CN115232206B

CN115232206B - anti-CD 2v protein monoclonal antibody and application thereof

Info

Publication number: CN115232206B
Application number: CN202210755791.3A
Authority: CN
Inventors: 王晶; 于鸣; 李新颖; 乔春霞; 罗龙龙; 肖鹤; 陈国江; 王志宏; 胡乃静; 冯健男; 沈倍奋
Original assignee: Academy of Military Medical Sciences AMMS of PLA
Current assignee: Academy of Military Medical Sciences AMMS of PLA
Priority date: 2022-06-30
Filing date: 2022-06-30
Publication date: 2023-04-11
Anticipated expiration: 2042-06-30
Also published as: CN115232206A

Abstract

anti-CD 2v protein monoclonal antibody and application thereof. The invention discloses an anti-CD 2v protein monoclonal antibody, which is selected from 7B1 or 11H7 antibody: the 7B1 or 11H7 antibody respectively comprises a heavy chain variable region CDR1-3 and a light chain variable region CDR1-3, wherein the amino acid sequences of the heavy chain variable region and the light chain variable region are respectively shown in a sequence table. The invention also discloses application of the antibody in preparing products for detecting, treating and/or preventing African swine fever virus. The antibody has high affinity for African swine fever virus.

Description

anti-CD 2v protein monoclonal antibody and application thereof

Technical Field

The invention relates to the technical field of biomedicine, in particular to an anti-CD 2v protein monoclonal antibody and application thereof.

Background

African Swine Fever (ASF) is a severe acute, hemorrhagic and virulent infectious disease caused by African Swine Fever Virus (ASFV) infecting domestic pigs and various wild pigs (such as African wild pigs, european wild pigs and the like), and the fatality rate can reach 100%. The African swine fever virus is a large nucleoplasmic double-stranded DNA virus, and among about 200 proteins encoded by the genome, there is a glycoprotein CD2v encoded by the EP402R gene, which plays an important role in invasion of the ASFV virus into the host and spread.

A Beijing animal husbandry veterinary research Juhongfei professor team carries out codon optimization on a full-length EP402R gene in an ASFV Geoga 2007/1 strain, is connected with a pET-28a (+) expression prokaryotic recombinant expression vector, is expressed by an escherichia coli system to obtain a CD2v recombinant protein, and finally, a polyclonal antibody is prepared by using the purified CD2v recombinant protein. China inspection and quarantine science research institute Linxiangmei professor team, the eukaryotic cell expression of CD2v extracellular domain is realized in a breakthrough way, and the murine polyclonal antibody is prepared by immunizing a mouse. However, polyclonal antibodies have poor specificity and are difficult to standardize, and the quality of the antibodies in different batches is greatly different. It is difficult to be used for the development of experimental and therapeutic agents requiring high specificity. Although monoclonal antibodies of CD2v have been identified, african swine fever virus has high mutability under the pressure of antibodies, and therefore, more novel antibodies against different epitopes are essential for treatment. The aim of the present invention is to develop an anti-CD 2v monoclonal antibody with high affinity to CD2v that recognizes a single epitope.

Disclosure of Invention

In order to solve the above technical problems, an object of the present invention is to provide a monoclonal antibody specifically recognizing CD2v protein, which is used for developing an african swine fever virus detection reagent; the other purpose of the invention is to use the monoclonal antibody as a medicament for preventing or treating African swine fever virus.

In order to achieve the purpose, the specific technical scheme of the invention is as follows:

in a first aspect, the invention provides a monoclonal antibody against the CD2v protein, said antibody being selected from the group consisting of the 7B1 or 11H7 antibodies:

the 7B1 antibody comprises a heavy chain variable region CDR1, CDR2, CDR3 and a light chain variable region CDR1, CDR2, CDR3; the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are respectively shown as amino acid sequences at 31-35, 50-66 and 99-103 in SEQ ID NO. 1; the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region are respectively shown as amino acid sequences at positions 24-39, 55-61 and 94-102 in SEQ ID NO. 3;

the 11H7 antibody comprises a heavy chain variable region CDR1, CDR2, CDR3 and a light chain variable region CDR1, CDR2, CDR3; the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are respectively shown as amino acid sequences at 31-36, 51-66 and 99-104 positions in SEQ ID NO. 5; the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region are respectively shown as amino acid sequences at positions 24-33, 49-55 and 88-96 in SEQ ID NO. 7.

Further, the 7B1 antibody further comprises heavy chain variable region FR1, FR2, FR3 and FR4 framework regions and light chain variable region FR1, FR2, FR3 and FR4 framework regions; the amino acid sequences of the heavy chain variable regions FR1, FR2, FR3 and FR4 are respectively shown as amino acid sequences at 1-30, 36-49, 67-98 and 104-114 in SEQ ID NO. 1; the amino acid sequences of the light chain variable regions FR1, FR2, FR3 and FR4 are respectively shown as amino acid sequences at 1-23, 40-54, 62-93 and 103-112 in SEQ ID NO. 3;

the 11H7 antibody further comprises heavy chain variable region FR1, FR2, FR3 and FR4 framework regions and light chain variable region FR1, FR2, FR3 and FR4 framework regions; the amino acid sequences of FR1, FR2, FR3 and FR4 of the heavy chain variable region are respectively shown as the amino acid sequences at 1-30, 37-50, 67-98 and 105-115 sites in SEQ ID NO. 5; the amino acid sequences of the light chain variable regions FR1, FR2, FR3 and FR4 are respectively shown as amino acid sequences at positions 1-23, 34-48, 56-87 and 97-106 in SEQ ID NO. 7.

