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

Abstract

The invention is applicable to the technical field of biology, and provides an L7 / L12-PADRE sequence-multi-linked B cell epitope recombinant protein and a kit for detecting an anti-Brucella antibody. The recombinant protein is a multi-epitope tandem recombinant protein composed of Brucella ribosome L7 / L12 protein, a PADRE polypeptide sequence and multiple B cell epitopes, the amino acid sequence of the recombinant protein is as shown in SEQ ID No.1, and the recombinant protein is good in antigenicity. The anti-brucella antibody indirect ELISA (iELISA) detection method and kit established by taking the recombinant protein as the coating antigen have the characteristics of high sensitivity, strong specificity and good repeatability, are suitable for detecting the condition of generating the anti-brucella antibody by an organism, can clarify the immune background of brucella infection or brucellosis vaccine, and can be used for detecting the brucella infection or brucellosis vaccine. Basic data is provided for prevention and control of the Brucella disease of humans and animals, and meanwhile, the kit is conveniently applied to large-scale sample detection and epidemiological monitoring.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a L7 / L12-PADRE sequence-multiple B cell epitope recombinant protein and kit for detecting anti-Brucella antibody. BACKGROUND

[0002] Brucellosis is a zoonotic bacterial infectious disease caused by Brucella spp., which is extremely harmful to human and animal health. It can infect animals such as cattle, pigs, sheep, goats, horses and dogs, and infected female animals will have miscarriage and stillbirth; and infected male animals mainly show orchitis, epididymitis, testicular enlargement and joint enlargement. Therefore, the disease is listed as one of the seven neglected zoonoses by the World Health Organization, and is also one of the zoonotic infectious diseases currently focused on prevention and control in China.

[0003] Due to the long incubation period of Brucella and the non-obvious early symptoms of infection, and there is currently no effective treatment method, the laboratory diagnosis of the disease is extremely important. Pathogenic detection is the gold standard for the diagnosis of Brucellosis, but this method has a high risk coefficient and needs to be operated by professionals in a BSL-3 laboratory, and the pathogen isolation and culture takes a long time and has a low isolation rate, and is not suitable for rapid detection in clinical practice. At present, the commonly used diagnostic methods in clinical practice mainly include: test tube agglutination test (SAT), rose bengal plate agglutination test (RBT), enzyme-linked immunosorbent assay (ELISA), whole milk ring test (MRT), etc. However, test tube agglutination test and rose bengal plate agglutination test are prone to false positive results, which may lead to misjudgment; whole milk ring test is mainly used for the initial screening of Brucellosis in lactating female animals. Compared with other methods, ELISA method has relatively high specificity and sensitivity, and can simultaneously detect a large number of antibody samples, but most of the existing kits on the market need to extract specific proteins or lipopolysaccharides from cultured Brucella, which not only has a high biological safety risk, but also is expensive.

[0004] In view of the above status, it is of practical significance to establish an economical and effective rapid detection method for anti-Brucella antibody for the rapid screening of epidemic diseases in primary breeding farms, the prevention and control of Brucellosis, and the purification. Therefore, the present application provides a L7 / L12-PADRE sequence-multiple B cell epitope recombinant protein for detecting anti-Brucella antibody, and a kit for detecting anti-Brucella antibody based on indirect ELISA (iELISA) method. SUMMARY

[0005] The application aims to provide an L7 / L12-PADRE sequence-multimeric B cell epitope recombinant protein and kit for detecting anti-Brucella antibodies, and aims to solve the problems in the background art.

[0006] The application aims to provide an L7 / L12-PADRE sequence-multimeric B cell epitope recombinant protein and kit for detecting anti-Brucella antibodies, and aims to solve the problems in the background art.

[0007] The application aims to provide an L7 / L12-PADRE sequence-multimeric B cell epitope recombinant protein and kit for detecting anti-Brucella antibodies, and aims to solve the problems in the background art.

