A method for preparing brucella antibodies
By preparing high-purity, high-specificity Brucella nanobodies, the problems of insufficient specificity and cross-reactivity of polyclonal antibodies in Brucella detection have been solved, achieving efficient and low-cost rapid detection of brucellosis.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- QINGDAO AGRI UNIV
- Filing Date
- 2026-04-17
- Publication Date
- 2026-06-26
Smart Images
Figure CN122277720A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of Brucella antibody preparation technology, and in particular to a method for preparing Brucella antibodies. Background Technology
[0002] Brucellosis is a global zoonotic infectious disease caused by Brucella bacteria. It is classified as a Class II animal disease in my country and is also one of the key occupational diseases requiring human prevention and control. Brucella can infect various livestock such as cattle, sheep, and pigs, causing serious reproductive disorders in female animals, including abortion, infertility, and orchitis, resulting in significant economic losses to the livestock industry.
[0003] In the prior art, patent document CN118184775A discloses a bovine Brucella nanobody, its preparation method, and its application, belonging to the field of disease antibody detection technology. The amino acid sequence of the bovine Brucella nanobody is shown in SEQ ID NO: 1. This nanobody, or its fusion protein with horseradish peroxidase, can be used as a detection reagent to establish a competitive ELISA for detecting anti-brucellosis antibodies in bovine serum. The operation is simple, the sample detection time is short, and it does not require the use of enzyme-labeled secondary antibodies. This simplifies the subsequent process for producing ELISA kits based on this method, greatly reducing production costs. Therefore, the use of nanobodies to establish animal disease detection technology has excellent market application prospects.
[0004] To address the issue that antibodies used for Brucella detection mainly consist of polyclonal and monoclonal antibodies, polyclonal antibodies have drawbacks. While polyclonal antibodies are simple to prepare and relatively inexpensive, they suffer from complex composition, inconsistent specificity, susceptibility to cross-reactivity, and significant batch-to-batch variability, making them unsuitable for accurate detection. Summary of the Invention
[0005] In order to overcome the above-mentioned defects in the prior art, the present invention provides a method for preparing Brucella antibodies.
[0006] The technical solution adopted by this invention to solve its technical problem is: a method for preparing Brucella antibodies, comprising the following steps: Step 1: Based on one or more combinations of Brucella outer membrane proteins Omp10, Omp19, and BP26, prepare recombinant antigens and optimize prokaryotic expression codons; Step 2: Immunize alpacas with recombinant antigen combined with adjuvant via subcutaneous injection at multiple sites, with booster injections every two weeks for three weeks, and isolate highly active lymphocytes. Step 3: Extract total RNA from lymphocytes, reverse transcribe to synthesize cDNA, and specifically amplify the VHH gene using nested PCR to accurately obtain the antibody coding sequence; Step 4: The VHH gene is cloned into a phage vector and electroporated to construct a high-capacity display library; Step 5: Solid-phase panning is performed using recombinant antigen as the target. Positive clones are identified by ELISA, and prokaryotic expression vectors are constructed after sequencing. Step 6: Induce expression of recombinant nanobodies, purify them using affinity chromatography, optimize elution conditions, and obtain high-purity, high-yield target protein; Step 7: The antibody activity was comprehensively identified by ELISA, Western blot, immunofluorescence and thermostability assays to confirm its high specificity, high affinity and excellent stability, which meet the application requirements.
[0007] Furthermore, in step 1, the antigen preparation process is as follows: The Brucella recombinant antigen is one or more combinations of Brucella outer membrane proteins Omp10, Omp19, and BP26, prepared by recombination using a prokaryotic expression system. The antigen has high purity and strong specificity, and can effectively reduce the background of non-specific antibody screening.
[0008] Furthermore, in step 2, the alpaca immunization process is as follows: The alpaca immunization program consists of a basic immunization plus three booster immunizations. The antigen dose is 0.5 mg per alpaca, and the immunization route is multiple subcutaneous injections in the neck and back. The immunization interval is 2 weeks, and blood is collected 7 days after the last immunization to ensure efficient stimulation of heavy chain antibody immune response.
[0009] Furthermore, in step 3, the gene acquisition method is as follows: The lymphocytes were isolated using density gradient centrifugation with a special separation solution for alpaca lymphocytes. The centrifugation parameters were 4℃, 2000r / min, and 20min, which can efficiently separate live lymphocytes and reduce RNA degradation.
