A Brucella OMP16 B cell epitope fusion protein and its application

Abstract

This invention belongs to the field of bioengineering technology, specifically relating to a Brucella OMP16 B-cell epitope fusion protein and its applications. The fusion protein comprises multiple repeating units linked by flexible linking peptides. Each repeating unit is formed by the tandem sequence of five Brucella OMP16 B-cell epitopes in their native order; the amino acid sequences of the five B-cell epitopes are: TLSKQAQW, LQRYPQY, GQRRAAAT, RDFLASRG, and VPTNRMRT. Based on this, this invention screened for the fusion protein with the best structural stability and theoretical solubility. It strongly binds to Brucella whole-cell polyclonal antibodies and OMP16 polyclonal antibodies, and is specifically recognized by five corresponding OMP16 monoclonal antibodies, indicating that the flexible linking peptides and tandem sequence effectively maintain the native conformation and spatial accessibility of each B-cell epitope.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a Brucella OMP16 B cell epitope fusion protein and its application. Background Technology

[0002] Brucellosis is a globally distributed and highly dangerous zoonotic infectious disease caused by Brucella bacteria. It not only seriously threatens public health but also causes enormous economic losses to the livestock industry. Strengthening surveillance, early detection, and culling infected animals are core measures for brucellosis control, while developing accurate, rapid, and specific detection technologies is a crucial prerequisite for implementing these control strategies.

[0003] Currently, rapid clinical detection of brucellosis mainly relies on serological methods. Traditional methods, such as the rose benzene plate test and tube agglutination test, are simple to perform, but they use complex antigenic components and have potential cross-reactivity with pathogens such as Escherichia coli, Salmonella, and Yersinia, leading to high false-positive rates and insufficient specificity. ELISA methods based on recombinant outer membrane proteins have improved specificity to some extent, but their detection performance may still be affected by incomplete epitope coverage or the presence of non-specific regions.

[0004] iELISA technology based on multi-epitope fusion proteins constructs detection antigens by rationally designing and cascading multiple screened, highly specific B-cell epitopes. This can minimize non-specific reactions and integrate multiple immune-dominant recognition sites, providing a foundation for developing next-generation serological detection reagents that combine high sensitivity and high specificity.

[0005] Brucella OMP16 possesses both high conservation and good immunogenicity, making it an ideal detection target. Therefore, to improve the specificity and accuracy of serological detection of brucellosis, a novel iELISA method with good detection performance was established. Using laboratory-prepared OMP16 monoclonal antibodies to identify corresponding B-cell epitopes, and through rational design and structural evaluation using bioinformatics tools, an OMP16 multi-epitope fusion protein was constructed and expressed. Based on this, an iELISA method for the OMP16 multi-epitope fusion protein was established, aiming to provide a new and promising technical means for the serological detection of brucellosis, and also to provide a technical reference for the development and application of multi-epitope antigens of other important pathogens. Summary of the Invention

[0006] Brucella outer membrane protein 16 (OMP16) is highly conserved and possesses good immunogenicity, making it an important candidate antigen for vaccines and a detection target. To improve the specificity and accuracy of serological detection of brucellosis, this invention utilizes the corresponding B-cell epitopes identified by five laboratory-prepared OMP16 monoclonal antibodies (mAbs). Through rational design and bioinformatics analysis, a multi-epitope fusion protein, OMP16Es6×, was constructed and expressed. Based on this, an iELISA method was established.

[0007] The specific technical solution provided by this invention is as follows: In a first aspect, the present invention provides a Brucella OMP16 B cell epitope fusion protein, the fusion protein comprising a plurality of repeating units tandemly linked by flexible linker peptides, each repeating unit being composed of five B cell epitopes of Brucella OMP16 tandemly linked in their natural order; the amino acid sequences of the five B cell epitopes are: TLSKQAQW, LQRYPQY, GQRRAAAT, RDFLASRG, and VPTNRMRT.

[0008] In a preferred embodiment of the present invention, the amino acid sequence of the flexible linker peptide is GGGSG; the repeating unit is repeated 2, 4, 6, 8 or 10 times.

[0009] More preferably, the repeating unit is repeated 6 times, and the amino acid sequence of the fusion protein is: TLSKQAQWLQRYPQYGQRRAAATRDFLASRGVPTNRMRT (B cell epitope tandem unit). GGGSG (Flexible Connecting Peptide) TLSKQAQWLQRYPQYGQRRAAATRDFLASRGVPTNRMRT GGGSG TLSKQAQWLQRYPQYGQRRAAATRDFLASRGVPTNRMRT GGGSG TLSKQAQWLQRYPQYGQRRAAATRDFLASRGVPTNRMRT GGGSG TLSKQAQWLQRYPQYGQRRAAATRDFLASRGVPTNRMRT GGGS GTLSKQAQWLQRYPQYGQRRAAATRDFLASRGVPTNRMRT.

[0010] In a second aspect, the present invention provides a nucleic acid molecule encoding the fusion protein.

[0011] In a third aspect, the present invention provides a recombinant expression vector containing the nucleic acid molecule.

[0012] In a fourth aspect, the present invention provides a host cell containing the recombinant expression vector.

[0013] In a fifth aspect, the present invention provides an indirect ELISA detection method for non-diagnostic purposes, using the fusion protein as a coating antigen, comprising the following steps: The fusion protein was coated onto a solid support; Add sealing solution to seal; Add the serum sample to be tested and incubate; Add enzyme-labeled secondary antibody and incubate; Add the substrate for color development and read the OD value; The read OD value is compared with a preset threshold, and the comparison result is output. The non-diagnostic purposes include monitoring antibody levels after vaccination, detecting Brucella antibodies for research purposes, or conducting epidemiological surveys.

[0014] In a preferred embodiment of the present invention, the concentration of the coating antigen is 0.01~0.02 μg / mL, the dilution factor of the serum to be tested is 1:5~20, and the enzyme-labeled secondary antibody is HRP-labeled rabbit anti-bovine IgG with a dilution factor of 1:2000~10000.

[0015] More preferably, the concentration of the coating antigen is 0.01~0.02 μg / mL, and the coating conditions are incubation at 37℃ for 1~3 hours followed by overnight incubation at 4℃, or direct overnight incubation at 4℃; the dilution factor of the serum to be tested is 1:5~1:20, and the incubation conditions are incubation at 37℃ for 0.5~2 hours; the enzyme-labeled secondary antibody is HRP-labeled rabbit anti-bovine IgG, the dilution factor is 1:2000~1:10000, and the incubation conditions are incubation at 37℃ for 0.5~2 hours; the color development conditions are incubation at 37℃ for 15~45 minutes.

