L7 / L12-PADRE sequence-polylink B cell epitope recombinant protein and kit for brucella antibody detection

Abstract

The application belongs to the technical field of biotechnology, and provides a L7 / L12-PADRE sequence-multiple B cell epitope recombinant protein and kit for detecting anti-Brucella antibody. The recombinant protein is a multi-epitope tandem recombinant protein composed of Brucella ribosome L7 / L12 protein, PADRE polypeptide sequence and multiple B cell epitopes, and the amino acid sequence of the recombinant protein is shown as SEQ ID No. 1, and the antigenicity is good. The anti-Brucella antibody indirect ELISA (iELISA) detection method and kit established by taking the recombinant protein as a coating antigen have the characteristics of high sensitivity, strong specificity and good repeatability, are suitable for detecting the anti-Brucella antibody produced by the organism, can clearly determine the Brucella infection or Brucella vaccine immunization background, provide basic data for preventing and controlling the human-animal shared Brucella disease, and are simultaneously convenient for being applied to large-scale sample detection and epidemiological monitoring.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to a recombinant protein of L7 / L12-PADRE sequence-multiplexed B-cell epitopes for the detection of anti-brucell antibodies and a kit thereof. Background Technology

[0002] Brucellosis is a zoonotic bacterial infectious disease caused by Brucella spp., posing a significant threat to human and animal health. It can infect cattle, pigs, sheep, goats, horses, dogs, and other animals. Infected female animals may experience abortion or stillbirth; while infected male animals primarily exhibit orchitis, epididymitis, testicular swelling, and joint swelling. For these reasons, the World Health Organization lists it as one of the seven neglected zoonotic diseases, and it is also one of the key zoonotic infectious diseases currently under control in my country.

[0003] Because Brucella has a long incubation period, early symptoms are not obvious, and there is currently no effective treatment, laboratory diagnosis of the disease is extremely important. Etiological detection is the gold standard for brucellosis diagnosis, but this method has a high risk factor, requires a BSL-3 laboratory and must be performed by professionals. Furthermore, pathogen isolation and culture are time-consuming and have low isolation rates, making it unsuitable for rapid clinical testing. Currently, commonly used clinical diagnostic methods include: Standard Agglutination Test (SAT), Rose Bengal Plate Test (RBT), Enzyme-Linked Immunosorbent Assay (ELISA), and Massive Reproductive Technology (MRT). However, SAT and RBT are prone to false positives, leading to misdiagnosis; the MRT is mainly used for initial screening of lactating animals for brucellosis. Compared to other methods, ELISA has relatively high specificity and sensitivity and can simultaneously detect a large number of antibody samples. However, most commercially available kits require extraction of specific proteins or lipopolysaccharides from cultured Brucella, which not only poses a high biosafety risk but is also expensive.

[0004] In view of the above situation, establishing an economical and effective rapid detection method for anti-brucellosis antibodies is of practical significance for the rapid screening of diseases, prevention and control of brucellosis, and eradication in grassroots farms. Therefore, this invention proposes an L7 / L12-PADRE sequence-multiplexed B-cell epitope recombinant protein for anti-brucellosis antibody detection, and a kit for detecting anti-brucellosis antibodies based on the indirect ELISA (iELISA) method. Summary of the Invention

[0005] The purpose of this invention is to provide an L7 / L12-PADRE sequence-multiplexed B-cell epitope recombinant protein and kit for detecting anti-brucell antibodies, aiming to solve the problems mentioned in the background art.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A recombinant protein consisting of an L7 / L12-PADRE sequence and multiple B-cell epitopes, wherein the L7 / L12-PADRE sequence and multiple B-cell epitopes are composed of Brucella ribosomal L7 / L12 protein, PADRE polypeptide sequence, and multiple B-cell epitopes linked by EAAAK linker and KK linker, respectively, and its amino acid sequence is shown in SEQ ID No. 1.

[0008] A gene encoding a recombinant protein based on the L7 / L12-PADRE sequence-multiplexed B-cell epitopes described above, the nucleotide sequence of which is shown in SEQ ID No. 2.