[0010] Furthermore, the total RNA extraction employed the Trizol one-step method. After extraction, the concentration and purity were detected using a nucleic acid quantification instrument, and the OD260 / OD280 ratio was controlled between 1.8 and 2.0 to ensure RNA integrity and reverse transcription efficiency. The cDNA synthesis used reverse transcriptase and random hexamer primers in a 20 μL reaction system. The reaction conditions were set at 25℃ for 10 min, 42℃ for 60 min, and 85℃ for 5 min to obtain a high-quality cDNA template. The VHH gene amplification employed a two-round nested PCR. The first round amplified the full-length variable region of the heavy chain antibody, and the second round amplified the VHH core fragment. Primers were designed to target the conserved regions of alpaca IgG2 and IgG3, and the amplified fragment size was approximately 400 bp, exhibiting high specificity and high yield.
[0011] Furthermore, step 4 is constructed as follows: The phage display library was constructed using the pCANTAB-5E phage vector, with TG1 competent cells as the host bacteria. The library was constructed through double enzyme digestion, ligation, and electroporation, with a library volume of not less than 1.0 × 10⁸ CFU to meet the requirements for high-affinity clone screening.
[0012] Furthermore, in step 5, the screening method is as follows: The positive clone screening was performed using an indirect ELISA method, with Brucella recombinant antigen as the coating antigen and Escherichia coli lysate as the negative control. Clones with an OD450 value three times or more than that of the negative control were selected as positive clones, and the VHH gene sequence was obtained by sequencing. The prokaryotic expression was performed using the pET-28a vector, with BL21 as the host bacterium, a final IPTG concentration of 0.5 mmol / L, an induction temperature of 37℃, and an induction time of 4 h.
[0013] Furthermore, the prokaryotic expression was performed using pET-28a as the vector, BL21 as the host bacterium, with a final IPTG concentration of 0.5 mmol / L, an induction temperature of 37°C, and an induction time of 4 h.
[0014] Furthermore, step 6 adopts the following process: The affinity purification was performed using a Ni-NTA agarose gel chromatography column with a binding buffer pH of 8.0 and an elution buffer containing 250 mmol / L imidazole. After the elution product was desalted by PBS, high-purity Brucella nanobody was obtained.
[0015] Furthermore, the activity identification scheme for step 7 is as follows: The activity identification includes determining the titer by indirect ELISA, verifying specificity by Western blotting, identifying natural antigen binding activity by indirect immunofluorescence assay, and assessing anti-denaturation ability by thermal stability test, thereby comprehensively characterizing the performance of nanobodies.
[0016] The beneficial effects of this invention are: 1. This invention uses recombinant Brucella-specific antigen for immunization, replacing traditional whole-bacterial antigen, which significantly reduces non-specific immune responses, improves screening specificity, and reduces cross-reactivity.
[0017] 2. This invention optimizes the nested PCR amplification system, resulting in high VHH gene amplification efficiency and strong specificity. The constructed phage library has a large capacity and good diversity, ensuring the screening probability of high-affinity clones.
[0018] 3. This invention establishes a three-round gradient solid-phase panning strategy, which gradually improves the rigor of screening, efficiently enriches specific clones, and shortens the screening cycle. The entire screening process only takes 2 to 3 weeks.
[0019] 4. This invention optimizes the prokaryotic expression and purification process, resulting in nanoantibodies with high soluble expression rate, high purification yield, and stable activity, enabling large-scale, low-cost preparation.
[0020] 5. The Brucella nanoantibody prepared by this invention has a small molecular weight, is resistant to high temperature and acid and alkali, has strong specificity and high sensitivity, and is suitable for various rapid detection platforms such as colloidal gold, fluorescence immunoassay, and ELISA, with broad application prospects. Attached Figure Description
[0021] The present invention will be further described below with reference to the accompanying drawings and embodiments.
[0022] Figure 1 This is a schematic diagram of the preparation process of the present invention; Detailed Implementation
[0023] To more clearly illustrate the technical solution of the present invention, the present invention will be further described below with reference to the accompanying drawings. Obviously, the drawings described below are only one embodiment of the present invention. For those skilled in the art, other embodiments can be obtained based on these drawings and embodiments without creative effort, and all of them fall within the protection scope of the present invention.
[0024] according to Figure 1 As shown, a method for preparing Brucella antibodies involves the following steps: First, step 1 is performed to obtain the target gene. In this operation, the Brucella Omp19 gene is selected as the immunogen gene. Then, a recombinant vector is constructed. During construction, the PCR product is recovered by agarose gel electrophoresis, and the ligation product is transformed into Escherichia coli BL21 competent cells. The cells are then plated on LB solid medium containing ampicillin resistance and cultured at 37°C for 12 hours. Next, positive clones are identified by picking single colonies for colony PCR and double enzyme digestion identification.
