Brucella sulfur-containing domain protein EntF and its application

Abstract

The present invention provides a new protein antigen EntF for diagnosing brucellosis, whose amino acid sequence is SEQ ID No: 1, and the corresponding nucleotide sequence is SEQ ID NO: 2. The new protein antigen EntF for diagnosing brucellosis provided by the present invention and the indirect ELISA detection method established based on the EntF antigen do not cross-react with other common pathogenic bacteria sera of cattle and sheep, and the indirect ELISA based on the EntF antigen has high specificity and good repeatability. The antigen protein EntF provided by the present invention can be used for serological diagnosis of bovine brucellosis, and 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 sulfur-containing domain protein EntF 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. It is an animal epidemic disease that must be reported by the World Organization for Animal Health (WOAH). The Law of the People's Republic of my country on the Prevention and Control of Infectious Diseases stipulates that it is a Class B infectious disease. Brucellosis has been reported in all provinces (autonomous regions and municipalities) in China, but the positive rate in northern provinces is higher and the positive rate in southern provinces is relatively low. In recent years, with the expansion of breeding scale and the increasing frequency of livestock and poultry transportation, the epidemic of brucellosis in animals has shown a trend of spreading from north to south. At the same time, human brucellosis has become an important public health issue. The prevention and control of brucellosis in animals is crucial to the safety of my country's animal husbandry production, public health safety and national biosecurity.

[0003] Serological methods for diagnosing brucellosis include red tiger 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 the staff. According to the approval requirements 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 novel protein antigen EntF for diagnosing brucellosis and application of the antigen in serological diagnosis of bovine brucellosis.

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

[0006] MHHHHHHKTQQAAMPPNATSPHQADVYLLRGFADIFSTGIDEIGAELQAAGVNAHVQGHAAWRLVLNRIVADQQKNGHLPVVLIGHSLGANAAIYIAEELERRGIAVDYMATFAATGPDPLPGNVRRVVNFYFKQHGWGLPLVPGPRFHGHLENRDFSNAKDVGHFNIEKQRPLQAEVVRDVLAVVNAD;

[0007] The present invention also provides a nucleotide sequence corresponding to EntF, SEQ ID NO: 2:

[0008] .

[0009] In another aspect, the present invention further provides a recombinant expression vector, into which the nucleic acid fragment of the antigen gene is inserted;

[0010] As a specific description of the embodiment, the recombinant expression vector is a prokaryotic expression vector pET-30(a).

[0011] In another aspect, the present invention further provides a recombinant engineering strain, wherein the recombinant engineering strain is transfected with the above-mentioned recombinant expression vector;

[0012] The present invention also provides an application of the antigen protein, which is application in the preparation of Brucella serological detection products.

[0013] The serological detection product, as a specific description of the embodiment, is an indirect ELISA detection product.

[0014] The new protein antigen EntF for diagnosing brucellosis provided by the present invention and the indirect ELISA detection method established based on the EntF 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), and the indirect ELISA based on the EntF antigen has high specificity and good repeatability. The antigen protein EntF 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

[0015] Figure 1 : New protein antigen rEntF for diagnosis of brucellosis prokaryotic expression (A) and purification (B)

[0016] (A) Figure 1 is the electrophoresis of rEntF before induction; 2 is the electrophoresis of the supernatant after rEntF induced by 20℃ IPTG; 3 is the electrophoresis of the precipitate after rEntF induced by 20℃ IPTG; 4 is the electrophoresis of the supernatant after rEntF induced by 37℃ IPTG; 5 is the electrophoresis of the precipitate after rEntF induced by 37℃ IPTG; M is the protein molecular weight standard. (B) Figure 1 is the purification of EntF protein, and M is the protein molecular weight standard.

