A method for rapid detection of BMAA bound to marine diatom protein

Abstract

The present invention discloses a method for rapidly detecting protein-bound BMAA in marine diatoms, comprising the following steps: firstly, full-wavelength scanning of the expressed and purified cysK enzyme is performed to determine the absorbance of the cysK protease; then, a method for detecting the cysK enzyme in diatom cells based on absorbance is established, and the concentration of the cysK enzyme in the diatom cells is detected at a specific wavelength (450nm); and the content of BMAA in the corresponding diatoms is determined by the LC-MS / MS method; finally, the linear relationship between the cysK enzyme concentration in the diatom cells and the amount of BMAA synthesis in the diatom cells is evaluated, so as to achieve the purpose of rapidly estimating the protein-bound BMAA in the diatom cells based on the absorbance method. There is no method for rapidly quantifying algal toxins using important synthases in algae, and the present invention can solve the problems of low efficiency and high cost of detecting BMAA toxins in diatoms to a certain extent.

Description

Technical Field

[0001] The invention relates to a method for rapidly detecting protein-bound β-nitrogen-methylamino-L-alanine (BMAA) synthesized by marine diatoms. Specifically, the method finds that the level of neurotoxin BMAA synthesized by marine diatoms is linearly correlated with the content of diatom cysteine ​​synthetase (cysK). By detecting the absorbance of diatom cysK enzyme, the content of protein-bound BMAA toxin in diatom cells can be estimated. The method belongs to the technical field of marine biotoxin detection. Background Art

[0002] Recent studies have found that diatoms, cyanobacteria and other organisms widely distributed in the aquatic environment can produce β-nitrogen-methylamino-L-alanine (BMAA), which has a selective damaging effect on motor neurons in humans and animals and is considered to be an important environmental factor inducing neurodegenerative diseases such as Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), and Parkinson's dementia syndrome (PDC). BMAA is commonly detected in a variety of mollusks (especially benthic mollusks) in the coastal waters of northern my country, and BMAA can be transmitted and enriched along the marine food chain, threatening the safety of marine aquatic products. It is worth noting that BMAA detected in laboratory-cultured marine diatoms mainly exists in a protein-bound form.

[0003] Given that BMAA is a small molecule amino acid derivative, how to accurately identify and quantify BMAA toxins in different samples has become a challenge for researchers. Currently, the detection methods for BMAA toxins mainly include: gas chromatography-mass spectrometry, high performance liquid chromatography-fluorescence detection, high performance liquid chromatography-mass spectrometry, capillary electrophoresis, etc. However, researchers still have disputes over the accuracy of different methods for quantitative detection of BMAA in environmental samples. In the past decade, chemical analysis methods based on optical detection or mass spectrometry have been widely used for the detection of BMAA in environmental samples. Since the BMAA molecule itself does not have fluorescent luminescence characteristics, it needs to be derivatized first to generate derivatives with fluorescent luminescent groups. The derivatives obtained by this pre-column derivatization method are more hydrophobic, which is conducive to the separation of reversed-phase chromatography columns, but because the derivatization agent has a broad spectrum of amino acid molecules, the selectivity of this method is not strong. Mass spectrometry, especially liquid chromatography tandem mass spectrometry (LC-MS / MS), is widely used for BMAA analysis in environmental samples. This method usually does not require pre-column derivatization when analyzing BMAA, but because BMAA is a small polar molecule, it is difficult to separate it from other amino acid molecules on a reversed-phase column, and a hydrophilic interaction chromatography column (HILIC) is needed to enhance the separation of the target compound. Some scholars have also combined the pre-column derivatization method with the LC-MS / MS method, using a reversed-phase column for separation, for the analysis of BMAA toxins. At present, high-performance liquid chromatography-mass spectrometry and high-performance liquid chromatography-fluorescence detection are commonly used internationally to analyze BMAA in environmental samples, but both have technical defects to varying degrees.

[0004] The applicant's research found that cysteine ​​synthetase (cysK), as a nucleophile, plays a catalytic role in the production of non-natural β-substituted L-α-amino acids. In multiple diatom strains that can synthesize BMAA, qRT-PCR verification found that the BMAA production in different strains of diatoms was positively correlated with the expression level of the cysK gene. In vitro biochemical experiments confirmed that the cysteine ​​residues in the peptide chain can be used as substrates for nucleophilic reactions catalyzed by cysK, allowing it to react with methylamine to form the chemical structure of BMAA. Therefore, cysK is an important prerequisite and rate-limiting factor for the synthesis of protein-bound BMAA by diatoms.

[0005] At present, HPLC-MS and HPLC-fluorescence detection are used to analyze BMAA toxins. Not only is the sample preparation process cumbersome, but the instrument price is also very expensive. This situation seriously restricts the detection efficiency of BMAA toxins and the development of related research work. Therefore, the concentration of cysK enzyme in diatom cells is detected by absorbance method, and the content of BMAA toxin in diatom samples is estimated based on the linear relationship between BMAA synthesis and cysK enzyme concentration. The method of the present invention solves the problems of low detection efficiency and high testing cost of BMAA toxin in diatom samples to a certain extent, and provides a rapid detection method for BMAA analysis of phytoplankton samples, which has significant economic and social benefits. Summary of the invention

[0006] The technical problem to be solved by the present invention is to address the deficiencies in the prior art. Based on the new discovery of the molecular mechanism of diatoms synthesizing protein-bound BMAA, and according to the linear relationship between the content of cysteine ​​synthase in diatom cells and the BMAA production, a method for rapidly detecting protein-bound BMAA in marine diatoms is proposed; since the level of protein-bound BMAA synthesized by marine diatoms is linearly correlated with the content of diatom cysK, the content of protein-bound BMAA toxin in diatom cells can be estimated by detecting the absorbance of diatom cysK enzyme. The method of the present invention has the advantages of quick detection method, low test cost and accurate quantitative results, etc.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] The present invention provides a method for rapidly detecting β-nitrogen-methylamino-L-alanine (BMAA) bound to marine diatom synthetic proteins, comprising the following steps:

[0009] (1) Establish a standard curve of cysK protease concentration and absorbance:

[0010] The prokaryotic expressed and purified cysK protease was subjected to full wavelength scanning to determine the maximum absorption wavelength of the cysK protease, the prokaryotic expressed and purified cysK protease was diluted to different concentrations, and the absorbance of the cysK protease of different concentrations was measured under the condition of the maximum absorption wavelength, and a standard curve of the cysK protease concentration and the absorbance was established, and there was a linear relationship between the concentration of the cysK protease of different dilutions and the absorbance;

[0011] (2) Detection of cysK protease concentration in BMAA-producing diatom cells:

[0012] When a certain type of BMAA-producing diatom cells grows to a stable period, a suction pump is used to collect part of the diatom cells onto a water filter membrane, and then PBS is used to transfer all the diatom cells on the water filter membrane to a centrifuge tube, the diatom cells in the centrifuge tube are washed twice with PBS and then re-dissolved with PBS, and then the re-dissolved algae cells are quickly frozen in liquid nitrogen for a few minutes and then thawed at room temperature, and then the algae cells are ultrasonically disrupted, and finally the absorbance of cysK in the diatom cells is detected at the maximum absorption wavelength of cysK protease, and the absorbance is brought into the standard curve obtained in step (1) to calculate the concentration of cysK protease in the BMAA-producing diatom cells, and by calculating the number of algae cells in the corresponding collection volume, the unit of cysK enzyme can be converted to obtain the content of cysK enzyme in the unit diatom cell;

