CN117209596A

CN117209596A - Monoclonal antibody 2C9 for neutralizing activity of P72 protein of anti-African swine fever virus and application thereof

Info

Publication number: CN117209596A
Application number: CN202311124301.0A
Authority: CN
Inventors: 王�琦; 曹丽艳; 郑海学; 孔祥雨; 万颖; 张宇; 孙茂文; 段月月; 袁聪
Original assignee: Lanzhou Veterinary Research Institute of CAAS
Current assignee: Lanzhou Veterinary Research Institute of CAAS
Priority date: 2023-09-01
Filing date: 2023-09-01
Publication date: 2023-12-12

Abstract

The invention belongs to the technical field of biology, and particularly relates to an anti-African Swine Fever Virus (ASFV) P72 protein neutralizing active antibody 2C9 and application thereof. The invention successfully obtains a monoclonal antibody 2C9,2C9 with specific neutralizing activity against ASFV P72 protein by utilizing hybridoma cell fusion technology, is a conformation dependent monoclonal antibody, has neutralizing function for preventing ASFV from infecting sensitive cells in vitro, has strong affinity for antigen, and can be applied to the research of antiviral drugs.

Description

Monoclonal antibody 2C9 for neutralizing activity of P72 protein of anti-African swine fever virus and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an anti-African Swine Fever Virus (ASFV) P72 protein neutralizing activity monoclonal antibody 2C9 and application thereof.

Background

African swine fever (African swine fever, ASF) is an acute, high-contact infectious disease of pigs caused by African swine fever virus (African swine fever virus, ASFV), and has a short disease course and a death rate of 100%. ASFV is a virus that is difficult to remove, and at present, inactivated vaccine, attenuated vaccine, subunit vaccine and DNA vaccine of ASF are not sufficient to protect pigs from ASFV. Little is known about the immune mechanism and its effective immune components that are effective in protecting swine herds from ASFV infection. Thus, in addition to expanding understanding of ASFV proteome and single protein functions, there is a need to find an appropriate balance between antibody and cell-mediated ASFV immune responses, which are of great importance for rational design of targeted vaccines.

ASFV structural proteins are more, wherein P72 is one of the main structural proteins, the protein sequence is more conservative, the protein is encoded by B646L gene, and the protein accounts for 31-33% of the virus particles, and is the most content of the structural proteins. P72 exists on the surface of a virus capsid, has better immunogenicity and antigenicity, and can induce the organism to generate neutralizing antibodies. The action mechanism of the neutralizing antibody mainly changes the configuration of the surface of the virus, prevents the virus from adsorbing to the susceptible cells, and ensures that the virus cannot penetrate into the susceptible cells for proliferation. Screening neutralizing antibodies against P72 protein will increase our understanding and understanding of pig humoral immune response, providing theoretical basis for designing novel vaccine of ASFV and effectively preventing and controlling ASF.

Disclosure of Invention

The invention aims to provide a neutralizing activity monoclonal antibody (2C 9) of an ASFV P72 protein and application thereof. The invention takes P72 protein obtained in the invention patent with publication number of CN112979765A as an immune source to immunize mice, and monoclonal antibody 2C9 with neutralizing activity against the P72 protein is obtained through cell fusion and subcellular screening technology.

In a first aspect of the present invention, there is provided a monoclonal antibody 2C9 against ASFV P72 protein, said monoclonal antibody having the following amino acid sequences of the heavy and light chain variable regions CDR1, CDR2 and CDR3:

CDR1 of heavy chain VH: GFNIKDTY;

CDR2 of heavy chain VH: IDPANGYT;

CDR3 of heavy chain VH: TKGRGWYFDV.

CDR1 of light chain VL: ESVDTYGNSF;

CDR2 of light chain VL: RAS;

CDR3 of light chain VL: QQGNEDPWT.

The amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody 2C9 are respectively shown as SEQ ID NO. 1 and SEQ ID NO. 2.

