Brucella BP26 protein, preparation method thereof, and preparation method and application of monoclonal antibody of Brucella BP26 protein

Abstract

The invention relates to a Brucella BP26 protein, a preparation method thereof, and a preparation method and application of a monoclonal antibody of the Brucella BP26 protein. The amino acid sequence of the BP26 protein is shown as SEQ ID NO: 1; brucella BP26 protein is selected as an antigen, a homologous recombination technology is utilized to construct a BP26 prokaryotic expression vector, IPTG (isopropyl-beta-d-thiogalactoside) induced expression is carried out, and purification is carried out to obtain the recombinant BP26. An animal is immunized with the Brucella BP26 protein; fusing the tumor cells with splenocytes of an immunized animal, taking a cell supernatant culture solution to detect titer, and screening hybridoma cells; and collecting and purifying the monoclonal antibody secreted by the hybridoma cells. According to the invention, efficient soluble expression is realized by optimizing a BP26 gene sequence, and high-immunogenicity protein is obtained through purification; the protein is used for immunizing and screening a high-affinity monoclonal antibody. The BP26 protein and the monoclonal antibody can improve the Brucella detection sensitivity and specificity, are suitable for vaccine development, and have important clinical application value.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a Brucella BP26 protein, a preparation method thereof, a preparation method of a monoclonal antibody thereof, and applications thereof. Background Art

[0002] Brucella is a Gram-negative intracellular parasitic bacterium that can infect humans, livestock, and various wild animals, and cause a zoonotic disease called brucellosis. Brucellosis is also known as brucellosis. Although brucellosis poses a serious threat to human health and the development of animal husbandry, and also causes significant losses to the global economy, due to the insufficient popularization of relevant health education, this disease is often ignored by the public.

[0003] In the detection methods of brucellosis, immunoserological detection is widely used as an important means, especially enzyme-linked immunosorbent assay, such as indirect enzyme-linked immunosorbent assay (I-ELISA) and competitive enzyme-linked immunosorbent assay (C-ELISA). However, most immunoserological detection methods mainly use lipopolysaccharide (LPS) antigen to detect LPS antibody. Due to some limitations of LPS, such as the established serological methods based on LPS can only detect smooth-type Brucella infection, cannot detect rough-type Brucella infection, and cannot distinguish natural infection from vaccine immunization. After the serum of immunized livestock is detected as positive, because it is impossible to judge whether it is a natural infection, a purification strategy has to be implemented. Once misdiagnosed, it will bring huge economic losses. Therefore, researchers began to look for new antigens that can distinguish natural infection and immune serum. Outer membrane protein (OMP) has become a research hotspot. Outer membrane proteins are exposed on the bacterial surface and are closely related to the immune response mechanism of the host. In past studies, outer membrane proteins, as important virulence factors, have played a crucial role in the immunoserological detection of brucellosis. Due to the good reactogenicity of outer membrane proteins, they can stimulate the body to produce a strong immune response, so they are regarded as important antigens for the diagnosis of brucellosis and candidates for Brucella subunit vaccines.

[0004] Brucella BP26 protein is a periplasmic protein with a molecular weight of 26 kDa, also known as OMP28 (Outer Membrane Protein 28). It has been recognized as a Brucella surface antigen with strong immunogenicity. Its gene sequence is highly conserved, capable of generating antibody responses in animals infected with Brucella, and has high specificity and sensitivity in the detection of brucellosis. Compared with the LPS of Brucella, although the antibody response induced by BP26 is relatively weak in animals, its specificity shown in the detection of brucellosis exceeds that of the LPS antigen. Due to its high immunogenicity, BP26 rarely cross-reacts with sera from other bacterial pathogen infections, making it an ideal candidate outer membrane protein antigen for detecting brucellosis caused by rough Brucella.

[0005] Currently, BP26 protein is mainly obtained by extracting from whole cell lysates or prokaryotic expression of a single gene, but there are problems such as low purity and incomplete epitope exposure, resulting in insufficient sensitivity of antibody reagents developed based on it (such as high false positive rates in traditional ELISA detection). Therefore, developing technologies for highly efficient expression and purification of BP26 protein and preparation of high-affinity monoclonal antibodies is of great significance for improving the diagnosis and treatment levels of brucellosis. Summary of the Invention

[0006] The object of the present invention is to provide a method for preparing a high-purity and high-immunogenic Brucella BP26 protein, as well as a technology for highly efficient monoclonal antibody preparation based on this protein, and to expand its applications in the detection, vaccine development and treatment of brucellosis.

[0007] To achieve the object of the invention, the present invention provides a Brucella BP26 protein, and the amino acid sequence of the BP26 protein is as shown in SEQ ID NO:1.

[0008] To achieve the object of the invention, the present invention also provides a method for preparing the above-mentioned Brucella BP26 protein. Select the above-mentioned Brucella BP26 protein as an antigen, use homologous recombination technology to construct a BP26 prokaryotic expression vector, perform IPTG-induced expression, and prepare the BP26 protein after purification.

[0009] Further, the specific steps are as follows:

[0010] S1. According to the amino acid sequence of the BP26 protein, reverse deduce the codons, construct the BP26 prokaryotic expression gene sequence, and the synthesized target gene sequence is as shown in SEQ ID NO:2;

[0011] S2. Transfer the synthesized target gene sequence into the expression vector pET30a-GST to obtain the pET30a recombinant plasmid;

[0012] S3. Prokaryotic expression and purification of BP26 protein: The recombinant plasmid pET30a was transferred into the expression vector BL21 for IPTG-induced expression. This process specifically includes transformation, small-scale expression, large-scale expression, protein purification (washing inclusion bodies), and protein renaturation.

