Brucella bacillus bacA protein purification and preparation and application of corresponding monoclonal antibody

Abstract

The present application relates to the field of monoclonal antibody preparation technology and biological detection technology, in particular to a kind of brucella BacA protein purification and corresponding monoclonal antibody preparation and application.The brucella BacA protein is purified in the present application, and a positive hybridoma cell strain is obtained by hybridoma cell technology, and is named 1A7.The heavy chain variable region of the monoclonal antibody 1A7 includes CDR1 with the amino acid sequence GYTLARRGMQCV, CDR2 with the amino acid sequence IDGCDSYISG and CDR3 with the amino acid sequence AREGLLR.The light chain variable region of the monoclonal antibody 1A7 includes CDR1 with the amino acid sequence QDVGNAVAWYQQ, CDR2 with the amino acid sequence WASTRHTGVP and CDR3 with the amino acid sequence QQYSSS.A competitive ELSA detection method is established by using the monoclonal antibody, and the monoclonal antibody can specifically react with swine, bovine and ovine brucella;It does not specifically react with escherichia coli, salmonella and yersinia enterocolitica, which provides support for the detection technology development of brucellosis in China.

Description

Technical Field

[0001] This invention relates to the fields of monoclonal antibody preparation technology and biodetection technology, specifically to the purification of Brucella BacA protein and the preparation and application of corresponding monoclonal antibodies. Background Technology

[0002] Brucellosis is a zoonotic disease caused by Brucella bacteria, characterized by abortion and fever. The most prominent symptom after infection is undulant fever, also known as Mediterranean remittent fever or madali's fever. Currently, the World Organisation for Animal Health (WOAH) lists brucellosis as a notifiable animal disease.

[0003] Brucella is classified into six classic species: *Brucella melitensis*, *Brucella abortus*, *Brucella suis*, *Brucella canis*, *Brucella ovis*, and *Brucella neotomae*. The main pathogenic strains to humans are *Brucella melitensis*, *Brucella bovis*, *Brucella suis*, and *Brucella canis*. *Brucella melitensis*, *Brucella bovis*, *Brucella suis*, and *Brucella canis* are smooth-strained strains, highly pathogenic to humans, and are the main pathogens of brucellosis in livestock, receiving widespread attention. *Brucella canis*, however, is a rough-strained strain with weak pathogenicity to humans, and has been the subject of relatively few studies and reports on it for a long time.

[0004] The outer membrane of Brucella consists of three parts: outer membrane proteins (OMPs), lipopolysaccharide (LPS), and phospholipids. Most antibodies produced after Brucella infection target LPS. Brucella LPS is composed of lipid A, a core oligosaccharide, and an O chain, and is classified into smooth and rough types based on the presence or absence of the O chain. The protein encoded by the Brucella bacA gene is called BacA protein. BacA protein is widely distributed on the Brucella cell membrane and is a cytoplasmic membrane transport protein. It is an important virulence factor of Brucella, mainly involved in bacterial survival and infection processes. During LPS synthesis, BacA participates in the generation of unusual ultra-long-chain fatty acids (C28) in lipid A biosynthesis, affecting its specific modification.

[0005] Currently, serological testing is the primary method for detecting brucellosis in animals, including rose benzene plate agglutination assay, tube agglutination assay, and enzyme-linked immunosorbent assay (ELISA). However, because Brucella lipopolysaccharide (LPS) shares the same antigenic epitope with LPS from other Gram-negative bacteria, serological testing can produce false positive results. Therefore, the preparation of specific monoclonal antibodies against Brucella BacA and the establishment of detection methods based on these specific monoclonal antibodies are crucial for brucellosis detection. Summary of the Invention

[0006] The purpose of this invention is to provide the purification of Brucella BacA protein and the preparation and application of corresponding monoclonal antibodies, thereby improving the accuracy of Brucella detection. The monoclonal antibody against Brucella BacA prepared by this invention has the advantages of good specificity and strong affinity, providing a solid foundation for the development of serological detection technology for Brucella in my country.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] This invention provides a monoclonal antibody 1A7 against Brucella BacA protein. The heavy chain variable region of monoclonal antibody 1A7 includes CDR1 with the amino acid sequence GYTLARRGMQCV, CDR2 with the amino acid sequence IDGCDSYISG, and CDR3 with the amino acid sequence AREGLLR. The light chain variable region of monoclonal antibody 1A7 includes CDR1 with the amino acid sequence QDVGNAVAWYQQ, CDR2 with the amino acid sequence WASTRHTGVP, and CDR3 with the amino acid sequence QQYSSS.

