Preparation method and application of brucella polyepitope fusion protein-based igy

Abstract

The application discloses a preparation method and application of IgY based on a Brucella multiepitope fusion protein, the fusion protein containing 11 cell epitopes of VirB1-VirB11 and being connected in series through a flexible connecting peptide, and the amino acid sequence of the fusion protein is shown as SEQ ID NO:1; the method is to screen and connect key antigen epitopes of Brucella, construct a high-efficiency expression vector, and obtain high-affinity IgY after immunizing poultry. The IgY can significantly improve the sensitivity and specificity of Brucella detection, has a potential for passive immunotherapy, and is suitable for the fields of diagnostic reagents, drug development and livestock prevention and control.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for preparing IgY based on Brucella multi-epitope fusion protein and its application. Background Technology

[0002] Brucella is a Gram-negative intracellular parasitic bacterium that can infect humans, livestock, and various wild animals, causing a zoonotic disease called brucellosis. Although brucellosis poses a serious threat to human health and livestock development and causes significant losses to the global economy, it is often overlooked by the public due to insufficient health education.

[0003] Currently, Brucella detection mainly relies on serological methods (such as ELISA and agglutination assays), but existing antibody reagents lack sufficient sensitivity and specificity, and traditional monoclonal antibody preparation is costly and time-consuming. IgY, an antibody extracted from avian egg yolks, offers advantages such as painless collection, high yield, and low cross-reactivity; however, its titer and specificity are highly dependent on antigen design. Traditional antigens are mostly single epitope proteins or inactivated whole bacteria, resulting in weak immunogenicity and incomplete epitope coverage. Therefore, developing a highly efficient IgY preparation technology based on multi-epitope fusion proteins is of great significance for improving the diagnosis and control of brucellosis. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing IgY based on Brucella multi-epitope fusion protein and its application. By optimizing antigen design, the potency and specificity of IgY are significantly improved, and its application in the detection, prevention or treatment of brucellosis is expanded.

[0005] To achieve the purpose of the invention, the present invention provides a Brucella multi-epitope fusion protein containing 11 cellular epitopes of VirB1-VirB11, which are linked together by flexible linker peptides, and its amino acid sequence is shown in SEQ ID NO:1.

[0006] To achieve the purpose of the invention, the present invention also provides a method for preparing IgY, wherein the above-mentioned fusion protein is used to immunize poultry, and IgY is extracted and purified from the egg yolk.

[0007] To achieve the purpose of the invention, the present invention also provides the application of the above-mentioned IgY in a brucellosis detection kit.

[0008] To achieve the purpose of the invention, the present invention also provides the application of the above-mentioned IgY in the preparation of brucellosis prevention or treatment drugs.

[0009] Compared with the prior art, the present invention has the following advantages:

[0010] (1) Multi-epitope fusion design: Immunogenicity is enhanced by combining epitopes, covering multiple Brucella serotypes;

[0011] (2) High-efficiency expression system: Optimize the host and vector to increase the yield of fusion protein (e.g., ≥50 mg / L fermentation broth);

[0012] (3) Low-cost IgY process: simplified purification steps, IgY yield ≥10mg / egg yolk;

[0013] (4) Multifunctional application: It has both diagnostic sensitivity (detection limit ≤1ng / mL) and therapeutic potential. Attached Figure Description

[0014] Figure 1 Electrophoretic identification of low-level expression of fusion protein; where M: Marker; 1: Uninduced control (BL21); 2-3: IPTG induced (BL21); gel: 12% SDS-PAGE;

[0015] Figure 2 Electrophoretic identification of fusion protein in large quantities; where M: Marker; 1: whole bacteria after sonication; 2: supernatant after sonication; 3: precipitate after sonication; gel: 12% SDS-PAGE;

[0016] Figure 3 Electrophoresis image for purification (soluble nickel column) of fusion protein after high-level expression; M: Marker; 1: Original protein; 2: Flow-through; 3: 15mM imidazole elution; 4: 60mM imidazole elution; 5-7: 300mM imidazole elution; Gel: 12% SDS-PAGE;

