Application of Rv0394c protein as stimulating antigen for detecting mycobacterium bovis infection

Abstract

The invention discloses application of Rv0394c protein as a stimulating antigen for detecting mycobacterium bovis infection, and belongs to the technical field of biology. The invention discloses a mycobacterium tuberculosis immunodominance antigen Rv0394c for the first time, and the mycobacterium tuberculosis immunodominance antigen Rv0394c has high sensitivity when being used as a bovine mycobacterium (M. bovis) infected gamma-interferon release test stimulating antigen and has the potential of being used as a skin test stimulating antigen.

Description

Technical Field

[0001] The invention relates to application of Rv0394c protein as a stimulating antigen for detecting bovine mycobacterium infection, and belongs to the field of biotechnology. Background Art

[0002] Bovine tuberculosis caused by Mycobacterium bovis (M. bovis) is a chronic wasting zoonosis. Although tuberculosis caused by Mycobacterium bovis accounts for a small proportion of tuberculosis, a large amount of data shows that the burden of Mycobacterium bovis on humans may be underestimated, especially in developing countries. Therefore, early diagnosis of bovine tuberculosis is crucial to controlling the spread of bovine tuberculosis.

[0003] At present, the main method for testing bovine tuberculosis at home and abroad is cellular immunology detection, such as intradermal hypersensitivity (TST), but it is cumbersome and time-consuming, and can no longer meet the requirements of modern clinical diagnosis and quarantine. Therefore, the method with the most application value and prospects is the bovine tuberculosis peripheral blood γ-interferon test. The stimulating antigen currently used in the bovine tuberculosis peripheral blood γ-interferon test is still the tuberculin purified protein derivative (PPD), which has complex antigen composition, poor specificity, and cumbersome preparation process; in recent years, a large number of studies have used Mycobacterium tuberculosis specific antigen proteins CFP10 and ESAT6 as stimuli, and the specificity has been improved, but the diagnostic sensitivity is poor. Therefore, it is imperative to explore and develop bovine tuberculosis cell immune stimulating antigen proteins with high sensitivity and specificity. Summary of the invention

[0004] Purpose of the invention: The purpose of the present invention is to provide an application of Rv0394c protein as a stimulating antigen for detecting bovine Mycobacterium infection.

[0005] Technical solution: The present invention provides the use of Rv0394c protein as a stimulating antigen in a γ-interferon release test for detecting Mycobacterium bovis (M. bovis) infection.

[0006] Furthermore, the amino acid sequence of the Rv0394c protein is shown in SEQ ID NO: 1.

[0007] Furthermore, the nucleotide sequence encoding the Rv0394c protein is as shown in SEQ ID NO:2.

[0008] The present invention also provides a stimulation antigen, which includes the above-mentioned Rv0394c protein.

[0009] Furthermore, the stimulation antigen is obtained by connecting the nucleotide sequence as described in SEQ ID NO: 2 to the pCold vector, transferring it into competent cells DH5α, screening for carboxybenzyl resistance, and then transferring it into competent cells BL21 (DE3).

[0010] Furthermore, the primer sequences for amplifying the nucleotide sequence described in SEQ ID NO: 2 are shown in SEQ ID NO: 3-4.

[0011] The present invention also provides a bovine peripheral blood interferon-γ stimulating antigen reagent, which comprises the above-mentioned stimulating antigen.

[0012] Furthermore, the reagent also includes CFP10-ESAT6 fusion protein.

[0013] The present invention also provides a skin test detection reagent for bovine tuberculosis, wherein the reagent comprises the above-mentioned stimulation antigen.

[0014] Furthermore, the concentration of the stimulation antigen is 25-50 μg.