[0008] The application aims to provide an L7 / L12-PADRE sequence-multimeric B cell epitope recombinant protein and kit for detecting anti-Brucella antibodies, and aims to solve the problems in the background art.

[0009] The application aims to provide an L7 / L12-PADRE sequence-multimeric B cell epitope recombinant protein and kit for detecting anti-Brucella antibodies, and aims to solve the problems in the background art.

[0010] Further, the L7 / L12-PADRE sequence-multimeric B cell epitope recombinant protein is used as a coating antigen, and an iELISA method is established to detect anti-Brucella antibodies.

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

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

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

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

[0015] Further, the kit determines whether the serum of the sample to be measured contains anti-Brucella antibody by measuring the OD 450nm value of the serum of the sample to be measured, and the judgment standard is: when the OD 450nm value is <0.6864, it is determined that the serum sample to be measured does not contain anti-Brucella antibody, and is negative; when the OD 450nm value is ≥0.6864, it is determined that the serum sample to be measured contains anti-Brucella antibody, and is positive.

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

[0017] The present application connects the Brucella ribosome L7 / L12 protein, the PADRE sequence and the multiple B cell epitopes through the EAAAK linker and the KK linker, constructs the L7 / L12-PADRE sequence-multiple B cell epitope recombinant protein, and establishes the anti-Brucella antibody iELISA detection method and kit by taking the recombinant protein as the coating antigen. The prepared recombinant protein antigen is good, and the anti-Brucella antibody iELISA detection method and kit constructed by the recombinant protein 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 the Brucella vaccine immunization background, and provide basic data for the prevention and control of the Brucella disease caused by the human-animal coexistence. Meanwhile, the method and kit can be applied to large-scale sample detection and epidemiological monitoring, and have good popularization and use prospect and value. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 is the L7 / L12-PADRE sequence-multiple B cell epitope recombinant protein induced expression; wherein M is an 18-250 kDa protein molecular weight marker Marker; lane 1 is a recombinant expression vector control added with an inducer (IPTG); lane 2 is a result of the bacterial body after induction of the recombinant expression bacteria; lane 3 is a result of the supernatant after 4h induction of the recombinant expression bacteria; lane 4 is a result of the precipitate after 4h induction of the recombinant expression bacteria; and lane 5 is a result after purification of the recombinant expression bacteria after 4h induction.

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

[0020] Figure 3 Western blot identification results of L7 / L12-PADRE sequence-multiple B cell epitope recombinant protein; M is molecular weight protein marker; 1 is the purified L7 / L12-PADRE sequence-multiple B cell epitope recombinant protein. DETAILED DESCRIPTION

[0021] In order to have a clearer understanding of the technical features, objectives and beneficial effects of the present application, the technical solutions of the present application are described in detail as follows, but it should not be understood as limiting the scope of the present application.

[0022] The specific implementation of the present application is described in detail in combination with specific examples. The test methods in the following examples without specific experimental conditions are usually carried out according to conventional conditions.

