A method for rapid detection of sturgeon infected with Mycobacterium marinum

Abstract

The present invention provides a method for rapidly detecting sturgeon infection with Mycobacterium marinum, belonging to the field of biological detection technology. The method uses the Ag85B and Rpf virulence proteins of Mycobacterium marinum as Mycobacterium marinum-specific antigens to stimulate sturgeon T cells. A commercial rabbit-derived polyclonal antibody for IFN-γ is used as a capture antibody, and a prepared mouse-derived polyclonal antibody for IFN-γ is used as a detection antibody. An indirect sandwich ELISA is used to detect IFN-γ secretion to determine whether the sturgeon is infected with Mycobacterium marinum. The detection method of the present invention is simple and easy to operate, suitable for routine laboratory use, and has high sensitivity and accuracy.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and particularly relates to a method for rapidly detecting sturgeons infected with Mycobacterium marinum. Background Art

[0002] Sturgeons, belonging to the class of bony fish, represent a crucial branch point in the evolutionary tree of the immune system, holding a crucial position in the evolution of fishes and, indeed, vertebrates as a whole. In 2010, they were listed as Critically Endangered on the IUCN Red List of Threatened Species. To protect this endangered species, sturgeon aquaculture has been developed in my country. With the increasing intensification of aquaculture, the threat of disease is increasing, seriously threatening the healthy and sustainable growth of sturgeon populations. Among these, mycobacterial disease in sturgeons has become a major constraint to the development of aquaculture. Mycobacterium marinum, Mycobacterium fortuitum, and Mycobacterium chelonae are common pathogens of fish. Under natural conditions, Mycobacterium marinum is a pathogen of over 150 species of fish and frogs, as well as freshwater eels and oysters. Mycobacterium marinum is often used as an excellent model for studying Mycobacterium tuberculosis in secondary laboratories due to its low virulence, low infectivity, and 85% nucleotide identity with Mycobacterium tuberculosis.

[0003] Currently, there is no unified standard for the diagnosis of Mycobacterium marinum. Common diagnosis of Mycobacterium marinum requires isolation and culture of mycobacteria from clinical samples, followed by identification of the bacterial species through molecular biological methods, ultimately identifying the pathogen. Isolation and culture still require at least one to two weeks, and the subsequent time-consuming identification of the bacterial species significantly delays diagnosis and treatment. DNA microarray chip technology cannot simultaneously detect pathogens and perform drug sensitivity testing, and cannot completely replace traditional culture and drug sensitivity testing. It requires specialized chips and scanners, which are relatively expensive. The use of an appropriate pre-treatment process for mass spectrometry identification is key to accuracy, and the pre-treatment requirements are high. The mass spectrometer database does not contain the corresponding fingerprint spectrum, and cannot accurately identify the three mycobacteria, including Mycobacterium marinum. Summary of the Invention

[0004] The present invention provides a method for rapidly detecting sturgeons infected with Mycobacterium marinum. The method is simple and easy to implement and can accurately and sensitively detect sturgeons infected with Mycobacterium marinum.

[0005] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0006] The present invention provides a method for rapidly detecting sturgeons infected with Mycobacterium marinum. The method comprises the following steps: inducing expression of Mycobacterium marinum virulence protein, mixing the virulence protein with sturgeon blood, using the mixed solution as a test sample, and using sturgeon blood without the virulence protein as a background control sample. The IFN-γ protein concentrations in the two samples are detected. If the difference between the test sample and the background control sample is greater than or equal to 14 pg / mL, the result is determined to be positive; if the difference between the test sample and the background control sample is less than 14 pg / mL, the result is determined to be negative.

[0007] Preferably, the virulence protein is one or more of Ag85B protein and Rpf protein.

[0008] Preferably, the method for inducing expression of the virulence protein of Mycobacterium marinum comprises: adding Ag85B-positive bacterial liquid and Rpf-positive bacterial liquid to LB medium containing kanamycin, culturing, and adding IPTG to induce expression of the recombinant protein.

[0009] Preferably, the amount of IPTG added is 0.4-1.2 mmol / L.

[0010] Preferably, the mixture of virulence protein and sturgeon blood and the sturgeon blood are placed in an incubator at 25 to 30° C. and incubated for 20 to 24 hours.