[0016] More preferably, the concentration of the coating antigen is 0.02 μg / mL, and the coating conditions are: incubation at 37°C for 2 hours followed by overnight incubation at 4°C; the dilution factor of the serum to be tested is 1:5, and the incubation conditions are: incubation at 37°C for 1 hour; the dilution factor of the enzyme-labeled secondary antibody is 1:5000, and the incubation conditions are: incubation at 37°C for 1 hour; the color development conditions are: incubation at 37°C for 30 minutes.

[0017] In a preferred embodiment of the present invention, the preset threshold is that when the OD value at 450nm is ≥ 0.392, the comparison result is output as higher than the threshold, and when the OD value is < 0.392, the comparison result is output as lower than the threshold.

[0018] In a sixth aspect, the present invention provides the use of the fusion protein in the preparation of brucellosis serological detection reagents or kits.

[0019] Compared with the prior art, the present invention has the following advantages: (1) This invention is based on the corresponding B-cell epitopes identified by five OMP16 monoclonal antibodies. These epitopes are tandemly linked in their natural order as basic fusion epitope units, and then linked by flexible linker peptides GGGSG. Using AlphaFold2 structure prediction and bioinformatics analysis (solubility, hydrogen bond network, hydrophobic clusters, etc.), the 6× repeat design (OMP16Es6×) with the best structural stability and theoretical solubility was selected from five candidate schemes (2×, 4×, 6×, 8×, and 10×). After prokaryotic expression and NTA affinity purification, a highly pure soluble fusion protein was successfully obtained. This rational design strategy avoids the problems of structural instability and epitope inactivation commonly found in traditional multi-epitope fusion proteins.

[0020] (2) Immunoreactivity assays confirmed that the OMP16Es6× fusion protein could not only bind strongly to Brucella whole-cell polyclonal antibody and OMP16 polyclonal antibody, but also be specifically recognized by the five corresponding OMP16 monoclonal antibodies. This indicates that the flexible linker peptide and tandem sequence effectively maintained the native conformation and spatial accessibility of each B cell epitope, and the key antigenic sites were not masked due to fusion. Each epitope maintained good antigenicity and accessibility in the fusion protein.

[0021] (3) This invention uses OMP16Es6× as the coating antigen and optimizes key parameters such as antigen coating concentration (0.02 μg / mL), serum dilution (1:5), secondary antibody dilution (1:5000), coating time (37℃ for 2 h followed by overnight incubation at 4℃), blocking conditions (5% skim milk, 37℃ for 2 h), serum / secondary antibody incubation time (37℃ for 1 h), and color development time (30 min) using a matrix titration method, establishing a standardized iELISA operating procedure. This method has a detection sensitivity of 1:100 and can effectively detect low-titer antibodies; it shows no cross-reactivity with positive sera of bovine viral diarrhea virus, foot-and-mouth disease virus, bovine tuberculosis mycobacterium, and Salmonella, demonstrating high detection specificity.

[0022] (4) Parallel testing results of 1318 clinical bovine serum samples using the national standard method, the Rose Bengal Plate Agglutination Test (RBPT), showed that the positive concordance rate of this method was 73.36%, the negative concordance rate was 89.36%, and the overall concordance rate was 82.30%. In preliminary applications at three large-scale farms, the two methods showed good consistency in the context of complex actual samples. Compared to RBPT, this method, because it detects antibodies against the OMP16-specific B-cell epitope, theoretically has higher specificity and can effectively reduce the false positive problem of traditional whole-cell antigen detection.

[0023] (5) The rational design process of “epitope identification → bioinformatics structure prediction → solubility and stability assessment → multiple screenings → prokaryotic expression and purification” adopted in this invention provides a reference and theoretical basis for the development of multi-epitope diagnostic antigens or vaccines for other pathogens (such as viruses, bacteria, and parasites).

[0024] In summary, this invention successfully constructed the Brucella OMP16 multi-epitope fusion protein OMP16Es6×, and established a high-performance iELISA detection method using this protein as an antigen. This provides a new technical means for the serological diagnosis of brucellosis that combines high sensitivity, high specificity, and good clinical application potential. Attached Figure Description

[0025] Figure 1 This is the plasmid map of the recombinant expression plasmid pET-28a-OMP16Es6×.

[0026] Figure 2 This involves the design and property analysis of OMP16 epitope fusion proteins. (A) Structural analysis of repeat fusion proteins with different epitopes. (B) Solubility analysis of repeat fusion proteins with different epitopes. (C) Stability analysis of repeat fusion proteins with different epitopes.

[0027] Figure 3 This document presents the expression, purification, and immunoreactivity analysis of the OMP16Es 6× epitope fusion protein. (A) Design of the OMP16Es 6× epitope fusion protein. (B) Schematic diagram of the construction of the OMP16Es 6× epitope fusion protein expression vector. (C) Relative positions of the OMP16Es epitope unit and linker in the fusion protein; red represents the linker, and blue represents OMP16Es. (D) Expression and purification of the OMP16Es 6× epitope fusion protein. (E) Immunoreactivity analysis of the OMP16Es 6× epitope fusion protein.

[0028] Figure 4 This is the optimal secondary antibody dilution factor for the iELISA method of epitope fusion proteins.

[0029] Figure 5 This is the optimal coating time for the iELISA method of epitope fusion proteins.

[0030] Figure 6 These are the optimal blocking conditions for the iELISA method for epitope fusion proteins.

[0031] Figure 7 This is the optimal reaction time for the iELISA method for epitope fusion proteins.

[0032] Figure 8 This is the optimal color development time for the iELISA method of epitope fusion proteins.

[0033] Figure 9 This is a frequency count of negative bovine clinical serum sample test results.

[0034] Figure 10 This relates to the sensitivity and specificity of the iELISA method for epitope fusion proteins. Detailed Implementation

[0035] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0036] iELISA based on multi-epitope fusion proteins is an emerging serological detection method. Its core lies in constructing a multi-epitope fusion protein as the detection antigen by linking multiple specific B-cell epitopes through connecting peptides. Compared to traditional whole-cell antigens or protein antigens, this method can enrich highly specific recognition sites, minimize cross-reactivity, and significantly improve the specificity and sensitivity of the detection, representing an important development direction in pathogen serological detection technology.