[0013] To achieve the invention purpose, the present invention also provides a preparation method of the monoclonal antibody against Brucella BP26 protein as described above. The specific steps are as follows: Immunize an animal with the above-mentioned Brucella BP26 protein; fuse tumor cells with the spleen cells of the immunized animal, and take the cell supernatant culture solution to detect the titer and screen hybridoma cells; collect and purify the monoclonal antibody secreted by the hybridoma cells, which is the monoclonal antibody against Brucella BP26 protein.

[0014] Furthermore, immunize BALB / c mice with the above-mentioned Brucella BP26 protein, fuse myeloma cells SP2 / 0 with the spleen cells of the immunized mice, take the cell supernatant culture solution to detect the titer and screen hybridoma cells, then prepare mouse ascites by the in vivo induction method, collect and purify the ascites to prepare the BP26 monoclonal antibody.

[0015] To achieve the invention purpose, the present invention also provides a monoclonal antibody against Brucella BP26 protein, which is secreted by the hybridoma cell line BP26-7E12, with the heavy chain being IgG1 and the light chain being of the κ type; the hybridoma cell line BP26-7E12 was deposited at the China Center for Type Culture Collection on February 15, 2023. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: C202330, and the taxonomic name is Hybridoma cell line Bp26-7E12.

[0016] To achieve the invention purpose, the present invention also provides an application of the above-mentioned monoclonal antibody in the preparation of drugs for diagnosing, preventing or treating brucellosis.

[0017] Compared with the prior art, the present invention has the following advantages:

[0018] The present invention realizes high-efficiency soluble expression by optimizing the BP26 gene sequence and purifies a highly immunogenic protein; uses this protein to immunize and screen out a monoclonal antibody with high affinity. The BP26 protein and the monoclonal antibody can significantly improve the detection sensitivity and specificity of Brucella and are applicable to vaccine development and targeted therapy, having important clinical application value. Brief Description of the Drawings

[0019] Figure 1 It is a comparative analysis diagram of the homologous recombinant sequence of BP26 amino acids;

[0020] Figure 2Electrophoresis identification diagram of small-scale expression of BP26 protein; where M: Marker; 1: Uninduced control (BL21); 2-3: IPTG-induced (BL21); Gel: 15% SDS-PAGE;

[0021] Figure 3 Electrophoresis identification diagram of large-scale expression of BP26 protein; where M: Marker; 1: Whole bacteria after sonication; 2: Supernatant after sonication; 3: Precipitate after sonication; Gel: 15% SDS-PAGE;

[0022] Figure 4 Electrophoresis identification diagram of purification of BP26 protein after large-scale expression; M: Marker; 12: Protein sample; 3: Flow-through; 4: Elution with 15 mM imidazole; 5: Elution with 60 mM imidazole; 6-9: Elution with 500 mM imidazole; Gel: 12% SDS-PAGE;

[0023] Figure 5 Schematic diagram of the specific reaction results of BP26 recombinant protein with the supernatants of 12 monoclonal antibody cells immunized in mice. Detailed implementation mode

[0024] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0025] The materials used in the present invention are as follows:

[0026] Serum samples

[0027] The 521 animal sera used in the present invention were all donated by the China Animal Health and Epidemiology Center (Qingdao), including 52 bovine negative sera, 44 positive sera, 54 ovine negative sera, 42 naturally infected sera (positive sera), 231 vaccine-immunized sera, 46 canine negative sera, 7 positive sera, 35 porcine negative sera, and 10 positive sera. All Brucella positive serum samples were confirmed by Rose Bengal plate agglutination test (RBPT) and tube agglutination test (SAT) according to the national standard (GB / T 18646-2018); while the negative serum samples were all collected from areas in China without Brucella infection. All of the above serum samples can be traced back to their background sources. Rabbit sera (rabbit anti-Vibrio parahaemolyticus, Escherichia coli, Salmonella, Vibrio cholerae, Yersinia enterocolitica, Listeria) were purchased from Tianjin Biochip Technology Co., Ltd.

[0028] Experimental animals, cell lines, strains

[0029] The SP2 / 0 cells, BL21(DE3) competent cells, and 8-10-week-old SPF-grade female BALB / c mice used in the present invention were all purchased from Beijing BGI Protein R & D Center Co., Ltd.

[0030] Reagent

[0031]

[0032]

[0033] Example 1

[0034] A Brucella BP26 protein. The BP26 amino acid sequence of B. melitensis 16S strain was obtained from the NCBI website. The MegAlin software was used to analyze and compare the BP26 amino acid homologous recombination sequences, and it was found that BP26 Brucella has a high degree of conservation (such as Figure 1 ). According to NCBI BP26 BLAST, Brucella melitensis biovar strain BP26 gene, namely AY166768.1, was selected, and the protein sequence is as shown in SEQ ID NO:1:

[0035] Example 2

[0036] A method for preparing a Brucella BP26 protein, the specific steps are as follows:

[0037] S1. According to the amino acid sequence of the BP26 protein, the codons were deduced backward, and the prokaryotic expression gene sequence of BP26 was constructed. The synthesized target gene sequence is as shown in SEQ ID NO:2;

[0038] S2. The synthesized target gene sequence was transferred into the expression vector pET30a-GST to obtain the pET30a recombinant plasmid;

[0039] S3. Prokaryotic expression and purification of the BP26 protein: The pET30a recombinant plasmid was transferred into the expression vector BL21, and IPTG induction expression was carried out. This process specifically includes transformation, small-scale expression, large-scale expression, protein purification (washing inclusion bodies), and protein renaturation.

