A nanobody against brucella omp25 protein and its application in competitive elisa detection

Abstract

The application belongs to the field of biotechnology, and provides a nano antibody against Brucella Omp25 protein and application thereof in competitive ELISA detection. The amino acid sequence of the nano antibody is shown as SEQ ID NO. 3. The application immunizes a llama with prokaryotic expressed Brucella Omp25 protein as an immunogen, extracts total RNA of peripheral blood lymphocytes, performs reverse transcription and two rounds of nested PCR to amplify a VHH fragment, performs enzyme digestion and connection on the VHH fragment and a pComb3Xss vector, and transforms the VHH fragment into TG1 competent cells. Through multiple rounds of panning by using phage display technology, the nano antibody against Brucella Omp25 protein is screened. The competitive ELISA detection method established by using the nano antibody can effectively detect the Brucella antibody level in sheep serum, has low detection limit and high sensitivity; and the developed matching kit has high specificity and greatly shortens the detection time.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a nanobody against Brucella Omp25 protein and application thereof in competitive ELISA detection. BACKGROUND

[0002] Brucellosis is a globally distributed zoonosis, causing over 500,000 infections annually. Its pathogen is Brucella spp., a gram-negative intracellular parasite, which can be transmitted through the digestive tract, respiratory tract, skin and mucosal contact, etc. The disease is widespread in the world, and the high-risk population includes people living or traveling in the epidemic area, and professional people exposed to Brucella. The clinical symptoms after infection include repeated high fever, heavy sweating, fatigue, joint pain, etc.

[0003] Brucella lacks classic virulence factors, and its pathogenicity is highly dependent on the immune escape mechanism mediated by outer membrane proteins. Among them, outer membrane protein Omp25 has become the focus of research due to its key role in pathogen-host interaction. Omp25 The gene is located on chromosome I of Brucella, which encodes a transmembrane protein Omp25 composed of 214 amino acids. Structural studies have shown that Omp25 forms a typical β-barrel conformation, and its extracellular region 4 loop structures (Loop 1-4) are directly involved in host cell adhesion, in which Loop3 mediates the internalization process by binding to the host cell surface heparin sulfate. This protein is highly conserved in Brucella, but in B. canis, the 52nd arginine is mutated to cysteine, resulting in a significant decrease in virulence, indicating that this protein is not only a structural protein, but also a key factor in regulating the virulence of Brucella. Experiments have confirmed that the Omp25 deletion mutant strain reduces the spleen bacterial load by 100 times in a mouse model, which has potential for subsequent detection and treatment.

[0004] Currently, the gold standard for Brucella detection is still based on bacterial isolation and culture. However, due to its limited sensitivity to bacterial load in the sample, the positive rate of chronic infection or antibiotic treatment drops to less than 20%; the culture period is as long as 2-4 weeks, and the operator is at high risk of exposure; and some atypical strains have difficulty growing in conventional culture medium, which limits the rapid diagnosis of Brucella. In immunological detection, the tube agglutination test (SAT) relies on the visible agglutination reaction of Brucella whole bacterial antigen and serum antibody. This method is simple and low in cost, but its sensitivity decreases to 60-70% in chronic infection, and has 15-30% cross-reaction with Yersinia, Salmonella and the like. In addition, the high titer serum of Brucella is false negative due to excessive antibodies, which needs gradient dilution and recheck, delaying the diagnosis opportunity. Enzyme-linked immunosorbent assay (ELISA) can improve the detection specificity by using recombinant protein antigens. The indirect ELISA detection of IgG / IgM has a sensitivity of 92-96%, and has shown good detection performance. However, the current immunological detection methods including ELISA still mainly rely on traditional monoclonal or polyclonal antibodies, which have large molecular weight, low stability and high cost, and a large number of experimental animals need to be immunized in the production process, which does not comply with the animal welfare principle. Therefore, it is necessary to develop new antibodies with high stability, strong specificity, high affinity and simple preparation process.

[0005] In llama, dromedary and other camelids, and in sharks, stingrays and other cartilaginous fishes, there is a natural heavy chain antibody (HCAbs) that lacks light chains, and the variable region of the heavy chain is called nanobody (Nb) or single domain antibody (variable domain of heavy chain of heavy-chain antibody, VHH). The molecular weight of nanobody is only 15 kDa, and compared with traditional IgG antibodies, nanobody has the advantages of easy expression, easy modification, good stability, strong specificity and high affinity. Most importantly, nanobody can recognize hidden antigen epitopes that conventional antibodies cannot recognize. The extended CDR1 region and longer CDR3 region of nanobody make it have outstanding antigenic determinants, which can deeply fold into the gap of the protein. This structural change allows nanobody to bind to the surface and crevice of the antigen, and has better recognition ability to the antigen. At present, there is no report on nanobody targeting Brucella Omp25 protein and its application in serological detection. Therefore, the present application provides a nanobody against Brucella Omp25 protein and its application in competitive ELISA detection. SUMMARY

[0006] The application aims to provide a nano-antibody against Brucella Omp25 protein and its application in competitive ELISA detection, aiming to solve the problems in the background art.

