CN111333725B - Monoclonal antibody YG11-2 for resisting staphylococcus aureus enterotoxin B and application thereof - Google Patents

Abstract

The invention discloses a monoclonal antibody YG11-2 against Staphylococcus aureus enterotoxin B and its application. The present invention protects an IgG antibody (named as YG11-2 antibody), which is composed of a light chain and a heavy chain; the heavy chain variable region in the heavy chain is defined by Sequence 1 of the sequence listing from the 21-145th amino acid at the N-terminus. residue composition; the light chain variable region in the light chain is composed of amino acid residues at positions 21-128 from the N-terminus of Sequence 3 of the sequence listing. The present invention also protects the application of the YG11-2 antibody in the preparation of a drug for treating enterotoxin B poisoning, in the preparation of a drug for antagonizing enterotoxin B, and in the preparation of a drug for preventing and/or treating Staphylococcus aureus-induced The use of medicines for diseases. The invention has important application prospects for treating enterotoxin B poisoning and preventing or treating diseases caused by Staphylococcus aureus.

Description

Monoclonal antibody YG11-2 for resisting staphylococcus aureus enterotoxin B and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a monoclonal antibody YG11-2 for resisting staphylococcus aureus enterotoxin B and application thereof, in particular to application of the monoclonal antibody in preparing a medicament for preventing and/or treating diseases caused by staphylococcus aureus.

Background

Staphylococcus aureus (Staphylococcus aureus) is called Staphylococcus aureus for short, and is a gram-positive pathogenic bacterium widely distributed in nature. It is estimated that 20-30% of the population carries this pathogen, which is mainly present in the mucosa, skin, and particularly the nasopharynx of the human body. Staphylococcus aureus can cause a range of diseases, from mild skin infections, to scalded skin syndrome, to abscesses, to life-threatening pneumonia, meningitis, endocarditis, osteomyelitis, toxic shock syndrome, and the like. Staphylococcus aureus infections affect a wide range of conditions involving the skin, soft tissues, respiratory tract, bone marrow, joints, blood vessels, etc.

Enterotoxins (SEs) are bacterial exotoxins secreted by staphylococci, and at present, more than twenty serotypes are reported, such as SEA, SEB, SEC1-3, SED, SEE, SEF, SEG and the like, are collectively called staphylococcus SE superantigens, and are important toxins for bacterial food poisoning, pyogenic infection and nosocomial infection. Enterotoxin b (seb), a toxin that is the most toxic of SEs, can cause food poisoning, and if directly entered the blood circulation, can activate a large number of T cells to release cytokines (IFN- γ, TNF- α, etc.), even causing death of the patient.

Enterotoxin B as a superantigen can activate T cells by binding specific TCR V beta region and MHC-II molecules on the T cells, directly activate T lymphocytes and release a series of cytokines, mainly including IFN-gamma, TNF-alpha and the like, thereby causing hyperpyrexia, nausea, diarrhea and Toxin Shock Syndrome (TSS). The mortality rate of TSS caused by Staphylococcus aureus enterotoxin B can reach 50%, and the mortality rate of SEB infection concurrent with influenza is higher than 90%, so SEB is listed as a conventionally stored "standard" ("classical") biological warfare agent by U.S. special warfare departments. At present, no effective vaccine and medicament aiming at SEB poisoning exists clinically.

Disclosure of Invention

The invention aims to provide a monoclonal antibody YG11-2 for resisting staphylococcus aureus enterotoxin B and application thereof.

The invention firstly protects an IgG antibody (named YG11-2 antibody) which consists of a light chain and a heavy chain; the heavy chain variable region in the heavy chain consists of 21 st to 145 th amino acid residues from the tail end of N of a sequence 1 in a sequence table; the light chain variable region in the light chain consists of 21 th-128 th amino acid residues from the N tail end of a sequence 3 in a sequence table.

The heavy chain is (a) or (b) as follows: (a) protein consisting of 21 st-475 th amino acid residues from the N tail end of a sequence 1 in a sequence table; (b) a protein shown in a sequence 1 in a sequence table.

The light chain is (c) or (d) as follows: (c) protein consisting of 21 st to 235 th amino acid residues from the tail end of N in a sequence 3 of a sequence table; (d) a protein shown in sequence 3 of the sequence table.

The gene encoding YG11-2 antibody also falls within the scope of the present invention.

The gene for coding the heavy chain is a DNA molecule shown by nucleotides 681-2108 from the 5' end of the sequence 2 of the sequence table. The gene for coding the light chain is a DNA molecule shown by nucleotides 681-1388 from the 5' end of the sequence 4 of the sequence table.

The invention also protects the application of the YG11-2 antibody in preparing medicines for treating diseases caused by enterotoxin B.

The invention also provides the application of the YG11-2 antibody in preparing a medicament for antagonizing enterotoxin B.