Further, the heavy chain variable region of the 7B1 antibody has an amino acid sequence shown as SEQ ID No.1, and the light chain variable region of the 7B1 antibody has an amino acid sequence shown as SEQ ID No. 3;

the heavy chain variable region of the 11H7 antibody has an amino acid sequence shown as SEQ ID NO.5, and the light chain variable region of the 11H7 antibody has an amino acid sequence shown as SEQ ID NO. 7.

Further, the monoclonal antibody further comprises an antibody heavy chain constant region and/or an antibody light chain constant region.

In a second aspect, the invention provides a nucleic acid molecule comprising a nucleic acid encoding a monoclonal antibody as defined in any one of the preceding claims.

Further, the nucleic acid molecule encoding the heavy chain variable region of the 7B1 antibody has a nucleotide sequence shown in SEQ ID NO. 2; the nucleic acid molecule for encoding the variable region of the light chain of the 7B1 antibody has a nucleotide sequence shown as SEQ ID NO. 4;

further, the nucleic acid molecule encoding the 11H7 antibody heavy chain variable region has a nucleotide sequence shown in SEQ ID NO. 6; the nucleic acid molecule for encoding the 11H7 antibody light chain variable region has a nucleotide sequence shown as SEQ ID NO. 8.

In a third aspect, the present invention provides an expression vector comprising the nucleic acid molecule described above.

Further, the expression vector further comprises a signal peptide linked to the light chain or the heavy chain of the monoclonal antibody.

Further, the amino acid sequence of the signal peptide linked to the heavy chain of the 7B1 antibody is shown in SEQ ID NO.9, and preferably, the nucleotide sequence encoding the signal peptide linked to the heavy chain thereof is shown in SEQ ID NO. 10; the amino acid sequence of the signal peptide connected with the light chain of the 7B1 antibody is shown as SEQ ID NO.11, and preferably, the nucleotide sequence of the signal peptide connected with the light chain of the signal peptide is shown as SEQ ID NO. 12;

the amino acid sequence of the signal peptide connected with the heavy chain of the 11H7 antibody is shown as SEQ ID NO.13, and preferably, the nucleotide sequence of the signal peptide connected with the heavy chain is coded as SEQ ID NO. 14; the amino acid sequence of the signal peptide linked to the light chain of the 11H7 antibody is shown in SEQ ID No.15, and preferably, the nucleotide sequence encoding the signal peptide linked to the light chain thereof is shown in SEQ ID No. 16.

In a fourth aspect, the invention provides a cell comprising an expression vector as described above.

In a fifth aspect, the invention provides a pharmaceutical composition comprising a monoclonal antibody as described in any one of the above.

In a sixth aspect, the invention provides a product comprising a monoclonal antibody according to any one of the above; or comprising a nucleic acid molecule as described in any of the above.

Further, the products include, but are not limited to, products for detecting antigen-antibody binding by enzyme-linked immunosorbent assay, colloidal gold immunochromatography, immunoblotting, immunofluorescence detection, and luminescence immunoassay.

Further, the product comprises a reagent, a test strip, a kit or a medicament.

Further, the kit comprises: colloidal gold immunoassay kit, chemiluminescence kit, enzyme-linked immunosorbent assay (ELISA) kit and immunofluorescence kit.

A seventh aspect of the invention provides use in any one of:

(1) The monoclonal antibody is used for preparing a reagent for detecting CD2v protein;

(2) The monoclonal antibody is used for preparing a kit for detecting or diagnosing African swine fever virus; preferably, the kit comprises an african swine fever virus antibody enzyme-linked immunization kit;

(3) The monoclonal antibody is used for separating, purifying or detecting the African swine fever virus CD2v protein which is not used for diagnosis and treatment;

(4) The monoclonal antibody is used for separating, purifying or detecting the African swine fever virus antibody for non-diagnosis and treatment purposes;

(5) Use of said monoclonal antibody or said nucleic acid molecule or said expression vector or said cell or said product or said pharmaceutical composition for the preparation of a medicament for the prevention and/or treatment of african swine fever virus.

Based on the technical scheme, the invention has the following beneficial effects:

the invention discovers that 2 new monoclonal antibodies can be specifically combined with CD2v for the first time, and the affinity reaches 10nM level. Further experiments show that the monoclonal antibody provided by the invention can recognize a single site, has stronger specificity, and can be used for detecting and treating African swine fever viruses.

Drawings

FIG. 1 shows the results of SDS-PAGE detection of the purity of eukaryotic expression CD2V-N protein, FIG. 1A shows the glycosylated modified CD2V protein, and FIG. 1B shows the non-glycosylated CD2V protein.

FIG. 2 shows the results of ELISA testing the binding properties of monoclonal antibodies to CD2 v-N.

FIG. 3 is the experimental result of the molecular interaction system for detecting the affinity of the monoclonal antibody and the CD2v-N antigen.

FIG. 4 shows the result of Western Blot to detect the binding of monoclonal antibody to 293T cell-expressed full-length CD2v-HA protein.

FIG. 5 shows the results of indirect immunofluorescence assay to detect the binding of monoclonal antibodies to CD2v-GFP protein expressed by 293T cells.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

All materials, reagents and the like in the following examples are commercially available unless otherwise specified.

Example 1 eukaryotic expression and purification of CD2v-N fusion proteins

1. Construction of expression vector for CD2v-N protein

According to the GenBank, QGM12912.2 CD2v protein sequence, signal peptide, CD2v protein extracellular region sequence (amino acid sequence is shown as SEQ ID NO. 17) and 6 XHis label are cloned into pCDNA3.1 eukaryotic expression vector to construct eukaryotic recombinant expression pCDNA3.1-CD2v-N-His, pCD2v-N-His vector for short. Putting 293T cells in an Opti-MEM complete medium, culturing at 37 ℃ for 2h, adding pCD2v-N-His 1 mu g into 200 mu L jet-pRIME buffer, vortexing for 5s, placing in a super clean bench for 10min, adding 293T cells, and gently mixing; at 6h after transfection, the transfection complex was replaced with Opti-MEM medium, and the cells were incubated at 37 ℃ for another 72-96h, and cell supernatants were collected.