[0040] S3-1. Transformation;

[0041] (1) Prepare competent cells: First, take out the BL21 competent cells stored at -80°C and thaw them slowly on ice.

[0042] (2) Add plasmid DNA: Then, add plasmid DNA to the thawed cells, ensure uniform mixing, and then keep it on ice for 30 minutes.

[0043] (3) Heat shock treatment: The mixture was heat shocked at 42°C for 90 seconds.

[0044] (4) Cool down and add culture medium: Immediately after heat shock, place on ice bath for 2 minutes and add 800 μL of antibiotic-free LB culture medium.

[0045] (5) Recovery culture: Incubate the mixture at 37 °C for 45 minutes to recover the cells.

[0046] (6) Centrifuge and resuspend: Centrifuge at 5000 rpm for 3 minutes, discard a portion of the supernatant, retain approximately 100 - 150 μL, and resuspend the bacterial cells.

[0047] (7) Plate coating and culture: Spread the resuspended bacterial cells on an LB plate containing the corresponding antibiotic. After spreading evenly, incubate the plate upside down in an incubator at 37 °C overnight.

[0048] S3-2, Small-scale expression;

[0049] (1) Initial stage of culture: Select a single colony that has undergone transformation treatment and inoculate it into 1.5 mL of LB liquid culture medium supplemented with the corresponding antibiotic. Incubate at 37 °C and 200 rpm.

[0050] (2) Induced expression: When the bacteria in the culture medium grow to an OD value of 0.6 - 0.8, add IPTG (0.5 mM) for induction and continue to incubate at the same temperature and rotation speed for 2 hours.

[0051] (3) Protein extraction: Take 1 mL of the bacterial liquid from the induced culture medium, centrifuge at a high speed of 12000 rpm for 1 min, discard the supernatant, resuspend the precipitate in 50 - 100 μL of 10 mM Tris-HCl (pH 8.0) buffer. The specific amount of buffer added depends on the amount of bacterial cells. Then, add an equal volume of 2-fold concentrated loading buffer, mix well, and heat at 100 °C for 5 minutes for subsequent electrophoresis analysis. The protein expression results are as Figure 2 shown.

[0052] S3-3, Large-scale expression;

[0053] (1) Start of culture: Add 50 μL of activated bacterial liquid to 5 mL of LB liquid culture medium containing a specific antibiotic, and then incubate at 37 °C and 200 rpm.

[0054] (2) Scale-up culture: Transfer the cultured bacterial liquid to 200 mL of LB liquid culture medium containing the same antibiotic, and continue to incubate at 37 °C and 200 rpm until the OD value reaches 0.6 - 0.8. At this time, add IPTG (0.5 mM) and induce overnight at 16 °C.

[0055] (3) Collect bacteria: After induction, centrifuge at 6000 rpm for 8 min, discard the supernatant and collect the bacteria.

[0056] (4) Lyse bacteria by ultrasound: Resuspend the bacterial cells with 20 - 30 mL of 10 mM Tris-HCl (pH 8.0) solution and lyse them using a 500 W ultrasonic wave for a total of 180 times, 5 seconds each time with a 5-second interval between each time. The protein expression results are as Figure 3 shown.

[0057] S2-2-4. Protein purification (washing inclusion bodies);

[0058] (1) Resuspend inclusion bodies: Resuspend the precipitate obtained by ultrasonic disruption and centrifugation with 20 - 30 mL of 10 mM Tris-HCl (pH 8.0) solution, and then let it stand for 10 minutes.

[0059] (2) First centrifugation: Centrifuge at 12000 rpm for 10 min, transfer the supernatant to another tube for storage.

[0060] (3) Resuspend again: Resuspend the precipitate with the same volume of 20 - 30 mL of 10 mM Tris-HCl (pH 8.0) solution again and let it stand for 10 min.

[0061] (4) Second centrifugation: Centrifuge at 12000 rpm for 10 minutes again and discard the supernatant.

[0062] (5) Repeat washing: Repeat steps 3 and 4 once to ensure more thorough removal of impurities.

[0063] (6) Dissolve inclusion bodies: First, resuspend the precipitate with a small amount of 10 mM Tris-HCl (pH 8.0) solution, and then add 5 - 10 mL of 10 mM Tris-HCl (pH 8.0) solution containing 8 M urea to dissolve the protein.

[0064] (7) Centrifugation: Centrifuge at 12000 rpm for 10 minutes and collect the supernatant.

[0065] (8) Protein electrophoresis: Take 50 μL of the supernatant for SDS-PAGE electrophoresis to check the purity and size of the protein.

[0066] S3-4. Protein renaturation;

[0067] (1) Gradually reduce the urea concentration using dialysis buffer: Prepare the dialysis buffer, which consists of 1% glycine, 0.1% SDS, 5% glycerol, and 10 mM Tris-HCl (pH 8.0), and set the urea concentration gradient to 6 M, 4 M, and 2 M. Dialyze the sample at 4°C with 20 times its volume of dialysis buffer to gradually reduce the urea concentration. Dialyze for 3 hours at each urea concentration, and perform overnight dialysis for the last stage (2 M urea concentration).