The second aspect of the invention provides a gene for encoding the monoclonal antibody 2C9 of the African swine fever virus P72 protein, wherein the gene sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody 2C9 are respectively shown as SEQ ID NO. 3 and SEQ ID NO. 4.

The heavy chain constant region of the monoclonal antibody 2C9 is of the IgG2a type.

The light chain constant region of the monoclonal antibody 2C9 is Kappa type.

The invention discovers that the antigen epitope of the monoclonal antibody 2C9 is a conformation dependent epitope.

In a third aspect, the present invention provides a recombinant expression vector comprising the heavy chain variable region and light chain variable region genes of monoclonal antibody 2C9.

In a fourth aspect the invention provides a host cell comprising a recombinant expression vector as described above.

The monoclonal antibody 2C9 has at least one of the following functions:

(1) Detecting ASFV;

(2) ASFV P72 protein was detected.

In a fifth aspect of the invention there is provided the use of monoclonal antibody 2C9 in any one of the following aspects:

(1) For the preparation of a product for the treatment of diseases related to ASFV infections and infections thereof; the product may be a medicament or vaccine.

(2) Is used for preparing ASFV detection reagent or kit.

The beneficial effects of the invention are as follows:

the invention successfully obtains a monoclonal antibody 2C9 with neutralization activity specific to ASFV P72 protein by utilizing cell fusion and subcellular screening technologies. The monoclonal antibody has the neutralizing function of preventing ASFV from infecting sensitive cells in vitro, has strong affinity to P72 protein, and provides a reliable research tool for further exploring the function of the P72 protein.

The monoclonal antibody 2C9 provided by the invention can be obtained by using a conventional genetic engineering or protein engineering method, so that the long-term freezing storage antibody loss of hybridoma cells is avoided, the optimization of the antibody on the gene and protein level is facilitated, and the specificity and affinity of the antibody are improved.

The monoclonal antibody 2C9 prepared by the invention can specifically identify and neutralize ASFV, so the antibody provides a new raw material for researching the pathogenic mechanism of ASFV, early detection kit and developing therapeutic antibody or vaccine.

Drawings

FIG. 1 shows ELISA detection of mouse antibody titers after P72 protein immunization;

1 to 3: 1. immunized mice, no. 2 and No. 3, NC: negative control.

FIG. 2 is a graph showing the reactivity of IFA to identify monoclonal antibodies;

a.2c9 monoclonal antibody;

SP2/0 cell culture broth.

FIG. 3 is a diagram showing the reactivity of Westernblot to identify 2C9 monoclonal antibodies;

m: protein Marker, WT: ASFV virus group; mock: a non-toxic group; GAPDH is an internal control.

Fig. 4 shows the result of subclass identification.

FIG. 5 is a SADS-PAGE identification after ascites purification;

m: protein Marker,2C9: and purifying the ascites.

FIG. 6 is the detection of titers after 2C9 antibody purification;

NC: ascites with the irrelevant monoclonal antibody after purification.

FIG. 7 is a graph depicting the neutralization activity of a red blood cell adsorption assay to identify 2C9 monoclonal antibodies;

mock: a non-toxic group; mouseser-WT: murine negative serum and ASFV combination; 1640-WT:1640 medium and ASFV mix; 2C9-WT:2C9 and ASFV mix.

FIG. 8 is a graph of qPCR identification of the neutralizing activity of 2C9 monoclonal antibodies;

FIG. 9 shows the results of variable region PCR amplification;

A. heavy chain variable region PCR amplification results. M: DL2000 marker; VH:2C9 heavy chain variable region PCR amplification.

B. Results of PCR amplification of the light chain kappa variable region. M: DL2000 marker; VH:2C9 light chain variable region PCR amplification.

Detailed Description

The following detailed description of the present invention is provided to facilitate understanding of the technical solution of the present invention, but is not intended to limit the scope of the present invention.