[0007] The application achieves the above-mentioned purposes by the following technical solutions.

[0008] The nano-antibody against Brucella Omp25 protein has an amino acid sequence as shown in SEQ ID NO. 3.

[0009] The gene encoding the nano-antibody has a nucleotide sequence as shown in SEQ ID NO. 2.

[0010] The recombinant expression vector comprises the gene.

[0011] The recombinant host cell comprises the recombinant expression vector.

[0012] The preparation method of the nano-antibody comprises the following steps: transforming the recombinant expression vector into the recombinant host cell, purifying the nano-antibody after induced expression.

[0013] The application of the nano-antibody in preparing a reagent or kit for detecting Brucella antibody.

[0014] Further, the kit is a competitive ELISA detection kit.

[0015] The competitive ELISA detection kit for detecting Brucella antibody comprises the nano-antibody.

[0016] Further, the kit further comprises an enzyme-labeled plate, HRP-labeled Omp25 protein, washing solution, color developing solution and / or termination solution; and the nano-antibody is coated on the enzyme-labeled plate.

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

[0018] The present application immunizes alpacas with Omp25 protein of prokaryotic expressed Brucella as immunogen, extracts total RNA of peripheral blood lymphocytes, amplifies VHH fragment through reverse transcription and two rounds of nested PCR, digests and links the VHH fragment and pComb3Xss vector, and transforms to TG1 competent cells, successfully screens nanobody Omp25Nb5E9 against Brucella Omp25 protein through multiple rounds of panning based on phage display technology. Based on this, the prokaryotic recombinant expression vector pET28a-Omp25Nb5E9 is constructed, and is transformed into E. coli BL21 competent cells by heat shock method, and after IPTG induction, the recombinant protein is affinity purified, and it is verified by Western Blot that the recombinant protein is nanobody Omp25Nb5E9 which can specifically recognize Brucella Omp25 protein, and has high specificity, strong stability and good sensitivity.

[0019] Based on nanobody Omp25Nb5E9, the present application establishes a competitive ELISA antibody detection method, determines that the critical value of inhibition rate is 25.101%, and can effectively detect the Brucella antibody level in sheep serum, has low detection limit and high sensitivity. Further, the present application develops a corresponding competitive ELISA detection kit, and when the kit is used for detecting Brucella, there is no cross reaction with positive serum of other human and animal shared disease bacteria such as Yersinia enterocolitica O9 and Escherichia coli O157, has strong specificity, and greatly shortens the detection time, thereby providing a reliable solution for rapid and accurate screening of Brucella. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 It is an SDS-PAGE protein electrophoresis identification chart of Omp25 protein before and after induction, wherein M represents Marker, 1 represents Omp25 protein after induction, and 2 represents Omp25 protein before induction.

[0021] Figure 2 It is an electrophoresis identification chart of first round of amplification of nested PCR, wherein M represents Marker, and 1-2 represent VH and VL regions of heavy chains of IgG1, IgG2 and IgG3 antibodies.

[0022] Figure 3 It is an electrophoresis identification chart of second round of amplification of nested PCR, wherein M represents Marker, 1 represents VHH sequence 1, and 2 represents VHH sequence 2.

[0023] Figure 4 It is an electrophoresis identification chart of positive rate of nanobody library insertion, wherein M represents Marker, and 1-15 represent PCR products of 15 randomly picked single colonies.

[0024] 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

[0025] 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.

[0026] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0027] Example 1: Preparation and screening of nanobodies against Brucella Omp25 protein;

[0028] 1. Preparation of antigens;

[0029] 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.

[0030] SEQ ID NO.1:

[0031] MRTLKSLVIVSAALLPFSATAFAADAIQEQPPVPAPVEVAPQYSWAGGYTGLYLGYGWNKAKTSTVGSIKPDDWKAGAFAGWNFQQDQIVYGVEGDAGYSWAKKSKDGLEVKQGFEGSLRARVGYDLNPVMPYLTAGIAGSQIKLNNGLDDESKFRVGWTAGAGLEAKLTDNILGRVEYRYTQYGNKNYDLAGTTVRNKLDTQDFRVGIGYKF.