The invention also protects the application of the YG11-2 antibody in preparing a medicament for neutralizing enterotoxin B.

The invention also discloses application of the YG11-2 antibody in preparing a medicament for inhibiting staphylococcus aureus.

The invention also discloses application of the YG11-2 antibody in preparing a medicament for preventing and/or treating diseases caused by staphylococcus aureus.

The invention also provides a medicament, the active ingredient of which is YG11-2 antibody.

The application of the medicine is (a) or (b) or (c) or (d) or (e) as follows:

(a) treating enterotoxin B-induced diseases;

(b) antagonizing enterotoxin B;

(c) neutralizing enterotoxin B;

(d) inhibiting staphylococcus aureus;

(e) preventing and/or treating diseases caused by staphylococcus aureus.

Any of the enterotoxins B may specifically be staphylococcus aureus enterotoxin B.

Any enterotoxin B is a protein shown in a sequence 5 in a sequence table.

Any one of the enterotoxins B is a protein which sequentially consists of the following two sections from the N end to the C end: a segment shown in sequence 7 of the sequence table, and a segment shown in sequence 5 of the sequence table.

The disease caused by enterotoxin B may be lethal shock syndrome (SEBILS) caused by enterotoxin B.

The disease caused by any staphylococcus aureus can be specifically a disease caused by enterotoxin B secreted by staphylococcus aureus.

The disease caused by any staphylococcus aureus can be specifically lethal shock syndrome (SEBILS) caused by enterotoxin B secreted by staphylococcus aureus.

The invention takes enterotoxin B as a target antigen, and quickly screens out a fully human monoclonal antibody of the enterotoxin B from a phage antibody library. The monoclonal antibody is a brand-new antibody, has higher affinity, inhibits the hemolytic activity of enterotoxin B, and can improve the survival rate of test animals infected with staphylococcus aureus. The antibody provided by the invention has important application value for prevention and control of staphylococcus aureus.

Drawings

FIG. 1 is an SDS-PAGE electrophoresis of His-SEB protein solution.

FIG. 2 is a graph showing the results of the promotion of lymphocyte proliferation by His-SEB protein.

FIG. 3 is a graph showing the results of a mouse lethal experiment.

FIG. 4 is a polyacrylamide gel electrophoresis image of YG11-2 antibody solution.

FIG. 5 is a graph showing the results of the detection of the binding activity of YG11-2 antibody by ELISA.

FIG. 6 is a graph showing the results of BIAcore assay for the affinity of YG11-2 antibody.

FIG. 7 is a graph showing the results of verifying the function of YG11-2 antibody in a mouse lethal model.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged. Unless otherwise specified, all PBS buffers in the examples were PBS buffer of 0.2M at pH 7.4. Lymphocyte separation solution: tianjin is tertiary. CFSE (fluorescent dye): invitrogen. PHA (phytohemagglutinin): sigma. D-galactosamine (D-GlaN): sigma.

The staphylococcus aureus enterotoxin B is also called SEB protein, and is shown as a sequence 5 in a sequence table, and a coding sequence of the staphylococcus aureus enterotoxin B is shown as a sequence 6 in the sequence table.

Example 1 preparation of His-SEB protein and functional study thereof

Preparation of His-SEB protein

1. The DNA molecule shown in the sequence 6 of the sequence table is used for replacing a small fragment between NdeI and XhoI enzyme cutting sites of a vector pET28a (+), so as to obtain a recombinant plasmid pET28 a-SEB. Sequencing verification is carried out.

In the recombinant plasmid pET28a-SEB, a DNA molecule shown in the sequence 6 of the sequence table and a DNA molecule coding His tag on a vector skeleton are fused to form a fusion gene, and a fusion protein is expressed.

The fusion gene is composed of the following two segments from upstream to downstream in sequence: DNA molecules shown in a sequence 8 of the sequence table and DNA molecules shown in a sequence 6 of the sequence table.

The fusion protein is composed of the following two segments from N end to C end in sequence: a segment shown in sequence 7 of the sequence table, and a segment shown in sequence 5 of the sequence table. The fusion protein is also called His-SEB protein.

2. The recombinant plasmid pET28a-SEB was introduced into Escherichia coli BL21(DE3) to obtain a recombinant strain, which was designated E.coli BL21(pET28 a-SEB).

3. Coli BL21(pET28a-SEB) was inoculated into a liquid LB medium containing 50. mu.g/mL kanamycin, and cultured at 37 ℃ with shaking at 200rpm until OD_600nmThe concentration was 0.6, IPTG was added thereto to 0.05mmol/L, and the mixture was cultured at 37 ℃ for 5 hours with shaking at 200rpm, and then centrifuged at 4 ℃ for 15min at 8000rpm, and the pellet was collected.

4. And (3) taking the thallus precipitate obtained in the step (3), carrying out heavy suspension by using a binding buffer solution, carrying out ultrasonic crushing on an ice bath, centrifuging at 8000rpm for 10min at 4 ℃, and collecting a supernatant.