[0013] (3) Analysis of BMAA toxins in BMAA-producing diatom cells using LC-MS / MS:

[0014] When a certain type of BMAA-producing diatom cells in step (2) grows to a stable period, part of the diatom cells are transferred to a centrifuge tube and algae mud containing the algae cells is collected by centrifugation, the protein-bound BMAA in the algae mud is extracted and resuspended with HCl to obtain a BMAA toxin extract, the BMAA toxin extract is purified by a solid phase extraction column and then analyzed by LC-MS / MS for the BMAA toxin in the BMAA-producing diatom cells, and the BMAA toxin is qualitatively and quantitatively analyzed by multiple reaction monitoring mode MRM according to the retention time and mass spectrometry parameters of the BMAA toxin; through qualitative and quantitative analysis, the concentration of protein-bound BMAA per diatom cell can be known;

[0015] (4) Linear evaluation of the cysK enzyme concentration in diatom cells and the BMAA content per unit algal cell protein:

[0016] According to the operations of step (2) and step (3), a plurality of BMAA-producing diatom cells are respectively detected to obtain the cysK enzyme content per diatom cell and the BMAA concentration per diatom cell protein-bound diatom cell of the plurality of diatom cells. A linear equation is established with the cysK enzyme content per diatom cell as the horizontal coordinate and the BMAA concentration per diatom cell protein-bound diatom cell as the vertical coordinate. The results show that the concentration of cysK enzyme has a good linear relationship with the content of BMAA, and the linear equation is a linear equation. The absorbance of the diatom cells grown to the stable phase is detected, and the BMAA concentration per diatom cell protein-bound diatom cell of the diatom cell can be obtained by calculation and conversion.

[0017] In the above technical scheme, in step (1), the prokaryotic expression and purification of cysK protease refers to recombinant diatom cysK protease with a purity of >90%; the maximum absorption wavelength of the cysK protease is 450nm. The prokaryotic expression and purification of cysK protease in the present invention is obtained by constructing a plasmid by total gene synthesis and cloning it into the expression vector pET-22b, further transforming it into Escherichia coli competent cells, culturing, inducing expression, collecting bacteria, purifying proteins, and finally verifying it by SDS-PAGE electrophoresis and Western Blot.

[0018] In the above technical scheme, in step (1), the prokaryotic expressed and purified cysK protease is diluted to different concentrations, and the concentrations of cysK protease are 0.005 mg / mL, 0.01 mg / mL, 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL, and 0.05 mg / mL, respectively.

[0019] In the above technical scheme, in step (2), after the BMAA-producing diatom cells grow to a stable period, 200 mL of diatom cells are collected onto a 0.45 μm water filter membrane using a suction pump, and then the diatom cells on the water filter membrane are rinsed into a 50 mL centrifuge tube using 5 mL of PBS for three times to ensure that all the diatom cells on the water filter membrane are transferred to the centrifuge tube; the collected diatom cells are then centrifuged at 8000 rpm, 8 min, and 4°C. After the centrifugation, the supernatant is removed and the diatom cells at the bottom of the centrifuge tube are collected; 10 mL of PBS is continued to be added to the collected diatom cells for centrifugal washing, and the centrifugal washing is performed twice (excluding the centrifugation of the collected diatom cells) at 8000 rpm, 8 min, and 4°C. After the two centrifugal washings, the supernatant is removed and the diatom cells at the bottom of the centrifuge tube are re-dissolved with 15 mL of PBS.

[0020] In the above technical scheme, in step (2), the reconstituted algae cells are quickly frozen in liquid nitrogen for 6 minutes, then thawed at room temperature, and after thawing, the algae cells are ultrasonically disrupted in an ice water bath, and the conditions for ultrasonic disruption are: ultrasonic power 50W, ultrasonic frequency set to 2S on, 10S off, and cycled 30 times; then a Multiskan Sky full-wavelength microplate reader is used to detect the absorbance of cysK in the diatom cells at a wavelength of 450nm, and the absorbance is brought into the standard curve obtained in step (1) to calculate the cysK concentration of the diatom cells, the unit of which is mg / mL, and the cell density of the diatom cells after reaching the stable period can be measured by flow cytometry; the volume of the collected diatom cells is 200mL, and the unit of cysK enzyme can be converted from mg / mL to fg / cell by calculating the number of algae cells in the corresponding collected volume, so as to know the content of cysK enzyme in the unit cell.

[0021] In the above technical solution, in step (2) and step (3), the method for culturing the BMAA-producing diatom cells is as follows: filtering natural seawater with a 0.45 μm mixed fiber filter membrane, mixing the seawater and the culture medium at a volume ratio of 1000:1 and then placing them in a conical flask, wherein the culture medium is an iron-restricted Guillard's f / 2 medium, that is, the concentration of FeCl3.6H2O in the Guillard's f / 2 medium formula is reduced by 3 times (1 / 3×Fe); inoculating BMAA-producing diatoms, and the initial inoculation density of the BMAA-producing diatoms is 1.0×10 4 cells mL -1 , and then placed it in an incubator for culture. The light intensity of the incubator was 6000 lx, the temperature was 20 °C, and the light-dark ratio was 12h:12h. The conical flask was gently shaken three times a day. During the culture period, 1 mL of algae solution was taken from each bottle and placed in a 5 mL centrifuge tube. The algae cell density in the stable period was detected by flow cytometry (the algae cell density in the stable period was detected by flow cytometry, where chlorophyll a (Chl a) fluorescence was used to measure the density of diatom samples, and the injection flow rate was 66 μL min -1 ), when the diatom cell density reaches 5.0×10 6 cells mL -1 Around 500 Å, it means that the BMAA-producing diatom cells have grown to a stable stage, that is, the algae density in the stable stage is about 5.0×10 6 cells mL -1 .

[0022] In the above technical solution, in step (3), 400 mL of algae liquid containing diatom cells is transferred to a centrifuge tube, centrifuged at 8000 rpm for 10 min at 4°C, and the supernatant is removed to obtain algae mud containing algae cells. It should be noted that the algae cell collection and sampling equipment must be sterilized and should be carried out in a sterile environment as much as possible.

