Mycobacterium bovis nano antibody and application thereof

Abstract

The invention provides a mycobacterium bovis nano antibody and application thereof, and the amino acid sequence of the nano antibody is SEQ ID NO: 1. The nucleotide sequence of the coding gene is SEQ ID NO: 2. The invention provides a nano antibody capable of specifically recognizing and neutralizing mycobacterium bovis, and the provided nano antibody has good recognition and binding capacity for mycobacterium bovis and has application prospects in preparation of a treatment or detection reagent for mycobacterium bovis.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioactive products, and particularly relates to a nanobody against Mycobacterium bovis and its application. Background Art

[0002] Bovine tuberculosis is a zoonotic infectious disease caused by Mycobacterium bovis, which not only causes significant economic losses to the livestock industry but also seriously threatens human health. Mycobacterium bovis can be transmitted to humans through air, food chain and other routes, leading to tuberculosis. At present, the diagnosis of bovine tuberculosis mainly relies on traditional bacteriological methods and immunological methods. However, these methods have problems such as long time consumption, low sensitivity and poor specificity, and it is difficult to meet the requirements of rapid and accurate diagnosis.

[0003] In terms of treatment, bovine tuberculosis mainly relies on antibiotics, but the long-term use has led to the emergence of drug-resistant strains, and the treatment effect has decreased significantly. In addition, antibiotic treatment has a long cycle, high cost, and may pose potential risks to the environment and food safety. Therefore, the development of new diagnostic and treatment means has become the focus of current research.

[0004] In recent years, nanobodies, as a new type of biomolecule, have shown great potential in disease diagnosis and treatment. Nanobodies are single-domain antibodies derived from camelid animals (such as alpacas and camels), and have the advantages of small molecular weight, high stability, easy expression and modification, etc. Compared with traditional antibodies, nanobodies can better penetrate tissues, and have stronger specificity and affinity for antigen binding. In addition, nanobodies can be mass-produced by genetic engineering with lower cost, and are suitable for industrial application.

[0005] In the field of bovine tuberculosis, the application research of nanobodies is still in its infancy. At present, there is no mature nanobody product for the diagnosis and treatment of bovine tuberculosis. Therefore, the development of a nanobody with strong specificity, high sensitivity and good stability is of great significance for improving the diagnostic accuracy and treatment of bovine tuberculosis. Summary of the Invention

[0006] The present invention provides a nanobody against Mycobacterium bovis and its application, so as to make up for the deficiencies of the prior art.

[0007] The present invention first provides a nanobody that specifically binds to Mycobacterium bovis, and the amino acid sequence of the nanobody is as follows:

[0008] GAAGGVWGRLGAAGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWV SAISGGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAKLL GSSYAMDYWGQGTLVTVSS(SEQ ID NO:1).

[0009] The nucleotide sequence of one of its encoding genes is as follows:

[0010] GGAGCTGCTGGAGGTGTTTGGGGACGGCTCGGAGCTGCTGGAGGATC

[0011] GCTGCGGCTCAGCTGTGCTGCTTCCGGCTTCACCTTCTCCAGCTACGCTAT

[0012] GAGCTGGGTACGGCAGGCTCCTGGCAAGGGACTGGAATGGGTATCTGCTA

[0013] TCAGCGGCGGAGGAAGCACCTACTACGCTGATAGCGTTAAGGGCCGTTTC

[0014] ACCATCAGCCGGGATAACAGCAAGAACACTCTGTACCTCCAGATGAACTC

[0015] CCTGCGGGCTGAGGACACCGCTGTTTATTACTGTGCTGCTAAGCTGCTCGG

[0016] ATCCTCCTACGCTATGGACTATTGGGGACAGGGAACTCTGGTTACTGTGAG

[0017] CAGC(SEQ ID NO:2);

[0018] Another aspect of the present invention further provides a recombinant expression vector, into which the above nucleic acid fragment is inserted.

[0019] The present invention also provides a host cell containing the above expression vector.

[0020] The present invention also provides the use of the above nanobody in the preparation of products for preventing or treating Mycobacterium bovis.

[0021] The present invention provides a nanobody capable of specifically recognizing Mycobacterium bovis. The provided nanobody has good recognition and binding capabilities for Mycobacterium bovis, and has the application prospect of preparing therapeutic or detection reagents for Mycobacterium bovis. Description of the Drawings

[0022] Figure 1 : Agarose gel electrophoresis detection result diagram;

[0023] Figure 2 : Recombinant protein Western blot analysis result diagram;

[0024] Figure 3 : SDS-PAGE identification diagram of nanobody purification;

[0025] Figure 4 : Nanobody specificity identification result diagram;

[0026] Figure 5 : Nanobody neutralization experiment result diagram. Detailed Embodiments

[0027] The object of the present invention is to provide a nanobody that specifically binds to Mycobacterium bovis. The provided nanobody has good recognition and binding capabilities for Mycobacterium bovis.

