Neutralizing monoclonal antibody 5D2 against African swine fever virus P72 protein and its application

Abstract

The present invention belongs to the field of biotechnology and specifically relates to a monoclonal antibody 5D2 that neutralizes the P72 protein of African swine fever virus (ASFV) and its applications. The heavy chain variable region of the monoclonal antibody or antigen-binding fragment of the present invention comprises a CDR1 with the amino acid sequence GYTFLTYW, a CDR2 with the amino acid sequence IFPASGST, and a CDR3 with the amino acid sequence ARSRDPSGPLT; the light chain variable region of the monoclonal antibody or antigen-binding fragment comprises a CDR1 with the amino acid sequence QTLVHSNGNTY, a light chain variable region with the amino acid sequence KVS, and a CDR3 with the amino acid sequence SQSTHVPPT. The monoclonal antibody or antigen-binding fragment exhibits in vitro neutralization function to prevent ASFV infection of sensitive cells, has strong affinity for antigens, and can be used in the research of antiviral drugs.

Description

Technical Field

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

[0002] African swine fever (ASF) is an acute, highly contagious disease of pigs caused by the African swine fever virus (ASFV). It has a short course and a mortality rate of up to 100%. ASFV is a difficult virus to eradicate. Currently, inactivated, attenuated, subunit, and DNA vaccines are insufficient to protect pigs against ASFV infection. The immune mechanisms and components that effectively protect pigs against ASFV infection are poorly understood. Therefore, in addition to expanding our understanding of the ASFV proteome and the functions of individual proteins, finding the right balance between antibody-mediated and cell-mediated immune responses to ASFV is crucial for the rational design of targeted vaccines.

[0003] ASFV has numerous structural proteins, of which P72 is a major one. This protein, encoded by the B646L gene and a relatively conserved sequence, accounts for 31-33% of the viral particle, making it the most abundant structural protein. P72 is present on the surface of the viral capsid and exhibits strong immunogenicity and antigenicity, inducing the production of neutralizing antibodies. Neutralizing antibodies primarily alter the conformation of the viral surface, preventing the virus from adsorbing to susceptible cells and thus preventing it from penetrating and proliferating. Screening for neutralizing antibodies targeting the P72 protein will enhance our understanding of the humoral immune response in pigs and provide a theoretical foundation for the design of new ASFV vaccines and effective ASF prevention and control efforts. Summary of the Invention

[0004] The present invention aims to provide a neutralizing monoclonal antibody (5D2) against ASFV P72 protein and its applications. The present invention uses the P72 protein obtained in the invention patent publication number CN112979765A as an immunogen to immunize mice. Through cell fusion and subcellular screening, the neutralizing monoclonal antibody 5D2 against P72 protein is obtained.

[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 and light chain variable regions CDR1, CDR2, and CDR3 of the monoclonal antibody or antigen-binding fragment are as follows:

[0006] CDR1 of heavy chain VH: GYTFLTYW;

[0007] CDR2 of heavy chain VH: IFPASGST;

[0008] CDR3 of heavy chain VH: ARSRDPSGPLT.

[0009] CDR1 of light chain VL: QTLVHSNGNTY;

[0010] CDR2 of light chain VL: KVS;

[0011] CDR3 of light chain VL: SQSTHVPPT.

[0012] 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.

[0013] 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 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.

[0014] The heavy chain constant region of the monoclonal antibody or antigen-binding fragment is of IgG1 type.

[0015] The light chain constant region of the monoclonal antibody or antigen-binding fragment is Kappa type.

[0016] The third aspect of the present invention provides a recombinant expression vector comprising a nucleic acid encoding the monoclonal antibody or antigen-binding fragment.

[0017] The fourth aspect of the present invention provides a host cell comprising the above-mentioned recombinant expression vector.

[0018] The monoclonal antibody or antigen-binding fragment has at least one of the following functions:

[0019] (1) Detection of ASFV;

[0020] (2) Detection of ASFV P72 protein.

[0021] In a fifth aspect, the present invention provides use of a monoclonal antibody or antigen-binding fragment in any of the following aspects:

[0022] (1) A product for the preparation of a product for the treatment of ASFV infection; the product may be a drug.

[0023] (2) Used to prepare ASFV detection reagents or kits.

[0024] 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.