[0011] Preferably, the IFN-γ protein concentration is detected by indirect sandwich ELISA.

[0012] Preferably, the indirect sandwich ELISA method uses rabbit-derived IFN-γ polyclonal antibody as the capture antibody and mouse-derived IFN-γ polyclonal antibody as the detection antibody.

[0013] Preferably, the preparation method of the mouse-derived IFN-γ polyclonal antibody comprises the following steps: immunizing mice three times with IFN-γ protein, collecting mouse serum samples on the 25th to 30th day, and purifying the IFN-γ polyclonal antibody by ammonium sulfate precipitation method to obtain the mouse-derived IFN-γ polyclonal antibody.

[0014] Preferably, the mouse IFN-γ polyclonal antibody needs to be diluted to 1:1000 to 1:3000.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The present invention utilizes Ag85B and Rpf virulence proteins as Mycobacterium marinum-specific antigens to stimulate sturgeon T cells for the first time, and detects IFN-γ secreted by the sturgeon after being stimulated by the antigens to determine whether the sturgeon is infected with Mycobacterium marinum.

[0017] The detection method of the present invention is simple and easy to implement, suitable for routine laboratory implementation, and can accurately and sensitively detect sturgeons infected with Mycobacterium marinum. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Cloning diagram of virulence genes of Mycobacterium marinum; wherein, 1: Marker; 2-4: Ag85B gene (978bp); 5-7: Rpf gene (414bp).

[0019] Figure 2 Identification diagram of positive bacteria transformed with recombinant plasmid into Escherichia coli BL21; 1 and 6: Markers; 2-5: Ag85B gene; 7-10: Rpf gene.

[0020] Figure 3 SDS-PAGE diagram after induction of recombinant protein; 1: Marker; 2: uninduced whole bacteria; 3: Ag85B-induced whole bacteria (34kDa); 4: Rpf-induced whole bacteria (14kDa).

[0021] Figure 4 Western Blot image of recombinant protein; 1: Marker; 2: Ag85B protein; 3: Rpf protein.

[0022] Figure 5 IFN-γ protein SDS-PAGE image; 1: Marker; 2: uninduced whole bacteria; 3: IFN-γ induced whole bacteria (19kDa).

[0023] Figure 6 Western Blot image of IFN-γ protein; 1: Marker; 2: IFN-γ protein.

[0024] Figure 7 ELISA test data of serum of mice immunized with IFN-γ antigen.

[0025] Figure 8 Specificity diagram of IFN-γ polyclonal antibody Western Blot detection; where: 1: Marker; 2: IFN-γ protein; 3: induced whole bacteria.

[0026] Figure 9 Standard curve of IFN-γ protein concentration and OD value. DETAILED DESCRIPTION

[0027] The present invention provides a method for rapidly detecting sturgeons infected with Mycobacterium marinum, comprising the following steps: inducing expression of a virulence protein of Mycobacterium marinum, mixing the virulence protein with sturgeon blood, using the mixture as a test sample, and using sturgeon blood without the virulence protein as a background control sample. Detecting the IFN-γ protein concentration in both samples, determining a positive result if the difference between the test sample and the background control sample is ≥14 pg / mL, and a negative result if the difference between the test sample and the background control sample is <14 pg / mL. The virulence protein of the present invention can stimulate T cells of sturgeons infected with Mycobacterium marinum to secrete IFN-γ, thereby activating macrophages to phagocytose and destroy the mycobacteria. After the sturgeons of the present invention are infected with Mycobacterium marinum, naive T cells begin to become sensitive to Mycobacterium marinum-specific antigens and develop into Mycobacterium marinum-specific effector T cells.

[0028] In the present invention, the virulence protein is one or more of Ag85B protein and Rpf protein, preferably Ag85B protein and Rpf protein. The method of inducing expression of virulence protein of Mycobacterium marinum according to the present invention comprises: adding Ag85B-positive bacterial solution and Rpf-positive bacterial solution to LB culture medium containing kanamycin, culturing, and adding IPTG to induce expression of recombinant protein. The culture conditions of the present invention are 35-40°C, 150-250r / min shaking culture for 2-3h (OD value is 0.6-0.8). The amount of IPTG added according to the present invention is 0.4-1.2mmol / L, preferably 1.0mmol / L when it is an Ag85B-positive bacterial solution, and preferably 0.5mmol / L when it is an Rpf-positive bacterial solution.

