Neutralizing monoclonal antibody 5D2 for resisting African swine fever virus P72 protein and application of neutralizing monoclonal antibody 5D2

Abstract

The invention belongs to the technical field of biology, and particularly relates to an anti-African swine fever virus (ASFV) P72 protein neutralizing monoclonal antibody 5D2 and application thereof. A heavy chain variable region of the monoclonal antibody or the antigen binding fragment comprises a CDR1 of which the amino acid sequence is GYTFLTYW, a CDR2 of which the amino acid sequence is IFPASGST, and a CDR3 of which the amino acid sequence is ARRSRDPSGPLT; a light chain variable region of the monoclonal antibody or the antigen binding fragment comprises a CDR1 with an amino acid sequence of QTLVHSNGNTY, a light chain variable region with an amino acid sequence of KVS and a CDR3 with an amino acid sequence of SQSTHVPPT, and the monoclonal antibody or the antigen binding fragment has a neutralizing function of preventing ASFV from infecting sensitive cells in vitro, is strong in antigen affinity and can be applied to research of antiviral drugs.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to an anti-African swine fever virus (ASFV) P72 protein neutralizing monoclonal antibody 5D2 and an application thereof. Background Art

[0002] African swine fever (ASF) is an acute, highly contagious disease of pigs caused by African swine fever virus (ASFV). It has a short course and a mortality rate of up to 100%. ASFV is a virus that is difficult to eliminate. Currently, inactivated vaccines, attenuated vaccines, subunit vaccines and DNA vaccines for ASF are not sufficient to protect pigs from ASFV. Little is known about the immune mechanisms that effectively protect pigs from ASFV infection and their effective immune components. Therefore, in addition to expanding the understanding of the ASFV proteome and the functions of individual proteins, it is also necessary to find an appropriate balance between antibody and cell-mediated ASFV immune responses, which are of great significance for the rational design of targeted vaccines.

[0003] ASFV has many structural proteins, among which P72 is one of the main structural proteins. The protein sequence is relatively conservative and is encoded by the B646L gene. It accounts for 31-33% of the virus particles and is the most abundant structural protein. P72 exists on the surface of the virus capsid, has good immunogenicity and antigenicity, and can induce the body to produce neutralizing antibodies. The mechanism of action of neutralizing antibodies is mainly to change the configuration of the virus surface, prevent the virus from adsorbing to susceptible cells, and prevent the virus from penetrating susceptible cells for proliferation. Screening for neutralizing antibodies against the P72 protein will increase our understanding and knowledge of the humoral immune response of pigs and provide a theoretical basis for the design of new ASFV vaccines and the effective prevention and control of ASF. Summary of the invention

[0004] The purpose of the present invention is to provide a neutralizing monoclonal antibody (5D2) against ASFV P72 protein and its application. The present invention uses the P72 protein obtained in the invention patent with publication number CN112979765A as an immune source, immunizes mice, and obtains the monoclonal antibody 5D2 against P72 protein with neutralizing activity through cell fusion and subcellular screening technology.

[0005] In a first aspect, the present invention provides a monoclonal antibody or antigen-binding fragment against ASFV P72 protein, wherein the amino acid sequences of the heavy chain and light chain variable regions CDR1, CDR2 and CDR3 of the monoclonal antibody or antigen-binding fragment are as follows: CDR1 of heavy chain VH: GYTFLTYW; CDR2 of heavy chain VH: IFPASGST; CDR3 of heavy chain VH: ARSRDPSGPLT.

[0006] CDR1 of light chain VL: QTLVHSNGNTY; CDR2 of light chain VL: KVS; CDR3 of light chain VL: SQSTHVPPT.

[0007] The amino acid sequence of the heavy chain variable region and the amino acid sequence of the light chain variable region of the monoclonal antibody or antigen-binding fragment are shown in SEQ ID NO: 1 or SEQ ID NO: 2, respectively.

[0008] The second aspect of the present invention provides a nucleic acid encoding the monoclonal antibody or antigen-binding fragment against the African swine fever virus P72 protein, and the nucleic acid sequences encoding the heavy chain variable region and the light chain variable region of the monoclonal antibody or antigen-binding fragment are shown in SEQ ID NO: 3 or SEQ ID NO: 4, respectively.

