Multi-link b cell epitope recombinant protein for detecting anti-brucella antibody and application thereof

Abstract

The application discloses a kind of detection anti-Brucella antibody multi-link B cell epitope recombinant protein and its application, it belongs to the field of biotechnology and immunodiagnosis technique, wherein, multi-link B cell epitope recombinant protein is formed by the B cell epitope of seven key outer membrane proteins Omp10, Omp16, Omp19, Omp25, Omp31, Omp2b, BP26 of Brucella, its amino acid sequence is as shown in sequence table SEQ ID NO:1.The multi-link B cell epitope recombinant protein has good antigenicity and reactivity, and the deer serum indirect ELISA detection system established with it as coating antigen shows excellent detection performance, has high sensitivity, strong specificity and good repeatability, and can accurately detect the level of anti-Brucella antibody in deer serum sample.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and immunodiagnostic technology, specifically to a multi-linked B-cell epitope recombinant protein for detecting anti-brucell antibodies and its applications. Background Technology

[0002] Brucellosis is a global zoonotic infectious disease caused by Brucella bacteria, posing a serious threat to livestock development and human public health. Deer brucellosis, a significant disease in deer farming, can cause symptoms such as abortion, stillbirth, orchitis in male deer, and stunted growth, resulting in severe economic losses. Furthermore, as deer are one of the hosts of Brucella, infection can be transmitted to humans through contact with contaminated meat and dairy products, posing a public health risk.

[0003] Currently, the main methods for detecting brucellosis in deer include etiological detection and serological detection. While etiological detection (such as bacterial isolation and culture, PCR) is the gold standard for diagnosis, it requires a BSL-3 biosafety laboratory, is technically demanding, time-consuming (culture cycle typically 5-7 days), and has a low isolation rate, making it difficult to meet the rapid detection needs of deer farms at the grassroots level. Serological detection, due to its ease of operation and lower cost, has become the mainstream method for detecting brucellosis in deer. Commonly used methods include the Satiocytogenetic Agglutination Test (SAT), the Rose Bengal Plate Test (RBT), and the Enzyme-Linked Immunosorbent Assay (ELISA).

[0004] However, existing serological detection methods have significant limitations: SAT and RBT are easily interfered with by non-specific antibodies in deer serum, resulting in a high false-positive rate and inability to accurately quantify the infection; commercially available ELISA kits are mostly based on whole Brucella proteins or single outer membrane proteins (such as Omp31 and BP26) as antigens, and are not optimized for the characteristics of prevalent Brucella strains and the deer immune system, resulting in insufficient antigen specificity, low sensitivity, and low specificity. Furthermore, there is currently no detection technology specifically for Brucella in deer that utilizes highly conserved B-cell epitope tandem recombinant protein antigens from various Brucella core outer membrane proteins, leading to a lack of dedicated tools for the accurate detection of Brucella in deer. Summary of the Invention

[0005] The purpose of this invention is to provide a multi-linked B-cell epitope recombinant protein for detecting anti-brucell antibodies and its application, in order to solve the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A recombinant protein for detecting anti-brucell antibodies is composed of B-cell epitopes linked together from seven key outer membrane proteins of Brucella: Omp10, Omp16, Omp19, Omp25, Omp31, Omp2b, and BP26. Its amino acid sequence is shown in SEQ ID NO:1.

[0008] Another object of the present invention is to provide a gene encoding the above-mentioned multi-B cell epitope recombinant protein for detecting anti-brucell antibodies, the nucleotide sequence of which is shown in the sequence listing SEQ ID NO:2.

[0009] Another object of the present invention is to provide a recombinant expression plasmid containing the encoding gene of the above-mentioned multi-linked B-cell epitope recombinant protein for detecting anti-brucell antibodies.

[0010] Another object of the present invention is to provide a host cell comprising the encoding gene of the above-mentioned multi-B cell epitope recombinant protein for detecting anti-brucell antibodies or the above-mentioned recombinant expression plasmid.

[0011] Another object of the present invention is to provide the application of the above-mentioned multi-B cell epitope recombinant protein for detecting anti-brucell antibodies in the preparation of a kit for detecting anti-brucell antibodies.

[0012] Furthermore, the method for detecting anti-brucellosis antibodies is as follows: using the recombinant B-cell epitope protein as the coating antigen, an indirect ELISA (iELISA) method is established to detect anti-brucellosis antibodies in deer serum.

[0013] Another object of the present invention is to provide a kit for detecting anti-brucellosis antibodies, comprising a vector coated with the above-described recombinant B-cell epitope protein for detecting anti-brucellosis antibodies.

[0014] Furthermore, the carrier is an enzyme-labeled plate; the optimal coating concentration of the multi-linked B-cell epitope recombinant protein is 0.3125 μg / mL.

[0015] Furthermore, the kit also includes one or more of the following: blocking solution, washing solution, diluent, enzyme-labeled reagent, substrate chromogenic solution, stop solution, positive standard serum, and negative standard serum.

[0016] Furthermore, the blocking solution is skim milk powder blocking solution; the washing solution is PBST solution; the diluent is PBS solution; the enzyme labeling reagent is horseradish peroxidase (HRP) labeled streptococcal protein G; the substrate chromogenic solution is 3,3',5,5'-tetramethylbenzidine solution; the stop solution is 2M sulfuric acid solution or 1-2M sodium hydroxide solution; the positive standard serum is anti-brucellosis antibody positive serum; and the negative standard serum is anti-brucellosis antibody negative serum.

