Antibody combinations for discriminating between brucella wild strains and s2 vaccine and uses thereof

Abstract

This invention discloses an antibody combination and its application for differentiating between wild-type Brucella strains and the S2 vaccine, comprising anti-Omp28 Fab clone L4-7 and anti-IcIR-1 Fab clone Y3-16. The anti-Omp28 Fab clone L4-7 is a fully assembled Fab fragment, with the light chain κ amino acid sequence as shown in SEQ ID NO.3 and the heavy chain Fd amino acid sequence as shown in SEQ ID NO.4. The anti-IcIR-1 Fab clone Y3-16 is a fully assembled Fab fragment, with the light chain κ amino acid sequence as shown in SEQ ID NO.5 and the heavy chain Fd amino acid sequence as shown in SEQ ID NO.6. This invention successfully prepared specific target antigens Omp28 and IcIR-1, and used phage display technology to screen for anti-Omp28 Fab and anti-IcIR-1 Fab with good specificity. The anti-Omp28 Fab can be used for specific detection of Brucella, while the anti-IcIR-1 Fab has the potential to distinguish between S2 vaccine strains and virulent strains. This study provides new antibody materials and ideas for developing rapid diagnostic reagents that can differentiate between Brucella infection and S2 vaccine immunization.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, specifically to an antibody combination and its application for identifying Brucella wild-type strains and S2 vaccine. Background Technology

[0002] Brucella is a Gram-negative bacterium belonging to the genus Brucella. It is the pathogen that causes brucellosis, a zoonotic infectious disease, and is classified as a Class B infectious disease in my country. The genus Brucella currently comprises 10 species, including Brucella bovis (…). Brucella.abortus ), Brucella mesenteriae ( Brucella.melitensis ), swine brucellosis ( Brucella.suis ), canine brucellosis ( Brucella.canis Brucella epididymis of sheep ( Brucella.ovis ), desert forest rodent species Brucella ( Brucella.neotomae Brucella vulgaris () Brucella.microti ), Brucella cetacea ( Brucella.ceti (isolated from whales and dolphins), pinniped Brucella ( Brucella.pinnipedialis (isolated from pinnipeds) and Brucella australis ( Brucella.inopinata (isolated from patients). Among them, the first three types are relatively widespread and can infect humans, seriously affecting the development of animal husbandry and human health in my country.

[0003] The diagnosis of brucellosis relies on routine serological testing and bacterial culture isolation, which is considered the "gold standard" for brucellosis diagnosis. Brucellosis is a zoonotic infectious disease. Etiological testing requires a high level of environmental and laboratory safety, necessitating a biosafety level 3 laboratory. Furthermore, bacteriological methods are time-consuming and dangerous, potentially causing infection among laboratory personnel and serious consequences, thus limiting their application. Currently, laboratories primarily use serological testing, but this technology is not perfect, lacking in sensitivity and specificity. The sensitivity of serological tests ranges from 65% to 95%. The main limitation of serological techniques is their low specificity, due to serological cross-reactivity, particularly between Brucella and Yersinia enterocolitica. In addition, molecular biological diagnostics are also commonly used to diagnose brucellosis. Routine PCR, real-time quantitative PCR, and loop-mediated isothermal amplification assay (LAMP)-PCR have been introduced as rapid diagnostic tools. Multiplex PCR has become a universal technique, capable of simultaneously amplifying multiple sites of one or more different genes. As costs and procedures become increasingly affordable and simple, molecular techniques are being used more and more for rapid microbial diagnostics. Due to their high sensitivity, molecular diagnostics has now become a useful tool for diagnosing many viral, bacterial, and fungal infections. Therefore, providing a detection method that can determine the presence of Brucella wild-type infection, perform typing, shorten experimental cycles, and improve detection efficiency has become a research topic we need to study. Summary of the Invention

[0004] To address the aforementioned technical problems, the first objective of this invention is to provide an antibody combination for identifying Brucella wild-type strains and S2 vaccine immunization, and the second objective is to provide its application. This provides a basis for the development of accurate rapid immunodiagnostic reagents for Brucella.

[0005] To achieve the first objective mentioned above, the present invention is implemented through the following technical solution: an antibody combination for identifying Brucella wild-type strains and S2 vaccine immunization, characterized in that it comprises anti-Omp28 Fab clone L4-7 and anti-IcIR-1 Fab clone Y3-16;

[0006] The anti-Omp28 Fab clone L4-7 is a fully assembled Fab fragment, with the light chain κ amino acid sequence as shown in SEQ ID NO.3 and the heavy chain Fd amino acid sequence as shown in SEQ ID NO.4;

[0007] The anti-IcIR-1 Fab clone Y3-16 is a fully assembled Fab fragment, with the light chain κ amino acid sequence shown in SEQ ID NO.5 and the heavy chain Fd amino acid sequence shown in SEQ ID NO.6.

[0008] The L4-7 clone specifically binds to the Omp28 protein of SEQ ID NO.1, exhibiting no cross-reactivity with *Escherichia coli*, *Staphylococcus aureus*, *Yersinia*, and *Salmonella*, and can recognize the S2 vaccine, A19 vaccine, and wild-type *Brucella* virus. The Y3-16 Fab specifically binds to the IcIR-1 polypeptide of SEQ ID NO.2, recognizing only the A19 vaccine and wild-type *Brucella* virus, but not the S2 vaccine antigen. It shows no specific reaction with the S2 vaccine, no cross-reactivity with *Escherichia coli* and *Staphylococcus aureus*, and extremely weak cross-reactivity with *Yersinia* and *Salmonella*.

[0009] The application of the antibody combination used to distinguish between Brucella wild-type strains and S vaccine immunization in the preparation of a veterinary diagnostic kit to differentiate between Brucella S2 vaccine immunization and wild-type strain infection.

[0010] It also contains two specific antigens:

[0011] (1) Brucella conserved mature outer membrane protein Omp28, the amino acid sequence of which is shown in SEQ ID NO.1;

[0012] (2) The S2 vaccine-specific deletion polypeptide IcIR-1, the amino acid sequence of which is shown in SEQ ID NO.2;

[0013] The Omp28 protein, with an amino acid sequence as shown in SEQ ID NO.1, is present in all bovine Brucella species ( Brucella abortus, B. abortus ), Brucella mesenteriae ( Brucella.melitensis, B.melitensis ), swine brucellosis ( Brucella.suis, B.suis The IcIR-1 polypeptide is stably expressed in Brucella wild-type virus and A19 vaccine, and the S2 vaccine genome lacks this coding segment.

[0014] The A19 vaccine is made from an attenuated strain of Brucella bovis, while the S2 vaccine is made from an attenuated strain of Brucella suis.

[0015] The preparation method of the target antigen is as follows: based on the conserved amino acid sequence Omp28 of Brucella obtained by screening, a recombinant expression strain of the conserved protein Omp28 was constructed, and expressed and purified; based on the S2 vaccine missing amino acid sequence IcIR-1 obtained by screening, the S2 vaccine missing peptide IcIR-1 was chemically synthesized.

[0016] The kit also includes blocking solution, washing solution, colorimetric solution, etc., which is existing technology.

[0017] Test kit interpretation criteria: In scenarios where livestock and poultry have been vaccinated with S2 vaccine, Omp28 positivity and IcIR-1 positivity indicate wild-type virus infection; Omp28 positivity and IcIR-1 negativity indicate S2 vaccine immunization only; double negativity indicates no infection and no immunization.

[0018] Application of Omp28 protein, with amino acid sequence as shown in SEQ ID NO.1, in the preparation of antibody detection and diagnostic reagents for Brucella serum samples.

[0019] The application of an Omp28 protein with an amino acid sequence as shown in SEQ ID NO.1 and a polypeptide IcIR-1 with an amino acid sequence as shown in SEQ ID NO.2 in the preparation of a reagent for distinguishing whether a Brucella serum sample is derived from a virulent strain or an S2 vaccine strain.

[0020] Bioinformatics analysis was used to compare and screen Brucella conserved protein Omp28 and S2 vaccine-deficient peptide IcIR-1, both of which were verified to have good immunogenicity. Omp28, a highly conserved Brucella protein, can be used for antibody detection and diagnosis in Brucella serum samples. In cases where the livestock immune background is unknown, the combined use of IcIR-1 peptide can further differentiate between antibodies originating from virulent strains or the S2 vaccine strain. If other anti-brucellosis vaccines are involved, the corresponding vaccine-deficient protein should be used in conjunction to improve the specificity and accuracy of the detection. In cases of early infection or low serum antibody levels, this method needs to be combined with nucleic acid detection methods.

[0021] Beneficial effects:

[0022] This invention successfully prepared specific target antigens Omp28 and IcIR-1, and used phage display technology to screen for anti-Omp28 Fab and anti-IcIR-1 Fab with good specificity. The anti-Omp28 Fab can be used for specific detection of Brucella, while the anti-IcIR-1 Fab has the potential to distinguish between S2 vaccine strains and virulent strains. This research provides new antibody materials and ideas for developing rapid diagnostic reagents that can differentiate between Brucella infection and S2 vaccine immunization. Attached Figure Description

[0023] Figure 1 The results of PCR identification of monoclonal colonies in the Brucella S2 vaccine Fab antibody library.

[0024] Figure 2 The results of PCR identification of single-clonal colonies in the Omp28 Fab antibody library.

[0025] Figure 3 Phage-ELISA results for anti-Omp28 Fab.

[0026] Figure 4 The molecular weight of the anti-Omp28 Fab antibody under reducing conditions is determined.

[0027] Figure 5The molecular weight determination results of the anti-Omp28 Fab antibody under non-reducing conditions.

[0028] Figure 6 The results show the specificity of the anti-Omp28 Fab assay.

[0029] Figure 7 The results are from the PCR identification of anti-IcIR-1 Fab.

[0030] Figure 8 To detect the interaction between the IcIR-1 peptide and each clone Fab using indirect ELISA.

[0031] Figure 9 The graph shows the molecular weight identification results of the anti-IcIR-1 Fab antibody.

[0032] Figure 10 The image shows the results of specific detection of anti-IcIR-1 Fab antibody. Detailed Implementation

[0033] The present invention will be further described below with reference to embodiments.