[0015] Beneficial effects: Compared with the prior art, the present invention has the following outstanding and significant advantages: The present invention discloses for the first time an immunodominant antigen Rv0394c of Mycobacterium tuberculosis, which has high sensitivity when used as a stimulating antigen for Mycobacterium bovis (M. bovis) infection, and has the potential to be used as a stimulatory antigen for skin test. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 : Target gene PCR amplification results and protein expression diagram. Figure 1 A is the PCR amplification result of Mycobacterium tuberculosis gene Rv0394c`, lane M is Maker DL15000, and lane 1 is the amplification result of Rv0394c; Figure 1 B is a PAGE image of the Mycobacterium tuberculosis gene Rv0394c, lane M is the Protein Marker, lane 2 is the empty vector control induced by IPTG; lane 3 is the precipitate induced by the recombinant strain; lane 4 is the supernatant of the induced strain; lane 5 is the purified Mycobacterium tuberculosis antigen protein Rv0394c; Figure 1 C is Western blot analysis of protein expression of Mycobacterium tuberculosis gene Rv0394c.

[0017] Figure 2 :The level of IFN-γ release was detected by stimulating spleen lymphocytes of H37Ra infected mice with protein. Figure 2 A is the IFN-γ response level after stimulation with CFP10-ESAT6 fusion protein; Figure 2 B is the IFN-γ response level after stimulation with Rv0394c protein alone; Figure 2 C is the IFN-γ response level after mixed stimulation of Rv0394c protein and CFP10-ESAT6 fusion protein.

[0018] Figure 3:Results of IFN-γ release test in peripheral blood of bovine tuberculosis.

[0019] Figure 4 :Results of Rv0394c protein as irritant in guinea pig skin test. Figure 4 A is the average diameter of the reaction after injection of bovine tuberculin PPDB; Figure 4 B is the average diameter result of the reaction after Rv0394c injection.

[0020] Figure 5 :Results of Rv0394c protein as irritant in guinea pig skin test. Figure 5 A is the result of local redness and swelling 24 hours after PBS injection. Figure 5 B is the result of local redness and swelling 48 hours after PBS injection; Figure 5 C is the result of local redness and swelling 24 hours after injection of bovine tuberculin PPDB (5 IU on the left and 10 IU on the right); Figure 5 D is the result of local redness and swelling 48 hours after injection of bovine tuberculin PPDB (5 IU on the left and 10 IU on the right); Figure 5 E is the result of local redness and swelling 24 hours after Rv0394c injection (25 μg on the left and 50 μg on the right); Figure 5 F is the result of local redness and swelling 48 hours after Rv0394c injection (25 μg on the left and 50 μg on the right). DETAILED DESCRIPTION

[0021] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0022] Example 1 Screening and cloning and expression of bovine tuberculosis stimulating antigen protein

[0023] 1. PCR amplification of Rv0394c gene

[0024] By using bioinformatics methods, the genome sequences of pathogenic mycobacteria and non-pathogenic mycobacteria (including pathogenic bacteria: Mycobacterium bovis, Mycobacterium tuberculosis, Mycobacterium caprae; non-pathogenic bacteria: Mycobacterium avium, Mycobacterium paratuberculosis, Mycobacterium smegmatis, Mycobacterium marinum, Mycobacterium kansasii, Mycobacterium abscessus, Mycobacterium intracellulare) were compared, and 149 differential antigens were screened out. The protein secretion, antigenicity, immunogenicity and other properties were predicted by combining UniProt and VaxiJ methods, and the Rv0394c protein (NCBI reference sequence is WP_003401942.1, SE Q ID NO:1:MTEPRPVFAVVISAGLSAIPMVGGPLQTVFDAIEERTRHRAETTTREICES) was determined. VGGADTVLSRIDKNPELEPLLSQAIEAATRTSMEAKRRLLAQAAAAALEDDQKVEPASLIVATLSQLEPVHIHALVRLAKAAKSSPDQDEIQRREVMRAASKVEPVPVLAALIQTGVAIATTTVWHGNGTGTPAEESGHILIHDVSDFGHRLLAYLRAADAGAELLILPSGGSAPTGDHPTPHPSTSR) is the target protein antigen.

[0025] Using the genomic DNA of the standard strain of Mycobacterium tuberculosis (MTB) H37Rv as a template, primmer5.0 was used to design overlapping primers (Table 1). The following reaction system and reaction procedure were used to amplify the Rv0394c gene (GenBank accession number is CP136197.1, SEQ ID NO: 2: atgact) using primers F and R (SEQ ID NO: 3-4).