[0023] In the above technical scheme, in step (3), when extracting protein-bound BMAA from algae mud, the operation steps are as follows: add 5 mL of 0.1 M trichloroacetic acid to the centrifuge tube containing the algae mud and vortex mix; after vortex mixing, place the sample in liquid nitrogen for 10 minutes for freeze-thaw crushing, thaw at room temperature and freeze-thaw crushing again, and repeat this step three times; after freeze-thaw crushing, ultrasonically crush the algae mud in an ice water bath for 10 minutes, the ultrasonic power is 50 W, the ultrasonic frequency is set to 2S on, 10S off, and the cycle is repeated 30 times; after the ultrasonication is completed, the algae mud is centrifuged at 4°C and 8000 rpm for 10 minutes, the supernatant is removed, 2 mL of 6 M HCl is added to the algae mud, and vortex mix is ​​performed; then it is transferred to a 4 mL injection bottle, and then 1 mL is taken. The centrifuge tube was rinsed with 6M HCl, vortexed and transferred to the 4mL injection bottle; the sample in the 4mL injection bottle was hydrolyzed at 110°C for 24h, cooled to room temperature and transferred to a 10mL centrifuge tube. At the same time, 1mL of 6M HCl was taken to rinse the 4mL injection bottle, and the rinsing solution was also transferred to the 10mL centrifuge tube. After centrifugation, 2mL of the supernatant was taken and dried with nitrogen at 55°C. After drying, 1mL of 20mM HCl was added to resuspend the particles to obtain the BMAA toxin extract.

[0024] In the above technical solution, in step (3), when the BMAA toxin extract is purified by the solid phase extraction column, the operation steps are as follows: Oasis MCX (3cc, 60mg) cation exchange solid phase extraction column is used for sample purification, first 3mL of 100% methanol is taken to activate the solid phase extraction column, and then 2mL of ultrapure water is added to balance the solid phase extraction column; after the solid phase extraction column is stabilized, 1mL of BMAA toxin extract is injected; after all the samples flow out of the solid phase extraction column, 2mL of 0.1N HCl and 2mL of 100% methanol are added in sequence to clean the impurities in the extract; finally, 3mL of 5% ammonia water (NH4OH) is used to elute the toxin extract enriched by the solid phase extraction column, the eluate is collected and the eluate is placed at 55°C and dried with nitrogen, after drying, 1mL of 20mM HCl is added to resuspend the particles, and finally filtered through a 0.22μm water filter membrane to obtain a purified BMAA toxin extract for LC-MS / MS toxin analysis.

[0025] In the above technical solution, in step (3), when performing LC-MS / MS analysis, the operation steps are as follows: using AB SCIEX Qtrap 4500 triple quadrupole / tandem linear ion trap mass spectrometer, the content of BMAA in algal cells is directly determined without derivatization, and the specific parameters are as follows:

[0026] Liquid chromatography conditions: Liquid chromatography using ZIC-HILIC column (150mm×2.1mm, 5μm), column temperature was set to 30℃, flow rate was 0.35mL / min, injection volume was 5μL, mobile phase A was an aqueous solution containing 50mM formic acid, mobile phase B was an acetonitrile solution containing 50mM formic acid, and the gradient elution mode was as follows: 0.00min mobile phase A 5%, mobile phase B 95%, 19.00min mobile phase A 40%, mobile phase B 60%, 25.00min mobile phase A 60%, mobile phase B 40%, 27.01min mobile phase A 5%, mobile phase B 95%, 30.00min mobile phase A 5%, mobile phase B 95%, and the percentages here refer to volume percentages.

[0027] The mass spectrometry parameters are as follows: Qtrap 4500 triple quadrupole / tandem linear ion trap mass spectrometer is used, and the connection interface is an electrospray ionization source (ESI). The analysis conditions of the mass spectrometry are as follows: electrospray voltage (IS) 5500V, nebulization temperature (TEM) 350℃, nebulization gas pressure (GS1) 50psi, auxiliary gas pressure (GS2) 50psi, curtain gas pressure (CUR) 40psi, collision gas pressure (CAD) Medium, entrance voltage (EP) 10V, collision chamber ejection voltage (CXP) 12V.

[0028] In the above technical solution, in step (3), when the toxin is analyzed in multiple reaction monitoring mode (MRM), the operation steps are as follows: the transition pattern with high abundance of product ions is used as the quantitative basis, and the transition pattern unique to each toxin is used for qualitative analysis. Four transition patterns are used, including m / z 119->102, 88, 56 and 44, the declustering potential (DP) is 30V, and the collision energy (CE) is 13, 17, 24 and 24eV respectively. The quantification of the target protein-bound BMAA adopts m / z 119->88; in addition, the detection signal intensity ratio between different transition ions is used as an auxiliary standard for the qualitative analysis of these toxin analogs; through this qualitative and quantitative analysis, the concentration of protein-bound BMAA in unit diatom cells can be calculated.

[0029] In the above technical scheme, in step (4), according to the operations of step (2) and step (3), 7 diatoms grown to the stable period are used as samples, namely, T. allenii, T. sinica, T. andamanica, T. minima, T. gravida, C. diadema, and T. flabella; cy in diatom cells of these 7 samples is measured respectively. The absorbance of cysK protease can be calculated and converted to know the content of cysK protease per diatom cell. Then, the concentration of protein-bound BMAA per diatom cell of these seven sample species was detected by LC-MS / MS. A linear equation was established with the content of cysK enzyme per diatom cell as the horizontal axis and the concentration of protein-bound BMAA per diatom cell as the vertical axis. The results showed that the concentration of cysK enzyme had a good linear relationship with the content of BMAA. The linear equation was the linear equation y=0.0062x+0.0032, R 2 =0.9598; detect the absorbance of diatom cells that have grown to the stable period, and the unit diatom cell protein-bound BMAA concentration of this type of diatom cells can be obtained through calculation and conversion.

[0030] Compared with the existing technology, it has the following characteristics:

[0031] The method of the present invention has the advantages of quick detection method, low test cost and accurate quantitative results. Specifically, the content of protein-bound BMAA in a unit diatom cell can be calculated by substituting the concentration of the detected cysK enzyme into the relevant linear equation, while the extraction process is cumbersome and the detection and verification work is relatively expensive when analyzing BMAA toxin by LC-MS / MS. The method of quickly detecting the protein-bound BMAA synthesized by marine diatoms by using the absorbance of the cysK expression enzyme in diatom cells is both time-saving and accurate. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1a : The recombinant plasmid restriction enzyme map of the cysK protease after prokaryotic expression and purification used in Example 1 of the present invention during the prokaryotic expression and purification process (wherein 1: double restriction enzyme digestion target gene; 2: Plasmid);

[0033] Figure 1b: SDS-PAGE analysis of the fusion protein expression test of cysK protease after prokaryotic expression and purification used in Example 1 of the present invention (wherein M: Protein Marker (Cat. No.: C610013); 1: total protein before induction; 2: 20°C supernatant; 3: 20°C precipitate; 4: 37°C supernatant; 5: 37°C precipitate);

[0034] Figure 1c : SDS-PAGE analysis of the nickel agarose affinity chromatography purification of the fusion protein of the cysK protease after prokaryotic expression and purification used in Example 1 of the present invention (wherein M: Protein marker (Cat. No.: C610013); 1: loading; 2: flow-through; 3: 20mM Imidazole elution fraction; 4: 50mM Imidazole elution fraction; 5: 500mM Imidazole elution fraction);

[0035] Figure 1d : SDS-PAGE analysis of the final purified protein of the cysK protease after prokaryotic expression and purification used in Example 1 of the present invention during the prokaryotic expression and purification process of the cysK protease (wherein M: Protein marker (Cat. No.: C610013); I: fusion target protein);

