Brucella CN hydrolase YxeI and its application

Abstract

The present invention provides a protein antigen YexI for diagnosing brucellosis, whose amino acid sequence is SEQ ID No: 1, and the nucleotide sequence of the encoding gene is SEQ ID NO: 2. The new protein antigen YexI for diagnosing brucellosis provided by the present invention and the indirect ELISA established based on the YexI antigen do not cross-react with other common pathogenic bacteria sera of cattle and sheep, and the indirect ELISA based on the YexI antigen has high specificity and good repeatability, and has a high compliance rate with imported commercial detection kits, which can reach 90.5%. The antigen protein YexI provided by the present invention can be used for serological diagnosis of bovine brucellosis, has the characteristics of high specificity, good repeatability, easy preparation of antigens and no biosafety risks, and a variety of serological diagnostic products can be developed based on the protein, providing new ideas and new means for the prevention and control of bovine brucellosis.

Description

Technical Field

[0001] The invention belongs to the technical field of human and animal disease detection, and specifically relates to a Brucella CN hydrolase YxeI and an application thereof in the diagnosis of bovine brucellosis. Background Art

[0002] Brucellosis (hereinafter referred to as brucellosis) is an important zoonotic infectious disease caused by Brucella and is an animal epidemic disease that must be reported according to the World Organization for Animal Health (WOAH).

[0003] Serological methods for diagnosing brucellosis include red bengal plate agglutination test (RBT), tube agglutination test (SAT), enzyme-linked immunosorbent assay (ELISA), fluorescence polarization test (FPA) and immunochromatography (ICA). Most of the above serological methods are based on lipopolysaccharide antigens. However, there are many problems with lipopolysaccharide as a diagnostic antigen. First, a large number of Brucella bacteria need to be cultured to extract Brucella lipopolysaccharide. The extraction process is complicated, time-consuming and labor-intensive, and there is also a risk of infection for workers. According to the approval requirements of the Ministry of Agriculture and Rural Affairs for highly pathogenic pathogenic microorganisms, experimental activities must be carried out in a biosafety level 3 laboratory (BSL-3), which greatly limits the application of this method. Second, Brucella lipopolysaccharide antigens are prone to cross-reaction with pathogens such as Yersinia enterocolitica O9 and Escherichia coli O157:H7 in serology, resulting in misdiagnosis. Therefore, the defects of traditional serological detection methods have prompted us to urgently find new and better antigens for the diagnosis of brucellosis. Summary of the invention

[0004] The invention provides a new protein antigen YxeI for diagnosing brucellosis and application of the antigen in serological diagnosis of bovine brucellosis.

[0005] The present invention first provides a new protein antigen YxeI for diagnosing brucellosis, whose amino acid sequence is SEQ ID No: 1:

[0006] MHHHHHHCTSFVLPTSDGGMVYGRTMEFGFNLKSDMIAIPRNYTITASGPDGAAGKKWKGKYATIGMNAFGIVALTDGMNEKGLAGGLLYFPEYAKYQDPSTAKPEDSLAPWDFLTWALANFSTVAEVKDALSTISIVDVKQKDLGFTPPAHYTLHDATGASIVIEPID GKLKVYDNKLGVMNTNSPSFDWHMTNLRNYVYLSRENPKPLQILGETIQSFGQGAGMHGIPGDTTPPSRFVRASAYVLSAKKVPSGLESVRLAEHIANNFDIPKGWSEEQNMFEYTQWTAFADMKNDVYYIKTYDDQVLRSFSFKDFDVDSKDILTIKFEPKLDAPSLKK;

[0007] The present invention also provides a gene encoding the above antigen, a specific nucleotide sequence of which is SEQ ID NO: 2;

[0008]

[0009] The present invention also provides a recombinant expression vector and an expression strain of the YxeI antigen protein;

[0010] The invention also provides application of the YxeI antigen protein in serological diagnosis of bovine brucellosis.