[0068] (2) Dialyze to remove small molecule impurities: Perform two dialysis steps using dialysis buffer without urea (1% glycine, 10 mM Tris-HCl, pH 8.0), each for 3 hours.

[0069] (3) Perform the final dialysis using 1×PBS: Perform two dialysis steps using 1×PBS (phosphate buffered saline), each for 3 hours.

[0070] (4) Centrifuge and collect the supernatant: Centrifuge at 12,000 rpm for 10 minutes, and then collect the supernatant.

[0071] (5) Detect the protein concentration and purity by electrophoresis: Take the supernatant for SDS-PAGE electrophoresis to detect the protein concentration and purity and confirm the refolding effect.

[0072] After protein purification, the results are as Figure 4 shown. The Western-Blot results show that the molecular weight of the BP26 protein is 53 kDa, the purity is approximately 90%, and the concentration is 1 mg / mL.

[0073] Example 3

[0074] A method for preparing a monoclonal antibody against Brucella BP26 protein, the specific steps are as follows:

[0075] S1. Mouse immunization;

[0076] S1-1. Number 4 BALB / c female mice as 1, 2, 3, and 4. Before each immunization, collect 2 μL of blood from the tail vein of each mouse and add it to an EP tube that has already been added with 198 μL of PBS. Mix well, mark the date, etc., and store it at -20°C for subsequent ELISA testing of the immune serum titer.

[0077] S1-2. Take a 1 mL syringe and perform multi-point immunization at the back subcutaneous, inguinal, and footpad sites. The immunization dose is 30 μg of protein per mouse each time. The immunization interval is 5 - 10 days. Take blood from the tail vein. After the serum titer results are ideal by ELISA, for the last two immunizations, perform intraperitoneal injection immunization with the bacterial solution (especially for the last immunization, immunize 4 days before cell fusion).

[0078] S2. Immunopotency I-ELISA detection: Coat overnight at 4°C with BP26 antigen protein at 2 μg / mL; Block at 37°C for 2 h with 2% milk; Dilute the serum serially 2-fold starting from 200-fold dilution. The blank control is 1×PBS, and the negative control is the negative serum diluted 200-fold. The specific procedure is as follows:

[0079] S2-1. Coat the ELISA plate with BP26 antigen. The ratio of coating solution to antigen is 200:1, 400:1, 800:1, and so on until 102400:1. Add 100 μL per well and incubate overnight at 4°C.

[0080] S2-2. Discard the coating solution, tap the plate, add 300 μL / well of PBST (add 1 mL of Tween-20% to 1000 mL of PBS) and wash 3 times, 3 min each time, and mix well by shaking.

[0081] S2-3. Pre-add 100 μL of PBS to each well. In the first row, add 100 μL of the serum collected when immunizing the mice to each well. Each mouse can be added to 2 wells each time. Then mix the first row well with a multichannel pipette, take 100 μL and add it to the empty wells in the second row, and so on, with 2-fold serial dilution. Discard 100 μL from the last row. Incubate at 37°C for 1 h. The negative control can be PBS, and for the positive control, take 2 μL of positive blood and add it to the well containing 200 μL of PBS.

[0082] S2-4. Discard the liquid, tap the plate, wash 3 times with 300 μL / well of PBST, 3 min each time. Add 100 μL of the solution containing the secondary antibody to each well (secondary antibody:PBS = 1:15000), and incubate at 37°C for 1 h.

[0083] S2-5. Discard the liquid, tap the plate, wash 3 times with 300 μL / well of PBST, 3 min each time. Add 100 μL of TMB chromogenic solution to each well and react in the dark at room temperature for 15 min.

[0084] S2-6. Add 50 μL of stop solution to each well and measure the OD with an ELISA reader 450nm .

[0085] The test results are shown in Table 2. It can be seen that the potencies of all 4 mice reached over 100,000, indicating that they can be used for subsequent experiments.

[0086] Table 1 Measurement of serum potency of mice after immunization

[0087]

[0088] S3. Preparation of monoclonal antibodies

[0089] S3-1. Preparation of myeloma cells SP2 / 0;

[0090] Take the cryopreserved myeloma SP2 / 0 cells, quickly dissolve them within 1 minute, centrifuge at 1500 rpm for 5 minutes, discard the liquid, resuspend with 1640 medium, and transfer them into a cell culture flask pre-added with 1640 culture medium containing 20% serum. Place them in a constant temperature incubator at 37°C with 5% CO2 and observe the cell attachment situation overnight.

[0091] S3-2. Preparation of feeder cells;

[0092] (1) Take healthy non-immunized BABL / c mice, collect blood by eye socket puncture, and store it at -20°C for use as a negative control.

[0093] (2) After sacrificing the mice by cervical dislocation, immerse the whole body in 75% alcohol for disinfection for 5 minutes.

[0094] (3) Irradiate with ultraviolet light in the laminar flow hood for 30 minutes, and prepare 1640 culture medium containing 2% HAT and 20% serum.

[0095] (4) Place the soaked mice in a glass dish in the laminar flow hood. Gently cut a small opening in the abdominal skin of the mice with scissors (note not to cut the peritoneum). Tear the abdominal skin of the mice with both hands to fully expose the abdominal cavity.