[0017] The recombinant B-cell epitope protein for detecting anti-brucellosis antibodies provided by this invention possesses excellent antigenicity and reactivity. An indirect ELISA detection system for deer serum established using this protein as the coating antigen exhibits superior detection performance, demonstrating high sensitivity, strong specificity, and good reproducibility, accurately detecting anti-brucellosis antibody levels in deer serum samples. The seven key outer membrane proteins selected in this invention function complementaryly in the Brucella-host immune recognition process: Omp10 and Omp16 participate in immune regulation; Omp19 has strong immunogenicity; Omp25 is closely related to bacterial virulence and intracellular survival; Omp31 has genus specificity, contributing to broader detection scope; Omp2b is highly conserved among strains, enhancing detection stability; and BP26 is a recognized dominant immune target, significantly improving detection sensitivity. By using various bioinformatics tools to screen and obtain specific, conserved, and highly affinity dominant antigenic epitopes of these seven key outer membrane proteins, and by using genetic engineering technology to fuse and express multi-linked B-cell epitope recombinant proteins for anti-brucell antibody detection, this invention achieves systematic coverage of multi-dimensional characterization of brucell antigens, thereby improving the sensitivity, specificity, and reliability of detection at the molecular level. Attached Figure Description

[0018] Figure 1 The SDS-PAGE electrophoresis analysis results of the recombinant B-cell epitope proteins provided in the embodiments of the present invention under different induction expression conditions are shown. Among them, lane M is a protein molecular weight standard marker of 10-180kDa; lanes 1-8 correspond to the following samples respectively: 1 is the supernatant of bacterial culture after induction at 16℃, 2 is the supernatant of bacterial culture without induction at 16℃, 3 is the bacterial culture precipitate after induction at 16℃, 4 is the bacterial culture precipitate without induction at 16℃, 5 is the supernatant of bacterial culture after induction at 37℃, 6 is the supernatant of bacterial culture without induction at 37℃, 7 is the bacterial culture precipitate after induction at 37℃, and 8 is the bacterial culture precipitate without induction at 37℃.

[0019] Figure 2The SDS-PAGE analysis results of the recombinant B-cell epitope protein provided in the embodiments of the present invention during the purification process are shown. Lane M is a protein molecular weight standard marker of 10-180kDa. Lanes 1 to 9 correspond to samples at different purification stages: 1 is the supernatant after recombinant bacterial induction expression, 2 and 3 are the flow-through liquid during affinity chromatography, 4 is the binding buffer (Buffer A) control, and 5 to 9 are the protein fractions collected by elution with imidazole concentrations of 40mmol / L, 60mmol / L, 80mmol / L, 150mmol / L, and 200mmol / L, respectively.

[0020] Figure 3 The results of SDS-PAGE electrophoresis analysis of the purified recombinant B-cell epitope protein provided in the embodiments of the present invention are shown; wherein, lane M is a 10-180kDa protein molecular weight standard marker, and lane 1 is the purified recombinant protein sample.

[0021] Figure 4 The Western blot identification results of the recombinant B-cell epitope protein provided in the embodiments of the present invention are shown. Lane M represents a 10-250 kDa protein molecular weight standard marker; lane 1 represents the purified recombinant protein.

[0022] Figure 5 The figure shows the hydrophilicity analysis results of the multi-linked B-cell epitope recombinant protein provided in the embodiments of the present invention.

[0023] Figure 6 The diagram shows the in vitro immunosimulation results of the multi-linked B-cell epitope recombinant protein provided in the embodiments of the present invention. Detailed Implementation

[0024] 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.

[0025] This invention selects conserved B-cell epitopes of seven key Brucella outer membrane proteins (Omp10, Omp16, Omp19, Omp25, Omp31, Omp2b, and BP26) and constructs a multi-epitope recombinant protein using a GS linker. These proteins are complementary in immune recognition: Omp10 and Omp16 participate in immune regulation; Omp19 has strong immunogenicity; Omp25 is closely related to bacterial virulence and intracellular survival; Omp31 has genus specificity, which helps to improve the broad spectrum of detection; Omp2b is highly conserved among strains, which can enhance detection stability; and BP26 is a recognized dominant immune target, which can significantly improve detection sensitivity. Integrating the above B-cell epitopes can comprehensively cover Brucella antigen characterization and improve the broad spectrum of detection. Based on this multi-epitope recombinant protein, this invention also establishes an iELISA detection method and kit for brucellosis in deer, which has high specificity and high sensitivity, providing reliable technical support for the prevention and control of brucellosis.

[0026] Specifically, in one embodiment of the present invention, a multi-linked B-cell epitope recombinant protein for detecting anti-brucell antibodies is provided, which is composed of B-cell epitopes of seven key outer membrane proteins of Brucella, namely Omp10, Omp16, Omp19, Omp25, Omp31, Omp2b and BP26, in tandem, and its amino acid sequence is shown in the sequence listing SEQ ID NO:1.

[0027] In another embodiment of the present invention, a gene encoding the above-mentioned multi-B cell epitope recombinant protein for detecting anti-brucell antibodies is also provided, the nucleotide sequence of which is shown in SEQ ID NO:2 of the sequence listing.

[0028] In another embodiment of the present invention, a recombinant expression plasmid comprising the encoding gene of the multi-linked B-cell epitope recombinant protein for detecting anti-brucell antibodies is also provided.

[0029] In another embodiment of the present invention, a host cell is also provided comprising the encoding gene of the above-described multi-B cell epitope recombinant protein for detecting anti-brucell antibodies or the above-described recombinant expression plasmid.

[0030] In another embodiment of the present invention, the application of the above-described multi-linked B-cell epitope recombinant protein for detecting anti-brucell antibodies is also provided in the preparation of a kit for detecting anti-brucell antibodies. Preferably, the kit is a deer serum indirect ELISA detection kit.

[0031] Specifically, the method for detecting anti-brucellosis antibodies is as follows: using the recombinant B-cell epitope protein as the coating antigen, an indirect ELISA method is established to detect anti-brucellosis antibodies in deer serum.

[0032] In another embodiment of the present invention, a kit for detecting anti-brucellosis antibodies is also provided, comprising a vector coated with the above-described recombinant B-cell epitope protein for detecting anti-brucellosis antibodies.

[0033] Preferably, the carrier is an enzyme-labeled plate; the optimal coating concentration of the multi-linked B-cell epitope recombinant protein is 0.3125 μg / mL, and the coating condition is incubation at 37°C for 2 hours.

[0034] In practical applications, the kit also includes one or more of the following: blocking solution, washing solution, diluent, enzyme-labeled reagent, substrate chromogenic solution, stop solution, positive standard serum, and negative standard serum.