2. Eukaryotic expression and purification of CD2v-N fusion protein

(1) The culture supernatant of the expressed CD2v protein collected above was centrifuged at 12000r/min for 15min. The supernatant was filtered through a 0.45 μm filter head and placed in an ice box for further use.

(2) Sucking 2mL of Ni-NTA filler by a liquid shifter, adding the Ni-NTA filler to the installed protein purification column, and carefully adding an upper gasket after the Ni filler is settled; the control valve was opened to allow 20% ethanol to flow out and the column was continued to be washed with 10mL of deionized water.

(3) The flow rate was adjusted and the column was washed with 10mL and above volumes of equilibration buffer at a rate of 1 mL/min.

(4) Dripping the cell culture supernatant filtered by the filter membrane into the well balanced column in batches, 5mL each time, adjusting the control valve to ensure that the flow rate does not exceed 1mL/min, quickly collecting the filtrate and repeatedly loading the filtrate for 2-3 times, collecting the filtrate of the last time, and standing at-20 ℃ for later use.

(5) The column was washed with washing buffer to remove contaminating proteins.

(6) Adding 10mL of elution buffer solution into the column to elute the target protein; reducing the flow rate; collecting the eluted solution into an EP tube, wherein each tube contains 1mL of the eluted solution, measuring the concentration of protein in each tube by using an enzyme-labeling instrument, marking the protein, and standing the protein at the temperature of minus 20 ℃ for later use.

(7) 5 XLoading Buffer (with or without mercaptoethanol) was added to the cell culture supernatant before purification and the transudate, eluate and eluate collected after purification, and boiled for 10min, and the results of purification were analyzed by 12% SDS-PAGE.

The CD2v-N protein obtained was eluted and analyzed by SDS-PAGE under reducing (reducing) and Non-reducing (Non-reducing) conditions. The results showed a diffuse band of interest at around 55-110kDa (FIG. 1A).

(8) The purified CD2v-N protein was deglycosylated under denaturing conditions with the glycosidase PNGase F (NEB BioLabs, # P0711S) or Endo H (NEB BioLabs, # P0702S) (see tables 1-3 for reaction systems). SDS-PAGE analysis showed that the CD2v protein had varying degrees of glycosylation, and after desugaring, the molecular weight was significantly reduced and the band was single (FIG. 1B). The dispersed bands at 55-110kDa are CD2v protein and have higher purity.

a. Deglycosylation modification (PNGase F enzyme)

TABLE 1 protein deglycosylation reaction System

Incubate at 50 ℃ for 10min.

b. Deglycosylation modification (Endo H enzyme)

TABLE 2 protein denaturation reaction System

Heating and reacting for 10min at 100 ℃;

table 3 protein desugarization reaction system:

incubate at 37 ℃ for 1h.

EXAMPLE 2 screening and preparation of monoclonal antibodies

1. CD2v-N protein immune Balb/c mouse

First immunization: 0.25mL Freund's complete adjuvant and 100. Mu.g CD2v-N protein (0.25 mL) were emulsified in water-in-oil and injected subcutaneously in the back of Balb/c mice at five points, 0.1 mL/mouse, time: 1 month.

And (3) boosting immunity: 0.25mL of Freund's incomplete adjuvant and 100. Mu.g of CD2v-N protein (0.25 mL) were emulsified in water-in-oil and injected subcutaneously in the back of Balb/c mice in five spots, 0.1 mL/mouse, time: and 15 days.

Second booster immunization: 0.25mL Freund's incomplete adjuvant and 100. Mu.g CD2v-N protein (0.25 mL) were emulsified in water-in-oil and injected subcutaneously into the back of Balb/c mice at five points, 0.1 mL/mouse, time: and 15 days.

And (3) determining the serum titer of Balb/c mice:

coating: diluting the antigen with coating solution, coating transversely, adding 100 μ L antigen into each well, and coating overnight at 4 deg.C; washing the plate: washing the plate with a full-automatic plate washing machine, wherein the washing liquid for 3 times is 1 xPBST; and (3) sealing: adding 200 μ L of 4% skimmed milk prepared with PBS into each well, sealing, and sealing at 37 deg.C for 1 hr; washing the plate: washing the plate for 3 times by using a full-automatic plate washing machine, wherein the washing liquid is 1 multiplied by PBST; sample adding: diluting the serum sample, vertically adding the diluted serum sample, adding 100 mu L of the diluted serum sample into each hole, adding 100 mu L of PBS into a blank control hole, and incubating for 1h at 37 ℃; washing the plate: washing the plate for 3 times by using a full-automatic plate washing machine, wherein the washing liquid is 1 multiplied by PBST; secondary antibody: the secondary antibody (coat anti-mouse IgG-HRP, # A16072) was diluted with PBS at 1; washing the plate: washing the plate for 5 times with a full-automatic plate washing machine, wherein the washing liquid is 1 × PBST; color development: preparing a TMB color developing solution, adding 100 mu L of the TMB color developing solution into each hole, and developing for 90s at room temperature in a dark place; and (4) terminating: the wells were stopped by adding 100. Mu.L of stop buffer and the absorbance at OD450 of each well was measured using a microplate reader.