[0011] The new protein antigen YxeI for diagnosing brucellosis provided by the present invention and the indirect ELISA established based on the YxeI antigen do not cross-react with the sera of other common pathogenic bacteria of cattle and sheep (such as Escherichia coli, Salmonella, Vibrio cholerae, Legionella, and Yersinia intestinalis). The indirect ELISA based on the YxeI antigen has high specificity and good repeatability, and has a high compliance rate with imported commercial detection kits, which can reach 90.5%. The antigen protein YxeI provided by the present invention can be used for serological diagnosis of bovine brucellosis, has the characteristics of high specificity, good repeatability, easy preparation of antigens and no biosafety risks, and a variety of serological diagnostic products can be developed based on the protein, providing new ideas and new means for the prevention and control of bovine brucellosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 :Prokaryotic expression (A) and purification (B) of the new protein antigen rYxeI for diagnosis of brucellosis, (A) 1 is the electrophoresis diagram of rYxeI before induction; 2 is the electrophoresis diagram of the supernatant after rYxeI induced with IPTG at 20℃; 3 is the electrophoresis diagram of the precipitate after rYxeI induced with IPTG at 20℃; 4 is the electrophoresis diagram of the supernatant after rYxeI induced with IPTG at 37℃; 5 is the electrophoresis diagram of the precipitate after rYxeI induced with IPTG at 37℃; M is the protein molecular weight standard. (B) 1 is the YxeI protein purification diagram, M is the protein molecular weight standard.

[0013] Figure 2 :Western blot of the new protein antigen rYxeI for brucellosis diagnosis and the bovine brucellosis serum reaction. 1 is the reaction of rYxeI with bovine Brucella positive serum, 2 is the reaction of rYxeI with bovine Brucella negative serum. DETAILED DESCRIPTION

[0014] The new protein antigen for diagnosing brucellosis provided by the present invention is the CN hydrolase protein YxeI of the toxicity-related substrate of the twin-arginine transport system of Brucella. The present invention uses molecular biological means to construct a YxeI prokaryotic expression vector, transfers the vector into the expression strain Rosetta (DE3), obtains a positive recombinant, performs induction expression and purification, and obtains the YxeI protein antigen. The protein antigen can react with positive serum of bovine brucellosis and has no reaction with negative serum. A serological diagnosis method for bovine brucellosis is established based on the good antigenicity of YxeI. The indirect ELISA based on YxeI has a compliance rate of 90.5% with the imported commercial ELISA kit (ID-VET). Compared with other serological methods, the compliance rates with the red bengal plate agglutination test (RBT) are 91.2%, the compliance rates with the fluorescence polarization test (FPA) are 89.8%, and the compliance rates with the test tube agglutination test (SAT) are 83.4%. The new brucellosis diagnostic protein antigen YxeI provided by the present invention can be used for the serological diagnosis of bovine brucellosis, has the characteristics of high specificity, good repeatability, easy antigen preparation and no biosafety risk, and a variety of serological diagnostic products can be developed based on the protein, which can provide new ideas and new means for the prevention and control of bovine brucellosis.

[0015] The present invention is described in detail below in conjunction with embodiments and drawings.

[0016] Example 1: Expression and purification of Brucella CN hydrolase YxeI

[0017] Based on the Brucella M28 genome sequence (chromosome 1: NC_017244.1), the amino acid sequence (SEQ ID NO: 1) and nucleotide sequence (SEQ ID NO: 2) of the Brucella twin-arginine transport system virulence-related substrate CN hydrolase protein YxeI were determined. The target fragment was synthesized, and the prokaryotic expression vector pET30(a)-YxeI was constructed. The vector was transferred into the expression strain, and the positive recombinant was obtained for induced expression and purification to obtain the YxeI protein antigen. The specific steps are as follows:

[0018] (1) Transformation: The recombinant expression vector pET30(a)-YexI was transformed into Rosetta (DE3) competent cells and cultured on LB solid plates containing a final concentration of 30 μg / mL kanamycin and 34 μg / mL chloramphenicol;

[0019] (2) Activation: The selected single clones were cultured in a liquid culture medium containing a final concentration of 30 μg / mL kanamycin and 34 μg / mL chloramphenicol;

[0020] (3) Induction: When the OD value reached 0.6, the inducer 0.5 mM IPTG was added and the culture was continued. The culture was carried out at 20°C overnight and at 37°C for 6 h. The culture without the addition of the inducer was used as the negative control.

[0021] (4) Collecting bacteria: centrifuge, discard the supernatant, and collect the bacteria;

[0022] (5) Expression detection: PBS was added to the collected bacteria for suspension, and the cells were fully dissolved using an ultrasonic disruptor and centrifuged. The precipitate after centrifugation was dissolved using PBS, and the supernatant and precipitate were processed separately, and samples were prepared for SDS-PAGE detection.