[0025] The beneficial effects of the present invention are:

[0026] The present invention utilizes cell fusion and subcellular screening techniques to successfully obtain a monoclonal antibody specifically targeting the ASFV P72 protein with neutralizing activity. This monoclonal antibody has the ability to neutralize ASFV in vitro, preventing it from infecting susceptible cells and exhibits strong affinity for the P72 protein, providing a reliable research tool for further exploring the function of the P72 protein.

[0027] The monoclonal antibodies provided by the present invention can be obtained using conventional genetic engineering or protein engineering methods, which avoids 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.

[0028] The monoclonal antibodies prepared by the present invention can specifically recognize and neutralize ASFV. Therefore, the antibodies provide new raw materials 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

[0029] 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.

[0030] Figure 2 IFA was used to identify the reactivity of monoclonal antibodies; A. 5D2 cell supernatant group; B. SP2 / 0 cell supernatant group.

[0031] Figure 3 Western blot was used to identify the reactivity of 5D2 monoclonal antibody; M: protein marker, WT: ASFV-infected group; Mock: uninfected control group; GAPDH was the internal reference control.

[0032] Figure 4 SADS-PAGE identification of ascites after purification; M: protein marker, 5D2: purified ascites.

[0033] Figure 5 Detection of the potency of the purified 5D2 antibody; NC: ascites of an unrelated monoclonal antibody after purification.

[0034] Figure 6The 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.

[0035] Figure 7 The 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.

[0036] Figure 8 Variable region PCR amplification results; A. Heavy chain variable region PCR amplification results. M: DL2000 marker; 1: 5D2 heavy chain variable region PCR amplification; 2: Negative water control. B. Light chain K variable region PCR amplification results. M: DL2000 marker; 1: 5D2 light chain variable region PCR amplification; 2: Negative water control. DETAILED DESCRIPTION

[0037] The present invention is described in more detail below through specific implementation methods to facilitate understanding of the technical solution of the present invention, but is not intended to limit the scope of protection of the present invention.

[0038] Example 1 Screening and identification of monoclonal antibodies against ASFV P72 protein

[0039] 1. Animal immunization

[0040] Three 6-week-old female BALB / c mice were immunized with 30 μg of purified recombinant P72 protein (kindly provided by Professor Xiang Ye of Tsinghua University; for preparation, refer to the invention patent application publication number CN112979765A). For the first immunization, the P72 protein was mixed with equal volumes of complete Freund's adjuvant, emulsified, and injected subcutaneously at multiple sites (one on the back and two on the abdomen). Booster immunizations were performed every two weeks for a total of four immunizations. Booster immunizations consisted of an equal volume of emulsified P72 recombinant protein mixed with incomplete Freund's adjuvant, using the same immunization method as the first immunization. Seven days after the fourth immunization, tail blood was collected for antibody titer determination.

[0041] Method for detecting polyclonal antibody titer

[0042] The purified P72 protein was diluted to 1 μg / mL with coating solution and added to the ELISA plate at a rate of 50 μL / well. The plate was coated at 37°C for 1 hour and washed four times with PBST (K2HPO4 0.26g, Na2HPO4·12H2O 2.89g, NaCl 8.50g, Tween-20 0.5mL, and diluted to 1L with water). The plate was then blocked with 5% skim milk powder at 37°C for 1 hour. Positive and negative serum were diluted with PBST in a series of 12 dilution steps from 1:1000 to 1:2048000. The plate was incubated at 37°C for 30 minutes. After washing four times with PBST, HRP-labeled goat anti-mouse IgG (1:20000 dilution) was added. After washing four times with PBST, the plate was developed with TMB and the OD was measured on a microplate reader. 450 .

[0043] The results are as follows Figure 1 As shown, when the serum was diluted to 1:2048000, the OD values ​​of immunized mice No. 1, 2, and 3 were 450 It was still greater than that of the serum of non-immune mice (NC group), indicating that the antibody titer could reach above 1:2048000.

[0044] Preparation of monoclonal antibodies

[0045] Splenocytes from a mouse immunized with P72 protein were mixed with SP20 cells at a ratio of 5:1 and fused using the fusogenic agent PEG. The fused cells were plated in a 96-well plate and cultured in a 37°C, CO2 incubator. When the hybridoma cells reached 1 / 10 of the bottom of the plate, the supernatant was aspirated and analyzed by ELISA (the ELISA coating method is the same as in 2. ELISA for polyclonal antibody titer). Wells positive by ELISA were then verified by indirect immunofluorescence assay (IFA). Hybridoma cells that were positive by both ELISA and IFA were subcloned using limiting dilution. Wells with single clones were observed under an inverted microscope, and the supernatant was removed for antibody detection using the ELISA and IFA methods described above. Positive cells were then subcloned three times.