2. Hybridoma cell fusion

And (3) boosting immunity: balb/c mice were boosted three days prior to fusion and were injected intraperitoneally with 0.5mL of 100. Mu.g/CD 2v-N protein.

Preparing trophoblast cells: removing neck, killing normal Balb/c mouse, soaking skin in 75% alcohol for sterilization, aseptically peeling skin, extracting 6mL 1640 culture solution with syringe, injecting into mouse abdominal cavity, repeatedly washing, sucking out abdominal cavity washing solution, counting, and adjusting cell concentration to 2 × 10 ⁵ Per mL, addition to 96-well plate, 0.1 mL/well, 37 ℃,5.0% CO ₂ The incubator is ready for use.

50% PEG preparation: taking a 10mL test tube, weighing 0.7g of PEG, dissolving in 0.7mL of serum-free 1640 culture solution, covering, sealing with white adhesive cloth, inserting a syringe needle, and placing into a beaker with 100 mL. Boiling with electromagnetic oven, timing for 30min, removing needle, decocting for 2min, and standing at room temperature.

Cell fusion:

taking an immunized Balb/c mouse, picking eyeballs and bleeding (taking serum to be reserved as a positive control), removing the neck and killing the mouse, soaking the skin in 75% alcohol for disinfection, aseptically taking the spleen, squeezing out spleen cells, adding 20mL 1640 culture solution into a plate, blowing away spleen cell masses through a 10mL suction tube, transferring cell suspension to a 50mL centrifuge tube, and washing once.

Taking myeloma cells in another centrifuge tube, centrifuging at 1200rpm/min for 4min, discarding supernatant, adding 40mL 1640 culture solution respectively for counting, and adjusting cell proportion according to counting results: splenocytes myeloma cells = 6: 1.

After mixing the two cells, centrifugation was carried out at 1200rpm/min for 4min to discard the supernatant, the cells at the bottom of the tube were flicked off, the tube was placed in a 37 ℃ water bath, 50% PEG 1mL was slowly added over 1min (gentle stirring while adding), the tube was left to stand 40s, 1mL 1640 culture solution was added over 1min (gentle stirring while adding), and then, 5mL 1640 culture solution was added over 2min and 10mL 1640 culture solution was added over 2min in the same manner. Centrifuging the cell suspension at 800rpm/min for 5min, discarding supernatant, gently flicking the tube bottom cells, adding 20% FBS-containing 1640 culture solution, mixing, and adding into 96-well plate (2 × 10) with trophoblast ⁶ seed/mL), at 37 ℃,5.0% CO ₂ Culturing in an incubator.

Liquid changing: the selection was carried out on day 8 by changing the medium at 20% FBS 1640 medium + HT once a day and 3 times a day before selection.

3. Screening of antigen-positive cells

Taking the supernatant of the monoclonal cell strain, screening positive clones by an ELISA method, and measuring the specific operation steps and the serum titer. The positive clones screened in the first round are transferred into a 24-pore plate for culture, and the positive clones are selected for subcloning after retesting.

4. Hybridoma cell cloning

Preparing trophoblast cells: the procedure is as in the "cell fusion" experiment to prepare trophoblast cells.

And (3) repeatedly and uniformly mixing the hybridoma cells to be cloned in the 24-well plate by using a sample injector, and then taking a small number of cell suspensions to another sterile test tube for accurate counting. 200 cells were suspended in 10mL of culture medium (about 2 cells/0.1 mL) and seeded into 96-well plates at 0.1mL per well for 1 block. 100 cells were suspended in 10mL of culture medium (about 1 cell/0.1 mL) and seeded into 96-well plates at 0.1mL per well for 2 blocks. The plates were incubated at 37 ℃ 5.5% CO ₂ Cultivation in incubators, 5Cell clones were observed under a microscope around day. And (3) determining a monoclonal cell strain, replacing liquid at proper time, detecting, and transferring the positive monoclonal cell strain into a 24-pore plate for expanded culture. And repeating the operation until the cloning rate reaches 100 percent, namely cloning.

Cell liquid change principle:

15 days, 20% of FBS 1640 culture medium + HAT

15 days, 20% of FBS 1640 culture broth + HT

Thereafter, 10% of FBS 1640 culture medium.

5. Mass production of monoclonal antibodies

Balb/c mice were injected intraperitoneally with 0.5mL paraffin oil and used one week after injection.

Hybridoma cells in logarithmic growth phase were harvested, washed twice with physiological saline and centrifuged at 1500rpm/min for 5min. Trypan blue staining counting, preparing 3X 10 with normal saline again ⁶ Cell suspension per mL.

Balb/c mice injected with paraffin oil were inoculated with hybridoma cells, 0.5mL per intraperitoneal injection. The abdomen of the mouse is obviously enlarged 7-12 days after inoculation, and the ascites can be extracted from the abdominal cavity at the moment. The extracted ascites is centrifuged at 3000rpm/min for 30min, and the supernatant is collected and purified by a Protein A chromatographic column to obtain the antibody for the subsequent experiment.

Example 3 identification of candidate antibody subtypes

Coating: the capture antibody (250X) using coating buffer diluted to 1X, ELISA plate each hole with 100 u L for coating, 4 degrees C coated overnight; washing the plate: washing the plate for 3 times by using a full-automatic plate washing machine, wherein the washing liquid is 1 multiplied by PBST; and (3) sealing: adding 250 mu L of blocking buffer solution into each hole, and blocking for 2h at room temperature; washing the plate: washing the plate for 3 times by using a full-automatic plate washing machine, wherein the washing liquid is 1 multiplied by PBST; sample adding: adding 50 mu L of analysis buffer solution into each hole, then adding 50 mu L of sample, and incubating for 1h at room temperature; washing the plate: washing the plate for 3 times by using a full-automatic plate washing machine, wherein the washing liquid is 1 multiplied by PBST; secondary antibody: diluting the detection antibody (250 ×) to 1 ×, adding 100 μ L detection antibody per well with assay buffer and incubating for 1h at room temperature; washing the plate: washing the plate for 5 times by using a full-automatic plate washing machine, wherein the washing liquid is 1 multiplied by PBST; color development: adding 100 mu L of color development liquid into each hole, and incubating for 15min at room temperature; and (4) terminating: adding 100 mu L of stop solution into each wellLine stop, assay of each well at OD with microplate reader ₄₅₀ The absorbance value of (c).

