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

Technical Field

[0001] The invention relates to the technical field of brucellosis detection, in particular to a specific polypeptide derived from Brucella BMEI1871 protein, and also to application of the polypeptide in an ELISA detection kit. Background Art

[0002] Brucellosis is a zoonotic infectious disease caused by Brucella, which is widely distributed all over the world. The most commonly used method for quarantine of brucellosis is to detect antibodies by ELISA method, but Brucella and common Yersinia, Ochrobacterium, Rhizobium, Escherichia coli have many similar antigens, and false positives often occur during the detection process, which seriously interferes with the sensitivity of the detection. In order to solve this defect, Brucella lipopolysaccharide is now commonly used as an antigen for coating, which can obtain better results. For example, patent application CN 106556701A discloses a Guiyang Brucella clip ELISA antibody detection kit, which is coated with 96-well enzyme label plates after quantitative purification of Brucella lipopolysaccharide (LPS), and positive serum is prepared by artificial immune healthy sheep, and finally a kit is obtained. However, Brucella lipopolysaccharide or surface protein has cross-antigenic epitopes with certain other genera of bacteria (such as Ochrobacterium, Escherichia coli and Yersinia), which reduces the specificity of detection. On the other hand, obtaining lipopolysaccharide requires the cultivation of Brucella and the extraction of lipopolysaccharide, which is a complicated process with high production costs and is prone to safety risks.

[0003] Invasion associated locus B (IalB) is the main virulence factor of Bartonella, which belongs to the order Rhizobiales together with Brucella, and plays a key role in the invasion of Bartonella into epithelial cells, dermal fibroblasts, erythrocytes and endothelial cells. IalB polyclonal antibody can inhibit the invasion of Bartonella into erythrocytes. After IalB knockout, the ability of Bartonella to invade cells is reduced by about 50%, which can be restored after complementation.

[0004] The protein encoded by Brucella BMEI1871 belongs to the IalB family. The inventors of the present invention have found through research that in the antibody spectrum after infection with virulent Brucella, the level of BMEI1871 antibodies is second only to Bp26, while in the weak strain A19ΔvirB12, only a very low level of BMEI1871 antibodies can be induced, and the difference between the two is extremely significant.

[0005] However, there is no method for detecting brucellosis based on Brucella BMEI1871 protein in the prior art. Summary of the invention

[0006] The purpose of the present invention is to overcome the existing defects and provide a new specific polypeptide, which is expected to be used for detecting brucellosis.

[0007] Based on this, the present invention provides a Brucella-specific polypeptide, the amino acid sequence of the specific polypeptide is shown in SEQ ID No.1.

[0008] In the present invention, the specific polypeptide is derived from Brucella BMEI1871 protein.

[0009] The present invention also provides a recombinant vector or expression box for expressing the above-mentioned specific polypeptide.

[0010] The present invention also provides a recombinant cell or a recombinant bacterium expressing the above-mentioned specific polypeptide.

[0011] The present invention also verifies the application of the specific polypeptide in preparing medicines or reagents for preventing or diagnosing brucellosis.

[0012] Particularly, the reagent is an ELISA detection kit or a colloidal gold detection kit.

[0013] Specifically, the present invention provides an ELISA detection kit for detecting brucellosis, which comprises a pre-coated plate, an enzyme-labeled antibody, a standard, a buffer, an antibody diluent, a color writer and a stop solution, wherein the pre-coated plate is coated with the specific polypeptide.

[0014] Through optimization experiments, the present invention determines that the final concentration of the pre-coated plate with 100 μL pH 9.6 is 1 μg ml -1 The specific polypeptide solution was coated, and then 200 μl of PBST containing 1% bovine serum albumin was added for blocking, followed by incubation 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.

[0015] Preferably, the dilution factor of the standard positive and negative serum in the kit is 100 times.

[0016] When preparing pre-coated plates, the coating time was overnight at 4°C, followed by blocking with 0.5% BSA for 1 hour.

[0017] Preferably, the incubation time of serum and secondary antibody is 30 minutes.

[0018] Preferably, the diluent for serum and secondary antibody is 0.5% BSA.

[0019] Preferably, each step of incubation is followed by washing with 300 μl of TBST solution, and each step is washed 5 times.

[0020] Preferably, the color developing solution is 100 μl of TMB solution, and the color is developed for 15 min in the dark, and then 50 μl of 10% sulfuric acid solution is added to terminate the reaction.

[0021] Preferably, the judgment threshold of the result is 0.3. When the OD value is greater than or equal to 0.3, it is judged as positive, otherwise it is negative.