[0035] Preferably, the blocking solution is 5% skim milk powder blocking solution; the washing solution is PBST solution; the diluent is PBS solution; the enzyme-labeled reagent is HRP-labeled Protein G; the substrate chromogenic solution is 3,3',5,5'-tetramethylbenzidine solution; the stop solution is 2M sulfuric acid solution or 1-2M sodium hydroxide solution; the positive standard serum is anti-brucellosis antibody-positive serum; and the negative standard serum is anti-brucellosis antibody-negative serum. The optimal dilution ratio of the enzyme-labeled reagent is 1:5000; the optimal reaction time of the substrate chromogenic solution is 15 min; and the optimal dilution ratio of the positive and negative standard sera is 1:100.

[0036] The kit measures the OD of the serum sample to be tested. 450nm The OD value is used to determine whether anti-brucellosis antibodies are present. The criterion is: when the OD value is... 450nm If the OD value is ≥0.7214, the serum sample contains anti-brucellosis antibodies and is considered positive; when the OD value is ≥0.7214, the serum sample contains anti-brucellosis antibodies and is considered positive. 450nm If the value is <0.7214, the serum sample to be tested does not contain anti-brucellosis antibodies and is judged as negative.

[0037] The indirect ELISA detection system established in this invention uses deer serum samples as the monitoring target. It features simple operation and high throughput, making it particularly suitable for large-scale clinical sample screening and epidemiological monitoring. This provides reliable technical support for the effective prevention and control of brucellosis, especially deer brucellosis. Furthermore, by using HRP-labeled Protein G instead of traditional enzyme-labeled secondary antibodies, and leveraging the specific binding characteristic of Protein G to antibodies, this kit can detect anti-brucellosis antibodies in the serum of various animals, demonstrating good and broad applicability and promising application prospects.

[0038] The following embodiments are implementation examples of the technical solution of the present invention in practical applications, but are not limited thereto. Unless otherwise specified, all materials and reagents involved are purchased from commercial channels; unless otherwise specified, all experimental methods used are conventional methods.

[0039] Example 1: Design of a multi-linked B-cell epitope recombinant protein for detecting anti-brucell antibodies based on immunoinformatics, as detailed below:

[0040] 1. Determination of the amino acid sequences of seven key Brucella outer membrane proteins: Omp10, Omp16, Omp19, Omp25, Omp31, Omp2b, and BP26. This invention retrieved and collected the amino acid sequences of seven Brucella outer membrane proteins (Omp10, Omp16, Omp19, Omp25, Omp2b, OMP31, and BP26) from public databases such as NCBI (https: / / www.ncbi.nlm.nih.gov / guide / proteins / ) and UniProt (http: / / www.uniprot.org) in Brucella malata, Brucella abortus, Brucella suis, Brucella canis, and Brucella ovis. The GenBank accession numbers corresponding to the selected sequences are: Omp10 (AHG55080.1), Omp16 (AAA59360.1), Omp19 (ALR81059.1), Omp25 (AFJ79953.1), Omp2b (SUW28200.1), OMP31 (ACS50328.1), and BP26 (AHG55076.1). All sequences were downloaded and saved in FASTA format as template datasets for subsequent conservation analysis.

[0041] 2. Cell Epitope Screening: Linear B-cell epitopes of seven Brucella outer membrane proteins (Omp10, Omp16, Omp19, Omp25, Omp2b, OMP31, and BP26) were systematically predicted using online prediction tools such as IEDB (https: / / www.iedb.org / ), SVMTriP (http: / / sysbio.unl.edu / SVMTriP / ), Bepipred (http: / / www.cbs.dtu.dk), and ABCpred (http: / / www.imtech.res). To further screen for high-confidence epitopes, the predicted linear B-cell epitopes were scored using DeepLBCEPred (http: / / www.biolscience.cn / DeepLBCEPred / ) and ABCpred, and epitopes with scores higher than 0.8 were retained as candidate epitopes. The candidate epitopes were analyzed for protein physicochemical properties using Expasy-ProtParam (https: / / web.expasy.org / cgi-bin / protparam / protparam), including instability index, total average hydrophilicity, and aliphatic index. Based on the comprehensive analysis results, dominant linear B cell epitopes were initially selected. The dominant epitopes of each outer membrane protein are shown in Table 1 below:

[0042] Table 1

[0043]

[0044] Based on the general recommendations of the Expasy-ProtParam tool, this invention sets the following epitope screening criteria: the instability index should be less than 40, and the average total hydrophilicity should be less than 0. According to these criteria, peptides P6, P7, P13, P14, and P21 were eliminated because they did not meet the requirements.

[0045] To further ensure that the epitope is located in the hydrophilic region, this embodiment of the invention utilizes the NovoPro (https: / / www.novopro.cn / tools / ) online tool for protein hydrophobicity analysis. The results of this tool are presented in curve form, and peptides with scores less than 0 in most regions are selected as candidates. According to the prediction results, the hydrophobicity analysis curve of peptide P17 is mostly above the threshold, indicating that its overall hydrophobicity is relatively strong, and therefore it is also rejected.

[0046] To further enhance the immunoreactivity of recombinant proteins, this invention used the DeepTMHMM (https: / / dtu.biolib.com / deeptmhmm) online tool to predict the transmembrane domains and subcellular localization of candidate B-cell epitopes. Based on the prediction results, peptide P3 was predicted to be located in the intracellular region and was therefore eliminated. This invention prioritizes screening extracellular epitopes primarily based on the following: extracellular epitopes are more easily recognized by the immune system during infection, exhibiting higher accessibility and immunogenicity; using them as recombinant antigens accurately mimics innate immune recognition, which is crucial for ensuring high sensitivity and specificity in serological detection; simultaneously, extracellular epitopes are generally more hydrophilic, which is beneficial for the soluble expression and correct folding of recombinant proteins. Therefore, eliminating intracellular epitopes based on transmembrane domain prediction is a key step in optimizing the diagnostic efficacy of recombinant proteins.

[0047] The B-cell dominant epitopes screened in this invention, particularly those derived from the Omp2b and BP26 proteins, show significant differences in amino acid sequence compared to the epitopes contained in the L7 / L12-PADRE sequence-multiplexed B-cell epitope recombinant protein disclosed in prior art CN120795181A. Except for polypeptide P19, the overall sequence identity of the remaining epitopes with the aforementioned recombinant protein is less than 50%. Although some individual amino acid residues are identical, their core immunogenic regions and spatial conformations are different.

