Method for distinguishing wild virulent strain of vibrio alginolyticus and cya gene deletion type attenuated strain based on proteomics

Abstract

The invention discloses a method for distinguishing a vibrio alginolyticus wild type virulent strain and a cya gene deletion type low virulent strain based on proteomics. According to the method, a peak area ratio of ATP synthase subunit alpha (atpA) to phosphopentose mutase (deoB) in vibrio alginolyticus, which is measured by proteomics, is utilized to judge an adenylate cyclase coding gene cya-deleted attenuated strain and a wild virulent strain, the vibrio alginolyticus cya gene deleted attenuated strain with a result of atpA / deoB in a sample to be detected being less than 0.2, and the vibrio alginolyticus cya gene deleted attenuated strain with a result of atpA / deoB in the sample to be detected being less than 0.2 is a wild virulent strain with a result of atpA / deoB in the sample to be detected being less than 0.2. The vibrio alginolyticus strain with the result of atpA / deoB being 1.2 or above is a vibrio alginolyticus wild type virulent strain. The distinguishing method is easy to operate, high in detection speed and reliable in result.

Description

Technical Field

[0001] The present invention belongs to the field of analytical chemistry and is a proteomics-based method for analyzing the relationship between the wild-type and virulent strains of Vibrio alginolyticus and of Method for identifying gene-deficient attenuated strains. Background Art

[0002] Vibrio alginolyticus is one of the most common, large-scale and serious conditional pathogens in aquaculture. It is widely distributed in coastal, river and aquatic ecological environments around the world. Its characteristic virulence factors include motility, biofilm, extracellular protease, adhesin, secretion system, quorum sensing system, etc. Marine aquaculture animals such as fish, shellfish, crabs, shrimps, etc. are easily infected by Vibrio alginolyticus, causing sepsis, anemia, gill ulceration, and even death, which brings huge economic losses to the marine aquaculture industry.

[0003] of Is the gene encoding adenylate cyclase, bacterial of When it is missing, the bacteria will lose the ability to generate cAMP and affect various physiological activities and metabolism of bacteria to a certain extent. For example, of The deletion strains have pleiotropic defects not only in carbohydrate metabolism but also in other inducible enzyme systems. of It also regulates the synthesis of specific capsular polysaccharides, biofilms, flagella, and pathogenicity of bacteria, such as the lack of Salmonella typhimurium of It will affect the expression of pili and flagella, significantly reducing their adhesion and invasion abilities; Salmonella choleraesuis of S. Typhimurium Δ of / crp The double mutation attenuated strain also has good immunogenicity and low toxicity.

[0004] Proteomics can help us better understand the interaction between proteins, changes in protein expression, and post-translational modifications of proteins. atpA and deoB identified in the present invention are relatively important functional proteins in Vibrio alginolyticus. atpA is a constituent subunit of ATP synthase, and plays a regulatory role when ATP synthase converts ADP into ATP; deoB is an isomerase that catalyzes the conversion of deoxyribose 1-phosphate and ribose 1-phosphate into deoxyribose 5-phosphate and ribose 5-phosphate, respectively, and plays an important role in the pentose phosphate pathway. So far, no one has proposed to discriminate the adenylate cyclase encoding gene of Vibrio alginolyticus by the peak area ratio of atpA and deoB of Vibrio alginolyticus in the results of proteomics determination. of of strong and weak strains. Summary of the invention

[0005] The purpose of the present invention is to provide a proteomics-based method for identifying a strong strain of wild-type Vibrio alginolyticus and of The method for distinguishing gene-deficient attenuated strains is to use the ratio of atpA and deoB peak areas in Vibrio alginolyticus to identify the gene encoding adenylate cyclase of Vibrio alginolyticus. of The discrimination of strong and weak strains of the virus provides a basis for sample discrimination in proteomics research.

[0006] In order to achieve the above object, the technical solution provided by the present invention is as follows: Protein samples of Vibrio alginolyticus were prepared, and relative quantitative analysis of atpA and deoB in the samples was performed using proteomics technology based on UPLC-MS / MS.

[0007] The preparation method of the Vibrio alginolyticus protein sample is as follows: Vibrio alginolyticus WT, of The deletion strains were inoculated into LBS medium and activated at 30°C and 200 rpm for 12 h. 600 = 1 was inoculated into a new 250 mL LBS medium and cultured for 9 h. The collected WT and Δ of The bacterial sludge was washed three times with 0.9% saline and once with ultrapure water. 6 Each cell was added with 400 μL lysis buffer (7M urea, 2M thiourea, 0.1% PMSF protease inhibitor, 6mM DDT), and ultrasonicated on ice. After ultrasonication, it was placed on ice for 40 minutes, centrifuged (14000rpm, 4℃, 30 min), and the supernatant was collected.