[0017] Figure 2 :Western blot of the new protein antigen rEntF for brucellosis diagnosis reacting with bovine brucellosis serum, where 1 is the reaction of rEntF with bovine Brucella positive serum, and 2 is the reaction of rEntF with bovine Brucella negative serum. DETAILED DESCRIPTION

[0018] The new protein antigen for diagnosing brucellosis provided by the present invention is the sulfur-containing domain protein EntF of the toxicity-related substrate of the twin-arginine transport system of Brucella. The present invention uses molecular biological means to construct an EntF prokaryotic expression vector, transfers the vector into the expression strain Rosetta (DE3), obtains a positive recombinant, performs induction expression and purification, and obtains the EntF protein antigen. The protein antigen can react with positive serum of bovine brucellosis, but not with negative serum, and a serological diagnosis method for bovine brucellosis is established based on the good antigenicity of EntF. The indirect ELISA based on EntF has a compliance rate of 92.6% 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 92.2%, the compliance rates with the fluorescence polarization test (FPA) are 91.2%, and the compliance rates with the test tube agglutination test (SAT) are 89.0%. The new brucellosis diagnostic protein antigen EntF 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.

[0019] The present invention is described in detail below in conjunction with embodiments and drawings.

[0020] Example 1: Expression and purification of Brucella sulfur-containing domain protein EntF

[0021] 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 sulfur-containing domain protein EntF were determined. The target fragment was synthesized, and the prokaryotic expression vector pET30(a)-EntF was constructed. The vector was transferred into the expression strain, and the positive recombinant was obtained for induced expression and purification to obtain the EntF protein antigen. The specific steps are as follows:

[0022] (1) Transformation: The recombinant expression vector pET30(a)-EntF 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;

[0023] (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;

[0024] (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.

[0025] (4) Collecting bacteria: centrifuge, discard the supernatant, and collect the bacteria;

[0026] (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.

[0027] (6) Large-scale expression: The Brucella recombinant protein was expressed in large quantities according to the above conditions.

[0028] (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 rEntF protein concentration is determined to be 0.82 mg / mL. Aliquot and store at -80°C.

[0029] Example 2: Antigenicity Identification of Brucella Sulfur-containing Domain Protein EntF

[0030] The purified rEntF 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 cells were incubated at 4°C overnight, washed 3 times with PBST, and SPG-HRP (1:10000) was added as the secondary antibody. The cells were incubated at 25°C for 2h, washed 3 times with PBST, and developed with ECL chemiluminescence detection reagent. The results are shown in Figure 2 As shown, rEntF can react well with bovine brucellosis positive serum, but not with negative serum, which proves that the Brucella sulfur domain protein EntF has good antigenicity.

[0031] Example 3: Establishment of a serological diagnostic method for bovine brucellosis based on EntF

[0032] The specific steps for establishing the serological diagnostic method for bovine brucellosis ELISA (Entf-iELISA) based on EntF are as follows:

[0033] 3.1 Determination of Entf-i ELISA antigen coating concentration and serum dilution

[0034] (1) Coating: The rEntF 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 to each well of a 96-well ELISA plate horizontally, with 2 replicates per group, and the coating was carried out at 4°C overnight.

[0035] (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.

[0036] (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.

[0037] (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.

[0038] (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.

[0039] (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.

[0040] 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 rEntF coating concentration was 0.78μg / mL and the serum dilution was 1∶400, the P / N value was the largest, reaching 5.27. Therefore, the optimal antigen coating concentration and serum dilution were 0.78μg / mL and 1∶400, respectively.

[0041] Table 1: Determination of optimal antigen coating concentration and serum dilution

[0042]

[0043] 3.2 Optimization of reaction conditions of EntF-iELISA method

[0044] 3.2.1 Optimization of antigen coating conditions

[0045] rEntF was coated under four conditions: 37℃2h plus 4℃ overnight incubation, 4℃ overnight incubation, 37℃2h incubation and 37℃1h incubation. Bovine Brucella positive and negative sera were set at the same time, each with 3 replicates, and the OD was read by microplate 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.

[0046] Table 2: Determination of optimal antigen coating conditions

[0047]

[0048]

[0049] 3.2.2 Optimization of closure time

[0050] 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.

[0051] Table 3: Determination of the optimal closure time

[0052]

[0053] 3.2.3 Optimization of blocking solution

[0054] 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.

[0055] Table 4: Determination of the best blocking solution

[0056]

[0057] 3.2.4 Optimization of serum reaction time

[0058] 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 60 minutes was the optimal serum reaction time.

[0059] Table 5: Determination of optimal serum reaction time

[0060]

[0061] 3.2.5 Optimization of secondary antibody concentration

[0062] 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 1:10000.