[0048] Based on the above screening criteria, the final dominant linear B-cell epitopes were determined as shown in Table 2 below:

[0049] Table 2

[0050]

[0051] 3. Construction of a multi-linked B-cell epitope recombinant protein for detecting anti-brucellosis antibodies: Based on the dominant B-cell epitopes of Brucella outer membrane proteins screened by bioinformatics analysis, this embodiment of the invention constructs a multi-linked B-cell epitope recombinant protein using a GS linker. Specifically, the B-cell epitope of Omp31 is located at the N-terminus of the protein sequence, the B-cell epitope of Omp25 is located at the C-terminus, and the remaining epitopes (Omp10, Omp16, Omp19, BP26, and Omp2b) are sequentially linked together using a GS linker. This embodiment of the invention evaluates this linking method using the online tools PROSOII (http: / / mips.helmholtz-muenchen.de / prosoII) and SOLpro (http: / / scratch.proteomics.ics.uci.edu). Compared with other linking methods, the linking method selected in this embodiment of the invention has the best hydrophilicity and maximum solubility.

[0052] In this embodiment of the invention, the hydrophilicity of the recombinant protein's amino acid sequence was analyzed using the NovoPro online tool. The results are as follows: Figure 5 As shown in the analysis curves, the hydrophobicity score of this protein is below 0 in most sequence regions, exhibiting strong hydrophilic properties overall. This property has multiple advantages: strong hydrophilicity helps the protein achieve soluble expression during expression, reduces inclusion body formation, and increases yield; the hydrophilic regions are mostly located on the protein surface, resulting in high antigen accessibility and facilitating antibody recognition, which is crucial for ensuring the sensitivity of the detection method; at the same time, this property also helps maintain the conformational stability of the protein, facilitating the long-term storage and application of diagnostic reagents.

[0053] To evaluate the potential of the multi-linked B-cell epitope recombinant protein designed in this invention as a diagnostic antigen, in vitro immune simulation was performed using the online tool C-IMMSIM (https: / / kraken.iac.rm.cnr.it / C-IMMSIM / index.php?page=1). The results are as follows: Figure 6 As shown, this recombinant protein can elicit a strong humoral immune response, manifested by a significant increase in specific IgG antibody levels and the sustained formation of immune complexes. This demonstrates that its contained B-cell epitopes have good immunogenicity and can effectively induce high levels of specific antibodies. These simulation results indicate that when used as an iELISA coating antigen, this protein possesses the ability to bind efficiently and specifically to anti-brucellosis antibodies in serum, providing a crucial basis for establishing a highly sensitive and specific detection method.

[0054] Furthermore, the introduction of the GS linker helps maintain the independence of the spatial conformation of each epitope, thereby ensuring that its immunogenicity is not interfered with. The final constructed multi-linked B-cell epitope recombinant protein for detecting anti-brucell antibodies has the amino acid sequence shown in SEQ ID NO:1, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO:2.

[0055] Example 2: This example provides a prokaryotic expression and purification method for a multi-linked B-cell epitope recombinant protein for detecting anti-brucell antibodies, as detailed below:

[0056] 1. Prokaryotic Expression and SDS-PAGE Analysis: The optimized target gene (as shown in SEQ ID NO:2) was ligated into the pET-32a expression vector to construct the recombinant expression plasmid pET-32a-multiplexed B-cell epitope recombinant protein; this recombinant expression plasmid was transformed into E. coli Rosetta (DE3) competent cells (host cells); positive clones were screened and verified by bacterial PCR and sequencing. Single colonies that were verified were selected for expansion culture, and the bacterial culture was cultured until the OD... 600nmWhen the protein concentration reached 0.6-1.0, IPTG was added to a final concentration of 0.5 mM, and the cells were induced at 16℃ and 120 rpm for 22 h, and at 37℃ and 180 rpm for 4 h, respectively. After induction, the bacterial cells were collected by centrifugation at 8000 rpm for 10 min. The bacterial cells were then sonicated for 40 min, and centrifuged again at 8000 rpm for 40 min. The supernatant and precipitate were collected separately for subsequent analysis. Recombinant bacteria without IPTG induction were included as a negative control. SDS-PAGE electrophoresis was used to analyze the expression of the recombinant protein in the samples.

[0057] SDS-PAGE results are as follows Figure 1 As shown in the figure, lane 5 exhibits a clear specific band at the expected molecular weight position, indicating successful expression of the recombinant protein. While lane 5 shows a clear band at the expected molecular weight position, lane 7 shows no corresponding band, indicating that the recombinant protein is mainly present in the supernatant. The results indicate that the target protein expression level is highest in the supernatant of the bacterial culture induced by IPTG at 37℃ (lane 5), and it exists primarily in a soluble form. Therefore, this condition was selected as the preferred method for subsequent purification. In summary, the multi-linked B-cell epitope recombinant protein for detecting anti-brucellosis antibodies was efficiently expressed in *E. coli*, and it exists primarily in a soluble form in the cytoplasmic supernatant.

[0058] 2. Purification and incubation activity identification of the recombinant multi-linked B-cell epitope protein for detecting anti-brucell antibodies: The supernatant containing the recombinant multi-linked B-cell epitope protein for detecting anti-brucell antibodies was filtered through a 0.45 µm filter membrane and purified using a Ni-NTA affinity chromatography column. Stepwise elution was performed using imidazole solutions of varying concentrations (40, 60, 80, 150, 200, 220, 250, 300 mmol / L), and the flow-through and eluted fractions at each concentration were collected. The purification efficiency of the recombinant protein was analyzed after SDS-PAGE electrophoresis and Coomassie brilliant blue staining.