The subtypes of the two candidate antibodies were determined using a mouse antibody subtype identification kit (sequoyifei, # a 38550). The results show that: the heavy chains of 7B1 and 11H7 are IgG1 class antibodies, and the light chains are of the kappa subtype.

Example 4 binding assay of candidate antibodies 7B1 and 11H7 to antigen

Coating: diluting the antigen to 2 mug/mL by the coating solution, adding 100 muL of diluted antigen into each hole of the enzyme-linked plate, and coating overnight at 4 ℃;

washing the plate: washing the plate for 3 times by using a full-automatic plate washing machine, wherein the washing liquid is 1 multiplied by PBST;

and (3) sealing: adding 200 μ L of 4% skimmed milk prepared with PBS into each well, sealing, and sealing at 37 deg.C for 1 hr;

sample adding: diluting the sample with PBS, adding 100 μ L of sample into each well, adding 100 μ L of PBS into blank control wells, and incubating for 1h at 37 ℃; washing the plate for 3 times;

secondary antibody: adding 100 mu L/well diluted PBS into the enzyme-linked plate, and incubating for 40min at 37 ℃;

washing the plate: washing the plate for 5 times by using a full-automatic plate washing machine, wherein the washing liquid is 1 multiplied by PBST;

color development: adding 100 mu L of TMB into each hole of the prepared TMB color development solution, and developing for 1min in a dark place at room temperature;

and (4) terminating: adding 100 μ L of stop solution into each well for termination, and detecting the OD of each well with enzyme-labeling instrument ₄₅₀ The absorbance of (b).

The binding properties of monoclonal antibodies to the antigen CD2v are shown in figure 2:

and detecting the binding property of the candidate antibodies 7B1 and 11H7 and the African swine fever virus CD2v protein by ELISA. The results show that both 7B1 and 11H7 antibodies recognize and bind to CD2v and have a concentration-dependent, EC ₅₀ The values are shown in Table 4.

TABLE 4 detection of binding Capacity of candidate antibodies to CD2v (EC) ₅₀ Value)

Example 5 analysis of 7B1 and 11H7 antibody specificity by ELISA assay

The literature shows that CD2V and CD2 molecules are highly homologous in structure, so that the inventors select hCD2 molecules and detect the binding condition of two monoclonal antibodies obtained by screening and the CD2 molecules.

Coated human hCD2 (His-tagged) protein (purchased from Beijing Yinqiao Shenzhou, inc. # 10982-H08H) at 2. Mu.g/mL, 7B1 and 11H7 were added at 0.1. Mu.g/mL, respectively, and the secondary antibody was purified using a Goat anti-mouse IgG-HRP (Saimer fly, # A16072) (1. The results in table 5 show that the two monoclonal antibodies only bind specifically to the CD2V molecule, but not to the hCD2 molecule, indicating that the antibodies screened have better specificity.

TABLE 5ELISA analysis of binding of mAbs 7B1 and 11H7 to hCD2 (OD) ₄₅₀ )

Example 6 determination of affinity of candidate antibodies 7B1, 11H7 Using an Octet molecular interaction System

Setting a sample plate distribution, wherein the concentration of the candidate antibody is 200nM, and the antigen concentration is diluted from 800nM to 12.5nM in a multiple ratio;

the program was set up as shown in table 6:

TABLE 6 setup procedure

The type and position of the probe are selected and the program is initiated for analysis.

The affinity assay results for the monoclonal antibody and the antigen CD2v are shown in fig. 3:

the affinity of the candidate antibodies 7B1 and 11H7 was determined using the Octet molecular interaction system from ForteBio. The results show that: the KD value of 7B1 was 3.27X 10 ^-08 KD value of M,11H7 is 7.59X 10 ^-08 M is shown in Table 7.

TABLE 7 kinetic constants for binding of 7B1, 11H7 to CD2v proteins respectively

Example 7 Western blot assay of the monoclonal antibody for expression of CD2v-HA protein in 293T cells

Cell plating: laying 1X 10 in six-hole plate ⁵ Each cell/well, and transfection is carried out when the cells grow to about 70% on the next day;

transfection: mixing 2 mu g of pCDNA3.1-CD2v-HA plasmid with 200 mu L of jetPRIME buffer, carrying out vortex oscillation for 10s, adding 4 mu L of jetPRIME reagent, carrying out vortex oscillation for 1s, standing at room temperature for 10min, adding the mixture into a six-hole plate, and changing the liquid after 4h; experimental grouping: blank control group (no plasmid transfected), control group (plasmid transfected), deglycosylated modified group (plasmid transfected +10 μ g/mL tunicamycin treatment 24h sigma, # T7765.

Collecting a protein sample: collecting cells 36h after transfection, adding 150 μ l RAPI cell lysate into each well, lysing for 10min on ice, then centrifuging for 15min at 12000rpm and 4 ℃, taking supernatant, placing the supernatant into a clean 1.5mL EP tube, quantifying protein by using a Bradford method, and adjusting the concentration of the protein sample;

SDS-PAGE and transfer: adding 4 Xprotein loading buffer, boiling water bath for 10min, loading 25 μ L of sample to each hole, and performing 120V electrophoresis for 1h; proteins were transferred to PVDF membranes.