[0034] Example 1 Antigen Analysis Screening

[0035] Gene sequences of *Brucella suis*, *Brucella melitensis*, *Brucella bovis*, and the anti-brucellosis vaccine S2 were obtained from the NCBI website. Gene alignment was performed using SnapGene, BioEdit, DNAMAN, and the BLAST function of the NCBI website to obtain the conserved amino acid sequences of *Brucella suis*, *Brucella melitensis*, and *Brucella bovis*. The deleted amino acid sequence of the brucellosis vaccine S2 was obtained by comparing the gene sequences of virulent *Brucella* strains and the S2 vaccine strain. Regions with good conservation, numerous dominant epitopes, and strong immunogenicity were selected as the conserved antigen sequences for this study; the deleted region of the S2 vaccine, which has strong immunogenicity and encodes a protein, was selected as the deleted antigen sequence for this study.

[0036] The bead deletion sequence of the S2 vaccine and the conserved sequences of Brucella suis, Brucella mesenteriae, and Brucella bovis were analyzed. Signal peptides were analyzed using the online website http: / / www.detaibio.com / tools / signal-peptide.htmL, hydrophobicity was analyzed using http: / / www.detaibio.com / tools / hydropathy-analysis.htmL, B-cell dominant epitopes were analyzed using http: / / sysbio.unl.edu / SVMTriP / prediction.php, and transmembrane regions were analyzed using http: / / www.detaibio.com / tools / transmembrane.htmL.

[0037] 1. Omp28 sequence analysis

[0038] Through gene alignment and screening, Omp28, a strain of Brucella with relatively good conservation, was selected for sequence feature analysis. Its amino acid sequence is as follows:

[0039] MNTRASNFLAASFSTIMLVGAFSLPAFAQENQMTTQPARIAVTGEGMMTASPDMAILNLSVLRQAKTAREAMTANNEAMTKVLDAMKKAGIEDRDLQTGGINIQPIYVYPDDKNNLKEPTITGYS VSTSLTVRVRELANVGKILDESVTLGVNQGGDLNLVNDNPSAVINEARKRAVANAIAKAKTLADAAGVGLGRVVEISELSRPPMPMPIARGQFRTMLAAAPDNSVPIAAGENSYNVSVNVVFEIK.

[0040] The transmembrane region, signal peptide, hydrophobicity, and B-cell dominant epitopes of the Omp28 amino acid sequence were analyzed. The results showed that the Omp28 amino acid sequence has a transmembrane structure in the N-terminal 7-29 peptide segment, containing multiple hydrophobic and hydrophilic regions, consistent with the typical hydrophobic distribution characteristics of membrane proteins. A signal peptide and multiple B-cell dominant epitopes are present in the N-terminal 1-28 peptide segment. Based on these analysis results, the segment lacking a signal peptide, transmembrane region, and rich in antigenic epitopes was selected as the conserved protein for this study. The amino acid sequence of the mature Omp28 protein was optimized as shown in SEQ ID NO. 1:

[0041] AQENQMTTQPARIAVTGEGMMTASPDMAILNLSVLRQAKTAREAMTANNEAMTKVLDAMKKAGIEDRDLQTGGINIQPIYVYPDDKNNLKEPTITGYSVSTSLTVRVRELAN VGKILDESVTLGVNQGGDLNLVNDNPSAVINEARKRAVANAIAKAKTLADAAGVGLGRVVEISELSRPPMPMPIARGQFRTMLAAAPDNSVPIAAGENSYNVSVNVVFEIK.

[0042] 2. IcIR-1 sequence analysis

[0043] Through gene alignment and screening, the deletion protein IcIR of the anti-brucellosis S2 vaccine was selected for sequence feature analysis. IcIR exists in different species of Brucella (e.g., ...). B. abortus, B. melitensis, B. suis It is highly conserved in the group, and its amino acid sequence is as follows:

[0044] MREPTLETDDRYRAPALDKGLDILELLASVDGGLTQAEIAKHLDRSPNEFYRMLDRLVKRGYVTKLDGDRYSLTLKLFGLAQLHAPVRRLASFATPFMRELADRSKQANQLAVFDRGSVVVIAQQEAPDYWG ISIRVGSHISLFDTGSGHVLLAFRSPEEREMMIAAHVKSRDEVNLDQDFYDRLDQIRERGYEMMASAQMAGAYNLSAPILGPDGTCIAALTCPYITLVNPSSAPDITQTISLLQKTVRDLSKLVGADVGVTG.

[0045] The N-terminal 1-52aa peptide segment of the S2 vaccine IcIR is deleted, and the sequence is: MREPTTLETDDRYRAPALDKGLDILELLASVDGGLTQAEIAKHLDRSPNEFYR.

[0046] Analysis of the transmembrane region, signal peptide, hydrophobicity, and B-cell dominant epitope of the deleted peptide amino acid sequence revealed that the deleted peptide contains a B-cell dominant epitope. Based on the above sequence analysis results, a highly conserved deletion region containing a dominant epitope from virulent Brucella strains was selected as the target antigen for this study. The optimized amino acid sequence of the S2 vaccine-specific deleted peptide IcIR-1 is shown in SEQ ID NO.2: MREPTLE TDDRYRAPALDKGLDILE.

[0047] Example 2: Preparation of recombinant Omp28 protein

[0048] 1. Preparation of Transetta competent cells

[0049] A single colony of activated *E. coli* Transetta was picked from LB-C (25 μg / mL Chl) solid medium and inoculated into 5 mL of LB-C (25 μg / mL Chl) liquid medium, and incubated overnight at 37°C with shaking. The next day, 1 mL of the bacterial culture was transferred to 100 mL of LB liquid medium and incubated at 37°C and 250 rpm until OD reached. 600 =Approximately 0.38. Transfer the bacterial culture to a sterile, pre-chilled centrifuge tube and place on ice for 10 min. Centrifuge at 4000 rpm for 10 min at 4°C, discard the supernatant, and invert the tube for 1 min. Gently resuspend the cells in pre-chilled 0.1 mol / L sterile CaCl2 solution and place on ice for 30 min. Centrifuge at 4000 rpm for 10 min at 4°C, discard the supernatant. Add 8 mL of pre-chilled 0.1 mol / L CaCl2 solution, gently resuspend the cells, and place on ice for 14 h. Add an equal volume of 30% glycerol to the prepared competent cells, aliquot into 100 μL tubes, and freeze at -80°C.

[0050] 2. Preparation of prokaryotic expression strain of recombinant Omp28

[0051] The pET28a(+)-Omp28 plasmid was diluted to a concentration of 50 ng / μL. 1 μL of the diluted plasmid mixture was added to one tube of Transetta competent cells, gently mixed, and incubated on ice for 5 min. After incubating at 42°C for 1.5 min, it was immediately removed and placed on ice for 5 min. 900 μL of LB liquid culture medium was added to the transformation medium, and the cells were incubated at 37°C with shaking at 250 rpm for 1 h. Then, 50 μL of the transformation medium was plated onto LB-K (30 μg / mL Kan) solid medium and incubated overnight at 37°C. The next day, single clones were picked from the culture plate for colony PCR identification. Positive single clones were used for protein expression.

[0052] 3. Optimization of expression conditions for recombinant Omp28 protein

[0053] Selected positive monoclonal clones were inoculated into 20 mL of LB-K (30 μg / mL Kan) liquid medium and cultured overnight at 37°C with shaking at 250 rpm. The next day, the seed culture was added to 60 mL of LB-K (30 μg / mL Kan) liquid medium at a ratio of 1:100 and cultured at 37°C until OD (Organic Growth Rate) was reached. 600When the concentration of the protein reaches 0.8-0.9, add 24 μL of 1M IPTG (final concentration 0.4 mM). The temperature is 37℃, the IPTG concentration is 0.4 mM, and the induction time is 5 h. After induction, collect the cells by centrifugation at 4000 rpm for 15 min. Resuspend the precipitate in 20 mL of 1×PBS and centrifuge at 4000 rpm for 15 min. Repeat this process and weigh the precipitate. Resuspend the collected sample at a ratio of cell wet weight to lysis buffer of 1:10, add PMSF to a final concentration of 0.1 mM, place the resuspended sample on ice, set the sonication intensity to 30%, and perform sonication for 3 seconds followed by a 6-second interval for 15 min to lyse the cells. After sonication, centrifuge at 10000 rpm for 15 min using a refrigerated centrifuge to separate the supernatant. Mix the supernatant with 3×SDS-PAGE Loading Buffer at a ratio of 1:2 and incubate at 100℃ for 10 min. SDS-PAGE gels with a separating gel concentration of 12% and a stacking gel concentration of 5% were used for electrophoresis at a constant current of 30mA. After electrophoresis, the gels were cut off, stained with Coomassie Brilliant Blue for 1 hour, and then destained overnight. The gels were photographed and developed using a gel imaging system to observe the expression of the target protein. The recombinant Omp28 protein produced the highest content of the target protein under the conditions of a temperature of 37℃, an IPTG concentration of 0.4mM, and an induction time of 5 hours, and was expressed as intracellular soluble protein.

[0054] 4. Purification of Omp28 protein

[0055] Single colonies of pET28a(+)-Omp28 / Transetta were picked and inoculated into 20 mL of LB-K (30 μg / mL Kan) liquid medium and cultured overnight at 37°C with shaking at 250 rpm. The next day, the seed culture was added to 1 L of LB-K (30 μg / mL Kan) liquid medium at a ratio of 1:100 and cultured at 37°C until OD280 was reached. 600 When the concentration of the protein inoculum reaches 0.8-0.9, add 400 μL of 1M IPTG (final concentration 0.4 mM). Incubate at 37℃, 250 rpm for 5 h with shaking. After induction, collect the bacterial cells by centrifugation at 4000 rpm for 15 min. Resuspend the precipitate in 20 mL of 1×PBS and centrifuge at 4000 rpm for 15 min. Repeat this process and weigh the precipitate. Resuspend the precipitate at a wet weight to lysis buffer ratio of 1:10 and add PMSF to a final concentration of 0.1 mM. Perform cell disruption using a high-pressure homogenizer. After disruption, centrifuge at 4℃, 10000 rpm for 15 min to obtain the supernatant. Remove impurities from the protein supernatant using a 0.45 μm microporous membrane and store on ice. Purify the Omp28 protein using a gravity flow nickel affinity chromatography column. The specific steps are as follows:

[0056] (1) Pour the equilibration buffer into the pre-packed column and roll the regulator pulley to the top to prevent liquid from flowing out. Rinse the pad with ultrapure water and place the lower pad at the bottom of the pre-packed column with tweezers, being careful not to generate air bubbles.