[0036] Figure 1e : Western Blot analysis of the fusion protein of cysK protease after prokaryotic expression and purification used in Example 1 of the present invention during the prokaryotic expression and purification process (wherein M: Prestained Marker; I: target protein);

[0037] Figure 2a : Full wavelength scanning diagram of cysK enzyme in step (1) of Example 1 of the present invention;

[0038] Figure 2b : Linear relationship diagram between cysK concentration and absorbance at different dilution ratios in step (1) of Example 1 of the present invention;

[0039] Figure 3 : Flow chart of detecting the cysk enzyme content in diatom cells in step (2) of Example 1 of the present invention;

[0040] Figure 4 : Linear relationship diagram between the cysK enzyme concentration in diatom cells and the BMAA concentration per diatom cell in step (4) of Example 1 of the present invention. DETAILED DESCRIPTION

[0041] The specific implementation of the technical solution of the present invention is described in detail below, but the present invention is not limited to the following description:

[0042] The present invention firstly expresses and purifies the key target protein cysK in Escherichia coli, then performs full wavelength scanning on the expressed and purified cysK enzyme to determine the absorbance of cysK protease, then establishes a method for detecting cysK enzyme in diatom cells based on absorbance method, and uses LC-MS / MS method to determine the content of BMAA in the corresponding diatom, and finally evaluates the linear relationship between the concentration of cysK enzyme in diatom cells and the amount of BMAA synthesized in diatom cells, thereby achieving the purpose of rapid detection of protein-bound BMAA in diatom cells based on absorbance method. Thus, the problem of high cost and low efficiency of BMAA analysis and detection is solved.

[0043] The prokaryotic expression and purification of cysK protease described in the embodiment of the present invention was completed by Shanghai Shenggong Biotechnology Co., Ltd. (it can also be performed by other units or individuals. This is a common operation technology of biochemical companies. As long as the purity of the prokaryotic expression and purification of cysK protease is >90% and the maximum absorption wavelength is 450nm, it can be used in the present invention). The plasmid was constructed by total gene synthesis and cloned into the expression vector pET-22b, and further transformed into Escherichia coli competent cells. After cultivation, induction of expression, collection of bacteria, and protein purification, 1 mg of recombinant diatom cysK protein with a purity of >90% was obtained by SDS-PAGE and Western Blot verification. The specific steps are as follows:

[0044] (1) Plasmid construction and verification: First, the signal peptide, transmembrane domain, and basic structure of the diatom cysK protein sequence were theoretically evaluated. 1-18AA is a signal peptide, without a transmembrane region. The signal peptide is removed, and the 19-365AA ligation product is transformed into the receptive E. coli TOP10 by heat stimulation. Positive clones are detected and sequenced (e.g. Figure 1a As shown), obtain the recombinant plasmid;

[0045] (2) Protein expression: The obtained recombinant plasmid was transferred into Escherichia coli BL21 (DE3) recipient cells, and after heat shock, it was spread on a plate containing 50 μg / mL ampicillin for culture. Then a single clone was picked and cultured in a liquid culture medium containing 50 μg / mL ampicillin. When the OD value reached 0.6, the inducer IPTG was added to continue the culture, cultured overnight at 20°C and cultured for 6 hours at 37°C. No inducer was added as a negative control. The bacterial liquid was collected and centrifuged at 6000×g for 20 minutes. The supernatant was discarded and the bacteria were collected. The collected bacteria were suspended in buffer A (PBS, pH 7.4), completely destroyed by ultrasound, centrifuged, and the centrifugal precipitate was dissolved in buffer B (8M Urea, 50mM Tris-HCl, 300mM NaCl, pH 8.0). The supernatant and precipitate were treated separately, and the samples were prepared and prepared for SDS-PAGE analysis (such as Figure 1b After SDS-PAGE analysis, the bacteria were -1 When the OD value reached 0.6, 0.5 mM IPTG was added and cultured overnight at 20°C to allow for large-scale expression. Cells were then collected by centrifugation.

[0046] (3) Protein purification: Dissolve the cell membrane with buffer C (50 mM Tris-HCl, 300 mM NaCl, 0.2 mM PMSF, 0.1% Triton X-100, pH 8.0), disrupt by ultrasound, and collect the crude protein supernatant by centrifugation. Rinse the equilibrated column with 5 mL Ni-NTA in a volume 5 times the column bed binding buffer at a flow rate of 5 mL min -1 . Incubate the crude protein with the equilibrated column filler for 1 hour. Wash the equilibrated column with binding buffer (50mM Tris, 300mM NaCl, pH 8.0). Wash the column with wash buffer (50mM Tris, 300mM NaCl, 20 / 50mM Imidazole, pH 8.0) and collect the effluent. Elute the column with elution buffer (50mM Tris, 300mM NaCl, 500mM Imidazole, pH 8.0) and collect the effluent. Treat the crude protein, eluate and elution effluent separately to prepare SDS-PAGE samples (such as Figure 1c shown);

[0047] (4) Protein purification: Finally, SDS-PAGE (such as Figure 1d As shown) and Western Blot (as shown Figure 1e As shown in the figure, 3 mL of cysK recombinant diatom protein with a concentration of 0.45 mg / mL and a purity of >90% was obtained.

[0048] The present invention will be described below in conjunction with specific embodiments:

[0049] Embodiment 1:

[0050] A method for rapidly detecting β-nitrogen-methylamino-L-alanine (BMAA) bound to marine diatom synthetic proteins comprises the following steps:

[0051] (1) Establish a standard curve of cysK protease concentration and absorbance:

[0052] The full wavelength scanning of prokaryotic expressed and purified cysK protease was performed using Multiskan Sky full wavelength microplate reader (Thermo Fisher Scientific, Shanghai, China), and the maximum absorption wavelength of cysK protease was determined to be 450 nm (cysK concentration was 0.45 mg / mL). The full wavelength scanning of cysK enzyme is shown in Figure 2. Figure 2a As shown; the prokaryotic expressed and purified cysK protease was diluted to different concentrations, the concentrations of cysK protease were 0.005 mg / mL, 0.01 mg / mL, 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL, 0.05 mg / mL, respectively, the absorbance of cysK protease of different concentrations was measured under the condition of maximum absorption wavelength (450 nm), and the standard curve of cysK protease concentration and absorbance was established. There was a linear relationship between the concentration of cysK protease of different dilutions and the absorbance, y=7.9578x+0.0087, R2=0.9966, as shown Figure 2b shown.

