibody according to claim 7 or 8 in the preparation of a reagent for detecting peste des petits ruminants virus.

10. The use according to claim 9, characterized in that The reagent is used for detecting peste des petits ruminants virus by direct immunofluorescence method.

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