[0046] IFA test: 1×10 4MA104 cells were plated in 96-well plates. After confluence, ASFV was inoculated at 0.1 MOI. 48 hours after inoculation, cells were fixed with 4% paraformaldehyde for 30 minutes at room temperature and washed three times with 0.01 mol / L PBS (K₂HPO₄ 0.26 g, Na₂HPO₄·12H₂O 2.89 g, NaCl 8.50 g, dilute to 1 L with water, pH 7.4). The cells were then permeabilized with 0.1% Triton X-100 for 10 minutes at room temperature and washed three times with PBS. Hybridoma supernatant and anti-mouse FITC-conjugated goat anti-mouse IgG were then incubated. After completion of the reaction, the cells were observed under a fluorescence microscope.

[0047] Monoclonal antibody specificity identification

[0048] 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 in ASFV-infected MA104 cells ( Figure 2 A), while no fluorescence signal was observed when the supernatant of SP2 / 0 cells was used to treat ASFV-infected cells ( Figure 2 B). Virus-infected and uninfected MA104 cells were collected, lysed, and subjected to SDS-PAGE. The protein gel was then transferred to a NC membrane for Western blot verification. 5D2 cell supernatant was used as the primary antibody and incubated at room temperature for 1 hour. Anti-mouse HRP-IgG was used as the secondary antibody and incubated at room temperature for 1 hour. After each incubation, the cells were washed four times with PBST and then exposed to light for color development. Western blot results showed that 5D2 cell supernatant could recognize P72 protein in virus-infected cells, indicating that 5D2 cell supernatant can specifically recognize P72 protein ( Figure 3 ).

[0049] Monoclonal antibody subclass identification

[0050] According to Southern Biotech's SBA Clonotyping TM System / HRP Antibody Subclass Identification Kit Operating Instructions: Use the monoclonal antibody obtained from the supernatant of 5D2 cells to identify its subclass. The heavy chain constant region of the monoclonal antibody is IgG1, and the light chain constant region is kappa.

[0051] Preparation, purification and titer detection of ascites

[0052] 6.1 Preparation of ascites

[0053] Balb / c mice aged 10 to 12 weeks were injected intraperitoneally with 0.5 mL of Freund's incomplete adjuvant. One week later, each mouse was injected intraperitoneally with 5×105 7-10 days after the mouse abdominal cavity was obviously bulging, the ascites was collected, aliquoted, and stored at -80℃.

[0054] Purification of ascites

[0055] According to PIERCE NAb TM Protein G Spin Purification Kit was used for affinity chromatography purification, and then the purity was determined by SDS-PAGE electrophoresis. 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. This monoclonal antibody is named 5D2.

[0056] Ascites titer test

[0057] The monoclonal antibody 5D2 purified from ascites was serially diluted with PBST at 40 ng / μL for 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 (1:20,000 dilution) was added and incubated at 37°C for 30 min. After washing four times with PBST, TMB was used for color development and the OD was measured on a microplate reader. 450 .

[0058] The results are as follows Figure 5 As shown, when the 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.

[0059] Example 2 Identification of the neutralizing activity of anti-ASFV P72 protein monoclonal antibody 5D2

[0060] 2.1 Red blood cell adsorption experiment

[0061] In order to verify whether the monoclonal antibody 5D2 has neutralizing activity, a red blood cell adsorption test was performed. The specific steps are as follows:

[0062] (1) Preparation of 1% porcine red blood cells: Aseptically collect healthy porcine blood into a heparin anticoagulant tube and mix thoroughly. Take 1 mL of anticoagulated blood and resuspend it in 9 mL of 0.01 mol / L pH 7.4 sterile PBS. Centrifuge at 500 g for 5 min and discard the supernatant. Resuspend the red blood cells in 9 mL of 0.01 mol / L pH 7.4 sterile PBS and repeat the washing process 4 times until the supernatant becomes clear. Discard the supernatant and prepare a 1% porcine red blood cell volume fraction with 0.01 mol / L pH 7.4 sterile PBS.

[0063] (2) Take 1 tube of PAM cells frozen in liquid nitrogen, thaw quickly in a 37°C water bath, centrifuge at 500g for 5 minutes, 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.