[0059] The SDS-PAGE analysis results of the multi-B cell epitope recombinant protein during the purification process are as follows: Figure 2 As shown, the electrophoresis diagram clearly reflects the presence and elution characteristics of the target protein at different purification stages, providing a basis for optimizing the purification process. Specifically, lane 1 shows the target band at the expected molecular weight position, indicating good expression of the recombinant protein. The target protein content in lane 2 is significantly reduced compared to lane 3, indicating good adsorption of protein by the nickel column, with most of the target protein being adsorbed onto the column. From lane 6 onwards, the content of impurity proteins decreases significantly, while the target protein content remains relatively high. In summary, the eluent was collected starting from 60 mmol / L imidazole and concentrated in a dialysis bag. This electrophoresis diagram clearly reflects the presence and elution characteristics of the target protein at different purification stages, providing a basis for optimizing the purification process.

[0060] The concentrated recombinant protein was analyzed by SDS-PAGE electrophoresis, and the results are as follows: Figure 3 As shown, lane 1 shows a clear target band at the expected molecular weight position. The recombinant protein exhibits a single, clear band at the expected molecular weight, and the lane background is clean with no residual impurities, indicating that the protein has good purification effect, high purity and uniformity, and meets the requirements for subsequent immunoassay applications.

[0061] Western blot identification results of recombinant B-cell epitope proteins are as follows: Figure 4 As shown; lane 1 is the purified recombinant protein, which shows a clear specific reaction band at the expected molecular weight, indicating that the recombinant protein can be specifically recognized by brucellosis positive serum, proving that it has good immunoreactivity and antigen specificity.

[0062] Example 3: Optimization of iELISA reaction conditions, as detailed below:

[0063] 1. Determination of the optimal coating concentration and serum dilution of recombinant protein antigen: The coating concentration and serum dilution of the above-mentioned multi-linked B-cell epitope recombinant protein were optimized using a checkerboard titration method to determine the optimal reaction conditions for the indirect ELISA detection system. The specific procedures were as follows: The recombinant protein was serially diluted with carbonate buffer to obtain a coating concentration gradient (40, 20, 10, 5, 2.5, 1.25, 0.625, 0.3125 μg / mL). 100 μL was added to each well of the ELISA plate, and the plate was incubated overnight at 4°C. After washing three times with PBST (2 min each time), 200 μL of 5% BSA blocking buffer was added, and the plate was blocked at 37°C for 2 h. After blocking, wash in the same manner. Serially dilute Brucella positive and negative sera with PBS at 1:50, 1:100, 1:200, 1:400, 1:800, and 1:1600, respectively, and add them to the corresponding wells in a square matrix layout (100 μL / well). Incubate at 37°C for 1 h. After washing with PBST, add 1:5000 diluted HRP-labeled Protein G (original concentration 1 mg / mL, 100 μL / well) as the enzyme-labeled secondary antibody. Wash again, add 100 μL of TMB substrate chromogenic solution to each well, and react at room temperature in the dark for 15 min. Then add stop solution (50 μL / well) to terminate the reaction. Read the OD using a microplate reader. 450nm Absorbance value, by comparing the OD values ​​of positive and negative serum 450nm The optimal conditions are determined by the value and the P / N ratio. The criterion is: positive serum OD 450nm >1.0, P / N>2.1, and negative control OD 450nm The values ​​were low. Based on the results in Table 3, the optimal antigen coating concentration was finally determined to be 0.3125 μg / mL, and the optimal serum dilution factor was 1:100.

[0064] Table 3. Determination of optimal antigen coating concentration and optimal serum dilution (checkerboard method)

[0065]

[0066] 2. Determination of Optimal Antigen Coating Conditions: To determine the optimal antigen coating conditions, ELISA plates (100 μL / well) were coated with the optimized optimal antigen concentration (0.3125 μg / mL). Three coating protocols were set up for comparison: Group I: incubation at 37℃ for 2 h; Group II: incubation at 37℃ for 2 h followed by overnight incubation at 4℃; Group III: direct incubation at 4℃ overnight. Each group had 3 replicates. After coating, the plates were washed 3 times with PBST (2 min each time), patted dry, and 5% BSA blocking buffer (200 μL) was added to each well, with blocking at 37℃ for 2 h. After washing and patting dry again, negative and positive sera at the optimal dilution (1:100) (100 μL / well) were added, and incubation was carried out at 37℃ for 1 h. After washing, HRP-labeled Protein G diluted 1:5000 (100 μL / well) was added, and incubation was carried out at 37℃ for 1 h. Wash again, add 100 μL of TMB substrate chromogenic solution to each well, and incubate at room temperature in the dark for 15 min. Then add stop solution (50 μL / well). Read the OD value using a microplate reader within 15 min. 450nm Absorbance. OD values ​​of positive and negative serum. 450nm The maximum P / N ratio was used as the criterion to determine the optimal coating conditions. As shown in Table 4, Group I (incubated at 37°C for 2 hours) had the highest P / N value, and was therefore determined to be the optimal antigen coating condition.

[0067] Table 4 Determination of Optimal Antigen Coating Conditions

[0068]

[0069] 3. Determination of the optimal blocking solution: To determine the optimal blocking solution, the ELISA plate was coated with the optimal antigen concentration (100 μL / well) and incubated at 37°C for 2 h. The plate was washed three times with PBST (2 min each time) and then blotted dry. Five groups of blocking solutions were set up for screening: Group I: 5% skim milk powder blocking solution; Group II: 5% BSA blocking solution; Group III: 1% BSA blocking solution; Group IV: 1% gelatin blocking solution; and Group V: 1M ammonium chloride blocking solution, with three replicates for each group. 200 μL of the corresponding blocking solution was added to each well, and the plate was blocked at 37°C for 2 h. After washing and blotting dry, negative and positive sera at the optimal dilution (1:100) (100 μL / well) were added, and the plate was incubated at 37°C for 1 h. After washing, HRP-labeled Protein G diluted 1:5000 (100 μL / well) was added, and the plate was incubated at 37°C for 1 h. Wash again, add 100 μL of TMB substrate chromogenic solution to each well, and incubate at room temperature in the dark for 15 min. Then add stop solution (50 μL / well). Read the OD value using a microplate reader within 15 min. 450nm Absorbance. OD values ​​of positive and negative serum. 450nm The optimal sealing solution was determined by maximizing the ratio of the average values ​​(P / N value). As shown in Table 5, Group I (5% skim milk powder) had the highest P / N value and was therefore selected as the optimal sealing solution.