[0023] Example 1: selection of the target fragment to be expressed

[0024] Firstly, the conservative amino acid sequences of surA (GenBank: XMD05064), OMP31 (GenBank: ACS50328), BP26 (GenBank: AAO39771), Trigger Factor (GenBank: AIJ68576) in Brucella melitensis, Brucella abortus, Brucella suis were collected from NCBI (https: / / www.ncbi.nlm.nih.gov / guide / proteins / ) database and analyzed. The MHC-I class epitopes, i.e. CTL epitopes, of surA, OMP31, BP26, Trigger Factor were predicted by IEDB (https: / / www.iedb.org / ), Rankpep (http: / / imed.med.ucm.es / Tools / rankpep.html), Syfpeithi (http: / / www.syfpeithi.de / ). The MHC-II class epitopes, i.e. HTL epitopes, of surA, OMP31, BP26, Trigger Factor were predicted by IEDB, Rankpep, NetMHCII (https: / / services.healthtech.dtu.dk / services / NetMHCIIpan-4.0 / ). The linear B cell epitopes of surA, OMP31, BP26, Trigger Factor were predicted by IEDB, SVMTriP (http: / / sysbio.unl.edu / SVMTriP / ). The predicted linear B cell epitopes were scored by DeepLBCEPred (http: / / www.biolscience.cn / DeepLBCEPred / ), and the epitopes with scores greater than 0.5 were selected as candidate epitopes.The instability index, hydrophilicity index, antigen index, toxicity, and allergenicity of the 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), ToxinPred2 (https: / / webs.iiitd.edu.in / raghava / toxinpred2 / index.html), and the like, and finally the dominant linear B cell epitopes were selected. The coding DNA of the B cell dominant epitopes derived from the four proteins of Brucella SurA, OMP31, BP26, and Trigger factor were connected using the coding DNA of the KK linker to construct the DNA encoding the concatenated B cell epitopes, and the nucleotide sequence is shown as SEQ ID No. 2: 433-1101 bp, and the corresponding amino acid sequence is shown as SEQ ID No. 1: 145-367 aa. The coding DNA of the ribosomal protein L7 / L12 derived from Brucella and the coding DNA of the PADRE sequence were connected using the coding DNA of the EAAAK linker, and the nucleotide sequences are shown as SEQ ID No. 2: 1-372 bp, 388-426 bp, respectively, and the corresponding amino acid sequences are shown as SEQ ID No. 1: 1-124 aa, 130-142 aa, respectively. Finally, the L7 / L12-PADRE sequence-catenated B cell epitope recombinant protein was formed, and the amino acid sequence is shown as SEQ ID No. 1, and the nucleotide sequence of the encoding gene is shown as SEQ ID No. 2.

[0025] SEQ ID No. 1:

[0026] MADLAKIVEDLSALTVLEAAELSKLLEEKWGVSAAAPVAVAAAGGAAPAAAAEEKTEFDVVLADGGANKINVIKEVRALTGLGLKEAKDLVEGAPKAVKEGASKDEAEKIKAQLEAAGAKVELKEAAAKAKFVAAWTLKAAAKKSEDEKEAEKVLDGKADKKVVDISDEEVDEQVKRIASSTRTFETKKGKAENEDRVTIDYLGKLDGEPFEGGADNDAQKKNVTDKKVSKEELTAEDEDAASEAKPAKKAAAKKKAEEGKSEEAKKPFSSFDKEDNEQVSGSLDVTAKKIYVYPDDKNNLKEPTITGYSVSKKSKDVKAAGVNRTTKPHDTEKGVEKKEGADSPAGQEKKAEELSKKYVQELREKA.

[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 to the pET-28a expression vector to construct the recombinant expression plasmid pET-28a-L7 / L12-PADRE sequence-multimeric B cell epitope. The recombinant plasmid was transformed into E. coli Rosetta (DE3) competent cells. The single colony with correct PCR and sequencing was selected for expansion culture. When the OD 600nm of the bacterial solution was about 0.6-1.0, 0.5 mM IPTG was added, and the expression was induced at 37°C and 180 rpm for 4 h. The bacterial cells were collected by centrifugation at 8000 rpm for 10 min, and then were disrupted by ultrasonication for 40 min. The supernatant was collected by centrifugation at 8000 rpm for 40 min, and the precipitate was resuspended. The recombinant bacteria without IPTG induction were set as a negative control. SDS-PAGE electrophoresis was performed on all samples to detect the expression of the recombinant protein.

[0032] The SDS-PAGE results Figure 1 showed that lane 2 had obvious specific bands at the expected molecular weight position, indicating that the recombinant protein was successfully expressed. Lane 3 had clear bands at the same position, while lane 4 had no obvious corresponding bands, indicating that the recombinant protein mainly existed in the supernatant. Lane 5 showed a single clear band at the target position, suggesting that a high-purity recombinant protein was obtained. In addition, lane 1 as a control had obvious target bands, which might be caused by the leakage expression of T7 RNA polymerase in the host bacteria. In summary, the L7 / L12-PADRE sequence-multimeric B cell epitope recombinant protein was efficiently expressed in E. coli, and mainly existed in the cytoplasmic supernatant in a soluble form.