[0009] Furthermore, the amino acid sequence of the heavy chain variable region of the monoclonal antibody 1A7 is shown in SEQ ID NO: 2; the amino acid sequence of the light chain variable region of the monoclonal antibody 1A7 is shown in SEQ ID NO: 4.

[0010] The present invention also provides a nucleic acid molecule comprising a nucleic acid sequence capable of encoding a monoclonal antibody against Brucella BacA protein, wherein the nucleic acid sequence comprises amino acid sequences CDR1, CDR2, and CDR3, which are respectively heavy chain variable regions; and amino acid sequences CDR1, CDR2, and CDR3, which are respectively light chain variable regions.

[0011] The present invention also includes a carrier comprising the aforementioned nucleic acid molecule.

[0012] The present invention also provides a host cell, wherein the host cell comprises the aforementioned nucleic acid molecule or the aforementioned vector.

[0013] Furthermore, the monoclonal antibody 1A7 can produce specific immune responses to Brucella suis, Brucella bovis, and Brucella ovis, but does not produce specific responses to Escherichia coli, Salmonella, or Yersinia enterocolitica.

[0014] Furthermore, the heavy chain of the monoclonal antibody 1A7 is IgG2b, and the light chain is Kappa.

[0015] The present invention also provides an application of the monoclonal antibody in the Brucella antibody competitive ELISA detection method.

[0016] Furthermore, the Brucella antibody competitive ELISA detection method specifically includes: diluting Brucella BacA protein to 0.5 μg / mL, coating an ELISA plate at 100 μl / well, incubating at 4°C for 15 hours, discarding the solution in the wells, and patting dry; adding 200 μl of 5% BSA as blocking solution to each well, incubating at 37°C for 1 hour, discarding the solution in the wells, and patting dry; adding 1:5 diluted serum for testing and 1:200 diluted monoclonal antibody. Add 50 μl of cloning antibody to each well and incubate at 37°C for 45 minutes. Discard the liquid in the wells and blot dry. Add 100 μl of 1:5000 diluted enzyme-labeled secondary antibody to each well and incubate at 37°C for 30 minutes. Discard the liquid in the wells and wash the plate 5 times with washing buffer (300 μl per well). After the last wash, blot dry on absorbent paper. Add 100 μl of substrate chromogenic solution to each well and incubate in the dark for 20 minutes. Add 50 μl of stop solution to each well and measure the OD of each well using a microplate reader. 450nm value.

[0017] Furthermore, the criteria for determining the effectiveness of the Brucella competitive ELISA antibody detection method are as follows:

[0018] When the inhibition rate (PI) of the serum sample to be tested is ≥60%, the Brucella antibody test result is judged as positive;

[0019] When the inhibition rate (PI) of the serum sample to be tested is <60%, the Brucella antibody test result is judged as negative.

[0020] This invention also provides a method for expressing and purifying BacA protein, specifically including: (1) using the genomic DNA of Brucella suis as a template, PCR amplification of the target gene fragment to construct the recombinant plasmid pET28a-bacA. (2) transforming the plasmid into Escherichia coli BL21 competent cells to induce the expression of Brucella protein BacA. (3) after the expressed inclusion body protein is lysed, denatured and renatured, and then purified using nickel column affinity chromatography.

[0021] The present invention discloses the following technical effects:

[0022] The immunogen used in this invention is purified Brucella BacA protein, which has strong immunogenicity and can effectively stimulate the body to produce specific antibodies. It does not contain live bacteria and has high safety.