[0009] The application of the L7 / L12-PADRE sequence-multiplexed B-cell epitope recombinant protein or the encoding gene described above in the preparation of a product for detecting anti-brucellosis antibodies.

[0010] Furthermore, using the L7 / L12-PADRE sequence-multiplexed B-cell epitope recombinant protein as the coating antigen, an iELISA method was established to detect anti-brucell antibodies.

[0011] A kit for detecting anti-brucellosis antibodies based on iELISA, the kit comprising: an enzyme-labeled plate coated with the L7 / L12-PADRE sequence-multiplexed B-cell epitope recombinant protein described above; blocking buffer; washing buffer; diluent; enzyme-labeled reagent; substrate chromogenic solution; positive standard serum; and negative standard serum.

[0012] Furthermore, the optimal coating concentration of the L7 / L12-PADRE sequence-multiplexed B-cell epitope recombinant protein is 4.15 μg / mL, and the coating condition is overnight at 4°C.

[0013] Furthermore, the blocking solution is 1M ammonium chloride; the washing solution is PBST solution; the diluent is PBS solution; the enzyme labeling reagent is horseradish peroxidase-labeled rabbit anti-sheep antibody; the substrate chromogenic solution is 3,3',5,5'-tetramethylbenzidine solution; the positive standard serum is anti-brucellosis positive serum; and the negative standard serum is anti-brucellosis negative serum.

[0014] Furthermore, the optimal dilution ratio of the enzyme-labeled reagent is 1:8000; the optimal reaction time of the substrate chromogenic solution is 10 min; and the optimal dilution ratio of the positive standard serum and the negative standard serum is 1:400.

[0015] Furthermore, 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 When the OD value is <0.6864, the serum sample being tested does not contain anti-brucellosis antibodies and is therefore considered negative; when the OD value is <0.6864, the serum sample is considered negative. 450nm If the value is ≥0.6864, the serum sample to be tested contains anti-brucellosis antibodies and is judged as positive.

[0016] Compared with the prior art, the beneficial effects of the present invention are:

[0017] This invention connects Brucella ribosomal L7 / L12 protein, PADRE sequence, and multiple B-cell epitopes using EAAAK and KK linkers to construct a recombinant protein of L7 / L12-PADRE sequence-multiple B-cell epitopes. Using this recombinant protein as a coating antigen, an iELISA method and kit for detecting anti-brucellosis antibodies were established. The prepared recombinant protein exhibits good antigenicity, and the iELISA method and kit for detecting anti-brucellosis antibodies constructed using this protein are characterized by high sensitivity, strong specificity, and good reproducibility. It is suitable for detecting the production of anti-brucellosis antibodies in the body, clarifying the background of Brucella infection or brucellosis vaccine immunity, and providing fundamental data for the prevention and control of zoonotic brucellosis. Furthermore, it can be applied to large-scale sample testing and epidemiological surveillance, showing great potential for widespread application and value. Attached Figure Description

[0018] Figure 1 The L7 / L12-PADRE sequence-multiplexed B-cell epitope recombinant protein was induced to express; M is a standard marker for protein molecular weight of 18-250 kDa; lane 1 is the control of the recombinant expression vector with added inducer (IPTG); lane 2 is the bacterial cell result after induction of recombinant expression bacteria; lane 3 is the supernatant result 4 h after induction of recombinant expression bacteria; lane 4 is the precipitation result 4 h after induction of recombinant expression bacteria; lane 5 is the purified result 4 h after induction of recombinant expression bacteria.

[0019] Figure 2Purification results of L7 / L12-PADRE sequence-multiplexed B-cell epitope recombinant protein: M is the standard marker for protein molecular weight of 18-250 kDa; lane 1 is the supernatant result 4 h after induction of recombinant expression bacteria; lane 2 is the flow-through buffer; lane 3 is 40 mmol / L imidazole elution buffer; lane 4 is 60 mmol / L imidazole elution buffer; lane 5 is 80 mmol / L imidazole elution buffer; lane 6 is 150 mmol / L imidazole elution buffer; lane 7 is 200 mmol / L imidazole elution buffer; lane 8 is 220 mmol / L imidazole elution buffer; lane 9 is 250 mmol / L imidazole elution buffer; lane 10 is 300 mmol / L imidazole elution buffer.