[0070] Table 5 Determination of the optimal sealing solution

[0071]

[0072] 4. Determination of Optimal Blocking Time: To determine the optimal blocking time, the ELISA plate was coated with the optimal antigen concentration (100 μL / well) and incubated at 37°C for 2 h. After washing three times with PBST (2 min each time), the plate was patted dry and then blocked with 5% skim milk blocking buffer. Four different blocking times were set up and optimized at 37°C: Group I 30 min, Group II 60 min, Group III 90 min, and Group IV 120 min, with three replicates for each group. After blocking, the plate was washed and patted dry, and the optimal dilution (1:100) of negative and positive serum (100 μL / well) was added, and the plate was incubated at 37°C for 1 h. After washing, HRP-labeled Protein G diluted 1:5000 (100 μL / well) was added, and the plate was incubated at 37°C for 1 h. After washing again, 100 μL of TMB substrate chromogenic solution was added to each well, and the plate was reacted at room temperature in the dark for 15 min before adding stop solution (50 μL / well). Read OD values ​​using an ELISA reader within 15 minutes. 450nm Absorbance. OD values ​​of positive and negative serum. 450nm The maximum P / N ratio was used as the criterion to determine the optimal sealing time. As shown in Table 6, Group II (sealed for 60 min) had the highest P / N value, and therefore was determined to be the optimal sealing time.

[0073] Table 6 Determination of Optimal Closure Time

[0074]

[0075] 5. Determination of Optimal Serum Incubation Time: To determine the optimal serum incubation time, the ELISA plate was coated with the optimal antigen concentration (100 μL / well) and incubated at 37°C for 2 h. After washing three times with PBST (2 min each time), the plate was patted dry and 5% skim milk blocking buffer was added. The plate was then blocked at 37°C for 60 min. After blocking, the plate was washed and patted dry. Optimal dilutions (1:100) of negative and positive serum (100 μL / well) were added, and four different incubation times were set up at 37°C for optimization: Group I 30 min, Group II 60 min, Group III 90 min, and Group IV 120 min, with three replicates per group. After incubation, the plate was washed and 1:5000 dilution of HRP-labeled Protein G (100 μL / well) was added. The plate was incubated at 37°C for 1 h. After washing again, 100 μL of TMB substrate chromogenic solution was added to each well, and the plate was incubated at room temperature in the dark for 15 min before adding stop solution (50 μL / well). Read OD values ​​using an ELISA reader within 15 minutes. 450nm Absorbance. OD values ​​of positive and negative serum. 450nm The maximum P / N ratio was used as the criterion to determine the optimal serum incubation time. As shown in Table 7, Group IV (incubation for 120 min) had the highest P / N value, and was therefore determined to be the optimal serum incubation time.

[0076] Table 7 Determination of Optimal Serum Incubation Time

[0077]

[0078] 6. Determination of the optimal dilution factor for enzyme-labeled secondary antibodies: To determine the optimal dilution factor for enzyme-labeled secondary antibodies, the ELISA plate was coated with the optimal antigen concentration (100 μL / well) and incubated at 37°C for 2 h. After washing three times with PBST (2 min each time), the plate was patted dry and 5% skim milk blocking buffer was added. The plate was then blocked at 37°C for 60 min. After blocking, the plate was washed and patted dry, and the optimal dilution (1:100) of negative and positive serum (100 μL / well) was added separately. The plate was incubated at 37°C for 120 min. After washing again, four dilution gradients of HRP-labeled Protein G were set up for screening: Group I 1:5000, Group II 1:10000, Group III 1:15000, and Group IV 1:20000. 100 μL was added to each well and the plate was incubated at 37°C for 1 h. Each group had 3 replicates. After washing, add 100 μL of TMB substrate chromogenic solution to each well and incubate at room temperature in the dark for 15 min. Then add stop solution (50 μL / well). Read the OD values ​​using a microplate reader within 15 min. 450nm Absorbance. OD values ​​of positive and negative serum. 450nmThe maximum P / N ratio was used as the criterion to determine the optimal dilution of the enzyme-labeled secondary antibody. As shown in Table 8, although group III (1:15000) had the highest P / N value, its positive serum OD... 450nm The value was below 1.0, which did not meet the detection sensitivity requirements, so it was not adopted. Among the other groups that met the conditions, Group I (1:5000) showed the highest P / N value, and was therefore determined to be the optimal dilution factor for the enzyme-labeled secondary antibody.

[0079] Table 8 Determination of the optimal dilution factor for enzyme-labeled secondary antibodies

[0080]

[0081] 7. Determination of Optimal Incubation Time for Enzyme-Labeled Secondary Antibody: To determine the optimal incubation time for enzyme-labeled secondary antibody, the plate was coated with the optimal antigen concentration (100 μL / well) and incubated at 37°C for 2 h. After washing three times with PBST (2 min each time), the plate was patted dry and 5% skim milk blocking buffer was added. The plate was then blocked at 37°C for 60 min. After blocking, the plate was washed and patted dry, and the optimal dilution (1:100) of negative and positive serum (100 μL / well) was added, respectively. The plate was then incubated at 37°C for 120 min. After washing, the optimal working concentration (1:5000) of HRP-labeled Protein G (100 μL / well) was added, and four incubation times were set at 37°C for optimization: Group I 30 min, Group II 60 min, Group III 90 min, and Group IV 120 min, with three replicates for each group. After incubation, wash the cells and add 100 μL of TMB substrate chromogenic solution to each well. Incubate at room temperature in the dark for 15 min, then add stop solution (50 μL / well). Read the OD values ​​using a microplate reader within 15 min. 450nm Absorbance. OD values ​​of positive and negative serum. 450nm The maximum P / N ratio was used as the criterion to determine the optimal incubation time for enzyme-labeled secondary antibodies. As shown in Table 9, Group II (incubation for 60 min) had the highest P / N value and was therefore determined to be the optimal incubation time for enzyme-labeled secondary antibodies.

