BMEI1871 protein specific polypeptide for diagnosing brucellosis and detection kit containing polypeptide

Abstract

The invention provides a specific polypeptide of Brucella, the amino acid sequence of the specific polypeptide is as shown in SEQ ID No.1, and the specific polypeptide is derived from BMEI1871 protein of Brucella. The invention also provides a recombinant vector or an expression cassette for expressing the specific polypeptide, and a recombinant cell or a recombinant bacterium. The invention verifies the application of the specific polypeptide in preparation of drugs or reagents for preventing, detecting or diagnosing brucellosis, and also provides an ELISA (enzyme-linked immuno sorbent assay) detection kit for detecting brucellosis. According to the present invention, the specific polypeptide of the brucella BMEI1871 protein is adopted as the antigen to establish the ELISA method for detecting the brucella antibody, such that the accuracy is high, and the powerful support is provided for brucellosis purification. The antigen source is convenient, the stability is high, the repeatability is strong, and the defects of using whole bacteria or lipopolysaccharide as the antigen are overcome.

Description

[0001] This invention claims a domestic priority claim, with application number CN 2024113861479, application date September 30, 2024, entitled "A BMEI1871 protein-specific polypeptide for diagnosing brucellosis and a detection kit containing said polypeptide". Technical Field

[0002] This invention relates to the field of brucellosis detection technology, and particularly to a specific polypeptide derived from Brucella BMEI1871 protein, and the application of said polypeptide in an ELISA detection kit. Background Technology

[0003] Brucellosis is a zoonotic infectious disease caused by Brucella bacteria, widely distributed throughout the world. The most common method for quarantining brucellosis is antibody detection via ELISA. However, Brucella shares many similar antigens with common bacteria such as Yersinia, Paleobacterium, Rhizobium, and Escherichia coli, often resulting in false positives and severely interfering with the sensitivity of the test. To address this deficiency, Brucella lipopolysaccharide is now commonly used as an antigen for coating, achieving better results. For example, Chinese inventor patent application CN 106556701A discloses a Guiyang Brucella clipped ELISA antibody detection kit, which uses purified Brucella lipopolysaccharide (LPS) to coat a 96-well ELISA plate after quantification, and prepares positive serum by artificially immunizing healthy sheep to obtain the final kit. However, Brucella lipopolysaccharide or surface protein has cross-antigenic epitopes with some other bacterial genera (such as Aristobacterium, Escherichia coli and Yersinia), which reduces the specificity of detection. On the other hand, obtaining lipopolysaccharide requires Brucella culture and lipopolysaccharide extraction, which are complex processes with high production costs and are prone to safety risks.

[0004] Invasion-associated locus B (IalB) is a major virulence factor of Bartonella, which belongs to the order Rhizobium (similar to Brucella) and plays a crucial role in Bartonella's invasion of epithelial cells, dermal fibroblasts, erythrocytes, and endothelial cells. IalB polyclonal antibodies can inhibit Bartonella's invasion of erythrocytes. IalB knockout reduces Bartonella's ability to invade cells by approximately 50%, but this ability can be restored upon complementation.

[0005] Brucella BMEI1871 encodes a protein belonging to the IalB family. The inventors of this invention have found that in the antibody profile following infection with virulent Brucella, the level of BMEI1871 antibody is second only to Bp26, while only a very low level of BMEI1871 antibody can be induced in the attenuated strain A19ΔvirB12, showing a highly significant difference between the two.

[0006] However, there is currently no method for detecting brucellosis based on the Brucella BMEI1871 protein. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a new specific polypeptide, which is expected to be used for the detection of brucellosis.

[0008] Based on this, the present invention provides a specific polypeptide of Brucella, the amino acid sequence of which is shown in SEQ ID No.1.

[0009] In this invention, the specific polypeptide is derived from Brucella BMEI1871 protein.

[0010] The present invention also provides recombinant vectors or expression cassettes for expressing the above-mentioned specific polypeptides.