[0020] Figure 3 The Western blot results show the L7 / L12-PADRE sequence-multiple B-cell epitope recombinant protein; where M is the molecular weight protein marker; and 1 is the purified L7 / L12-PADRE sequence-multiple B-cell epitope recombinant protein. Detailed Implementation

[0021] In order to provide a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention will now be described in detail below, but it should not be construed as limiting the scope of implementation of the present invention.

[0022] The specific implementation of the present invention will be described in detail below with reference to specific embodiments. Experimental methods in the following embodiments that do not specify specific experimental conditions are generally performed under conventional conditions.

[0023] Example 1: Selecting the target segment to be expressed;

[0024] First, conserved amino acid sequences of four proteins—surA (GenBank: XMD05064), OMP31 (GenBank: ACS50328), BP26 (GenBank: AAO39771), and Trigger Factor (GenBank: AIJ68576)—collected from the NCBI database (https: / / www.ncbi.nlm.nih.gov / guide / proteins / ) in *Brucella malata*, *Brucella abortus*, and *Brucella suis* were analyzed. MHC class I epitopes (CTL epitopes) for surA, OMP31, BP26, and Trigger Factor were predicted using IEDB (https: / / www.iedb.org / ), Rankpep (http: / / imed.med.ucm.es / Tools / rankpep.html), and Syfpeithi (http: / / www.syfpeithi.de / ). MHC-II epitopes (HTL epitopes) of four proteins—surA, OMP31, BP26, and Trigger Factor—were predicted using IEDB, Rankpep, and NetMHCII (https: / / services.healthtech.dtu.dk / services / NetMHCIIpan-4.0 / ). Linear B-cell epitopes of these four proteins were predicted using IEDB and SVMTriP (http: / / sysbio.unl.edu / SVMTriP / ). DeepLBCEPred (http: / / www.biolscience.cn / DeepLBCEPred / ) was used to score the predicted linear B-cell epitopes, and epitopes with scores greater than 0.5 were selected as candidate epitopes.The instability index, hydrophilicity index, antigenicity index, toxicity, and allergenicity of candidate epitopes were analyzed using software such as Expasy-ProtParam (https: / / web.expasy.org / cgi-bin / protparam / protparam), vaxijen v2.0 (https: / / ddg-pharmfac.net / vaxijen / VaxiJen / VaxiJen.html), and ToxinPred2 (https: / / webs.iiitd.edu.in / raghava / toxinpred2 / index.html), and the dominant linear B-cell epitopes were finally selected. The DNA encoding B-cell dominant epitopes derived from four Brucella proteins (SurA, OMP31, BP26, and Trigger factor) was ligated using the KK linker to construct a DNA encoding multiple B-cell epitopes. The nucleotide sequences are shown in SEQ ID NO. 2 (433-1101 bp) and the corresponding amino acid sequences are shown in SEQ ID NO. 1 (145-367 aa). The DNA encoding the ribosomal protein L7 / L12 derived from Brucella was ligated using the EAAAK linker to ligate the DNA encoding the PADRE sequence. The nucleotide sequences are shown in SEQ ID NO. 2 (1-372 bp) and SEQ ID NO. 2 (388-426 bp), and the corresponding amino acid sequences are shown in SEQ ID NO. 1 (1-124 aa) and SEQ ID NO. 1 (130-142 aa), respectively. This resulted in the L7 / L12-PADRE sequence-multiple B-cell epitope recombinant protein, with the amino acid sequence shown in SEQ ID NO. 1 and the nucleotide sequence of its encoding gene shown in SEQ ID NO. 2.