[0057] (2) Add 3 mL of nickel affinity agarose gel medium to the pre-packed column. After 30 min of natural sedimentation, use tweezers to place the upper gasket on top of the medium. Be careful not to generate air bubbles during the column packing process.

[0058] (3) Connect the gravity flow nickel affinity chromatography column to the nucleic acid protein detector, and roll the regulator pulley to make the drop rate of the equilibration buffer reach 7 seconds / drop. When the transmittance (T%) reaches 100, adjust the absorbance (A) value to 0.

[0059] (4) After the values ​​stabilize, load the sample. When the level of the equilibration buffer is close to the upper pad, slowly pour the treated protein supernatant into the gravity flow nickel affinity chromatography column. Roll the regulator pulley to make the protein supernatant drop rate reach 8 seconds / drop. Load the sample in batches. Use a 1.5 mL EP tube to collect the percolation solution and place it on ice.

[0060] (5) After the sample loading is completed, add equilibration buffer. When the absorbance value on the nucleic acid protein detector does not change, elute with 40mM, 100mM, 200mM and 400mM imidazole elution buffer respectively. Determine the peak condition by the absorbance value and collect the elution buffer at different peak values ​​and place it on ice.

[0061] (6) After elution, add equilibration buffer until the absorbance value does not change, add 20% ethanol to seal the purification column, and place it in a 4°C refrigerator.

[0062] (7) The protein concentration of the column permeate and elution buffer was determined by BCA method. The collected sample was mixed with 3× Loading Buffer and incubated in a water bath at 100℃ for 10 min. The purification status of the target protein was checked by SDS-PAGE gel electrophoresis with a separating gel concentration of 12% and a stacking gel concentration of 5%. The protein purity was analyzed by ImageJ software. The calculation formula is: Purity (%) = gray value of target protein band / total gray value of all bands in the lane × 100%.

[0063] 5. Identification of the molecular weight of recombinant Omp28 protein

[0064] Prepare SDS-PAGE gels with a separating gel concentration of 12% and a stacking gel concentration of 5%. Mix the expressed protein with 3× Loading Buffer and incubate at 100°C for 10 min. Electrophoresis conditions: constant voltage of 65V until the protein marker enters the separating gel, then change the voltage to 120V and stop electrophoresis when bromophenol blue migrates to the bottom of the separating gel. After electrophoresis, assemble the transfer clips, avoiding air bubbles between the membrane and the gel during assembly. Place the clips in pre-cooled transfer buffer and transfer the membrane at a constant current of 200mA for 1 h 30 min. After transfer, wash with TBST for 10 min, then block with 5% skim milk powder-TBST at room temperature for 1 h. After blocking, wash three times with TBST for 10 min each time. Dilute 6×His-Tag monoclonal antibody with blocking buffer at a ratio of 1:5000 and incubate at 4°C for 14 h. The following day, HRP-labeled Goat Anti-Mouse IgG antibody was diluted 1:10000 and incubated at room temperature for 1 hour. The cells were washed three times with TBST for 10 minutes each time. The developing solution was prepared according to the instructions of the ultrasensitive ECL chromogenic kit, and the cells were developed and exposed using a gel imaging system to determine the molecular weight of the protein.

[0065] Recombinant Omp28 protein was expressed in large quantities at 37℃, IPTG concentration of 0.4 mM, and induction time of 5 h. Purification was performed using a gravity flow nickel affinity chromatography column with gradient elution using 40 mM, 100 mM, 200 mM, and 400 mM imidazole elution buffers. SDS-PAGE electrophoresis showed that recombinant Omp28 protein was eluted in large quantities at imidazole concentrations of 200 mM and 400 mM, with purities of 95% and 98%, respectively, forming a single target band with a molecular weight of 28 kDa.

[0066] Recombinant Omp28 protein was analyzed by Western blot after SDS-PAGE electrophoresis. The molecular weight of the protein was determined using 6×His-Tag monoclonal antibody as the primary antibody and HRP-labeled Goat Anti-Mouse IgG as the secondary antibody. The results showed a distinct band around 28 kDa for the recombinant Omp28 protein, indicating a molecular weight of 28 kDa, consistent with the theoretical value.

[0067] Example 3: Construction of the Fab phage antibody library for the anti-brucellosis S2 vaccine

[0068] 1. Animal immunization and serum sample collection

[0069] Nine healthy female BALB / c mice aged 7-8 weeks were randomly divided into three groups of three each. One group served as a negative control, while the other two groups were administered 3.14 × 10⁻⁶ mg / L via gavage and intraperitoneal injection, respectively. 6 CFU / each, 1.57×10 6Mice were immunized with CFU / mouse of the anti-brucellosis S2 vaccine. The specific procedure was as follows: Anti-brucellosis S2 vaccine powder was weighed and added to a 10mL centrifuge tube, dissolved in physiological saline, and serially diluted. The dosage was determined based on the mouse's body weight. A second immunization was administered three weeks after the first immunization. Blood and spleen samples were collected one week after the second booster immunization. Serum antibodies in immunized mice were detected using a Brucella competitive ELISA kit from Luoyang Laipusheng Information Technology Co., Ltd., to assess the mice's immune status. Results showed that all six immunized mice produced antibodies (S / N value less than 0.5), with a lower S / N value in the gavage group. The antibody titers in the gavage-immunized mice were higher than those in the injection group.

[0070] 2. Extraction of total RNA from mouse spleen

[0071] Total RNA was extracted from the spleen of immunized mice using the Ultrapure RNA Kit (DNase I) from Kangwei Century Company. The specific procedures are as follows:

[0072] (1) Sample processing

[0073] After thoroughly grinding the spleens of immunized mice in liquid nitrogen, 3 mL of Trlzon Reagent was added in three portions and ground in the same way. The ground samples were then placed in RNase-Free centrifuge tubes and frozen in liquid nitrogen.

[0074] (2) Take the processed spleen out of the -80℃ freezer, thaw it at room temperature, blow it repeatedly to ensure that the spleen is fully lysed, and leave it at room temperature for 5 minutes.

[0075] (3) Add 600 μL of chloroform, shake vigorously for 15 seconds, and let stand at room temperature for 2 minutes.

[0076] (4) Centrifuge at 12000 rpm and 4℃ for 10 min, and take the upper aqueous phase and add an equal volume of 70% ethanol to mix.

[0077] (5) Add the above solution to the collection tube equipped with the adsorption column, centrifuge at 12000 rpm for 20 s and discard the filtrate.

[0078] (6) Add 350 μL Buffer RW1, centrifuge at 12000 rpm for 20 s and discard the filtrate.

[0079] (7) DNase I mixture: Take 52 μL RNase-Free Water, 8 μL 10×Reaction Buffer and 20 μL DNase I (1U / μL) and mix them evenly in a 1.5 mL centrifuge tube.

[0080] (8) Add 350 μL Buffer RW1 to the adsorption column, centrifuge at 12000 rpm for 1 min and discard the filtrate.

[0081] (9) Add 500 μL Buffer RW2, centrifuge at 12000 rpm for 20 s and discard the filtrate.

[0082] (10) Repeat operation 9.

[0083] (11) Centrifuge at 12000 rpm for 2 min, discard the filtrate and evaporate the ethanol.

[0084] (12) Add 40 μL of RNase-Free Water to the adsorption column, place at room temperature for 2 min, centrifuge at 12000 rpm for 1 min, recover the solution and centrifuge again, collect and store at -80℃.

[0085] The total RNA in the spleen of immunized mice was examined by agarose gel electrophoresis. The results showed that the RNA bands in the gavage group were clearer, brighter, and had higher concentrations than those in the injection group. Combined with the results of competitive ELISA, the average S / N value of the gavage group was 0.13, while that of the injection group was 0.18, indicating that the gavage group produced higher antibody titers. Therefore, the RNA from the gavage group was selected as the template for the next step of Fab amplification.

[0086] 3. cDNA strand synthesis

[0087] The extracted RNA was reverse transcribed into cDNA using the HiFi Script cDNA Synthesis Kit from Kangwei Century Company. The specific steps are as follows:

[0088] (1) Dissolve the RNA template, Primer Mix, dNTP Mix, DTT, RT Buffer, HiFiScript and RNase-FreeWater and place them on ice for later use to prepare reaction system 1 (see Table 1).

[0089] Table 1. cDNA strand synthesis reaction system 1

[0090]

[0091] (2) React at 70℃ for 10 min, then in an ice bath for 2 min, and centrifuge.

[0092] (3) Add the following reagents to the reaction solution, see Table 2.

[0093] Table 2 cDNA strand synthesis reaction system 2

[0094]

[0095] (4) React at 50℃ for 50 min; or at 85℃ for 5 min.

[0096] (5) After a short centrifugation, cool on ice and store at -80°C.

[0097] 4. Amplification of the Fab gene in the anti-brucellosis S2 vaccine

[0098] 4.1 Amplification of the κ gene and Fd segment gene

[0099] Using primers designed by the Scripps Research Institute (Table 3), the full-length genes of the anti-brucellosis S2 vaccine antibody κ and Fd segments were amplified using cDNA as templates. The amplification systems are shown in Tables 4 and 5. After amplification, the PCR product bands were examined using a 1.5% agarose gel.

[0100] Table 3 Primers for Fab antibody gene amplification

[0101]

[0102] Note: CTC GAG is the Xho I restriction site at the 5' end of the heavy chain, and ACT AGT is the Spe I restriction site at the 3' end of the heavy chain; GAG CTC is the Sac I restriction site at the 5' end of the light chain, and TCT AGA is the Xba I restriction site at the 3' end of the light chain. R represents A / G, K represents T / G, and W represents A / T.

[0103] Table 4 PCR amplification reaction system for the κ gene

[0104]

[0105] The amplification conditions were: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 1 min, 65℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 10 min.

[0106] Table 5. PCR amplification reaction system for Fd gene

[0107]

[0108] The amplification conditions were: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 1 min; 63℃ annealing for 30 s; 72℃ extension for 30 s; 35 cycles; 72℃ extension for 10 min.