[0011] The present invention also provides recombinant cells or recombinant bacteria expressing the above-mentioned specific polypeptides.

[0012] The present invention also verifies the application of the specific polypeptide in the preparation of drugs or reagents for the prevention or diagnosis of brucellosis.

[0013] Specifically, the reagent is an ELISA kit or a colloidal gold kit.

[0014] Specifically, the present invention provides an ELISA detection kit for detecting brucellosis, the detection kit comprising a pre-coated plate, an enzyme-labeled antibody, a standard, a buffer, an antibody diluent, a writing agent, and a stop solution, wherein the pre-coated plate is coated with the specific polypeptide described in claim 1.

[0015] Through optimization experiments, this invention determined that the final concentration of the pre-coated plate using 100 μL of pH 9.6 solution is 1 μg·ml. -1 The sample was coated with the specific polypeptide solution of claim 1, and then blocked with 200 μl of PBST containing 1% bovine serum albumin. Subsequently, it was incubated with 100 μl of 100-fold diluted standard positive and negative serum and 100 μl of 5000-fold diluted recombinant protein G labeled with horseradish peroxidase.

[0016] Preferably, the standard positive and negative serum in the kit is diluted 100 times.

[0017] When preparing the pre-coated plate, the coating time was overnight at 4°C, and then it was blocked with 0.5% BSA for 1 hour.

[0018] Preferably, the incubation time for both serum and secondary antibody is 30 minutes.

[0019] Preferably, the serum and secondary antibody are diluted with 0.5% BSA.

[0020] Preferably, the incubation is followed by washing with 300 μl TBST solution after each incubation step, and the number of washes per step is 5.

[0021] Preferably, the colorimetric solution is 100 μl of TMB solution, and the reaction is carried out in the dark for 15 min. Then, 50 μl of 10% sulfuric acid solution is added to terminate the reaction.

[0022] Preferably, the threshold for determining the result is 0.3. A result is considered positive when the OD value is greater than or equal to 0.3, and negative otherwise.

[0023] This invention utilizes a specific polypeptide of Brucella BMEI1871 protein as an antigen to establish an ELISA method for detecting Brucella antibodies. This method is highly accurate and provides strong support for brucellosis eradication. The antigen is readily available, highly stable, and reproducible, overcoming the shortcomings of using whole bacteria or lipopolysaccharide as antigens. Attached Figure Description

[0024] Figure 1 The results of PredictProtein analysis of BMEI1871 protein in Example 1;

[0025] Figure 2 The linear epitope of the BMEI1871 protein was obtained from Bepipred 2.0 analysis in Example 1;

[0026] Figure 3 The results of the reactivity analysis of the peptide in Example 1;

[0027] Figure 4 Results of a BMEI1871 protein-specific peptide reaction;

[0028] Figure 5 The ROC curves for ELISA and positive / negative sera in Example 3 are shown. Detailed Implementation

[0029] The following examples are used to explain the technical solutions of the present invention in a non-limiting manner.

[0030] In this invention, unless otherwise specified, “%” for concentration refers to mass percentage and “:” refers to mass ratio.

[0031] Example 1: Analysis and Screening of BMEI1871 Protein-Specific Peptides

[0032] The Brucella BMEI1871 protein sequence was downloaded from NCBI. First, PredictProtein was used to predict and analyze the protein's secondary structure, solvent accessibility, transmembrane regions, coiled-coil regions, disordered regions, binding regions to proteins, DNA, and RNA, subcellular localization, disulfide bonds, and sequence conserved regions. Figure 1 .

[0033] from Figure 1 It is known that amino acids 1-63 of the Brucella BMEI1871 protein are located in the intracellular or transmembrane region, making them difficult to use as antigenic sites. Therefore, in the subsequent antigenic epitope analysis, the epitope in the extracellular, non-transmembrane region 64-245AA was selected as the antigen.