[0037] Based on five identified OMP16 monoclonal antibody epitopes, this invention constructs and expresses an OMP16 multi-epitope fusion protein through rational design and bioinformatics analysis. Subsequently, using this fusion protein as the coating antigen, a novel iELISA method is established through systematic optimization of reaction conditions, providing a novel serological detection method with both high sensitivity and specificity for the clinical prevention and control of brucellosis.

[0038] 1. Method 1.1 Design and Bioinformatics Analysis of OMP16 Epitope Fusion Protein To construct a Brucella OMP16 multi-epitope fusion protein detection antigen with high immunogenicity, good solubility, and structural stability, this invention utilizes five previously identified linear monoclonal antibody epitopes: 77-TLSKQAQW-84, 85-LQRYPQY-91, 112-GQRRAAAT-119, 120-RDFLASRG-127, and 128-VPTNRMRT-135 (see application number 202410716374.7). The numbers represent the B-cell epitopes' positions within the full-length amino acid sequence of the Brucella OMP16 protein. 77-TLSKQAQW-84 represents the peptide segment from amino acid 77 to amino acid 84, 85-LQRYPQY-91 represents positions 85 to 91, and 112-GQRRAAAT-119 represents the peptide segment from amino acid 77 to amino acid 84. Based on the following (represented by positions 112 to 119, 120-RDFLASRG-127, 120 to 127, and 128-VPTNRMRT-135, 128 to 135), a systematic and rational design and comprehensive bioinformatics evaluation were conducted. First, according to the order of the five epitopes in the natural amino acid sequence of OMP16 (i.e., the order of appearance of each epitope from the N-terminus to the C-terminus in the OMP16 protein amino acid sequence), they were sequentially tandem to form basic epitope units. To reduce steric hindrance between adjacent epitope units, the above basic tandem unit was treated as a repeating module and repeatedly linked 2, 4, 6, 8, and 10 times respectively using a flexible linker (GGGSG), thereby obtaining five candidate fusion proteins of different lengths, named OMP16Es2×, OMP16Es4×, OMP16Es6×, OMP16Es8×, and OMP16Es10×, respectively. Its sequence is as follows:

[0039] OMP16Es2×, SEQ ID NO.1: TLSKQAQWLQRYPQYGQRRAAATRDFLASRGVPTNRMRTGGGSGTLSKQAQWLQRYPQYGQRRAAATRDFLASRGVPTNRMRT OMP16Es4×, SEQ ID NO.2: TLSKQAQWLQRYPQYGQRRAAATRDFLASRGVPTNRMRTGGGSGTLSKQAQWLQRYPQYGQRRAAATRDFLASRGVPTNRMRTGGGSGTLSKQAQWLQRYPQYGQRRAAATRDFLASRGVPTNRMRTGGGSGTLSKQAQWLQRYPQYGQRRAAATRDFLASRGVPTNRMRT OMP16Es6×,SEQ ID NO.3:TLSKQAQWLQRYPQYGQRRAAATRDFLASRGVPTNRMRTGGGSGTLSKQAQWLQRYPQYGQRRAAATRDFLASRGVPTNRMRTGGGSGTLSKQAQWLQRYPQYGQRRAAATRDFLASRGVPTNRMRTGGGSGTLSKQAQWLQRYPQYGQRRAAATRDFLASRGVPTNRMRTGGGSGTLSKQAQWLQRYPQYGQRRAAATRDFLASRGVPTNRMRTGGGSGTLSKQAQWLQRYPQYGQRRAAATRDFLASRGVPTNRMRT OMP16Es8×,SEQ ID NO.4:TLSKQAQWLQRYPQYGQRRAAATRDFLASRGVPTNRMRTGGGSGTLSKQAQWLQRYPQYGQRRAAATRDFLASRGVPTNRMRTGGGSGTLSKQAQWLQRYPQYGQRRAAATRDFLASRGVPTNRMRTGGGSGTLSKQAQWLQRYPQYGQRRAAATRDFLASRGVPTNRMRTGGGSGTLSKQAQWLQRYPQYGQRRAAATRDFLASRGVPTNRMRTGGGSGTLSKQAQWLQRYPQYGQRRAAATRDFLASRGVPTNRMRTGGGSGTLSKQAQWLQRYPQYGQRRAAATRDFLASRGVPTNRMRTGGGSGTLSKQAQWLQRYPQYGQRRAAATRDFLASRGVPTNRMRT OMP16Es10×, SEQ ID NO.5: TLSKQAQWLQRYPQYGQRRAAATRDFLASRGVPTNRMRTGGGSGTLSKQAQWLQRYPQYGQRRAAATRDFLASRGVPTNRMRTGGGSGTLSKQAQWLQRYPQYGQ RRAAATRDFLASRGVPTNRMRTGGGSGTLSKQAQWLQRYPQYGQRRAAATRDFLASRGVPTNRMRTGGGSGTLSKQAQWLQRYPQYGQRRAAATRDFLASRGVPTNRMRT GGGSGTLSKQAQWLQRYPQYGQRRAAATRDFLASRGVPTNRMRTGGGSGTLSKQAQWLQRYPQYGQRRAAATRDFLASRGVPTNRMRTGGGSGTLSKQAQWLQRYPQYGQ RRAAATRDFLASRGVPTNRMRTGGGSGTLSKQAQWLQRYPQYGQRRAAATRDFLASRGVPTNRMRTGGGSGTLSKQAQWLQRYPQYGQRRAAATRDFLASRGVPTNRMRT Subsequently, the amino acid sequences of the five candidate fusion proteins were submitted to the AlphaFold2 protein structure prediction tool for high-precision three-dimensional structure prediction, obtaining predicted structural models for each protein to assess overall folding compactness, epitope spatial accessibility, and potential spatial conflicts. Finally, the SOLpro tool was used to predict the theoretical solubility probability of each protein in the *E. coli* expression system, and the ProteinTools tool was used to predict the number of hydrogen bond networks, salt bridges, and hydrophobic clusters for each protein to assess its structural stability. Based on a comprehensive evaluation of the solubility and stability of each protein, a suitable design scheme was selected.