[0025] SEQ ID No. 1:

[0026] MADLAKIVEDLSALTVLEAAELSKLLEEKWGVSAAAPVAVAAAGGAAAAAAEEKTEFDVVLADGGANKINVIKEVRALTGLGLKEAKDLVEGAPKAVKEGASKDEAEKIKAQLEAAGAVELKEAAAKAKFVAAWTLKAAAKKSEDEKEEAEKVLDGKADKKVVDISDEEVDEQVKRIASSTRT FETKKGKAENEDRVTIDYLGKLDGEPFEGGADNDAQKKNVTDKKVSKEELTAEDEDAASEAKPAKKAAKKKAEEGKSEEAKKPFSSFDKEDNEQVSGSLDVTAKKIYVYPDDKNNLKEPTITGYSVSKKSKDVKAAGVNRTTPKPHDTEKGVEKKEGADSPAGKEKKAEELSKKYVQELREKA。

[0027] SEQ ID No.2:

[0028]

[0029] Example 2: Prokaryotic expression and purification;

[0030] 1. Prokaryotic expression and SDS-PAGE analysis;

[0031] The optimized target gene was ligated into the pET-28a expression vector to construct the recombinant expression plasmid pET-28a-L7 / L12-PADRE sequence-multiplexed B-cell epitopes; the recombinant plasmid was transformed into E. coli Rosetta(DE3) competent cells; single colonies with correct PCR and sequencing results were selected for amplification culture until the bacterial culture OD... 600nm When the protein concentration was approximately 0.6–1.0, IPTG was added to a final concentration of 0.5 mM. Expression was induced for 4 h at 37 °C and 180 rpm. The cells were collected by centrifugation at 8000 rpm for 10 min, sonicated for 40 min, and then centrifuged again at 8000 rpm for 40 min. The supernatant was collected and the precipitate resuspended. A negative control of recombinant bacteria without IPTG induction was also included. All samples were analyzed by SDS-PAGE electrophoresis to detect the expression of the recombinant protein.

[0032] SDS-PAGE results ( Figure 1 The results showed that lane 2 exhibited a clear, specific band at the expected molecular weight position, indicating successful expression of the recombinant protein; lane 3 showed a clear band at the same position, while lane 4 showed no corresponding band, indicating that the recombinant protein was mainly present in the supernatant; lane 5 showed a single clear band at the target position, suggesting that a high purity of the recombinant protein was obtained; additionally, the clear target band in lane 1, used as a control, may be due to leakage expression of T7 RNA polymerase in the host bacteria. In summary, the L7 / L12-PADRE sequence-multiplexed B-cell epitope recombinant protein was efficiently expressed in *E. coli*, and mainly existed in a soluble form in the cytoplasmic supernatant.

[0033] 2. Purification and activity identification of L7 / L12-PADRE sequence-multiplexed B-cell epitope recombinant protein;

[0034] The supernatant of the L7 / L12-PADRE sequence-multiplexed B-cell epitope recombinant protein was filtered through a 0.45µm syringe filter, and the target protein was purified using a Ni-NTA protein purification gravity column. Gradient elutions were performed at concentrations of 40 mmol / L imidazole, 60 mmol / L imidazole, 80 mmol / L imidazole, 150 mmol / L imidazole, 200 mmol / L imidazole, 220 mmol / L imidazole, 250 mmol / L imidazole, and 300 mmol / L imidazole. The flow-through and eluent were collected and prepared separately for SDS-PAGE electrophoresis and Coomassie brilliant blue staining to observe the purification effect.

[0035] The results show that ( Figure 2 The presence of the target band at the expected molecular weight position in lane 1 indicates good recombinant protein expression. The significantly reduced target protein content in lane 2 compared to lane 1 indicates good adsorption of the target protein by the nickel column, with most of the target protein being adsorbed onto the column. From lane 5 onwards, the content of other proteins decreases, while the target protein content remains relatively high. In summary, collection began with 80 mmol / L imidazole eluent, which was then placed in a dialysis bag for concentration.