[0053] (2) Detection of cysK protease concentration in BMAA-producing diatom cells:

[0054] Natural seawater was filtered with a 0.45 μm mixed fiber filter membrane, and 200 mL of filtered seawater and 0.2 mL of iron-restricted Guillard's f / 2 medium (the content of FeCl3.6H2O in the medium was reduced to reduce the concentration of FeCl3.6H2O by 3 times (1 / 3×Fe)) were added to the culture medium at a volume ratio of 1000:1 in a 500 mL conical flask; BMAA-producing diatoms were inoculated, and the initial inoculation density of BMAA-producing diatoms was 1.0×10 4 cells mL -1 Then, it was placed in an incubator for cultivation. The light intensity of the incubator was 6000 lx, the temperature was 20 °C, and the light-dark ratio was 12h:12h. The conical flask was gently shaken three times a day. During the cultivation period, 1 mL of algae solution was taken from each bottle and placed in a 5 mL centrifuge tube. The density of algae cells in the stable period was detected by flow cytometry. When the diatom cell density reached 5.0×10 6 cells mL -1Around 30 s, it means that the BMAA-producing diatom cells have grown to the stable phase.

[0055] like Figure 3 As shown, when the BMAA-producing diatom cells grow to a stable period, a suction pump is used to collect 200 mL of diatom cells onto a 0.45 μm water filter membrane, and then 5 mL of PBS is used to rinse the diatom cells on the water filter membrane into a 50 mL centrifuge tube for 3 times to ensure that all the diatom cells on the water filter membrane are transferred to the centrifuge tube; the collected diatom cells are then centrifuged at 8000 rpm, 8 min, and 4°C. After the centrifugation, the supernatant is removed and the diatom cells at the bottom of the centrifuge tube are collected; 10 mL of PBS is continued to be added to the collected diatom cells for centrifugal washing, and the centrifugal washing is performed twice at 8000 rpm, 8 min, and 4°C. After the 2 centrifugal washings, the supernatant is removed and the diatom cells at the bottom of the centrifuge tube are re-dissolved with 15 mL of PBS. The reconstituted algae cells were quickly frozen in liquid nitrogen for 6 minutes, then thawed at room temperature, and then ultrasonically disrupted in an ice-water bath. The conditions for ultrasonic disruption were as follows: ultrasonic power 50W, ultrasonic frequency set to 2S on, 10S off, and cycled 30 times; then the absorbance of cysK in diatom cells was detected at a wavelength of 450nm using a Multiskan Sky full-wavelength microplate reader;

[0056] The absorbance is substituted into the standard curve obtained in step (1) to calculate the cysK concentration of the diatom cells, which is expressed in mg / mL. The cell density of the diatom cells after reaching the stable period is known. The volume of the collected diatom cells is 200 mL. By calculating the number of algae cells in the corresponding collected volume, the unit of cysK enzyme can be converted from mg / mL to fg / cell, and the content of cysK enzyme per unit cell can be known.

[0057] (3) Analysis of BMAA toxins in BMAA-producing diatom cells using LC-MS / MS:

[0058] When the BMAA-producing diatom cells in step (2) grow to a stable stage, the algae liquid with a volume of 400 mL is centrifuged at 8000 rpm for 10 min at 4°C to collect the algae cells, and the supernatant is removed to obtain the algae mud containing the algae cells. It should be noted that the algae cells and sampling equipment must be sterilized and should be carried out in a sterile environment as much as possible.

[0059] Extraction of protein-bound BMAA from algal mud: Add 5 mL of 0.1 M trichloroacetic acid to the centrifuge tube containing the algal mud, vortex mix, place the sample in liquid nitrogen and freeze for 10 min, thaw at room temperature and freeze-thaw again, repeat this step three times; after freeze-thaw, ultrasonically crush the algal mud in an ice water bath for 10 min, with an ultrasonic power of 50 W and an ultrasonic frequency set to 2S on and 10S off, and cycle 30 times; after the ultrasonication, centrifuge the algal mud at 4°C and 8000 rpm for 10 min, remove the supernatant, add 2 mL of 6 M HCl to the algal mud, vortex mix; then transfer it to a 4 mL injection bottle, then take 1 mL of 6 M HCl to rinse the centrifuge tube, vortex mix and transfer it to the 4 mL injection bottle; hydrolyze the sample in the injection bottle at 110°C for 24 h, cool it to room temperature and transfer it to a 10 mL centrifuge tube, and take 1 mL The sample bottle was rinsed with 6M HCl, and the rinsing solution was also transferred to the 10mL centrifuge tube. After centrifugation, 2mL of the supernatant was blown dry with nitrogen at 55°C. After drying, 1mL of 20mM HCl was added to resuspend the particles to obtain the BMAA toxin extract.

[0060] Purification of BMAA toxin extract by solid phase extraction column: Oasis MCX (3cc, 60mg) cation exchange solid phase extraction column was used for sample purification. First, 3mL of 100% methanol was taken to activate the solid phase extraction column, and then 2mL of ultrapure water was added to equilibrate the solid phase extraction column. After the solid phase extraction column was stabilized, 1mL of BMAA toxin extract was injected. After all the samples flowed out of the solid phase extraction column, 2mL of 0.1N HCl and 2mL of 100% methanol were added in sequence to clean the impurities in the extract. Finally, 3mL of 5% ammonia water (NH4OH) was used to elute the toxin extract enriched by the solid phase extraction column, and the eluate was collected and dried with nitrogen at 55°C. After drying, 1mL of 20mM HCl was added to resuspend the particles, and finally filtered through a 0.22μm water filter membrane to obtain the purified BMAA toxin extract for LC-MS / MS toxin analysis.

[0061] When performing LC-MS / MS analysis, the operating steps are as follows: AB SCIEX Qtrap 4500 triple quadrupole / tandem linear ion trap mass spectrometer is used to directly determine the content of BMAA in algal cells without derivatization. The specific parameters are as follows:

[0062] Liquid chromatography conditions: Liquid chromatography using ZIC-HILIC column (150 mm × 2.1 mm, 5 μm), column temperature set to 30 ° C, flow rate of 0.35 mL / min, injection volume of 5 μ L, mobile phase A is an aqueous solution containing 50 mM formic acid, mobile phase B is an acetonitrile solution containing 50 mM formic acid, gradient elution mode is as follows: 0.00 min mobile phase A 5%, mobile phase B 95%, 19.00 min mobile phase A 40%, mobile phase B 60%, 25.00 min mobile phase A 60%, mobile phase B 40%, 27.01 min mobile phase A 5%, mobile phase B 95%, 30.00 min mobile phase A 5%, mobile phase B 95%, the percentage here refers to the volume percentage;

[0063] The mass spectrometry parameters are as follows: Qtrap 4500 triple quadrupole / tandem linear ion trap mass spectrometer is used, and the connection interface is an electrospray ionization source (ESI). The analysis conditions of the mass spectrometry are shown in the table below: electrospray voltage (IS) 5500V, nebulization temperature (TEM) 350℃, nebulization gas pressure (GS1) 50psi, auxiliary gas pressure (GS2) 50psi, curtain gas pressure (CUR) 40psi, collision gas pressure (CAD) Medium, entrance voltage (EP) 10V, collision chamber ejection voltage (CXP) 12V.