[0009] The heavy chain constant region of the monoclonal antibody or antigen-binding fragment is of IgG1 type.

[0010] The light chain constant region of the monoclonal antibody or antigen-binding fragment is of Kappa type.

[0011] The third aspect of the present invention provides a recombinant expression vector comprising a nucleic acid encoding the monoclonal antibody or antigen-binding fragment.

[0012] The fourth aspect of the present invention provides a host cell comprising the above-mentioned recombinant expression vector.

[0013] The monoclonal antibody or antigen-binding fragment has at least one of the following functions: (1) Detection of ASFV; (2) Detection of ASFV P72 protein.

[0014] In a fifth aspect, the present invention provides the use of a monoclonal antibody or an antigen-binding fragment in any of the following aspects: (1) A product for the preparation of a product for the treatment of ASFV infection; the product may be a drug.

[0015] (2) Used to prepare ASFV detection reagents or kits.

[0016] In addition, the present invention also provides a drug, an African swine fever detection reagent or a kit containing the above-mentioned monoclonal antibody or antigen-binding fragment.

[0017] The beneficial effects of the present invention are: The present invention uses cell fusion and subcellular screening technology to successfully obtain a monoclonal antibody with neutralizing activity that is specific to the ASFV P72 protein. The monoclonal antibody has a neutralizing function in vitro that prevents ASFV from infecting sensitive cells, and has a strong affinity for the P72 protein, providing a reliable research tool for further exploring the function of the P72 protein.

[0018] The monoclonal antibodies provided by the present invention can be obtained by conventional genetic engineering or protein engineering methods, thereby avoiding the loss of antibodies during long-term cryopreservation of hybridoma cells. It is also beneficial to optimize the antibodies at the gene and protein levels, thereby improving the specificity and affinity of the antibodies.

[0019] The monoclonal antibody prepared by the present invention can specifically recognize and neutralize ASFV. Therefore, the antibody provides a new raw material for the research on the pathogenic mechanism of ASFV, the development of early detection kits and therapeutic antibodies or vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 ELISA was used to detect the antibody titer of mice after immunization with P72 protein; 1-3#: mice immunized with No. 1, 2 and 3, NC: negative control.

[0021] Figure 2 Identification of monoclonal antibody reactivity for IFA; A. 5D2 cell supernatant group; B. SP2 / 0 cell supernatant group.

[0022] Figure 3 Western blot was used to identify the reactivity of 5D2 monoclonal antibody; M: protein marker, WT: ASFV-infected group; Mock: non-infected control group; GAPDH was the internal reference control.

[0023] Figure 4 SADS-PAGE identification after ascites purification; M: protein marker, 5D2: ascites after purification.

[0024] Figure 5 Detection of the potency of 5D2 antibody after purification; NC: ascites of irrelevant monoclonal antibody after purification.

[0025] Figure 6 The neutralizing activity of 5D2 monoclonal antibody was identified by hemocyte adsorption test; Mock: uninfected group; Mouseserum-WT: mouse negative serum mixed with ASFV group; 1640-WT: 1640 culture medium mixed with ASFV group; 5D2-WT: 5D2 mixed with ASFV group.

[0026] Figure 7The neutralizing activity of 5D2 monoclonal antibody was identified by qPCR; Mock: uninfected group; Mouseserum-WT: mouse negative serum mixed with ASFV group; 1640-WT: 1640 culture medium mixed with ASFV group; 5D2-WT: 5D2 mixed with ASFV group.

[0027] Figure 8 The results of PCR amplification of variable regions; A. PCR amplification of heavy chain variable region. M: DL2000 marker; 1: PCR amplification of 5D2 heavy chain variable region; 2: negative water control. B. PCR amplification of light chain K variable region. M: DL2000 marker; 1: PCR amplification of 5D2 light chain variable region; 2: negative water control. DETAILED DESCRIPTION

[0028] The present invention is described in more detail below through specific implementation modes to facilitate understanding of the technical solution of the present invention, but is not intended to limit the protection scope of the present invention.