[0023] The monoclonal antibody provided by this invention can produce specific immunological responses to both swine Brucella antigen and clinical isolate antigen, but does not react with Yersinia enterocolitica, Escherichia coli, and Salmonella Dublin, which have strong cross-reactivity with Brucella.

[0024] The monoclonal antibody provided by this invention has good affinity, strong binding ability, and good specificity. In competitive ELISA, Brucella positive serum can efficiently inhibit the binding of this monoclonal antibody to Brucella BacA, indicating that this monoclonal antibody can be used to establish a Brucella competitive ELISA antibody detection method, which can lay a good experimental foundation for the future development of Brucella pathogen and antibody detection methods. Attached Figure Description

[0025] Figure 1 This is a graph showing the polyacrylamide gel electrophoresis analysis of the purified protein.

[0026] Figure 2 The results of ELISA detection of antibody titers in immunized mice;

[0027] Figure 3 The image shows the purification effect of monoclonal antibodies as detected by Western blotting.

[0028] Figure 4 To identify the specificity of Brucella monoclonal antibody 1A7 using Western blotting;

[0029] Figure 5 This is a ROC curve. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described in further detail below. It should be noted that, unless otherwise specified, all reagents used in the examples are commercially available.

[0031] Example 1: Expression and purification of Brucella BacA protein

[0032] Brucella suis S2 strain, stored at -80℃, was revived and inoculated onto tryptic soy agar (TSA) plates, and incubated upside down in a 37℃ / humidity incubator for 72 h. A single, well-grown colony was picked and inoculated onto tryptic soy liquid medium (TSB), and incubated on a shaker for 48 h. 20 mL of the culture was incubated at 100℃ for 20 min to inactivate the bacteria, and Brucella genomic DNA (gDNA) was extracted according to the bacterial genome extraction kit instructions. Using the genome as a template, the target gene fragment was amplified by PCR, and the recombinant plasmid pET28a-bacA was constructed via homologous recombination. The plasmid was transformed into competent E. coli BL21 cells, induced by shaking, and subjected to polyacrylamide gel electrophoresis. The results showed successful expression of the Brucella protein BacA. The expressed protein was an inclusion body protein, and purification required protein denaturation and renaturation.

[0033] The bacterial culture was induced in large quantities, centrifuged, resuspended in Hisbinding Buffer, and lysed. After lysing, the bacterial precipitate was centrifuged again, and Buffer A, 20% SKL, and DTT were added. The precipitate was dissolved until clear, and then incubated at room temperature for 1 hour. The supernatant was collected by centrifugation. 20% PEG-4000, 50 mmol / L oxidized glutathione, and 100 mmol / L reduced glutathione were added to the supernatant, mixed thoroughly, and incubated at room temperature for 1 hour. Samples were taken for polyacrylamide gel electrophoresis. The protein refolding solution was then dialyzed and concentrated.

[0034] The concentrated protein refolding solution was filtered through a 0.45 μm filter. The supernatant was then bound to a nickel column, and the protein was purified by nickel column affinity chromatography. After purification, the eluent, washings, and elution buffer were analyzed by polyacrylamide gel electrophoresis to determine the purification effect. Figure 1 The purified protein was dialyzed, concentrated, and its concentration was determined by the BCA method. After aliquoting, it was stored at -80°C.

[0035] Example 2: Preparation of Brucella monoclonal antibody hybridoma cell line 1A7

[0036] 1. Animal immunization

[0037] 100 μg of purified Brucella BacA protein per mouse was mixed with an equal volume of Freund's complete adjuvant and thoroughly emulsified. The mixture was then injected subcutaneously at multiple sites, 0.3 mL per mouse. A second immunization was administered two weeks after the initial immunization, followed by a third and fourth immunization every week with Freund's incomplete adjuvant emulsified antigen. Three days before fusion, a booster immunization of 200 μg of protein per mouse was administered intraperitoneally. Blood was collected from the tail vein of the mice, and serum antibody titers were detected using an indirect ELISA method. Serum titers of all immunized mice reached above 1:12800, with mouse number 3 exhibiting the highest dilution titer. Figure 2 Therefore, mice were boosted with immunization 3 days before fusion, and each mouse was injected intraperitoneally with 200 μg of protein for cell fusion.