[0064] When the toxins were analyzed using the multiple reaction monitoring (MRM) mode, the operating steps were as follows: the transition pattern with high product ion abundance was used as the quantitative basis, and the transition pattern unique to each toxin was used for qualitative analysis. Four transition patterns were used, including m / z119->102, 88, 56 and 44, with a declustering potential (DP) of 30 V and a collision energy (CE) of 13, 17, 24 and 24 eV, respectively. The target protein-bound BMAA was quantified using m / z 119->88; in addition, the detection signal intensity ratio between different transition ions was used as an auxiliary standard for the qualitative analysis of these toxin analogs; through this qualitative and quantitative analysis, the concentration of protein-bound BMAA in unit diatom cells can be calculated.

[0065] (4) Linear evaluation of the cysK enzyme concentration in diatom cells and the BMAA content per unit algal cell protein:

[0066] Through step (2) and step (3), the concentration of cysK protease in a certain BMAA-producing diatom cell and the concentration of BMAA bound to the algae cell protein per unit can be known; for different types of marine diatoms producing BMAA, the concentration of cysK protease in their algae cells and the concentration of BMAA bound to the algae cell protein per unit can be measured according to the operations of step (2) and step (3), respectively, so that the concentration of cysK protease in the cells of multiple BMAA-producing diatoms and the concentration of BMAA bound to the algae cell protein per unit can be known;

[0067] In the embodiment of the present invention, 7 marine diatoms grown to a stable period were selected for cysK enzyme absorbance detection in diatom cells, namely: T. allenii, T. sinica, T. andamanica, T. minima, T. gravida, C. diadema, and T. flabella. These 7 samples were cultured using iron-restricted Guillard's f / 2 medium, and the content of FeCl3.6H2O in the f / 2 medium formula (preservative solution 2) was changed to reduce the concentration of FeCl3.6H2O by 3 times (1 / 3×Fe), while other preservative solutions in the medium were not changed; in addition to being cultured in an iron-restricted medium, the above 7 diatoms were also cultured normally in a normal Guillard's f / 2 (1×Fe) medium.

[0068] The diatom cells of the 7 strains grown to the stable phase and cultured normally and cultured with reduced iron content (1 / 3×Fe) were collected and extracted, and the absorbance of cysK protease in the diatom cells was measured. The cysK protease content in the diatom cells was measured and calculated, and then the concentration of protein-bound BMAA per diatom cell was detected by LC-MS / MS. The results are shown in Table 1.

[0069] Table 1: Changes in BMAA production and intracellular cysK enzyme concentration in different diatom cells

[0070]

[0071]

[0072] Based on the above data, the linear evaluation was performed on the intracellular cysK protease concentration and the protein-bound BMAA concentration per algal cell of seven BMAA-producing diatoms, namely, T. allenii, T. sinica, T. andamanica, T. minima, T. gravida, C. diadema, and T. flabella, which were cultured to the stable phase with the FeCl3.6H2O concentration in the f / 2 medium reduced by 3 times (1 / 3×Fe): the intracellular cysK protease content per diatom cell protein of these seven diatoms cultured under iron-limited conditions (fg cell) was used. -1 ) as the horizontal axis, and the concentration of BMAA per diatom cell detected by LC-MS / MS (fg cell -1 ) was used as the ordinate to establish a linear equation. The results showed that the content of cysK enzyme had a good linear relationship with the concentration of BMAA. The related linear equation was y = 0.0062x + 0.0032, R 2 =0.9598, such as Figure 4 shown.

[0073] It is worth noting that the absorbance value of the blank group using Guillard's f / 2 medium at a wavelength of 450nm was about 0.04, while in the detection of T. sinica under the selected iron stress (1 / 3×Fe), the BMAA production per unit cell was about 0.026fg cell -1 The absorbance of cysK enzyme is also very low, about 0.064. In addition, the BMAA production per unit cell is less than 0.026fg cell -1 The absorbance detection value of cysK enzyme in diatom cells is very low, close to f / 2 culture medium (0.04), which shows that this method has a lower limit for the detection effectiveness of cysK enzyme concentration. Therefore, only when the BMAA content per unit diatom cell is >0.026fg cell -1 The diatom cells can be used to detect the cysK enzyme concentration using the absorbance method, and then the linear equation (y = 0.0062x + 0.0032, R 2 =0.9598) to directly estimate the concentration of protein-bound BMAA.

[0074] Verification Example 1:

[0075] In order to further verify whether the linear equation in Example 1 of the present invention is universal, the applicant detected the content of cysK enzyme per unit cell of normal growth of T. andamanica, T. gravida and T. flabella in the normal f / 2 medium formula (the medium formula was not changed) when they grew to the stable stage, and the content was 2.95 fg cell -1 , 5.40fg cell -1 and 4.67fg cell -1 , substitute into the linear equation (y=0.0062x+0.0032, R 2 =0.9598) directly calculated the concentration of protein-bound BMAA per unit cell to be 0.027 fg cell -1 , 0.037fg cell -1 and 0.032fg cell -1 The concentrations of protein-bound BMAA detected by LC-MS / MS in these three diatoms were 0.027 fg cell -1 , 0.034fg cell -1 and 0.033fg cell -1 The results showed that the calculated value was basically consistent with the result of our subsequent LC-MS / MS detection.

[0076] Verification Example 2:

[0077] The applicant also selected two other diatoms that can produce BMAA, Planktoniella blanda and Thalassiosiratealata, which were cultured to the stable phase by reducing the iron content (1 / 3×Fe) in the f / 2 culture medium formula to further verify the linear result. The results showed that the corresponding cysK concentrations detected by step (2) for Planktoniella blanda and Thalassiosiratealata were 4.16 fg cell -1 and 5.13fg cell -1 , from the linear equation (y = 0.0062x + 0.0032, R 2 =0.9598) directly estimated the concentration of protein-bound BMAA to be 0.029fgcell -1 and 0.035fg cell -1This is basically consistent with the results of subsequent LC-MS / MS detection. The content of protein-bound BMAA per unit cell in P. blanda and T. tealata detected by LC-MS / MS was 0.031 fg cell -1 and 0.036fg cell -1 Therefore, this also confirms the feasibility of linear evaluation using the intracellular cysK protease concentration of BMAA-producing diatom cells and the unit algal cell protein-bound BMAA concentration.

[0078] Application Example 1:

[0079] The applicant randomly selected three diatoms producing BMAA isolated from the coastal waters of China (mainly the East China Sea and the South China Sea), which were from the Diatom Collection Center of South China Normal University. These three diatoms were cultured by changing the content of FeCl3.6H2O in the f / 2 culture medium formula (preservation solution 2) to reduce the concentration of FeCl3.6H2O by 3 times (1 / 3×Fe), while other preservation solutions in the culture medium remained unchanged. They are: Planktoniella blanda, Thalassiosira tealata and Chaetoceros hirtisetus. They are cultured to the stable phase as in step (2) and the absorbance is detected. The absorbance of cysK enzyme is calculated to be 0.0785, 0.2518 and 0.1786 respectively. By substituting it into the standard curve in step (1), it can be found that the concentration of cysK protease in this diatom cell or several diatoms is 4.16 fg cell. -1 , 5.13fg cell -1 and 4.56fg cell -1 Then, the cysK protease concentration is substituted into the linear equation of step (4), and the BMAA toxin content of the diatom sample can be quickly estimated to be 0.029 fg cell -1 , 0.035fg cell -1 and 0.031fg cell -1 .