[0040] 1.2 Construction, expression, and purification of the OMP16 epitope fusion protein Based on the above bioinformatics analysis, OMP16Es6× was determined to be the optimal candidate antigen (it should be noted that the subsequent schemes of this invention only use OMP16Es6× as the optimal candidate antigen for illustrative purposes, but the other four antigens can also achieve the expected results of this invention), and its design process is as follows. Figure 3 As shown in Figure A, the relative positions of the OMP16Es 6× epitope unit and the linker in the fusion protein are as follows: Figure 3As shown in Figure C. To obtain a recombinant protein applicable to subsequent immunological detection, the OMP16Es6× fusion protein encoding gene was codon optimized based on E. coli codon preference, synthesized at Nanjing GenScript Biotech Co., Ltd., and cloned into the prokaryotic expression vector pET-28a(+), constructing the recombinant expression plasmid pET-28a-OMP16Es6×. The plasmid map is shown below. Figure 1 and Figure 3 As shown in B.

[0041] Subsequently, the recombinant plasmid, verified by sequencing, was transformed into E. coli BL21(DE3) expression strain and cultured in LB medium containing kanamycin at 37°C with shaking until OD. 600 The concentration was approximately 0.8. Subsequently, IPTG was added to a final concentration of 1.0 mM, and expression was induced at 16°C and 180 rpm for 18 h to promote soluble protein expression. After induction, the bacterial cells were collected by centrifugation, sonicated (300W, 3 seconds on, 3 seconds off, 30 min), and centrifuged again. The supernatant was collected. The His-tagged soluble fusion protein was purified using an NTA affinity kit. SDS-PAGE was used to analyze protein expression and purification, and BCA assay was used to determine protein concentration. The protein was then aliquoted and stored at -80°C.

[0042] 1.3 Immunoreactivity Analysis of OMP16 Epitope Fusion Protein To confirm whether the obtained multi-epitope fusion protein OMP16Es6× successfully retained the natural antigenicity of its constituent epitopes, this invention used an iELISA assay to analyze its immunoreactivity. The purified OMP16Es6× fusion protein was diluted to 1 μg / well with carbonate coating buffer and then coated onto 96-well microplates. Primary antibodies were prepared using laboratory-prepared Brucella mouse polyclonal antibody (1:1000), OMP16 mouse polyclonal antibody (1:1000), and OMP16 monoclonal antibodies D3, H4, F5, E6, and B7 (1:3000). Mouse negative serum (1:1000) was added as a control, and HRP-labeled goat anti-mouse IgG antibody (1:5000) was used as a secondary antibody. Incubation was performed, followed by TMB chromogenic reaction, and the reaction was terminated with 2M H2SO4 solution. The results were then analyzed using an iELISA reader. 450nm Reading at the location.

[0043] Among them, the Brucella mouse polyclonal antibody and OMP16 monoclonal antibody D3, H4, F5, E6, B7 are described in "Zhai Yunyi. Establishment of Brucella outer membrane protein 16 antibody competitive ELISA method [D]. Northwest A&F University, 2022. DOI:10.27409 / d.cnki.gxbnu.2022.001369."

[0044] 1.4 iELISA Method Construction (1) Coating: The above fusion protein was diluted with carbonate coating buffer (pH 9.6), and 100 μL / well was added to the microplate. The plate was incubated at 37°C and then transferred to 4°C and left to stand overnight to allow the fusion protein to be fully adsorbed onto the surface of the solid support. (2) Washing: Discard the coating solution, add PBST washing solution (PBS containing 0.05% Tween-20) to each well, let stand for 30 seconds and then discard, repeat washing 3 to 5 times, and pat dry on the last wash; (3) Blocking: Add blocking solution to each well and block the sites on the solid support that are not bound to proteins at 37°C; after blocking, wash again according to the method in step (2); (4) Add the serum to be tested: Dilute the serum sample to be tested with PBS, add 100 μL of diluted serum to each well, and incubate to allow the specific antibodies in the serum to bind to the coating antigen; (5) Washing: Wash according to the method in step (2); (6) Add enzyme-labeled secondary antibody: Dilute HRP-labeled rabbit anti-bovine IgG secondary antibody with PBS, add 100 μL to each well, and incubate; (7) Washing: Wash according to the method in step (2); (8) Color development: Add 100 μL of TMB color development solution to each well and incubate in the dark; (9) Termination: Add 50 μL of 2M H2SO4 stop solution to each well and gently shake to mix; (10) Reading and judgment: Use an enzyme-linked immunosorbent assay (ELISA) reader to read the OD value of each well at a wavelength of 450 nm.

[0045] 1.4.1 Optimization of optimal antigen coating concentration and serum dilution factor To establish an iELISA method based on the OMP16 multi-epitope fusion protein, this invention first determined and optimized two key parameters in the reaction system—antigen coating concentration and serum dilution factor—using a checkerboard titration method. The purified OMP16Es6× antigen was serially diluted with carbonate coating buffer at concentration gradients of 0.4, 0.2, 0.1, 0.02, 0.01, and 0.002 μg / mL, and 100 μL / well was used to coat 96-well microplates, incubating overnight at 4°C. Simultaneously, Brucella-positive and Brucella-negative bovine hyperimmune serum were serially diluted with PBS at dilutions of 1:5, 1:10, 1:20, 1:40, 1:60, 1:80, 1:100, and 1:120. After washing with PBST and blocking with 5% skim milk, different dilutions of positive and negative sera were added sequentially, 100 μL / well, and incubated at 37°C for 1 h. After washing with PBST, 100 μL / well of HRP-labeled rabbit anti-bovine IgG secondary antibody (1:5000) was added, and incubated at 37°C for 1 h. Finally, 100 μL of TMB substrate was added to each well for reaction, and the reaction was terminated with 2M H2SO4. The absorbance of each well was read at 450 nm. The optimal reaction conditions were evaluated by calculating the ratio of OD values ​​of positive to negative sera for each combination of antigen concentration and serum dilution (P / N value).

