Nanometer antibody for resisting brucella Omp25 protein and application of nanometer antibody in competitive ELISA (enzyme-linked immuno sorbent assay) detection

Abstract

The invention is applicable to the technical field of biology, and provides a nano antibody for resisting Brucella Omp25 protein and application of the nano antibody in competitive ELISA (enzyme-linked immuno sorbent assay) detection. The amino acid sequence of the nano antibody is as shown in SEQ ID NO. 3. Omp25 protein of prokaryotic expression brucella is used as an immunogen to immunize alpaca, peripheral blood lymphocyte total RNA of the alpaca is extracted, a VHH fragment is amplified through reverse transcription and two rounds of nested PCR, the VHH fragment and a pComb3Xss vector are subjected to enzyme digestion and connection and transformed into a TG1 competent cell, a phage display technology is utilized to perform multiple rounds of panning, and the recombinant protein is obtained. And screening out the nano antibody for resisting the Brucella Omp25 protein. The competitive ELISA detection method established by using the nano antibody can effectively detect the level of the Brucella antibody in sheep serum, and is low in detection limit and high in sensitivity; the developed matched kit is high in specificity, and the detection time is greatly shortened.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to a nanobody against Brucella Omp25 protein and its application in competitive ELISA detection. Background Technology

[0002] Brucellosis is a globally prevalent zoonotic disease, causing over 500,000 infections annually. The causative agent is Brucella spp., a Gram-negative intracellular parasite that can be transmitted through the digestive tract, respiratory tract, skin, and mucous membranes. The disease is widespread globally, with high-risk groups including those residing in or traveling to endemic areas, and those with occupational exposure to Brucella. Clinical symptoms after infection include recurrent high fever, profuse sweating, fatigue, and joint pain.

[0003] Brucella lacks classic virulence factors, and its pathogenicity is highly dependent on immune escape mechanisms mediated by outer membrane proteins. Among them, the outer membrane protein Omp25 has become a focus of research due to its central role in pathogen-host interactions. Omp25 The gene, located on Brucella chromosome I, encodes Omp25, a transmembrane protein composed of 214 amino acids. Structural studies show that Omp25 forms a typical β-barrel conformation, with its four extracellular loop structures (Loop 1-4) directly involved in host cell adhesion. Among them, Loop 3 mediates bacterial internalization by binding heparin sulfate to the host cell surface. This protein is highly conserved in Brucella species, but in Brucella canis, the mutation at position 52 (arginine to cysteine) significantly reduces its virulence, indicating that this protein is not only a structural protein but also a key factor regulating Brucella virulence. Experiments have confirmed that the Omp25 deletion mutant strain exhibits a 100-fold decrease in bacterial load in the spleen of a mouse model, demonstrating potential for future detection and treatment.

[0004] Currently, bacterial isolation and culture remain the gold standard for Brucella detection. However, its sensitivity is limited by the bacterial load in the sample, with the positive rate plummeting to below 20% after chronic infection or antibiotic treatment. The long culture period (2-4 weeks) and high risk of operator exposure, along with the difficulty of growing some atypical strains in conventional media, limit the rapid diagnosis of Brucella. In immunological testing, the SAT test relies on the visible agglutination reaction between Brucella whole-cell antigens and serum antibodies. This method is simple and inexpensive, but its sensitivity drops to 60-70% in chronic infections and exhibits 15-30% cross-reactivity with Yersinia and Salmonella. Furthermore, high-titer Brucella sera can produce false negatives due to antibody excess, requiring serial dilution and retesting, delaying diagnosis. Enzyme-linked immunosorbent assay (ELISA) can improve detection specificity by using recombinant protein antigens. Indirect ELISA for IgG / IgM has demonstrated good detection performance with a sensitivity of 92-96%. However, current immunological detection methods, including ELISA, still mainly rely on traditional monoclonal or polyclonal antibodies. These antibodies have large molecular weights, low stability, and high costs. Moreover, the production process requires immunizing a large number of experimental animals, which does not comply with animal welfare principles. Therefore, their application in existing detection methods still cannot meet the requirements of production practice. Thus, it is particularly necessary to develop novel antibodies with high stability, high specificity, high affinity, and simple preparation process.