[0029] In the present invention, the mixture of virulence protein and sturgeon blood and the sturgeon blood are placed in an incubator at 25-30°C and incubated for 20-24 hours, preferably in an incubator at 28°C for 22 hours. After the mixture of virulence protein and sturgeon blood and the sturgeon blood are incubated, they are centrifuged at 3000-5000 rpm for 2-4 minutes to separate the supernatant (plasma).

[0030] In the present invention, the IFN-γ protein concentration is detected by an indirect sandwich ELISA method. The indirect sandwich ELISA method of the present invention uses a rabbit-derived IFN-γ polyclonal antibody as a capture antibody and a mouse-derived IFN-γ polyclonal antibody as a detection antibody. The preparation method of the mouse-derived IFN-γ polyclonal antibody of the present invention comprises the following steps: immunizing mice three times with IFN-γ protein, collecting mouse serum samples on the 25th to 30th day, and purifying the IFN-γ polyclonal antibody by ammonium sulfate precipitation to obtain the mouse-derived IFN-γ polyclonal antibody. The present invention performs a first immunization of mice using Freund's complete adjuvant mixed with 80-120 μg IFN-γ protein in a 1:1 ratio and then injected subcutaneously; 12-16 days after the first immunization, a second immunization is performed using Freund's incomplete adjuvant mixed with 30-70 μg antigen in a 1:1 ratio and then injected intraperitoneally; 5-9 days after the second immunization, a third immunization is performed using Freund's incomplete adjuvant mixed with 30-70 μg antigen in a 1:1 ratio and then injected intraperitoneally; one week after the third booster immunization, a serum test is performed, and antisera from all mice are collected when the ratio (P / N) of the serum to be tested to the known negative serum is ≥2.5. The mouse-derived IFN-γ polyclonal antibody needs to be diluted to 1:1000-1:3000, preferably 1:2000.

[0031] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0032] Example 1 Acquisition of Ag85B and Rpf Virulence Proteins

[0033] 1. Construction of prokaryotic expression vectors for Mycobacterium marinum virulence factors

[0034] The Ag85B and Rpf gene sequences of Mycobacterium marinum were downloaded, and primers were designed using Primer Premier 5.0 (Table 1). The 5' ends of the primers contained restriction endonuclease sites (underlined) for NheI, XhoI, and BamHI, as well as protective bases. PCR amplification was performed using Mycobacterium marinum DNA as a template. The amplification conditions for Ag85B were: 94°C denaturation for 5 min, 94°C denaturation for 30 s, 64°C annealing for 30 s, 72°C extension for 1 min 30 s, for a total of 30 cycles, with a final extension at 72°C for 10 min, and a 4°C incubation period. The amplification conditions for Rfp were: 94°C denaturation for 5 min, 94°C denaturation for 30 s, 61°C annealing for 30 s, 72°C extension for 30 s, for a total of 30 cycles, with a final extension at 72°C for 10 min, and a 4°C incubation period. PCR products were detected by electrophoresis ( Figure 1) and recovered using a DNA recovery kit. The recovered target gene and pET-28a(+) vector were simultaneously digested with double enzymes, the digestion products were ligated with T4 DNA ligase, and the recombinant plasmid was transformed into Escherichia coli BL21 competent cells. PCR identification was performed to determine whether the bacteria were positive and sequencing verification was performed ( Figure 2 ) to obtain Ag85B-positive bacterial liquid and Rpf-positive bacterial liquid, respectively.

[0035] Table 1 Primers for Ag85B and Rpf genes

[0036]

[0037] 2. Induced Expression and Purification of Recombinant Fusion Protein

[0038] The positive bacterial cultures of the two genes obtained in the above steps were added to LB medium containing kanamycin at a ratio of 1:100, and cultured at 37°C, 200 rpm for 3 h (OD value was about 0.6). IPTG was added to Ag85B and Rpf to a final concentration of 1.0 mmol / L and 0.5 mmol / L, respectively, and cultured at 37°C, 200 rpm for 6 h to induce the expression of the two recombinant proteins. The results were detected by SDS-PAGE and Western blotting ( Figure 3 、 Figure 4 ); Ni-NTAResin was used to purify the target proteins. Ag85B and Rpf virulence proteins were obtained respectively.