[0046] 1.4.2 Optimization of the optimal secondary antibody dilution factor for the iELISA method Based on the determination of the optimal antigen coating concentration and serum dilution factor, this invention optimized the dilution factor of HRP-labeled rabbit anti-bovine IgG antibody to determine the optimal working concentration of the enzyme-labeled secondary antibody. In a system with fixed antigen and primary antibody reaction conditions, the rabbit anti-bovine IgG antibody was serially diluted with PBS solution, setting eight concentration gradients: 1:1000, 1:2500, 1:5000, 1:10000, 1:15000, 1:20000, 1:25000, and 1:30000. Subsequent reactions were then carried out according to the standardized procedure described above. The optimal working concentration of the secondary antibody was evaluated by comparing the OD450 values ​​and P / N values ​​of positive and negative sera at different secondary antibody dilutions.

[0047] 1.4.3 Optimization of the optimal antigen coating time for the iELISA method To determine the optimal antigen coating time and further improve the stability and reproducibility of the method, this invention systematically compared the effects of different temperature and time combinations on the coating effect based on the optimized antigen coating concentration. First, the fusion protein antigen was diluted according to the above-mentioned antigen coating concentration and added to a 96-well ELISA plate. Then, five different coating conditions were set: incubation at 37°C for 2 h followed by overnight incubation at 4°C; incubation at 4°C overnight; incubation at 37°C for 1 h; incubation at 37°C for 2 h; and incubation at 37°C for 3 h. After coating, subsequent experiments were conducted according to the established conditions and procedures. By comparing the P / N values ​​of each reaction system, the optimal antigen coating conditions were determined.

[0048] 1.4.4 Optimization of the best blocking solution and blocking time for the iELISA method Based on the established reaction conditions, this invention further evaluated the impact of blocking solution type and blocking time on detection performance. First, to screen for the optimal blocking solution, this invention compared four commonly used blocking reagents: 5% (v / v) skim milk solution, 10% skim milk solution, 3% gelatin solution, and 1% BSA solution. All blocking was performed by incubation at 37°C for 2 hours. Based on this, to determine the optimal blocking time, this invention used the screened optimal blocking solution and further set four blocking time points: overnight incubation at 4°C, incubation at 37°C for 1 hour, incubation at 37°C for 2 hours, and incubation at 37°C for 3 hours, to evaluate the optimal antigen blocking time. The results were used to determine the optimal antigen blocking solution and blocking time by comparing the P / N values ​​of each reaction group.

[0049] 1.4.5 Optimization of optimal serum and secondary antibody incubation time for iELISA method To determine the optimal incubation time for serum and secondary antibody, this invention optimized the incubation time of serum and enzyme-labeled secondary antibody based on the above reaction conditions. First, to determine the optimal serum incubation time, under optimized antigen coating and blocking conditions, diluted positive hyperimmune serum and negative control serum (1:5) were added to the reaction wells, and five time gradients (15 min, 30 min, 1 h, 2 h, and 3 h) were set at 37°C for comparison. After determining the serum incubation time, to further optimize the secondary antibody reaction conditions, HRP-labeled rabbit anti-bovine IgG secondary antibody was added to the reaction wells, and five time gradients (15 min, 30 min, 1 h, 2 h, and 3 h) were set at 37°C for comparison to evaluate the optimal incubation time for the secondary antibody. The optimal serum and secondary antibody incubation time was determined by calculating the P / N value of the reaction at different incubation times.

[0050] 1.4.6 Optimization of the optimal color development time for the iELISA method To ensure the colorimetric signal is within the ideal detection range of the microplate reader, the incubation time of the TMB chromogenic substrate was optimized based on the aforementioned optimized conditions. According to the established optimal reaction conditions, antigen coating, blocking, serum and secondary antibody incubation were performed, followed by thorough washing. 100 μL of TMB chromogenic solution was added to each well, and the microplate was immediately transferred to a 37°C incubator for light-protected reaction. During this process, four time gradients of 15 min, 30 min, 45 min, and 60 min were set for comparison. At each time point, an equal volume of 2M H₂SO₄ stop solution was immediately added to each well to terminate the reaction. The absorbance of each well was then read at 450 nm using a microplate reader, and the optimal colorimetric time was evaluated by comparing the P / N values ​​of each reaction group.

[0051] 1.5 Determination of the cutoff value for the iELISA method After establishing and optimizing the iELISA method based on the OMP16Es6× antigen, to determine the critical value of this method, this invention tested 240 clinically negative bovine serum samples, confirmed by national standard methods and without a history of Brucella infection, stored in the laboratory, under all the reaction conditions optimized in the above system. Each sample had three replicate wells. After the test, GraphPad software was used to perform frequency distribution analysis and normality test on the OD450 values ​​of the 240 samples. Based on the data conforming to or close to a normal distribution, the mean and standard deviation were calculated. The specificity of the detection method was set at a 95% statistical confidence level, and the critical value of this method was calculated and determined to be Mean ± 2 × SD. Finally, based on the calculated critical value, a clinical test result interpretation standard was established: if the mean OD450 of the tested serum sample is greater than or equal to this critical value, it is judged as Brucella antibody positive; otherwise, it is judged as negative.

[0052] 1.6 Sensitivity Evaluation of the iELISA Method To evaluate the analytical performance of the established iELISA method based on the OMP16Es6× antigen and determine its detection sensitivity, Brucella bovine positive hyperimmune serum was serially diluted to 1:5, 1:10, 1:20, 1:40, 1:60, 1:80, 1:100, and 1:120. All dilutions were then tested according to the established optimal iELISA conditions and procedures. The results were analyzed using GraphPad software. When a dilution test result was negative, the previous positive dilution was considered the detection sensitivity of the method.

[0053] 1.7 Evaluation of iELISA method specificity To evaluate the analytical performance of the established method and determine its specificity, bovine serum containing common pathogens that are prone to cross-reactivity with Brucella, such as bovine viral diarrhea virus, foot-and-mouth disease virus, Mycobacterium bovis tuberculosis, and Salmonella, was selected. The serum was diluted 1:5 and then subjected to subsequent experiments under the optimized conditions and procedures described above. Brucella-positive and Brucella-negative bovine serum were also included as control groups. The potential cross-reactivity was analyzed by comparing the reaction signals of each pathogen-positive serum with Brucella-positive and negative serum.

[0054] 1.8 Evaluation of iELISA Method Conformity To evaluate the diagnostic accuracy and clinical applicability of the established method, this invention uses the widely used national standard method, the Rose Bengal Plate Agglutination Test, as a comparative method, and conducts large-scale parallel testing of clinical samples to calculate the concordance rate between the two methods. First, 1318 bovine clinical serum samples with clear backgrounds were randomly selected. Each sample was simultaneously tested in parallel and independently using both the iELISA method and the RBPT method established in this invention. All operations and result interpretations were performed without knowledge of the results of the other method. After testing, the positive concordance rate, negative concordance rate, and overall concordance rate of the two methods were calculated.