[0082] Table 9 Determination of the optimal incubation time for enzyme-labeled secondary antibodies

[0083]

[0084] 8. Determination of the optimal color development time for the substrate chromogenic solution: Based on the optimized coating and blocking conditions, Brucella recombinant antigen was diluted to 0.3125 μg / mL with carbonate buffer, and 100 μL / well was used to coat the ELISA plate. The plate was incubated at 37°C for 2 h. After washing three times with PBST, 200 μL / well of 5% skim milk blocking buffer was added, and the plate was blocked at 37°C for 60 min. After washing again, 100 μL / well of Brucella positive and negative sera (1:100 dilution) were added, and the plate was incubated at 37°C for 120 min. After washing, 100 μL of HRP-labeled Protein G (1:5000 dilution) was added, and the plate was incubated at 37°C for 60 min. After washing, 100 μL of TMB substrate chromogenic solution was added to each well, and the reaction was carried out at room temperature in the dark. Four color development times (5, 10, 15, and 20 min) were set for comparison. After the reaction was complete, 50 μL of stop solution was added to each well, and the OD values ​​were read using a microplate reader at the corresponding time points. 450nm Absorbance. OD values ​​of positive and negative serum. 450nm The maximum P / N ratio is used as the judgment criterion, combined with the positive serum OD. 450nm The requirement of ≥1.0 was used to determine the optimal development time. As shown in Table 10, although the 5-minute group had the highest P / N value, the positive OD... 450nm The value was below 1.0, which did not meet the sensitivity requirements; among the other groups, the 15-minute group had the highest P / N value and was therefore determined to be the optimal color development time.

[0085] Table 10 Determination of the optimal color development time for the substrate and developing solution

[0086]

[0087] 9. Determination of positive and negative cutoff values: The iELISA test was performed under the optimal reaction conditions determined above. 44 anti-brucellosis negative sera were tested, with each serum sample tested three times. The average of the three tests was taken, and the results were analyzed based on OD values. 450nm The mean (X) of 44 anti-brucellosis negative sera was calculated to be 0.4232, and the standard deviation (SD) was 0.0993. Therefore, the cutoff point for positive and negative seroconversion was determined as C = X + 3SD = 0.7214, which is the OD value of the serum to be tested. 450nm A value ≥ 0.7214 is considered positive, and a value less than 0.7214 is considered negative (as shown in Table 11).

[0088] Table 11 Determination of Critical Values ​​for Negative and Positive Sex

[0089]

[0090] 10. Specificity test: Perform iELISA test according to the optimal reaction conditions determined above. Test three serum samples positive for deer tuberculosis (DTB), with each sample tested three times. Results are expressed as OD.450nm The average value was expressed and compared with the positive / negative cutoff value. Simultaneously, negative deer serum uninfected with Brucella and deer tuberculosis was used as a control, and the P / N value was calculated. Furthermore, under the same conditions, HRP-labeled goat anti-mouse secondary antibody was used to detect positive mouse serum against Brucella epididymis (as a positive control), as well as positive serum against enterotoxigenic Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii, Pseudomonas aeruginosa, Salmonella, and Yersinia enterocolitica, and their corresponding negative controls. Each sample was repeated three times, and the results were averaged. The OD values ​​of each positive serum were compared. 450nm Mean and P / N values. Table 12 shows that the OD values ​​of all tuberculosis-positive serum ferrous sulfate in deer... 450nm All values ​​were below 0.7214, and the P / N ratios were all less than 2.1, indicating a negative result. Table 13 shows that only the OD values ​​of the Brucella-positive control serum were negative. 450nm A mean value greater than 0.7214 and a P / N value higher than 2.1 are considered positive. Other non-target bacteria positive sera are also considered positive. 450nm All values ​​were less than 0.7214, and all P / N values ​​were less than 2.1, indicating a negative result. These results demonstrate that the iELISA method established in this embodiment of the invention has good specificity and shows no cross-reactivity with other common pathogens.

[0091] Table 12 Specificity Detection

[0092]

[0093] Table 13 Specificity Detection

[0094]

[0095] 11. Sensitivity Test: Following the optimal reaction conditions determined above, an iELISA test was performed. Brucellosis-positive control deer serum was serially diluted at 1:50, 1:100, 1:200, 1:400, 1:800, and 1:1600, with three replicate wells for each dilution. The established iELISA method was used for detection. As shown in Table 14, when the serum was diluted to 1:100, its OD... 450nm The mean value is still higher than 0.7214, and the P / N value is greater than 2.1, so the result is positive, indicating that the solution provided by the embodiment of the present invention has good detection sensitivity.

[0096] Table 14 Sensitivity Detection

[0097]

[0098] 12. Intra-assay and Inter-assay Repeatability Tests: To evaluate the repeatability of the iELISA method established in this embodiment, 24 deer serum samples were selected and iELISA tests were performed according to the optimal reaction conditions determined above. Intra-assay repeatability was assessed by setting up 3 replicate wells for each sample within the same batch and measuring OD. 450nm The values ​​were calculated, and the mean (X) and standard deviation (SD) were used for evaluation, with the coefficient of variation (CV = SD / X × 100%). Inter-batch repeatability was assessed by testing the same sample in different batches (a total of 3 batches), also based on OD. 450nm The CV values ​​of each sample were calculated between different batches. The results (as shown in Table 15) showed that the intra-batch CV range of the 24 serum samples was 0.65%-6.60%, and the inter-batch CV range was 4.04%-9.97%, all below 10%, indicating that the scheme provided by the embodiments of the present invention has good intra-batch and inter-batch repeatability and detection stability.

[0099] Table 15 Intra-batch and Inter-batch Repeatability Tests

[0100]

[0101] Example 4: Clinical sample testing and comparative analysis with the Rose Bengal plate agglutination test, as detailed below:

[0102] Following the optimal reaction conditions determined in Example 3 above, 475 deer serum samples were tested using the recombinant B-cell epitope protein and iELISA method provided in this embodiment of the invention, with the Rose Bengal plate agglutination assay used as a reference method for comparison. The results showed a high degree of consistency between the two methods: 448 out of 475 samples showed consistent results, resulting in an overall concordance rate of 94.32%.