[0026] Table 1. Primer sequences of fusion gene Rv0394c

[0027]

[0028] The PCR reaction system included: 2× Taq Master mix 12.5 μL, upstream primer 1.0 μL, downstream primer 1.0 μL, template (dsDNA) 2.0 μL, H 2 2. 4% dHO 8.5 μL.

[0029] PCR reaction program: 95℃ pre-denaturation for 5min; 95℃ denaturation for 50s; 58℃ annealing for 50s; 72℃ extension for 1min; 30 cycles; final extension at 72℃ for 10min; storage at 4℃. Figure 1 As shown in A. 2. Construction of pCold-Rv0394c recombinant plasmid

[0030] Take 5 μL of Rv0394c gene PCR product and perform 1% agarose gel electrophoresis. After the band is correctly identified, use gel recovery kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., item number DP219-03) to recover the target fragment. Use Xho I and XbaI to double-digest the prokaryotic expression vector plasmid empty vector pColdI (purchased from takara, item number 3360-3364), the digestion time is 3 hours, and the temperature is 37°C. The digestion recovery system is shown in Table 2.

[0031] Table 2. Enzyme digestion recovery system

[0032] Components volume XOt 1μL Xgf 1μL 10×MBuffer 2μL pCold Plasmid 10μL <![CDATA[H 2 The]]> 6μL

[0033] The target gene fragment was connected to the vector after restriction digestion using ClonExpress Ultra One Step Cloning Kit. The connection system is shown in Table 3.

[0034] Table 3. Ligation reaction system

[0035]

[0036] The ligation product was heat-transferred into 100 μL of competent cells DH5α in a volume not exceeding 10 μL, and spread on an LB plate containing carboxybenzyl resistance. After a single colony grows on the carboxybenzyl plate (about 12 hours), a single colony was aseptically picked and transferred to 5mL LB liquid culture medium. After shaking and culturing for 14 hours, 2mL of bacterial solution was aseptically aspirated for preservation. A portion of the bacterial solution was subjected to PCR identification, and the rest was extracted with conventional methods for plasmids, and double enzyme digestion was performed with Xho I and Xba I for identification. Send positive clones for sequencing. After culturing at 37°C overnight, a single colony was picked for culture and recombinant plasmids were extracted. PCR identification and enzyme digestion identification were performed using the recombinant plasmid as a template. The verified correct plasmid was sent to Qingke (Nanjing) Biological Company for sequencing. After sequencing was correct (720bp), the bacterial solution was preserved ( Figure 1 A). Enzyme digestion identification system is shown in Table 4.

[0037] Table 4. Enzyme digestion identification system

[0038] Components volume XOt 1μL Xgf 1μL 10×MBuffer 2μL Recombinant plasmid 10μL SW 6μL

[0039] 3. Expression and purification of recombinant protein Rv0394c

[0040] The recombinant plasmids that were sequenced and identified correctly were transferred into BL21 (DE3) competent cells, and PCR identification was performed to screen positive clones. The correct colonies were identified for overnight culture and inoculated into carb LB liquid culture medium at a ratio of 1:100 the next day, cultured at 37°C for 3-4h until the OD value reached 0.4-0.6, and then 1.0mM IPTG was added and cultured at 15°C and 150rpm for 24h. The induced bacterial solution was collected by centrifugation at 4°C and 8000rpm for 10min, washed three times with PBS, resuspended and lysed with an ultrasonic disruptor, and the supernatant and precipitate were collected at 4°C and 10000rpm, respectively, and the precipitate was resuspended with 40μL PBSC, 20μL of each supernatant and precipitate were added to 5μL 5x loading buffer, boiled in boiling water for 10min, and analyzed by 12% SDS-PAGE. After confirming the expression of the recombinant prokaryotic protein, 1L of recombinant bacteria was induced again, and after being crushed by a high-pressure crusher, centrifuged at 12000r / min for 10min, the supernatant and the precipitate were boiled at the same time, and the sample was tested by 12% SDS-PAGE. After confirming the expression of the recombinant prokaryotic protein, 1L of recombinant bacteria was induced again, and after being crushed by a high-pressure crusher, centrifuged at 12000r / min for 10min, the supernatant and the precipitate were boiled at the same time, and the sample was tested by 12% SDS-PAGE to determine the expression form of the recombinant protein. As a result, BL21 (DE3) pET32a-Rv0394c expressed soluble protein.