[0055] 1.9 Preliminary Clinical Application of iELISA Method To evaluate the reliability of the established method in practical applications, this invention selected three large-scale farms (A, B, and C) with a Brucella infection background in a livestock breeding community in Lingwu City, Ningxia Hui Autonomous Region, for serum sample collection and testing. A total of 62 tail vein blood samples were collected from the three farms, and the serum was separated, aliquoted, and frozen. Subsequently, the 62 clinical serum samples were independently and parallelly tested under the same conditions and environment, following both the established iELISA procedure and the RBPT method, by different researchers under double-blind conditions for both samples and results. Finally, the judgment results of the two methods on the same sample were compared and statistically analyzed one by one to evaluate the consistency of the iELISA method with existing national standard methods in complex field sample environments.

[0056] 2. Results 2.1 Design and Analysis of OMP16 Epitope Fusion Protein This invention utilizes the linear sequence of five epitopes (77-TLSKQAQW-84, 85-LQRYPQY-91, 112-GQRRAAAT-119, 120-RDFLASRG-127, 128-VPTNRMRT-135) in the natural amino acid sequence of OMP16, tandemly to form basic epitope units. Subsequently, these basic tandem epitope units were used as repeating modules and repeatedly linked 2, 4, 6, 8, and 10 times using a flexible linker (GGGSG), thereby obtaining five candidate fusion proteins of different lengths. Figure 2 (A)

[0057] This invention utilizes the AlphaFold2 deep learning model to perform high-precision three-dimensional structure prediction for five fusion proteins. The results show that with the increase of tandem repeat count, the regularity and compactness of the overall protein structure exhibit a trend of first increasing and then stabilizing. Specifically, the 6×, 8×, and 10× models show more stable global folding conformations.

[0058] Furthermore, this invention utilized various bioinformatics tools to quantitatively analyze the solubility and stability of these models. Solubility analysis showed that protein solubility gradually decreased with increasing tandem repeat counts, with 2×, 4×, and 6× models exhibiting higher solubility. Figure 2 Stability analysis showed that the 6× tandem repeat epitope had the highest number of hydrogen bond networks and hydrophobic clusters, while the 10× tandem repeat epitope had the highest number of salt bridges and charge separations, followed by the 6× tandem repeat epitope fusion protein. Figure 2 (C). Based on the above analysis, the 6× fusion protein exhibits the best balance between structural stability and theoretical solubility. Therefore, this invention selects the 6× fusion protein, which is a fusion protein composed of 6 repeating 5-epitope units tandemly, as a candidate antigen for subsequent prokaryotic expression and purification.

[0059] 2.2 Construction, expression, and purification of the OMP16 epitope fusion protein The results are as follows Figure 3 As shown in Figure D, SDS-PAGE analysis revealed that the purified 6× epitope fusion protein exhibited a single, clear main band at approximately 45 kDa, consistent with the theoretical molecular weight, and possessed high purity. This indicates that the present invention successfully obtained a high-purity recombinant multi-epitope fusion protein, named OMP16Es6×.

[0060] This invention uses laboratory-prepared Brucella mouse polyclonal antibody, OMP16 mouse polyclonal antibody (for details on Brucella mouse polyclonal antibody and OMP16 mouse polyclonal antibody, please refer to the literature "Zhai Yunyi. Establishment of Brucella outer membrane protein 16 antibody competitive ELISA method [D]. Northwest A&F University, 2022. DOI:10.27409 / d.cnki.gxbnu.2022.001369."), and five specific monoclonal antibodies corresponding to the epitopes as primary antibodies for detection. The results show that the OMP16Es6× fusion protein produces strong specific reaction signals with both the Brucella polyclonal antibody and the OMP16 polyclonal antibody, indicating that it covers the main immunodominant epitopes of Brucella and OMP16 proteins. More importantly, this fusion protein can specifically bind to all five monoclonal antibodies targeting different epitopes. Figure 3 The results (E) confirm that the tandem fusion design and flexible linker peptides of this invention effectively maintain the epitope accessibility and immunoreactivity of each B cell epitope in its fusion protein, without masking or conformational changes of key antigen sites due to tandem fusion. These results demonstrate that this invention successfully prepared the OMP16 multi-epitope fusion protein antigen, laying the foundation for the subsequent establishment of an iELISA method based on this antigen.

[0061] 2.3 Optimization of optimal antigen coating concentration and serum dilution factor for iELISA method The results (Table 1) showed that the reaction system exhibited the highest P / N value when the antigen coating concentration was 0.01 μg / mL and the serum dilution was 1:5, and the OD of the positive serum wells was also the highest at this time. 450 The value is close to 1.0, which is within the ideal linear range for ELISA reader detection. This condition ensures a high detection signal while maximally distinguishing between positive and negative samples, guaranteeing the sensitivity and specificity of the detection method.

[0062] Table 1. Optimal antigen coating concentration and serum dilution factor matrix Supplementary Table 1: Optimal Antigen Coating Concentration and Serum Dilution Factors Matrix Supplementary Table 1: Optimal Antigen Coating Concentration and Serum Dilution Factors (square matrix) 2.4 Optimization of the optimal secondary antibody dilution concentration for the iELISA method The results showed that ( Figure 4When the secondary antibody dilution is 1:5000, the reaction system exhibits the highest P / N value. At this point, the OD value of the positive wells is within the suitable range for microplate reader detection, while the OD value of the negative wells remains at a low level. Therefore, this invention determines 1:5000 as the optimal working concentration of the secondary antibody in this method.

[0063] 2.5 Optimal Antigen Coating Time Optimization for iELISA Method The results showed that ( Figure 5 The reaction system exhibited the highest P / N value under the coating conditions of 37℃ for 2 h followed by overnight incubation at 4℃. Therefore, the optimal antigen coating conditions determined by this invention are: using 0.01 μg / mL of OMP16Es6× fusion protein, incubating at 37℃ for 2 h, and then transferring to 4℃ for overnight incubation.