[0036] The concentrated recombinant protein was transferred onto a PVDF membrane and blocked with 5% skim milk powder at 37°C for 1 hour. A sheep brucellosis-positive serum was used as the primary antibody, diluted 1:400, and incubated overnight at 4°C. HRP (horseradish peroxidase)-labeled rabbit anti-sheep antibody was used as the secondary antibody, diluted 1:5000, and incubated at room temperature for 2 hours. Finally, the reactivity was assessed using a horseradish peroxidase DAB colorimetric assay kit.

[0037] The results show that ( Figure 3 The presence of a distinct specific chromogenic band at approximately 48 kDa in lane 1 indicates that the recombinant protein can undergo a specific immune response with Brucella ovis positive serum, demonstrating good reactivity.

[0038] Example 3: Optimization of iELISA reaction conditions;

[0039] 1. Determination of the optimal coating concentration of recombinant protein antigen and the dilution concentration of the serum to be tested;

[0040] The optimal coating concentration and serum dilution of the L7 / L12-PADRE sequence-multiplexed B-cell epitope recombinant protein were determined using a checkerboard method. The specific procedures were as follows: The recombinant protein was serially diluted with carbonate buffer to coating concentrations of 16.6 μg / mL, 8.3 μg / mL, 4.15 μg / mL, 2.075 μg / mL, 1.0375 μg / mL, and 0.51875 μg / mL, 100 μL / well, and incubated overnight at 4°C; washed three times with PBST; 1% BSA blocking buffer (200 μL / well) was added and incubated at 37°C for 2 h; after blocking, the protein was washed as before, and the anti-brucellosis positive protein was isolated using PBS. Serum and negative sera were serially diluted at 1:50, 1:100, 1:200, 1:400, 1:800, and 1:1600, respectively, and arranged in a square matrix. 100 μL / well was incubated at 37°C for 1 h. After washing three times with PBST, 100 μL / well of rabbit anti-goat HRP (working concentration 1:5000) was added. After washing three times, 100 μL / well of chromogenic buffer was added, and the mixture was incubated at 37°C for 15 min. The reaction was terminated by adding 50 μL of 2MH2SO4. OD values ​​were read using a microplate reader. 450nmValues; by comparing the OD values ​​of positive and negative sera. 450nm The optimal antigen coating concentration and serum dilution are determined using the OD value and P / N value. When the positive serum OD... 450nm When the value is greater than 1, P / N > 2.1, and the negative value is low while the P / N value is high, the corresponding antigen concentration and serum working concentration are the optimal antigen coating concentration and the optimal dilution concentration of the serum to be tested. Table 1 shows that the optimal antigen coating concentration is 4.15 μg / mL, and the optimal dilution ratio for positive and negative sera is 1:400.

[0041] Table 1. Determination of optimal antigen coating concentration and optimal serum dilution (checkerboard method)

[0042]

[0043] 2. Determination of the sealing solution;

[0044] Coat the ELISA plate with the optimal antigen concentration and incubate overnight at 4°C. The next day, wash three times with PBST solution for 2 minutes each time, and pat dry. Divide the blocking solution into four groups: Group I: 5% skim milk powder; Group II: 1% BSA; Group III: 1% gelatin; Group IV: 1M ammonium chloride. Add 200 μL to each well of the coated ELISA plate and block at 37°C for 2 hours. Then wash three times with PBST solution for 2 minutes each time, pat dry, and block at 37°C for 2 hours. Perform three parallel wells for each group. After blocking and washing, add 100 μL of the optimal concentration of negative and positive serum per well and incubate at 37°C for 1 hour. After washing, add 100 μL of enzyme-labeled antibody (HRP-labeled rabbit anti-goat secondary antibody) diluted 1:5000 per well and incubate at 37°C for 1 hour. After washing, add 100 μL of substrate chromogenic solution (3,3',5,5'-tetramethylbenzidine, abbreviated as TMB) to each well and incubate at 37°C in the dark for 15 min; add 50 μL of stop solution to each well and measure the OD on a microplate reader within 15 min. 450nm Value. Select positive OD. 450nm Mean and negative OD 450nm The type of sealing solution with the highest P / N ratio is considered the optimal sealing solution. As shown in Table 2, group IV (1M ammonium chloride) has the highest P / N value and is therefore the optimal sealing solution.