[0005] In camel species such as alpacas and dromedary camels, as well as cartilaginous fish such as sharks and rays, there exist naturally occurring heavy-chain antibodies (HCAbs) lacking the light chain. The variable domain of this heavy chain is called a nanobody (Nbs), also known as a variable domain of heavy chain of heavy-chain antibody (VHH). Nanobodies have a molecular weight of only 15 kDa. Compared to traditional IgG antibodies, nanobodies offer advantages such as ease of expression, ease of modification, good stability, high specificity, and high affinity. Most importantly, nanobodies can recognize hidden antigenic epitopes that conventional antibodies cannot. The extended CDR1 region and longer CDR3 region of nanobodies give them prominent antigenic determinants, allowing them to penetrate deep into the gaps in folded proteins. This structural change allows nanobodies to bind to the surface and gaps of antigens, resulting in better antigen recognition. Currently, there are no reports on nanobodies targeting Brucella Omp25 protein and their application in serological detection. Therefore, this invention proposes a nanobody against Brucella Omp25 protein and its application in competitive ELISA detection. Summary of the Invention

[0006] The purpose of this invention is to provide a nanobody against Brucella Omp25 protein and its application in competitive ELISA detection, aiming to solve the problems mentioned in the background art.

[0007] The objective of this invention is achieved through the following technical solution: A nanobody against Brucella Omp25 protein, the amino acid sequence of which is shown in SEQ ID NO.3.

[0008] A gene encoding a nanobody as described above, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0009] A recombinant expression vector comprising the genes described above.

[0010] A recombinant host cell comprising the recombinant expression vector described above.

[0011] A method for preparing a nanobody as described above includes: transforming the recombinant expression vector described above into the recombinant host cell described above, purifying it after inducing expression, and obtaining the nanobody.

[0012] The application of a nanobody as described above in the preparation of a reagent or kit for detecting Brucella ovis.

[0013] Furthermore, the kit is a competitive ELISA detection kit.

[0014] A competitive ELISA kit for detecting antibodies against Brucella mesenteriae, comprising the nanobody described above.

[0015] Furthermore, it also includes an ELISA plate, HRP-labeled Omp25 protein, washing solution, chromogenic solution, and / or stop solution; the nanobody is coated on the ELISA plate.

[0016] Compared with the prior art, the beneficial effects of the present invention are: This invention utilizes prokaryotically expressed Brucella Omp25 protein as an immunogen to immunize alpacas. Total RNA was extracted from their peripheral blood lymphocytes, and the VHH fragment was amplified by reverse transcription and two rounds of nested PCR. The VHH fragment was then digested and ligated with the pComb3Xss vector and transformed into TG1 competent cells. Using phage display technology, after multiple rounds of screening, the anti-Brucella Omp25 protein nanobody Omp25Nb5E9 was successfully screened. Based on this, the prokaryotic recombinant expression vector pET28a-Omp25Nb5E9 was constructed and transformed into Escherichia coli BL21 competent cells by heat shock. After IPTG induction, the recombinant protein was affinity purified, and Western blotting confirmed that the recombinant protein was indeed the Brucella Omp25 protein nanobody Omp25Nb5E9, exhibiting high specificity, strong stability, and good sensitivity.

[0017] Based on the nanobody Omp25Nb5E9, this invention establishes a competitive ELISA antibody detection method with an inhibition rate critical value of 25.101%, which can effectively detect Brucella antibody levels in sheep serum with low detection limit and high sensitivity. Furthermore, this invention develops a corresponding competitive ELISA detection kit. When using this kit to detect Brucella, there is no cross-reactivity with positive sera from other zoonotic bacteria such as Yersinia enterocolitica O9 and Escherichia coli O157, demonstrating high specificity and significantly shortening the detection time, providing a reliable solution for rapid and accurate Brucella screening. Attached Figure Description

[0018] Figure 1 SDS-PAGE electrophoresis images of Omp25 protein before and after induction are shown; where M: Marker; 1: Omp25 protein after induction; 2: Omp25 protein before induction.

[0019] Figure 2 This is an electrophoresis identification image of the first round of nested PCR amplification; where M: Marker; 1-2: VH and VL regions of IgG1, IgG2, and IgG3 antibody heavy chains.

[0020] Figure 3 This is an electrophoresis image of the second round of nested PCR amplification and identification; where M: Marker; 1: VHH sequence 1; 2: VHH sequence 2.

[0021] Figure 4 Electrophoretic identification of the insertion positivity rate of nanobody library; where M: Marker; 1-15: PCR products of 15 randomly selected monoclonal colonies.