[0041] Affinity chromatography is based on the principle that the target protein is retained by the specific binding of the solid-phase ligand, while other impurities flow through the column. Since the fusion protein carries 6 histidines at the end, the histidine side chain can be affinity with metal ions such as nickel, zinc and cobalt. Under neutral and weakly alkaline conditions, the target protein with histidine tag binds to the Ni-NTA agarose resin column, allowing the fusion protein to be directly purified. The operation process is as follows:

[0042] (1) Take a Ni-NTA prepacked column of corresponding specifications according to the required loading capacity and let the storage buffer flow out by gravity.

[0043] (2) Equilibrate the column with two column volumes of Binding / Wash Buffer and use a flow rate of 1 mL / min to allow the Buffer to slowly drain from the resin.

[0044] (3) Prepare the sample solution by mixing the protein extract (lysate supernatant, which has been filtered) with Binding / Wash Buffer at a ratio of 1:1 so that the total volume of the sample solution is twice the column volume.

[0045] (4) Add the sample solution to the column and collect the flow-through into a centrifuge tube. If there is excess sample, re-load it. Re-circulating once can improve the binding force between the sample and the filler.

[0046] (5) Wash the column with two column volumes of Binding / Wash Buffer and collect the flow-through. Repeat this step using a new collection tube until the absorbance of the flow-through at 280 nm is close to the baseline.

[0047] (6) Elute the histidine-tagged protein on the column with twice the column volume of Elution Buffer. Repeat this step twice and store the eluate separately until the absorbance of the eluate at 280 nm is close to the baseline.

[0048] The purified protein sample was mixed with protein loading buffer at a ratio of 4:1, and the sample was boiled at 100℃ for 10 minutes before SDS-PAGE electrophoresis; the protein band was transferred to the NC membrane and blocked with PBST containing 5% skim milk for 2 hours; the blocking solution was discarded, and PBST was washed three times, each time for 5 minutes; PBST was discarded, and His primary antibody liquid diluted 1:1000 was added, and incubated at 4℃ overnight; His primary antibody was recovered, and NC membrane was washed 5 times with PBST, each time for 5 minutes; PBST was discarded, and goat anti-mouse secondary antibody diluted with 5% skim milk (1:5000 dilution) was added and incubated at room temperature for 2 hours; secondary antibody was recovered, and PBST was washed 5 times, each time for 5 minutes; West Pico PLUS chemiluminescent substrate was used for color development for 1 minute, and the membrane was exposed and imaged in a chemiluminescent imaging system and the image was saved. The results are as follows Figure 1 As shown in B, the target band of the recombinant protein was visible at around 26.3 kD.

[0049] Example 2 Evaluation of the immunological properties of Rv0394c protein

[0050] 1. Establishment of H37Ra infection mouse model

[0051] Female C57BL / 6 mice aged 6-8 weeks were purchased from Vital River Company. All animal experiments were approved by the Animal Welfare and Ethics Committee of Yangzhou University and were performed in accordance with the experimental guidance of the Institutional Management Committee and the Experimental Animal Ethics Committee. The mouse carcasses after the experiment were sent to the Animal Carcass Harmless Disposal Center of Yangzhou University for harmless destruction.

[0052] Take H37Ra with good growth and adjust the bacterial solution concentration to 5x10 6CFU / ml, 6-8 week old BALB / c female mice were injected through the tail vein, 100 μL per mouse, and a PBS control group (100 μL / mouse) was set up at the same time, with 6 mice in each group, to establish a tuberculosis infection model. Four weeks after infection, the mice were killed and spleen cells were taken for experiments.

[0053] 2. Evaluation of the immune characteristics of recombinant proteins using H37Ra infected mouse model

[0054] Four weeks after infection, mice were euthanized, and the spleens of mice were removed by aseptic dissection in a biosafety cabinet and placed in a plate with ice-bathed CM (complete RPMI 1640 medium). The spleens were ground with a frosted glass slide and the cells were filtered through a 70 μm cell filter; an appropriate amount of CM was added and centrifuged at 4°C, 1000 rpm for 10 min; the supernatant was discarded, 3 mL of red blood cell lysis buffer was added and the cell pellet was resuspended, and the lysis was carried out at room temperature for 5 min; 2 volumes of DPBS were immediately added to terminate the reaction; centrifuged at 4°C, 1000 rpm for 10 min; the cell pellet was resuspended in CM and continued to be centrifuged at 4°C, 1000 rpm for 10 min for washing, and this process was repeated twice; finally, 3 mL of fresh CM was added to resuspend the cells, and the cells of each group were counted using a cell counter and kept on ice for later use.