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

[0034] The supernatant of the L7 / L12-PADRE sequence-multimeric B cell epitope recombinant protein was filtered with a 0.45 µm needle filter, and the target protein was purified using a Ni-NTA protein purification gravity column. Gradient elution was performed at 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 eluate were collected for sample preparation, SDS-PAGE electrophoresis, and Coomassie blue staining to observe the purification effect.

[0035] The results show that ( Figure 2 ): Lane 1 shows a target band at the expected molecular weight, indicating good recombinant protein expression. Lane 2 shows significantly less target protein than Lane 1, indicating effective protein adsorption on the nickel column, with most of the target protein adsorbed to the column. Starting in Lane 5, the amount of contaminants decreases, while the target protein remains high. In summary, starting with the 80 mmol / L imidazole eluate, the eluate was collected and placed in a dialysis bag for concentration.

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

[0037] The results show that ( Figure 3 ): An obvious specific color band appeared in lane 1 at about 48kD, which indicated that the recombinant protein could produce a specific immune reaction with the positive serum of Brucella ovis and had good reactivity.

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

[0039] 1. Determination of the optimal coating concentration of the 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-multiple B cell epitope recombinant protein were determined using the checkerboard method. The specific operation was as follows: the recombinant protein was serially diluted with carbonate buffer to a coating concentration of 16.6μg / mL, 8.3μg / mL, 4.15μg / mL, 2.075μg / mL, 1.0375μg / mL, and 0.51875μg / mL, 100μL / well, and incubated at 4°C overnight; washed 3 times with PBST; added 1% BSA blocking solution, 200μL / well, and incubated at 37°C for 2h; after blocking, washed as above, and anti-Brucella positive cells were blocked with PBS. Serum and negative serum were diluted in series at 1:50, 1:100, 1:200, 1:400, 1:800, and 1:1600, respectively, and formed into square arrays, 100 μL / well, and incubated at 37°C for 1 hour; after washing 3 times with PBST, rabbit anti-sheep HRP with a working concentration of 1:5000 was added at 100 μL / well; after washing 3 times, 100 μL / well of color development solution was added and color was developed at 37°C for 15 minutes; 2M H2SO4 was added to terminate the reaction at 50 μL / well; the OD was read on a microplate reader. 450nmValues; by comparing the OD 450nm Values and P / N values to determine the optimal antigen coating concentration and serum dilution. When the positive serum OD 450nm value is greater than 1, P / N > 2.1, and the negative value is low, P / N value is high, the corresponding antigen concentration and serum working concentration are the optimal antigen coating concentration and the best dilution concentration of the serum to be tested. As can be seen from Table 1, the optimal antigen coating concentration is 4.15 μg / mL, and the best dilution multiple of positive and negative serum is 1:400.

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

[0042]

[0043] 2, determination of blocking solution;

[0044] The enzyme-labeled plate was coated with the optimal antigen concentration, incubated at 4°C overnight, washed with PBST solution the next day, 3 times, 2 min each time, and dried. The blocking solution was divided into 4 groups: group I was 5% skim milk; group II was 1% BSA; group III was 1% gelatin; group IV was 1M ammonium chloride. 200 μL of each group was added to the coated enzyme-labeled plate, and the plate was blocked at 37°C for 2 h, then washed with PBST solution 3 times, 2 min each time, and dried. Each group was made in triplicate. After blocking and washing, the optimal concentration of negative serum and positive serum was added, 100 μL / well, and incubated at 37°C for 1 h. After washing, 1:5000 diluted enzyme-labeled antibody (HRP-labeled rabbit anti-sheep secondary antibody) was added, 100 μL / well, and incubated at 37°C for 1 h. After washing, 100 μL of substrate developing solution (3,3',5,5'-tetramethylbenzidine, abbreviated as TMB) was added to each well, and the plate was developed at 37°C for 15 min in the dark; 50 μL of stop solution was added to each well, and the OD 450nm value was measured on the enzyme-labeled instrument within 15 min. The ratio of the mean OD 450nm value of the positive to the mean OD 450nm value of the negative (P / N value) was the highest, and the type of blocking solution was selected as the best blocking solution. As can be seen from Table 2, the P / N value of group IV (1M ammonium chloride) is the highest, which is the best blocking solution.