[0038] 2. Cell fusion

[0039] a. Preparation of feeder cells

[0040] One day prior to fusion, one blank Balb / c mouse was selected, and blood was excised from the orbital cavity. The blood was collected, and the serum was separated to obtain the negative serum. After euthanizing the mouse by dislocation, it was immersed in 75% ethanol for 5 minutes and then transferred to a sterile room. The mouse was placed in a right lateral decubitus position, the ethanol was dried, and a set of sterile surgical instruments was used. Forceps were used to gently lift the abdominal skin upwards, and ophthalmic scissors were used to make a small incision vertically below the midline of the abdomen. The skin was then firmly grasped with ophthalmic forceps and peeled off to fully expose the peritoneum. Using a different set of sterile surgical instruments, the abdominal wall was lifted with ophthalmic forceps, and the abdominal wall was cut open with ophthalmic scissors to expose the spleen located on the left side of the abdominal cavity. The spleen was removed and placed in a small dish, the connective tissue was carefully removed, and the spleen was repeatedly rinsed with RPM-1640 basal culture medium. The spleen was then minced with ophthalmic scissors and ground in a cell strainer. During the grinding process, the cells were repeatedly rinsed with RPM-1640 basal medium, and the rinsing solution was collected in 50 mL centrifuge tubes, with a total of 20 mL of RPM-1640 basal medium added. The cell suspension in the centrifuge tubes was centrifuged at 2500 rpm for 10 minutes, and the supernatant was discarded. The cells were gently tapped at the bottom of the tube to loosen them, and the cells were resuspended in RPM-1640 complete medium. The feeder cells were resuspended in 30 mL of HAT medium (50 μL per well in a 96-well plate) and cultured in a cell culture incubator (37°C, 5% CO2).

[0041] b. Preparation of immune spleen cells

[0042] Mice that have undergone shock immunization were subjected to orbital bleeding. Blood was collected and serum was separated, which is the positive serum. The preparation method of immune spleen cells is the same as that of feeder cells. The cells were resuspended in 10 mL of RPM-1640 basal medium, passed through a cell sieve again to remove tissue fragments, and 10 μL of cell suspension was collected for counting and later use.

[0043] c. Preparation of myeloma cells

[0044] SP2 / 0 mouse myeloma cells were revived and cultured in advance to ensure they were in the logarithmic phase on the day of fusion. At fusion, the adherent SP2 / 0 mouse myeloma cells were resuspended in RPM-1640 basal medium, and the cell number was adjusted to maintain a ratio of approximately 1:5 between SP2 / 0 mouse myeloma cells and mouse spleen cells.

[0045] d. Cell fusion

[0046] Resuspended spleen cells and myeloma cells (5:1 ratio) were mixed in a 50 mL centrifuge tube and centrifuged at 1000 rpm for 10 minutes. The supernatant was discarded, and the supernatant was completely absorbed with absorbent paper. The mixture was then shaken to thoroughly mix the cell pellets into a paste. 0.8 mL of preheated (37°C) 50% PEG 1500 solution was slowly added dropwise to the cell pellet, gently tapping and rotating the centrifuge tube as it was added. The addition was completed within one minute, with a slower rate of approximately one drop every 5 seconds for the first 30 seconds, and the remainder added over the next 30 seconds. After standing for 1 minute, 20 mL of preheated (37°C) RPM-1640 basal medium was added dropwise to terminate the PEG effect. The first 1 mL of RPM-1640 basal medium was added slowly over 30 seconds, followed by 2 mL of RPM-1640 basal medium over the next 30 seconds. Add the remaining RPM-1640 basal medium over 1 minute. Centrifuge at 1000 rpm for 10 minutes and discard the supernatant. Incubate the centrifuge tubes in a 37°C cell culture incubator for 5 minutes. Then, gently resuspend the pelleted cells in 1640 basal medium containing 20% ​​fetal bovine serum and 1% HAT, and add 100 μL to each well of a 96-well culture plate containing feeder cells. Incubate in a cell culture incubator (37°C, 5% CO2).