[0096] (5) Use a 5 mL syringe to inject 1640 medium without serum into the abdominal cavity of the mice along the bottom fat layer, injecting 10 mL.

[0097] (6) Use the syringe piston to gently massage the abdomen of the mice to fully mix the macrophages in the 1640 medium.

[0098] (7) Use the syringe to gently draw out the injected 1640 medium along the injection site and place it in a centrifuge tube. Centrifuge at 1500 rpm for 5 minutes, resuspend with 1640 containing 2% HAT and 20% serum, and count the cells.

[0099] (8) Adjust the cell concentration to 10 5 / mL, and calculate the volume of 1640 culture medium containing 2% HAT and 20% serum to be added.

[0100] (9) Plate the cells in a 96-well plate, 100 μL per well, and place it in a constant temperature incubator at 37°C with 5% CO2. Observe the cell attachment situation overnight.

[0101] S3-3. Cell fusion;

[0102] (1) Prepare a suspension of spleen cells from immunized mice

[0103] Four days before cell fusion, BALB / c mice with high antibody titers in serum were given a final booster immunization (using the bacterial solution), and then blood was collected from the eye sockets to isolate positive serum. Subsequently, the mice were sacrificed by cervical dislocation and immersed in 75% alcohol for 5 minutes for disinfection. The spleens of the mice were removed on a super clean bench under sterile conditions and placed in a petri dish containing serum-free RPMI-1640 medium for washing. Then, the spleen tissue was minced and ground in the petri dish, and at the same time, it was rinsed and filtered with RPMI-1640 medium to collect the cell suspension. The suspension was transferred to a 50 mL centrifuge tube and centrifuged at 1500 rpm for 10 minutes. After removing the supernatant, serum-free medium was added again. Finally, the cell concentration was adjusted to 10 7 cells / mL for later use.

[0104] (2) Preparation of myeloma cells

[0105] SP2 / 0 cells in the logarithmic growth phase were collected and centrifuged at 1500 rpm for 10 minutes, and the supernatant was discarded. Then, the cells were appropriately diluted with 1640 medium and the cell concentration was calculated. The cell concentration was adjusted to 10 6 cells / mL for standby.

[0106] (3) Cell fusion

[0107] 1) According to the required number of feeder cells, prepare the corresponding number of spleen cells and SP2 / 0 cells, with the ratio of spleen cells to SP2 / 0 cells being 10:1.

[0108] 2) After mixing these two types of cells, centrifuge and discard the supernatant. To ensure uniform cell mixing, this step can be repeated twice.

[0109] 3) After the last centrifugation to discard the supernatant, gently flick the bottom of the centrifuge tube with your finger to form a uniform cell slurry.

[0110] 4) Slowly add 1 mL of 50% PEG (polyethylene glycol) preheated to 37°C, and gently shake while adding to ensure it is added within 1 min.

[0111] 5) After standing for 90 s, slowly add preheated incomplete culture medium to terminate the action of PEG. The liquid addition speed is: add 1 mL in the first minute, 2 mL in the second minute, and 3 mL in the third minute. After slowly adding about 20 mL of culture medium, it can be gently mixed, and then centrifuged at 1500 rpm for 5 minutes and the supernatant is discarded.

[0112] 6) Add 2% HAT culture medium, mix gently to ensure that the cells are dispersed into a single-cell suspension. Be extremely careful during this process to avoid damaging the fused cells. Subsequently, add sufficient 2% HAT culture medium to the required volume and mix thoroughly.

[0113] 7) Add 100 μL of the cell suspension to each well of the pre-prepared 96-well culture plate containing feeder cells. After labeling, place the culture plate in an incubator at 37 °C with 5% CO₂ and incubate for 5 days without moving.

[0114] (4) Screening of hybridoma positive cell lines

[0115] 1) Observe the cell growth status: Starting from the 5th day after cell fusion, observe the cell growth in the 96-well plate every day. Pay particular attention to the wells where fused cells are present, i.e., the situation where the cells have covered approximately 1 / 3 to 1 / 4 of the well bottom.

[0116] 2) Screen positive hybridoma cells by I-ELISA method: For the wells where fused cell growth is observed, use the I-ELISA method to screen for positive hybridoma cells, and this needs to be repeated 2 to 3 times.

[0117] 3) Semi-quantitatively replace 2% HT culture medium: During the screening process, to accelerate the growth of cell colonies, start to semi-quantitatively replace 2% HT (Hypoxanthine and Thymidine) culture medium.

[0118] 4) Perform a semi-media change on the 13th day: Starting from the 13th day after cell fusion, perform a semi-media change operation, that is, remove half of the old culture medium and add the same volume of fresh 2% HT culture medium.

[0119] After fusing mouse spleen cells and SP2 / 0 cells, use a semi-solid medium containing HAT for screening culture. After two rounds of screening, a total of 45 hybridoma cell lines with positive reactions were obtained. Plate the 45 positive cell lines with BP26 and use the ELISA method again for the third screening. As shown in Table 2, 12 positive hybridoma cell lines were obtained. The bold numbers represent the positive hybridoma cell lines.

[0120] Table 2: 12 positive hybridoma cells were obtained after the third screening of monoclonal cells

[0121]

[0122]

[0123] (5) Cloning of positive hybridoma cells

[0124] After identifying the positive hybridoma cells, the limited dilution method is used to subclone and culture these cells to ensure the acquisition of monoclonal cell lines. The specific operation process is as follows:

[0125] 1) Prepare feeder cells: One day before starting the cloning of hybridoma cells, prepare the feeder cells in advance.