[0029] Example 1 Screening and identification of anti-ASFV P72 protein monoclonal antibodies 1. Animal immunization Three 6-week-old female BALB / c mice were selected and the purified P72 recombinant protein (gifted by Professor Xiang Ye of Tsinghua University, the preparation method refers to the invention patent application with application publication number CN112979765A) was used to immunize the mice at a dose of 30 μg / mouse. For the first immunization, the P72 protein was mixed with an equal volume of Freund's complete adjuvant and emulsified, and multiple subcutaneous injections were performed into the mice (one point on the back and two points on the abdomen). Reinforcement immunization was performed every 2 weeks, for a total of four immunizations. The reinforcing immunization was to mix an equal volume of P72 recombinant protein and Freund's incomplete adjuvant and emulsify them. The immunization method was the same as the first immunization. Blood was collected from the tail 7 days after the fourth immunization to determine the antibody titer.

[0030] Methods for detecting polyclonal antibody titer The purified P72 protein was diluted to 1 μg / mL with coating solution, added to the ELISA plate at 50 μL / well, coated at 37°C for 1 hour, washed four times with PBST (K2HPO4 0.26g, Na2HPO4·12H2O 2.89g, NaCl 8.50g, Tween-20 0.5mL, dilute to 1L with water), and blocked at 37°C for 1 hour with 5% skim milk powder. The positive and negative sera were diluted with PBST in multiple ratios, with a dilution gradient from 1:1000 to 1:2048000, 12 dilution gradients, and incubated at 37°C for 30 minutes. After washing four times with PBST, HRP-labeled goat anti-mouse IgG (1:20000 dilution) was added, washed four times with PBST, and TMB was used for color development, and the OD was measured on an enzyme reader. 450 .

[0031] The results are as follows Figure 1 As shown, when the serum was diluted to 1:2048000, the OD of mice immunized with No. 1, 2 and 3 was 450 It was still greater than that of non-immune mouse serum (NC group), indicating that the antibody titer could reach above 1:2048000.

[0032] Preparation of monoclonal antibodies Take the spleen cells of a mouse immunized with P72 protein and mix them with SP20 cells in a ratio of 5:1, and then perform cell fusion under the action of fusion agent PEG. The fused cells are plated in a 96-well cell plate and cultured in a CO2 incubator at 37°C. When the hybridoma cells cover 1 / 10 of the bottom of the cell plate, take the supernatant for ELISA detection (ELISA coating method is the same as 2. ELISA method to detect polyclonal antibody titer). The wells identified as positive by ELISA are then verified by indirect immunofluorescence test (IFA). Select the ELISA and IFA positive wells, and use the limiting dilution method to subclone the screened positive hybridoma cells. Observe under an inverted microscope, mark the wells where only a single clone grows, take the supernatant, and use the above ELISA and IFA methods for antibody detection. Positive cells enter the next round of subcloning, which is performed three times in total.

[0033] IFA test: 1×10 4 MA104 cells were plated in a 96-well cell plate, and after the cells were fully plated, 0.1 MOI ASFV was inoculated. 48 hours after inoculation, the cells were fixed with 4% paraformaldehyde at room temperature for 30 minutes, washed three times with 0.01 mol / L PBS (K2HPO4 0.26g, Na2HPO4·12H2O 2.89g, NaCl 8.50g, water to 1L, pH 7.4); then permeabilized with 0.1% TritonX-100, acted at room temperature for 10 minutes, and washed three times with PBS; then incubated with hybridoma cell supernatant and anti-mouse FITC-labeled goat anti-mouse IgG, and the results were observed under a fluorescence microscope after the reaction was completed.