Binding buffer (ph 7.4): containing 20mmol/L Na₃PO₄0.5mol/L NaCl, 20mmol/L imidazole and the balance of water.

5. Taking the supernatant obtained in the step 4, and using Ni²⁺And (5) purifying by using an affinity column. And (3) purification process: after loading, the column is washed with binding buffer to remove contaminating proteins, and then eluted with an eluent to collect the protein of interest (post-column collection solution).

Eluent (ph 7.4): containing 20mmol/L Na₃PO₄0.5mol/L NaCl and 500mmol/L imidazole, and the balance being water.

6. And (4) transferring the solution after column chromatography obtained in the step (5) into a dialysis bag, then putting the dialysis bag into PBS buffer solution for dialysis, and then collecting the liquid phase in the dialysis bag, namely the His-SEB protein solution.

7. And (3) detecting the protein concentration of the His-SEB protein solution by using a BCA protein quantitative method. The protein concentration of the His-SEB protein solution is 1 mg/ml.

8. The SDS-PAGE electrophoresis of the His-SEB protein solution is shown in figure 1. In FIG. 1, the left lane is marker and the right lane is His-SEB protein solution.

II, His-SEB protein for promoting lymphocyte proliferation

1. Preparation of lymphocytes

(1) Taking fresh healthy people for resisting blood coagulation, and diluting with PBS buffer solution with the same volume to obtain blood diluent.

(2) Two 15mL centrifuge tubes were taken, 3mL of lymphocyte separation medium was added to each tube, 5mL of blood dilution was added to each tube, 500g of the mixture was centrifuged for 20min, and the lymphocyte layer was aspirated and washed 2 times with PBS buffer.

(3) CFSE-labeled cells

Resuspend lymphocytes with PBSA solution and count to give 1X 10⁶Individual cells/mL of cell suspension; CFSE was added to the cell suspension to a concentration of 2. mu.M, and the cells were stained at 37 ℃ for 10min in the dark, then 1/4 volumes of precooled calf serum were added and incubated on ice for 5min, and then centrifuged at 1000rpm for 10min, and the cell pellet was collected and washed 2 times with PBS buffer.

PBSA solution: PBS buffer containing 1g/100ml BSA.

2. His-SEB protein for promoting lymphocyte proliferation

Resuspending the cells obtained in step 1 in 1640 medium to obtain 5X 10⁵Individual cells/mL of cell suspension; and (3) taking a 24-well plate, adding 1mL of cell suspension into each well, then adding a His-SEB protein solution (the concentration of the His-SEB protein in the system is 1 mu g/mL), culturing for 7 days, then collecting cells, washing for 3 times by using PBS buffer solution, and detecting by using a flow cytometer. A negative control treatment with an equal volume of PBS buffer instead of His-SEB protein solution was set. Positive control treatment was set by replacing the His-SEB protein solution with an equal volume of PHA solution (to give a PHA concentration of 1. mu.g/ml in the system).

The results are shown in FIG. 2. The result shows that the His-SEB protein can obviously stimulate the proliferation of lymphocytes.

Third, lethal experiment in mice

4 test protein solutions: diluting the His-SEB protein solution with PBS buffer solution to obtain protein solutions with the protein concentrations of 75 mug/mL, 50 mug/mL or 25 mug/mL respectively; PBS buffer was used as 0-concentration protein solution.

Sensitizer solution: d-galactosamine was prepared into a solution of 50mg/mL using PBS buffer.

BALB/c female mice, 6-8 weeks old, were randomly divided into 4 groups of 8 mice each.

A first group: injecting sensitizer solution (0.4 mL/mouse) into abdominal cavity, injecting 0 concentration protein solution (0.4 mL/mouse) into abdominal cavity after 30min, observing death condition of mice in each group, and counting survival rate every 12 hr;

second group: injecting sensitizer solution (0.4 mL/mouse) into abdominal cavity, injecting 25 μ g/mL protein solution (0.4 mL/mouse) into abdominal cavity after 30min, observing death condition of mice in each group, and counting survival rate every 12 hr;

third group: injecting sensitizer solution (0.4 mL/mouse) into abdominal cavity, injecting 50 μ g/mL protein solution (0.4 mL/mouse) into abdominal cavity after 30min, observing death condition of mice in each group, and counting survival rate every 12 hr;

and a fourth group: the sensitization solution (0.4 mL/mouse) is injected into the abdominal cavity, 75 mug/mL protein solution (0.4 mL/mouse) is injected into the abdominal cavity after 30min, the death condition of each group of mice is observed, and the survival rate is counted every 12 hours.

The survival results are shown in FIG. 3. The results indicate that His-SEB protein can cause lethal shock syndrome (SEB induced residual shock SEBILS), resulting in death of mice. The effect of the His-SEB protein is caused by the SEB protein, and the effect of the His tag is only purification in the preparation process of the protein.