[0022] The present invention uses the specific polypeptide of Brucella BMEI1871 protein as an antigen to establish an ELISA method for detecting Brucella antibodies, which has high accuracy and provides strong support for brucellosis purification. The antigen source is convenient, stable, and reproducible, overcoming the shortcomings of whole bacteria or lipopolysaccharide as antigens. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The PredictProtein analysis results of the BMEI1871 protein in Example 1;

[0024] Figure 2 The linear epitope of BMEI1871 protein obtained by Bepipred 2.0 analysis in Example 1;

[0025] Figure 3 The results of the reactivity analysis of the polypeptide in Example 1;

[0026] Figure 4 This is the ROC curve of ELISA and positive and negative serum in Example 3. DETAILED DESCRIPTION

[0027] The following examples are used to explain the technical solutions of the present invention in a non-limiting manner.

[0028] In the present invention, unless otherwise specified, "%" used to describe concentration refers to mass percentage, and ":" refers to mass ratio.

[0029] Example 1 Analysis and screening of BMEI1871 protein-specific polypeptides

[0030] The protein sequence of Brucella BMEI1871 was downloaded from NCBI. The secondary structure, solvent accessibility, transmembrane region, coiled-coil region, disordered region, binding region with protein, DNA and RNA, subcellular localization, disulfide bond and sequence conservation region of the protein were first predicted and analyzed by PredictProtein. Figure 1 .

[0031] from Figure 1 It can be seen that the amino acids 1-63 of the Brucella BMEI1871 protein are located in the intracellular or transmembrane region, which is difficult to serve as an antigenic site. Therefore, in the subsequent antigen epitope analysis, the epitope in the extracellular, non-transmembrane 64-245AA region was selected as an antigen.

[0032] Then select Bepipred 2.0 on the IDEB website for epitope analysis to obtain the epitope Figure 2 and Table 1.

[0033] Table 1 Linear epitopes of BMEI1871 protein obtained by Bepipred 2.0 analysis

[0034]

[0035]

[0036] At the same time, the PDB file of BMEI1871 protein was downloaded, and linear epitopes and conformational epitopes were predicted in the Ellipro program on the IDEB website, and the linear epitope table 2 and conformational epitope table 3 of BMEI1871 protein were obtained. Through comparative analysis, it can be seen that the peptide No. 3 in Table 1, MRKNKQPPAPPSVYTQTPGFAVKSTEIMVP, covers the linear epitopes and conformational epitopes of the two prediction methods, and has a high antigen score, so it is selected as the target peptide.

[0037] Table 2 Linear epitopes of BMEI1871 protein obtained by Ellipro analysis

[0038]

[0039] Table 3 Conformational epitopes of BMEI1871 protein analyzed by Ellipro

[0040]

[0041] BLAST analysis of the polypeptide MRKNKQPPAPPSVYTQTPGFAVKSTEIMVP confirmed that it has good specificity, is highly conserved among various Brucella species, and has obvious differences from other bacteria. Therefore, it was selected as an antigen and sent to Shanghai Biotech Co., Ltd. for biosynthesis.

[0042] Example 2 Identification of BMEI1871 protein-specific polypeptide reactivity

[0043] The polypeptide synthesized in Example 1 was diluted to a 10 μg / ml polypeptide solution, 2 μL was spotted on a nitrocellulose (NC) membrane, and after drying, it was placed in PBS (pH 7.4) containing 1% (W / V) BSA and blocked at 37°C for 1 hour. After blocking, it was washed and then incubated with a 1:2000 dilution of standard positive and negative serum and a 1:5000 HRP-labeled recombinant protein G at 37°C for 0.5 hours, respectively. After each incubation step, it was rinsed 3 times with PBST, and finally incubated with a substrate solution for color development, and the reaction was terminated when the spots appeared.

[0044] The results are as follows Figure 3 , Brucella-positive serum showed obvious spots (A), while Brucella-negative serum did not (B).