[0045] Table 2 Determination of the optimal sealing solution

[0046]

[0047] 3. Determination of the optimal working concentration of enzyme-labeled secondary antibody;

[0048] Dilute Brucella antigen protein to 4.15 μg / mL with carbonate buffer, add 100 μL / well to each well of the ELISA plate, and incubate overnight at 4°C. The next day, wash three times with PBST solution for 2 min each time, and blot dry. Add 200 μL of 1M ammonium chloride solution to each well of the coated ELISA plate, block at 37°C for 2 h, then wash three times with PBST solution for 2 min each time, and blot dry. Add 100 μL / well of anti-brucellosis positive serum diluted 1:400 with PBS solution as a positive control, and add 100 μL / well of anti-brucellosis negative serum diluted 1:400 with PBS solution as a negative control. For serum incubation, the conditions were set at 37°C for 1 hour, followed by washing three times with PBST solution for 2 minutes each time, and then patting dry. Taking HRP-labeled rabbit anti-goat secondary antibody as an example, the optimal working concentration of the enzyme-labeled secondary antibody was determined. The enzyme-labeled secondary antibody was diluted with PBS solution at 1:2000, 1:3000, 1:4000, 1:5000, 1:6000, 1:7000, 1:8000, and 1:9000, and added to the ELISA plate at 100 μL / well. Incubation was performed at 37°C for 1 hour, with three parallel wells for each group. The plates were then washed three times with PBST solution for 2 minutes each time, and patted dry. 100 μL of TMB substrate chromogenic solution was added to each well, and incubation was performed at 37°C in the dark for 15 minutes. 50 μL of stop solution was added to each well, and the OD was measured on a microplate reader within 15 minutes. 450nm Value, select positive OD 450nm Mean and negative OD 450nm The optimal working concentration of enzyme-labeled antibody is the enzyme-labeled antibody dilution ratio that maximizes the ratio of the mean (P / N value). As shown in Table 3, 1:8000 is the optimal enzyme-labeled secondary antibody dilution ratio.

[0049] Table 3 Determination of the optimal working concentration of enzyme-labeled secondary antibodies

[0050]

[0051] 4. Determining the optimal development time of the substrate and developing solution;

[0052] Dilute Brucella antigen protein to 4.15 μg / mL with carbonate buffer, add 100 μL / well to each well of the ELISA plate, and incubate overnight at 4°C. The next day, wash three times with PBST solution for 2 min each time, and blot dry. Add 200 μL of 1M ammonium chloride solution to each well of the coated ELISA plate, and block at 37°C for 2 h. Perform three parallel wells for each group, then wash three times with PBST solution for 2 min each time, and blot dry. Add 100 μL / well of anti-brucellosis positive serum diluted 1:400 with PBS solution as a pretreatment. For the positive control, 100 μL / well of anti-brucellosis negative serum diluted 1:400 with PBS was added. The serum was incubated at 37°C for 1 h, followed by washing three times with PBST solution for 2 min each time, and then patted dry. For the enzyme-labeled antibody (HRP-labeled rabbit anti-goat secondary antibody), 100 μL / well was added to the ELISA plate after a 1:8000 dilution with PBS. The plate was incubated at 37°C for 1 h, followed by washing three times with PBST solution for 2 min each time, and then patted dry. 100 μL of TMB substrate chromogenic solution was added to each well, and the plate was incubated at 37°C in the dark for 10, 15, 20, 25, and 30 min. 50 μL of stop solution was added to each well, and the OD was measured on a microplate reader within 15 min. 450nm Value, select positive OD 450nm Mean and negative OD 450nm The optimal color development time is defined as the time when the ratio of the mean values ​​(P / N value) is maximized. As shown in Table 4, 10 minutes is the optimal color development time.