[0008] Mix the protein sample with 2× loading buffer at a volume ratio of 5:1 and heat at 100°C for 8 min to denature the protein. Add 30µL of sample to each electrophoresis lane in sequence, with a voltage of 150-160V. When bromophenol blue migrates to 0.5cm from the bottom of the separation gel, turn off the power. Remove the gel glass plate from the electrophoresis device, stain with Coomassie brilliant blue for 1h, and then decolorize.

[0009] After decolorization, add 50-100 μL of trypsin (0.01 μg / μL) to the gel and place it at 4°C for 30 min. After the enzyme solution is completely absorbed, add 15-20 μL of enzymatic buffer (25 mM NH4HCO3) to completely immerse the gel. Keep it at 37°C for more than 15 hours or overnight, add 200 μL of extraction solution I (5% TFA), bathe it at 40°C for 1 hour, sonicate for 3 minutes every 30 minutes, and aspirate the extraction solution into another clean tube; add 200 μL of extraction solution II (50% acetonitrile, 2.5% TFA) to the gel block, keep it at 30°C for 1 hour, sonicate for 3 minutes every 30 minutes, and finally combine the extraction solutions, dry the peptides, and prepare for mass spectrometry detection.

[0010] The detection conditions of the liquid chromatography of the proteomics technology of UPLC-MS / MS are: The peptides were loaded onto a 2 cm self-assembled precolumn (100 μm inner diameter; 1.9 μm C18-AQ filler; DrMaisch) using mobile phase A as an aqueous solution containing 0.1% formic acid by volume and separated using a homemade analytical column with a 75 μm inner diameter and a 20 cm column length (ReproSil-Pur C18-AQ filler; 1.9 μm; DrMaisch). The analytical mobile phase B was acetonitrile containing 0.1% formic acid by volume; the gradient elution conditions were: 0-13 min, 7%-15% B, 13-59 min 15%-28% B, 59-83 min 42% B, 83-94 min 95% B, 94-98 min 30%-96% B, 98-105 min, 95% B. The pump flow rate was 5 μL / min and the injection volume was 5 μL. The mass spectrometry conditions of the UPLC-MS / MS proteomics technique are: The mass spectrometry scanning method was positive ion scanning mode, the ion transfer tube temperature was set to 320 °C, and the spray voltage was 2.2 kV. The first-level full scan detection range of the mass spectrometer was 350-1500 m / z, the resolution was 60000, the m / z was 200, the maximum injection time was 50 ms, and the target AGC was set to 300%; the resolution of the second-level spectrum was set to 15000, the m / z was 200, the maximum injection time was 22 ms, and the second-level only selected peptides with charges of 2-6 for high-energy collision dissociation, the energy level was set to 27%, the isolation window was set to 1.6 Th, the target AGC was set to 75%, and the intensity threshold was 2e4; the scanning cycle of the full scan spectrum and multiple second-level spectra was set to 1.3 s; the dynamic exclusion time was 45 s; the FAIMS compensation voltage was set to -45 V and -65 V, and the other parameters were set as default.

[0011] Compared with the prior art, the present invention has the following beneficial effects: The present invention utilizes the ratio of atpA and deoB peak areas in Vibrio alginolyticus samples and the correlation between different genotype strains to identify the adenylate cyclase encoding gene of Vibrio alginolyticus. of The discrimination method of the present invention has the following characteristics: simple operation, accurate and reliable results, and small sample consumption. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 is the ratio of the atpA and deoB peak areas in the embodiment. DETAILED DESCRIPTION

[0013] The present invention will be further described in detail below in conjunction with specific examples, but the implementation methods of the present invention include but are not limited to the scope represented by the following examples. Example

[0014] A proteomics-based analysis of the relationship between a wild-type and virulent strain of Vibrio alginolyticus and of The method for distinguishing the gene-deficient attenuated strains is to detect the atpA and deoB contents in Vibrio alginolyticus and calculate their ratio. of The relevant strong and weak strains are distinguished: the atpA / deoB result in the tested sample is below 0.2, which is Vibrio alginolyticus of The gene-deficient weak strain and the atpA / deoB result above 1.2 are the wild-type strong strains of Vibrio alginolyticus.

[0015] The specific method is: The atpA and deoB contents in Vibrio alginolyticus samples were determined by UPLC-MS / MS-based proteomics technology.

[0016] The samples to be tested include wild-type WT, of The deletion strain Δ of .

[0017] The wild strain WT is specifically Vibrio alginolyticus ( Vibrio alginolyticus ) EPGS020401, strain accession number CCTCC No. AB209306, deposited in China Center for Type Culture Collection, Wuhan, China.

[0018] The deletion strain Δ of The specifics are constructed and preserved by the laboratory.

