Monoclonal antibody 3F1 with neutralizing activity against African swine fever virus P72 protein and its application

Abstract

The present invention belongs to the field of biotechnology, and specifically relates to an anti-African swine fever virus (ASFV) P72 protein neutralizing active antibody 3F1 and its application. The present invention uses hybridoma cell fusion technology to successfully obtain a neutralizing active monoclonal antibody 3F1 specifically targeting ASF VP72 protein, which has a neutralizing function in vitro to prevent ASFV from infecting sensitive cells, has a strong affinity for antigens, and can be applied to the research of antiviral drugs.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a monoclonal antibody 3F1 with neutralizing activity against African swine fever virus (ASFV) P72 protein 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), with 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 into 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 (3F1) 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 3F1 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 3F1 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 are as follows:

[0006] CDR1 of heavy chain VH: GFSLTSYG;

[0007] CDR2 of heavy chain VH: IWRGGNTD;

[0008] CDR3 of heavy chain VH: AKDGEVITVLMDY.

[0009] CDR1 of light chain VL: QSLLYSNTQKIY;

[0010] CDR2 of light chain VL: WAS;

[0011] CDR3 of light chain VL: QQYYSYPLT.

[0012] The amino acid sequence of the heavy chain variable region and the amino acid sequence of the light chain variable region of monoclonal antibody 3F1 are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0013] The second aspect of the present invention provides a gene encoding the monoclonal antibody 3F1 against the African swine fever virus P72 protein, and the gene sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody 3F1 are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

[0014] The heavy chain constant region of the monoclonal antibody 3F1 is of IgG1 type.

[0015] The light chain constant region of the monoclonal antibody 3F1 is of Kappa type.

[0016] The third aspect of the present invention provides a recombinant expression vector comprising the heavy chain variable region and light chain variable region genes of the monoclonal antibody 3F1.

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

[0018] The monoclonal antibody 3F1 has at least one of the following functions:

[0019] (1) Detection of ASFV;

[0020] (2) Detect ASFVP72 protein.

[0021] In a fifth aspect, the present invention provides the use of monoclonal antibody 3F1 in any of the following aspects:

[0022] (1) Used to prepare products for treating ASFV infection and related diseases caused by infection; the product may be a drug or a vaccine.

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

[0024] The beneficial effects of the present invention are:

[0025] The present invention uses cell fusion and subcellular screening technology to successfully obtain a monoclonal antibody 3F1 that is specific to ASFV P72 protein and has neutralizing activity. This monoclonal antibody has a neutralizing function in vitro to prevent ASFV from infecting sensitive cells, and has a strong affinity for P72 protein, providing a reliable research tool for further exploring the function of P72 protein.

[0026] The monoclonal antibody 3F1 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, and is also conducive to optimizing the antibodies at the gene and protein levels, thereby improving the specificity and affinity of the antibodies.

[0027] The monoclonal antibody 3F1 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

[0028] Figure 1 ELISA was used to detect the antibody titer of mice after immunization with P72 protein;

[0029] 1-3: mice immunized with No. 1, 2 and 3, NC: negative control.

[0030] Figure 2 Identify the reactivity of monoclonal antibodies for IFA;

[0031] A. 3F1 monoclonal antibody;

[0032] B.SP2 / 0 cell culture medium.

[0033] Figure 3 To identify the reactivity of 3F1 monoclonal antibody for Western blot;

[0034] M: protein marker, WT: ASFV-infected group; Mock: uninfected control group; GAPDH is the internal reference control.

[0035] Figure 4 The results of subclass identification.

[0036] Figure 5 SADS-PAGE identification after ascites purification;

[0037] M: protein marker, 3F1: ascites after purification.

[0038] Figure 6 To detect the potency of 3F1 antibody after purification;

[0039] NC: ascites of unrelated monoclonal antibodies after purification.

[0040] Figure 7 To identify the neutralizing activity of 3F1 monoclonal antibody for hemadsorption assay;

[0041] Mock: uninfected group; Mouseserum-WT: mouse negative serum mixed with ASFV group; 1640-WT: 1640 culture medium mixed with ASFV group; 3F1-WT: 3F1 mixed with ASFV group.

[0042] Figure 8 To identify the neutralizing activity of 3F1 monoclonal antibody by qPCR;

[0043] Fig. 9 is the result of PCR amplification of the variable region;

[0044] A. PCR amplification results of heavy chain variable region. M: DL2000 marker; VH: PCR amplification of 3F1 heavy chain variable region.