Example 2 screening of monoclonal antibodies against SEB protein by phage antibody library

Biological panning of anti-SEB monoclonal antibody

SEB group

1. First round affinity panning

(1) The immune tubes were coated with 500. mu.L of His-SEB protein solution (protein concentration adjusted with PBS buffer) at a protein concentration of 10. mu.g/mL, overnight at 4 ℃.

(2) And (3) taking the immune tube, adding PBST buffer solution containing 5g/100mL of skimmed milk powder, and sealing at room temperature for 1 h.

(3) The phage antibody library was added with PBST buffer containing 5g/100mL skim milk powder and blocked at room temperature for 1 h.

(4) Taking the immune tube which completes the step (2), adding sterile PBS buffer solution for washing for 3 times, and then adding the phage antibody library which completes the step (3) (the input amount of phage is about 1.2 multiplied by 10)¹²) And standing at room temperature for 1 h.

(5) After the step (4) is completed, the immune tube is taken, a proper amount of sterile PBS buffer solution is added for washing (in order to wash away the unbound phage), 500 mu L of HCl-Glycine with pH2.2 and 0.1M is added for eluting the phase-Abs, the eluent is collected, and 1.5M Tris-HCl (pH8.8) is added for adjusting the pH value to 7.4.

(6) Inoculating Escherichia coli TG1 to LB liquid culture medium, and performing shaking culture at 37 deg.C and 200rpm to logarithmic phase to obtain culture solution.

(7) Mixing 500 μ L of the eluate obtained in step (5) with 10mL of the culture broth obtained in step (6), standing at 37 deg.C for 30min (for infection), centrifuging at 4000rpm for 15min, collecting the thallus, uniformly spreading on a 2YTAG plate, and culturing at 37 deg.C overnight.

(8) And (4) after the step (7) is completed, scraping the colony on the 2YTAG plate, then inoculating the colony in a 2YTAG culture medium for phage display, and precipitating by using PEG/NaCl to obtain the phage. The phage is obtained after one round of panning.

2. Second round of affinity panning

Replacing the phage antibody library obtained in the step 1 (3) with the phage obtained in the step 1 (8), and obtaining the phage without changing other steps. The phage is obtained after two rounds of panning.

3. Third round of affinity panning

And (3) replacing the phage antibody library obtained in the step (3) with the phage obtained in the step (2), and obtaining the phage without changing other steps. The phage is obtained after three rounds of panning.

(II) control group

According to the SEB group three rounds of affinity panning, His-SEB protein solution was replaced by PBS buffer solution, and other steps were not changed, so as to obtain the corresponding phage (as a control).

The number of phage in each round of affinity panning is shown in table 1. The results show that after three rounds of panning, the number of recombinant phages obtained from the SEB group is obviously increased, while the number of phages in the control group is not changed greatly. This indicates that phage binding to SEB was significantly enriched.

TABLE 1

Number of panning rounds	His-SEB protein (mu g/mL)	Amount of phage input	Phage output of SEB group	Control phage output
					One round of elutriation	10	1.2×1012	1.6×104	5.6×103
Two rounds of panning	10	2.5×1012	3.6×104	1.2×103
					Three-wheel elutriation	10	3.2×1012	5.5×106	2.4×103

Screening of anti-SEB monoclonal antibody positive clone

1. After the first step is completed, a plurality of clones obtained after three rounds of panning of the SEB group are respectively taken and inoculated in 1mL of 2YTAG culture medium, and shaking culture is carried out at 37 ℃ and 220rpm overnight, so as to obtain culture bacterial liquid.

2. Inoculating 30 μ L of culture bacterial liquid into 900 μ L of 2YTAG culture medium, performing shake culture at 37 deg.C and 220rpm to OD_600nmThe value reaches 0.6-0.8, then 5X 10 is added¹⁰The helper phage M13KO7 was allowed to stand at 37 ℃ for 30 min.

3. After completion of step 2, centrifugation at 4000rpm for 15min at 4 ℃ was performed, the pellet was collected and resuspended in 1mL of 2YTAK, incubated overnight at 220rpm at 28 ℃.

4. After step 3 was completed, the positive clones were identified by phase-ELISA and sequenced.

The results showed that the ELISA signal values of 79 clones out of 176 clones showed positive, with a positive rate of 81.25%. The 12 clones with higher ELISA signal values were selected and sequenced to obtain 1 antibody sequence, which was named YG11-2 antibody.

The YG11-2 antibody is IgG, the heavy chain is shown in sequence 1 of the sequence table (the 21 st to 145 th amino acid residues in the sequence 1 constitute the heavy chain variable region), and the light chain is shown in sequence 3 of the sequence table (the 21 st to 128 th amino acid residues in the sequence 3 constitute the light chain variable region).