[0034] Monoclonal antibody specificity characterization After three subclonings, the cell supernatants secreted by the obtained single cell lines were subjected to IFA and western blot detection. The IFA identification results showed that the 5D2 cell supernatant could detect specific green fluorescence signals ( Figure 2 A), while no fluorescence signal was observed when the supernatant of SP2 / 0 cells was used to treat ASFV-infected cells ( Figure 2B). Virus-infected and uninfected MA104 cells were collected, and SDS-PAGE was performed after cell lysis. The protein gel was then transferred to the NC membrane for western blot verification. 5D2 cell supernatant was used as the primary antibody and acted at room temperature for 1 hour. Anti-mouse HRP-IgG was used as the secondary antibody and acted at room temperature for 1 hour. After each action, it was washed 4 times with PBST and then exposed for color development. Western blot results showed that 5D2 cell supernatant could recognize P72 protein in virus-infected cells, indicating that 5D2 cell supernatant could specifically recognize P72 protein ( Figure 3 ).

[0035] Monoclonal antibody subclass identification According to Southern Biotech's SBA Clonotyping TM System / HRP Antibody Subclass Identification Kit Operating Instructions: The monoclonal antibody in the supernatant of 5D2 cells was identified as subclass. The heavy chain constant region of the monoclonal antibody was IgG1 type, and the light chain constant region was Kappa type.

[0036] Preparation, purification and titer detection of ascites 6.1 Preparation of ascites Balb / c mice aged 10 to 12 weeks were selected and each mouse was intraperitoneally injected with 0.5 mL of Freund's incomplete adjuvant. One week later, each mouse was intraperitoneally injected with 5×10 5 The positive hybridoma cells (0.2 mL) prepared in "3. Preparation of monoclonal antibodies" were added. After 7 to 10 days, the abdominal cavity of the mouse was obviously bulging. The ascites was collected, packaged, and stored at -80 ℃.

[0037] Purification of ascites According to PIERCE NAb TM Protein G Spin Purification Kit was used for affinity chromatography purification, and then SDS-PAGE electrophoresis was used to identify the purity. Figure 4 It can be concluded that the heavy chain of the monoclonal antibody obtained after ascites purification is about 55KD, and the light chain is about 25KD. The monoclonal antibody was named 5D2.

[0038] Ascites titer test The monoclonal antibody 5D2 purified from ascites was diluted with PBST at 40 ng / μL, with a total of 12 dilutions. 50 μL / well of the diluted antibody was added to the ELISA plate coated with P72 protein and incubated at 37°C for 30 min. After washing four times with PBST, HRP-labeled goat anti-mouse IgG (1:20000 dilution) was added and incubated at 37°C for 30 min. After washing four times with PBST, TMB was used for color development and OD was measured on an ELISA plate reader.450 .

[0039] The results are as follows Figure 5 As shown, when monoclonal antibody 5D2 was diluted to 0.0390625 ng / μL, its OD 450 It was still greater than the irrelevant monoclonal antibody group (NC group), indicating that the antibody titer could reach above 0.0390625 ng / μL.

[0040] Example 2 Identification of the neutralizing activity of anti-ASFV P72 protein monoclonal antibody 5D2 2.1 Red blood cell adsorption experiment In order to verify whether the monoclonal antibody 5D2 has neutralizing activity, a red blood cell adsorption test was performed, and the specific operation steps are as follows: (1) Preparation of 1% porcine red blood cells: Aseptically collect healthy porcine blood into a heparin anticoagulation tube and mix thoroughly. Take 1 mL of anticoagulated blood, resuspend it with 9 mL of 0.01 mol / L pH7.4 sterile PBS, centrifuge at 500 g for 5 min, and discard the supernatant; resuspend the red blood cells with 9 mL of 0.01 mol / L pH7.4 sterile PBS, and repeat the washing 4 times until the supernatant becomes clear. Discard the supernatant and use 0.01 mol / L pH7.4 sterile PBS to prepare a volume fraction of 1% porcine red blood cells.

[0041] (2) Take a tube of PAM cells frozen in liquid nitrogen, thaw quickly in a 37°C water bath, centrifuge at 500 g for 5 min, discard the supernatant, and resuspend the cells in 1 mL of RPMI-1640 medium containing 10% FBS and 2% double antibody at a concentration of 1×10 5 The cells were plated in 96-well cell culture plates and cultured at 37°C for 24 h.