[0047] 3. Hybridoma cell screening and subcloning

[0048] On day 5 after cell fusion, 50 μL of HAT medium was added to each well. Starting from day 7, half of the medium was discarded and HAT medium was added daily. When the cell colonies reached 1 / 4 of the culture well size, the cell supernatant was collected for indirect ELISA detection to screen for positive hybridoma cells. Purified BacA protein was used as the antigen. 100 μL of the cell culture supernatant from each well was used as the primary antibody, and horseradish peroxidase-labeled goat anti-mouse IgG (H+L) was used as the secondary antibody. SP2 / 0 cell culture supernatant was used as a negative control. ELISA was used to detect OD... 450nm Higher values ​​indicate positive hybridoma cells. The corresponding positive hybridoma cells are labeled, supplemented with HAT medium, and cultured on a larger scale. The selected positive hybridoma cells are subcloned three times using limiting dilution to obtain a stable hybridoma cell line 1A7 that secretes specific monoclonal antibodies.

[0049] Example 3. PCR amplification and sequencing of the variable region gene of anti-brucellosis BacA protein monoclonal antibody 1A7

[0050] Total RNA was extracted from hybridoma cells secreting monoclonal antibody 1A7 against Brucella BacA protein using the Trizol lysis method, and then cDNA was synthesized by reverse transcription. Degenerate primers were designed for amplification of the heavy and light chain variable regions of monoclonal antibody 1A7. After amplification, agarose gel electrophoresis was performed, and successfully amplified PCR products were sequenced.

[0051] Table 1 Primer sequences for amplifying the variable regions of the heavy and light chains.

[0052]

[0053] Sequencing results show that the nucleotide sequence of the heavy chain variable region of monoclonal antibody 1A7 against Brucella BacA protein is shown in SEQ ID NO.1:

[0054] CCGTTTTGGCGAAAGTGACTGAGGCTCTGGACCAGTAGCCCACCACGTGTAGATGCCAAATTCGAATCTTGGACCAGGATGTACTTGGAATTCCTCAGATGACATGCTGCTCAGGCCTTCACTGTGTCGGTGAAGGACGGGTCTACCTGCCTGACTACTGGCCCTTAACCTTTCCATT GCCACTAATATAACTATCACAACCATCAATCACTCCAATCCACTCAAGGCCTTGCCCAAGGCTCTGCTTCACACATTGCATCCCCCTCCTGGCTAAGGTGTACCCCACCCACTCCTGAATCTTCACTCAGGCCCCAGGCTTCACCAGCTCAGCCCCAGACTCCTGCGCTGCACCTCA

[0055] The amino acid sequence of the variable region of the heavy chain of the monoclonal antibody 1A7 against Brucella BacA protein is shown in SEQ ID NO.2:

[0056] RCSAGVWGAGEAWGLSEDSGVGWGYTLARRGMQCVKQSLGQGLEWIGVIDGCDSY ISGNGKVKGQSGRTRPSPTQRPEQAREGLLRLL

[0057] The nucleotide sequence of the variable region of the light chain of the monoclonal antibody 1A7 against Brucella BacA protein is shown in SEQ ID NO.3:

[0058] GTGAACGGGAGAGCTGCTATATTGCTGACAGAAATAATCTGCCAGGTCCTCAGACTGCACATTGCTAATGGTGAGAATGAAATCTGTCCCAGATCCACTGCCTGTGAAGCGATCAGGGACTCCAGTATGCCGGGTGGATGCCCAGTAAAT CAGTAATTTTGGAGATTGCCCTGGTTTCTGTTGATACCAGGCTACAGCATTACCCACATCCTGACTGGCCTTGCAGGTGATGCTGACCCTGTCTCCTACTGATGTGGACATGATTTGTGAGACTGGGCATCTGGATGCAATGTCCAAAACC

[0059] The amino acid sequence of the variable region of the light chain of the monoclonal antibody 1A7 against Brucella BacA protein is shown in SEQ ID NO.4:

[0060] IASRCPVSQIMSTSVGDRVSITCKASQDVGNAVAWYQQKPGQSPKLLIYWASTRHTGV PDRFTGSGSGTDFILTISNVQSEDLADYFCQQYSSS

[0061] The sequences of the heavy and light chain variable regions of monoclonal antibody 1A7 against Brucella BacA protein obtained by sequencing were analyzed. The amino acid sequences of the three CDR regions of the heavy chain variable region were as follows:

[0062] CDR1: GYTLARRGMQCV (shown in SEQ ID NO.5);

[0063] CDR2: IDGCDSYISG (shown in SEQ ID NO. 6);

[0064] CDR3: AREGLLRLL (shown in SEQ ID NO.7).

[0065] The amino acid sequences of the three CDR regions in the light chain variable region are as follows:

[0066] CDR1: QDVGNAVAWYQQ (as shown in SEQ ID NO.8);

[0067] CDR2: WASTRHTGVP (shown in SEQ ID NO. 9);

[0068] CDR3: QQYSSS (as shown in SEQ ID NO.10).

[0069] Example 4. Preparation, subtype identification and purification of Brucella monoclonal antibody 1A7

[0070] 1. Preparation of ascites

[0071] Six- to eight-week-old female Balb / c mice were selected and sensitized by intraperitoneal injection of 500 μL Freund's incomplete adjuvant. Seven days later, the mice were injected intraperitoneally with 500 μL of positive hybridoma cells per mouse, approximately 10 5 ~10 6 1 cell. Observe mice 7-10 days after cell injection. When the abdomen is swollen, collect ascites fluid, centrifuge at 5000 r / min for 10 min, and collect the supernatant.

[0072] 2. Monoclonal antibody subtype identification

[0073] Antibody typing was performed using a mouse monoclonal antibody subtype identification kit. Hybridoma cell supernatant was diluted 1:100 with PBST and added 50 μL to each well of the strip sample. 50 μL of 1× goat anti-mouse IgM + IgG-HRP was added to each well. The mixture was gently mixed, and the plates were sealed with a sealing film and incubated at room temperature for 1 h. The liquid in the wells was discarded, and the plates were washed three times with 1×PBST. The plates were patted dry on absorbent paper, and 100 μL of freshly prepared chromogenic buffer was added to each well. The plates were incubated at room temperature in the dark for 10-20 min. 50 μL of stop solution was added to each well, and the OD values ​​were read using a microplate reader. 450 The pore with the highest OD value corresponds to the corresponding subtype.

[0074] The results are shown in Table 1. The heavy chain of monoclonal antibody 1A7 is IgG2b, and the light chain is Kappa.

[0075] Table 2. Identification of Monoclonal Antibody Subtypes

[0076]

[0077] 3. Monoclonal antibody purification

[0078] Prepare neutralization buffer (1M Tris-HCl, pH 8.8), elution buffer (50mM glycine, pH 2.7), and phosphate-buffered saline (PBS). Filter the solution using a 0.45μm pore size filter. Dilute the ascites supernatant sample to be purified 1:1 with PBS. Pack a purification column with an appropriate amount of Beyotime Protein A+G Agarose. Wash and equilibrate the column with 10-20 column volumes of PBS. Load the antibody to be purified onto the purification column and collect the eluent. Wash with 10-20 column volumes of PBS to remove unbound and non-specifically bound proteins, and collect the wash buffer. After washing, add 100μL of neutralization buffer per mL of eluent. Pre-add an appropriate amount of neutralization buffer to the collection tube, then elute the bound antibody with 10mL of elution buffer. Collect the eluted antibody in separate tubes. Take 20 μL of sample from each collection tube, add 25 μL of 2×SDS-PAGE Loading Buffer and 5 μL of DTT, mix well, and perform SDS-PAGE electrophoresis; after staining with Coomassie Brilliant Blue and destaining, the antibody purification effect is detected. Figure 4 Afterwards, the purified monoclonal antibody was dialyzed, concentrated, and its concentration was measured. It was then aliquoted and stored at -80°C.