[0034] Then, on the IDEB website, select Bepipred 2.0 to perform antigenic epitope analysis and obtain the antigenic epitopes. Figure 2 See Table 1.

[0035] Table 1 shows the linear epitopes of the BMEI1871 protein obtained by Bepipred 2.0 analysis.

[0036]

[0037] Simultaneously, the PDB file of the BMEI1871 protein was downloaded, and linear and conformational epitopes were predicted using the Ellipro program on the IDEB website, resulting in Table 2 (linear epitopes) and Table 3 (conformational epitopes) for the BMEI1871 protein. Comparative analysis showed that peptide MRKNKQPPAPPSVYTQTPGFAVKSTEIMVP, number 3 in Table 1, covered both linear and conformational epitopes predicted by the two methods and had a high antigen score; therefore, it was selected as the target peptide.

[0038] Table 2. Linear epitopes of BMEI1871 protein obtained by Ellipro analysis.

[0039]

[0040] Table 3. Conformational epitopes of BMEI1871 protein analyzed by Ellipro

[0041]

[0042]

[0043] BLAST analysis of the peptide MRKNKQPPAPPSVYTQTPGFAVKSTEIMVP confirmed its good specificity, high conservation among various Brucella species, and significant differences from other bacteria. Figure 3Therefore, it was chosen as the antigen and sent to Sangon Biotech (Shanghai) Co., Ltd. for biosynthesis.

[0044] Example 2 Identification of BMEI1871 protein-specific peptide reactivity

[0045] The polypeptide synthesized in Example 1 was diluted to a polypeptide solution of 10 μg / ml. 2 μL of the solution was spotted onto a nitrocellulose (NC) membrane, dried, and then blocked in PBS (pH 7.4) containing 1% (w / v) BSA at 37°C for 1 hour. After blocking, the membrane was washed and then incubated at 37°C for 0.5 hours with standard positive and negative sera diluted 1:2000 and HRP-labeled recombinant protein G diluted 1:5000, respectively. After each incubation, the membrane was washed three times with PBST. Finally, the membrane was incubated with the substrate solution for color development, and the reaction was terminated when spots appeared.

[0046] The results are as follows Figure 4 Brucella-positive serum showed obvious spots (A), while negative serum did not (B).

[0047] Example 3: Establishment of a polypeptide ELISA antibody detection method

[0048] Determination of the optimal coating concentration

[0049] The peptides from Example 1 were diluted with carbonate buffer at pH 9.6 to a final concentration of 1 μg / ml. -1 0.1 μg·ml -1 0.05 μg·ml -1 Add 100 μL of the solution to the microplate and incubate at 37°C for 1 hour for coating. Then add 200 μL of PBST containing 1% bovine serum albumin and block at 37°C for 2 hours. Subsequently, add 100 μL of 50-fold diluted standard positive and negative sera, and 100 μL of 5000-fold diluted recombinant protein G labeled with horseradish peroxidase, and incubate at 37°C for 30 minutes. After each incubation, wash the plate 6 times with PBST. Finally, add 100 μL of TMB solution and react in the dark for 15 minutes. Stop the reaction by adding 50 μL of 10% sulfuric acid solution, and read the OD using a microplate reader. 450 Value. Compare the OD values ​​(P / N) of positive and negative serum, and select the value with the highest P / N for 1 μg / ml. -1 The peptide concentration will be used as the antigen coating concentration for subsequent experiments.