[0055] The H37Ra-infected mouse spleen lymphocytes were stimulated with protein, and the cell supernatant was collected 48 hours later. The IFN-γ release level was detected by IFN-γ ELISA kit (Hexu (Zhengzhou) Biotechnology Co., Ltd.). The specific steps were to adjust the number of mouse spleen lymphocytes to 1×10 6 200 μL of each well was added to a 96-well plate, and 20 μL of protein diluted with complete RPMI1640 medium (20 μg / mL, respectively, Rv0394c protein prepared in Example 1, CFP10-ESAT6 fusion protein (constructed in this laboratory), Rv0394c protein + CFP10-ESAT6 fusion protein (1:1) was added. A negative control (1640 medium) was also set up. The cell plate was placed at 37°C and 5% CO 2 After 48 h of culture in a cell culture incubator, the cell supernatant was collected and the following operations were performed:

[0056] (1) One day before the experiment, coat the ELISA plate with mouse IFN-γ capture antibody, dilute 1:250, 100 μL / well, and incubate at 4°C overnight;

[0057] (2) The next day, the cells were washed three times with PBST and blocked with PBS containing 2% BSA at 37°C for 2 h;

[0058] (3) Wash 4 times with PBST, add the supernatant of each cell to be tested, 100 μL / well (50 μL sample + 50 μL PBS dilution), and add the commercial standard rIFN-γ starting from 2000 pg / mL to make a serial dilution as a standard curve control, and incubate at 37°C for 2 h;

[0059] (4) Wash 5 times with PBST, add biotinylated detection antibody, dilute 1:250 (add 1:250 secondary antibody and HRP to the diluent at the same time, add in dark), 100 μL / well, and place at 37°C for 2 h;

[0060] (5) Wash 7 times with PBST, add TMB substrate for color development, and 2M H 2 SO 4 The reaction was terminated and the OD was read by enzyme-linked immunosorbent reader. 450 and OD 570 The content of IFN-γ cytokine (pg / mL) in the cell supernatant of each group was calculated according to the standard curve.

[0061] The results are as follows Figure 2 As shown in the results, the IFN-γ level was significantly increased by Rv0394c protein stimulation, and the infected group and the uninfected group could be distinguished (P < 0.05). At the same time, when the Rv0394c protein was mixed with CFP10-ESAT6 fusion protein for stimulation, the infected group and the uninfected group could be distinguished, and the response level was significantly higher than that of the CFP10-ESAT6 fusion protein alone stimulation group (P < 0.05).

[0062] Example 3 Evaluation and identification of the stimulating effect of Rv0394c protein

[0063] The IFN-γ release test of bovine tuberculosis peripheral blood was carried out on Rv0394c protein, CFP10-ESAT6 protein and CFP10-ESAT6+Rv0394c protein to evaluate their stimulating effects.

[0064] 1. Collection and preparation of blood samples

[0065] (1) Sample collection

[0066] Fourteen bovine blood samples were collected from four cattle farms. 5 ml of bovine tail venous blood was collected and added to a sodium heparin anticoagulation vacuum tube. The tube was gently inverted several times to mix evenly, and 250 μL of the anticoagulated blood was added to each well of a 96-well cell culture plate.

[0067] (2) Antigen stimulation

[0068] In a 96-well cell culture plate with bovine blood, 25 μL PBS, 25 μL avian PPD (PPDA), 25 μL bovine PPD (PPDB), a final concentration of 10 μg / mL of Mycobacterium tuberculosis fusion protein Rv0394c, and a final concentration of 10 μg / mL CFP10-ESAT6+Rv0394c protein were aseptically added, and the mixture was fully shaken and mixed with a micro-oscillator to completely mix the stimulating antigen with the blood, and incubated in a 37°C wet incubator for 16 hours. Centrifuge for 10 minutes at 4°C and 2000 rpm, collect the upper plasma, and store at -80°C for subsequent testing. 14 bovine plasma samples were tested using a commercial bovine tuberculosis detection kit (Hexu (Zhengzhou) Biotechnology Co., Ltd.), all of which were positive and the protein stimulation effect was evaluated.