[0025] Then, recombinant protein expression was induced. Positive recombinant bacteria were inoculated into LB liquid medium and cultured at 37°C with shaking until the OD600 reached 0.6–0.8. IPTG was added to a final concentration of 0.5 mmol / L, and induction was performed at 37°C for 4 h. The bacterial cells were collected, washed with PBS, resuspended, sonicated, and centrifuged at 12000 r / min for 10 min. The supernatant and precipitate were collected separately for SDS-PAGE analysis. Finally, the recombinant protein was inactivated. The recombinant Omp19 protein in the supernatant was purified using a Ni-NTA affinity chromatography column. Impurities were washed sequentially with 10 mmol / L, 20 mmol / L, and 50 mmol / L imidazole, and the target protein was eluted with 250 mmol / L imidazole. The eluent was dialyzed against PBS for 48 h, with the dialysate changed three times, to obtain high-purity Brucella recombinant antigen. The protein concentration was determined by the BCA method, adjusted to 1 mg / mL, and stored at -80°C for later use.
[0026] Step 2: Isolation of alpaca immunization and lymphocytes. Two healthy alpacas with no history of disease, aged 1-2 years, were selected and housed in a clean-grade animal facility. After one week of acclimatization, immunization began. Immunogen emulsification was then performed by mixing recombinant Omp19 antigen with an equal volume of Freund's complete adjuvant and emulsifying thoroughly under ice bath conditions until stable and non-dispersible in a water droplet test. During the immunization process, basic immunization was performed on day 0, with multiple subcutaneous injections of emulsified antigen at a dose of 0.5 mg per alpaca into the neck and back. Booster immunizations were performed on days 14, 28, and 42 using Freund's incomplete adjuvant emulsified antigen, with the same dosage and route as the basic immunization. Seven days after immunization, blood was collected from the marginal ear vein, and serum was separated. Antiserum titer was detected by indirect ELISA; a titer of 1:105 or higher was considered immunization successful. Finally, lymphocytes were separated. During separation, 50 mL of blood was collected from the jugular vein of the immunized alpaca and anticoagulated with heparin sodium. Dilute the anticoagulated blood with an equal volume of PBS and slowly layer it onto the upper layer of the alpaca lymphocyte separation medium, maintaining a clear interface. Centrifuge horizontally at 2000 rpm for 20 min at 4°C. Aspirate the lymphocytes from the middle white membrane layer, add 5 times the volume of PBS, mix well, centrifuge at 1500 rpm for 10 min, discard the supernatant, repeat the washing twice, and finally resuspend the lymphocytes in 1 mL of PBS. Stain with trypan blue and count the cells. Cells with a viability greater than 95% can be used for subsequent experiments.
[0027] Step 3, gene acquisition: First, total RNA extraction is performed. During extraction, approximately 1 × 10⁷ freshly isolated lymphocytes are taken, 1 mL of Trizol reagent is added, and the cells are thoroughly lysed by pipetting. After standing at room temperature for 5 min, add 200 μL of chloroform, shake vigorously for 15 s, stand at room temperature for 2 min, centrifuge at 12000 r / min for 15 min at 4 °C, transfer the upper aqueous phase to a new centrifuge tube, add an equal volume of isopropanol, mix well, stand at room temperature for 10 min, centrifuge at 12000 r / min for 10 min at 4 °C, discard the supernatant, add 1 mL of 75% ethanol to wash the precipitate, centrifuge at 7500 r / min for 5 min at 4 °C, discard the supernatant, dry at room temperature for 5 min, add 50 μL of RNase-free ddH2O to dissolve the precipitate, detect the concentration and OD value using a nucleic acid quantification instrument, and store at -80 °C.