And (3) sealing: sealing with 5% skimmed milk prepared by 1 × TBST at room temperature for 1 hr;

primary anti-incubation: 7B1, 11H7 and mouse anti-HA-tag antibody (purchased from MBL company, # M132-3) were diluted to 1. Mu.g/mL with 5% skim milk prepared with 1 XTSST, respectively, overnight at 4 ℃,1 XTSST washed PVDF membrane for 10min, and repeated 3 times;

and (3) secondary antibody incubation: the secondary antibody (coat anti-mouse Ig G-HRP, # A16072) was diluted with 5% skim milk in 1 XTSSL at a ratio of 1;

and (3) developing: ECL (from GE, # RPN 2106) was developed and the developer was ready to use.

The Western blot results of the CD2v protein expressed by the monoclonal antibody and 293T cells are shown in FIG. 4:

to further confirm the function of the candidate antibodies 7B1 and 11H7, CD2v-HA fusion protein plasmids were transfected by 293T cells for transient expression, then tunicamycin (Sigma, # T7765) was used to inhibit glycosylation modification, and cell lysates were collected for Western blot. The results show that the antibodies 7B1 and 11H7 can not recognize the 43kDa deglycosylated modified CD2v protein, but can well recognize the 95kDa glycosylation modified CD2v full-length protein.

Example 8 immunofluorescence assay of CD2v-GFP protein expression by 293T cells

1. Construction of CD2v-GFP vector

The fragments of interest were PCR amplified as shown in Table 8:

TABLE 8 amplification System

PCR program, as shown in table 9:

TABLE 9 amplification procedure

Agarose gel electrophoresis: the heavy and light chain fragments amplified by PCR were subjected to DNA gel electrophoresis at 200V,30min. The agarose gel was placed in an ultraviolet analyzer for gel cutting.

And (3) glue recovery: the gel was cut and placed in a 2mL EP tube, 700. Mu.L of the sol was added, water bath at 56 ℃ was carried out, and 300. Mu.L of isopropanol was added after the gel was dissolved. The solution is fully mixed evenly and added into an adsorption column by two times, and the solution is centrifuged at 12000rpm for 30s each time to discard waste liquid. Adding 600 μ L PE column washing twice, discarding waste liquid, air-separating at 12000rpm for 2min, standing at room temperature for 1min, and air-drying the excessive PE. The column was placed in a clean 1.5mL EP tube, 40. Mu.L EB was added, and the column was allowed to stand at room temperature for 2min, centrifuged at 12000rpm for 30s and eluted.

Enzyme digestion, as shown in table 10:

TABLE 10 enzyme digestion System

Agarose gel electrophoresis and recovery of DNA fragments.

Ligation, as shown in table 11:

TABLE 11 connection system

Ligation was carried out overnight at 16 ℃.

And (3) transformation: the competence was thawed on ice, 10. Mu.L of the ligation product was placed in 50. Mu.L of competence, and allowed to stand on ice for 30min. Placing the competence in a water bath at 42 ℃ for heat shock for 45s, immediately taking out the competence after the heat shock is finished, placing the competence on ice for cooling for 2min, adding 700 mu L of non-resistant 2YT culture medium, and placing the mixture in a shaking table at 37 ℃ for 220r/min for culturing for 50min. After the culture is finished, centrifuging at 5000rpm for 1min, discarding 400 mu L of culture medium, uniformly mixing, coating in a kanamycin-added solid 2YT culture plate, and culturing overnight at 37 ℃ in an incubator.

Bacterial liquid PCR: taking a bacterial plate which is cultured overnight, picking 10 single bacterial colonies and placing the single bacterial colonies in 1mL kanamycin-containing 2YT culture medium for shaking bacteria for 4h. 100 mu L of bacterial liquid is taken, and is centrifuged for 1min in 100 ℃ boiling water bath and 10min,5000rmp, and the supernatant is taken as a template.

The PCR system is shown in Table 12; the PCR procedure is shown in Table 13.

TABLE 12PCR System

TABLE 13PCR procedure

Taking positive clone for enzyme digestion identification.

2. Indirect immunofluorescence

Cell plating: spreading 1X 10 in a confocal small dish ⁵ Each cell/well, and transfection is carried out when the cells grow to about 70% on the next day; after 36h of cell transfection, the cell culture supernatant was aspirated and washed once with physiological saline, and fixed with 4% paraformaldehyde at 4 ℃ for 24h; 4% paraformaldehyde fixing solution in the pipette was blotted with 2mL 50nM NH ₄ The cells were washed three times with Cl for 5min each time; permeabilization: permeabilization at room temperature for 20min with 1mL of permeabilization solution (1% BSA +0.1% The Triton X-100/PBS buffer); and (3) sealing: pipetting, adding 100. Mu.L of blocking buffer (5% goat serum +0.1% Triton X-100/PBS buffer) to the middle part of the glass-bottomed dish, and incubating at room temperature for 1h; primary antibody incubation: diluting the monoclonal antibody with an antibody diluent to 1 mu g/mL, completely sucking the confining liquid, adding 100 mu L primary antibody in the middle of a glass bottom dish, and incubating overnight at 4 ℃; 0.1% by volume of Triton in 2mL ^TM Washing with X-100 PBS buffer solution for three times, each time for 5min; and (3) secondary antibody incubation: fluorescent secondary antibody (F (ab') ₂ -coat anti-Mo use IgG (H + L), alexa Fluor 594, purchased from Samerafei, # A-11020) was diluted as described, 100. Mu.L of fluorescent secondary antibody was added to the middle part of the glass plate, and incubated for 1H at room temperature in the dark; 0.1% of Triton in 2mL ^TM Washing with PB S buffer solution X-100 for 5min three times; two drops of anti-quenching blocking agent containing DAPI were added dropwise and observed directly under a fluorescent microscope.