Example 3 functional characterization of YG11-2 antibody

Preparation of the YG11-2 antibody

1. Construction of recombinant plasmid

The DNA molecule shown in the sequence 2 of the sequence table is used for replacing a small fragment between Hind III and XhoI enzyme cutting sites in the vector pCDNA3.1(+), so as to obtain the heavy chain expression vector. In the sequence 2 of the sequence table, the 1 st-584 th nucleotide constitutes a promoter, the 681 nd and 2108 th nucleotides encode the full-length heavy chain, and the 2141 nd and 2196 th nucleotides constitute a terminator.

The small fragment between Hind III and XhoI enzyme cutting sites in the vector pCDNA3.1(+) is replaced by the DNA molecule shown in the sequence 4 of the sequence table to obtain the light chain expression vector. In the sequence 4 of the sequence table, the nucleotides 1-584 constitute a promoter, the nucleotides 681-1388 encode a full-length light chain, and the nucleotides 1421-1476 constitute a terminator.

2. Preparation of antibodies

(1) The heavy chain expression vector and the light chain expression vector were mixed equimolar, 293T cells were CO-transfected with liposome 2000 (as described in the specification), followed by high glucose DMEM medium (Gibco) at 37 ℃ with 5% CO₂Cultured for 3 days under the condition, and the supernatant was collected.

(2) And (2) taking the supernatant obtained in the step (1), purifying protein A, and collecting a purified product.

(3) And (3) taking the purified product obtained in the step (2), performing ultrafiltration concentration, changing the solution, and changing the system into a PBS buffer solution to obtain the YG11-2 antibody solution.

The YG11-2 antibody solution was assayed for antibody concentration using A280nm UV absorption. The antibody concentration in the YG11-2 antibody solution was 2 mg/ml.

The polyacrylamide gel electrophoresis pattern of the YG11-2 antibody solution is shown in FIG. 4. In FIG. 4, A is reductive electrophoresis and B is non-reductive electrophoresis.

Second, ELISA detection of binding Activity of YG11-2 antibody

1. The plate was then labeled with a coating solution (100. mu.l/well) and incubated overnight at 4 ℃.

The coating solution consists of a coating source and a coating buffer solution, and the concentration of the coating source in the coating solution is 10 mug/mL. The coating was the His-SEB protein prepared in example 1. Coating buffer (ph 9.6): na (Na)₂CO₃ 1.59g、NaHCO₃2.94g and the balance of water.

2. After completion of step 1, the microplate was removed and washed three times with PBST buffer.

3. After step 2 is completed, the ELISA plate is taken out, PBST buffer solution containing 5g/100mL of skimmed milk powder is added, and the plate is blocked for 1h at 37 ℃.

4. Taking the YG11-2 antibody solution prepared in the first step, preparing a mother solution with an antibody concentration of 10 μ g/mL by using a PBS buffer solution, and then performing three-time gradient dilution (total 10 dilution gradients) by using the PBS buffer solution to obtain YG11-2 antibody solutions with different concentrations.

5. The plate after completion of step 3 was added with PBS buffer (as a control) or YG11-2 antibody solution (100. mu.L per well) at different concentrations obtained in step 4, and incubated at 37 ℃ for 1 h. 3 multiple wells were set for each concentration.

6. After completion of step 5, the microplate was removed and washed three times with PBST buffer (250. mu.L per well).

7. After completion of step 6, the microplate was taken, and a dilution of a goat anti-human IgG secondary HRP-labeled antibody (the goat anti-human IgG secondary HRP-labeled antibody was diluted 1:40000 in PBST buffer containing 5g/100mL skim milk powder) was added thereto, followed by incubation at 37 ℃ for 30 min.

8. After step 7, the microplate is removed, and a chromogenic reagent (100. mu.L per well) is added for development at room temperature for 5 min. The color reagent is a component in the TMB color development kit.

9. After the step 8 is finished, taking the ELISA plate, and adding 10% H₂SO₄The solution was stopped from developing (50. mu.L per well) and then the OD at 450nm was measured.

The results are shown in FIG. 5 (R)²0.9896). The results show that the YG11-2 antibody can be combined with His-SEB protein, and the combination activity is higher, and the EC50 value is 46.25 ng/mL.

Three, BIAcore determination of affinity of YG11-2 antibody

The method for measuring the affinity of the antibody by adopting a capture method comprises the following specific steps: will resist human F_CThe fragmented antibodies were coupled to the surface of the chip CM5, and the YG11-2 antibody solution prepared in step one was diluted to an antibody concentration of 0.5. mu.g/mL, ensuring that about 100RU of the antibody was captured by the anti-human Fc antibody. The affinity of the antibody was determined by running a series of concentration gradients (concentrations 33nM, 13.2nM, 5.3nM, 2.1nM and 0.85nM, respectively) over the stationary phase surface through the His-SEB protein.