[0022] Figure 5SDS-PAGE protein electrophoresis identification of the prokaryotically expressed and purified nanobody Omp25Nb5E9; where M: Marker; 1-2: Omp25Nb5E9 nanobody before purification; 3-4: Omp25Nb5E9 nanobody after purification. Detailed Implementation

[0023] To provide a clearer understanding of the technical features, objectives, and beneficial effects of this invention, the technical solution of this invention is described in detail below, but this should not be construed as limiting the scope of implementation of this invention. In this invention, materials, reagents, or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0024] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0025] Example 1: Preparation and screening of nanobodies against Brucella Omp25 protein; 1. Preparation of antigens; In this experiment, the Omp25 protein (amino acid sequence shown in SEQ ID NO.1), unique to Brucella, was selected as the immunogen. The strain expressing the Omp25 protein was a pre-constructed BL21-pET28a-Omp25 prokaryotic expression strain (construction process: gene encoding Omp25 protein was expressed as follows). Omp25 Gene synthesis was performed, and two restriction enzyme sites, BamHI and XhoI, were introduced. The gene was directionally cloned into the pET28a(+) vector and transformed into the BL21 expression strain, ultimately successfully constructing the prokaryotic expression strain BL21-pET28a-Omp25. Kanamycin was added to a final concentration of 100 μg / mL, and the culture was carried out at 220 rpm in a shaker at 37°C until the OD value reached 0.5. Isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration, and the culture was induced at 37°C and 180 rpm for 6 h. After induction, the bacterial precipitate was collected and resuspended in a urea-free binding medium (containing Na3PO4·12H2O, NaCl, imidazole, and distilled water). The obtained Omp25 protein was identified by SDS-PAGE electrophoresis, and the results are shown below. Figure 1 As shown, the induced Omp25 protein exhibits the expected target band at around 25 kDa, indicating successful Omp25 protein expression.

[0026] SEQ ID NO.1: MRTLKSLVIVSAALLPFSATAFAADAIQEQPPVPAPVEVAPQYSWAGGYTGLYLGYGWNKAKTSTVGSIKPDDWKAGAFAGWNFQQDQIVYGVEGDAGYSWAKKSKD GLEVKQGFEGSLRARVGYDLNPVMPYLTAGIAGSQIKLNNGLDDESKFRVGWTAGAGLEAKLTDNILGRVEYRYTQYGNKNYDLAGTTVRNKLDTQDFRVGIGYKF.

[0027] 2. Alpaca immunization; Take 1 mL of Omp25 protein (concentration 1 mg / mL), mix it with an equal volume of Freund's complete adjuvant, emulsify thoroughly, and inject it around the cervical lymph nodes of healthy adult male alpacas to complete the initial immunization. After the initial immunization, booster immunizations are administered every 14 days for a total of five immunizations. For the last four booster immunizations, Freund's incomplete adjuvant is used instead of Freund's complete adjuvant. Seven days after each immunization, alpaca venous serum is collected, and antibody titers are monitored by ELISA. When the antibody titer reaches 1:51200, a nanobody library is constructed.

[0028] 3. Construction of nanobody libraries; When the alpaca serum antibody titer reached 1:51200, 75 mL of alpaca peripheral blood was collected. Total RNA was extracted from peripheral blood lymphocytes using a kit and reverse transcribed into cDNA. Specific primers were designed (the nested PCR primer sequences used are shown in Table 1). Two rounds of nested PCR amplification were performed (the first round used cDNA as a template, amplifying antibody-related genes with primers CALL001 and CALL002; the second round used the amplification product from the first round as a template, amplifying antibody-related genes with VHH-...). Sac I-F and VHH- Spe (I-R primer amplification), introducing two sticky ends, Sac I and Spe I, finally obtaining a pure VHH sequence band (e.g., Figure 2 The electrophoresis results after the first round of amplification shown indicate that lanes 1-2 contain multiple amplified bands including the VH and VL regions of the IgG1, IgG2, and IgG3 antibody heavy chains, successfully amplifying the antibody-related gene regions; Figure 3The electrophoresis results after the second round of amplification shown indicate that lane 1 (VHH sequence 1) and lane 2 (VHH sequence 2) show clear, single bands at approximately 470 bp, indicating successful introduction of SacI and SpeI sticky ends, resulting in a pure VHH sequence band. The obtained VHH target band was gel-cleaved and recovered. The VHH target fragment and the pComb3Xss vector (purchased from Beijing BioBio Biotechnology Co., Ltd.) were then double-digested using Fast Digest SacI and Fast Digest SpeI enzymes (e.g.,...). Figure 3 As shown in the figure, the VHH fragment was ligated into the pComb3Xss vector using T4 DNA ligase, and then transformed into E. coli TG-1 competent cells by heat shock to construct a nanobody library. The library size was found to be 3 × 10⁻⁶. 8 Fifteen single-clone colonies were randomly selected for PCR identification, and the electrophoresis results showed (e.g.) Figure 4 As shown in the figure, clear bands appeared in lanes 1-15 at positions corresponding to the size of the VHH fragment, indicating a positive insertion rate of 93.33%, and a high-quality nanobody library was successfully constructed.