[0053] Table 4. Determination of color development time

[0054]

[0055] 5. Determining the critical values ​​for positive and negative sex;

[0056] The iELISA test was performed under the optimal reaction conditions determined above. Forty-five 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... 450nm The mean (X) of 45 anti-brucellosis negative sera was calculated to be 0.42491, and the standard deviation (SD) was 0.0872. Therefore, the cutoff point for positive and negative seroconversion was determined as C = X + 3SD = 0.6864, which is the OD value of the serum to be tested. 450nm A value ≥ 0.6864 is considered positive, and a value below 0.6864 is considered negative (Table 5).

[0057] Table 5. Determination of Critical Values ​​for Positive and Negative Sex

[0058]

[0059] 6. Specificity test;

[0060] iELISA tests were performed under the optimal reaction conditions determined above. Positive sera for sheep peste des petits ruminants (PPR), foot-and-mouth disease (FMD), and infectious coryza (CCPP) were detected. Each serum sample was tested three times, and the average of the three tests was compared with the cutoff values. Simultaneously, iELISA tests were performed using HRP-labeled sheep anti-mouse secondary antibodies under the same optimal reaction conditions. Positive sera for mice against Brucella abortus A19 (anti-brucellosis antibody positive control group), enterotoxigenic Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii, Pseudomonas aeruginosa, Salmonella, and Yersinia enterocolitica were detected, along with their corresponding negative sera for mice. Each serum sample was tested three times, and the average of the three tests was compared. The OD values ​​of the mouse positive sera were compared. 450nm Mean and P / N value. Table 6-1 shows the OD values ​​of PPR, FMD, and CCPP positive sera. 450nm A mean value <0.6864 indicates a negative result. Table 6-2 shows the OD values ​​of serum positive for Brucella abortus strain A19 (anti-brucellosis antibody positive control group). 450nm A mean > 1 and P / N > 2.1 are considered positive. The OD values ​​of positive sera against enterotoxigenic Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii, Pseudomonas aeruginosa, Salmonella, and Yersinia enterocolitica are also considered positive. 450nm All values ​​were less than 1, and the P / N ratio was less than 2.1, indicating a negative result. In conclusion, the established iELISA method exhibits good specificity.

[0061] Table 6-1 Specificity Detection

[0062]

[0063] Table 6-2 Specificity Detection

[0064]

[0065] 7. Sensitivity test;

[0066] Brucellosis-positive control sheep serum was collected and diluted with sample diluent at 1:160, 1:320, 1:640, 1:1280, 1:2560, 1:5120, 1:10240, and 1:120480. Each group was run in triplicate. iELISA was then performed on the positive serum at different dilutions. The results (Table 7) showed that even at a serum dilution of 1:1280, the test result was still positive (OD). 450nm The mean value is >0.6864 and the P / N ratio is >2.1, indicating that the method has good sensitivity.

[0067] Table 7 Sensitivity Detection

[0068]

[0069] 8. Intra-batch and inter-batch replication tests;

[0070] Sixteen sheep serum samples were collected and tested under the optimal reaction conditions determined above. Each sample was tested in triplicate, and its OD value was measured. 450nm The values ​​were calculated, and their mean (x) and standard deviation (SD) were determined. The intra-batch repeatability of the detection method was determined by the range of variation of the coefficient of variation (standard deviation / mean × 100%). Then, 16 sheep serum samples were tested in different batches according to the optimal reaction conditions determined above, for a total of 3 batches, and their OD values ​​were measured. 450nm The mean (x) and standard deviation (SD) of the sample from different batches were calculated. The inter-batch repeatability of the detection method was determined by the range of variation of the coefficient of variation (standard deviation / mean × 100%). The coefficient of variation was calculated based on the test results: CV = (SD ÷ X) × 100%. The results (Table 8) show that the intra-batch coefficient of variation for the 16 sheep serum samples ranged from 0.90 to 5.54%, all less than 10%; the inter-batch repeatability coefficient of variation ranged from 2.41 to 9.01%, all less than 10%. This indicates that the established iELISA method has good intra-batch and inter-batch repeatability and stability.