[0028] Then, cDNA was synthesized using a reverse transcription kit. The reaction mixture consisted of 20 μL of total RNA (2 μg) and RNase-free ddH2O added to a final volume of 20 μL. Reaction conditions: 25℃ for 10 min, 42℃ for 60 min, 85℃ for 5 min, and the product was stored at -20℃. Then, the VHH gene was amplified. During amplification, cDNA was used as a template to amplify the variable region of the heavy chain antibody. The primers were CALL001 and CALL002. The reaction system was 50 μL: 25 μL of 2×PCRMix, 2 μL of cDNA, 1 μL each of forward and reverse primers, and 1 μL of ddH2O2. Reaction conditions: 95℃ for 5 min; 95℃ for 30 s, 55℃ for 30 s, 72℃ for 1 min, 30 cycles; 72℃ for 10 min. Then, using the above PCR product as a template, the VHH gene was amplified with primers VHH-F and VHH-R, introducing SfiⅠ and NotⅠ restriction enzyme sites. The reaction system and conditions were the same as the first round, with the annealing temperature adjusted to 58℃. Finally, the PCR product was subjected to 1.5% agarose gel electrophoresis, and a specific band of about 400 bp was observed using the gel imaging system. The VHH gene was purified using a gel extraction kit, and the concentration was determined for later use.
[0029] Step 4, Library Construction: The purified VHH gene and pCANTAB-5E phage vector were double-digested with Sfi I and Not I, respectively. The digestion system (50 μL) consisted of 5 μL 10×Buffer, 20 μL vector / gene, 1 μL each of restriction endonucleases, and 3 μL ddH2O. Digestion was carried out at 37°C for 4 h. Then, 10 μL of the culture medium was serially diluted and plated onto LB-AMP solid medium. The medium was incubated overnight at 37°C. Single colonies were counted, and the library volume was calculated. In this example, the library volume reached 2.3 × 10⁸ °C. FU was then randomly selected, and 20 single colonies were subjected to colony PCR. Electrophoresis showed that the inserted fragments were uniform and the positive rate was greater than 95%. Sequencing showed that the VHH gene sequence had good diversity and the library quality was qualified. Finally, the remaining bacterial culture was inoculated into 200 mL of LB-AMP medium, and M13KO7 helper phage was added. The culture was incubated at 37°C for 30 min, then shaken for 12 h. The supernatant was collected by centrifugation, and PEG-NaCl was added to precipitate the phage. The phage was resuspended in PBS to obtain the Brucella nanobody primary phage library, which was stored at -80°C.
[0030] like Figure 1As shown, based on the above embodiments, the present invention provides a technical solution. Step 5 involves screening clones. First, each well of the ELISA plate is coated with Brucella recombinant Omp19 antigen. The coating concentration is 10 μg / mL in the first round, 5 μg / mL in the second round, and 1 μg / mL in the third round, with 100 μL per well. The plate is incubated overnight at 4°C. Then, the plate is washed three times with PBST. 200 μL of 5% skim milk is added to each well, and the plate is blocked at 37°C for 2 hours. The phage is then bound, and the number of washes with PBST is increased in each round: 10 times in the first round, 15 times in the second round, and 20 times in the third round. Add 100 μL of 0.2 mol / L glycine-hydrochloric acid elution buffer (pH 2.2) to each well and elute for 10 min at room temperature. Immediately add 50 μL of 1 mol / L Tris-HCl to neutralize at pH 9.0. Then, elute the phage to infect logarithmic-phase TG1 bacteria and amplify at 37°C to prepare the next round of panning phages. After completing three rounds of panning, single clones are screened, and finally, six positive clones with strong ELISA signals are selected for sequencing to obtain the complete VHH gene sequence. Sequence alignment and homology analysis identify three highly specific nanobody clones with unique sequences.
[0031] Step 6, Expression Inactivation: First, the optimal positive clone VHH gene was selected, primers with NcoⅠ and XhoⅠ restriction sites were designed, PCR amplification was performed, double digestion was performed, and the mixture was ligated into the pET-28a vector and transformed into BL21 competent cells. Colony PCR and restriction enzyme digestion were used to identify positive recombinant bacteria. Then, expression was induced. The positive recombinant bacteria were inoculated into LB-KAN liquid medium, cultured at 37°C with shaking, and IPTG was added to a final concentration of 0.5 mmol / L. The mixture was induced at 37°C for 4 h. Bacterial cells were collected before and after induction. SDS-PAGE analysis was performed to detect expression levels, with the expression level exceeding 30% of total protein. The expression was predominantly soluble in the supernatant. Bacterial cells were then collected, washed twice with PBS, resuspended in binding buffer, and sonicated on ice at 300W for 3 seconds followed by 5-second intervals (99 times total). The cells were then centrifuged at 12000 rpm for 20 min at 4°C. The supernatant was collected and equilibrated using a Ni-NTA chromatography column with binding buffer. The supernatant was loaded onto the column at a flow rate of 1 mL / min and washed with binding buffer until the baseline stabilized. Elution was then performed with elution buffer, and the elution peak was collected. Purity was determined by SDS-PAGE, with the target protein purity exceeding 90%. The eluted product was transferred to a dialysis bag and dialyzed against PBS at 4°C for 24 h, with the dialysate changed three times to remove imidazole and salt ions. The nanobody was then concentrated using ultrafiltration, and the concentration was determined by the BCA method. The concentration was adjusted to 1 mg / mL and aliquoted at -20°C for storage.