[0080] The applicant verified the above evaluation results. Liquid chromatography tandem mass spectrometry (LC-MS / MS) analyzed that under iron-restricted conditions (1 / 3×Fe), the contents of BMAA toxins per unit cell of Planktoniella blanda, Thalassiosira tealata and Chaetoceros hirtisetus were 0.031fg cell-1, 0.036 and 0.032fg cell-1 respectively when the three strains grew to the stable period. The results showed that the accuracy of the BMAA toxins evaluated by the method of the present invention was close to the results of LC-MS / MS analysis and detection. The method of the present invention using the absorbance of cysK expression enzyme in diatom cells to quickly detect the BMAA bound to marine diatom synthetic proteins is both time-saving and accurate, and can solve the problem that when using LC-MS / MS to analyze the BMAA content produced by a large number of diatoms, not only the extraction process is cumbersome but also the detection and verification work is relatively expensive.

[0081] The above examples are only for illustrating the technical concept and technical features of the present invention, and cannot be used to limit the protection scope of the present invention. Any equivalent transformation or modification made according to the essence of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for rapid detection of β-nitrogen-methylamino-L-alanine (BMAA) bound to marine diatom synthetic proteins, characterized in that: The following steps are involved: (1) Establish a standard curve of cysK protease concentration and absorbance: The prokaryotic expressed and purified cysK protease was subjected to full wavelength scanning to determine the maximum absorption wavelength of the cysK protease, the prokaryotic expressed and purified cysK protease was diluted to different concentrations, and the absorbance of the cysK protease of different concentrations was measured under the condition of the maximum absorption wavelength, and a standard curve of the cysK protease concentration and the absorbance was established, and there was a linear relationship between the concentration of the cysK protease of different dilutions and the absorbance; (2) Detection of cysK protease concentration in BMAA-producing diatom cells: When a certain type of BMAA-producing diatom cells grows to a stable period, a suction pump is used to collect part of the diatom cells onto a water filter membrane, and then all the diatom cells on the water filter membrane are transferred to a centrifuge tube using PBS. The diatom cells in the centrifuge tube are washed twice with PBS and then re-dissolved with PBS. The re-dissolved diatom cells are then quickly frozen in liquid nitrogen for a few minutes and then thawed at room temperature. The diatom cells are then ultrasonically disrupted, and finally the absorbance of the cysK protease in the diatom cells is detected at the maximum absorption wavelength of the cysK protease. The absorbance is brought into the standard curve obtained in step (1) to calculate the concentration of the cysK protease in the BMAA-producing diatom cells. By calculating the number of diatom cells in the corresponding collection volume, the unit of the cysK protease can be converted to obtain the content of the cysK protease in the unit diatom cells. (3) Analysis of BMAA toxins in BMAA-producing diatom cells using LC-MS / MS: When a certain type of BMAA-producing diatom cells in step (2) grows to a stable period, part of the diatom cells are transferred to a centrifuge tube and algae mud containing the diatom cells is collected by centrifugation, the protein-bound BMAA in the algae mud is extracted and resuspended with HCl to obtain a BMAA toxin extract, the BMAA toxin extract is purified by a solid phase extraction column and then analyzed by LC-MS / MS for the BMAA toxin in the BMAA-producing diatom cells, and the BMAA toxin is qualitatively and quantitatively analyzed by multiple reaction monitoring mode MRM according to the retention time and mass spectrometry parameters of the BMAA toxin; through qualitative and quantitative analysis, the concentration of protein-bound BMAA in a unit diatom cell can be known; (4) Linear evaluation of the cysK protease concentration in diatom cells and the BMAA content per unit diatom cell protein: According to the operations of step (2) and step (3), various BMAA-producing diatom cells are respectively detected to obtain the cysK protease content per diatom cell and the BMAA concentration per diatom cell protein-bound diatom cell of the various BMAA-producing diatom cells. A linear equation is established with the cysK protease content per diatom cell as the horizontal axis and the BMAA concentration per diatom cell protein-bound diatom cell as the vertical axis. The results show that the concentration of cysK protease has a good linear relationship with the content of BMAA, and the linear equation is a linear equation. The absorbance of the diatom cells grown to the stable period is detected, and the BMAA concentration per diatom cell protein-bound diatom cell of the diatom cell can be obtained by calculation and conversion.

2. The method according to claim 1, characterized in that: In step (1), the prokaryotically expressed and purified cysK protease refers to a recombinant diatom cysK protease with a purity of >90%, and the maximum absorption wavelength of the cysK protease is 450 nm; the prokaryotically expressed and purified cysK protease is diluted to different concentrations, and the concentrations of the cysK protease are 0.005 mg / mL, 0.01 mg / mL, 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL, and 0.05 mg / mL, respectively.

3. The method according to claim 1, characterized in that: In step (2), when the BMAA-producing diatom cells grow to a stable period, 200 mL of diatom cells are collected onto a 0.45 μm water filter membrane using a suction pump, and then the diatom cells on the water filter membrane are rinsed into a 50 mL centrifuge tube using 5 mL of PBS for three times to ensure that all the diatom cells on the water filter membrane are transferred to the centrifuge tube; the collected diatom cells are then centrifuged at 8000 rpm, 8 min, and 4°C. After the centrifugation, the supernatant is removed and the diatom cells at the bottom of the centrifuge tube are collected; 10 mL of PBS is continued to be added to the collected diatom cells for centrifugal washing, and the centrifugal washing is performed twice at 8000 rpm, 8 min, and 4°C. After the two centrifugal washings, the supernatant is removed and the diatom cells at the bottom of the centrifuge tube are re-dissolved with 15 mL of PBS.

4. The method according to claim 1, characterized in that: In step (2), the reconstituted diatom cells are quickly frozen in liquid nitrogen for 6 min, then thawed at room temperature, and after thawing, the algae cells are ultrasonically disrupted in an ice water bath, the ultrasonic disruption conditions are: ultrasonic power 50 W, ultrasonic frequency set to 2 s on, 10 s off, and cycled 30 times; then a Multiskan Sky full-wavelength microplate reader is used to detect the absorbance of cysK protease in the diatom cells at a wavelength of 450 nm, and the absorbance is brought into the standard curve obtained in step (1) to calculate the cysK protease concentration of the diatom cells, the unit of which is mg / mL, and the cell density of the diatom cells after reaching the stable period can be measured by flow cytometry; the volume of the collected diatom cells is 200 mL, and the unit of cysK protease can be converted from mg / mL to fg / cell by calculating the number of diatom cells in the corresponding collected volume, so as to know the content of cysK protease per unit cell.