[0028] The present invention will be described in detail below in conjunction with the embodiments and the drawings.

[0029] Example 1: Preparation of Mycobacterium bovis nanobody

[0030] 1. Preparation of immunogen

[0031] Take the inactivated Mycobacterium bovis (ATCC 19210) bacterial solution, and adjust the concentration to 1×10 8 CFU / mL with PBS buffer. Mix the bacterial solution with Freund's complete adjuvant (1:1 volume ratio), and use the emulsion as the immunogen to immunize alpacas.

[0032] 2. Immunization of alpacas

[0033] Subcutaneously inject 1 mL of the immunogen (containing 5×10^7 CFU) into the neck of the alpaca, and inject at multiple points (0.2 mL per point). Boost immunization is carried out every 2 weeks for a total of 4 times. Freund's incomplete adjuvant is used during boost immunization. One week after the last immunization, collect 50 mL of the alpaca's peripheral blood.

[0034] 3. Isolation of peripheral blood lymphocytes

[0035] Mix the collected peripheral blood with an equal volume of PBS buffer. Slowly add the diluted blood to the lymphocyte separation solution at a ratio of 2:1. Centrifuge at 400 g for 30 min at room temperature. Collect the lymphocyte layer at the interface and wash it twice with PBS (300 g, 10 min).

[0036] 4. Total RNA extraction and cDNA synthesis

[0037] Extract total RNA from lymphocytes using TRIzol reagent. The specific operation is as follows:

[0038] Add 1 mL of TRIzol to lyse the cells and let it stand at room temperature for 5 minutes; add 0.2 mL of chloroform, shake vigorously for 15 seconds, and let it stand at room temperature for 3 minutes; centrifuge at 12,000×g for 15 minutes at 4℃; transfer the supernatant to a new tube, add 0.5 mL of isopropanol, and let it stand at room temperature for 10 minutes; centrifuge at 12,000×g for 10 minutes at 4℃ and discard the supernatant; wash the precipitate with 75% ethanol and centrifuge at 7,500×g for 5 minutes at 4℃; dissolve it in 50 μL of nuclease-free pure water after air drying.

[0039] Synthesize cDNA using M-MLV reverse transcriptase:

[0040] Reaction system: 5 μg of total RNA, 1 μL of random primer (50 μM), 1 μL of dNTPs (10 mM), 4 μL of 5× buffer, 1 μL of M-MLV reverse transcriptase, and make up to 20 μL with nuclease-free water.

[0041] Reaction conditions: 25℃ for 10 minutes, 37℃ for 50 minutes, 70℃ for 15 minutes.

[0042] 5. Nanobody gene amplification

[0043] Design specific primers:

[0044] VHH-F: 5'-GATGTGCAGCTGCAGGAGTCTGGRGGAGG-3';

[0045] VHH-R: 5'-GGACTAGTGCGGCCGCTGGAGACGGTGACCTGGGT-3';

[0046] PCR reaction system: 2 μL of cDNA template, 1 μL of VHH-F (10 μM), 1 μL of VHH-R (10 μM), 0.5 μL of Phusion polymerase, 4 μL of dNTPs (2.5 mM), 10 μL of 5× buffer, and make up to 50 μL with nuclease-free water.

[0047] PCR reaction conditions: 98°C, 30 seconds; 98°C, 10 seconds, 55°C, 30 seconds, 72°C, 30 seconds, 35 cycles; 72°C extension for 10 minutes.

[0048] 6. Construction of the nanobody gene library

[0049] Clone the PCR product into the pComb3X vector and transform competent Escherichia coli TG1 cells. Pick monoclonal colonies and inoculate them into 2×YT medium (containing 100 μg / mL ampicillin), and culture overnight at 37°C. Collect the bacterial solution, extract the plasmid and perform PCR identification. The results of agarose gel electrophoresis are shown in Figure 1 , proving the successful construction of the recombinant plasmid and constructing the nanobody gene library.

[0050] Example 2: Expression and purification of a specific nanobody against the MPB70 protein

[0051] 1. Expression of the MPB70 protein

[0052] Pick positive clones and inoculate them into 5 mL of LB medium (containing 50 μg / mL kanamycin), and culture with shaking overnight at 37°C. Inoculate the overnight culture into 50 mL of LB medium at a ratio of 1:100 and culture at 37°C until the OD600 reaches 0.6. Add IPTG (final concentration 0.5 mM) and induce expression at 16°C for 16 hours. Centrifuge at 4,000×g for 10 minutes at 4°C to collect the bacteria. Resuspend the bacteria in 10 mL of PBS buffer (containing 1 mM PMSF), sonicate, and centrifuge at 12,000×g for 30 minutes at 4°C. The results ( Figure 2 ) show that specific bands appear at 43 kDa in the induced strain, soluble and inclusion body proteins, and there is none in the control group, indicating that the recombinant protein p-MPB70 binds specifically to the antibody and has good immunogenicity.