[0071] Table 8. Intra-batch and inter-batch repeatability tests

[0072]

[0073] Example 4: Clinical sample testing and comparative analysis with the Rose Bengal plate agglutination test;

[0074] The iELISA test was performed under the optimal reaction conditions determined above, and the Rose Bengal plate agglutination test was used to detect 48 serum samples. The results (Table 9) show that the iELISA method of this invention and the Rose Bengal plate agglutination test results are highly consistent. Of the 48 serum samples, only 2 samples (numbered 4 and 37) showed inconsistent results between the two methods, while the remaining 46 samples were consistent. The concordance rate was 95.83%, demonstrating that the iELISA method of this invention has good reliability and applicability in clinical sample testing, and can accurately reflect the level of anti-brucellosis antibodies in serum samples, providing an effective detection method for the clinical diagnosis of brucellosis and showing good application prospects.

[0075] Table 9 Comparison of results from the Tiger Bronze Plate Agglutination Test and the iELISA method of this invention.

[0076]

[0077]

[0078] The above clinical sample testing uses sheep serum as an example. Other types of samples, such as milk, tissue, and organ samples, can also be detected by this kit after sample pretreatment to detect anti-brucellosis antibodies. For testing other animal samples, simply change the animal-labeled secondary antibody.

[0079] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent.

Claims

1. A recombinant protein consisting of an L7 / L12-PADRE sequence and multiple B-cell epitopes, characterized in that, The L7 / L12-PADRE sequence-multiple B-cell epitope recombinant protein is composed of Brucella ribosomal L7 / L12 protein, PADRE polypeptide sequence, and multiple B-cell epitopes linked by EAAAK linker and KK linker, respectively, and its amino acid sequence is shown in SEQ ID No.

1.

2. A gene encoding the L7 / L12-PADRE sequence-multiplexed B-cell epitope recombinant protein according to claim 1, characterized in that, The nucleotide sequence of the encoding gene is shown in SEQ ID No.

2.

3. The use of the L7 / L12-PADRE sequence-multiplexed B-cell epitope recombinant protein according to claim 1 or the encoding gene according to claim 2 in the preparation of a product for detecting anti-brucellosis antibodies.

4. The application according to claim 3, characterized in that, Using the L7 / L12-PADRE sequence-multiplexed B-cell epitope recombinant protein as the coating antigen, an iELISA method was established to detect anti-brucell antibodies.

5. A kit for detecting anti-brucellosis antibodies based on iELISA, characterized in that, The kit comprises: an enzyme-labeled plate coated with the L7 / L12-PADRE sequence-multiplexed B-cell epitope recombinant protein as described in claim 1; blocking buffer; washing buffer; diluent; enzyme-labeled reagent; substrate chromogenic solution; positive standard serum; and negative standard serum.

6. The reagent kit according to claim 5, characterized in that, The coating concentration of the L7 / L12-PADRE sequence-multiplexed B-cell epitope recombinant protein was 4.15 μg / mL, and the coating condition was overnight at 4°C.

7. The reagent kit according to claim 5, characterized in that, The blocking solution is 1M ammonium chloride; the washing solution is PBST solution; the diluent is PBS solution; the enzyme labeling reagent is horseradish peroxidase-labeled rabbit anti-sheep antibody; the substrate chromogenic solution is 3,3',5,5'-tetramethylbenzidine solution; the positive standard serum is anti-brucellosis positive serum; and the negative standard serum is anti-brucellosis negative serum.

8. The reagent kit according to claim 5, characterized in that, The enzyme-labeled reagent is diluted at a ratio of 1:8000; the reaction time of the substrate chromogenic solution is 10 min; and the dilution ratio of the positive standard serum and the negative standard serum is 1:

400.

9. The reagent kit according to claim 5, characterized in that, 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 When the OD value is <0.6864, the serum sample being tested does not contain anti-brucellosis antibodies and is therefore considered negative; when the OD value is <0.6864, the serum sample is considered negative. 450nm If the value is ≥0.6864, the serum sample to be tested contains anti-brucellosis antibodies and is judged as positive.

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