[0079] Example 5. Titer determination and specificity identification of monoclonal antibodies

[0080] 1. Monoclonal antibody titer determination

[0081] The purified monoclonal antibody was serially diluted at 1:100, 1:200, 1:400, and 1:800, and its titer was determined by indirect ELISA. The results are shown in Table 2. The titer of the monoclonal antibody was 1:12800.

[0082] Table 3. Monoclonal Antibody Titer Determination

[0083]

[0084] 2. Monoclonal antibody specificity identification

[0085] pET-28a empty vector bacterial solution, BacA protein, *Brucella bovis*, *Brucella melioides*, *Brucella suis*, *Escherichia coli* O157, and *Salmonella* were diluted in PBS, added with SDS-PAGE Loading Buffer, boiled for 10 minutes, and transferred to nitrocellulose membranes after polyacrylamide gel electrophoresis. The membranes were blocked with 5% skim milk prepared with TBST at 37°C for 2 hours. After blocking, the membranes were incubated with diluted monoclonal antibodies for 2 hours, washed 4 times with TBST, and incubated with HRP-labeled goat anti-mouse IgG at 37°C for 1 hour. After 5 washes with TBST, an appropriate amount of ECL chemiluminescent substrate was added to the membranes, and the membranes were exposed and analyzed using a gel imaging system. Results ( Figure 4 It can be seen that the monoclonal antibody specifically reacts with swine, bovine, and ovine Brucella and BacA protein, but does not specifically react with pET-28a empty vector bacterial solution, Escherichia coli, and Salmonella.

[0086] Example 6. Establishment of a competitive ELISA antibody detection method for Brucella.

[0087] Dilute Brucella BacA protein to 0.5 μg / mL and coat each well with 100 μl of the solution. Incubate at 4°C for 15 hours; discard the solution and blot dry. Add 200 μl of 5% BSA as blocking solution to each well and incubate at 37°C for 1 hour; discard the solution and blot dry. Add 50 μl of 1:5 diluted test serum and 50 μl of 1:200 diluted monoclonal antibody to each well, incubate at 37°C for 45 minutes, discard the liquid, and blot dry. Add 100 μl of 1:5000 diluted enzyme-labeled secondary antibody to each well, incubate at 37°C for 30 minutes, discard the liquid, wash the plate 5 times with washing buffer (300 μl per well), and blot dry on absorbent paper after the last wash. Add 100 μl of substrate development solution to each well and develop in the dark for 20 minutes. Add 50 μl of stop solution to each well. Measure the OD of each well using a microplate reader. 450nm Value. Calculate its inhibition rate (PI%), and import the detection results and serum background information into SPSS 26.0 software to plot the ROC curve. Figure 5 ), analyze the curve results, and determine the judgment criteria.

[0088] The criteria for determining the Brucella competitive ELISA antibody detection method are as follows:

[0089] When the inhibition rate (PI) of the serum sample to be tested is ≥60%, the Brucella antibody test result is judged as positive;

[0090] When the inhibition rate (PI) of the serum sample to be tested is <60%, the Brucella antibody test result is judged as negative.

[0091] Example 7. Performance testing of the Brucella competitive ELISA antibody detection method

[0092] 1. Sensitivity test

[0093] Brucella national standard positive serum (1000 IU / mL) was diluted 5-fold, 10-fold, 20-fold, and 40-fold with sample diluent for testing, and the inhibition rate (PI) value of the serum samples was calculated. The results are shown in Table 3 below. When the Brucella national standard positive serum was diluted 320 times, the competitive ELISA test result was positive.

[0094] Table 3. Sensitivity Test Results

[0095]

[0096]

[0097] 2. Competitive ELISA detection of serum anti-Yersinia enterocolitica O:9

[0098] The above-described Brucella competitive ELISA antibody detection method was used to detect clinical bovine antibody-positive serum, bovine negative serum, and bovine enterocolitis Yersin-positive serum. The results are shown in Table 4 below. The detection result for bovine enterocolitis Yersin-positive serum was negative, indicating that the established method has good specificity.