[0064] (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℃ for 2 hours, and then inoculated with cells in (2) and cultured for 24 hours. The group of healthy mouse serum (mouseserum) and 1640 culture medium mixed with ASFV served as a positive control; the uninfected group served as a blank cell control.

[0065] (4) Pig red blood cells were diluted to 1×10 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.

[0066] The experimental results are as follows Figure 6 As shown, compared with the healthy mouse serum (mouse serum) and ASFV mixed group, and the 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.

[0067] Quantitative PCR (qPCR)

[0068] To further validate the ability of mAb 5D2 to neutralize ASFV, qPCR was used to measure the ASFV genome copy number after the different treatments in step 2.1. The cells from step 2.1 were frozen and thawed three times at -80°C. DNA was extracted from 200 μL of the aliquot according to the Qiagen DNA kit instructions and used as a template for qPCR. The 20 μL reaction system included: 10 μL of Premix Ex Taq™ (Probe qPCR) (2×); 0.5 μL of upstream primer (10 μM); 0.5 μL of downstream primer (10 μM); 3 μL of test sample DNA; 0.5 μL of fluorescent probe; and 4.5 μL of ddH2O. The upstream primer was 5'-CTGCTCATGGTATCAATCTTATCGA-3'; the downstream primer was 5'-GATACCACAAGATCAGCCGT-3' (see King DP, Reid SM, Hutchings GH, Grierson SS, Wilkinson PJ, Dixon LK, Bastos AD, Drew TW. Development of a TaqMan PCR assay with internal amplification control for the detection of African swine fever virus. J Virol Methods.). 2003 Jan;107(1):53-61. pMT18-P72 was diluted 10-fold and used as a standard to generate a standard curve. The reaction conditions were: initial denaturation at 95°C for 30 seconds (the first cycle); 40 cycles of 95°C for 5 seconds and 60°C for 30 seconds (the second cycle). Fluorescence signal detection was performed at the end of each extension cycle in the second step.

[0069] The viral copy number was calculated based on the standard curve. Figure 7 As shown, compared with the healthy mouse serum (mouseserum) and ASFV mixed group, and the 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.

[0070] Example 3 PCR amplification and sequence determination of the variable region gene of the anti-ASFV P72 protein monoclonal antibody 5D2

[0071] 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).

[0072] The antibody variable region genes were amplified using nested PCR. First, the variable region genes were amplified using the aforementioned cDNA as a template using the first round of mouse IgG and κ light chain primers. Then, the first round product was used as a template for the second round of mouse IgG and κ light chain primers. The PCR reaction system consisted of 25 μL of PrimeSTAR Max Premix (2×), 1 μL each of P1 and P2, 1 μL of cDNA, and ddH2O to 50 μL. The reaction program was as follows: pre-denaturation at 98°C for 2 min; 30 cycles of denaturation at 98°C for 10 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s; and finally, extension at 72°C for 10 min. Primers for antibody variable region gene amplification refer to the literature (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.)

[0073] 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 The target fragment was recovered by gel excision. The recovered target fragment was inserted into the pMD-18T vector and sequenced. The sequencing results were compared with the antibody gene library (IMGT). Sequencing confirmed that the amplified sequence was the DNA of the heavy and light chain variable regions of the monoclonal antibody.

[0074] 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.

[0075] The amino acid sequence of the heavy chain variable region of the murine 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 murine anti-ASFV P72 protein monoclonal antibody 5D2 is shown in SEQ ID NO: 2. The amino acid sequences of the heavy and light chain variable regions CDR1, CDR2, and CDR3 of the 5D2 monoclonal antibody are shown in Table 1 below.

[0076] Table 1. Amino acid sequences of CDR1, CDR2, and CDR3 of the heavy and light chain variable regions of monoclonal antibody 5D2

[0077] CDR1 CDR2 CDR3 Heavy chain VH GYTFLTYW IFPASGST ARSRDPSGPLT Light chain VL QTLVHSNGNTY KVS SQSTHVPPT

[0078] The embodiments described above are only preferred embodiments of the present invention and do not limit the scope of implementation of the present invention. Therefore, any equivalent changes or modifications made according to the structure, characteristics 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 with an amino acid sequence of GYTFLTYW, a CDR2 with an amino acid sequence of IFPASGST, and a CDR3 with an amino acid sequence of ARSRDPSGPLT; The light chain variable region of the monoclonal antibody or antigen-binding fragment includes a CDR1 with an amino acid sequence of QTLVHSNGNTY, a CDR2 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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