[0019] The sample analysis steps are as follows: 1. Preparation of Vibrio alginolyticus protein samples Vibrio alginolyticus WT, of The deletion strains were inoculated into LBS medium and activated at 30 °C and 200 rpm for 12 h. 600 = 1 was inoculated into a new 250 mL LBS medium and cultured for 9 h. The collected WT and Δ of The bacterial sludge was washed three times with 0.9% saline and once with ultrapure water. 6 Each cell was added with 400 μL lysis buffer (7M urea, 2M thiourea, 0.1% PMSF protease inhibitor, 6mM DDT), and ultrasonicated on ice. After ultrasonication, it was placed on ice for 40 minutes, centrifuged (14000rpm, 4℃, 30 min), and the supernatant was collected.

[0020] Mix the protein sample with 2× loading buffer at a volume ratio of 5:1 and heat at 100°C for 8 min to denature the protein. Add 30µL of sample to each electrophoresis lane in sequence, with a voltage of 150-160V. When bromophenol blue migrates to 0.5cm from the bottom of the separation gel, turn off the power. Remove the gel glass plate from the electrophoresis device, stain with Coomassie brilliant blue for 1h, and then decolorize.

[0021] After decolorization, add 50-100 μL of trypsin (0.01 μg / μL) to the gel and place it at 4°C for 30 min. After the enzyme solution is completely absorbed, add 15-20 μL of enzymatic buffer (25 mM NH4HCO3) to completely immerse the gel. Keep it at 37°C for more than 15 hours or overnight, add 200 μL of extraction solution I (5% TFA), bathe it at 40°C for 1 hour, sonicate for 3 minutes every 30 minutes, and aspirate the extraction solution into another clean tube; add 200 μL of extraction solution II (50% acetonitrile, 2.5% TFA) to the gel block, keep it at 30°C for 1 hour, sonicate for 3 minutes every 30 minutes, and finally combine the extraction solutions, dry the peptides, and prepare for mass spectrometry detection.

[0022] 2.atpA and deoB determination method: The liquid chromatography detection conditions of the proteomics technology of UPLC-MS / MS are as follows: the peptides are loaded on a 2 cm self-assembled pre-column (100 μm inner diameter; 1.9 μm C18-AQ filler; DrMaisch) with a mobile phase A of 0.1% formic acid by volume, and separated using a homemade analytical column (ReproSil-PurC18-AQ filler; 1.9 μm; DrMaisch) with a 75 μm inner diameter and a 20 cm column length. The analytical mobile phase B is acetonitrile containing 0.1% formic acid by volume; the gradient elution conditions are: 0-13 min, 7%-15% B, 13-59 min 15%-28% B, 59-83 min 42% B, 83-94 min 95% B, 94-98 min 30%-96% B, 98-105min, 95% B. The flow rate of the pump is 5 μL / min and the injection volume is 5 μL. The mass spectrometry conditions of UPLC-MS / MS proteomics technology are as follows: the mass spectrometry scanning method is the positive ion scanning mode, the ion transfer tube temperature is set to 320 °C, and the spray voltage is 2.2 kV. The first-level full scan detection range of the mass spectrometer is 350-1500 m / z, the resolution is 60000, the m / z is 200, the maximum injection time is 50 ms, and the target AGC is set to 300%; the resolution of the second-level spectrum is set to 15000, the m / z is 200, the maximum injection time is 22 ms, and the second-level uses only peptides with charges of 2-6 for high-energy collision dissociation, the energy level is set to 27%, the isolation window is set to 1.6 Th, the target AGC is set to 75%, and the intensity threshold is 2e4; the scanning cycle of the full scan spectrum and multiple second-level spectra is set to 1.3 s; the dynamic exclusion time is 45 s; the FAIMS compensation voltage is set to -45 V and -65 V, and the other parameters are set as default.

[0023] Under this analytical condition, the peak areas of atpA and deoB in the above-mentioned Vibrio alginolyticus were obtained, and it was found that the ratio of atpA to deoB changed significantly, as shown in Table 1; the ratio of atpA to deoB peak area is shown in Figure 1 (WT indicates wild type, Δ of express of mutant strain).

[0024] Table 1 Ratio of atpA and deoB peak areas The present invention uses the ratio of atpA and deoB peak areas as a marker for distinguishing the adenylate cyclase encoding gene of Vibrio alginolyticus. of The criteria for distinguishing between the weak strain of the deletion strain and the strong strain of the wild type. That is, the result of atpA / deoB in the tested sample is below 0.2, which is Vibrio alginolyticus. of The gene-deficient weak strain and the atpA / deoB result above 1.2 are the wild-type strong strains of Vibrio alginolyticus.