[0109] 4.2 Purification and Recovery of PCR Products

[0110] After electrophoresis, the target band was excised, and the PCR product was purified and recovered using the OMEGA agarose DNA gel extraction kit. The specific procedures are as follows:

[0111] (1) The target gene fragment was separated by 1.5% agarose gel and the target band was cut into a centrifuge tube.

[0112] (2) Add XP2 Binding Buffer of the same volume as the target band, and incubate in a water bath at 55°C for 10 minutes until the gel is completely melted.

[0113] (3) Take 700 μL of liquid into a collection tube containing an adsorption column, centrifuge at 10000g for 1 min and discard the filtrate.

[0114] (4) Add 300 μL XP2 Binding Buffer, centrifuge at 13000g for 1 min, and discard the filtrate.

[0115] (5) Add 700 μL SPW Wash Buffer, centrifuge at 13000g for 1 min, and discard the filtrate.

[0116] (6) Repeat step 5.

[0117] (7) Centrifuge for 2 min, discard the filtrate, and let it stand at room temperature for 15 min to evaporate the ethanol.

[0118] (8) Add 20 μL of Elution Buffer, let stand at room temperature for 2 min, centrifuge at 13000g for 1 min, and store in a -20℃ refrigerator.

[0119] 5. Extraction of pComb3XSS plasmid

[0120] The pComb3XSS / TG1 strain, stored at -80℃, was streaked onto 2×YT-A (100 μg / mL Amp) solid medium and incubated overnight at 37℃. The next day, single colonies were picked and inoculated into 20 mL of 2×YT-A (100 μg / mL Amp) liquid medium and incubated at 37℃ and 250 rpm for 14 h. Plasmids were extracted using the OMEGA plasmid DNA Mini Purification Kit. The purity and concentration of the plasmids were determined using a General Electric micro-spectrophotometer and stored at -20℃.

[0121] 6. Preparation of Escherichia coli XL1-Blue competent cells

[0122] 6.1 Preparation of XL1-Blue competent cells by electroporation

[0123] (1) Take out the XL1-Blue strain, streak it on LB-T (50 μg / mL Tet) plate, and incubate overnight at 37°C.

[0124] (2) The next day, single clones were picked and cultured in 20 mL SB-T (10 μg / mL Tet) liquid medium at 37℃ and 250 rpm for 14 h.

[0125] (3) The next day, the bacterial culture was added to 500 mL of SB-T (10 μg / mL Tet) liquid culture medium at a ratio of 1:50, and cultured at 37°C and 250 rpm until OD was obtained. 600 When the concentration is 0.7-0.8, immediately remove the bacterial solution and place it on ice for 30 minutes.

[0126] (4) Transfer the bacterial culture to a pre-cooled centrifuge bottle and centrifuge at 3500 rpm and 4°C for 15 min;

[0127] (5) Discard the supernatant, add 200 mL of pre-cooled 10% glycerol to gently resuspend the cells. The entire process must be carried out on ice. After the cells are completely dissolved, centrifuge at 3500 rpm and 4°C for 15 min.

[0128] (6) Discard the supernatant and add 100 mL of pre-cooled 10% glycerol to repeat the above operation.

[0129] (7) Discard the supernatant and add 50 mL of pre-cooled 10% glycerol to repeat the above operation.

[0130] (8) Discard the supernatant, add 1 mL of pre-cooled 10% glycerol and gently resuspend the cells in an ice-water bath, dispense into 80 μL / vial, freeze quickly with liquid nitrogen, and store at -80℃.

[0131] 6.2 Determination of XL1-Blue competent cell titer

[0132] (1) Take out one XL1-Blue competent cell, wait for it to melt and place it on ice. Place the pComb3XSS plasmid and the electroporation cup on ice for 5 minutes.

[0133] (2) Gently pipette 1 ng of pComb3XSS plasmid into one XL1-Blue competent cell, add the mixed liquid to an electroporation cup, and place on ice for 10 min.

[0134] (3) Use the Bio-Rad electroporator in Bacteria Ec2 mode. After the electroporation cup is wiped dry, immediately place it in the electroporation tank for electroporation. After the beep sounds, remove the electroporation cup and immediately add 1 mL of preheated SOC liquid culture medium to the electroporation cup. Mix well and transfer to a sterile centrifuge tube. Incubate at 37°C and 250 rpm for 1 h.

[0135] (4) Spread 10 μL of electroporation bacterial culture on LB-A (100 μg / mL Amp) plate for culture.

[0136] (5) The next day, the titer of XL1-Blue competent cells was calculated by single-clone counting.

[0137] 7. Preparation of the helper phage VCSM13

[0138] 7.1 Amplification of helper phage VCSM13

[0139] (1) Used sterile SB liquid medium to serially dilute stored helper phage VCSM13

[0140] (2) Take 1 mL of diluted helper phage VCSM13 to infect 100 mg of XL1-Blue cells (from 10 mL OD). 600 (Obtained by centrifuging XL1-Blue bacterial culture at 4000 rpm for 15 min with a concentration of 0.8-0.9), and then incubating at 37℃ with slow shaking at 100 rpm for 30 min.

[0141] (3) Add the infected XL1-Blue bacterial solution to 5 mL of LB semi-solid medium at 50℃, quickly invert and mix well, pour onto LB solid medium plates without resistance, and incubate at 37℃ overnight.

[0142] (4) The next day, take a 1cm×1cm phage plaque and inoculate it into 20mL OD 600 Incubate XL1-Blue bacterial culture at 0.8-0.9 g / mL for 2 hours at 37°C with shaking at 250 rpm.

[0143] (5) Add the above bacterial solution to 100 mL SB-TK (10 μg / mL Tet, 70 μg / mL Kan) liquid culture medium and culture overnight at 37 °C and 250 rpm with shaking.

[0144] (6) The next day, the bacterial solution was dispensed into sterile centrifuge tubes and centrifuged at 4000 rpm for 15 min at 4°C.

[0145] (7) The supernatant was lysed in a water bath at 70°C. After 20 min, it was centrifuged at 4°C and 4000 rpm for 15 min.

[0146] (8) Collect the supernatant, which is the amplified helper phage VCSM13. Filter it with a 0.45 μM microporous membrane to remove impurities, aliquot it into sterile 10 mL centrifuge tubes, and store at 4 °C.

[0147] 7.2 Determination of VCSM13 titer of auxiliary bacteriophage

[0148] The stored helper phages were serially diluted to 10⁻⁶ using sterile SB liquid medium. -9 10 -10 10 -11Take 100 μL of diluted helper phage to infect 500 μL of OD. 600 XL1-Blue bacterial suspension with a concentration of 0.8-0.9 was incubated at 37°C with shaking at 100 rpm for 30 min. The infected bacterial suspension was added to 5 mL of LB semi-solid medium incubated at 50°C, quickly inverted to mix, and then poured onto antibiotic-free LB solid medium. The medium was incubated overnight at 37°C. The following day, the number of plaques was calculated to estimate the titer of the prepared helper phage.

[0149] 8. Construction and identification of the anti-brucellosis S2 vaccine antibody κ library

[0150] 8.1 Double digestion of pComb3XSS plasmid and κ gene of anti-brucellosis S2 vaccine antibody

[0151] The 60 μg pComb3XSS plasmid and 12 μg κ chain mixture (equal mass of κ gene amplified by different primers) were digested with SacI and XbaI. The double digestion reaction system is shown in Tables 6 and 7.

[0152] Table 6. Sac I / Xba I double digestion of pComb3XSS

[0153]

[0154] Table 7. Sac I / Xba I double enzyme digestion of κ gene

[0155]

[0156] 8.2 Recovery and purification of double enzyme digestion products

[0157] The double digestion products of pComb3XSS plasmid and κ chain mixture were separated by 0.7% and 1.5% agarose gels. The target bands of both were excised and the DNA fragments were recovered and purified using a gel recovery kit.

[0158] 8.3 Connecting pComb3XS to the κ gene

[0159] The κ gene and the double digestion product of the pComb3XSS plasmid were ligated using DNA ligase. The ligation system is shown in Table 8, where the molar ratio of fragment to vector is 3:1. The reaction conditions were 16℃ for 14 h.

[0160] Table 8. Antibrucellosis S2 vaccine antibody κ gene and vector linkage system

[0161]

[0162] 8.4. Recovery and purification of ligation products

[0163] The ligation product was purified and recovered using the kit. The ligation product was briefly centrifuged, and the volume was brought up to 100 μL with sterile water. An equal volume of XP2 Binding Buffer was then added and mixed. The purification steps are as described in 4.2.

[0164] 8.5 Electroconversion Connection Products

[0165] (1) Add 4 μL of the above ligation product to 5 tubes of 80 μL XL1-Blue electroporation competent cells and mix gently. Add the mixed liquid to a 0.2 cm electroporation cuvette, let it stand for 10 min, then place the electroporation cuvette in the electroporator and set the electroporation mode to Bacteria Ec2.

[0166] (2) After the prompt sound, remove the electroporation cup, add 1 mL of 37°C SOC liquid culture medium to the electroporation cup immediately, mix the liquid in the electroporation cup, and transfer it to a sterile EP tube. Incubate at 37°C and 250 rpm for 1 h.

[0167] (3) After mixing by pipetting, 8 μL, 4 μL, 2 μL and 1 μL of the electroconversion solution were respectively spread on LB-A (100 μg / mL Amp) solid medium, incubated overnight at 37°C, and the number of single clones was recorded and the conversion efficiency was calculated.

[0168] (4) Take the remaining electroconversion solution into 10 mL of SB-AT (20 μg / mL Amp and 10 μg / mL Tet) liquid medium and incubate at 37 °C and 250 rpm for 1 h.

[0169] (5) Add 4.5 μL of 100 mg / mL Amp (final concentration 50 μg / mL), and incubate at 37℃ and 250 rpm for 1 h with shaking.

[0170] (6) Add the above culture solution to 100 mL SB-AT (50 μg / mL Amp and 10 μg / mL Tet) liquid culture medium, shake overnight at 37 °C and 250 rpm.

[0171] (7) The next day, a recombinant plasmid containing the κ gene was extracted using a plasmid extraction kit, named pComb3XS-κ, and the DNA content and purity were determined.

[0172] 8.6. Large-scale extraction of recombinant plasmid pComb3XS-κ

[0173] The pComb3XS-κ recombinant plasmid was extracted using a plasmid extraction kit from Nanjing Novizan Pharmaceutical Co., Ltd.