5. The method according to claim 1, characterized in that: In step (2) and step (3), the method for culturing the BMAA-producing diatom cells is as follows: filtering natural seawater with a 0.45 μm mixed fiber filter membrane, mixing seawater and culture medium at a volume ratio of 1000:1 and placing the mixture in a conical flask, wherein the culture medium is an iron-restricted Guillard's f / 2 medium, i.e., the concentration of FeCl3.6H2O in the Guillard's f / 2 medium formula is reduced by 3 times; inoculating BMAA-producing diatoms, and the initial inoculation density of the BMAA-producing diatoms is 1.0×10 4 cells mL -1 Then, it was placed in an incubator for cultivation. The light intensity of the incubator was 6000 lx, the temperature was 20℃, and the light-dark ratio was 12 h: 12 h. The conical flask was gently shaken three times a day. During the cultivation period, 1 mL of algae solution was taken from each bottle and placed in a 5 mL centrifuge tube. The algae cell density in the stable period was detected by flow cytometry. When the diatom cell density reached 5.0× 10 6 cells mL -1 When the BMAA-producing diatom cells grow to the stable stage, the density of algae in the stable stage is about 5.0× 10 6 cells mL -1 .

6. The method according to claim 1, characterized in that: In step (3), 400 mL of algae liquid containing diatom cells is transferred to a centrifuge tube, centrifuged at 8000 rmp for 10 min at 4°C, and the supernatant is removed to collect the algae mud containing diatom cells; 5 mL of 0.1 M trichloroacetic acid is added to the centrifuge tube containing the algae mud, and after vortexing, the sample is placed in liquid nitrogen for 10 min, thawed at room temperature, and freeze-thawed again, and repeated three times; after freeze-thaw, the algae mud is ultrasonically broken in an ice water bath for 10 min, the ultrasonic power is 50 W, the ultrasonic frequency is set to 2 s on and 10 s off, and the cycle is repeated 30 times; after the ultrasonication, the algae mud is centrifuged at 4°C and 8000 rmp for 10 min, the supernatant is removed, 2 mL of 6 M HCl is added to the algae mud, and vortexed; then it is transferred to a 4 mL injection bottle, and then 1 mL of 6 M HCl is taken to rinse the centrifuge tube, and after vortexing, it is also transferred to the 4 mL injection bottle; the sample in the injection bottle was hydrolyzed at 110 ° C for 24 h, cooled to room temperature and transferred to a 10 mL centrifuge tube, and 1 mL of 6 M HCl was taken to rinse the injection bottle, and the rinsing solution was also transferred to the 10 mL centrifuge tube. After centrifugation, 2 mL of the supernatant was taken and dried with nitrogen at 55 ° C. After drying, 1 mL of 20 mM HCl was added to resuspend the particles to obtain the BMAA toxin extract.

7. The method according to claim 1, characterized in that: In step (3), when the BMAA toxin extract is purified by solid phase extraction column, the operation steps are as follows: Oasis MCX cation exchange solid phase extraction column is used for sample purification, and the specifications of Oasis MCX are 3 cc, 60 mg; first, 3 mL of 100% methanol is taken to activate the solid phase extraction column, and then 2 mL of ultrapure water is added to equilibrate the solid phase extraction column; after the solid phase extraction column is stabilized, 1 mL of BMAA toxin extract is injected; after all the samples flow out of the solid phase extraction column, 2 mL of 0.1 N HCl and 2 mL of 100% methanol are added in sequence to wash the impurities in the extract; finally, 3 mL of 5% ammonia water (NH4OH) is used to elute the toxin extract enriched by the solid phase extraction column, the eluate is collected and dried under nitrogen at 55°C, and after drying, 1 mL of 20 mM HCl is added to resuspend the particles, and finally filtered through a 0.22 µm water filter membrane to obtain the purified BMAA toxin extract for LC-MS / MS toxin analysis.

8. The method according to claim 1, characterized in that: In step (3), when performing LC-MS / MS analysis, the operation steps are as follows: Use AB SCIEX Qtrap 4500 triple quadrupole / tandem linear ion trap mass spectrometer to directly measure the content of BMAA in algal cells without derivatization. The specific parameters are as follows: Liquid chromatography conditions: The liquid chromatography used SeQuant® ZIC-HILIC column with a column size of 150 mm × 2.1 mm, 5 µm, the column temperature was set to 30 °C, the flow rate was 0.35 mL / min, the injection volume was 5 µL, the mobile phase A was an aqueous solution containing 50 mM formic acid, the mobile phase B was an acetonitrile solution containing 50 mM formic acid, and the gradient elution mode was as follows: 0.00 min mobile phase A 5%, mobile phase B 95%, 19.00 min mobile phase A 40%, mobile phase B 60%, 25.00 min mobile phase A 60%, mobile phase B 40%, 27.01 min mobile phase A 5%, mobile phase B 95%, 30.00 min mobile phase A 5%, mobile phase B 95%, the percentages here refer to volume percentages; The mass spectrometry parameters are as follows: a Qtrap 4500 triple quadrupole / tandem linear ion trap mass spectrometer was used, and the connection interface was an electrospray ionization source (ESI). The mass spectrometry analysis conditions are as follows: electrospray voltage (IS) 5500V, nebulization temperature (TEM) 350℃, nebulization gas pressure (GS1) 50 psi, auxiliary gas pressure (GS2) 50 psi, curtain gas pressure (CUR) 40 psi, collision gas pressure (CAD) Medium, entrance voltage (EP) 10V, collision chamber ejection voltage (CXP) 12V.

9. The method according to claim 1, characterized in that: In step (3), when the toxin is analyzed using the multiple reaction monitoring mode (MRM), the operation steps are as follows: the transition mode with high product ion abundance is used as the quantitative basis, and the transition mode unique to each toxin is used for qualitative analysis. Four transition modes are used, including m / z 119 -> 102, 88, 56 and 44, with a declustering potential (DP) of 30 V and a collision energy (CE) of 13, 17, 24 and 24 eV, respectively. The target protein-bound BMAA is quantified using m / z 119 -> 88; in addition, the detection signal intensity ratio between different transition ions is used as an auxiliary standard for the qualitative analysis of these toxin analogs; through this qualitative and quantitative analysis, the concentration of protein-bound BMAA per unit diatom cell can be calculated.

10. The method according to claim 1, characterized in that: In step (4), according to the operation of step (2) and step (3), 7 diatoms grown to the stable stage are used as samples, namely, Thalassiosira ellensis ( T. allenii )、Thalassosina sinensis( T. sinica )、Andaman Sea Thalassiosira ( T. andamanica )、Thalassosella miltiorrhiza( T. minima )、Thalassosella latticeata( T. gravida )、Crown horn algae( C. diadema )、Thalassosina fan-shaped( T. flabella ); the absorbance of cysK protease in diatom cells of these seven samples was determined respectively, and the content of cysK protease per diatom cell could be known by calculation and conversion respectively. Then, the concentration of protein-bound BMAA per diatom cell of these seven samples was detected by LC-MS / MS respectively, and a linear equation was established with the content of cysK enzyme per diatom cell as the horizontal coordinate and the concentration of protein-bound BMAA per diatom cell as the vertical coordinate. The results showed that the concentration of cysK enzyme had a good linear relationship with the content of BMAA, and the linear equation was the linear equation y=0.0062x + 0.0032, R 2 =0.9598; detect the absorbance of diatom cells that have grown to the stable period, and the unit diatom cell protein-bound BMAA concentration of the diatom cells can be obtained through calculation and conversion.

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