Example 1 screening and identification of monoclonal antibodies against ASFV P72 protein

1. Immunization of animals

3 female BALB/c mice of 6W age were selected, and the purified P72 recombinant protein (which was given away from the university of Qinghai to the teachings of the light, preparation method was referred to the patent application of the invention with application publication No. CN 112979765A) was immunized in an amount of 30. Mu.g/mouse. For the first immunization, P72 protein was mixed with Freund's complete adjuvant in equal volumes and emulsified, and mice were injected subcutaneously with multiple spots (one spot on the back and two spots on the abdomen). Boosting is carried out every 2W for four times, wherein the boosting is to uniformly mix P72 recombinant protein and Freund's incomplete adjuvant in equal volume for emulsification, and the immunization method is the same as the first immunization. And after four days, 7d tail blood collection is carried out to determine the antibody titer.

ELISA method for detecting polyclonal antibody titer

The purified P72 protein was diluted to 1. Mu.g/mL with the coating solution, and the mixture was added to ELISA plates at 50. Mu.L/well, coated at 37℃for 1 hour, and then treated with PBST (K ₂ HPO ₄ 0.26g、Na ₂ HPO ₄ ·12H ₂ O2.89 g, naCl 8.50g, tween-20.5 mL, water was added to fix the volume to 1L) and the mixture was washed four times, and then 5% skimmed milk powder was added thereto and the mixture was blocked at 37℃for 1 hour. Positive serum and negative serum were diluted with a PBST-fold ratio, the dilution gradient was from 1:1000 to 1:2048000, the dilution was 12, the reaction was carried out at 37 ℃ for 30min, HRP-labeled goat anti-mouse IgG (dilution of 1:20000) was added after four PBST washes, TMB developed after four PBST washes, and OD was measured on a microplate reader ₄₅₀ 。

The results are shown in FIG. 1, where the serum was diluted to 1:2048000 and the mice were immunized with OD numbers 1, 2 and 3 ₄₅₀ Still larger than the serum of the non-immune mice (NC group), which shows that the antibody titer can reach more than 1:2048000.

3. Preparation of monoclonal antibodies

Spleen cells of a P72 protein immunized mouse are taken and mixed with SP20 cells according to the proportion of 5:1, and then cell fusion is carried out under the action of fusion agent PEG. The fused cells were plated in 96-well cell plates at 37℃with CO ₂ Culturing in an incubator. After hybridoma cells were confluent 1/10 of the bottom of the cell plate, the supernatant was aspirated for ELISA detection (ELISA coating method and 2.ELISA method for polyclonal antibody titer detection). Wells identified as positive by ELISA were then validated by indirect immunofluorescence assay (IFA). ELISA and IFA positive holes are selected, and the selected positive hybridoma cells are subcloned by a limiting dilution method. Wells were visualized under an inverted microscope, where only individual clones were grown, and supernatants were taken for antibody detection using the ELISA and IFA methods described above. Positive cells entered the next round of subcloning, performed three times in total.

IFA test: will be 1X 10 ⁴ Individual MA104 cells were plated in 96-well cell plates and, after confluence of monolayers, inoculated with 0.1MOI ASFV. 48h after inoculation, cells were fixed with 4% paraformaldehyde at room temperature for 30min, and with 0.01mol/L PBS (K ₂ HPO ₄ 0.26g、Na ₂ HPO ₄ ·12H ₂ 2.89g of O, 8.50g of NaCl, water to 1L, pH 7.4) for 3 times; then 0.1% Triton X-100 is used for permeation, and the reaction is carried out for 10min at room temperature, and PBS is used for washing 3 times; hybridoma cell supernatants were then incubated with anti-mouse FITC labeled goat anti-mouse IgG and the results were observed under a fluorescence microscope after completion of the reaction.