[0045] Table 2 Determination of optimal blocking solution

[0046]

[0047] 3, determination of the optimal working concentration of enzyme-labeled secondary antibody;

[0048] Brucella antigen protein was diluted to 4.15 μg / mL with carbonate buffer, added to the ELISA plate at 100 μL / well, incubated at 4°C overnight, washed 3 times with PBST solution for 2 minutes each time the next day, and patted dry; 200 μL of 1M ammonium chloride solution was added to each well of the coated ELISA plate, blocked at 37°C for 2 hours, then washed 3 times with PBST solution for 2 minutes each time, and patted dry; 100 μL / well of anti-Brucella positive serum diluted 1:400 with PBS solution was added as a positive control, and 100 μL / well of anti-Brucella negative serum diluted 1:400 with PBS solution was added as a negative control The serum reaction conditions were set at 37°C for 1 hour, then washed 3 times with PBST solution for 2 minutes each time, and patted dry; taking HRP-labeled rabbit anti-goat secondary antibody as an example, the optimal working concentration of the enzyme-labeled secondary antibody was determined, and 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 times, and added to the enzyme-labeled plate at 100 μL / well, and incubated at 37°C for 1 hour. Three parallel wells were made for each group, and then washed 3 times with PBST solution for 2 minutes each time, and patted dry; 100 μL TMB substrate colorimetric solution was added to each well, and the color was developed at 37°C in the dark for 15 minutes; 50 μL stop solution was added to each well, and the OD was measured on the microplate reader within 15 minutes. 450nm value, select positive OD 450nm Mean and negative OD 450nm The enzyme-labeled antibody dilution ratio at which the mean ratio (P / N value) is maximized is taken as the optimal working concentration. 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. Determination of the optimal development time of the substrate color developing solution;

[0052] Dilute the Brucella antigen protein with carbonate buffer to 4.15 μg / mL, add 100 μL / well to the enzyme-labeled plate, incubate at 4°C overnight, wash 3 times with PBST solution, 2 min each time, and pat dry; add 200 μL of 1M ammonium chloride solution to each well of the coated enzyme-labeled plate, block at 37°C for 2 h, and then wash 3 times with PBST solution, 2 min each time, and pat dry; add 100 μL / well of anti-Brucella positive serum diluted 1:400 with PBS solution as a positive control, add 100 μL / well of anti-Brucella negative serum diluted 1:400 with PBS solution as a negative control, and set the serum action conditions at 37°C for 1 h, then wash 3 times with PBST solution, 2 min each time, and pat dry; dilute the enzyme-labeled antibody (HRP-labeled rabbit anti-goat secondary antibody) 1:8000 with PBS solution, add 100 μL / well to the enzyme-labeled plate, and act at 37°C for 1 h, then wash 3 times with PBST solution, 2 min each time, and pat dry; add 100 μL of TMB substrate color developing liquid to each well, develop color at 37°C for 10, 15, 20, 25, and 30 min in the dark; add 50 μL of stop solution to each well, and measure the OD 450nm values on the enzyme-labeled instrument within 15 min, and select the ratio of the positive OD 450nm mean value to the negative OD 450nm mean value (P / N value) when the color development time is the maximum. As shown in Table 4, 10 min is the optimal color development time.