[0126] 2) Collect and count positive cells: The cells in the positive culture wells screened by the I-ELISA method are gently blown with 1640 culture medium containing 20% serum to make the cells evenly mixed, and then all are transferred to a sterile container. The specific number of cells is determined by hemocytometry. Then, a part of the cells is used for cloning culture, and the remaining cells are transferred to a 24-well plate for expansion culture.

[0127] 3) Perform cell dilution and culture: The screened positive cells are serially diluted with 1640 culture medium containing 20% fresh bovine serum until the concentration reaches 1 cell / 100 μL, and then these diluted cells are dropped into a 96-well plate, with each well containing as close to one cell as possible, and cultured under the conditions of 37°C and 5% CO2 to form monoclonal clones. During the cloning process, pay attention to the expansion culture and cryopreservation of the positive hybridoma cell line.

[0128] (6) Expansion culture of hybridoma cells

[0129] 1) Select monoclonal cell wells: When all the hybridoma cell wells show positive reactions (positive rate reaches 100%) by I-ELISA detection, select the monoclonal cell wells with higher absorbance (OD 450 value).

[0130] 2) Transfer from 96-well plate to 24-well plate: When the selected monoclonal cells grow to completely cover the bottom of the wells in the 96-well plate, transfer these cells to a 24-well plate.

[0131] 3) Subculture: Continue to observe and culture the cells in the 24-well plate until the cells cover the bottom of 4 to 6 wells. At this time, there are enough cells to be transferred to a larger cell culture flask for more extensive expansion culture.

[0132] 4) Continuously evaluate antibody expression: During the expansion culture process, it is recommended to regularly use the I-ELISA method to evaluate the strength of the antibodies produced by the cells. It should be noted that with the increase in the number of passages, the antibody expression of some positive clones may weaken or even disappear. Therefore, regular detection should be carried out to ensure the acquisition of high-quality antibody-producing cell lines.

[0133] (7) Cryopreservation of hybridoma cells

[0134] 1) Cell collection: First, gently detach the well-cultured hybridoma cells from the culture flask and then transfer them to a sterile centrifuge tube.

[0135] 2) Centrifugation and supernatant discard: Centrifuge the centrifuge tube containing the cells at 1500 rpm for 5 minutes to precipitate the cells. After centrifugation, carefully discard the supernatant, leaving the cell pellet.

[0136] 3) Adding cryopreservation solution: Add an appropriate amount of cell cryopreservation solution (usually containing a cryoprotectant such as DMSO and a high concentration of serum to protect the cells from damage during cryopreservation) to the cell pellet, and gently pipette or gently shake to evenly disperse the cells in the cryopreservation solution.

[0137] 4) Aliquoting into cryotubes: Evenly aliquot the treated cell cryopreservation solution into labeled cryotubes. The volume of each tube depends on the specific situation of the laboratory, usually 1 - 2 mL.

[0138] 5) Stepwise cooling: First, place the cryotubes containing the cells in a 4°C refrigerator for 0.5 hours to slowly reduce the temperature. Then, transfer them to a -20°C refrigerator for further cooling for 1.5 hours, and then transfer them to an -80°C ultra-low temperature refrigerator for storage.

[0139] 6) Long-term storage: Transfer the cells that have been in the -80°C refrigerator overnight to a liquid nitrogen tank for storage. The temperature of liquid nitrogen is even lower (-196°C), which can better maintain the cell viability and stability.

[0140] S3-4. Preparation of monoclonal antibody ascites

[0141] (1) Pretreatment of mice: One week before inoculating hybridoma cells, intraperitoneally inject 0.5 mL of liquid paraffin oil into healthy female BALB / c mice aged 8 - 12 weeks. This step aims to stimulate the formation of a microenvironment in the mouse peritoneal cavity that is favorable for the growth of hybridoma cells.

[0142] (2) Cell preparation:

[0143] Collect hybridoma cells: Gently detach the well-cultured positive hybridoma cells from the culture flask, transfer them to a sterile centrifuge tube, and centrifuge at 1500 rpm for 10 minutes; Resuspend the cells: Discard the supernatant and resuspend the cell pellet with phosphate-buffered saline (PBS). Adjust the cell density: Adjust the cell density to 1×10 7 cells / mL.

[0144] (3) Inoculating cells: Intraperitoneally inject 0.5 mL of cell suspension into each mouse, containing approximately 5×10 6 cells.

[0145] (4) Observation and collection of ascites: After inoculation, closely observe the status of the mice. When the abdomen of the mice is significantly enlarged and they move slowly, it indicates that ascites has accumulated. Collection of ascites: Use a 5 mL syringe to extract the liquid in the abdominal cavity. The color of the initially collected ascites may be light yellow or dark red. Processing and preservation: Let the collected ascites stand overnight at 4 °C, then centrifuge at 10,000 rpm for 30 minutes to remove blood cells and fat masses, and collect the supernatant. Aliquot and label the supernatant and store it at -20 °C.

[0146] (5) Repeated collection: In the next 2 to 3 days, repeat the above extraction process according to the recurrence of ascites in the mice. Each mouse can usually be collected 2 to 3 times, and the ascites are respectively labeled as the Ⅰ, Ⅱ, and Ⅲ generations of ascites.