[0053] Load the supernatant onto a nickel column and wash with PBS buffer until the baseline is stable. Elute the target protein with PBS buffer containing 250 mM imidazole. Load the eluate into a dialysis bag (10 kDa molecular weight cut-off) and dialyze with PBS buffer to remove imidazole overnight at 4°C. Determine the protein concentration, aliquot and store at -80°C.

[0054] 2. Screening for specific nanobodies

[0055] Coat a 96-well plate with MPB70 protein (10 μg / mL), 100 μL per well, overnight at 4°C. Discard the coating solution, add 5% skim milk (200 μL per well), and incubate at 37°C for 1 hour for blocking. Add the phage library of nanobodies (10^11 pfu / mL), 100 μL per well, and incubate at 37°C for 1 hour. Wash 3 times (with PBS-Tween20) to wash away unbound phages. Add acidic elution buffer (0.1 M glycine-HCl, pH 2.2), 100 μL per well, and incubate at room temperature for 10 minutes. Immediately add 1 M Tris-HCl (pH 9.1) to neutralize the elution buffer. Mix the eluate with log-phase Escherichia coli TG1 and let stand at 37°C for 30 minutes. Inoculate into 2×YT medium (containing 100 μg / mL ampicillin) and culture with shaking overnight at 37°C. Collect the bacterial solution, extract phages, and repeat the above steps for 3 rounds to obtain high-affinity nanobodies.

[0056] 3. Expression and purification of nanobodies

[0057] Clone the screened nanobody gene into the pET-28a(+) vector and transform competent Escherichia coli BL21(DE3) cells. Pick positive clones, inoculate into LB medium (containing 50 μg / mL kanamycin), and culture at 37°C until the OD600 reaches 0.6. Add IPTG (final concentration 0.5 mM) and induce expression at 16°C for 16 hours. Collect the bacterial cells, sonicate and centrifuge to take the supernatant. Purify the nanobody using nickel column affinity chromatography and perform SDS-PAGE analysis. The results are as Figure 3 , and the purification effect is good. After sequencing and sequence alignment analysis, the nanobody was obtained, and the sequence of its encoding gene is as follows:

[0058] GGAGCTGCTGGAGGTGTTTGGGGACGGCTCGGAGCTGCTGGAGGATC

[0059] GCTGCGGCTCAGCTGTGCTGCTTCCGGCTTCACCTTCTCCAGCTACGCTAT

[0060] GAGCTGGGTACGGCAGGCTCCTGGCAAGGGACTGGAATGGGTATCTGCTA

[0061] TCAGCGGCGGAGGAAGCACCTACTACGCTGATAGCGTTAAGGGCCGTTTC

[0062] ACCATCAGCCGGGATAACAGCAAGAACACTCTGTACCTCCAGATGAACTC

[0063] CCTGCGGGCTGAGGACACCGCTGTTTATTACTGTGCTGCTAAGCTGCTCGG

[0064] ATCCTCCTACGCTATGGACTATTGGGGACAGGGAACTCTGGTTACTGTGAG

[0065] CAGC(SEQ ID NO:2);

[0066] The amino acid sequence of the encoded protein is as follows:

[0067] GAAGGVWGRLGAAGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWV SAISGGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAKLL GSSYAMDYWGQGTLVTVSS(SEQ ID NO:1).

[0068] Example 3: Verification of the specificity and antibacterial effect of nanobodies

[0069] 1. Specificity detection

[0070] Coat Mycobacterium bovis, Mycobacterium tuberculosis, Brucella and Actinomyces (1 μg / mL) in 96-well plates, 100 μL / well, overnight at 4 °C. Discard the coating solution, add 5% skim milk (200 μL / well), and block at 37 °C for 1 hour. Add nanobody (100 nM), 100 μL / well, and incubate at 37 °C for 1 hour. Wash 3 times (PBS-Tween 20). Add HRP-labeled secondary antibody (diluted 1:5,000), 100 μL / well, and incubate at 37 °C for 1 hour. Wash 3 times. Add TMB substrate solution (100 μL / well), and develop color at room temperature for 10 minutes. Add 2M H2SO4 to terminate the reaction (50 μL / well), and measure the absorbance at 450 nm( Figure 4 ), The nanobody only binds to the Mycobacterium bovis antigen, indicating its high specificity.