[0017] Figure 4 Electrophoresis image for purification (denatured nickel column) of fusion protein after high-level expression; M: Marker; 1: Original protein; 2: Flow-through; 3: 15mM imidazole elution; 4: 60mM imidazole elution; 5-7: 500mM imidazole elution; Gel: 12% SDS-PAGE;

[0018] Figure 5 Electrophoretic identification image of purified fusion protein after high-level expression; M: Marker; 1: Purified protein renaturation; Gel: 12% SDS-PAGE;

[0019] Figure 6 SDS-PAGE analysis of purified IgY. Detailed Implementation

[0020] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0021] The materials used in this invention are as follows:

[0022] Example 1

[0023] A Brucella multi-epitope fusion protein comprising 11 cellular epitopes, VirB1-VirB11, linked together by a flexible linker peptide, the amino acid sequence of which is shown in SEQ ID NO:1.

[0024] Example 2

[0025] Preparation of multi-epitope fusion proteins

[0026] (1) Selected epitopes of Brucella: VirB1 (AA 156-170, AA 179-189, AA 220-234), VirB2 (AA 71-86, AA41-52, AA30-40), VirB3 (AA 12-31, AA 116-125, AA 36-47), VirB4 (AA 510-516, AA 175-185, AA 732-741, AA110-117), VirB5 (AA 189-195, AA 164-177, AA 144-154, AA137-142), and VirB6 (AA 305-319, AA 262-275, AA... Epitopes 23-31, AA326-334), VirB7 (AA 28-33, AA 34-53, AA 13-19), VirB8 (AA 7-26, AA 184-193, AA213-218, AA35-40), VirB9 (AA19-30, AA 261-275, AA 163-172, AA188-195), VirB10 (AA 67-81, AA 87-98, AA 324-330, AA251-256), and VirB11 (AA 227-239, AA 238-243, AA 48-55, AA138-150);

[0027] (2) Epitope genes were ligated via overlap PCR, inserted into the pET-28a vector, transformed into E. coli BL21, and induced to express by IPTG; the specific process is as follows:

[0028] (2-1) Transformation

[0029] (2-1-1) Take out the competent cells (BL21) stored at -80℃ and thaw them slowly on ice.

[0030] (2-1-2) Add competent cells to the ligation product, mix well, and place on ice for 30 min.

[0031] (2-1-3) Heat shock at 42℃ for 90 seconds.

[0032] (2-1-4) After ice bath for 2 min, add 800 μl of antibiotic-free LB medium.

[0033] (2-1-5) Incubate at 37℃ for 45 min.

[0034] (2-1-6) Centrifuge at 5000 rpm for 3 min, discard most of the supernatant, keep about 100-150 μl, resuspend the bacterial cells, select LB plates with corresponding resistance, and spread them on the plates.

[0035] (2-1-7) Dry the product and incubate it upside down in a 37℃ incubator overnight.

[0036] (2-2) Small-scale expression

[0037] (2-2-1) Pick single clones from the transformed plate and transfer them to 1.5 ml of LB liquid medium containing the corresponding resistance, and incubate at 37°C and 200 rpm.

[0038] (2-2-2) Cultured until OD = 0.6-0.8, induced with IPTG (0.5mM), cultured at 37℃ and 200rpm for 2h.

[0039] (2-2-3) Take 1 ml of the induced bacterial culture, centrifuge at 12000 rpm for 1 min, discard the supernatant, and disperse the precipitate with 50-100 μl of 10 mM Tris-HCl (pH 8.0) solution (the amount of buffer added depends on the amount of bacterial cells). Add an equal volume of 2× loading buffer, boil at 100℃ for 5 min, and detect by electrophoresis (e.g., ...). Figure 1 (As shown).

[0040] (2-3) Extensive expression

[0041] (2-3-1) Add 1-2 μl of activated bacterial solution to 5 ml of the corresponding resistant LB liquid medium and incubate at 37°C and 200 rpm.

[0042] (2-3-2) Transfer the cultured bacterial solution to 250 ml of the corresponding resistant LB liquid medium, incubate at 37°C and 200 rpm until OD = 0.6-0.8, and induce overnight with IPTG (0.5 mM) at 16°C.