[0039] Example 2: Obtaining mouse IFN-γ polyclonal antibody

[0040] 1. Induced expression and purification of IFN-γ protein

[0041] The positive bacterial suspension containing IFN-γ recombinant plasmid available in the Laboratory of Aquatic Diseases and Immunology, College of Animal Science, Yangtze University was added to LB medium containing kanamycin at a ratio of 1:100 and cultured at 37°C, 200 r / min for 3 h (OD value was about 0.6). IPTG was added to a final concentration of 1.0 mmol / L and cultured at 37°C, 200 r / min for 8 h to induce recombinant protein expression. SDS-PAGE and Western blotting were used for detection ( Figure 5 、 Figure 6 ); Ni-NTAResin was used to purify the target protein to obtain IFN-γ protein.

[0042] 2. Animal immunization

[0043] ICR mice weighing approximately 35g were selected. The first immunization was performed subcutaneously with a mixture of Freund's complete adjuvant and 100μg IFN-γ protein in a 1:1 ratio. A second immunization was performed 14 days after the first immunization, with a thorough mixture of Freund's incomplete adjuvant and 50μg antigen in a 1:1 ratio, followed by intraperitoneal injection. Seven days after the second immunization, a third immunization was performed, with a thorough mixture of Freund's incomplete adjuvant and 50μg antigen in a 1:1 ratio, followed by intraperitoneal injection. One week after the third booster immunization, serum was tested, and antisera from all mice were collected when the ratio of the test serum to the known negative serum (P / N) was ≥2.5.

[0044] Serum titer detection: ELISA was used to detect serum titer. IFN-γ antigen was coated at 4 μg / mL at 4°C overnight. When serum samples were added, a 1:2000 dilution of the test antiserum was added to the first well. The subsequent wells were diluted to 1:128,000. A negative reference solution (1:2000) was added to wells 8 and 9, and a blank control solution was added to well 10. 100 μL was added to each well. Finally, the OD value was measured at a wavelength of 450 nm using a spectrophotometer. The titer test results are shown in Tables 2 and 3. Figure 7 The IFN-γ polyclonal antibody was purified by ammonium sulfate precipitation method, and the protein concentration was 1.32 mg / mL by BCA quantitative detection. The antibody specificity was detected by Western blotting ( Figure 8 ).

[0045] Table 2 Serum titer test results

[0046]

[0047] Example 3 Detection Method for Sturgeons Infected with Mycobacterium Marinum

[0048] Sturgeon blood was drawn into an anticoagulant tube and gently mixed. Each blood sample was added to a test tube containing the specific antigen Ag85B and Rpf virulence protein (prepared in Example 1) and a non-stimulated background control tube, respectively. The tubes were incubated in a 28°C incubator for 22 h, centrifuged at 4000 rpm for 3 min, and the supernatant (plasma) was separated.

[0049] Rabbit IFN-γ polyclonal antibody was diluted at a volume ratio of 1:500 and 100 μL was added to the corresponding 96-well ELISA plate and incubated at 4°C overnight. The next day, the coating solution was discarded and the residual liquid was patted dry, and the plates were washed three times with washing solution for 5 minutes each time. 200 μL of blocking solution was added to each well and blocked at 37°C for 2 hours. The blocking solution was discarded and patted dry, and the plates were washed three times with washing solution for 5 minutes each time. The first row was the standard wells, and the IFN-γ protein was gradiently diluted (2 times) with the diluent. One replicate well was designed for each concentration. The second and third rows were the detection wells, and each detection sample was diluted 5 times with the diluent, with a total of 3 replicates. The fourth row was the background control wells, and each detection sample was diluted 5 times with the diluent. The fifth row was the blank control well. Only diluent was added, 100 μL per well, and the wells were incubated at 37°C for 2 h. The liquid was discarded and patted dry, and the wells were washed three times with washing solution for 5 min each time. 100 μL of mouse IFN-γ polyclonal antibody diluted 1:2000 was added to each well and incubated at 37°C for 1 h. The liquid was discarded and patted dry, and the wells were washed three times with washing solution for 5 min each time. 100 μL of goat anti-mouse enzyme-labeled secondary antibody diluted 1:5000 was added to each well and incubated at 37°C for 1 h. The liquid was discarded and patted dry, and the wells were washed three times with washing solution for 5 min each time. 100 μL of mixed color development solution was added to each well and the wells were developed at 37°C in the dark for 25 min. 50 μL of 1MH2SO4 was added to each well to stop the color development, and the OD value was immediately measured at a wavelength of 450 nm on a spectrophotometer.