[0174] 8.7 Identification and volume determination of the anti-brucellosis S2 vaccine antibody κ library

[0175] Twenty single clones were randomly selected from the LB-A plates cultured overnight in step 4.2.9.5 for PCR identification. The system is shown in Table 9. The PCR products were separated by 1.5% agarose gel. The recombination of the κ gene was checked based on the target band, and the library capacity of the light chain library was estimated.

[0176] Table 9. Colony PCR Identification κ Library Reaction System

[0177]

[0178] The amplification conditions were: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 1 min, 65℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 10 min.

[0179] 9. Construction of the primary phage antibody library for the anti-brucellosis S2 vaccine Fab

[0180] 9.1. Double enzyme digestion of pComb3XS-κ recombinant plasmid and Fd gene of anti-brucellosis S2 vaccine antibody

[0181] The pComb3XS-κ recombinant plasmid and 25 μg of anti-brucellosis S2 vaccine antibody Fd mixture (equal mass of Fd gene amplified by each primer pair) were digested with Spe I / Xho I. The double digestion reaction system is shown in Tables 10 and 11.

[0182] Table 10. pComb3XS-κ double enzyme digestion with Spe I / Xho I

[0183]

[0184] Table 11 Spe I / Xho I double enzyme digestion of Fd

[0185]

[0186] 9.2 Recovery and purification of double enzyme digestion products

[0187] The method is the same as step 8.2.

[0188] 9.3 Connect pComb3XS-κ and Fd

[0189] The Fd gene and the double digestion product of the pComb3XS-κ plasmid were ligated using DNA ligase. The ligation system is shown in Table 12, with a molar ratio of ligation fragment to vector of 3:1. The reaction conditions were 16℃ for 14 h.

[0190] Table 12. Antibrucellosis S2 vaccine antibody Fd gene and vector ligation system

[0191]

[0192] 9.4 Electroconversion Connection Products

[0193] The method is the same as steps 8.4 and 8.5.

[0194] 9.5 Identification of the Fab primary antibody library

[0195] Twenty single clones were randomly selected from overnight cultured LB-A plates for PCR identification. Primer sequences are shown in Table 13, and the system is shown in Table 14. PCR products were separated by 0.7% agarose gel, and the Fab target band was examined to identify the recombination rate of the Fab gene.

[0196] Table 13 Primers for Fab antibody gene fragment amplification

[0197]

[0198] Table 14 Reaction system for colony PCR identification of the Fab gene

[0199]

[0200] The amplification conditions were: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 1 min; 59℃ annealing for 30 s; 72℃ extension for 1 min; 35 cycles; 72℃ extension for 10 min.

[0201] 9.6 Packaging Fab Antibody Library

[0202] (1) Add 2 mL of electroconversion solution to 10 mL of SB-AT (20 μg / mL Amp and 10 μg / mL Tet) liquid culture medium, and culture at 37 °C and 250 rpm for 1 h.

[0203] (2) Add 3.6 μL of 100 mg / mL Amp to make the final concentration of the culture medium 50 μg / mL, and culture at 37℃ and 250 rpm for 1 h.

[0204] (3) Place 10 12 The pfu helper phage VCSM13 and the above bacterial culture were added to 100 mL of SB-AT (10 μg / mL Tet and 50 μg / mL Amp) and cultured at 37 °C with shaking at 250 rpm for 2 h.

[0205] (4) Add 261 μL of 30 mg / mL Kan (final concentration of 70 μg / mL), incubate overnight at 37°C and 250 rpm.

[0206] (5) The next day, centrifuge at 4000 rpm for 15 min at 4℃, and transfer the supernatant to a 500 mL sterile centrifuge bottle.

[0207] (6) Add PEG8000 / NaCl to make their final concentrations 40 g / L and 30 g / L respectively. After mixing the liquid, let it stand in an ice bath for 30 min to allow the phage to precipitate.

[0208] (7) Centrifuge at 4℃ and 9000rpm for 20min, and discard the supernatant. Invert the centrifuge tube for 10min to dry it thoroughly.

[0209] (8) Add 2 mL of sterile 1×PBS to resuspend the phage pellet, mix thoroughly, dispense into sterile EP tubes, centrifuge at 12000 rpm for 5 min at 4°C to remove residual cell debris, and the Fab primary phage antibody library is located in the supernatant. Store at 4°C.

[0210] (9) Dilute the packaged Fab antibody library to 10 μL using SB liquid medium. -8 10 -9 10 -10 10 -11 .

[0211] (10) Take 100 μL of each graded dilution solution and infect 1 mL of OD. 600 XL1-Blue bacterial suspension with a concentration of 0.8-0.9 was incubated at 37°C for 30 minutes. 100 μL of the infected bacterial suspension was spread onto LB-A (100 μg / mL Amp) solid medium and inverted at 37°C. The following day, the number of single clones was recorded, and the titer of the Fab primary phage antibody library was calculated.

[0212] Experimental results:

[0213] 1. Agarose gel electrophoresis was used to examine the amplification of the full-length κ and Fd segments of the anti-brucellosis S2 vaccine antibody. The results showed that one κ gene failed to amplify the target band, while the other κ and Fd segments exhibited specific bands between 500-750 bp. PCR products were recovered, and PCR products of κ and Fd amplified with different primers were mixed in equal masses for antibody library construction. The concentration of the κ gene after mixing was 126.3 ng / μL, and the concentration of the Fd gene after mixing was 318.2 ng / μL.

[0214] 2. The κ and Fd chains were sequentially inserted into the pComb3X vector via enzyme digestion and ligation. After transformation into XL1-Blue, single-clone counting was performed. The results showed that the pComb3X-anti-brucellosis S2 vaccine Fab / XL1-Blue had 1.54 × 10⁻⁶ clones. 7 One transformant. Twenty single colonies were randomly selected for PCR identification, and the results showed 14 positive clones (1400-1500 bp), with a gene recombination rate of 70%.

[0215] like Figure 1As shown, Figure 1 In the diagram, M represents the DL5000 DNA Maker, CK represents the negative control, and 1-20 represent the PCR identification results of 20 single-clone colonies. The arrows indicate the Fab band.

[0216] Using the helper phage VCSM13 to package the Fab antibody library for the anti-brucellosis S2 vaccine, a antibody with a titer of 2.87 × 10⁻⁶ was constructed. 13 pfu / mL of anti-brucellosis S2 vaccine Fab phage antibody library.

[0217] Example 4: Screening and Detection of Anti-Omp28 Fab

[0218] 1. Screening for Omp28 Fab clones

[0219] (1) Using Na2CO3-NaHCO3 as the coating solution, the purified Omp28 antigen and the laboratory-available Omp19 antigen were diluted and screened using 96-well plates. Each well was coated with 5 μg, 100 μL / well, and coated at 4℃ for 14-16 h.

[0220] (2) The next day, pat dry the coating solution, wash away the unadsorbed antigen in the plate with 0.1% PBST, wash 3 times, 200 μL / well, 3 min each time. Add 200 μL / well of 3% BSA-PBS, block at 37℃ for 1 h.

[0221] (3) After the blocking is completed, discard the blocking solution and wash the plate 3 times with 0.1% PBST, 200 μL / well, for 2 min each time.

[0222] (4) Add 100 μL / well of Brucella S2 vaccine Fab phage antibody library to the well plate coated with Omp19 antigen and incubate at room temperature on a shaker for 1 h. After incubation, use a pipette to aspirate the supernatant of anti-Brucella S2 vaccine Fab phage antibody that has not bound to Omp19 antigen and add it to the well plate coated with Omp28 antigen and incubate at room temperature on a shaker for 2 h.

[0223] (5) Discard the antibody supernatant and wash the plate 5 times with 0.1% PBST, 200 μL / well, for 3 min each time. (Wash 10 times for the second round of screening; wash 15 times for the third round of screening)

[0224] (6) Add 100 μL / well Gly-HCl-BSA elution buffer (pH=2.2), place the plate on a shaker and incubate at room temperature for 10 min. During the incubation, repeatedly blow the plate with the pipette tip several times, avoiding the generation of air bubbles.

[0225] (7) After incubation, add 7 μL of 2M Tris base to each well to neutralize the elution buffer, collect the elution in the well, filter and sterilize, and then dispense into sterile centrifuge tubes.

[0226] The results showed that after the third round of screening, the enrichment of Omp28 per microgram of antigen against the phage antibody library remained essentially unchanged.

[0227] 2. Determination of the titer of the eluent

[0228] Take 10 μL of the elution product and dilute it to 10 μL with SB liquid medium. -2 10 -3 Take 100 μL of the diluted solution to infect 1 mL of OD. 600 XL1-Blue bacterial suspension with a concentration of 0.8-0.9 was incubated at 37°C for 30 min. 100 μL of the infected bacterial suspension was spread onto LB-A (100 μg / mL Amp) solid medium and incubated overnight at 37°C. The number of single clones was recorded, and the titer after each round of screening and the enrichment effect of each μg of antigen on the phage antibody library after each round of screening were calculated.

[0229] Add 2mL OD 600 The remaining elution product was added to the XL1-Blue bacterial culture with a concentration of 0.8-0.9, and incubated at 37°C for 30 min. After incubation, the infected bacterial culture was added to 10 mL of SB-AT (20 μg / mL Amp and 10 μg / mL Tet) liquid medium and cultured at 37°C with shaking at 250 rpm for 1 h. The antibody library supernatant was obtained, which is the first round of enriched phage antibody library. 10 μL of the first round of enriched phage antibody library was used for titer determination, and the remaining antibody library was used for the next round of screening.

[0230] 3. PCR identification of positive clones from the third round of phage library

[0231] One hundred single clones were randomly selected from the third round of screening plates to determine the recombination rate of the Fab gene. Primer sequences are shown in Table 13, and colony PCR reaction systems are shown in Table 14. Positive clones after three rounds of screening were examined by 0.7% agarose gel electrophoresis.

[0232] The results are as follows Figure 2 As shown, Figure 2 M: DL10000 DNA Marker, 1-100: PCR identification results of 100 single colonies. The results show that 18 out of 100 single colonies were positive, with a target band of 1400-1500 bp.