[0050] Table 4 Determination of Optimal Antigen Coating Concentration

[0051]

[0052]

[0053] Determination of the optimal sealing fluid

[0054] 1 μg / ml diluted with carbonate buffer at pH 9.6 -1 100 μL of peptide solution was coated onto an ELISA plate at 37°C for 1 hour. Then, 200 μL of PBST containing 5% horse serum, 1% ovalbumin, 1% bovine serum albumin, and 5% skim milk powder were added, and the plate was blocked at 37°C for 2 hours. Subsequently, 100 μL of 50-fold diluted standard positive and negative sera, and 100 μL of 5000-fold diluted recombinant protein G labeled with horseradish peroxidase were added sequentially, and the plates were incubated at 37°C for 30 minutes. After each incubation, the plate was washed 6 times with PBST. Finally, 100 μL of TMB solution was added, and the reaction was stopped by adding 50 μL of 10% sulfuric acid solution. The OD450 value was then read using an ELISA reader. The OD values ​​(P / N) of positive and negative sera were compared, and the PBST containing 1% bovine serum albumin with the highest P / N value was selected as the blocking solution for subsequent experiments.

[0055] Table 5 Determination of the Optimal Sealing Fluid

[0056]

[0057] Determination of the optimal serum dilution factor

[0058] 100 μL of a 1 μg·ml⁻¹ polypeptide solution diluted with pH 9.6 carbonate buffer was used to coat the microplate at 37°C for 1 hour. Then, PBST containing 1% bovine serum albumin was added, and the plate was blocked at 37°C for 2 hours. Subsequently, 100 μL of standard positive and negative sera diluted 50, 100, and 200 times with blocking buffer, and 100 μL of recombinant protein G labeled with horseradish peroxidase diluted 5000 times were added, and the plates were incubated at 37°C for 30 minutes. After each incubation, the plate was washed 6 times with PBST. Finally, 100 μL of TMB solution was added, and the reaction was stopped by adding 50 μL of 10% sulfuric acid solution. The OD450 value was then read using a microplate reader. The OD values ​​(P / N) of positive and negative sera were compared, and the 100-fold serum dilution with the highest P / N value was selected as the blocking buffer for subsequent experiments.

[0059] Table 6 Determination of the optimal serum dilution factor

[0060]

[0061] Determination of the optimal enzyme marker dilution factor

[0062] 100 μL of a 1 μg·ml⁻¹ polypeptide solution diluted with pH 9.6 carbonate buffer was used to coat an ELISA plate at 37°C for 1 hour. Then, PBST containing 1% bovine serum albumin was added, and the plate was blocked at 37°C for 2 hours. Subsequently, 100 μL of standard positive and negative sera diluted 100-fold with blocking buffer, and 100 μL of recombinant protein G labeled with horseradish peroxidase diluted 500, 1000, 2000, and 5000-fold were added, and the plates were incubated at 37°C for 30 minutes. After each incubation, the plate was washed 6 times with PBST. Finally, 100 μL of TMB solution was added, and the reaction was stopped by adding 50 μL of 10% sulfuric acid solution. The OD450 value was then read using an ELISA reader. The OD values ​​(P / N) of positive and negative sera were compared, and the 5000-fold dilution of recombinant protein G with the highest P / N value was selected as the blocking buffer for subsequent experiments.

[0063] Table 7 Determination of the optimal dilution factor for enzyme markers

[0064]

[0065] Determination of critical values

[0066] The established ELISA method was used to measure multiple standard positive and negative serum samples, and the results are shown in Table 8.

[0067] Table 8. OD450 values ​​of standard serum

[0068]

[0069]

[0070] The OD450 values ​​of the detection results were used to plot ROC curves using Primer 9 software to determine the critical value, specificity, and sensitivity of the method. Figure 5 As shown.

[0071] The cutoff value was 0.3. An OD450 greater than or equal to 0.3 was considered positive, indicating that the animal had produced antibodies. An OD450 less than 0.3 was considered negative. At this cutoff value, the sensitivity was 93.75% and the specificity was 93.33%. The OD value of negative serum was significantly lower than that of positive serum, and the difference was statistically significant.

[0072] Table 9 Comparison of OD values ​​of positive and negative ELISA serum

[0073]

[0074] Example 4: Preparation and Validation of a Peptide ELISA Antibody Detection Kit

[0075] Preparation of peptide ELISA antibody kit

[0076] The peptide from Example 1 was diluted with carbonate buffer at pH 9.6 to a final concentration of 1 μg / ml. -1 Add 0.05% Proclin 300, take 100μL and add it to the microplate, and place it in a 37℃ incubator for 1 hour for coating.