The results of immunofluorescence experiments with monoclonal antibodies 7B1, 11H7 binding to CD2v-GFP protein expressed by 293T cells are shown in FIG. 5:

in order to further confirm the function of the candidate antibody, a CD2v-EGFP plasmid is constructed, 293T cells are used for transiently expressing CD2v-GFP protein, and immunofluorescence detection is carried out by using 7B1 and 11H7 monoclonal antibodies respectively. The results show that: the green fluorescence carried by the CD2v-GFP protein can be well co-localized with red fluorescence emitted by the combination of the candidate monoclonal antibody and the secondary antibody, and the results show that the 7B1 and 11H7 monoclonal antibodies can recognize the CD2v-GFP protein expressed by 293T.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

SEQUENCE LISTING

<110> military medical research institute of military science institute of people's liberation force of China

<120> anti-CD 2v protein monoclonal antibody and application thereof

<130> P220013

<160> 17

<170> PatentIn version 3.5

<210> 1

<211> 114

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 1

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Val Val Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Arg Pro Asp Gln Gly Leu Glu Trp Ile

35 40 45

Gly Arg Ile Asn Pro Tyr Asp Ser Glu Thr His Tyr Arg Gln Lys Phe

50 55 60

Lys Asp Lys Ala Ile Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Thr Val Tyr Tyr Cys

85 90 95

Ala Thr Ala Phe Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val

100 105 110

Ser Ser

<210> 2

<211> 342

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 2

caggtccaac tgcagcagcc tggggctgag gtggtgaggc ctggggcctc agtgaagctg 60

tcctgcaagg cttctggcta cacgttcacc agttactgga tgaactgggt tagacagagg 120

cctgaccaag gccttgagtg gattggaagg attaatcctt acgatagtga aactcactac 180

aggcagaagt tcaaggacaa ggccatattg actgtagaca aatcctccag cacagcctac 240

atgcaactca gcagcctgac atctgaggac tctacggtct attattgtgc aaccgcgttt 300

tttgactact ggggccaagg caccactctc acagtctcct ca 342

<210> 3

<211> 112

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 3

Asp Val Val Met Thr Gln Thr Pro Leu Thr Leu Ser Val Ile Ile Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser

20 25 30

Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser

35 40 45

Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro

50 55 60

Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Trp Gln Gly

85 90 95

Thr His Phe Pro Gln Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 4

<211> 336

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 4

gatgttgtga tgacccagac tccactcact ttgtcggtta tcattggaca accagcctcc 60

atctcgtgca agtcaagtca gagcctctta gatagtgatg gaaagacata tttgaattgg 120

ttgttacaga ggccaggcca gtctccaaag cgcctaatct atctggtgtc taaactggac 180

tctggagtcc ctgacaggtt cactggcagt ggatcaggga cggatttcac actgaaaatc 240

agcagagtgg aggcggagga tttgggagtt tattattgct ggcaaggtac acattttcct 300

cagacgttcg gtggaggcac caagctggaa atcaaa 336

<210> 5

<211> 115

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 5

Asp Val Gln Leu Gln Glu Ser Gly Pro Asp Leu Val Lys Pro Ser Gln

1 5 10 15

Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Gly

20 25 30

Tyr Ser Trp His Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp

35 40 45

Met Gly Tyr Met Leu Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe

65 70 75 80

Leu Gln Leu Ile Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Arg Leu Gly Gly Val Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr

100 105 110

Val Ser Ser

115

<210> 6

<211> 345

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 6

gatgtgcagc ttcaggagtc aggacctgac ctggtgaaac cttctcagtc actttcactc 60

acctgcactg tcactggcta ctccatcacc agtggttata gctggcactg gatccggcaa 120

tttccaggaa acaaactgga atggatgggc tatatgctct acagtggtag cactaactac 180

aacccatctc tcaaaagtcg aatctctatc actcgagaca catccaagaa ccagttcttc 240

ctgcagttga tttctgtgac tactgaggac acagccacat attactgtgc aagactgggg 300

ggggtggact actggggtca aggaacctca gtcaccgtct cctca 345

<210> 7

<211> 106

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 7

Gln Ile Val Leu Thr Gln Ser Pro Ala Leu Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Thr Tyr Met

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Arg Ser Ser Pro Lys Pro Trp Ile Tyr

35 40 45

Leu Thr Ser Asn Leu Thr Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Ser Asn Pro Phe Thr