[0023] (6) Large-scale expression: The Brucella recombinant protein was expressed in large quantities according to the above conditions.

[0024] (7) Purification: Take 5 mL of Ni-NTA filler and wash the equilibrated column with 25 mL of equilibration buffer at a flow rate of 5 mL / min. Add crude protein and incubate at 4°C for 1 h. Then, wash the column again with equilibration buffer. After equilibration, wash the column with elution buffer and collect the effluent. Place the eluted fraction in a dialysis bag and dialyze it into protein buffer. After concentration, use polyethylene glycol (PEG) 20000, filter at 0.45 μm, and quantify it using a BCA kit. The rYxeI protein concentration is determined to be 0.43 mg / mL. Aliquot and store at -80°C.

[0025] Example 2: Antigenicity Identification of Brucella CN Hydrolase YxeI

[0026] The purified rYxeI protein in Example 1 was subjected to SDS-PAGE gel electrophoresis, and the recombinant protein on the gel was transferred to a PVDF membrane using a fast wet transfer instrument, and blocked at 25°C for 1h with 5% skim milk powder. The positive serum of bovine brucellosis (1:100) was used as the primary antibody, and the negative serum of the control group was used as the primary antibody. The membrane was incubated at 4°C overnight, washed 3 times with PBST, and SPG-HRP (1:10000) was added as the secondary antibody. The membrane was incubated at 25°C for 2h, washed 3 times with PBST, and developed with ECL chemiluminescent detection reagent. The results are shown in the attached figure. Figure 2 As shown, rYxeI can react well with bovine brucellosis positive serum, but not with negative serum, which proves that Brucella CN hydrolase protein YxeI has good antigenicity.

[0027] Example 3: Establishment of a serological diagnostic method for bovine brucellosis based on YxeI

[0028] The specific steps for establishing the YxeI-based bovine brucellosis ELISA (YxeI-iELISA) serological diagnostic method are as follows:

[0029] 3.1 Determination of YxeI-iELISA antigen coating concentration and serum dilution

[0030] (1) Coating: The rYxeI protein obtained in Example 1 was diluted to 6.25, 3.125, 1.56, 0.78, and 0.39 μg / mL using coating solution, and 100 μL was added horizontally to each well of a 96-well ELISA plate, with 2 replicates per group, and the coating was carried out at 4°C overnight.

[0031] (2) Blocking: Discard the antigen coating solution, add 300 μL PBST to each well, shake for 3 min, and wash 3 times. After washing, add 100 μL 5% skim milk powder to each well and block at 37°C for 1 h.

[0032] (3) Incubation with primary antibody: discard the solution and wash the plate. Dilute the serum with sample diluent at 1:100, 1:200, 1:400 and 1:800, add 100 μL to each well, add vertically into the ELISA plate, and incubate at 37°C for 1 h.

[0033] (4) Incubation with secondary antibody: discard the solution and wash the plate, dilute HRP-goat anti-bovine IgG with PBST at 1:8000, add 100 μL to each well, and incubate at 37°C for 1 h.

[0034] (5) Color development: discard the solution and wash the plate, add 100 μL of single-component color development solution to each well, and develop the color for 10 min at room temperature in the dark.

[0035] (6) Stop reading: Add 50 μL ELISA stop solution to each well and read at OD 450nm The values ​​of each well were read using an enzyme-labeled instrument.

[0036] The results are shown in Table 1. The P / N value (P / N value = positive serum OD 450 Value / negative serum OD 450 The maximum value was taken as the optimal antigen coating concentration and serum dilution. The results showed that when the rYxeI coating concentration was 0.78μg / mL and the serum dilution was 1∶200, the P / N value was the largest, reaching 3.41, so the optimal antigen coating concentration and serum dilution were 0.78μg / mL and 1∶200 respectively.

[0037] Table 1: Determination of optimal antigen coating concentration and serum dilution Table 3.2 Optimization of reaction conditions of YxeI-i ELISA method

[0038]

[0039] 3.2.1 Optimization of antigen coating conditions

[0040] rYxeI was coated under four conditions: 37°C for 2 h plus 4°C overnight incubation, 4°C overnight incubation, 37°C for 2 h, and 37°C for 1 h. Bovine Brucella positive and negative sera were set at the same time, with 3 replicates in each well, and the OD was read by an ELISA reader. 450 The results are shown in Table 2. The condition with the largest P / N value was selected as the best coating condition. The results showed that incubation at 37℃ for 1h was the best coating condition.