4. Monoclonal antibody specificity identification

After three subcloning, the cell supernatant secreted by the obtained single cell strain is subjected to IFA and western blot detection. IFA assay showed that 2C9 cell supernatant acted on ASFV-infected MA104 cells to detect a specific green fluorescent signal (fig. 2A), whereas SP2/0 cell supernatant acted on ASFV-infected cells did not see a fluorescent signal (fig. 2B). Virus-infected and uninfected MA104 cells were collected, subjected to SDS-PAGE after cell lysis, and then protein gel was transferred to NC membrane for western blot verification. 2C9 cell supernatant was used as primary antibody, anti-mouse HRP-IgG was used as secondary antibody at room temperature for 1h, and after each reaction was completed, PBST was used for washing 4 times, followed by exposure and development. Westernblot results showed that 2C9 cell supernatants were unable to recognize P72 protein in virus-infected cells, indicating that 2C9 cell supernatants recognized epitopes of P72 protein as conformation-dependent (FIG. 3).

5. Monoclonal antibody subclass identification

SBA cloning according to Southern Biotech ^TM System/HRP antibody subclass identification kit procedure demonstrated the antibody subclass identification of the monoclonal antibodies in the 2C9 cell supernatant obtained. As a result, the heavy chain constant region of the monoclonal antibody was of the IgG2a type and the light chain constant region was of the Kappa type, as shown in FIG. 4.

6. Preparation, purification and potency detection of ascites

6.1 preparation of ascites

Taking 10-12W Balb/c mice, wherein each mouse is injected with Freund's incomplete adjuvant in the abdominal cavity of 0.5mL, and each mouse is injected with 5X 10 in the abdominal cavity after 1W ⁵ Positive hybridoma (0.2 mL) prepared in the preparation of monoclonal antibody, wherein after 7-10 days, the abdominal cavity of the mouse is obviously raised, ascites are collected, split charging is carried out, and the mice are preserved at-80 ℃.

6.2 purification of ascites

According to PIERCE Corp. NAb ^TM Protein G Spin Purification Kit and then purifying by affinity chromatography, and identifying the purity by SDS-PAGE electrophoresis, as can be seen from FIG. 5, the monoclonal antibody obtained after ascites purification has a heavy chain of about 55KD and a light chain of about 25KD, and is designated as 2C9.

6.3 ascites titer detection

Diluting the monoclonal antibody 2C9 obtained by ascites purification by PBST according to the ratio of 40 ng/mu L, diluting for 12 gradients altogether, adding 50 mu L/hole of the diluted antibody into a P72 protein coated ELISA plate, and performing the action for 30min at 37 ℃; after four times of PBST washing, HRP-labeled goat anti-mouse IgG (diluted 1:20000) was added, and the mixture was allowed to act at 37℃for 30min, after four times of PBST washing; TMB color development, and OD measurement on enzyme-labeled instrument ₄₅₀ 。

As shown in FIG. 6, when monoclonal antibody 2C9 was diluted to 0.01953125 ng/. Mu.L, the OD thereof was as follows ₄₅₀ Still larger than the irrelevant monoclonal antibody group (NC group), which shows that the antibody titer can reach more than 0.01953125 ng/. Mu.L.

EXAMPLE 2 identification of neutralizing Activity of anti-ASFVP 72 protein monoclonal antibody 2C9

2.1 erythrocyte adsorption experiments

To verify whether monoclonal antibody 2C9 has neutralizing activity, a red blood cell adsorption assay was performed, which was performed as follows:

(1) Preparation of 1% porcine erythrocytes: sterile sampling healthy pig blood into heparin anticoagulation tube, and mixing thoroughly. 1mL of anticoagulation was resuspended in 9mL of 0.01mol/L sterile PBS pH7.4, and then centrifuged at 500g for 5min, and the supernatant was discarded; 9mL of 0.01mol/L sterile PBS pH7.4 was used to resuspend the red blood cells, and the wash was repeated 4 times until the supernatant became clear. The supernatant was discarded and 1% by volume of porcine erythrocytes was prepared with 0.01mol/L sterile PBS pH 7.4.