[0071] 2. Neutralization test

[0072] Serial two-fold dilute 100 μL of nanobody, with a concentration range of 0.781 μM to 50 μM, and take PBS buffer as the negative control. Mix the diluted nanobodies with an equal volume of Mycobacterium bovis(1×10 6 CFU / mL) respectively, and incubate at 37 °C for 1 hour. Inoculate BAMs in 96-well plates, 1×10 per well 4Cells were cultured at 37 °C until they adhered to the wall. The nanobody M.bovis-Nb mixture was added to a 96-well plate, 100 μL per well, and cultured at 37 °C for 1 hour. The supernatant was discarded, and the cells were washed twice with PBS buffer. The medium was changed to fresh RPMI 1640 medium (containing 10% fetal bovine serum), and the cells were continuously cultured at 37 °C for 24 hours. The cells were fixed with 4% paraformaldehyde and incubated at room temperature for 15 minutes. 0.1% Triton X-100 was added to permeabilize the cells, and the cells were incubated at room temperature for 10 minutes. 5% skim milk was added and blocked at room temperature for 1 hour. HRP-labeled goat anti-rabbit secondary antibody (diluted 1:5,000) was added and incubated at room temperature for 1 hour. TMB chromogenic solution was added and developed at room temperature for 10 minutes. 2M H 2 SO 4 was added to terminate the reaction, and the absorbance at 450 nm was measured. The results Figure 5 showed that the nanobody concentration of 25 μM could completely neutralize Mycobacterium bovis.

[0073] 3. Animal experiments

[0074] Six healthy adult cows were selected, and each cow was inoculated nasally with 1 mL of Mycobacterium bovis bacterial solution (containing 1×10 6 CFU) for modeling, and then randomly divided into an experimental group and a control group, with 3 cows in each group. Treatment started on the 7th day after inoculation. Each cow in the experimental group was intravenously injected with a nanobody solution (1 mg / kg) once a week for 4 consecutive weeks. The control group was injected with an equal volume of PBS buffer. The clinical symptoms of the cows were recorded weekly (scoring criteria: 0 points: no clinical symptoms, 1 point: mild cough, normal appetite; 2 points: moderate cough, decreased appetite; 3 points: severe cough, fever, obvious decreased appetite). The tuberculin skin test (TST) and interferon-γ release assay (IGRA) were performed on the 0th, 14th, 28th, and 42nd days after inoculation. 0.1 mL of bovine tuberculin (PPD) was injected intradermally into the neck, and the skin thickening was measured 48 hours later (a skin thickening of ≥4 mm was judged as positive). Peripheral blood was collected, plasma was separated, and the interferon-γ level was detected using a kit. Lung tissue samples were collected in vivo using a thoracoscope and biopsy needle, and the bacterial count was determined using a bacterial load detection kit.

[0075] Table 1: Results of clinical manifestations of experimental animals (score)

[0076]

[0077] Table 2: Results of positive tuberculin skin test (TST / head)

[0078]

[0079] Table 3: Interferon-γ level of experimental animals (IGRA / (IU / mL))

[0080]

[0081] Table 4: Average bacterial load in the lungs of experimental animals on the 42nd day (CFU / g)

[0082]

[0083] As can be seen from the results in Tables 1 - 4, with the extension of the infection time, the clinical symptoms of the control group of cattle gradually worsened, while the symptoms of the experimental group of cattle improved significantly. The nanobody significantly improved the clinical symptoms of the infected cattle, significantly reduced the positive rate of the bovine tuberculin test, decreased the level of interferon-γ, indicating that it inhibited the immune response of Mycobacterium bovis, and decreased the bacterial load in the lungs, indicating that it had a significant antibacterial effect.

[0084] In summary, the present invention provides a nanobody that specifically binds to Mycobacterium bovis and a method for preparing the same. Through experimental verification, the nanobody has high specificity, high affinity and a significant antibacterial effect, and has broad application prospects in the diagnosis and treatment of bovine tuberculosis.

Claims

1. A nanobody that specifically binds to bovine Mycobacterium tuberculosis, characterized in that: The amino acid sequence of the nanobody is SEQ ID NO:

1.

2. A nucleic acid fragment, characterized in that The nucleic acid fragment encodes the nanobody according to claim 1.

3. The nucleic acid fragment according to claim 2, characterized in that The sequence of the nucleic acid fragment is SEQ ID NO:

2.

4. A recombinant expression vector, characterized in that: The nucleic acid fragment according to claim 2 is inserted into the recombinant expression vector.

5. A recombinant host cell, characterized in that The recombinant host cell carries the recombinant expression vector according to claim 4.

6. Use of the nanobody according to claim 1 in the preparation of a product for preventing or treating bovine tuberculosis.

7. A product for preventing or treating bovine tuberculosis Mycobacterium, characterized in that: The preparation contains the nanobody according to claim 1 at a pharmacologically effective concentration.

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