[0147] S4, Identification of BP26 monoclonal cell subtypes;

[0148] Use the Southern Biotech mouse antibody subtype identification kit to identify the selected positive cell lines. Refer to the instruction manual, and the specific steps are as follows:

[0149] S4-1, Coating: First, dilute the subclass coating antibody to 2 μg / mL with the coating solution, add 100 μL to each well, and after incubating overnight at 4 °C, wash 3 times with PBST.

[0150] S4-2, Blocking: Use 2% skim milk powder as the blocking solution for blocking, 200 μL / well, incubate in an incubator at 37 °C for 2 h, and wash the plate in the same way after incubation.

[0151] S4-3, Primary antibody: Add 100 μL of the primary antibody (cell culture supernatant) and negative control (SP2 / 0 culture supernatant) to each well, incubate in an incubator at 37 °C for 1 h, and wash the plate in the same way after incubation.

[0152] S4-4, Secondary antibody: Dilute each type of subclass secondary antibody (Goat Anti-Mouse IgM, IgG1, IgG2a, IgG2b, etc.) with PBS, 100 μL / well, add to the appropriate wells respectively, incubate in an incubator at 37 °C for 1 h, and wash the plate in the same way after incubation.

[0153] S4-5, Color development: Add 100 μL of the color development solution to each well, and the color development time is about 10 min.

[0154] S4-6, Termination: Add 50 μL / well of the termination solution to terminate.

[0155] S4-7, Measure the absorbance values using dual wavelengths (450 nm, 630 nm), record and save the data.

[0156] The 12 selected positive cell lines were subjected to subclass identification, and finally 7 IgG-type positive hybridoma cell lines were obtained. The experimental results are shown in Table 3. After identification, the heavy chain of monoclonal antibody No. 10 was IgG1 and the light chain was κ chain, as shown in Table 4.

[0157] Table 3 Subtype identification of 7 positive hybridoma cell lines

[0158]

[0159] Table 4 Subtype identification of monoclonal antibodies After identification

[0160]

[0161]

[0162] According to the instruction manual of Beyotime Western-Blot Kit, the BP26 recombinant protein was reacted with 12 monoclonal antibodies, and the results are as Figure 5 shown. The figure shows that the BP26 recombinant protein can specifically react with the supernatants of 12 monoclonal antibody cells from immunized mice.

[0163] S5. Purification by gravity column method of mouse ascites

[0164] S5-1. Purify Protein G in the ascites using a Protein G pre-packed gravity column from Sangon Biotech (Shanghai). First, equilibrate with 5 column volumes of Binding / Wash Buffer to make the volume of the buffer in the column the same as that of the target protein, and repeat 2 - 3 times.

[0165] S5-2. Add the sample to the equilibrated gravity column and ensure that the sample stays in the column for at least 2 minutes to allow the sample to fully contact the resin in the column. Then, collect the effluent. To improve the binding efficiency, the loading operation can be repeated.

[0166] S5-3. Wash with 10 - 15 column volumes of binding / washing buffer to remove non-specifically bound impurity proteins, and collect the washed effluent.

[0167] S5-4. Elute with 5 - 10 column volumes of elution buffer and collect the eluate in batches by column volume, collecting one column volume for each batch. Detecting these batches separately can not only ensure that all target proteins are eluted, but also obtain proteins with high purity and high concentration. The eluted fractions should be immediately adjusted to neutral pH value with the neutralizing solution Tris.

[0168] S5-5. Detect the concentration of the purified antibody using the BCA method, and determine the antibody titer and antibody subtype using I-ELISA.

[0169] The BCA detection method in Thermo Fisher's Nano Drop was used to detect the concentration of ascites, and the detection results are shown in Table 5 below. The concentration of all purified monoclonal antibodies reached over 2 mg / mL.

[0170] Table 5 Concentration of 12 Monoclonal Antibodies Detected by BCA Method

[0171]

[0172]

[0173] The titer of ascites was detected by ELISA method as shown in Table 6. The titer of all monoclonal antibodies reached over 20,000, indicating the successful purification of monoclonal antibodies.

[0174] Table 6 ELISA Measurement Results of Ascites Titers of 12 Monoclonal Antibodies

[0175]

[0176] S6. Establish I-ELISA for Epitope Recognition between Monoclonal Antibodies and BP26 Peptide

[0177] The BP26 peptide synthesized in our laboratory before was used and conjugated with Keyhole Limpet Hemocyanin (KLH). The I-ELISA method was used to verify the BP26 peptides corresponding to monoclonal antibodies 1 - 12. The coating concentration of KLH was 0.025 μg / μL, the coating concentration of BP26 was 0.03 μg / μL, the coating concentration of polypeptide peptide was 0.04 μg / μL, and 4% skim milk powder buffer was used as the blocking solution. It was incubated in an incubator at 37°C for 2 h. After washing the plate three times, 100 μL / well of the monoclonal antibody stock solution was added and incubated in the incubator at 37°C for 1 h. After washing the plate three times, rabbit anti-mouse IgG diluted 1:10000 was added and incubated in the incubator at 37°C for 1 h. After washing the plate three times, it was developed colorimetrically in the dark, and OD was measured. 450nm 。

[0178] The corresponding relationship between 12 monoclonal antibodies and BP26 peptide was detected by the above method, and the corresponding results of I-ELISA detection are shown in Table 7. Among them, monoclonal antibody 10 corresponds to P3 peptide, and monoclonal antibodies 22, 28, and 44 correspond to P6 peptide.