[0064] 2.6 Optimization of blocking solution and blocking time for iELISA method The results show that ( Figure 6 When using 5% skim milk powder for blocking, the detection system achieved the highest P / N value, indicating that this blocking solution effectively inhibited non-specific binding while minimizing interference with the antigen-antibody specific reaction. After determining the optimal blocking solution, this invention further optimized the blocking time. Under the same conditions, blocking was performed at four time points: overnight at 4°C, incubation at 37°C for 1 h, 2 h, and 3 h. The experimental results showed that the highest P / N value was obtained when the blocking time was 2 h at 37°C. Based on the above results, this invention determined the optimal blocking conditions for this iELISA method to be: using 5% skim milk and blocking at 37°C for 2 h.

[0065] 2.7 Optimization of serum and secondary antibody incubation time for iELISA method By comparing the P / N values ​​at different incubation times, it was found that antibody binding did not reach saturation at 15 min or 30 min of incubation. However, extending the incubation time to 2 h or 3 h did not significantly increase the P / N value, while the background value increased slightly. The highest P / N value was observed at a serum incubation time of 1 h. Therefore, the optimal serum incubation time was determined to be 37℃ for 1 h.

[0066] After determining the serum incubation time, this invention further optimized the incubation time of the secondary antibody. HRP-labeled rabbit anti-bovine IgG (1:5000) was added to the reaction wells, and incubated at 37°C for 15 min, 30 min, 1 h, 2 h, and 3 h, respectively. The results showed ( Figure 7 The highest P / N value can be obtained by incubating the secondary antibody for 1 hour. Based on the above results, this invention determines that the optimal incubation time for both serum and secondary antibody in the iELISA method is 37°C for 1 hour.

[0067] 2.8 Optimization of the optimal color development time for the iELISA method The results are as follows Figure 8 As shown, with the development time increasing from 15 min to 30 min, the OD value of the positive serum wells increased significantly, while the OD value of the negative serum wells only increased slightly. Therefore, the P / N value peaked at 30 min. When the development time was extended to 45 min and 1 h, the increase in OD value of the positive wells tended to level off, but the OD value of the negative wells showed a more significant increase, leading to a decrease in the P / N value. Therefore, the optimal development time for this iELISA method is incubation at 37°C for 30 min.

[0068] 2.9 Determination of the cutoff value for the iELISA method The results showed that ( Figure 9 The distribution of this data set basically conforms to a normal distribution. The calculated mean OD450 value of all negative samples was 0.232, and the standard deviation was 0.080. To cover 95% of the negative population and thus control specificity at a high level, this invention uses a 95% confidence interval, i.e., the critical value is set as "Mean ± 2 × SD". Therefore, the critical value of this method is 0.392, which is the OD450 value of the serum sample to be tested. 450 A value ≥ 0.392 is considered a positive result for Brucella antibodies; OD 450 A value < 0.392 is considered negative.

[0069] Based on the above experimental results, the final operating steps of this method are as follows: OMP16Es6× protein diluted to 0.01 μg / mL, 100 μL / well, incubated at 37℃ for 2 h, followed by overnight coating at 4℃; PBST 200 μL / well, held for 30 s, then discarded, patted dry, and washed 3 times; serum sample 5-fold diluted, 100 μL / well, incubated at 37℃ for 1 h; PBST 200 μL / well, held for 30 s, then discarded, patted dry, and washed 3 times; rabbit anti-bovine enzyme-labeled secondary antibody 5000-fold diluted, 100 μL / well, incubated at 37℃ for 1 h; PBST 200 μL / well, held for 30 s, then discarded, patted dry, and washed 3 times; TMB chromogenic solution, 100 μL / well, incubated at 37℃ for 30 min; 2M H2SO4 stop solution, 50 μL / well, to terminate the reaction. Results interpretation: OD... 450 A value ≥ 0.392 is considered positive; OD 450 <0.392, judged as negative.

[0070] 2.10 Evaluation of the sensitivity and specificity of the iELISA method Sensitivity test results as follows Figure 10As shown in Figure A, the OD value decreases gradually with increasing serum dilution. When the serum is diluted to 1:100, its OD value is still higher than the critical value, indicating a positive result; when diluted to 1:120, its OD value drops below the critical value, indicating a negative result. Therefore, the detection sensitivity of this method is determined to be 1:100, indicating that this method can effectively detect low concentrations of Brucella-specific antibodies and has good detection sensitivity.

[0071] Specificity evaluation results as follows Figure 10 As shown in Figure B, except for Brucella positive serum which showed a positive reaction, the positive serum test results for bovine viral diarrhea virus, foot-and-mouth disease virus, Mycobacterium tuberculosis, and Salmonella were all negative. This indicates that the method has good detection specificity and can effectively distinguish Brucella from other pathogens.

[0072] 2.11 Evaluation of iELISA Method Conformity This invention parallelly tested 1318 bovine clinical serum samples collected and preserved in the laboratory. The results are shown in Table 2. The RBPT method detected 428 positive samples and 890 negative samples, with a positive rate of 32.47%. The iELISA method detected 322 positive samples and 996 negative samples, with a positive rate of 24.43%. Further calculation of the concordance rate between the two methods revealed a positive concordance rate of 73.36% and a negative concordance rate of 89.36%, with an overall concordance rate of 82.30%. This indicates that the iELISA method established in this invention demonstrates high accuracy in large-scale clinical sample testing.

[0073] Table 2. Concordance rate between iELISA and RBPT for epitope fusion proteins 2.12 Preliminary Clinical Application of iELISA Method To preliminarily evaluate the applicability and reliability of the iELISA method established in this study in a real-world livestock farming environment, clinical serum samples were collected from three large-scale farms in a livestock farming community in Lingwu City, Ningxia Hui Autonomous Region. A total of 62 representative bovine serum samples were collected from farms A, B, and C, and the iELISA method and the Rose Bengal Plate Test (RBT) were used in parallel for detection and result comparison.

[0074] The results (Table 3) show that in the 18 samples tested at Ranch A, the RBPT and iELISA methods were completely consistent, with 9 positive and 9 negative samples respectively, achieving an overall concordance rate of 100%. In the 36 samples tested at Ranch B, RBPT detected 1 positive and 35 negative samples; iELISA detected 0 positive and 36 negative samples. In the 8 samples tested at Ranch C, neither method detected any positive results, showing complete consistency and a 100% concordance rate. The two methods differed only in one sample, with an overall concordance rate of 97.22% (35 / 36). This result demonstrates that the iELISA method for OMP16 multi-epitope fusion protein established in this invention has good consistency with the widely used RBPT method in field sample testing, especially showing highly consistent detection results in ranches with low positive rates and some ranches with high positive rates.