[0032] Step 7, Activity Identification, Test Content: as follows: Indirect ELISA titer assay: 100 μL of Brucella recombinant antigen was coated onto an enzyme-labeled plate and incubated overnight at 4°C. After blocking, serially diluted nanobodies were added and incubated at 37°C for 1 hour. After washing, enzyme-labeled secondary antibody was added and incubated at 37°C for 30 minutes. TMB was used for color development, and the OD450 value was measured after termination. The results showed that the nanobodies achieved a titer of over 1:1 × 10⁵, indicating high affinity.
[0033] Specificity identification: Brucella whole-cell protein was transferred to an NC membrane after SDS-PAGE electrophoresis and blocked with 5% skim milk; nanobody was added for 1:1000 dilution and then incubated overnight at 4°C; after washing with PBST, enzyme-labeled secondary antibody was added and incubated at room temperature for 1 h; DAB color development showed a specific target band, with no cross-reactivity with Escherichia coli, Salmonella, Pasteurella, etc., indicating good specificity.
[0034] Immunofluorescence assay: Brucella was inoculated onto a glass slide, fixed with 4% paraformaldehyde, permeabilized and blocked; nanobodies were added and incubated at 37°C for 1 h; FITC-labeled secondary antibody was incubated in the dark and observed under a fluorescence microscope. Clear bacterial-specific fluorescence was visible, indicating that the nanobodies can recognize natural Brucella antigens.
[0035] Thermal stability test: The nanobody was incubated at 37°C for 1 h, 60°C for 30 min, and 90°C for 10 min, then rapidly cooled in an ice bath, and the remaining activity was detected by indirect ELISA.
[0036] Acid-base stability test: The nanobody was placed in buffer solutions of pH 2.0, pH 4.0, pH 7.0, pH 10.0 and pH 12.0 at room temperature for 1 h, and its activity was tested after being adjusted back to neutral.
[0037] In this invention, based on the structural characteristics of natural heavy chain antibodies in animals, alpacas are immunized with Brucella-specific recombinant antigens to stimulate the production of Brucella heavy chain antibodies. Post-immunization lymphocytes are isolated and total RNA is extracted, which is then reverse transcribed to obtain cDNA. The VHH gene is specifically amplified using nested PCR and cloned into a phage vector to construct a high-capacity, high-diversity Brucella nanobody phage display library. Using Brucella recombinant antigens as targets, a three-round gradient solid-phase panning process is used to progressively improve the screening rigor and efficiently enrich high-affinity, high-specificity phage clones. The target VHH gene is obtained through ELISA screening and gene sequencing, and then cloned into a prokaryotic expression vector for efficient soluble expression in E. coli. High-purity nanobodies are obtained through Ni-NTA affinity chromatography purification. Multidimensional activity identification confirms that they possess high specificity, high affinity, and high stability, and can be directly used for Brucella immunological detection and related research.
[0038] This invention, through optimization of the entire process, solves the problems of long preparation cycle, poor stability, insufficient specificity of traditional antibodies, as well as low screening efficiency and complex processes of existing nanobodies. It realizes the rapid, efficient, and large-scale preparation of Brucella nanobodies, providing core reagent support for the research and development of rapid detection technology for brucellosis.
[0039] The above embodiments are merely exemplary embodiments of the present invention and are not intended to limit the present invention. The scope of protection of the present invention is defined by the claims. Those skilled in the art can make various modifications or equivalent substitutions to the present invention within its spirit and scope of protection, and such modifications or equivalent substitutions should also be considered to fall within the scope of protection of the present invention.