[0077] Then add 200 μl of PBST containing 1% bovine serum albumin and 0.05% Proclin 300, block at 37°C for 2 hours, air dry, seal, and store at 4°C.

[0078] A peptide ELISA antibody kit was prepared by adding 20% ​​glycerol and 0.05% Proclin 300 to positive and negative standard sera, and adding 0.05% Proclin 300 to other reagents. The kit was then subjected to consistency, repeatability, and specificity tests.

[0079] Consistency test

[0080] Forty-six unknown bovine serum samples were simultaneously tested using both the Brucella antibody ELISA kit sold by IDEXX and the polypeptide ELISA kit established in this invention. The concordance between the two kits was then compared. Results showed that the IDEXX ELISA kit detected 22 positive cases, while the Brucella antibody ELISA kit established in this invention detected 23 positive cases. Only one sample showed a discrepancy between the two kits, resulting in an overall concordance rate of 97.8%.

[0081] Table 10 Consistency Test

[0082]

[0083] Repeatability test

[0084] The polypeptide ELISA of this invention was tested using 12 bovine serum samples and standard positive serum as intra- and inter-assay assays. The intra- and inter-assay coefficients of variation were calculated for the three results to evaluate the stability of the method. The results are shown in Tables 11-12.

[0085] Table 11 Intra-batch Tests

[0086]

[0087] Table 12 Inter-batch Trial

[0088]

[0089]

[0090] The results showed that the OD values ​​of the 13 samples in the intra-batch and inter-batch tests were very close, and the intra-batch and inter-batch coefficients of variation were both less than 0.1, indicating that the kit of the present invention has very good stability.

[0091] Specificity test

[0092] The polypeptide ELISA of this invention was used to detect nine positive sera infected with bovine infectious rhinotracheitis, bovine foot-and-mouth disease, bovine viral diarrhea, bovine mycoplasmal pneumonia, bovine pasteurellosis, bovine salmonellosis, bovine colibacillosis, bovine Yersinia, bovine leukobacterium and Brucella, and to determine the specificity of the detection method. The results are shown in Table 13.

[0093] Table 13 Specificity Tests

[0094]

[0095]

[0096] The results showed that, except for Brucella-positive serum, the OD 450nm values ​​of other sera were all below the critical value, and were judged as negative. This demonstrates that the polypeptide ELISA detection method does not react with antibodies against common bovine diseases and similar pathogens, exhibiting good specificity (Table 12).

[0097] Example 5: Detection of Brucella Immunization Effect in Cattle Farms

[0098] Blood samples were collected from 97 cattle at a cattle farm that had been immunized with Brucella A19 vaccine for two months. Serum was obtained by centrifugation and tested using the kit in Example 4. The OD450 values ​​of the 97 samples are shown in Table 14. The values ​​of 96 samples were greater than 0.3, indicating a positive result. Only sample No. 55 was negative, indicating a good immunization effect.