(2) Taking 1 PAM cell frozen in liquid nitrogen tank, thawing rapidly in water bath at 37deg.C, centrifuging at 500g for 5min, discarding supernatant, and re-suspending the cell with 1mL RPMI-1640 medium containing 10% FBS and 2% double antibody at 1×10 ⁵ The cells/wells were plated on 96-well cell culture plates and incubated at 37℃for 24h.

(3) Wild type ASFV (2×10) ⁶ TCID 50/mL)The healthy mouse serum (mouseserum), 1640 medium and 2C9 antibody (1 mg/mL) were mixed in equal volumes respectively, incubated at 37℃for 2 hours, and then the cells in (2) were inoculated for further culture for 24 hours. Healthy mouse serum (mouseserum) and 1640 medium mixed with ASFV as positive control; the non-vaccinated group was a blank cell control.

(4) Pig erythrocytes were diluted to 1X 10 with 0.01mol/L sterile PBS pH7.4 ⁷ mu.L of the culture medium is taken and added into a 24-hole cell culture plate; after culturing for 2-3 d at 37 ℃, observing the red blood cell adsorption condition of ASFV by an inverted fluorescence microscope, photographing, recording and storing.

As shown in fig. 7, the number of rosette-like structures was significantly reduced after treatment of ASFV with monoclonal antibody 2C9 compared to healthy murine serum (mouseserum) and 1640 medium in combination with ASFV, indicating that monoclonal antibody 2C9 was effective in inhibiting replication of ASFV.

2.2 fluorescent quantitative PCR (qPCR)

To further verify the ability of monoclonal antibody 2C9 to neutralize ASFV, genomic copy numbers of ASFV after different antibody treatments were tested using qPCR assays. The cells in step 2.1 were repeatedly freeze-thawed 3 times at-80℃and 200. Mu.L of DNA was extracted according to the Qiagen DNA kit extraction protocol, and qPCR was performed using this as a template. The reaction system was 20. Mu.L comprising: premix Ex Taq ^TM (Probe qPCR) (2×) 10 μl; the upstream primer ((10. Mu.M) 0.5. Mu.L; downstream primer (10. Mu.M) 0.5. Mu.L; detection sample DNA 3. Mu.L; fluorescent probe: 0.5. Mu.L; ddH) ₂ O4.5. Mu.L. An upstream primer: 5'-CTGCTCATGGTATCAATCTTATCGA-3'; a downstream primer: 5'-GATACCACAAGATCAGCCGT-3' (cf. 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 viruses. J Virol methods.2003Jan;107 (1): 53-61). pMT18-P72 was diluted 10-fold as a standard, and a standard curve was drawn. The reaction conditions are as follows: pre-denaturation at 95 ℃ for 30s, which is the first cycle; 95℃for 5s and 60℃for 30s are 40 cycles of the second step, and fluorescence signal detection is performed at the end of extension of each cycle of the second step.

The results of the viral copy number calculation according to the standard curve are shown in fig. 8, and the monoclonal antibody 2C9 treated ASFV group significantly reduced in viral copy number and significantly different compared to the healthy murine serum (mouseserum) and 1640 medium mixed with ASFV group, further demonstrating that monoclonal antibody 2C9 inhibited replication of ASFV.

EXAMPLE 3 PCR amplification and sequencing of the anti-ASFVP 72 protein monoclonal antibody 2C9 variable region Gene

Monoclonal antibody 2C9 hybridoma RNA was extracted and reverse transcribed into cDNA (PrimeScript II 1st Strand cDNA Synthesis Kit,TAKARA,6210A) using Oligo-dt or random primers, respectively.