[0075] Table 3 Clinical applications of the iELISA method for epitope fusion proteins In summary, this invention tandemly strings together five epitopes—77-TLSKQAQW-84, 85-LQRYPQY-91, 112-GQRRAAAT-119, 120-RDFLASRG-127, and 128-VPTNRMRT-135—according to their natural sequence order to obtain basic epitope units. Multi-epitope fusion proteins often face problems such as structural instability, low expression solubility, and epitope inactivation due to their high sequence repetition and disruption of the original antigen structure. Therefore, to efficiently design and express multi-epitope fusion proteins and improve the success rate of protein construction, this invention employs bioinformatics methods to conduct preliminary analysis and screening of fusion protein design schemes. First, this invention uses AlphaFold2 to predict the structure and analyze the physicochemical properties of fusion proteins containing different repeat units (2×, 4×, 6×, 8×, 10×). It was found that the 6-repeat design (OMP16Es6×) achieves the optimal balance between structural stability and theoretical solubility, while ensuring the accessibility of each epitope. Furthermore, this invention obtains high-purity OMP16Es6× protein through prokaryotic expression and affinity chromatography purification in *E. coli*. This protein not only reacts with Brucella polyclonal antibodies and OMP16 polyclonal antibodies in iELISA assays, but is also effectively recognized by five corresponding OMP16 monoclonal antibodies. This result confirms that the tandem strategy and flexible linker used in this invention successfully preserve the natural antigenicity of each epitope, without causing destruction or masking of key conformational epitopes due to the fusion design.

[0076] Based on the obtained multi-epitope fusion protein antigen, this invention establishes a standardized iELISA detection procedure through systematic condition optimization. This invention also evaluates the sensitivity, specificity, and concordance rate of this method. The results show that its detection sensitivity reaches 1:100, indicating that it can effectively detect low-titer antibodies and possesses good detection capability. This indicator is significantly higher than the iELISA method established using the intact OMP16 protein as the diagnostic antigen. Specificity tests confirm that the OMP16Es6× antigen shows no cross-reactivity with positive sera from common clinical pathogens such as bovine viral diarrhea virus, foot-and-mouth disease virus, Mycobacterium bovis, and Salmonella, demonstrating high specificity. In a comparison of sample concordance rates with the current national standard method RBT, the overall concordance rate of this method was 82.30%, with a high negative concordance rate (89.36%) and a relatively low positive concordance rate (73.36%). This difference may stem from the fundamental difference in the principles of the two methods: RBPT detects agglutinating antibodies against broad-spectrum antigens such as lipopolysaccharide, exhibiting high sensitivity but relatively insufficient specificity, while this method detects antibodies against specific B-cell epitopes of OMP16, resulting in higher specificity. Therefore, some RBPT-positive samples detected by this method but not by iELISA may be due to false positives from RBPT. In subsequent preliminary clinical applications at three ranches, this method showed good consistency with RBT, confirming its clinical application value.

[0077] The above description is merely an example and illustration of the present invention. Those skilled in the art can make various modifications or additions to the specific embodiments described, or use similar methods to replace them, as long as they do not deviate from the invention or exceed the scope defined in the claims, all of which should fall within the protection scope of the present invention.

Claims

1. A Brucella OMP16 B cell epitope fusion protein, characterized in that, The fusion protein comprises multiple repeating units tandemly linked by flexible linker peptides, each repeating unit being composed of five B-cell epitopes of Brucella OMP16 tandemly linked in their natural sequence; the amino acid sequences of the five B-cell epitopes are: TLSKQAQW, LQRYPQY, GQRRAAAT, RDFLASRG, and VPTNRMRT.

2. The fusion protein of claim 1, wherein, The amino acid sequence of the flexible linker peptide is GGGSG; the repeating unit is repeated 2, 4, 6, 8 or 10 times.

3. The fusion protein of claim 2, wherein, The repeating unit is repeated 6 times, and the amino acid sequence of the fusion protein is: TLSKQAQWLQRYPQYGQRRAAATRDFLASRGVPTNRMRTGGGSGTLSKQAQWLQRYPQYGQRRAAATRDFLASRGVPTNRMRTGGGSGTLSKQAQWLQRYPQYGQRRAAATRDFLASRGVPTNRMRTGGGSGTLSKQAQWLQRYPQYGQRRAAATRDFLASRGVPTNRMRTGGGSGTLSKQAQWLQRYPQYGQRRAAATRDFLASRGVPTNRMRTGGGSGTLSKQAQWLQRYPQYGQRRAAATRDFLASRGVPTNRMRTGGGSGTLSKQAQWLQRYPQYGQRRAAATRDFLASRGVPTNRMRT.

4. A nucleic acid molecule encoding the fusion protein of any one of claims 1 to 3.

5. A recombinant expression vector containing the nucleic acid molecule of claim 4.

6. A host cell containing the recombinant expression vector of claim 5.

7. An indirect ELISA detection method for non-diagnostic purposes, characterized in that, Using the fusion protein as a coating antigen, the method includes the following steps: The fusion protein according to any one of claims 1 to 3 is coated onto a solid support; Add sealing solution to seal; Add the serum sample to be tested and incubate; Add enzyme-labeled secondary antibody and incubate; Add the substrate for color development and read the OD value; The read OD value is compared with a preset threshold, and the comparison result is output. The non-diagnostic purposes include monitoring antibody levels after vaccination, detecting Brucella antibodies for research purposes, or conducting epidemiological surveys.

8. The detection method according to claim 7, characterized in that, The concentration of the coating antigen is 0.01~0.02 μg / mL, the dilution factor of the serum to be tested is 1:5~20, and the enzyme-labeled secondary antibody is HRP-labeled rabbit anti-bovine IgG with a dilution factor of 1:2000~10000.

9. The detection method according to claim 7 or 8, characterized in that, The preset threshold is set so that when the OD value at 450nm is ≥0.392, the comparison result is output as higher than the threshold, and when the OD value is <0.392, the comparison result is output as lower than the threshold.

10. The use of the fusion protein according to any one of claims 1 to 3 in the preparation of a serological detection reagent or kit for brucellosis.

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