[0045] Example 3 Establishment of polypeptide ELISA antibody detection method

[0046] Determination of the optimal coating concentration

[0047] The polypeptide of Example 1 was diluted with pH 9.6 carbonate buffer to a final concentration of 1 μg ml -1 , 0.1μg·ml -1 , 0.05μg·ml -1 , take 100μL and add it to the ELISA plate, place it in a 37℃ incubator for 1 hour for coating. Then add 200μl PBST containing 1% bovine serum albumin and block at 37℃ for 2 hours. Then add 100μL of 50-fold diluted standard positive and negative serum and 100μl of 5000-fold diluted recombinant protein G labeled with horseradish peroxidase and incubate at 37℃ for 30 minutes. Wash the plate 6 times with PBST after each incubation step, and finally react with 100μl TMB solution in the dark for 15 minutes. After adding 50μl 10% sulfuric acid solution to terminate the reaction, read the OD with an ELISA reader. 450 Compare the OD value of the positive serum with the OD value of the negative serum (P / N), and select the 1μg·ml with the largest P / N value. -1 The peptide concentration was used as the antigen coating concentration in subsequent experiments.

[0048] Table 4 Determination of the optimal antigen coating concentration

[0049]

[0050] Determination of the optimal blocking solution

[0051] 1 μg ml diluted in carbonate buffer, pH 9.6 -1100 μL of peptide solution was coated on the ELISA plate at 37°C for 1 hour, and then 200 μL of PBST containing 5% horse serum, 1% ovalbumin, 1% bovine serum albumin, and 5% skimmed milk powder was added to block at 37°C for 2 hours. Subsequently, 100 μL of 50-fold diluted standard positive and negative serum and 100 μL of 5000-fold diluted recombinant protein G labeled with horseradish peroxidase were added and incubated at 37°C for 30 minutes. After each incubation step, the plate was washed 6 times with PBST, and finally 100 μL of TMB solution was reacted in the dark for 15 minutes. After adding 50 μL of 10% sulfuric acid solution to terminate the reaction, the OD450 value was read with an ELISA reader. Compare the OD value of the positive serum with the OD value of the negative serum (P / N), and select the PBST containing 1% bovine serum albumin with the largest P / N value as the blocking solution for the subsequent test.

[0052] Table 5 Determination of the optimal blocking solution

[0053]

[0054] Determination of the optimal serum dilution multiple

[0055] 100 μL of 1 μg·ml-1 peptide solution diluted with pH 9.6 carbonate buffer was used to coat the ELISA plate for 1 hour at 37°C, and then PBST containing 1% bovine serum albumin was added to block for 2 hours at 37°C. Subsequently, 100 μL of standard positive and negative serum diluted 50, 100, and 200 times with blocking solution and 100 μL of recombinant protein G labeled with horseradish peroxidase diluted 5000 times were added and incubated at 37°C for 30 minutes. After each incubation step, the plate was washed 6 times with PBST, and finally 100 μL of TMB solution was reacted in the dark for 15 minutes. After adding 50 μL of 10% sulfuric acid solution to terminate the reaction, the OD450 value was read with an ELISA reader. The OD value of the positive serum was compared with the OD value of the negative serum (P / N), and the 100-fold serum dilution with the largest P / N value was selected as the blocking solution for the subsequent test.

[0056] Table 6 Determination of the optimal dilution multiple of serum

[0057]

[0058] Determination of the Optimal Dilution Multiple of Enzyme Markers

[0059] 100 μL of 1 μg·ml-1 peptide solution diluted with pH 9.6 carbonate buffer was used to coat the ELISA plate for 1 hour at 37°C, and then PBST containing 1% bovine serum albumin was added to block for 2 hours at 37°C. Subsequently, 100 μL of standard positive and negative serum diluted 100 times with blocking solution and 100 μL of recombinant protein G labeled with horseradish peroxidase diluted 500, 1000, 2000, and 5000 times were added and incubated at 37°C for 30 minutes. After each incubation step, the plate was washed 6 times with PBST, and finally 100 μL of TMB solution was reacted in the dark for 15 minutes. After adding 50 μL of 10% sulfuric acid solution to terminate the reaction, the OD450 value was read with an ELISA reader. The OD value of the positive serum was compared with the OD value of the negative serum (P / N), and the 5000-fold diluted ELISA recombinant protein G with the largest P / N value was selected as the blocking solution for the subsequent test.

[0060] Table 7 Determination of the optimal enzyme marker dilution multiple

[0061]

[0062] Determination of critical value

[0063] The established ELISA method was used to test 58 standard positive and negative sera. The OD450 values ​​of the test results were plotted using Primer 9 software to draw ROC curves to determine the critical value, specificity, and sensitivity of the method. Figure 4 shown.

[0064] The critical value of the result is 0.3, and OD450 greater than or equal to 0.3 is positive, indicating that the test animal has produced antibodies. OD450 less than 0.3 is negative. At this critical value, the sensitivity is 100% and the specificity is 97.78%. The OD value of negative serum is significantly lower than that of positive serum, and the difference between the two is significant.