[0029] Table 1. Nested PCR Primer Sequences

[0030] 4. Rescue of Brucella nanobody libraries; Add 1 mL of the nanobody library to 200 mL of 2×YT medium, then add ampicillin to a final concentration of 100 μg / mL. Incubate at 37℃ and 220 rpm until the OD value reaches 0.6. Add helper phage M13K07 at a ratio of 50:1. Incubate for 30 min, then incubate at 37℃ and 180 rpm for 30 min. Centrifuge at 8000 rpm for 10 min, discard the supernatant, and resuspend the bacterial pellet in 200 mL of 2×YT medium pre-added with 100 μg / mL ampicillin and 75 μg / mL kanamycin. IPTG at a final concentration of 1 mM was used for induction at 28°C for 16 h. The induced bacterial culture was centrifuged at 8000 rpm for 30 min, and the supernatant was collected. Sterile polyethylene glycol / sodium chloride (PEG / NaCl) solution was added to 1 / 5 of the total volume, and the culture was incubated on ice for at least 4 h. After the ice bath, the culture was centrifuged at 12000 rpm for 1 h. The resulting precipitate was the recombinant phage containing the VHH sequence. The phage precipitate was resuspended in sterile PBS and centrifuged at 12000 rpm for 10 min. The residual bacterial precipitate at the bottom of the centrifuge tube was discarded, and the supernatant was the rescued phage.

[0031] 5. Screening for Brucella-specific recombinant phages; The optimal coating concentration of Omp25 protein was determined to be 15 μg / mL using the checkerboard method. At this concentration, 100 μL of Omp25 protein was coated onto each well of an ELISA plate and incubated at 37°C for 1 h. The plate was washed five times with 0.01% phosphate-buffered saline (PBST) at 5-minute intervals, and then blocked with 5% skim milk powder for 1 h. The plate was then washed five times with 0.01% PBST at 5-minute intervals and then blocked for use. The rescued phages were diluted to 1 × 10⁻⁶. 11 Incubate the plate with 200 μL of pfu / mL solution per well at 37°C for 2 hours. Then, shake at 200 rpm for 20 minutes at 25°C. Wash the plate three times with 0.01% PBST, three times with 0.02% PBST, and three times with 0.03% PBST, with 5-minute intervals between each wash. Finally, wash the plate 10 times with PBS, with 3-minute intervals between each wash. After washing, blot the plate clean and add 150 μL of glycine-hydrochloric acid buffer (pH 2.2) to each well. Shake at 400 rpm for 20 minutes at 37°C to elute the phages adsorbed on the plate. Collect the eluent and neutralize it with Tris-HCl solution (pH 8.0) to adjust the final pH to approximately 7.0. The neutralized recombinant phage was infecting competent *E. coli* TG-1 cells, incubated for 30 min, and then shaken at 28°C and 180 rpm for 30 min. The resulting bacterial suspension was resuspended in 500 μL of medium and spread entirely onto 2×YT solid medium containing ampicillin. The cells were incubated overnight at 37°C with the suspension inverted. Colonies were scraped off to prepare a bacterial suspension, and helper phage M13K07 was added again. The phage rescue and specific recombinant phage screening processes were repeated four times to obtain specific recombinant phages targeting the *Brucella* Omp25 protein. The obtained specific recombinant phage was then infecting competent *E. coli* TG-1 cells and spread onto 2×YT solid medium containing ampicillin. Single colonies were subjected to Phage-ELISA detection. A positive result was indicated by an OD value above the cut-off value. This positive TG-1 strain was preserved and named Omp25Nb5E9. The recombinant plasmid inside the Omp25Nb5E9 strain contains a specific VHH sequence against Brucella. After sequencing, the sequence was aligned and processed using MAGA7 software, and the sequence obtained is the anti-brucellosis VHH sequence. The Brucella nanobody Omp25Nb5E9 was obtained through sequencing, and its encoding gene nucleotide sequence is shown in SEQ ID NO.2, and its amino acid sequence is shown in SEQ ID NO.3.