[0099] Table 14 Antibody Detection in Immunized Cattle Farms

[0100] serial number OD450 serial number OD450 serial number OD450 serial number OD450 serial number OD450 1 0.420 21 0.535 41 0.389 61 0.894 81 0.557 2 0.481 22 0.578 42 0.412 62 0.305 82 0.721 3 0.327 23 0.444 43 0.376 63 0.398 83 0.432 4 0.599 24 0.389 44 0.333 64 0.418 84 0.546 5 0.545 25 0.497 45 0.311 65 0.559 85 0.331 6 0.416 26 0.488 46 0.359 66 0.514 86 0.580 7 0.407 27 0.315 47 0.537 67 0.845 87 0.458 8 0.401 28 0.612 48 0.403 68 0.373 88 0.319 9 0.345 29 0.487 49 0.649 69 0.533 89 0.345 10 0.689 30 0.501 50 0.465 70 0.854 90 0.411 11 0.467 31 0.355 51 0.392 71 0.603 91 0.366 12 0.528 32 0.383 52 0.426 72 0.394 92 0.343 13 0.350 33 0.502 53 0.370 73 0.455 93 0.521 14 0.422 34 0.463 54 0.394 74 0.342 94 0.419 15 0.521 35 0.376 55 0.228 75 0.397 95 0.573 16 0.403 36 0.357 56 0.498 76 0.487 96 0.434 17 0.512 37 0.448 57 0.621 77 0.325 97 0.574 18 0.514 38 0.572 58 0.381 78 0.502 19 0.472 39 0.590 59 0.467 79 0.485 20 0.334 40 0.579 60 0.449 80 0.601

[0101] Example 6: Detection of Brucella infection in cattle farms

[0102] Sixty-eight blood samples were collected from a cattle farm that had not been vaccinated against Brucella. Serum was obtained by centrifugation and tested using the kit described in Example 4. The OD450 values ​​of all samples are shown in Table 15. No sample had a value exceeding 0.3, and all were negative, indicating that the cattle on the farm were not infected with Brucella.

[0103] Table 15 Antibody Detection in Non-Vaccinated Cattle Farms

[0104] serial number OD450 serial number OD450 serial number OD450 serial number OD450 serial number OD450 1 0.209 15 0.289 29 0.254 43 0.221 57 0.161 2 0.163 16 0.199 30 0.167 44 0.135 58 0.266 3 0.201 17 0.255 31 0.176 45 0.142 59 0.185 4 0.159 18 0.184 32 0.133 46 0.165 60 0.215 5 0.214 19 0.263 33 0.241 47 0.288 61 0.139 6 0.182 20 0.149 34 0.171 48 0.122 62 0.233 7 0.238 21 0.249 35 0.203 49 0.274 63 0.157 8 0.117 22 0.191 36 0.152 50 0.179 64 0.264 9 0.143 23 0.098 37 0.187 51 0.156 65 0.141 10 0.276 24 0.245 38 0.212 52 0.268 66 0.175 11 0.118 25 0.212 39 0.229 53 0.281 67 0.244 12 0.231 26 0.272 40 0.208 54 0.293 68 0.253 13 0.127 27 0.195 41 0.154 55 0.172 14 0.257 28 0.206 42 0.225 56 0.279

[0105] Therefore, the detection method established by this invention has high accuracy, providing strong support for the detection and eradication of brucellosis.

Claims

1. A Brucella-specific polypeptide, the amino acid sequence of which is shown in SEQ ID No.

1.

2. The specific polypeptide according to claim 1, characterized in that... The specific polypeptide is derived from Brucella BMEI1871 protein.

3. A recombinant vector or expression cassette for expressing the specific polypeptide of claim 1.

4. Recombinant cells expressing the specific polypeptide of claim 1.

5. The use of the specific polypeptide of claim 1 in the preparation of reagents for detecting brucellosis.

6. The application according to claim 5, characterized in that... The specific polypeptide is used to prepare ELISA detection kits or colloidal gold detection kits.

7. An ELISA detection kit for detecting brucellosis, the ELISA detection kit comprising a pre-coated plate, enzyme-labeled antibody, standards, buffer solution, antibody dilution solution, chromogenic agent, and stop solution, characterized in that... The pre-coated plate is coated with the specific polypeptide of claim 1.

8. The detection kit according to claim 7, characterized in that... The pre-coated plate was prepared with 100 μL of pH 9.6 solution to a final concentration of 1 μg·ml. -1 The sample was coated with the specific polypeptide solution of claim 1, and then blocked with 200 μl of PBST containing 1% bovine serum albumin. Subsequently, it was incubated with 100 μl of 100-fold diluted standard positive and negative serum and 100 μl of 5000-fold diluted recombinant protein G labeled with horseradish peroxidase.

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