Claims
1. A method for the preparation of Brucella antibodies based on the method characterized by: Includes the following steps; Step 1: Based on one or more combinations of Brucella outer membrane proteins Omp10, Omp19, and BP26, prepare recombinant antigens and optimize prokaryotic expression codons; Step 2: Immunize alpacas with recombinant antigen combined with adjuvant via subcutaneous injection at multiple sites, with booster injections every two weeks for three weeks, and isolate highly active lymphocytes. Step 3: Extract total RNA from lymphocytes, reverse transcribe to synthesize cDNA, and specifically amplify the VHH gene using nested PCR to accurately obtain the antibody coding sequence; Step 4: The VHH gene is cloned into a phage vector and electroporated to construct a high-capacity display library; Step 5: Solid-phase panning is performed using recombinant antigen as the target. Positive clones are identified by ELISA, and prokaryotic expression vectors are constructed after sequencing. Step 6: Induce expression of recombinant nanobodies, purify them using affinity chromatography, optimize elution conditions, and obtain high-purity, high-yield target protein; Step 7: The antibody activity was comprehensively identified by ELISA, Western blot, immunofluorescence and thermostability assays to confirm its high specificity, high affinity and excellent stability, which meet the application requirements.
2. The method for preparing Brucella antibodies according to claim 1, characterized in that, In step 1, the antigen preparation process is as follows: The Brucella recombinant antigen is one or more combinations of Brucella outer membrane proteins Omp10, Omp19, and BP26, prepared by recombination using a prokaryotic expression system.
3. The method for preparing Brucella antibodies according to claim 1, characterized in that, Step 2, the alpaca immunization process, is as follows: The alpaca immunization program consists of a basic immunization plus three booster immunizations. The antigen dose is 0.5 mg per alpaca, and the immunization route is multiple subcutaneous injections in the neck and back. The immunization interval is 2 weeks, and blood is collected 7 days after the last immunization to ensure efficient stimulation of heavy chain antibody immune response.
4. The method for preparing Brucella antibodies according to claim 1, characterized in that, In step 3, the gene acquisition method is as follows: The lymphocytes were isolated using density gradient centrifugation, with alpaca lymphocyte-specific separation solution used. The centrifugation parameters were 4℃, 2000r / min, and 20min.
5. The method for preparing Brucella antibodies according to claim 1, characterized in that, The total RNA was extracted using the Trizol one-step method. After extraction, the concentration and purity were detected by a nucleic acid quantification instrument. The OD260 / OD280 ratio was controlled between 1.8 and 2.0 to ensure RNA integrity and reverse transcription efficiency. The cDNA synthesis used reverse transcriptase and random hexamer primers in a 20 μL reaction system. The reaction conditions were set at 25℃ for 10 min, 42℃ for 60 min, and 85℃ for 5 min to obtain a high-quality cDNA template. The VHH gene amplification was performed using two rounds of nested PCR. The first round amplified the full-length variable region of the heavy chain antibody, and the second round amplified the VHH core fragment. Primers were designed to target the conserved regions of alpaca IgG2 and IgG3.
6. The method for preparing Brucella antibodies according to claim 1, characterized in that, The construction method for step 4 is as follows: The phage display library was constructed using the pCANTAB-5E phage vector, with TG1 competent cells as the host bacteria. The library was constructed through double enzyme digestion, ligation, and electroporation, with a library volume of not less than 1.0 × 10⁸ CFU to meet the requirements for high-affinity clone screening.
7. The method for preparing Brucella antibodies according to claim 1, characterized in that, The screening method in step 5 is as follows: The positive clone screening was performed using an indirect ELISA method, with Brucella recombinant antigen as the coating antigen and Escherichia coli lysate as the negative control. Clones with an OD450 value three times or more than that of the negative control were selected as positive clones, and the VHH gene sequence was obtained by sequencing. The prokaryotic expression was performed using the pET-28a vector, with BL21 as the host bacterium, a final IPTG concentration of 0.5 mmol / L, an induction temperature of 37℃, and an induction time of 4 h.
8. The method for preparing Brucella antibodies according to claim 7, characterized in that, The prokaryotic expression uses pET-28a as the vector, BL21 as the host bacterium, and the final IPTG concentration is set at 0.5 mmol / L. The induction temperature is 37℃ and the induction time is 4h, which can achieve efficient and soluble expression of nanobodies.
9. The method for preparing Brucella antibodies according to claim 1, characterized in that, Step 6 adopts the following process: The affinity purification was performed using a Ni-NTA agarose gel chromatography column with a binding buffer pH of 8.0 and an elution buffer containing 250 mmol / L imidazole. After the elution product was desalted by PBS, high-purity Brucella nanobody was obtained.
10. The method for preparing Brucella antibodies according to claim 1, characterized in that, The activity identification scheme for step 7 is as follows: The activity identification includes determining the titer by indirect ELISA, verifying specificity by Western blotting, identifying natural antigen binding activity by indirect immunofluorescence assay, and assessing anti-denaturation ability by thermal stability test, thereby comprehensively characterizing the performance of nanobodies.