[0065] Table 8 Comparison of OD values ​​of positive and negative serum determined by ELISA

[0066]

[0067] Example 4 Preparation and verification of polypeptide ELISA antibody detection kit

[0068] Preparation of peptide ELISA antibody kit

[0069] The polypeptide of 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 100 μL and add it to the ELISA plate, and place it in a 37°C incubator for 1 hour for coating.

[0070] Then, 200 μl of PBST containing 1% bovine serum albumin and 0.05% Proclin 300 was added and blocked at 37°C for 2 hours. After drying in the shade, the tube was sealed and stored at 4°C.

[0071] 20% glycerol and 0.05% Proclin 300 were added to the positive and negative standard serum, and 0.05% Proclin 300 was added to other reagents to prepare a peptide ELISA antibody kit. The kit was then tested for consistency, repeatability and specificity.

[0072] Conformance test

[0073] 46 unknown bovine serum samples were tested simultaneously with the Brucella antibody ELISA test kit sold by IDEXX and the polypeptide ELISA test kit established by the present invention, and the conformity of the two test kits was tested. As a result, 22 positive cases were detected by the IDEXX ELISA test kit, and 23 positive cases were detected by the Brucella antibody ELISA test kit established by the present invention. The results of only one sample of the two test kits were inconsistent, and the overall conformity of the two test kits reached 97.8%.

[0074] Table 9 Consistency test

[0075]

[0076] Repeatability test

[0077] The polypeptide ELISA of the present invention was tested intra-batch and inter-batch using 12 bovine serum samples and standard positive serum, and the intra-batch and inter-batch coefficients of variation of the three results were calculated to evaluate the stability of the method, as shown in Tables 10-11.

[0078] Table 10 In-batch test

[0079]

[0080] Table 11 Batch test

[0081]

[0082] The results showed that the OD values ​​of the intra-batch test and the inter-batch test of the 13 samples were very close, and the intra-batch coefficient of variation and the inter-batch coefficient of variation were both less than 0.1, indicating that the kit of the present invention has very good stability.

[0083] Specificity test

[0084] The polypeptide ELISA of the present invention was used to detect 9 positive sera infected with bovine infectious rhinotracheitis, bovine foot-and-mouth disease, bovine viral diarrhea, bovine mycoplasma pneumonia, bovine pasteurellosis, bovine salmonellosis, bovine Escherichia coli disease, bovine Yersinia, bovine Ochrobacterium and Brucella to determine the specificity of the detection method. The results are shown in Table 12.

[0085] Table 12 Specificity test

[0086]

[0087]

[0088] The results showed that, except for the Brucella positive serum, the OD 450nm values ​​of other serum were all below the critical value and were judged as negative. This shows that the polypeptide ELISA detection method does not react with antibodies of common cattle diseases and similar pathogens, and has good specificity (Table 12).

[0089] Example 5 Detection of Brucella Immune Effect in Dairy Farms

[0090] Blood was collected from animals at a dairy farm in Benxi City, Liaoning Province 2 months after A19 vaccine immunization, and 97 serum samples were obtained by centrifugation. After detection by the kit of Example 4, 96 positive samples were obtained, indicating that the immunization effect was good.

[0091] Example 6 Detection of Brucella Immune Effect in Beef Cattle Farms

[0092] Blood was collected from animals at a beef cattle farm immunized with Brucella vaccine in Shenyang, Liaoning Province, and 68 serum samples were obtained by centrifugation. No positive samples were detected by the test kit of Example 4, indicating that no Brucella infection occurred in the beef cattle farm.

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 according to claim 1.

4. A recombinant cell or recombinant bacterium expressing the specific polypeptide according to claim 1.

5. Use of the specific polypeptide according to claim 1 in the preparation of a drug or reagent for preventing, detecting or diagnosing brucellosis.

6. The use according to claim 5, characterized in that The specific polypeptide is used for preparing an ELISA detection kit or a colloidal gold detection kit.

7. An ELISA test kit for detecting brucellosis, the ELISA test kit comprising a pre-coated plate, an enzyme-labeled antibody, a standard substance, a buffer, an antibody diluent, a color developer and a stop solution, characterized in that The pre-coated plate is coated with the specific polypeptide according to claim 1.

8. The detection kit according to claim 7, characterized in that The pre-coated plate was treated with 100 μL of pH 9.6 to a final concentration of 1 μg ml -1 The specific polypeptide solution of claim 1 is coated, and then 200 μl of PBST containing 1% bovine serum albumin is added for blocking, followed by incubation 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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