[0069] The premise of the kit is the negative control OD 450 When PPDB stimulated OD 450 Value - PBS stimulation OD 450 Value ≥ 0.1, and PPDB stimulates OD 450 Value - PPDA stimulation OD 450 A value ≥ 0.1 was considered positive, otherwise it was considered negative.

[0070] 2. Evaluation of the stimulatory effect of the Mycobacterium tuberculosis fusion protein Rv0394c in tuberculosis-positive cattle

[0071] The experiment included verification of differential expression of IFN-γ levels in serum of tuberculosis-positive bovines stimulated by Mycobacterium tuberculosis Rv0394c protein and CFP10-ESAT6+Rv0394c protein.

[0072] The indirect ELISA procedure for detecting the Mycobacterium tuberculosis fusion protein Rv0394c and CFP10-ESAT6+Rv0394c protein is as follows:

[0073] Coating antibody: purified monoclonal antibody 2G5 (purified and stored by GenScript), coating concentration is 5 μg / mL.

[0074] Enzyme-labeled antibody diluent: Use PBS containing 1% BSA to dilute HRP-5E11 (purified, labeled and stored by Nanjing Zhongding Biotechnology Co., Ltd.), 1:1000 dilution.

[0075] Coating solution (CBS): laboratory prepared, anhydrous Na 2 CO 3 :0.1696g, NaHCO 3 :0.2856g, add SW to make up to 100mL.

[0076] The negative control was fetal bovine serum, and the positive control was tuberculosis-positive bovine serum, both of which were stored in the laboratory.

[0077] (1) Take the ELISA plate one day before the experiment, add 100 μL of 5 μg / mL 2G5 to each well, and coat it at 4°C overnight;

[0078] (2) The next day, discard the coating solution, wash three times with PBST, pat dry, add 150 μL of blocking solution (PBS containing 2% BSA) to each well, and incubate at 37°C for 2 h;

[0079] (3) Discard the blocking solution, wash four times with PBST, pat dry, add 50 μL of sample diluent (PBS) and 50 μL of plasma stimulated with each protein for 16 h to each well, and incubate at 37°C for 2 h;

[0080] (4) Discard the sample solution, wash five times with PBST, pat dry, add 100 μL of enzyme-labeled secondary antibody 5E11 (1:1000 dilution) to each well, and incubate at 37°C for 2 h;

[0081] (5) Discard the enzyme-labeled secondary antibody, wash 7 times with PBST, pat dry, add 100 μL TMB colorimetric solution to each well, develop at 37°C in the dark for 10 min, and add 50 μL 2M H 2 SO 4 Stop the reaction and measure OD 450 The result is Figure 3 As shown. The OD mean of the PPDB stimulation result was 1.59 (PPDB-PBS), the OD mean of the CFP10-ESAT6 fusion protein alone stimulation group was 0.5655 (CE), and the OD mean of the Rv0394c alone stimulation group was 0.26, which had a better effect (Rv0394c-PBS). At the same time, the OD mean of the Rv0394c protein and CFP10-ESAT6 mixed stimulation was 1.12 (CE+Rv0394c-PBS), and the IFN-γ response of the mixed stimulation group was significantly increased compared with the CFP10-ESAT6 fusion protein alone stimulation group. The above results show that Rv0394c protein has good potential as a stimulator for the in vitro release test of IFN-γ in bovine tuberculosis peripheral blood.

[0082] Example 4 Evaluation and identification of the stimulating effect of Rv0394c protein as a guinea pig skin test irritant

[0083] 1. Establishment of a guinea pig model infected with Mycobacterium bovis

[0084] The experimental animals were three 6-8 week old female Hartley GuineaPig SPF grade guinea pigs. All animal experiments were approved by the Animal Welfare and Ethics Committee of Yangzhou University and were carried out in accordance with the experimental guidance of the Institutional Management Committee and the Experimental Animal Ethics Committee. The guinea pig carcasses after the experiment were handed over to the Animal Carcass Harmless Disposal Center of Yangzhou University for harmless destruction.