[0032] 2. Alpaca immunization;

[0033] Take 1 mL Omp25 protein (concentration 1 mg / mL), mixed with equal volume of Freund's complete adjuvant, after fully emulsified, injected in the neck lymph node around the healthy adult male alpaca, complete the first immunization. After the first immunization, once every 14 days, a total of five times. Among them, the last four times of booster immunization, use Freund's incomplete adjuvant instead of Freund's complete adjuvant. 7 days after each immunization, the serum of alpaca venous blood was collected, and the antibody titer was monitored by ELISA detection. When the antibody titer reached 1:51200, the construction of nanobody library was carried out.

[0034] 3. Construction of nanobody library;

[0035] When the antibody titer of alpaca serum reached 1:51200, 75 mL of peripheral blood of alpaca was collected, and the total RNA of peripheral blood lymphocytes was extracted using kit, and the cDNA was reverse transcribed. Design specific primers (the sequences of the primers used in the nested PCR are shown in Table 1), and amplify by two rounds of nested PCR (the first round uses cDNA as template, and CALL001 and CALL002 primers are used to amplify antibody related genes; the second round uses the first round amplification product as template, and VHH- Sac Ⅰ-F and VHH- Spe Ⅰ-R primers are used for amplification), introduce Sac I and Spe I two sticky ends, finally obtain pure VHH sequence band (as shown in the electrophoresis result after the first round of amplification, lanes 1-2 have multiple amplification bands containing IgG1, IgG2, IgG3 antibody heavy chain VH and VL region, successfully amplified antibody related gene region; as Figure 2 Figure 3 ​The second round of amplification electrophoresis results are shown in lane 1 (VHH sequence 1) and lane 2 (VHH sequence 2) at about 470 bp, indicating that the SacI and SpeI sticky ends are successfully introduced, and a pure VHH sequence band is obtained). The VHH target band obtained was gel-recovered, and Fast Digest Sac I and Fast Digest SpeI enzymes were used to double-digest the VHH target fragment and the pComb3Xss vector (purchased from Beijing Bao Bo Wei Biotechnology Co., Ltd.) (as shown in Figure 3 The VHH fragment was ligated to the pComb3Xss vector using T4 DNA ligase, and then heat shock transformation was performed on E. coli TG-1 competent cells, thereby constructing a nanobody library. After detection, the library capacity reached 3 x 10 8 PCR identification was performed on 15 randomly selected single colonies, and the electrophoresis results are shown in (as shown in Figure 4 Lanes 1-15 all showed clear bands at a position 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.

[0036] Table 1 Nest PCR primer sequences

[0037]

[0038] 4. Rescue of Brucella nanobody library;

[0039] Take 1 mL of the nanobody library and add 200 mL of 2xYT medium, then add ampicillin at a final concentration of 100 μg / mL, and incubate at 37°C, 220 rpm until the OD value is 0.6. Add helper phage M13K07 at a multiplicity of infection of 50:1, stand for 30 min, incubate at 37°C, 180 rpm for 30 min, centrifuge at 8000 rpm for 10 min, discard the supernatant, and resuspend the bacterial pellet in 200 mL of 2xYT medium. Add 100 μg / mL of ampicillin and 75 μg / mL of kanamycin to the medium, and then add IPTG at a final concentration of 1 mM. Induce at 28°C for 16 h, centrifuge the induced bacterial solution at 8000 rpm for 30 min, take the supernatant, add a total volume of 1 / 5 of sterile polyethylene glycol / sodium chloride (PEG / NaCl) solution, and ice-bath for 4 h or more. After ice-bathing, centrifuge at 12000 rpm for 1 h, and the obtained precipitate contains VHH sequence recombinant phage. Resuspend the phage precipitate with sterile PBS, centrifuge at 12000 rpm for 10 min, discard the residual bacterial pellet at the bottom of the centrifuge tube, and the supernatant is the rescued phage.

[0040] 5. Screening of anti-Brucella specific recombinant phage;