[0045] B. PCR amplification results of light chain kappa variable region. M: DL2000 marker; VH: PCR amplification of 3F1 light chain variable region. DETAILED DESCRIPTION

[0046] 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.

[0047] Example 1 Screening and identification of anti-ASFVP72 protein monoclonal antibodies

[0048] 1. Animal immunization

[0049] Three female BALB / c mice aged 6 weeks 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 immunized at an amount 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 on the mice (one point on the back and two points on the abdomen). Reinforced immunization was performed every 2 weeks, for a total of four immunizations. The reinforced 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 four immunizations to determine the antibody titer.

[0050] 2. ELISA method to detect polyclonal antibody titer

[0051] 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 acted 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 .

[0052] The results are as follows Figure 1 As shown, when the serum was diluted to 1:2048000, the OD of immunized mice No. 1, 2 and 3 was 450 It is still greater than the serum of non-immune mice (NC group), indicating that the antibody titer can reach above 1:2048000.

[0053] 3. Preparation of Monoclonal Antibodies

[0054] Take the spleen cells of a P72 protein immunized mouse and mix them with SP20 cells in a ratio of 5:1, and then perform cell fusion under the action of the fusion agent PEG. The fused cells are plated in a 96-well cell plate and cultured in a 37°C, CO2 incubator. 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.

[0055] IFA test: 1×10 4MA104 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, and 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.

[0056] 4. Monoclonal Antibody Specificity Identification

[0057] After three subclones, 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 3F1 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 2 B). 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. 3F1 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 3F1 cell supernatant could recognize P72 protein in virus-infected cells, indicating that the 3F1 monoclonal antibody in 3F1 cell supernatant could specifically recognize P72 protein ( Figure 3 ).

[0058] 5. Monoclonal Antibody Subclass Identification

[0059] According to Southern Biotech's SBA Clonotyping TM System / HRP Antibody Subclass Identification Kit Operating Instructions The monoclonal antibodies in the supernatant of 3F1 cells were identified for antibody subclasses. Figure 4 As shown, the heavy chain constant region of the 3F1 monoclonal antibody is of IgG1 type, and the light chain constant region is of Kappa type.

[0060] 6. Preparation, purification and titer detection of ascites

[0061] 6.1 Preparation of ascites

[0062] 10-12 week old Balb / c mice were selected and each mouse was intraperitoneally injected with 0.5 mL of Freund's incomplete adjuvant. After 1 week, 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°C.

[0063] 6.2 Purification of ascites

[0064] 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 5 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 is named 3F1.

[0065] 6.3 Ascites titer detection

[0066] The monoclonal antibody 3F1 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 allowed to act at 37°C for 30 min. After washing four times with PBST, HRP-labeled goat anti-mouse IgG (1:20000 dilution) was added and allowed to act at 37°C for 30 min. After washing four times with PBST, TMB was used for color development and the OD was measured on an ELISA plate reader. 450 .

[0067] The results are as follows Figure 6 As shown, when monoclonal antibody 3F1 was diluted to 0.01953125 ng / μL, its OD 450 It is still greater than the irrelevant monoclonal antibody group (NC group), indicating that the antibody titer can reach above 0.01953125 ng / μL.

[0068] Example 2 Identification of the neutralizing activity of anti-ASFVP72 protein monoclonal antibody 3F1

[0069] 2.1 Red blood cell adsorption experiment

[0070] In order to verify whether the monoclonal antibody 3F1 has neutralizing activity, a red blood cell adsorption test was performed, and the specific operation steps are as follows:

[0071] (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 for 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.

[0072] (2) Take one tube of PAM cells frozen in liquid nitrogen tank, 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.

[0073] (3) Wild-type ASFV (2×10 6 TCID50 / mL) was mixed with healthy mouse serum (mouseserum), 1640 culture medium and 3F1 antibody (1 mg / mL) in equal volumes, incubated at 37°C for 2 hours, and then inoculated with cells in (2) and cultured for 24 hours. The group mixed with healthy mouse serum (mouseserum) and 1640 culture medium and ASFV was used as a positive control; the uninfected group was a blank cell control.

[0074] (4) Porcine red blood cells were diluted to 1×10 in 0.01 mol / L pH 7.4 sterile PBS. 7 / mL, take 100 μL and add it to a 24-well cell culture plate; after culturing at 37°C for 2-3 days, observe the red blood cell adsorption of ASFV under an inverted fluorescence microscope, take pictures, record and save.