[0085] Take Mycobacterium bovis allergen (donated by China Veterinary Drug Administration), dilute each Mycobacterium bovis allergen with 1 mL of vaccine emulsified white oil preheated to 45°C, and inject subcutaneously into the inner side of the leg (thigh groin) of each guinea pig, about 0.1 mL / pig, to infect 4-week-old 6-8-week-old female Hartley Guinea Pigs, to establish a tuberculosis infection model.

[0086] 2. Evaluation and identification of the irritation effect of Rv0394c protein skin test

[0087] Four weeks after all guinea pigs were infected with bovine tuberculosis allergen, the hair on both sides of the spine of the guinea pigs was removed and the guinea pigs were randomly divided into three groups, namely the PBS group, the PPDB group, and the Rv0394c protein group. In the PPDB group, 1IU PPDB and 0.5IU PPDB were intradermally injected at 3 points on both sides of the spine. In the Rv0394c protein group, 50μg Rv0394c protein and 25μg Rv0394c protein were intradermally injected at 3 points on both sides of the spine. In the PBS group, 0.1mL PBS was intradermally injected at both sides of the spine. The longitudinal and transverse diameters of the local redness and swelling were observed and recorded 24 and 48 hours after the injection, and the average value was calculated (the sum of the longitudinal and transverse diameters divided by 2). The judgment criteria were: the average redness and swelling reaction diameter ≥5mm was judged as positive, and <5mm was judged as negative. The results are as follows Figure 4 As shown, the skin test results of the PBS injection group were negative, and the skin test results of the PPDB group on guinea pigs sensitized to Mycobacterium tuberculosis were all positive ( Figure 4 A), indicating that guinea pigs were successfully sensitized. At the same time, the skin test results of the 50μg Rv0394c protein group in the Mycobacterium tuberculosis sensitized guinea pigs were all positive. In the guinea pig model sensitized by bovine Mycobacterium tuberculosis, the reaction intensity of the 50μg Rv0394c protein group was significantly higher than that of the 25μg Rv0394c protein group, and there was a relatively obvious dose-effect relationship (the guinea pig redness and swelling results are shown in Figure 2). Figure 5 The above results show that although the effect of Rv0394c protein is not as good as PPDB, it can also be used as a skin test stimulant, and the preparation method is simple, so it has good application potential.

Claims

1. Application of Rv0394c protein as a stimulating antigen for detecting Mycobacterium bovis (M. bovis) infection.

2. The use according to claim 1, characterized in that: The amino acid sequence of the Rv0394c protein is shown in SEQ ID NO:

1.

3. The use according to claim 2, characterized in that: The nucleotide sequence encoding the Rv0394c protein is shown in SEQ ID NO:

2.

4. A stimulating antigen, characterized in that The stimulating antigen comprises the Rv0394c protein according to claim 1.

5. The stimulating antigen according to claim 4, characterized in that The stimulation antigen is obtained by connecting the nucleotide sequence as described in SEQ ID NO: 2 to the pCold vector, transferring it into competent cells DH5α, screening for carboxybenzyl resistance, and then transferring it into competent cells BL21 (DE3).

6. The stimulating antigen according to claim 5, characterized in that The primer sequences for amplifying the nucleotide sequence described in SEQ ID NO: 2 are shown in SEQ ID NOs: 3-4.

7. A bovine peripheral blood interferon-γ stimulating antigen reagent, characterized in that: The reagent comprises the stimulating antigen according to any one of claims 4 to 6.

8. The bovine peripheral blood interferon-γ stimulating antigen reagent according to claim 7, characterized in that: The reagents also include CFP10-ESAT6 fusion protein.

9. A skin test reagent for bovine tuberculosis, characterized in that: The reagent comprises the stimulating antigen according to any one of claims 4 to 6.

10. The skin test detection reagent for bovine tuberculosis according to claim 9, characterized in that: The concentration of the stimulation antigen is 25-50 μg.

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