[0041] The optimal coating concentration of Omp25 protein was determined to be 15 μg / mL by checkerboard verification. The Omp25 protein was coated on the enzyme-labeled plate at this concentration, 100 μL per well, incubated at 37°C for 1 h, washed with 0.01% phosphate buffered saline (PBST) for 5 times with 5 min interval, tapped to dry, blocked with 5% skimmed milk powder for 1 h, washed with 0.01% PBST for 5 times with 5 min interval, tapped to dry, and reserved. The rescued phage was diluted to 1 x 10 11 pfu / mL, 200 μL per well, incubated at 37°C for 2 h, then shaken at 25°C and 200 rpm for 20 min, sequentially washed with 0.01% PBST for 3 times, 0.02% PBST for 3 times, and 0.03% PBST for 3 times with 5 min interval, and finally washed with PBS for 10 times with 3 min interval. The washed enzyme-labeled plate was tapped to dry, 150 μL of glycine-hydrochloric acid buffer (Glycine-HCl) with pH of 2.2 was added to each well, 37°C, 400 rpm high-speed shaking for 20 min, the phage adsorbed on the enzyme-labeled plate was eluted, the eluate was collected, and neutralized with Tris-HCl neutralizing solution with pH of 8.0 to adjust the final concentration pH to about 7.0. The neutralized recombinant phage was used to infect E. coli TG-1 competent cells, stood for 30 min, shaken at 28°C and 180 rpm for 30 min, the obtained bacterial solution was resuspended with 500 μL of culture medium, and completely spread on 2 x YT solid medium containing ampicillin, incubated in a 37°C incubator overnight, scraped the colonies to prepare bacterial suspension, added helper phage M13K07 again, and repeated the above processes of phage rescue and specific recombinant phage screening for 4 times to obtain specific recombinant phage against Brucella Omp25 protein. The obtained specific recombinant phage was used to infect E. coli TG-1 competent cells, spread on 2 x YT solid medium containing ampicillin, and the obtained single colonies were detected by Phage-ELISA. The obtained OD value above the Cut-off was a positive result, the positive TG-1 strain was preserved, and named as Omp25Nb5E9. The recombinant plasmid in the Omp25Nb5E9 strain contained specific VHH sequence against Brucella, and the sequence was aligned and arranged by MAGA7 software after sequencing. The sequenced sequence was the VHH sequence against Brucella. The Brucella nanobody Omp25Nb5E9 was sequenced, the nucleotide sequence of the encoding gene was shown as SEQ ID NO. 2, and the amino acid sequence was shown as SEQ ID NO. 3.

[0042] SEQ ID NO. 2:

[0043] GATGTGCAGCTGGTGGAGTCTGGGGGGGGCTTGGTGCAGCCTGGGGGTTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCACCGTCGGCAGCAATTCCGTGGGCTGGTACCGCCAGACTCCAGAAAGATTGCGTGATTTGGTCGCGACTATTTGGCGTGATGGTGAAACGACTTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCGTGGACAACGCCAGGAAGACAGTGTATCTGCAAATGAGCAGCCTGAAACCTGAGGACACGGCCGTCTATTACTGTAATATGCGGCCATATGACACGCACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA.

[0044] SEQ ID NO. 3:

[0045] DVQLVESGGGLVQPGGSLRLSCAASGSTVGSNSVGWYRQTPERLRDLVATIWRDGETTYADSVKGRFTISVDNARKTVYLQMSSLKPEDTAVYYCNMRPYDTHYWGQGTQVTVSS.

[0046] 6. Prokaryotic expression and purification of the nanobody Omp25Nb5E9 of Brucella;

[0047] According to 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 to introduce two sticky ends of BamHI and XhoI, and PCR amplification was performed. The prokaryotic expression vector pET28a(+) (purchased from Yixing Biotech (Shanghai) Co., Ltd.) and the Omp25Nb5E9 fragment were double-digested using Fast Digest BamHI enzyme and Fast Digest XhoI enzyme, and then the Omp25Nb5E9 fragment was ligated to pET28a(+) using T4 DNA ligase to construct the recombinant expression plasmid pET28a-Omp25Nb5E9 (nucleotide sequence as shown in SEQ ID NO. 4). The recombinant expression plasmid pET28a-Omp25Nb5E9 was transformed into E. coli BL21 competent cells by heat shock method, and the recombinant expression strain BL21-pET28a-Omp25Nb5E9 was constructed. The recombinant expression strain BL21-pET28a-Omp25Nb5E9 was cultured to an OD value of 0.8, 1 mM IPTG was added, and induction was performed at 25°C for 20 h. The bacterial pellet was collected by centrifugation at 8000 rpm for 10 min, washed with sterile PBS for 3 times, resuspended with 15 mL PBS for the last time, and the pellet was subjected to ultrasonic treatment at 4°C. The ultrasonic program was set as follows: ultrasonic time 3 s, interval time 3 s, power 60%, and ultrasonic time 20 min. The ultrasonic supernatant was subjected to nickel column purification, and the purified protein eluate was added with 5x loading buffer, boiled thoroughly, and then subjected to Western Blot detection. The 15 kDa protein band obtained was the nanobody Omp25Nb5E9 against Brucella Omp25 protein purified (as shown in SEQ ID NO. 4). Figure 5

[0048] SEQ ID NO. 4:

[0049] ​

[0050] Example 2: Establishment of a competitive ELISA antibody detection method based on nanobody Omp25Nb5E9;

[0051] The specific steps for detecting Brucella using a competitive ELISA antibody detection method are as follows:

[0052] 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%.