[0032] SEQ ID NO.2: GATGTGCAGCTGGTGGAGTCTGGGGGGGGCTTGGTGCAGCCTGGGGGTTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCACCGTCGGCAGCAATTCCGTGGGCTGGTACCGCCAGACTCCAGAAAGATTGCGTGATTTGGTCGCGACTATTTGGCGTGATGGTGAAACGACTTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCGTGGACAACGCCAGGAAGACAGTGTATCTGCAAATGAGCAGCCTGAAACCTGAGGACACGGCCGTCTATTACTGTAATATGCGGCCATATGACACGCACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA。

[0033] SEQ ID NO.3: DVQLVESGGGLVQPGGSLRLSCAASGSTVGSNSVGWYRQTPERLRDLVATIWRDGETTYADSVKGRFTISVDNARKTVYLQMSSLKPEDTAVYYCNMRPYDTHYWGQGTQVTVSS。

[0034] 6. Prokaryotic expression and purification of the nanobody Omp25Nb5E9 of Brucella; Based on the sequencing results of the Omp25Nb5E9 positive strain, specific primers (Omp25Nb5E9-BamHI-F: GGATCCGATGTGCAGCTGGTGGA (as shown in SEQ ID NO. 9); Omp25Nb5E9-XhoI-R: CTCGAGTGAGGAGACGGTGACCT (as shown in SEQ ID NO. 10)) were designed, introducing two sticky ends, BamHI and XhoI, for PCR amplification. The prokaryotic expression vector pET28a(+) (purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.) and the Omp25Nb5E9 fragment were double-digested using Fast Digest BamHI and Fast Digest XhoI enzymes. Then, the Omp25Nb5E9 fragment was ligated to pET28a(+) using T4 DNA ligase to construct the recombinant expression plasmid pET28a-Omp25Nb5E9 (nucleotide sequence shown in SEQ ID NO. 4). The recombinant expression plasmid pET28a-Omp25Nb5E9 was transformed into *E. coli* BL21 competent cells using a heat shock method to construct the recombinant expression strain BL21-pET28a-Omp25Nb5E9. The recombinant expression strain BL21-pET28a-Omp25Nb5E9 was cultured until the OD value reached 0.8, then IPTG was added to a final concentration of 1 mM, and the cells were induced at 25°C for 20 h. After centrifugation at 8000 rpm for 10 min, the bacterial pellet was collected, washed three times with sterile PBS, and finally resuspended in 15 mL of PBS. The pellet was then sonicated at 4°C with the following sonication program: sonication time 3 s, interval 3 s, power 60%, sonication time 20 min. The sonicated supernatant was purified using a nickel column. The purified protein eluent was then added to 5× loading buffer. After thorough boiling, Western blotting was performed. The resulting 15 kDa protein band was the purified nanobody Omp25Nb5E9 targeting Brucella Omp25 protein (e.g., Omp25Nb5E9). Figure 5 (As shown).

[0035] SEQ ID NO.4:

[0036] Example 2: Establishment of a competitive ELISA antibody detection method based on nanobody Omp25Nb5E9; The specific steps for detecting Brucella using a competitive ELISA antibody detection method are as follows: Using 15 μg / mL Omp25Nb5E9 as the coating agent, 100 μL was coated onto each well of an ELISA plate and incubated at 37°C for 1 h. The plate was then washed five times with 0.01% PBST buffer for 5 min each time. 200 μL of 5% skim milk powder was added to each well, and the plate was incubated at 37°C for 1 h. The plate was then washed five times with 0.01% PBST buffer for 5 min each time. The serum to be tested was diluted 1:100, and 50 μL of the diluted serum was added to each well, along with 50 μL of 15 μg / mL horseradish peroxidase (HRP)-labeled Omp25 protein. The mixture was vortexed and incubated at 37°C for 1 h. The plate was washed five times with 0.01% PBST buffer. 100 μL of Omp25Nb5E9 was added to each well. Incubate the plate with 3,3',5,5'-tetramethylbenzidine (TMB) substrate solution in the dark for 15 min. Stop the reaction by adding 50 μL of 2 mol / L H₂SO₄ solution to each well. Place the plate in a microplate reader and read the OD value of each well at 450 nm. Calculate the inhibition rate (PI): Inhibition rate (PI) = (OD value of negative serum) / (OD value of negative serum). 450nm -Sample serum OD 450nm ) / Negative serum OD 450nm ×100%.