85 90 95

Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 8

<211> 318

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 8

caaattgttc tcacccagtc tccagcactc atgtctgcat ctccagggga gaaggtcacc 60

atgacctgca gtgccagctc aagtgttact tacatgtact ggtaccaaca gaagccaaga 120

tcctccccca aaccctggat ttatctcaca tccaacctga cttctggagt ccctgctcgc 180

ttcagtggca gtgggtctgg gacctcttac tctctcacaa tcagcagcat ggaggctgaa 240

gatgctgcca cttattactg ccagcagtgg actagtaacc cattcacgtt cggctcgggg 300

acaaagttgg aaataaaa 318

<210> 9

<211> 19

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 9

Met Gly Trp Ser Tyr Ile Ile Leu Phe Leu Leu Ala Thr Ala Thr Cys

1 5 10 15

Val His Ser

<210> 10

<211> 57

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 10

atgggatgga gctatatcat cctcttcttg ttagcaacag ctacatgtgt ccactcc 57

<210> 11

<211> 19

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 11

Met Ser Pro Ala Gln Phe Leu Phe Leu Leu Val Leu Trp Ile Arg Glu

1 5 10 15

Ser Asn Gly

<210> 12

<211> 57

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 12

atgagtcctg cccagttcct gtttctgtta gtgctctgga ttcgggaaag caacggt 57

<210> 13

<211> 18

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 13

Met Arg Val Leu Ile Leu Leu Cys Leu Leu Thr Ala Phe Pro Gly Ile

1 5 10 15

Leu Ser

<210> 14

<211> 54

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 14

atgagagtgc tgattctttt gtgcctgctc acagcctttc ctggtatcct gtct 54

<210> 15

<211> 22

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 15

Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Met Ser Ala Ser

1 5 10 15

Val Ile Met Ser Arg Gly

20

<210> 16

<211> 66

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 16

atggattttc aagtgcagat tttcagcttc ctgctaatga gtgcctcagt cataatgtcc 60

agggga 66

<210> 17

<211> 191

<212> PRT

<213> CD2v

<400> 17

Ile Asp Tyr Trp Val Ser Phe Asn Lys Thr Ile Ile Leu Asp Ser Asn

1 5 10 15

Ile Thr Asn Asp Asn Asn Asp Ile Asn Gly Val Ser Trp Asn Phe Phe

20 25 30

Asn Asn Ser Phe Asn Thr Leu Ala Thr Cys Gly Lys Ala Gly Asn Phe

35 40 45

Cys Glu Cys Ser Asn Tyr Ser Thr Ser Ile Tyr Asn Ile Thr Asn Asn

50 55 60

Cys Ser Leu Thr Ile Phe Pro His Asn Asp Val Phe Asp Thr Thr Tyr

65 70 75 80

Gln Val Val Trp Asn Gln Ile Ile Asn Tyr Thr Ile Lys Leu Leu Thr

85 90 95

Pro Ala Thr Pro Pro Asn Ile Thr Tyr Asn Cys Thr Asn Phe Leu Ile

100 105 110

Thr Cys Lys Lys Asn Asn Gly Thr Asn Thr Asn Ile Tyr Leu Asn Ile

115 120 125

Asn Asp Thr Phe Val Lys Tyr Thr Asn Glu Ser Ile Leu Glu Tyr Asn

130 135 140

Trp Asn Asn Ser Asn Ile Asn Asn Phe Thr Ala Thr Cys Ile Ile Asn

145 150 155 160

Asn Thr Ile Ser Thr Ser Asn Glu Thr Thr Leu Ile Asn Cys Thr Tyr

165 170 175

Leu Thr Leu Ser Ser Asn Tyr Phe Tyr Thr Phe Phe Lys Leu Tyr

180 185 190

Claims

1. An anti-CD 2v protein monoclonal antibody, wherein said antibody is selected from the group consisting of 7B1 or 11H7 antibodies:

2. The monoclonal antibody of claim 1, wherein the 7B1 antibody further comprises heavy chain variable region FR1, FR2, FR3 and FR4 framework regions and light chain variable region FR1, FR2, FR3 and FR4 framework regions; the amino acid sequences of the heavy chain variable regions FR1, FR2, FR3 and FR4 are respectively shown as amino acid sequences at 1-30, 36-49, 67-98 and 104-114 in SEQ ID NO. 1; the amino acid sequences of the light chain variable regions FR1, FR2, FR3 and FR4 are respectively shown as amino acid sequences at positions 1-23, 40-54, 62-93 and 103-112 in SEQ ID NO. 3;

the 11H7 antibody further comprises heavy chain variable region FR1, FR2, FR3 and FR4 framework regions and light chain variable region FR1, FR2, FR3 and FR4 framework regions; the amino acid sequences of FR1, FR2, FR3 and FR4 of the heavy chain variable region are respectively shown as the amino acid sequences at 1-30, 37-50, 67-98 and 105-115 sites in SEQ ID NO. 5; the amino acid sequences of the light chain variable regions FR1, FR2, FR3 and FR4 are respectively shown as amino acid sequences at 1-23, 34-48, 56-87 and 97-106 positions in SEQ ID NO. 7.

3. The monoclonal antibody of claim 2, wherein the heavy chain variable region of the 7B1 antibody has the amino acid sequence shown in SEQ ID No.1 and the light chain variable region of the 7B1 antibody has the amino acid sequence shown in SEQ ID No. 3;

4. A nucleic acid molecule encoding the monoclonal antibody of any one of claims 1-3.

5. An expression vector comprising the nucleic acid molecule of claim 4.

6. The expression vector of claim 5, further comprising a nucleotide sequence for a signal peptide linked to the light or heavy chain of the monoclonal antibody.

7. A cell comprising the expression vector of claim 6.

8. A test strip or reagent or kit comprising a monoclonal antibody according to any one of claims 1 to 3.

9. A pharmaceutical composition comprising a monoclonal antibody according to any one of claims 1 to 3, or comprising a nucleic acid molecule according to claim 4, or an expression vector according to claim 5.

10. Use in any one of:

(1) Use of a monoclonal antibody according to any one of claims 1 to 3 in the preparation of a reagent for the detection of CD2v protein;

(2) Use of a monoclonal antibody according to any one of claims 1-3 in the manufacture of a kit for the detection or diagnosis of African swine fever virus;

(3) Use of the monoclonal antibody according to any one of claims 1 to 3 for the isolation, purification or detection of the CD2v protein of african swine fever virus for non-diagnostic therapeutic purposes.

11. The use of claim 10, wherein the kit is an african swine fever virus antibody enzyme-linked immunization kit.