[0103] The results indicate that the recombinant B-cell epitope protein and the established iELISA system provided in this embodiment of the invention have good accuracy and reliability in clinical serum sample detection, and can be effectively used for the detection of anti-brucellosis antibodies, providing an efficient serological tool for the clinical diagnosis of brucellosis.

[0104] It should be noted that, since the embodiments of this invention use HRP-labeled Protein G as a universal secondary antibody, which can widely bind to IgG Fc fragments of various mammals, the above detection method does not require changing the secondary antibody and can be directly applied to the detection of anti-brucellosis antibodies in serum samples of various animals other than deer (such as cattle and sheep), significantly improving the versatility and ease of operation of the method. Furthermore, milk samples, tissue homogenates, and organ samples can also be detected using the multi-linked B-cell epitope recombinant protein and iELISA system provided in these embodiments of the invention after appropriate processing.

[0105] In summary, the embodiments of the present invention utilize GS linker to tandemly fuse highly conserved and efficient B-cell epitopes selected from seven key outer membrane proteins of Brucella (Omp10, Omp16, Omp19, Omp25, Omp31, Omp2b, and BP26) to construct a structurally stable and antigenically excellent multi-linked B-cell epitope recombinant protein.

[0106] Furthermore, this invention establishes an iELISA detection system for deer serum to detect anti-brucellosis antibodies using the recombinant B-cell epitope protein as the coating antigen. This detection system demonstrated high sensitivity, strong specificity, and good reproducibility in evaluation, accurately and stably detecting anti-brucellosis antibody levels in serum, providing a reliable tool for early diagnosis and immune status assessment of brucellosis infection. Moreover, this invention uses HRP-labeled Protein G instead of traditional enzyme-labeled secondary antibodies, enabling the detection of anti-brucellosis antibodies in multiple animals. It supports large-scale screening of anti-brucellosis antibodies in serum from various animal species without requiring changes to the enzyme-labeled secondary antibody for different animal species, demonstrating significant application value in brucellosis epidemiological monitoring and integrated prevention and control.

[0107] Compared with existing Brucella multi-antigen epitope fusion recombinant proteins (such as the L7 / L12-PADRE sequence-multi-B cell epitope recombinant protein disclosed in CN120795181A), the multi-B cell epitope recombinant protein constructed in this embodiment of the invention has significant differences and unique advantages in antigen composition and epitope selection. This multi-B cell epitope recombinant protein is composed of seven highly conserved B cell epitopes from key Brucella outer membrane proteins: Omp10, Omp16, Omp19, Omp25, Omp2b, Omp31, and BP26, covering a wider range of immune-related antigen regions. Specifically, this multi-B cell epitope recombinant protein includes epitopes derived from Omp10, Omp16, Omp19, Omp25, and Omp2b, which are not present in existing multi-B cell epitope recombinant proteins, thus providing a more comprehensive antigenic spectrum. Furthermore, regarding Omp31 and BP26, the B-cell epitopes screened through bioinformatics prediction in this invention showed low sequence identity and structural overlap with the corresponding epitopes in existing multi-epitope recombinant proteins, indicating that they are significantly different in antigen characterization and target different immunodominant regions. Therefore, the multi-antigen epitope fusion recombinant protein prepared in this invention demonstrates innovative structural design and functional advantages in terms of epitope diversity, antigen coverage, and potential detection specificity, making it more conducive to the development of specific, sensitive, stable detection kits capable of detecting various animal anti-brucellosis antibodies.

[0108] Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the inventive concept. The technical scope of this invention is not limited to the contents of the specification.

Claims

1. A recombinant B-cell epitope protein for detecting anti-brucellosis antibodies, characterized in that, The recombinant B-cell epitope protein is composed of B-cell epitopes of seven key outer membrane proteins of Brucella, namely Omp10, Omp16, Omp19, Omp25, Omp31, Omp2b, and BP26, linked together, and its amino acid sequence is shown in SEQ ID NO:

1.

2. A gene encoding a multi-linked B-cell epitope recombinant protein for detecting anti-brucell antibodies as described in claim 1, characterized in that, The nucleotide sequence of the encoding gene is shown in the sequence listing SEQ ID NO:

2.

3. A recombinant expression plasmid comprising the encoding gene of the multi-linked B-cell epitope recombinant protein for detecting anti-brucell antibodies as described in claim 2.

4. A host cell comprising the encoding gene of the recombinant B-cell epitope protein for detecting anti-brucell antibodies as described in claim 2 or the recombinant expression plasmid as described in claim 3.

5. The use of the multi-linked B-cell epitope recombinant protein for detecting anti-brucell antibodies as described in claim 1 in the preparation of a kit for detecting anti-brucell antibodies.

6. The application according to claim 5, characterized in that, The method for detecting anti-brucellosis antibodies is as follows: using the recombinant B-cell epitope protein as the coating antigen, an indirect ELISA method is established to detect anti-brucellosis antibodies in deer serum.

7. A kit for detecting anti-brucellosis antibodies, characterized in that, The vector includes a multi-linked B-cell epitope recombinant protein for detecting anti-brucell antibodies as described in claim 1.

8. The kit for detecting anti-brucellosis antibodies according to claim 7, characterized in that, The carrier is an enzyme-labeled plate; the optimal coating concentration of the multi-linked B-cell epitope recombinant protein is 0.3125 μg / mL.

9. The kit for detecting anti-brucellosis antibodies according to claim 7 or 8, characterized in that, The kit also includes one or more of the following: blocking solution, washing solution, diluent, enzyme-labeled reagent, substrate chromogenic solution, stop solution, positive standard serum, and negative standard serum.

10. The kit for detecting anti-brucellosis antibodies according to claim 9, characterized in that, The blocking solution is skim milk powder blocking solution; the washing solution is PBST solution; the diluent is PBS solution; the enzyme labeling reagent is horseradish peroxidase-labeled streptococcal protein G; the substrate chromogenic solution is 3,3',5,5'-tetramethylbenzidine solution; the stop solution is 2M sulfuric acid solution or 1-2M sodium hydroxide solution; the positive standard serum is anti-brucellosis antibody positive serum; the negative standard serum is anti-brucellosis antibody negative serum.

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