[0037] Example 3: Determination of the critical value of inhibition rate; Fifty sheep serum samples that tested negative for both the Rose Bengal plate agglutination test and the tube agglutination test were prepared. A competitive ELISA reaction was then performed according to the experimentally established method to measure the OD. 450nm The inhibitory concentration (PI) was calculated, followed by the average inhibition rate (X) and standard deviation (SD) of the serum samples. A positive / negative criterion was established: when PI ≥ X + 3SD, the serum was considered positive for *Brucella medullaris* antibodies; when PI ≤ X + 2SD, the serum was considered negative for *Brucella medullaris* antibodies; and when X + 2SD < PI < X + 3SD, the serum was considered questionable. The results showed that the average inhibition rate (X) of 50 negative sheep serum samples was 3.561%, and the standard deviation (SD) was 8.180%. Substituting these criteria, the critical inhibition rate for this method was calculated to be 25.101%. That is, when the inhibition rate is greater than 25.101%, the serum sample is considered positive for *Brucella medullaris* antibodies; when the inhibition rate is less than 19.921%, the sample is considered negative for *Brucella medullaris* antibodies; if the inhibition rate is between these two values, the test is repeated; if the inhibition rate is still less than 19.921%, the result is considered negative.

[0038] Example 4: Specificity test; The established and optimized competitive ELISA antibody detection method was used to detect Yersinia enterocolitica O9-positive serum and Escherichia coli O157-positive serum, with Brucella mesenteriae-positive serum added as a control to determine the specificity of the established competitive ELISA antibody detection method. As shown in Table 2, the competitive ELISA antibody detection method established in this invention detected negative results for both Yersinia enterocolitica O9-positive serum and Escherichia coli O157-positive serum, indicating that the competitive ELISA antibody detection method has good specificity.

[0039] Table 2 Specificity test results

[0040] Example 5: Sensitivity Test; Brucella medullaris standard positive serum was diluted at ratios of 1:50, 1:100, 1:200, 1:400, 1:800, 1:1000, 1:1200, and 1:1400, and tested using an established and optimized competitive ELISA antibody detection method to determine its sensitivity. Table 3 shows that even when the Brucella medullaris standard positive serum was diluted to 1:1200, the competitive ELISA antibody detection method could still detect a positive result, indicating that the method has good sensitivity.

[0041] Table 3 Sensitivity Test Results

[0042] Example 6: Brucella nanobody competitive ELISA detection kit; Based on established competitive ELISA antibody detection methods, this embodiment provides a Brucella nanobody competitive ELISA detection kit, comprising the following components: ELISA plate coated with nanobody Omp25Nb5E9; Antigen to be tested: serum sample; Enzyme-labeled antigen: HRP-labeled Omp25 protein; Washing buffer: 0.01% PBST buffer; Developing solution: TMB substrate developing solution; Termination solution: 2 mol / L H2SO4 solution.

[0043] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent.

Claims

1. A nanobody against Brucella Omp25 protein, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

3.

2. A gene encoding the nanobody as described in claim 1, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.

2.

3. A recombinant expression vector, characterized in that, Includes the gene described in claim 2.

4. A recombinant host cell, characterized in that, Includes the recombinant expression vector as described in claim 3.

5. A method for preparing nanobodies as described in claim 1, characterized in that, include: The recombinant expression vector of claim 3 is transformed into the recombinant host cell of claim 4, and after induction of expression and purification, the nanobody is obtained.

6. The use of the nanobody as described in claim 1 in the preparation of a reagent or kit for detecting Brucella ovis antibodies.

7. The application according to claim 6, characterized in that, The kit is a competitive ELISA detection kit.

8. A competitive ELISA kit for detecting antibodies against Brucella mesenteriae, characterized in that, It includes the nanobody as described in claim 1.

9. The competitive ELISA detection kit according to claim 8, characterized in that, It also includes an ELISA plate, HRP-labeled Omp25 protein, washing solution, chromogenic solution and / or stop solution; the nanobody is coated on the ELISA plate.

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