The antibody variable region gene was amplified by nested PCR. The cDNA is first used as template to amplify the variable region gene of antibody with the first round of mouse antibody IgG and kappa light chain primer, and the first round of product is then used as template to amplify the variable region gene of antibody with the second round of mouse antibody IgG and kappa light chain primer. The PCR reaction system is as follows: primeSTAR Max Premix (2X) 25. Mu.L, 1. Mu.L each of P1 and P2, 1. Mu.L cDNA, ddH ₂ O was added to 50. Mu.L. The reaction procedure is: pre-denaturation at 98℃for 2min; denaturation at 98℃for 10s, annealing at 55℃for 30s, elongation at 72℃for 30s,30 cycles; extending at 72℃for 10min. Primers for gene amplification of antibody variable regions are described in (von Boehmer, l., liu, c., ackerman, s., gitlin, a.d., wang, q., gazumyan, a., nussenzweig, m.c.,2016.Sequencing and cloning of antigen-specific antibodies from mouse memory B cells, nature protocols 11, 1908-1923.).

After the amplification, 1% agarose gel electrophoresis is carried out, the gene sizes of the heavy chain and kappa light chain variable regions of the monoclonal antibody 2C9 are about 300bp (figure 9), and the target fragment is recovered by cutting gel. The recovered target fragment was inserted into a pMD-18T vector, and sequence was determined. The sequencing results were compared with the antibody gene library (IMGT) and confirmed that the amplified sequences were DNA of the heavy and light chain variable regions of the monoclonal antibody.

Specifically, the DNA sequence of the heavy chain variable region of the mouse anti-ASFV P72 protein monoclonal antibody 2C9 is shown in SEQ ID NO. 3; the DNA sequence of the light chain variable region of the monoclonal antibody 2C9 of the mouse anti-ASFV P72 protein is shown as SEQ ID NO. 4.

The amino acid sequence of the heavy chain variable region of the mouse anti-ASFV P72 protein monoclonal antibody 2C9 is shown as SEQ ID NO. 1; the amino acid sequence of the light chain variable region of the mouse anti-ASFV P72 protein monoclonal antibody 2C9 is shown as SEQ ID NO. 2. The amino acid sequences of the variable regions CDR1, CDR2 and CDR3 of monoclonal antibody 2C9 heavy chain and light chain are shown in table 1 below.

TABLE 1 amino acid sequences of the variable regions CDR1, CDR2 and CDR3 of monoclonal antibody 2C9 heavy and light chain

——	CDR1	CDR2	CDR3
				Heavy chain VH	GFNIKDTY	IDPANGYT	TKGRGWYFDV
Light chain VL	ESVDTYGNSF	RAS	QQGNEDPWT

The above-described embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention, so that all equivalent changes or modifications of the structure, characteristics and principles described in the claims should be included in the scope of the present invention.

Claims

1. The monoclonal antibody 2C9 of the African swine fever virus P72 protein is characterized in that the heavy chain variable region of the monoclonal antibody 2C9 comprises a CDR1 with an amino acid sequence of GFNIKDTY, a CDR2 with an amino acid sequence of IDPANGYT and a CDR3 with an amino acid sequence of TKGRGWYFDV;

the light chain variable region of monoclonal antibody 2C9 comprises CDR1 with amino acid sequence ESVDTYGNSF, CDR2 with amino acid sequence RAS, CDR3 with amino acid sequence QQGNEDPWT.

2. The monoclonal antibody 2C9 against the P72 protein of African swine fever virus according to claim 1, wherein 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 2C9 are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively.

3. A gene encoding the monoclonal antibody 2C9 against the african swine fever virus P72 protein of claim 1.

4. The gene according to claim 3, wherein the heavy chain variable region and the light chain variable region of the monoclonal antibody 2C9 have the gene sequences shown in SEQ ID NO. 3 and SEQ ID NO. 4, respectively.

5. A recombinant expression vector comprising the gene of claim 3.

6. A host cell comprising the recombinant expression vector of claim 5.

7. The use of the monoclonal antibody 2C9 of claim 1 or 2, the gene of claim 3 or 4, the recombinant expression vector of claim 5, the host cell of claim 6 in any of the following aspects,

(1) The application in preparing products for treating diseases related to African swine fever virus infection and infection thereof; the product is a drug or vaccine;

(2) The application in preparing African swine fever detection reagent or kit.