[0233] 4. Phage-ELISA detection of anti-Omp28 Fab phage clones

[0234] 4.1 Preparation of monoclonal phage antibodies

[0235] Randomly selected monoclonal colonies identified positive by colony PCR were inoculated into 10 mL of SB-AT (50 μg / mL Amp, 10 μg / mL Tet) liquid medium and cultured at 37°C and 250 rpm for 14 h. Then, colonies were inoculated into 5 mL of SB-AT liquid medium at a 1:50 ratio and cultured at 37°C and 250 rpm until OD (Organic Cloning) was reached. 600 =0.8-0.9. According to 10:1 (10 10 The product was packaged by adding helper phage VCSM13 at a ratio of PFU (phosphorus urea nitrogen) and incubating with shaking for 2 hours. Then, 12 μL of 30 mg / mL Kan (final concentration 70 μg / mL) was added, and the mixture was incubated overnight at 37°C and 250 rpm. The next day, the mixture was centrifuged at 12,000 rpm for 10 minutes, and the supernatant was collected. The monoclonal phage antibody was located in the supernatant.

[0236] 4.2 Phage-ELISA Detection

[0237] (1) The purified Omp28 antigen diluted with Na2CO3-NaHCO3 was used to coat 96-well plates, 1 μg per well. The negative control was Na2CO3-NaHCO3 buffer. Two replicates were set for each sample and negative control, 100 μL / well. Coating was carried out at 4℃ for 14 h.

[0238] (2) The next day, the coating solution was dried, and 200 μL of 0.1% PBST was added to each well to wash away any unadsorbed antigen. 200 μL of 3% BSA-PBS was added to each well, and the plate was blocked at 37°C for 1.5 h. At the same time, the phage antibody was mixed with 3% BSA-PBS at a ratio of 4:1 and incubated at room temperature for 30 min to reduce non-specific binding.

[0239] (3) After blocking, wash the well plate 5 times with 0.1% PBST for 5 min each time, 200 μL / well. Add 100 μL of the prepared monoclonal phage antibody to the well plate and incubate in a constant temperature incubator for 2 h.

[0240] (4) Discard the supernatant and wash the plate 5 times with 0.1% PBST, 5 min each time, 200 μL / well.

[0241] (5) Add 100 μL / well of HRP-labeled Anti-M13 Antibady diluted with 3% BSA-PBS and incubate at 37°C for 1 h.

[0242] (6) Discard the supernatant and wash the plate 5 times with 0.1% PBST, 5 min each time, 200 μL / well.

[0243] (7) Perform color development using the TMB-ECL kit and measure the absorbance value at 450 nm. Calculate the ratio of the sample group to the negative group, and determine the positive clones if the ratio is greater than 2.1 (P / N>2.1).

[0244] 5. Sequencing analysis of positive clones

[0245] Five positive clones with the highest P / N values ​​from the Phage-ELISA results were selected and inoculated into 10 mL of LB-AT (50 μg / mL Amp, 10 μg / mL Tet) liquid medium. They were cultured overnight at 37°C and 250 rpm. The plasmids were extracted the next day and sent to the company for sequencing.

[0246] 6. Soluble expression of anti-Omp28 Fab antibody

[0247] (1) Take out the glycerol bacteria of Escherichia coli HB2151 and streak it into 2×YT-N (100μg / mL NaI) solid medium and incubate at 37℃ with the medium upside down.

[0248] (2) The next day, single clones were picked and inoculated into 20 mL of 2×YT-NG (100 μg / mL NaI, 5.6% 2M glucose) liquid culture medium and cultured overnight at 37°C and 250 rpm with shaking.

[0249] (3) Add the overnight cultured bacterial solution to 20 mL of SOB-NG (100 μg / mL NaI, 5.6% 2M glucose) liquid medium at a ratio of 1:100, and culture at 37°C with shaking at 250 rpm until OD. 600 =0.8.

[0250] (4) Dilute the monoclonal phage antibody supernatant with 2×YT liquid medium, add 100μL of the diluted monoclonal phage antibody supernatant to 1mL of HB2151 bacterial culture with OD600=0.8 for infection, and culture at 37℃, 250rpm for 1h with shaking.

[0251] (5) Take 100 μL of the infected bacterial solution and spread it on 2×YT-AG (100 μg / mL Amp, 5.6% 2M glucose) solid medium and incubate overnight at 37°C.

[0252] (6) The next day, single clones were selected for colony PCR identification. The primer sequences are shown in Table 13, and the colony PCR reaction system is shown in Table 14.

[0253] (7) Pick HB2151 single clones (negative control) and single clones identified as positive and inoculate them into 20 mL of 2×YT-NG (100 μg / mL NaI, 5.6% 2M glucose) and 20 mL of 2×YT-AG (100 μg / mL Amp, 5.6% 2M glucose) liquid medium, respectively, and culture overnight at 37℃ with shaking at 250 rpm.

[0254] (8) Inoculate 100 μL of seed culture into 100 mL of 2×YT-A (100 μg / mL Amp) liquid medium, and inoculate the negative control into 100 mL of 2×YT-N (100 μg / mL NaI) liquid medium. Incubate at 37℃ with shaking at 250 rpm until OD. 600 =0.8.

[0255] (9) Add 100 μL of 1M IPTG and induce at 30℃ and 250 rpm for 7 h.

[0256] (10) Centrifuge the bacterial culture at 4000 rpm for 15 min at 4℃ and retain the precipitate. Add 500 μL of pre-cooled 1×TES and 750 μL of pre-cooled 1 / 4×TES to resuspend the bacterial cells, mix well and place on ice for 30 min.

[0257] (11) Centrifuge at 12000 rpm for 10 min at 4℃. The supernatant is the periplasmic space expression product. After determining the concentration by BCA method, aliquot and store in a -80℃ refrigerator.

[0258] 7. Western blot analysis of Omp28 Fab resistance

[0259] 7.1 Identification of the molecular weight of anti-Omp28 Fab antibody

[0260] The recombinant Fab antibody expressed above was subjected to SDS-PAGE electrophoresis under both reducing and non-reducing conditions. Reducing conditions: the sample was mixed with reducing 3× Loading Buffer (containing β-mercaptoethanol) and incubated at 100°C for 10 min. Non-reducing conditions: the sample was mixed with non-reducing 3× Loading Buffer (without β-mercaptoethanol) and incubated at 100°C for 10 min. The separating gel and stacking gel concentrations were 5% and 12%, respectively. Electrophoresis conditions were: constant voltage of 65V until the protein marker entered the separating gel, then the voltage was increased to 120V until bromophenol blue completely migrated from the bottom of the separating gel. After electrophoresis, the transfer clamp was assembled, and the membrane was transferred at a constant current of 200mA for 1 h 30 min. After transfer, the membrane was washed with TBST for 5 min. Blocking was performed using 5% skim milk powder-TBST at room temperature for 1 h. After blocking, the membrane was washed three times with TBST for 10 min each time. The HA-Tag Mouse monoclonal antibody was diluted to 1:2000 with primary antibody dilution buffer and incubated overnight at 4°C. The next day, the HRP-labeled GoatAnti-Mouse IgG antibody was diluted 1:10000 with 3% BSA-TBST and incubated at room temperature for 1 hour. After incubation, the antibody was washed three times with TBST for 10 minutes each time. The developing solution was prepared according to the instructions of the ultrasensitive ECL chromogenic kit and developed and exposed on a gel imaging system.

[0261] 7.2 Specificity detection of anti-Omp28 Fab antibody

[0262] Based on sequencing results and molecular weight identification results, L4-4 and L4-7 Fab were selected for specificity detection. Recombinant antigen Omp28, Escherichia coli protein, Staphylococcus aureus protein, Yersinia germiae protein, Salmonella protein, S2 vaccine protein, and A19 vaccine protein were mixed with 3× Loading Buffer and then subjected to Western blot for specificity detection. Fab antibody diluted with 3% BSA-TBST was used as the primary antibody and incubated at 4°C for 16 h; HA-Tag Mouse monoclonal antibody was used as the secondary antibody and incubated at room temperature for 2 h; HRP-labeled Goat Anti-Mouse IgG antibody was used as the third antibody and incubated at room temperature for 1 h, as detailed in section 7.1.

[0263] Experimental results:

[0264] The purified recombinant protein Omp28 was coated onto a 96-well plate. Phage-ELISA was performed using phage antibodies against the 18 selected positive clones as primary antibodies and HRP-conjugated Anti-M13 as secondary antibodies. A negative control was the coating solution (Na2CO3-NaHCO3), and OD450 was measured. Clones with a sample value greater than 2.1 times the negative control value (P / N > 2.1) were considered positive clones. Results are as follows... Figure 3 As shown, seven Omp28-positive clones were obtained, and they were sorted from high to low P / N values ​​as L4-7, L4-8, L4-4, L4-1, L5-48, L5-25, and L5-24.

[0265] Plasmids were extracted and sequenced from the five positive clones with the highest P / N ratios (L4-1, L4-4, L4-7, L4-8, and L5-48). The Fd chains of all five clones could translate into complete proteins, with a homology of 90.75%. The κ chain of L4-7 could translate into a complete protein, while the κ chains of the other four positive clones showed small deletions, with a homology of 97.48%. Analysis of the complementarity-determining regions (CDRs) and backbone regions (FRs) of the antibody Fd chains revealed significant changes in the CDR-H3 sequence.

[0266] The five positive clones selected above (L4-1, L4-4, L4-7, L4-8, and L5-48) were subjected to soluble antibody expression. Their molecular weight and binding specificity were detected using SDS-PAGE and Western blot. The primary antibody was HA-Tag Mouse monoclonal antibody, and the secondary antibody was HRP-conjugated Goat Anti-Mouse monoclonal antibody.

[0267] like Figure 4 and 5 As shown: Figure 4 In the image, A is an SDS-PAGE electrophoresis image of anti-Omp28 Fab antibody under reducing conditions; B is a Western blot image of anti-Omp28 Fab antibody under reducing conditions; the arrows indicate the Fd band.

[0268] Figure 5 A. SDS-PAGE electrophoresis image of anti-Omp28 Fab antibody under non-reducing conditions; B. Western blot image of anti-Omp28 Fab antibody under non-reducing conditions.

[0269] The negative control HB2151 bacterial protein showed no bands. Under reducing conditions, interchain disulfide bonds were disrupted, and all five clones showed obvious Fd monomer bands in the 25-35 kDa range. Under non-reducing conditions, interchain disulfide bonds remained intact, and L4-4 and L4-7 both showed obvious Fab bands in the 40-55 kDa range. Figure 4 The other three clones only showed Fd bands ( Figure 5 This indicates that the heavy and light chains of L4-4 and L4-7 can be translated and self-assembled into Fab, and the expression level of Fab is relatively high.