[0053] Example 3: Determination of the critical value of inhibition rate;

[0054] 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. 450nmThe average inhibition rate (X) and standard deviation (SD) of the serum sample are calculated. The positive and negative judgment criteria are set: when PI≥X+3SD, the serum is judged to be Brucella melitensis antibody positive; when PI≤X+2SD, the serum is judged to be Brucella melitensis antibody negative; and when X+2SD<PI<X+3SD, the serum is judged to be suspicious. The results show that the average inhibition rate (X) of 50 negative sheep sera is 3.561%, and the standard deviation (SD) is 8.180%. According to the above judgment criteria, the inhibition rate critical value of the method is calculated to be 25.101%, that is, when the sample inhibition rate is greater than 25.101%, the sample serum is judged to be Brucella melitensis antibody positive; when the sample inhibition rate is less than 19.921%, the sample is judged to be Brucella melitensis antibody negative; and if the inhibition rate is between the two, the test is performed again, and if the inhibition rate of the test result is still less than 19.921%, it is determined to be negative.

[0055] Example 4: Specificity test

[0056] The established and optimized competitive ELISA antibody detection method was used to detect Yersinia enterocolitica O9 positive serum, Escherichia coli O157 positive serum, and Brucella melitensis positive serum as a control to determine the specificity of the established competitive ELISA antibody detection method. As shown in Table 2, the established competitive ELISA antibody detection method detects the above-mentioned sera (Yersinia enterocolitica O9 positive serum, Escherichia coli O157 positive serum) to be negative, indicating that the competitive ELISA antibody detection method has good specificity.

[0057] Table 2 Specificity test results

[0058]

[0059] Example 5: Sensitivity test

[0060] The Brucella melitensis standard positive serum was diluted at a ratio of 1:50, 1:100, 1:200, 1:400, 1:800, 1:1000, 1:1200, and 1:1400, and the established and optimized competitive ELISA antibody detection method was used for testing to determine the sensitivity of the competitive ELISA antibody detection method. As shown in Table 3, when the Brucella melitensis standard positive serum is diluted to 1:1200, the competitive ELISA antibody detection method can still detect positive, indicating that the competitive ELISA antibody detection method has good sensitivity.

[0061] Table 3 Sensitivity test results

[0062]

[0063] Example 6: Nanobody competitive ELISA detection kit for Brucella

[0064] Based on the established competitive ELISA antibody detection method, this embodiment provides a nanobody competitive ELISA detection kit for Brucella, which comprises the following components:

[0065] Coated nanobody Omp25Nb5E9 enzyme plate

[0066] Detection antigen: serum to be detected

[0067] Enzyme-labeled antigen: HRP-labeled Omp25 protein

[0068] Washing solution: 0.01% PBST buffer

[0069] Color developing solution: TMB substrate color developing solution

[0070] Termination solution: 2 mol / L H2SO4 solution

[0071] The above is only a preferred embodiment of the present application, and it should be noted that for those skilled in the art, without departing from the concept of the present application, several modifications and improvements can be made, which should be considered as the protection scope of the present application, and these will not affect the effect and practicality of the present application.

Claims

1. A Nanobody against Brucella Omp25 protein, characterized in that, The amino acid sequence is shown as SEQ ID NO.

3.

2. A gene encoding the Nanobody of claim 1, characterized in that, The nucleotide sequence is shown as SEQ ID NO.

2.

3. A recombinant expression vector, characterized in that, The gene of claim 2.

4. A recombinant host cell, characterized in that, The recombinant expression vector of claim 3.

5. A method of producing a Nanobody according to claim 1, characterized in that, The recombinant expression vector of claim 3. The recombinant expression vector of claim 3 is transformed into the recombinant host cell of claim 4, and the nanobody is obtained after purification after induced expression.

6. Use of the nanobody of claim 1 in the preparation of a reagent or kit for detecting antibody against O. ovilus.

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

8. A competitive ELISA test kit for detecting antibodies to Brucella melitensis characterized in that, The nanobody of claim 1.

9. The competitive ELISA test kit according to claim 8, characterized in that, The kit further comprises an enzyme-labeled plate, an HRP-labeled Omp25 protein, a washing solution, a color developing solution and / or a stop solution; and the nanobody is coated on the enzyme-labeled plate.

Images