The results are shown in FIG. 6 and Table 2. The result showed that the affinity of the YG11-2 antibody was 21.35 nM.

TABLE 2

Kon(1/MS)	Koff(1/S)	KD
			6.324E+4	0.001350	2.135E-8

Fourth, the function of YG11-2 antibody was verified by mouse lethal model

Test solution 1: mu.l of His-SEB protein solution (His-SEB protein solution prepared in example 1, protein concentration adjusted with PBS buffer) was mixed with 200. mu.l of YG11-2 antibody solution (YG 11-2 antibody solution prepared in step one, antibody concentration adjusted with PBS buffer) to obtain test solution 1. 200. mu.l of His-SEB protein solution contained 20. mu.g of His-SEB protein. 200 μ l of YG11-2 antibody solution contained 200 μ g of YG11-2 antibody.

Test solution 2: mu.l of His-SEB protein solution (His-SEB protein solution prepared in example 1, protein concentration adjusted with PBS buffer) was mixed with 200. mu.l of YG11-2 antibody solution (YG 11-2 antibody solution prepared in step one, antibody concentration adjusted with PBS buffer) to obtain sample solution 2. 200. mu.l of His-SEB protein solution contained 20. mu.g of His-SEB protein. 200 μ l of YG11-2 antibody solution contained 100 μ g of YG11-2 antibody.

Test solution 3: mu.l of His-SEB protein solution (His-SEB protein solution prepared in example 1, protein concentration adjusted with PBS buffer) was mixed with 200. mu.l of YG11-2 antibody solution (YG 11-2 antibody solution prepared in step one, antibody concentration adjusted with PBS buffer) to obtain test solution 3. 200. mu.l of His-SEB protein solution contained 20. mu.g of His-SEB protein. 200 μ l of YG11-2 antibody solution contained 50 μ g of YG11-2 antibody.

Test solution 4: mu.l of His-SEB protein solution (His-SEB protein solution prepared in example 1, protein concentration adjusted with PBS buffer) was mixed with 200. mu.l of control IgG solution (control IgG, product of ZDR-5001, manufactured by Beijing Zhonghua Kinghua Biotech Co., Ltd., antibody concentration adjusted with PBS buffer) to obtain sample solution 4. 200. mu.l of His-SEB protein solution contained 20. mu.g of His-SEB protein. 200 μ g of control IgG was contained in 200 μ l of control IgG solution.

Sensitizer solution: d-galactosamine was prepared into a solution of 50mg/mL using PBS buffer.

BALB/c female mice, 6-8 weeks old, were randomly divided into 4 groups of 8 mice each.

A first group: injecting sensitizer solution (0.4 mL/mouse) into abdominal cavity, injecting test solution 1(0.4 mL/mouse) into abdominal cavity after 30min, observing death condition of mice in each group, and counting survival rate every 24 hours;

second group: injecting sensitizer solution (0.4 mL/mouse) into abdominal cavity, injecting test solution 2(0.4 mL/mouse) into abdominal cavity after 30min, observing death condition of mice in each group, and counting survival rate every 24 hours;

third group: injecting sensitizer solution (0.4 mL/mouse) into abdominal cavity, injecting test solution 3(0.4 mL/mouse) into abdominal cavity after 30min, observing death condition of mice in each group, and counting survival rate every 24 hours;

and a fourth group: the sensitization solution (0.4 mL/mouse) is injected into the abdominal cavity, the test solution 4(0.4 mL/mouse) is injected into the abdominal cavity after 30min, the death condition of each group of mice is observed, and the survival rate is counted every 24 hours.

The results are shown in FIG. 7. The results show that after 3 days, the survival rate of the first group of mice is 100%, the survival rate of the second group of mice is 12.5%, and the survival rates of the third group and the fourth group of mice are both 0. The YG11-2 antibody can effectively neutralize His-SEB protein and improve the survival rate of mice.

SEQUENCE LISTING

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

<120> monoclonal antibody YG11-2 for resisting staphylococcus aureus enterotoxin B and application thereof

<130> GNCYX182095

<160> 8

<170> PatentIn version 3.5

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<213> Homo sapiens

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Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

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Gly Ser Thr Gly Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val

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Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser

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Leu Ser Asp His Ala Val His Trp Val Arg Gln Ala Pro Gly Lys Gly

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Leu Glu Trp Val Ala Leu Ile Ser Tyr Asp Gly Ser Asp Lys Tyr Tyr

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Asn Thr Leu Tyr Leu Gln Met Thr Ser Leu Lys Pro Glu Asp Thr Ala

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Val Tyr Tyr Cys Ala Arg Gln Gly Gly Asn Lys Asn Ile Asn Tyr Phe

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Phe Gln Tyr Leu Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser

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Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser

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Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp

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Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr

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Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr

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Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln

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Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp

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Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro

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Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro

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Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