[0270] Based on sequencing and molecular weight identification results, Western blot was used to detect the specificity of L4-4 and L4-7 Fab. Lysates of pET28a(+)-Omp28 / Transetta, S2, A19 vaccines, and common pathogens were used as antigens. Periplasmic extracts of L4-4 and L4-7 clones were used as primary antibodies, HA-Tag Mouse monoclonal antibody as secondary antibody, and HRP-conjugated Goat Anti-Mouse monoclonal antibody as triclonal antibody for detection. Results showed that a single, clear band existed in the supernatant of pET28a(+)-Omp28 / Transetta lysate, S2 vaccine lysate, and A19 vaccine lysate, with a molecular weight consistent with Omp28 (28 kDa). No bands were observed in the remaining lanes. Figure 6 A. L4-4 Fab specificity detection results; B. L4-7 Fab specificity detection results. 1: BSA, 2: pET28a(+)-Omp28 / Transetta lysis supernatant, 3: recombinant Omp28 purified protein, 4: Escherichia coli lysis supernatant, 5: Staphylococcus aureus lysis supernatant, 6: Yersinia pestis lysis supernatant, 7: Salmonella lysis supernatant, 8: S2 vaccine lysis supernatant, 9: A19 vaccine lysis supernatant (arrows indicate Omp28 bands), demonstrating that the screened Fab antibodies L4-4 and L4-7 can specifically bind to the Omp28 antigen and do not cross-react with Escherichia coli, Staphylococcus aureus, Yersinia pestis, or Salmonella.

[0271] L4-7 light chain kappa amino acid sequence as SEQ ID NO.3: VMTQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPHTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC.

[0272] Heavy chain Fd amino acid sequence as SEQ ID NO.4: SGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEINPDSSTINYTPSLKDKFIISRDNAKNTLYLQMSKVRSEDTALYYCASNWDVGFDYWGQGT TLTVSSATTTAPSVYPLVPGCSDTSGSSVTLGCLVKGYFPEPVTVKWNYGALSSGVRTVSSVLQSGFYSLSSLVTVPSSTWPSQTVICNVAHPASKTELIKRIEPRIPK.

[0273] Example 5: Screening and Identification of IcIR-1 Fab

[0274] (I) Construction of the A19 Fab phage antibody library

[0275] Brucella A19 vaccine was stored in the laboratory at -20℃; BALB / c mice were purchased from Chongqing Enswell; pComb3XSS / TG1 glycerol bacteria were purchased from Weichuang Biotechnology; strain E. coli XL1-Blue and helper phage VCSM13 were stored in the laboratory at -80℃.

[0276] The methods and steps for constructing A19-immunized mice and the Fab antibody library are the same as in Example 3.

[0277] Results and Analysis

[0278] 1. Competitive ELISA detection of mouse serum antibody concentration

[0279] Three 6-8 week old female BALB / c mice were immunized with the A19 vaccine. After the third immunization, a competitive ELISA assay was performed, with three replicates per mouse. A score (S / N) < 0.5 for all three mice indicated that the vaccine immunization had produced specific antibodies, and the titer met the criteria for collecting serum and RNA. Blood was collected from the iris of these mice at this stage, and serum was used for subsequent experiments.

[0280] 2. Total RNA extraction from mouse spleen

[0281] Total RNA was extracted from mouse spleens using a kit, and its concentration and purity were determined (see Table 15). The RNA concentration from all three mice was greater than 4 μg, and the A260 / A280 value was within the ideal RNA range of 1.8–2.2. The extracted total RNA was electrophoresed on a 1.5% agarose gel. The results showed that all three rRNA bands were intact, indicating that the RNA quality met the requirements for library construction and could be used as a template for the next step of Fab amplification.

[0282] Table 15. Purity and concentration of total RNA in the spleen of mice immunized with A19 vaccine

[0283]

[0284] 3. Amplification and recovery of antibody light chain κ gene and heavy chain Fd gene

[0285] RNA of high quality (RNAs 2 and 3) was selected as templates for A19 reverse transcription to synthesize the first strand of cDNA. Equal volumes of the first strand cDNA were mixed and used as templates for amplifying the A19 light chain κ gene and heavy chain Fd gene. Agarose gel electrophoresis of the amplification products showed that lanes 2-7 of the seven different light chain κ gene primers exhibited bright, specific bands between 500bp and 750bp (approximately 660bp), while no band was amplified in lane 1. For the heavy chain Fd, all three different primers showed bright, specific bands between 500bp and 750bp (approximately 660bp). The PCR products were recovered according to the kit instructions, and equal masses of the heavy chain Fd PCR products amplified by the three different combinations and the six light chain κ PCR products were mixed separately for antibody library construction. The concentration of the A19 κ gene after mixing was 387.8 ng / μL, and the concentration of the A19 Fd gene after mixing was 387.1 ng / μL.

[0286] 4. Construction of the light chain κ library

[0287] 4.1 Enzyme digestion of the κ gene and pComb3XSS plasmid

[0288] 10 μg of the purified mixture of six light chain κ molecules was double-digested with Sac I and Xba I. After 1.5% agarose gel electrophoresis, a band of approximately 680 bp was excised under UV light. The target fragment was recovered using a gel extraction kit, and the A19 κ concentration was determined to be 255.8 ng / μL. 40 μg of pComb3XSS plasmid was double-digested with Sac I and Xba I. After 0.7% agarose gel electrophoresis, two bands of approximately 3800 bp and 1200 bp were observed. The 3800 bp band was excised, and the vector DNA was recovered, with a concentration of 442.6 ng / μL.

[0289] 4.2 Identification of recombination rate of light chain κ library

[0290] The pComb3XSS vector with identical sticky ends was ligated to the A19κ gene at a molar ratio of 1:3. The recovered ligation products were then transformed into XL1-Blue competent cells via electroporation. 10 μL, 5 μL, and 2.5 μL of the electroporation buffer were plated onto LB-A solid medium. The following day, single-clone counting was performed, showing a pComb3XS-A19κ colony count of 7.46 × 10⁻⁶. 6Two transformants were randomly selected from each plate, and colony PCR electrophoresis was performed using rTaq polymerase. The results showed that all 20 single clones of the A19κ gene showed a band at approximately 680 bp, indicating a 100% recombination rate for the A19κ gene and a library size of 7.46 × 10⁻⁶. 6 .

[0291] 4.3 Extraction of pComb3XS-A19κ plasmid

[0292] After spreading a portion of the electroconversion solution, the remaining electroconversion solution was incubated overnight. The next day, the pComb3XS-κ plasmid was extracted and its concentration and purity were determined. The results are shown in Table 16.

[0293] Table 16 pComb3XS-A19 κ plasmid concentration and purity

[0294]

[0295] 5. Construction and quality testing of the A19 Fab phage antibody library

[0296] 5.1. Spe I / Xho I double digestion of heavy chain Fd with pComb3XS-A19κ plasmid

[0297] 10 μg of the purified Fd mixture was double-digested with Spe I / Xho I, and a band of approximately 680 bp was excised by 1.5% agarose gel electrophoresis. After purification, the A19 Fd concentration was measured to be 125.5 ng / μL. 40 μg of the pComb3XS-A19κ plasmid was double-digested with Spe I / Xho I, and a band of approximately 4100 bp was excised by 0.7% agarose gel electrophoresis under UV light. The concentration of the pComb3XS-A19κ plasmid after digestion was measured to be 243.5 ng / μL using an ultra-micro nucleic acid and protein analyzer.

[0298] 5.2 Electroconversion Results of the Connecting Products

[0299] A light chain vector with the same sticky ends was ligated to the Fd gene at a molar ratio of 1:3. The recovered ligation product was then transformed into XL1-Blue competent cells via electroporation. 10 μL, 5 μL, and 2.5 μL of the electroporation buffer were spread onto LB-A solid medium. The following day, single-clone counting was performed, and the results showed that pComb3X-A19 Fab / XL1-Blue had 5.2 × 10⁻⁶ cells. 6 A transformant.

[0300] 5.3 Quality Assurance of A19 Fab Phage Antibody Library

[0301] Electroporation buffer was added to helper phage VCSM13 for packaging to obtain the A19 Fab phage antibody library. The phage antibody library was serially diluted and used to infect XL1-Blue bacterial suspensions, then plated on LB-A solid medium. The next day, single clones were counted. Twenty single clones were randomly selected from each plate for colony PCR electrophoresis to identify the recombination rates of the A19 κ gene, A19 heavy chain Fd, and full-length Fab gene. The results showed that all 20 clones of the A19 κ gene showed a band at approximately 680 bp, 14 clones of the A19 Fd segment showed a band at approximately 680 bp, and 14 clones of the full-length A19 Fab showed a band at approximately 1500 bp. Therefore, the recombination rate of the A19 κ gene was 100%, the recombination rate of the A19 heavy chain Fd was 70%, and the recombination rate of the A19 Fab was 70%. Thus, the titer of the A19 Fab phage antibody library was 6.7 × 10⁻⁶. 12 pfu / mL.

[0302] (II) Screening and identification of anti-IcIR-1 Fab

[0303] 1. Screening of anti-IcIR-1 Fab phage antibody library

[0304] The synthesized IcIR-1 peptide was diluted and screened using 96-well plates with a coating amount of 2 μg per well and 100 μL / well. The screening was performed using the Fab phage antibody library for the anti-brucellosis A19 vaccine, following the same steps as in Example 4. The results showed that after the third round of screening, the enrichment of IcIR-1 per microgram of antigen against the phage antibody library increased significantly.

[0305] 2. Determination of the titer of the eluent

[0306] Same as Example 4

[0307] 3. PCR identification of Fab in the third round of screening

[0308] Fifty-nine single clones were randomly selected from the third round of screening plates to determine the recombination rate of the Fab gene. The procedure was the same as in Example 4.

[0309] 4. Soluble expression of anti-IcIR-1 Fab antibody

[0310] (1) Na2CO3-NaHCO3 was used as the coating solution to dilute the synthesized IcIR-1 peptide. After mixing, the solution was added to a 96-well plate with an antigen coating amount of 2 μg per well. The negative control was coated with Na2CO3-NaHCO3. Each sample and negative control was coated in two replicates, 100 μL / well, and coated overnight at 4°C.