[0053] Table 4 Color development time determination

[0054]

[0055] 5. Determination of positive and negative critical values;

[0056] According to the optimal reaction conditions determined above, perform iELISA tests on 45 anti-Brucella negative serum samples, repeat each serum sample 3 times, take the average of the 3 values, and calculate the average value (X) of the 45 anti-Brucella negative serum samples as 0.42491 and the standard deviation (SD) as 0.0872 according to the OD 450nm values, thereby determining the positive and negative critical value point C = X + 3SD = 0.6864, i.e., the OD 450nm value of the serum to be tested ≥ 0.6864 is judged to be positive, and less than 0.6864 is judged to be negative (Table 5).

[0057] Table 5 Determination of positive and negative critical values

[0058]

[0059] 6. Specificity test;

[0060] According to the above determined optimum reaction conditions, iELISA test was carried out to detect sheep PPR positive serum, sheep FMD positive serum, sheep CCPP positive serum, each serum sample was repeated 3 times, and the average value of 3 times was compared with the positive and negative critical value. At the same time, according to the above determined optimum reaction conditions, HRP labeled sheep anti mouse secondary antibody was used for iELISA test to detect mouse anti Brucella abortus A19 strain positive serum (anti Brucella antibody positive control group), anti enterotoxigenic E. coli positive serum, anti Staphylococcus aureus positive serum, anti Acinetobacter baumannii positive serum, anti Pseudomonas aeruginosa positive serum, anti Salmonella positive serum, anti Yersinia enterocolitica positive serum and their corresponding mouse negative serum, each serum sample was repeated 3 times, and the average value of 3 times was compared with the OD 450nm Average value and P / N value of mouse positive serum. As can be seen from table 6-1, the OD 450nm Average value of PPR, FMD and CCPP positive serum is less than 0.6864, which is judged as negative. As can be seen from table 6-2, the OD 450nm Average value of anti Brucella abortus A19 strain positive serum (anti Brucella antibody positive control group) is greater than 1, and P / N is greater than 2.1, which is judged as positive; the OD 450nm Average value of anti enterotoxigenic E. coli positive serum, anti Staphylococcus aureus positive serum, anti Acinetobacter baumannii positive serum, anti Pseudomonas aeruginosa positive serum, anti Salmonella positive serum and Yersinia enterocolitica positive serum is less than 1, and P / N is less than 2.1, which is judged as negative. In summary, the established iELISA method has good specificity.

[0061] Table 6-1 specific detection

[0062]

[0063] Table 6-2 specific detection

[0064]

[0065] 7. Sensitivity test;

[0066] The Brucellosis positive control sheep serum was diluted by sample diluent at 1:160, 1:320, 1:640, 1:1280, 1:2560, 1:5120, 1:10240 and 1:120480, and each group had 3 parallel holes, and then iELISA test was carried out on the positive serum of different dilution. The results showed that (table 7) when the serum dilution ratio was 1:1280, the detection result was still positive (OD 450nm Average value was greater than 0.6864, and P / N was greater than 2.1), which indicated that the sensitivity of the method was good.

[0067] Table 7 Sensitivity test

[0068]

[0069] 8. Intra-batch repeatability and inter-batch repeatability test;

[0070] Take 16 samples of sheep serum, and detect them according to the optimal reaction conditions determined above, with 3 repeats for each sample, measure the OD 450nm values, calculate the average (x) and standard deviation (SD), and determine the intra-batch repeatability of the detection method by the variation range of the coefficient of variation (standard deviation / average x 100%). Then use 16 samples of sheep serum to detect different batches according to the optimal reaction conditions determined above, a total of 3 batches, measure the OD 450nm values, calculate the average (x) and standard deviation (SD) of the samples in different batches, and determine the inter-batch repeatability of the detection method by the variation range of the coefficient of variation (standard deviation / average x 100%). According to the detection results, the coefficient of variation is calculated: CV=(SD÷X) x 100%. The results show (Table 8) that the intra-batch coefficient of variation of the 16 samples of sheep serum is in the range of 0.90-5.54%, all less than 10%; the inter-batch repeatability coefficient of variation is in the range of 2.41-9.01%, all less than 10%. It is shown that the intra-batch and inter-batch repeatability and stability of the established iELISA method are good.