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Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn

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Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

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Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

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Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

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Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

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Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu

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Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

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Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

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Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

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Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

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Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

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<213> Homo sapiens

<400> 2

gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 60

catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 120

acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 180

ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 240

aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 300

ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 360

tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc 420

ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt 480

ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa 540

tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag 600

aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctg 660

gctagggatc cgccgccacc atggagaccg acaccctgct gctctgggtg ctgctgctct 720

gggtgcccgg gtccaccggt caggtgcagc tggtggagtc tgggggaggc gtggtccagc 780

ctgggaggtc cctgagactc tcctgtgcag cctctggatt cagcctcagc gaccatgctg 840

tccactgggt ccgccaggct ccaggcaagg ggctggagtg ggtggctctt atctcatatg 900

atggaagtga taaatactac aaagactccg tgaagggccg gttcaccatc tccagagaca 960

atcccaagaa cacgctgtat ctgcagatga ccagtctgaa acctgaggac acggctgttt 1020

attactgtgc gagacaaggt gggaacaaaa acataaacta cttcttccag tacctggacg 1080

tctggggcca agggacaatg gtcaccgtct cttcagctag caccaagggc ccatcggtct 1140

tccccctggc accctcctcc aagagcacct ctgggggcac agcggccctg ggctgcctgg 1200

tcaaggacta cttccccgaa cccgtgacgg tgtcgtggaa ctcaggcgcc ctgaccagcg 1260

gcgtgcacac cttcccggct gtcctacagt cctcaggact ctactccctc agcagcgtgg 1320

tgaccgtgcc ctccagcagc ttgggcaccc agacctacat ctgcaacgtg aatcacaagc 1380

ccagcaacac caaggtggac aagagagttg agcccaaatc ttgtgacaaa actcacacat 1440

gcccaccgtg cccagcacct gaactcctgg ggggaccgtc agtcttcctc ttccccccaa 1500

aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg gtggtggacg 1560

tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg gaggtgcata 1620

atgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg gtcagcgtcc 1680

tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag gtctccaaca 1740

aagccctccc agcccccatc gagaaaacca tctccaaagc caaagggcag ccccgagaac 1800

cacaggtgta caccctgccc ccatcccggg aggagatgac caagaaccag gtcagcctga 1860

cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag agcaatgggc 1920

agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc tccttcttcc 1980

tctatagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc ttctcatgct 2040

ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc ctgtccccgg 2100

gtaaatagat ccaaagatcc cccgacctcg acctctggct aataaaggaa atttattttc 2160

attgcaatag tgtgttggaa ttttttgtgt ctctca 2196

<210> 3

<211> 235

<212> PRT

<213> Homo sapiens

<400> 3

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Thr Thr Leu Thr Gln Ser Pro Gly Thr Leu Ser