[0311] (2) The next day, pat dry the coating solution, wash away the unadsorbed antigen in the plate with 0.1% PBST, wash 3 times, 200 μL / well, 5 min each time. Add 200 μL / well of 3% BSA-PBS, block at 37℃ for 2 h.

[0312] (3) After the blocking is completed, discard the blocking solution and wash the plate 3 times with 0.1% PBST, 200 μL / well, for 5 min each time.

[0313] (4) Add 200 μL / well of IcIR-1 phage Fab antibody to the well plate and incubate at 37°C for 2 h.

[0314] (5) Discard the antibody and wash the plate 3 times with 0.1% PBST, 200 μL / well, for 5 min each time.

[0315] (6) Add the HA-Tag Mouse monoclonal antibody to the well plate, dilute it with 3% BSA-TBST at a ratio of 1:2000, 100 μL / well, and incubate at 37°C for 1 h.

[0316] (7) Discard the antibody and wash 3 times with 0.1% PBST, 200 μL / well, 5 min each time.

[0317] (8) Add 100 μL of HRP-labeled Goat Anti-MouselgG antibody diluted with 3% BSA-TBST at a ratio of 1:10000, and incubate at 37°C for 1 h.

[0318] (9) Discard the antibody and wash 3 times with 0.1% PBST, 200 μL / well, 5 min each time.

[0319] (10) Use the TMB-ECL kit for color development and measure the absorbance at 450 nm. Calculate the ratio of the sample group to the negative group, and determine the positive clones if the ratio is greater than 2.1 (P / N>2.1).

[0320] 5. Sequencing analysis of positive clones

[0321] Two positive clones with the highest P / N values ​​from the indirect ELISA results were selected and inoculated into 10 mL of LB-AT (50 μg / mL Amp, 10 μg / mL Tet) liquid medium. They were cultured overnight at 37°C and 250 rpm. The plasmids were extracted the next day and sent to the company for sequencing.

[0322] 7. Western blot analysis of anti-IclR-1 Fab

[0323] 7.1 Identification of the molecular weight of anti-IclR-1 Fab antibody

[0324] Y3-9 and Y3-16, which showed higher P / N ratios in the indirect ELISA results, were selected for molecular weight identification. See Example 4 for specific steps.

[0325] 7.2 Specificity detection of anti-IcIR-1 Fab antibody

[0326] Y3-9 and Y3-16, which showed higher p / n ratios in the indirect ELISA results, were selected for specificity testing. Western blot was used to determine whether Y3-9 and Y3-16 exhibited specific reactions with the S2 and A19 vaccines, and whether they showed cross-reactivity with *Escherichia coli*, *Staphylococcus aureus*, *Yersinia*, and *Salmonella*. Specific steps are detailed in Example 4.

[0327] like Figure 7 As shown, Figure 7 M: DL5000 DNA Marker, 1-59: PCR identification results of 59 single-clone colonies, with arrows indicating Fab bands.

[0328] Fifty-nine single clones were randomly selected from the third-round screening plates of the anti-IcIR-1 Fab phage antibody library and identified by colony PCR using universal primers on the vector. 0.7% agarose gel electrophoresis showed that 13 of the 59 single clones were positive with a target band of 1400-1500 bp. These 13 positive single clones were selected for soluble expression of Fab.

[0329] Absorbance measurements at 450 nm showed that clones with a P / N ratio greater than 2.1 (P / N > 2.1) were considered positive. Six anti-IcIR-1 positive clones were obtained, arranged in the following order according to their P / N values: Y3-9, Y3-16, Y3-14, Y3-11, Y3-17, and Y3-15. The results are as follows: Figure 8 As shown.

[0330] Plasmids were extracted and sequenced from Y3-9 and Y3-16, which had the highest P / N values, from the positive results of indirect ELISA. The results showed that the protein translated from the κ chain of the two positive monoclonal chains had a small deletion, with a homology of 99.32%. The Fd chain was translated into a complete protein with a homology of 76.10%. By locating the complementarity-determining regions (CDRs) and backbone regions (FRs) of the Fd chain, significant changes in the CDR-H2 sequence were found.

[0331] The two positive clones (Y3-9 and Y3-16) obtained from the above screening were subjected to soluble antibody expression. After SDS-PAGE electrophoresis, their molecular weight was determined by Western blot. The primary antibody was HA-Tag Mouse monoclonal antibody, and the secondary antibody was HRP-conjugated Goat Anti-Mouse monoclonal antibody. The results are as follows: Figure 9 As shown in the figure, Figure 9 A. SDS-PAGE electrophoresis of Y3-9; B. Western blot of Y3-9 under non-reducing conditions; C. Western blot of Y3-9 under reducing conditions; D. SDS-PAGE electrophoresis of Y3-16; E. Western blot of Y3-16 under non-reducing conditions; F. Western blot of Y3-16 under reducing conditions; M: Protein Marker; 1 and 6: Periplasmic space extract of HB2151; 2 and 5: Periplasmic space extract of HB2151 + mercaptoethanol; 3 and 7: Periplasmic space extracts of Y3-9 and Y3-16; 4 and 8: Periplasmic space extracts of Y3-9 and Y3-16 + mercaptoethanol.

[0332] The negative control HB2151 pericyte extract did not show any target bands. Under reducing conditions with the addition of mercaptoethanol, the disulfide bonds between the Fab chains were disrupted, and both Y3-9 and Y3-16 showed obvious monomeric bands in the 25-35 kDa range. Figure 9 C and F); Under non-reducing conditions without the addition of mercaptoethanol, the disulfide bonds between Fab chains remained intact, and both clones showed Fab bands at 40-55 kDa (C and F); Figure 9 The lighter color of the B and E bands indicates that the Fab expression levels of Y3-9 and Y3-16 are low and the number of heavy and light chain complete matches is small.

[0333] The specificity of Y3-9 and Y3-16 Fab was detected by Western blot. The supernatant from the lysate of S2 and A19 vaccines and common pathogens was used as the antigen. The primary antibody was the periplasmic extract of Y3-9 and Y3-16 clones, the secondary antibody was HA-Tag Mouse monoclonal antibody, and the tertiary antibody was HRP-conjugated Goat Anti-Mouse monoclonal antibody. Results are as follows: Figure 10 As shown, Figure 10 Specific detection results for A.Y3-9 and B.Y3-16. M: Protein Marker, 1: Escherichia coli lysate supernatant, 2: Staphylococcus aureus lysate supernatant, 3: Yersinia pestis lysate supernatant, 4: Salmonella lysate supernatant, 5: BSA lysate supernatant, 6: A19 vaccine lysate supernatant, 7: S2 vaccine lysate supernatant. The arrows indicate IcIR protein bands.

[0334] Both Y3-9 and Y3-16 exhibited specific bands in the supernatant channels of A19 vaccine lysate, with a molecular weight of 36 kDa. Y3-16 showed no specific band in the supernatant channels of S2 vaccine lysate. This demonstrates that the expressed Fab antibody Y3-16 can distinguish between A19 and S2 vaccines. Furthermore, Y3-9 showed no cross-reactivity with *E. coli*, but weak cross-reactivity with *Staphylococcus aureus*, *Yersinia*, and *Salmonella*. Y3-16 showed no cross-reactivity with *E. coli* and *Staphylococcus aureus*, but weak cross-reactivity with *Yersinia* and *Salmonella*, indicating that Y3-16 has good specificity. Y3-16, the light chain κ amino acid sequence is shown in SEQ ID NO.5: MVETLSNLLRSVVALSPPDLLPVLYLSLNRLGPPQQGLELGVGDGVLLKAVAQATGRQLESVRAEVAEKGDVGLVAENSRSTQRLMLPPPPLTISGVFTKFCDIARLTGSASMAKKMDIIKGLFVACRHSEARYIARSLSGRLRQHSR.

[0335] Heavy chain Fd amino acid sequence as SEQ ID Shown in NO.6: SGTELVRPGTSVKISCKASGYTFTNYWLAWVKQRPGHGLEWIGDIYPGRGNNNIEKFRGKATLTADTSSSTAYMQLKSLTFEDSAVYFCARSGLDYGNYGWFAYW GQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTLPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDC.

Claims

1. An antibody combination for identifying Brucella wild-type strains and S2 vaccine, characterized in that: Includes anti-Omp28 Fab clone L4-7 and anti-IcIR-1 Fab clone Y3-16; The anti-Omp28 Fab clone L4-7 is a fully assembled Fab fragment, with the light chain κ amino acid sequence as shown in SEQ ID NO.3 and the heavy chain Fd amino acid sequence as shown in SEQ ID NO.4; The anti-IcIR-1 Fab clone Y3-16 is a fully assembled Fab fragment, with the light chain κ amino acid sequence shown in SEQ ID NO.5 and the heavy chain Fd amino acid sequence shown in SEQ ID NO.

6.

2. The antibody combination for identifying Brucella wild-type strains and S2 vaccine according to claim 1, characterized in that: The clone L4-7 specifically binds to the Omp28 protein of SEQ ID NO.1 and shows no cross-reactivity with Escherichia coli, Staphylococcus aureus, Yersinia, and Salmonella.

3. The antibody combination for identifying Brucella wild-type strains and S2 vaccine according to claim 2, characterized in that: The Y3-16 Fab specifically binds to the IcIR-1 peptide of SEQ ID NO.

2.

4. The use of the antibody combination of claim 1 for identifying Brucella wild-type strains and S2 vaccine in the preparation of a veterinary diagnostic kit to distinguish between Brucella S2 vaccine immunization and wild-type strain infection.

5. The application according to claim 4, characterized in that, It also contains two specific antigens: (1) Brucella conserved mature outer membrane protein Omp28, the amino acid sequence of which is shown in SEQ ID NO.1; (2) S2 vaccine-specific deletion polypeptide IcIR-1, the amino acid sequence of which is shown in SEQ ID NO.

2.

6. The application according to claim 5, characterized in that: The Omp28 protein, with an amino acid sequence as shown in SEQ ID NO.1, is stably expressed in all species of Brucella abortus, Brucella melitensis, and Brucella suis. The IcIR-1 polypeptide is only present in wild-type Brucella strains and the A19 vaccine; the S2 vaccine genome lacks this coding region.

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