[0050] Result judgment: The concentration of IFN-γ protein in the standard wells and the OD value were used to establish a standard curve ( Figure 9 ), the standard curve is y=1370.7x-164.74(R 2 =0.9948). The OD values ​​of the test wells and background control wells were substituted into the standard curve to determine the IFN-γ protein concentration of each group. When the concentration of the test well minus the concentration of the background control well was ≥14 pg / mL, the group was considered positive, indicating a high probability of infection. When the concentration of the test well minus the concentration of the background control well was <14 pg / mL, the group was considered negative, indicating a low probability of infection.

[0051] To verify the feasibility of the above detection method, 100 μL of Mycobacterium marinum (1×10 7 Sturgeons were challenged with 100 CFU / mL of the virus and blood samples were collected 30 and 60 days after infection. Blood from healthy sturgeons served as a negative control. Both the negative control and the challenge samples were tested using the aforementioned assay. The results are shown in Table 3.

[0052] Table 3 Test results of poison-infected sturgeons and healthy sturgeons

[0053]

[0054] The results in Table 3 show that high concentrations of IFN-γ were detected in all sturgeons after infection, that is, the antigen stimulation group of the infection detection group minus the background control group was greater than 14 pg / mL, and the antigen stimulation group of the negative control minus the background control group was less than 14 pg / mL, proving that this detection method can correctly determine whether sturgeons are infected with Mycobacterium marinum.

[0055] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Application of Mycobacterium marinum toxicity protein in the preparation of a product for rapid detection of sturgeon infected with Mycobacterium marinum, characterized in that: The virulence protein is produced by inducing expression of Mycobacterium marinum. The virulence protein is mixed with sturgeon blood, and the mixture is used as a test sample. Sturgeon blood without virulence protein is used as a background control sample. The IFN-γ protein concentration in the two samples is tested. If the test sample minus the background control sample is ≥14 pg / mL, it is judged as positive. If the test sample minus the background control sample is less than 14 pg / mL, it is judged as negative. The virulence proteins of Mycobacterium marinum are Ag85B protein and Rpf protein.

2. The use according to claim 1, characterized in that The method for inducing expression of virulence protein of Mycobacterium marinum comprises: adding Ag85B-positive bacterial liquid and Rpf-positive bacterial liquid to LB culture medium containing kanamycin, culturing, and adding IPTG to induce expression of recombinant protein.

3. The use according to claim 2, characterized in that The amount of IPTG added is 0.4-1.2 mmol / L.

4. The use according to claim 1, wherein The toxic protein and sturgeon blood mixture and the sturgeon blood are placed in an incubator at 25 to 30° C. and incubated for 20 to 24 hours.

5. The use according to claim 1, characterized in that The IFN-γ protein concentration is detected by indirect sandwich ELISA.

6. The use according to claim 5, characterized in that The indirect sandwich ELISA method uses rabbit-derived IFN-γ polyclonal antibody as the capture antibody and mouse-derived IFN-γ polyclonal antibody as the detection antibody.

7. The use according to claim 6, characterized in that The preparation method of the mouse-derived IFN-γ polyclonal antibody comprises the following steps: immunizing mice with IFN-γ protein three times, collecting mouse serum samples on the 25th to 30th day, and purifying the IFN-γ polyclonal antibody by ammonium sulfate precipitation method to obtain the mouse-derived IFN-γ polyclonal antibody.

8. The use according to claim 6, characterized in that The mouse IFN-γ polyclonal antibody needs to be diluted to 1:1000 to 1:3000.

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