[0041] Table 2 Determination of optimal coating conditions

[0042]

[0043] 3.2.2 Optimization of closure time

[0044] The blocking time was selected as 60 min, 90 min and 120 min at 37 °C, and positive and negative sera of bovine brucellosis were set at the same time, with 3 replicates for each well, and the OD was read by the microplate reader. 450 The results are shown in Table 3. The condition with the maximum P / N value was selected as the optimal blocking time. The results showed that incubation at 37℃ for 120min was the optimal blocking time.

[0045] Table 3: Determination of the optimal closure time

[0046]

[0047] 3.2.3 Optimization of blocking solution

[0048] 1% skim milk powder, 5% skim milk powder, 1% BSA, 5% BSA and commercial blocking solution were selected as blocking solution, and positive and negative sera of bovine brucellosis were set at the same time, with 3 replicates in each well, and the OD was read by microplate reader. 450 The results are shown in Table 4. The optimal blocking solution was selected when the P / N value was the largest. The results showed that 5% skim milk powder was the optimal blocking solution.

[0049] Table 4: Determination of the best blocking solution

[0050]

[0051]

[0052] 3.2.4 Optimization of serum reaction time

[0053] 37℃30min, 60min, 90min and 120min were selected as the test serum reaction time, and positive and negative sera of bovine brucellosis were set at the same time, with 3 replicates for each, and the OD was read by the microplate reader. 450The results are shown in Table 5. The optimal reaction time was selected when the P / N value was the largest. The results showed that incubation at 37°C for 120 minutes was the optimal serum reaction time.

[0054] Table 5: Determination of optimal serum reaction time

[0055]

[0056] 3.2.5 Optimization of secondary antibody concentration

[0057] The enzyme-labeled antibody (HRP goat anti-bovine IgG from Abcam, concentration of 1 mg / mL) was diluted at 1:2000, 1:4000, 1:6000, 1:8000, 1:10000 and 1:12000, respectively. At the same time, positive and negative sera of bovine brucellosis were set up, each with 3 replicates, and the OD was read by a microplate reader. 450 The values ​​were averaged and the results are shown in Table 6. The conditional optimal secondary antibody dilution was selected when the P / N value was the largest. The results showed that the optimal secondary antibody dilution was 1:8000.

[0058] Table 6: Determination of optimal secondary antibody dilution

[0059]

[0060] 3.2.6 Optimization of secondary antibody reaction time

[0061] The reaction time of enzyme-labeled secondary antibody was 30min, 40min, 50min and 60min respectively. Positive and negative sera of bovine brucellosis were set at the same time. Three replicates were set for each well. OD was read by microplate reader. 450 The results are shown in Table 7. The optimal secondary antibody reaction time was selected when the P / N value was the largest. The results showed that incubation at 37°C for 50 minutes was the optimal secondary antibody reaction time.

[0062] Table 7: Determination of the optimal secondary antibody reaction time

[0063]

[0064] The indirect ELISA detection method for bovine brucellosis antibodies established according to the above optimized conditions is as follows: the purified Brucella recombinant protein YxeI was diluted to 0.78 μg / mL with coating solution, coated on the ELISA plate at 100 μL / well, coated at 37°C for 1 hour, washed 3 times with PBST, blocked with 5% skim milk powder 200 μL / well at 37°C for 2 hours, washed 3 times with PBST, serum was diluted with PBST at a dilution of 1:200 and added to the ELISA plate at 100 μL / well, incubated at 37°C for 120 minutes, washed 3 times with PBST, HRP-labeled goat anti-bovine IgG was diluted with PBST at 1:8000 and added to the ELISA plate at 100 μL / well, incubated at 37°C for 50 minutes, washed 3 times with PBST, added with color developing solution at 100 μL / well, developed at room temperature in the dark for 15 minutes, added 50 μL / well stop solution, and read the OD with an ELISA reader. 450 value.

[0065] 3.2.7 Determination of critical value of rYxeI-iELISA method

[0066] The established indirect ELISA method was used to test 50 negative bovine sera. The test results of the 50 negative sera are shown in Table 8. The average value of the rYxeI-iELISA method They are 0.3478 and the standard deviation SD is 0.0616 respectively. It is the critical judgment standard of yin and yang, and the critical value of this method is 0.4711.