[0075] The experimental results are as follows Figure 7 As shown, compared with the mixed groups of healthy mouse serum (mouserum) and 1640 culture medium with ASFV, the number of rosette-like structures was significantly reduced after ASFV was treated with monoclonal antibody 3F1, indicating that monoclonal antibody 3F1 can effectively inhibit the replication of ASFV.

[0076] 2.2 Quantitative PCR (qPCR)

[0077] To further verify the ability of monoclonal antibody 3F1 to neutralize ASFV, the qPCR test was used to detect the genome copy number of ASFV after treatment with different antibodies. 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: PremixEx Taq TM (Probe qPCR) (2×) 10μL; upstream primer ((10μM) 0.5μL; downstream primer (10μM) 0.5μL; test sample DNA 3μL; fluorescent probe: 0.5μL; ddH2O: 4.5μL. Upstream primer: 5'-CTGCTCATGGTATCAATCTTATCGA-3'; downstream primer: 5'-GATACCACAAGATCAGCCGT-3' (reference: King DP, Reid SM, Hutchings GH, Grierson SS, Wilkinson PJ, Dixon LK, Bastos AD, Drew TW. Development of a TaqManPCR assay with internal amplification control for the detection of Africanswine fever virus. J Virol Methods. 2003 Jan; 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.

[0078] The virus copy number was calculated based on the standard curve. Figure 8 As shown, compared with the group mixed with healthy mouse serum (mouserum) and 1640 culture medium and ASFV, the number of viral copies in the ASFV group treated with monoclonal antibody 3F1 was significantly reduced and the difference was extremely significant, further indicating that monoclonal antibody 3F1 inhibited the replication of ASFV.

[0079] Example 3 PCR amplification and sequence determination of the variable region gene of the anti-ASFVP72 protein monoclonal antibody 3F1

[0080] The monoclonal antibody 3F1 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).

[0081] 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×) 25μL, 1μL each of P1 and P2, 1μL of cDNA, and ddH2O to 50μL. The reaction procedure was: 98℃ pre-denaturation for 2min; 98℃ denaturation for 10s, 55℃ annealing for 30s, 72℃ extension for 30s, 30 cycles; 72℃ extension for 10min. 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.).

[0082] After amplification, 1% agarose gel electrophoresis was performed, and the heavy chain and κ light chain variable region gene size of monoclonal antibody 3F1 was approximately 300 bp ( Fig. 9 ), 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 and analyzed with the antibody gene library (IMGT). 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.

[0083] Specifically, the DNA sequence encoding the heavy chain variable region of the mouse anti-ASFV P72 protein monoclonal antibody 3F1 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 3F1 is shown in SEQ ID NO: 4.

[0084] The amino acid sequence of the heavy chain variable region of the mouse anti-ASFV P72 protein monoclonal antibody 3F1 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 3F1 is shown in SEQ ID NO: 2.

[0085] The amino acid sequences of the heavy chain and light chain variable regions CDR1, CDR2 and CDR3 of monoclonal antibody 3F1 are shown in Table 1 below.

[0086] Table 1 Amino acid sequences of CDR1, CDR2 and CDR3 of the heavy and light chain variable regions of monoclonal antibody 3F1

[0087] —— CDR1 CDR2 CDR3 Heavy chain VH GFSLTSYG IWRGGNTD AKDGEVITVLMDY Light chain VL QSLLYSNTQKIY WAS QQYYSYPLT

[0088] 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. Monoclonal antibody 3F1 against African swine fever virus P72 protein, characterized in that: The heavy chain variable region of the monoclonal antibody 3F1 includes a CDR1 with an amino acid sequence of GFSLTSYG, a CDR2 with an amino acid sequence of IWRGGNTD, and a CDR3 with an amino acid sequence of AKDGEVITVLMDY; The light chain variable region of the monoclonal antibody 3F1 includes a CDR1 with an amino acid sequence of QSLLYSNTQKIY, a CDR2 with an amino acid sequence of WAS, and a CDR3 with an amino acid sequence of QQYYSYPLT.

2. The monoclonal antibody 3F1 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 3F1 are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

3. A gene encoding the monoclonal antibody 3F1 against African swine fever virus P72 protein according to claim 1.

4. The gene according to claim 3, characterized in that The gene sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody 3F1 are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

5. A recombinant expression vector comprising the gene according to claim 3. A host cell comprising the recombinant expression vector according to claim 5.

7. Use of the monoclonal antibody 3F1 according to claim 1 or 2, the gene according to claim 3 or 4, the recombinant expression vector according to claim 5 or the host cell according to 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.

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