[0071] Table 8 Intra-batch repeatability and inter-batch repeatability test

[0072]

[0073] Example 4: Detection of clinical samples and comparative analysis with the Rose Bengal plate agglutination test;

[0074] According to the optimal reaction conditions determined above, the iELISA test is carried out, and at the same time, the Rose Bengal plate agglutination test is used to detect 48 serum samples. The results show (Table 9) that the detection results of the iELISA method of the application and the Rose Bengal plate agglutination test have high consistency. Among the 48 serum samples, only 2 samples (No. 4 and No. 37) have inconsistent results by the two detection methods, and the remaining 46 samples are consistent. The coincidence rate is 95.83%, which reflects that the iELISA method of the application has good reliability and applicability in the detection of clinical samples, and can more accurately reflect the content level of anti-Brucella antibody in serum samples, providing an effective detection means for the clinical diagnosis of brucellosis, and having good application prospect.

[0075] Table 9 Comparison of detection results of the Rose Bengal plate agglutination test and the iELISA method of the application

[0076]

[0077]

[0078] The above clinical sample detection is only taken as an example of sheep serum sample, other kinds of samples, such as milk sample, tissue sample, organ sample, etc., can be pretreated, and the reagent kit can also detect the anti-Brucella antibodies therein. For other animal sample detection, only different animal enzyme-labeled secondary antibodies need to be replaced.

[0079] The above is only a preferred embodiment of the present application, and it should be noted that, for those skilled in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which should also be considered as the protection scope of the present application, and these will not affect the effect of the present application and the practicability of the patent.

Claims

1. A L7 / L12-PADRE sequence-multiple B cell epitope recombinant protein, characterized in that: The L7 / L12-PADRE sequence-multi-linked B cell epitope recombinant protein is formed by connecting the Brucella ribosomal L7 / L12 protein, the PADRE polypeptide sequence, and the multi-linked B cell epitope through an EAAAK linker and a KK linker, respectively, and its amino acid sequence is shown in SEQ ID No.

1.

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

2.

3. Use of the L7 / L12-PADRE sequence-multi-linked 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-Brucella antibodies.

4. The use according to claim 3, characterized in that The L7 / L12-PADRE sequence-multi-linked B cell epitope recombinant protein was used as the coating antigen, and an iELISA method was established to detect anti-Brucella antibodies.

5. A kit for detecting anti-brucella antibodies based on iELISA, characterized in that, The kit comprises: an enzyme-labeled plate coated with the L7 / L12-PADRE sequence-multi-linked B cell epitope recombinant protein according to claim 1; a blocking solution; a washing solution; a diluent; an enzyme-labeled reagent; a substrate color development solution; a positive standard serum; and a negative standard serum.

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

7. The 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 rabbit anti-sheep antibody labeled with horseradish peroxidase; the substrate color developing solution is 3,3',5,5'-tetramethylbenzidine solution; the positive standard serum is anti-Brucella positive serum; and the negative standard serum is anti-Brucella negative serum.

8. The kit according to claim 5, wherein The optimal dilution ratio of the enzyme-labeled reagent is 1:8000; the optimal reaction time of the substrate color developing solution is 10 min, and the optimal dilution ratio of the positive standard serum and the negative standard serum is 1:

400.

9. The kit according to claim 5, wherein The kit measures the OD of the serum sample to be tested. 450nm The value is used to judge whether the anti-Brucella antibody is present. The judgment standard is: when OD 450nm When the OD value is less than 0.6864, the serum sample to be tested does not contain anti-Brucella antibodies and is judged to be negative; 450nm If the value is ≥0.6864, the serum sample to be tested contains anti-Brucella antibodies and is judged to be positive.

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