[0067] Table 8: OD of negative serum detected by rYxeI bovine brucellosis ELISA method 450 Value Table

[0068]

[0069]

[0070] Example 4: Evaluation of specificity, repeatability and sensitivity of Yxel-i ELISA

[0071] The optimized YxeI-iELISA was further evaluated for specificity, repeatability and sensitivity.

[0072] 4.1 Specificity evaluation

[0073] The optimized YxeI-iELISA was used to detect positive sera of Yersinia enterocolitica, Escherichia coli, Vibrio cholerae, Salmonella, and Legionella. Positive and negative sera of Brucella bovis were set as controls. Three replicates were set for each serum. The OD was read by an enzyme reader. 450 The results are shown in Table 9. The results show that the above positive serum and Brucella negative serum OD450 The ratios of the values ​​were all less than 2.1, indicating that the established bovine brucellosis antibody ELISA detection method had good specificity.

[0074] Table 9: Results of specific cross-experiment of ELISA test method for bovine brucellosis antibody

[0075]

[0076]

[0077] 4.2 Repeatability evaluation

[0078] The same batch of purified Brucella protein was coated on the ELISA plate, and 3 positive and negative sera for Brucellosis were tested, with 3 replicates for each serum, and the intra-plate coefficient of variation was tested. The results are shown in Table 10.

[0079] The same batch of purified Brucella protein was coated on three ELISA plates, and three positive and negative sera were tested for Brucellosis, with three replicates for each serum. The inter-plate coefficient of variation was tested, and the results are shown in Table 10.

[0080] Coefficient of variation (CV) = standard deviation (SD) / mean

[0081] The results showed that the intra-plate coefficient of variation was and the inter-plate coefficient of variation was, both less than 10%, indicating that the established bovine brucellosis antibody ELISA detection method has good repeatability.

[0082] Table 10: Repeatability results of bovine brucellosis antibody rYxeI-iELISA detection method

[0083]

[0084] 4.3 Sensitivity evaluation

[0085] Three brucellosis positive sera and one negative serum were taken and diluted at 1:50, 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200 and 1:6400, and tested with the established bovine brucellosis antibody detection method. The results are shown in Table 11. It shows that the serum sensitivity of the established ELISA method with rYxeI as the coating antigen can reach 1:400.

[0086] Table 11: Sensitivity test results of bovine brucellosis antibody ELISA test method

[0087]

[0088]

[0089] Example 5: Yxel-iELISA clinical serum validation and method comparison

[0090] YxeI-iELISA and imported commercial Brucella antibody detection kit (IDVET-iELISA), red bengal plate agglutination test (RBT), fluorescence polarization test (FPA), and tube agglutination test (SAT) were used to test 283 bovine serum samples, and the consistency of YxeI-iELISA and other methods was compared. From Tables 12 to 15, the consistency of YxeI-iELISA with IDVET-iELISA, RBT, FPA, and SAT methods was 90.5%, 91.2%, 89.8%, and 83.4%, respectively.

[0091] Table 12: Comparison of the test results of rYxeI-ELISA method and IDVET-ELISA kit

[0092]

[0093] Table 13: Comparison of rYxeI-ELISA and red tiger plate agglutination test results

[0094]

[0095] Table 14: Comparison of rYxeI-ELISA method and fluorescence polarization test results

[0096]

[0097] Table 15: Comparison of rYxeI-ELISA and tube agglutination test results

[0098]

[0099]

[0100] In summary, the new brucellosis diagnostic protein antigen YxeI and the indirect ELISA established based on the YxeI antigen provided by the present invention can be used for the serological diagnosis of bovine brucellosis, and have the characteristics of high specificity, good repeatability, easy preparation of antigens and no biosafety risks. In addition, a variety of serological diagnostic products can be developed based on the protein, providing new ideas and new means for the prevention and control of bovine brucellosis.

[0101] The above description is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, any modifications or improvements made without departing from the technical principles of the present invention are all within the scope of protection required by the present invention.

Claims

1. Use of an antigen protein having an amino acid sequence of SEQ ID No: 1 in the preparation of a product for detecting bovine Brucella.

2. The use according to claim 1, characterized in that The product is an ELISA detection product.

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