[0179] Table 7 Epitope Recognition of BP26 Peptide and 12 Monoclonal Antibodies

[0180]

[0181] S7. Determine the Optimal Monoclonal Antibody According to the Inhibitory Effect of Positive Serum of Animal Brucellosis on Monoclonal Antibodies.

[0182] A total of 290 serum samples were collected, including 52 bovine negative sera, 44 positive sera, 54 ovine negative sera, 42 positive sera, 46 canine negative sera, 7 positive sera, 35 porcine negative sera, and 10 positive sera. First, a C-ELISA kit was used to detect the inhibitory effect of positive sera from four animals, namely cattle, sheep, pigs, and dogs, on monoclonal antibodies. The inhibitory percentage (IP) against 12 monoclonal antibodies was calculated using the formula [100 - (OD value of positive serum / OD value of negative serum) × 100]%. According to the IP values, these positive sera were divided into 5 groups: 20%, 20% - 39%, 40% - 59%, 60% - 79%, and ≥80%. If the serum with an IP value greater than 20% was considered positive, the monoclonal antibody with the highest inhibition rate was selected to establish the C-ELISA method. 450 value / OD of negative serum 450 value) × 100]%

[0183] Positive sera of Brucella melitensis from four animals, namely cattle, sheep, pigs, and dogs, were used to detect 12 monoclonal antibodies by the C-ELISA method respectively. According to the results in Table 8, monoclonal antibody 10 was inhibited by 100% bovine Brucella melitensis serum, 97.62% ovine serum, 100% canine serum, and 100% porcine serum. For the same positive sera of Brucella melitensis, other monoclonal antibodies showed much lower inhibition percentages than monoclonal antibody 10. Similarly, if the IP threshold was increased to 40%, monoclonal antibody 10 was still inhibited by the highest percentages of positive sera of bovine, ovine, canine, and porcine Brucella melitensis, which were 38.64%, 57.14%, 100%, and 95.83% respectively. Therefore, monoclonal antibody 10 was the first choice for preparing the C-ELISA kit for BP26 protein. This monoclonal antibody 10 was named hybridoma cell line BP26-7E12, and the deposit number was CCTCC NO: C202330.

[0184] Table 8 Inhibition rate IP of positive sera from different animals against 12 monoclonal antibodies

[0185]

[0186]

[0187] In summary, the present invention achieved high-efficiency soluble expression by optimizing the BP26 gene sequence, and purified a highly immunogenic protein; a high-affinity monoclonal antibody was screened by immunizing with this protein. The BP26 protein and the monoclonal antibody can significantly improve the detection sensitivity and specificity of Brucella melitensis, and are applicable to vaccine development and targeted therapy, having important clinical application value.

Claims

1. A Brucella BP26 protein, characterized in that The amino acid sequence of the BP26 protein is shown in SEQ ID NO:

1.

2. A method for preparing the Brucella BP26 protein as claimed in claim 1, characterized in that: The Brucella BP26 protein as claimed in claim 1 is selected as an antigen, a BP26 prokaryotic expression vector is constructed using homologous recombination technology, IPTG-induced expression is performed, and the BP26 protein is prepared after purification.

3. A method for preparing Brucella BP26 protein according to claim 2, characterized in that, The specific steps are as follows: S1. According to the amino acid sequence of BP26 protein, the codons were reversed to construct the BP26 prokaryotic expression gene sequence. The synthesized target gene sequence is shown in SEQ ID NO: 2; S2, transferring the synthesized target gene sequence into the expression vector pET30a-GST to obtain the pET30a recombinant plasmid; S3. Prokaryotic expression and purification of BP26 protein: The pET30a recombinant plasmid was transferred into the expression vector BL21 and IPTG-induced expression was performed. The process specifically included transformation, small-scale expression, large-scale expression, protein purification (washing inclusion bodies), and protein renaturation.

4. A method for preparing a monoclonal antibody against Brucella BP26 protein, characterized in that: The specific steps are as follows: immunizing an animal with the Brucella BP26 protein described in claim 1 or the Brucella BP26 protein prepared in claim 2; fusing tumor cells with spleen cells of the immunized animal, and taking the cell supernatant culture fluid to detect the titer and screen hybridoma cells; collecting and purifying the monoclonal antibodies secreted by the hybridoma cells, which are monoclonal antibodies against the Brucella BP26 protein.

5. The method for preparing a monoclonal antibody against Brucella BP26 protein according to claim 4, characterized in that: BALB / c mice are immunized with the Brucella BP26 protein described in claim 1 or the Brucella BP26 protein prepared in claim 2, myeloma cells SP2 / 0 are fused with spleen cells of the immunized mice, and the cell supernatant culture fluid is taken to detect the titer, hybridoma cells are screened, and then mouse ascites is prepared by an in vivo induction method, the ascites is collected and purified to prepare BP26 monoclonal antibodies.

6. A monoclonal antibody against Brucella BP26 protein, secreted by hybridoma cell line BP26-7E12 with a deposit number of CCTCC NO: C202330, with a heavy chain of IgG1 and a light chain of κ type.

7. Use of the monoclonal antibody of claim 6 in the preparation of a drug for diagnosing, preventing or treating brucellosis.

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