[0042] (3) Wild-type ASFV (2×10 6 TCID 50 / mL) was mixed with healthy mouse serum (mouseserum), 1640 culture medium and 5D2 antibody (1 mg / mL) in equal volumes, incubated at 37°C for 2 h, and then inoculated with cells in (2) and cultured for 24 h. The group of healthy mouse serum (mouseserum) and 1640 culture medium mixed with ASFV was used as a positive control; the group without infection was used as a blank cell control.

[0043] (4) Porcine red blood cells were diluted to 1×10 in 0.01 mol / L sterile PBS at pH 7.4. 7 / mL, take 100 µL and add it to a 24-well cell culture plate; after culturing at 37℃ for 2-3 days, observe the red blood cell adsorption of ASFV under an inverted fluorescence microscope, take pictures, record and save.

[0044] The experimental results are as follows Figure 6As shown, compared with the healthy mouse serum (mouse serum) and ASFV mixed group, 1640 culture medium and ASFV mixed group, the number of rosette-like structures in the ASFV group treated with antibody 5D2 was significantly reduced, indicating that antibody 5D2 can effectively inhibit the replication of ASFV.

[0045] Quantitative PCR (qPCR) In order to further verify the ability of monoclonal antibody 5D2 to neutralize ASFV, the qPCR test was used to detect the genome copy number of ASFV after different treatments in 2.1. The cells in step 2.1 were repeatedly frozen and thawed at -80°C for 3 times, and 200 μL was taken to extract DNA according to the Qiagen DNA kit extraction instructions, and qPCR was performed using this as a template. The reaction system was 20 μL, including: Premix Ex Taq™ (Probe qPCR) (2×) 10 μL; upstream primer ((10 μM) 0.5 μL; downstream primer (10μM) 0.5 μL; test sample DNA 3 μL; fluorescent probe: 0.5μL; ddH2O: 4.5 μL. Upstream primer: 5'-CTGCTCATGGTATCAATCTTATCGA-3'; downstream primer: 5'-GATACCACAAGATCAGCCGT-3' (reference: King DP, Reid SM, Hutchings GH, Grierson SS, WilkinsonPJ, Dixon LK, BastosAD, Drew TW. Development of a TaqMan PCR assay with internal amplificationcontrol for the detection of African swine fever virus.J Virol Methods. 2003Jan;107(1):53-61.). pMT18-P72 was diluted 10 times as a standard and a standard curve was drawn. The reaction conditions were: pre-denaturation at 95°C for 30s as the first cycle; 95°C for 5s, 60°C for 30s as the second cycle for 40 cycles, and fluorescence signal detection was performed at the end of each extension cycle of the second step.

[0046] The virus copy number was calculated based on the standard curve. Figure 7 As shown, compared with the healthy mouse serum (mouserum) and ASFV mixed group, 1640 culture medium and ASFV mixed group, the virus copy number in the ASFV group treated with monoclonal antibody 5D2 was significantly reduced and the difference was extremely significant, further indicating that monoclonal antibody 5D2 inhibited the replication of ASFV.

[0047] Example 3 PCR amplification and sequence determination of the variable region gene of anti-ASFV P72 protein monoclonal antibody 5D2 The monoclonal antibody 5D2 hybridoma cell RNA was extracted and reverse transcribed into cDNA using Oligo-dt or random primers (PrimeScript II 1st Strand cDNA Synthesis Kit, TAKARA, 6210A).