[0099] Table 4 Results of Specificity Tests

[0100]

[0101] 3. Compliance Rate Test

[0102] Using the above-mentioned Brucella competitive ELISA detection method and the Brucella cELISA antibody detection kit from Wuhan Keqian Biotechnology Co., Ltd., 118 clinical serum samples were tested, and the concordance rates of the two methods were compared. The results are shown in Table 5 below. The overall concordance rate between the Brucella competitive ELISA detection method and the commercial Brucella cELISA antibody detection kit was 96.7%.

[0103] Table 5 Results of the Clinical Serum Concordance Rate Trial

[0104]

Claims

1. A monoclonal antibody 1A7 against Brucella BacA protein, characterized in that, The heavy chain variable region of the monoclonal antibody 1A7 includes CDR1 with the amino acid sequence GYTLARRGMQCV, CDR2 with the amino acid sequence IDGCDSYISG, and CDR3 with the amino acid sequence AREGLLR. The light chain variable region of the monoclonal antibody 1A7 includes CDR1 with the amino acid sequence QDVGNAVAWYQQ, CDR2 with the amino acid sequence WASTRHTGVP, and CDR3 with the amino acid sequence QQYSSS.

2. The monoclonal antibody 1A7 according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody 1A7 is shown in SEQ ID NO: 2; the amino acid sequence of the light chain variable region of the monoclonal antibody 1A7 is shown in SEQ ID NO:

4.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the nucleic acid molecule of the monoclonal antibody 1A7 as described in claim 1.

4. A host cell, characterized in that, The host cell comprises the nucleic acid molecule of claim 3 or the vector of claim 4.

5. The monoclonal antibody 1A7 according to claim 1, characterized in that, The monoclonal antibody 1A7 can produce specific immune responses to Brucella suis, Brucella bovis, and Brucella ovis, but does not produce specific responses to Escherichia coli, Salmonella, or Yersinia enterocolitica.

6. The monoclonal antibody 1A7 according to claim 1, characterized in that, The heavy chain of the monoclonal antibody 1A7 is IgG2b, and the light chain is Kappa.

7. The application of the monoclonal antibody against Brucella BacA protein according to claim 1 in the preparation of a Brucella competitive ELISA antibody detection method.

8. According to claim 8, the Brucella competitive ELISA antibody detection method is as follows: Brucella BacA protein is diluted to 0.5 μg / mL, 100 μl / well is used to coat an ELISA plate, and the plate is incubated at 4°C for 15 hours. The solution in the wells is discarded, and the plate is dried. 200 μl of 5% BSA is added to each well as blocking solution, and the plate is incubated at 37°C for 1 hour. The solution in the wells is discarded, and the plate is dried. A 1:5 dilution of the test serum and a 1:200 dilution of the test serum are added. Add 50 μl of monoclonal antibody to each well and incubate at 37°C for 45 minutes. Discard the liquid in the wells and blot dry. Add 100 μl of enzyme-labeled secondary antibody diluted 1:5000 to each well and incubate at 37°C for 30 minutes. Discard the liquid in the wells and wash the plate 5 times with washing buffer (300 μl per well). After the last wash, blot dry on absorbent paper. Add 100 μl of substrate chromogenic solution to each well and incubate in the dark for 20 minutes. Add 50 μl of stop solution to each well and measure the OD of each well using a microplate reader. 450nm value.

9. The Brucella competitive ELISA antibody detection method according to claim 9, characterized in that, The criteria for judgment are: When the inhibition rate (PI) of the serum sample to be tested is ≥60%, the Brucella antibody test result is judged as positive; When the inhibition rate (PI) of the serum sample to be tested is <60%, the Brucella antibody test result is judged as negative.

10. Steps for expressing and purifying Brucella BacA protein: (1) Using the Brucella suis genome as a template, the target gene fragment was amplified by PCR to construct the recombinant plasmid pET28a-bacA; (2) The plasmid was transformed into Escherichia coli BL21 competent cells to induce the expression of Brucella protein BacA; (3) The expressed inclusion body protein was cleaved and denatured and renatured, and then purified by nickel column affinity chromatography.

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