[0063] Table 6: Determination of optimal secondary antibody dilution

[0064]

[0065] 3.2.6 Optimization of secondary antibody reaction time

[0066] 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 60 minutes was the optimal secondary antibody reaction time.

[0067] Table 7: Determination of the optimal secondary antibody reaction time

[0068]

[0069] Optimized EntF-iELISA: Purified Brucella protein antigen rEntF was diluted to 0.78 μg / mL with coating solution and coated on the ELISA plate at 100 μL / well, coated at 37°C for 1 h, washed 3 times with PBST, blocked with 5% skim milk powder 200 μL / well at 37°C for 2 h, washed 3 times with PBST, serum was diluted 1:400 with PBST and added to the ELISA plate at 100 μL / well, incubated at 37°C for 120 min, washed 3 times with PBST, HRP-labeled goat anti-bovine IgG was diluted 1:10000 with PBST and added to the ELISA plate at 100 μL / well, incubated at 37°C for 60 min, washed 3 times with PBST, added color development solution at 100 μL / well, developed at room temperature in the dark for 15 min, added 50 μL / well stop solution, and read OD with an ELISA reader 450 value.

[0070] 3.2.7 Determination of critical value of rEntF-iELISA method

[0071] The optimized EntF-iELISA was used to test 50 negative bovine sera, and the test results of the 50 negative sera are shown in Table 8. The average values ​​of the rEntF-iELISA method were 0.2444, and the standard deviations SD were 0.0536. Based on ±2SD as the critical judgment standard for positive and negative, the critical value of this method was 0.3517.

[0072] Table 8: OD of negative serum detected by rEntF bovine brucellosis ELISA method 450 value

[0073]

[0074]

[0075] Example 4: Evaluation of EntF-iELISA specificity, repeatability and sensitivity

[0076] The optimized EntF-iELISA was further evaluated for specificity, repeatability and sensitivity.

[0077] 4.1 Specificity evaluation

[0078] The optimized EntF-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 OD 450 The ratios of the values ​​were all less than 2.1, indicating that EntF-iELISA had good specificity.

[0079] Table 9: Results of specific cross-experiment of ELISA test method for bovine brucellosis antibody

[0080]

[0081] 4.2 Repeatability evaluation

[0082] The same batch of purified rEntF was coated on an ELISA plate, and three positive and negative sera for brucellosis were detected, and three replicates were set for each serum. The intra-plate coefficient of variation was detected, and the results are shown in Table 10. The same batch of purified rEntF was coated on three ELISA plates, and three positive and negative sera for brucellosis were detected, and three replicates were set for each serum. The inter-plate coefficient of variation was detected, and the results are shown in Table 9.

[0083]

[0084] 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 EntF-iELISA had good reproducibility.

[0085] Table 10: Repeatability results of rEntF-iELISA test method for bovine brucellosis antibody

[0086]

[0087] 4.3 Sensitivity evaluation

[0088] 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 rEntF as the coating antigen can reach 1:800.

[0089] Table 11: Sensitivity test table of bovine brucellosis antibody ELISA test method

[0090]

[0091] Example 5: EntF-iELISA clinical serum validation and method comparison

[0092] EntF-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 at the same time, and the consistency of EntF-iELISA and other methods was compared. From Tables 12 to 15, it can be seen that EntF-iELISA has the highest consistency with the imported commercial Brucella antibody detection kit IDVET-iELISA, and the consistency rates of IDVET-iELISA, RBT, FPA, and SAT methods are 92.6%, 92.2%, 91.2%, and 89.0%, respectively.

[0093] Table 12: Comparison of rEntF-ELISA method and IDVET-ELISA kit test results

[0094]

[0095] Table 13: Comparison of rEntF-ELISA and red tiger plate agglutination test results

[0096]

[0097] Table 14: Comparison of rEntF-ELISA method and fluorescence polarization test results

[0098]

[0099] Table 15: Comparison of rEntF-ELISA and tube agglutination test results

[0100]

[0101] In summary, the new brucellosis diagnostic protein antigen EntF and the indirect ELISA detection product established based on the EntF 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 antigen preparation and no biosafety risk. Moreover, 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.

[0102] 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. Application of a protein antigen having an amino acid sequence of SEQ ID No: 1 in the preparation of Brucella serological detection products.

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

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