Claims

1. A proteomics-based analysis of the wild-type and virulent strains of Vibrio alginolyticus cya The method for distinguishing a gene-deficient attenuated strain is characterized by: By calculating the peak area ratio of atpA and deoB in the alginolytic Vibrio detected by proteomics, we can distinguish the wild-type and virulent strains of Vibrio alginolyticus from cya Gene-deficient weak strains, the results of atpA / deoB in the sample are below 0.2, which is Vibrio alginolyticus cya The gene-deficient weak strains, the atpA / deoB result of 1.2 or above is the wild-type strong strain of Vibrio alginolyticus; The wild-type strong strain of Vibrio alginolyticus is Vibrio alginolyticus EPGS020401, with strain collection number CCTCC No. AB209306; Vibrio alginolyticus cya The gene-deficient attenuated strain is an attenuated strain of Vibrio alginolyticus, specifically, the wild-type virulent strain of Vibrio alginolyticus lacks the coding gene related to the synthesis of adenylate cyclase. cya .

2. A proteomics-based method for identifying a virulent wild-type strain of Vibrio alginolyticus according to claim 1 cya The method for distinguishing a gene-deficient attenuated strain is characterized by: The specific method for detecting the content of atpA and deoB in Vibrio alginolyticus is: Protein samples of Vibrio alginolyticus were prepared, and relative quantitative analysis of atpA and deoB in the samples was performed using proteomics technology based on UPLC-MS / MS.

3. A proteomics-based method for identifying a virulent wild-type strain of Vibrio alginolyticus according to claim 2 cya The method for distinguishing a gene-deficient attenuated strain is characterized by: The detection conditions of the liquid chromatography of the proteomics technology of UPLC-MS / MS are: The mobile phase A was an aqueous solution containing 0.1% formic acid by volume. The peptides were loaded on a 2 cm self-assembled pre-column and separated using a homemade analytical column with an inner diameter of 75 μm and a column length of 20 cm. The analytical mobile phase B was acetonitrile containing 0.1% formic acid by volume. The gradient elution conditions were: 0-13 min, 7%-15% B, 13-59 min 15%-28% B, 59-83 min 42% B, 83-94 min 95% B, 94-98 min 30%-96% B, 98-105 min, 95% B. The flow rate of the pump was 5 μL / min and the injection volume was 5 μL.

4. A proteomics-based method for identifying a virulent wild-type strain of Vibrio alginolyticus according to claim 2 cya The method for distinguishing a gene-deficient attenuated strain is characterized by: The mass spectrometry conditions of the UPLC-MS / MS proteomics technique are: The mass spectrometry scanning method was positive ion scanning mode, the ion transfer tube temperature was set to 320 °C, and the spray voltage was 2.2 kV; the first-level full scan detection range of the mass spectrometer was 350-1500 m / z, the resolution was 60000, the m / z was 200, the maximum injection time was 50 ms, and the target AGC was set to 300%; the resolution of the secondary spectrum was set to 15000, the m / z was 200, the maximum injection time was 22 ms, and the secondary use only selected peptides with charges of 2-6 for high-energy collision dissociation, the energy level was set to 27%, the isolation window was set to 1.6Th, the target AGC was set to 75%, and the intensity threshold was 2e4; the scanning period of the full scan spectrum and multiple secondary spectra was set to 1.3 s; the dynamic exclusion time was 45 s; the FAIMS compensation voltage was set to -45 V and -65 V, and the other parameters were set as default.

5. A proteomics-based method for identifying a virulent wild-type strain of Vibrio alginolyticus according to claim 2 cya The method for distinguishing a gene-deficient attenuated strain is characterized by: The preparation method of the Vibrio alginolyticus protein sample is as follows: Collected WT and Δ cya The bacterial sludge was washed three times with 0.9% saline and once with ultrapure water for 10 6 Add 400 μL lysis buffer to each cell, sonicate in an ice bath, place on ice for 40 minutes after sonication, centrifuge, and take the supernatant; Mix the protein sample with 2× loading buffer at a volume ratio of 5:1, heat at 100°C for 8 min to denature the protein, add 30µL of sample to each electrophoresis lane in sequence, voltage 150-160V, wait until bromophenol blue migrates to 0.5cm from the bottom of the separation gel, turn off the power, remove the gel glass plate from the electrophoresis device, stain with Coomassie brilliant blue for 1h, and then decolorize; After decolorization, add 50-100 μL of trypsin to the gel and place it at 4°C for 30 min. When the enzyme solution is completely absorbed, add 15-20 μL of enzymatic buffer to completely immerse the gel. Keep it at 37°C for more than 15 hours or overnight, add 200 μL of extraction solution I, bathe it in 40°C water for 1 hour, sonicate for 3 minutes every 30 minutes, and aspirate the extraction solution into another clean tube; add 200 μL of extraction solution II to the gel block, keep it at 30°C for 1 hour, sonicate for 3 minutes every 30 minutes, and finally combine the extraction solutions, dry the peptides, and prepare for mass spectrometry detection.

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