20 25 30

Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser

35 40 45

Val Ser Ser Asn Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

50 55 60

Arg Leu Leu Ile Tyr Gly Ala Ser Thr Arg Ala Ala Gly Ile Pro Asp

65 70 75 80

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

85 90 95

Ser Leu Gln Ser Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Ile Asn

100 105 110

Asn Trp Pro Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

115 120 125

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

130 135 140

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

145 150 155 160

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

165 170 175

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

180 185 190

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

195 200 205

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

210 215 220

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210> 4

<211> 1476

<212> DNA

<213> Homo sapiens

<400> 4

gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 60

catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 120

acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 180

ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 240

aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 300

ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 360

tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc 420

ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt 480

ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa 540

tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag 600

aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctg 660

gctagggatc cgccgccacc atggagaccg acaccctgct gctctgggtg ctgctgctct 720

gggtgcccgg gtccaccggt gaaacgacac tcacgcagtc tccaggcacc ctgtctttgt 780

ctccagggga aagagccacc ctctcctgca gggccagtca gagtgtcagc agcaacgtag 840

cctggtacca gcagaaacct ggccaggctc ccaggctcct catctatggt gcatccacca 900

gggccgctgg tatcccagac aggttcagtg gcagtgggtc tgggacagac ttcactctca 960

ccatcagcag cctgcagtct gaagattttg cagtttattt ctgtcagcag attaataact 1020

ggcctccgct cactttcggc caagggacca agctcgagat caaacgtacg gtggctgcac 1080

catctgtctt catcttcccg ccatctgatg agcagttgaa atctggaact gccagcgttg 1140

tgtgcctgct gaataacttc tatcccagag aggccaaagt acagtggaag gtggataacg 1200

ccctccaatc gggtaactcc caggagagtg tcacagagca ggacagcaag gacagcacct 1260

acagcctcag cagcaccctg acgctgagca aagcagacta cgagaaacac aaagtctacg 1320

cctgcgaagt cacccatcag ggcctgagct cgcccgtcac aaagagcttc aacaggggag 1380

agtgttagat ccaaagatcc cccgacctcg acctctggct aataaaggaa atttattttc 1440

attgcaatag tgtgttggaa ttttttgtgt ctctca 1476

<210> 5

<211> 239

<212> PRT

<213> Artificial sequence

<400> 5

Glu Ser Gln Pro Asp Pro Lys Pro Asp Glu Leu His Lys Ser Ser Lys

1 5 10 15

Phe Thr Gly Leu Met Glu Asn Met Lys Val Leu Tyr Asp Asp Asn His

20 25 30

Val Ser Ala Ile Asn Val Lys Ser Ile Asp Gln Phe Leu Tyr Phe Asp

35 40 45

Leu Ile Tyr Ser Ile Lys Asp Thr Lys Leu Gly Asn Tyr Asp Asn Val

50 55 60

Arg Val Glu Phe Lys Asn Lys Asp Leu Ala Asp Lys Tyr Lys Asp Lys

65 70 75 80

Tyr Val Asp Val Phe Gly Ala Asn Tyr Tyr Tyr Gln Cys Tyr Phe Ser

85 90 95

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

100 105 110

Cys Met Tyr Gly Gly Val Thr Glu His Asn Gly Asn Gln Leu Asp Lys

115 120 125

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

130 135 140

Ser Phe Asp Val Gln Thr Asn Lys Lys Lys Val Thr Ala Gln Glu Leu

145 150 155 160

Asp Tyr Leu Thr Arg His Tyr Leu Val Lys Asn Lys Lys Leu Tyr Glu

165 170 175

Phe Asn Asn Ser Pro Tyr Glu Thr Gly Tyr Ile Lys Phe Ile Glu Asn

180 185 190

Glu Asn Ser Phe Trp Tyr Asp Met Met Pro Ala Pro Gly Asp Lys Phe

195 200 205

Asp Gln Ser Lys Tyr Leu Met Met Tyr Asn Asp Asn Lys Met Val Asp

210 215 220

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

225 230 235

<210> 6

<211> 720

<212> DNA

<213> Artificial sequence

<400> 6

gagagtcaac cagatcctaa accagatgag ttgcacaaat cgagtaaatt cactggtttg 60

atggaaaata tgaaagtttt gtatgatgat aatcatgtat cagcaataaa cgttaaatct 120

atagatcaat ttctatactt tgacttaata tattctatta aggacactaa gttagggaat 180

tatgataatg ttcgagtcga atttaaaaac aaagatttag ctgataaata caaagataaa 240

tacgtagatg tgtttggagc taattattat tatcaatgtt atttttctaa aaaaacgaat 300

gatattaatt cgcatcaaac tgacaaacga aaaacttgta tgtatggtgg tgtaactgag 360

cataatggaa accaattaga taaatataga agtattactg ttcgggtatt tgaagatggt 420

aaaaatttat tatcttttga cgtacaaact aataagaaaa aggtgactgc tcaagaatta 480

gattacctaa ctcgtcacta tttggtgaaa aataaaaaac tctatgaatt taacaactcg 540

ccttatgaaa cgggatatat taaatttata gaaaatgaga atagcttttg gtatgacatg 600

atgcctgcac caggagataa atttgaccaa tctaaatatt taatgatgta caatgacaat 660

aaaatggttg attctaaaga tgtgaagatt gaagtttatc ttacgacaaa gaaaaagtga 720

<210> 7

<211> 21

<212> PRT

<213> Artificial sequence

<400> 7

Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro

1 5 10 15

Arg Gly Ser His Met

20

<210> 8

<211> 63

<212> DNA

<213> Artificial sequence

<400> 8

atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60

atg 63

Claims (7)

1. An IgG antibody against staphylococcal enterotoxin B consists of a light chain and a heavy chain; the amino acid sequence of the heavy chain is shown as a sequence 1 in a sequence table; the amino acid sequence of the light chain is shown as a sequence 3 in a sequence table.

2. A gene encoding the IgG antibody of claim 1.

3. The gene of claim 2, wherein: the nucleotide sequence of the gene for coding the heavy chain is shown as the 681-2108 th nucleotide from the 5' end of the sequence 2 in the sequence table; the nucleotide sequence of the gene for coding the light chain is shown as the 681-1388 th nucleotide from the 5' end of the sequence 4 in the sequence table.

4. Use of an IgG antibody according to claim 1 for the preparation of a medicament for antagonizing enterotoxin B.

5. Use of the IgG antibody of claim 1 in the manufacture of a medicament for neutralizing enterotoxin B.

6. Use of the IgG antibody of claim 1 in the manufacture of a medicament for inhibiting staphylococcus aureus.

7. A medicament, the active ingredient of which is the IgG antibody of claim 1;

the application of the medicine is (b) or (c) or (d) as follows:

(b) antagonizing enterotoxin B;

(c) neutralizing enterotoxin B;

(d) inhibiting Staphylococcus aureus.

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