[0043] (2-3-3) Harvesting: Centrifuge at 8000 rpm for 6 min. Discard the supernatant.

[0044] (2-3-4) Ultrasonic disruption: The bacterial cells were dispersed by blowing with 20-30 ml of 10 mM Tris-HCl (pH 8.0) solution and then ultrasonically disrupted (500 W, 180 times, 5 seconds each time, with a 5-second interval).

[0045] (2-3-5) Electrophoresis to determine the expression pattern: Take 100 μl of the sonicated bacterial suspension, centrifuge at 12000 rpm for 10 min, transfer 50 μl of supernatant to another EP tube, remove the supernatant completely, and then disperse the precipitate with 50 μl of 10 mM Tris-HCl (pH 8.0) solution. Electrophoresis detection (e.g.) Figure 2 (As shown).

[0046] (3) After purification, a fusion protein with a purity >95% (molecular weight approximately 28 kDa) was obtained. The specific process is as follows:

[0047] (3-1) Protein purification (soluble nickel column)

[0048] (3-1-1) Wash the nickel column (Ni Sepharose 6Fast Flow, GE Healthcare) with deionized water until pH 7.0.

[0049] (3-1-2) Apply nickel until pH 2-3.

[0050] (3-1-3) Wash the column with deionized water until pH 7.0.

[0051] (3-1-4) Equilibrate the nickel column with 10mM Tris-HCl (pH 8.0) solution, approximately 100ml.

[0052] (3-1-5) Equilibrate the nickel column with approximately 50 ml of a 10 mM Tris-HCl (pH 8.0) solution containing 0.5 M sodium chloride.

[0053] (3-1-6) Dilute the sample and load it. The sample contains sodium chloride, with a final concentration of 0.5M.

[0054] (3-1-7) After loading the sample, wash the column with a 10mM Tris-HCl (pH 8.0) solution containing 0.5M sodium chloride.

[0055] (3-1-8) The protein peaks were collected by eluting with 10mM Tris-HCl (pH 8.0) (containing 0.5M sodium chloride) solutions containing 15mM imidazole, 60mM imidazole and 300mM imidazole respectively.

[0056] (3-1-9) Electrophoresis detection of protein purification effect (e.g.) Figure 3 (As shown).

[0057] (3-2) Protein purification (denaturing nickel column)

[0058] (3-2-1) Wash the nickel column (Ni Sepharose 6Fast Flow, GE Healthcare) with deionized water until pH 7.0.

[0059] (3-2-2) Apply nickel until pH 2-3.

[0060] (3-2-3) Wash the column with deionized water until pH 7.0.

[0061] (3-2-4) Equilibrate the nickel column with 10mM Tris-HCl (pH 8.0) solution, approximately 100ml.

[0062] (3-2-5) Equilibrate the nickel column with approximately 50 ml of a 10 mM Tris-HCl (pH 8.0) solution containing 8 M urea and 0.5 M sodium chloride.

[0063] (3-2-6) Dilute the sample and load it. The sample contains 0.5M sodium chloride, 8M urea, and 10mM Tris-HCl (pH 8.0).

[0064] (3-2-7) After the sample loading is completed, the column is washed with a 10mM Tris-HCl (pH 8.0) solution containing 8M urea and 0.5M sodium chloride.

[0065] (3-2-8) The protein peaks were collected by eluting with 10mM Tris-HCl (pH 8.0) solutions containing 15mM imidazole, 60mM imidazole and 500mM imidazole respectively.

[0066] (3-2-9) Electrophoresis detection of protein purification effect (e.g.) Figure 4 (As shown).

[0067] (3-3) Protein refolding

[0068] (3-3-1) Dialyze the sample with dialysis buffer (1% glycine, 0.1% SDS, 5% glycerol, 10 mM Tris-HCl, pH 8.0, with urea concentration gradients of 6 M, 4 M, and 2 M) at 4°C for refolding. Each urea concentration was used for 3 h, with the final stage being overnight dialysis.

[0069] (3-3-2) Dialyze twice with 1% glycine and 10mM Tris-HCl (pH 8.0), 3 hours each time.