[0048] The antibody variable region gene was amplified by nested PCR. First, the above cDNA was used as a template to amplify the antibody variable region gene using the first round of mouse antibody IgG and κ light chain primers. Then, the first round product was used as a template to amplify the antibody variable region gene using the second round of mouse antibody IgG and κ light chain primers. The PCR reaction system was: PrimeSTAR Max Premix (2×) 25 μL, P1 and P2 1 μL each, cDNA 1 μL, ddH2O to 50 μL. The reaction program was: 98 ℃ pre-denaturation for 2 min; 98 ℃ denaturation for 10 s, 55 ℃ annealing for 30 s, 72 ℃ extension for 30 s, 30 cycles; 72 ℃ extension for 10 min. References for primers for antibody variable region gene amplification (von Boehmer, L., Liu, C., Ackerman, S., Gitlin, AD, Wang, Q., Gazumyan, A., Nussenzweig, MC, 2016. Sequencing and cloning of antigen-specific antibodies from mouse memory B cells. Nature protocols 11, 1908-1923.) After amplification, 1% agarose gel electrophoresis was performed, and the gene size of the heavy chain and κ light chain variable region of monoclonal antibody 5D2 was approximately 300 bp ( Figure 8 ), gel cutting and recovery of target fragments. The recovered target fragments were inserted into the pMD-18T vector for sequence determination. The sequencing results were compared with the antibody gene library (IMGT) and the sequencing results confirmed that the amplified sequences were DNA of the heavy chain variable region and light chain variable region of the monoclonal antibody.

[0049] Specifically, the DNA sequence encoding the heavy chain variable region of the mouse anti-ASFV P72 protein monoclonal antibody 5D2 is shown in SEQ ID NO: 3; the DNA sequence encoding the light chain variable region of the mouse anti-ASFV P72 protein monoclonal antibody 5D2 is shown in SEQ ID NO: 4.

[0050] The amino acid sequence of the heavy chain variable region of the mouse anti-ASFV P72 protein monoclonal antibody 5D2 is shown in SEQ ID NO: 1; the amino acid sequence of the light chain variable region of the mouse anti-ASFV P72 protein monoclonal antibody 5D2 is shown in SEQ ID NO: 2. The amino acid sequences of the heavy chain and light chain variable regions CDR1, CDR2 and CDR3 of the 5D2 monoclonal antibody are shown in Table 1 below.

[0051] Table 1. Amino acid sequences of CDR1, CDR2 and CDR3 of the heavy and light chain variable regions of monoclonal antibody 5D2 CDR1 CDR2 CDR3 Heavy chain VH GYTFLTYW IFPASGST ARSRDPSGPLT Light chain VL QTLVHSNGNTY KVS SQSTHVPPT The embodiments described above are only preferred embodiments of the present invention and are not intended to limit the scope of implementation of the present invention. Therefore, any equivalent changes or modifications made according to the structures, features and principles described in the patent scope of the present invention should be included in the scope of the patent application of the present invention.

Claims

1. A monoclonal antibody or antigen-binding fragment against African swine fever virus P72 protein, characterized in that: The heavy chain variable region of the monoclonal antibody or antigen-binding fragment comprises a CDR1 having an amino acid sequence of GYTFLTYW, a CDR2 having an amino acid sequence of IFPASGST, and a CDR3 having an amino acid sequence of ARSRDPSGPLT; The light chain variable region of the monoclonal antibody or antigen-binding fragment comprises a CDR1 with an amino acid sequence of QTLVHSNGNTY, a light chain variable region with an amino acid sequence of KVS, and a CDR3 with an amino acid sequence of SQSTHVPPT.

2. The monoclonal antibody against African swine fever virus P72 protein according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region and the amino acid sequence of the light chain variable region of the monoclonal antibody or antigen-binding fragment are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

3. A nucleic acid encoding the monoclonal antibody or antigen-binding fragment against African swine fever virus P72 protein according to claim 1.

4. The nucleic acid according to claim 3, characterized in that The nucleic acid sequences encoding the heavy chain variable region and light chain variable region of the monoclonal antibody or antigen-binding fragment are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

5. A recombinant expression vector comprising the nucleic acid according to claim 3 or 4. A host cell comprising the recombinant expression vector according to claim 5.

7. Use of the monoclonal antibody or antigen-binding fragment of claim 1 or 2, the nucleic acid of claim 3 or 4, the recombinant expression vector of claim 5, or the host cell of claim 6 in any of the following aspects, characterized in that: (1) Use in the preparation of drugs for treating African swine fever virus infection; (2) Application in the preparation of African swine fever detection reagents or kits.

8. A drug, African swine fever detection reagent or kit containing the monoclonal antibody or antigen-binding fragment according to claim 1 or 2.

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