[0070] (3-3-3) Centrifuge at 12000 rpm for 10 min, collect the supernatant, and perform electrophoresis for detection (e.g.) Figure 5 (As shown).

[0071] Example 3: Preparation and potency determination of IgY

[0072] A method for preparing IgY involves immunizing poultry with the fusion protein from Example 1, and then extracting and purifying IgY from the egg yolk; the specific process is as follows:

[0073] (1) Immunization of chickens

[0074] SPF-treated laying hens (25 weeks old) were divided into three experimental groups, labeled 1A, 1B, 2A, 2B, 3A, and 3B. A negative control group received PBS (0.01 M, pH 7.4) as the solvent. The first group (1A, 1B) involved inoculating each hen with 10⁶ CFU / mL of *B. abortus* S66 and immunizing them with an equal volume of Freund's adjuvant. In the second group (2A, 2B) and the third group (3A, 3B), hen were inoculated with 2 mg / mL of laboratory-preserved fusion protein and 2 mg / mL of LPS, respectively, each mixed with Freund's adjuvant. Immunization was performed via intramuscular injection every two weeks for five rounds, with each injection being 300 μL. Freund's complete adjuvant (CFA) was used for initial immunization, while Freund's incomplete adjuvant (IFA) was used for subsequent immunizations. IgY antibody titers in hen serum and egg yolk were assessed at days 14, 28, 42, 56, and 70 post-immunization.

[0075] (2) Purification and identification of IgY

[0076] Eggs were collected for one week, 14 days after the last immunization. IgY was extracted from the egg yolks using the PEG6000 method, as shown below:

[0077] (2-1) The collected eggs were sterilized with 75% ethanol. Then, the eggshells were gently cracked, the egg whites were removed, and the yolks were collected using a common kitchen egg yolk separator. The yolks were placed on filter paper and gently stirred to remove excess egg white. Next, the yolks were punctured, and the yolk mixture was collected in a 50 mL centrifuge tube. The volume of the collected yolk mixture (V1, mL) was recorded.

[0078] (2-2) Add 2×V1 (mL) of PBS to the collected egg yolk liquid and record the total volume (V2, mL). Then, add 3.5% (w / v) of PEG6000 (the amount of PEG6000, g = 3.5% × V2). Gently rotate the mixture for 10 minutes to mix thoroughly. Centrifuge at 10000 × g for 20 minutes at 4°C. Filter the supernatant through filter paper, collect and record the volume (V3, mL).

[0079] (2-3) Add 8.5% PEG6000 to the collected supernatant (the amount of PEG6000, g = 8.5% × V3). Gently rotate the mixture for 10 minutes to mix thoroughly. Centrifuge at 10000 × g for 20 minutes at 4°C, discard the supernatant, and collect the precipitate.

[0080] (2-4) Resuspend the precipitate in 10 mL of PBS and add 1.2 g of PEG6000. Gently rotate the mixture for 10 minutes and mix thoroughly using a glass rod and a vortex mixer. Centrifuge at 10000×g for 20 minutes at 4 °C, discard the supernatant, collect the precipitate, and resuspend it in 1 mL of PBS.

[0081] (2-5) The finally collected IgY was placed in a dialysis bag (10 kDa) and dialyzed overnight with 0.1% saline, then transferred to PBS for dialysis for 4 hours. The dialysis samples were collected and stored at -20°C. The concentration of IgY was determined using a BCA protein quantification kit, and the purity of IgY was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (12% SDS-PAGE). Figure 6 (As shown).

Claims

1. A Brucella multi-epitope fusion protein, characterized in that, It contains 11 cellular epitopes of VirB1-VirB11, linked together by flexible linker peptides, and its amino acid sequence is shown in SEQ ID NO:

1.

2. A method for preparing IgY, characterized in that, IgY was obtained by immunizing poultry with the fusion protein of claim 1 and extracting and purifying it from the egg yolk.

3. The use of IgY as described in claim 2 in a brucellosis detection kit.

4. The use of the IgY according to claim 2 in the preparation of brucellosis prevention or treatment drugs.

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