Method for separating flagellum microfilaments and axial filaments of euglena gracilis
By performing flagellar shedding treatment and differential centrifugation technology on the thin naked algae flagella, the axle filaments and microfilaments were successfully isolated, solving the problem of inaccurate separation of flagellar components in the prior art, providing high-purity samples, supporting more in-depth structural biology research.
Patent Information
- Application Number
- CN202510003143.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-02
- Publication Date
- 2025-03-07
AI Technical Summary
The prior art is difficult to accurately and completely separate different components in the thin nude algae flagella, limiting the high-resolution structural biology research of protophytic flagella.
By resuspending the thin nude algae with buffer solution, adding flagella shedding solution for flagella shedding treatment, combining differential centrifugation and detergent extraction technology, the axes and microfilaments of the thin nude algae flagella were separated.
The effective separation of the axes and microfilaments of thin nude algae flagellum is achieved, and high-purity samples are provided, suitable for high-resolution structural biology research, and supplement the structural information of protozoa flagellum.
Smart Images
Figure CN119570631A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of microbial structure research, and in particular to a method for separating flagellar filaments and axonemes of slender euglena. Background Art
[0002] Microbial flagella are a special structure of microorganisms, which play an important role in the movement, infection and immunity of microorganisms, and classification and identification. The research on the structural biology of microbial flagella is a hot topic in current structural biology research. The research on flagella can help people understand the movement mechanism of microorganisms such as pathogens and the host infection process; use the chemotaxis of flagellar bacteria to detect specific chemicals in the environment; and provide inspiration for the development of microrobots and nanotechnology by studying the structure and movement mechanism of flagella.
[0003] At present, the structural biology research on microbial flagella mainly focuses on bacteria such as Escherichia coli and plant green algae such as Chlamydomonas reinhardtii, and there are few studies on flagella in protists. According to the inventors' understanding, the current research on protist flagella mainly focuses on the overall structural state of the whole flagellum of Euglena gracilis. By using samples with high content of Euglena gracilis for related separation, it is impossible to accurately and completely separate the different components in the flagellum, which is not conducive to the use of single-particle cryo-electron microscopy technology to perform high-resolution structural biology research on protist flagella. Summary of the invention
[0004] The technical problem to be solved by the present invention is that the slender euglena used in the prior art is relatively large in size, making it difficult to achieve accurate and complete separation of different components in the flagellum.
[0005] In order to solve the above problems, the present invention proposes the following technical solutions:
[0006] A method for separating flagellar filaments and axonemes of Euglena gracilis, comprising the following steps:
[0007] S1, after resuspending the gracilis with a first buffer solution, adding a flagella shedding solution to perform flagella shedding treatment to obtain a mixture of flagella and gracilis without flagella;
[0008] S2, separating the flagella in the mixture by a first differential centrifugation method to obtain crude extracted flagella of Euglena gracilis;
[0009] S3, resuspending the crudely extracted flagella of Euglena gracilis with a second buffer solution, performing a first centrifugation, and collecting the supernatant; adding a detergent to the supernatant for extraction, performing a second centrifugation, and recovering the precipitate to obtain a mixture of axonemes and microfilaments of Euglena gracilis flagella;
[0010] S4, resuspending the mixture of the axoneme and microfilaments of the flagella of Euglena gracilis with a third buffer solution, adding a coenzyme for incubation, and separating the axoneme and microfilaments of the flagella of Euglena gracilis by a second differential centrifugation method.
[0011] A further technical solution is that the detergent includes at least one of NP-40, Triton X-100 and DDM.
[0012] A further technical solution is that in step S3, the concentration of the added detergent is 0.1-2 wt%, and the extraction is performed at 0-4 degrees Celsius for 0.5-1.5 hours.
[0013] A further technical solution is that in step S4, the coenzyme added is adenosine triphosphate powder with a final concentration of 8 to 15 mM, and the mixture is incubated at room temperature for 0.5 to 1.5 hours.
[0014] A further technical solution is that before step S1, the method further comprises: taking 3 to 4 L of the culture of Euglena gracilis that has been cultured for at least 5 days, centrifuging to obtain Euglena gracilis. In a specific embodiment, the method is centrifuged at room temperature using a centrifugal force of 400 to 600 g for 15 to 30 minutes, and the desired Euglena gracilis is located at the bottom of the centrifuge bottle.
[0015] A further technical solution is that the first buffer solution is a sodium acetate solution with a concentration of 20 to 30 mM, a pH of 6.5 to 7.5, and a dosage of 30 to 50 mL; the second buffer solution is a tris(hydroxymethyl)aminomethane solution with a concentration of 30 to 50 mM, a pH of 7.5 to 8.5, and a dosage of 300 to 800 μL.
[0016] A further technical solution is that the third buffer solution contains 25-30 mM 4-hydroxyethylpiperazineethanesulfonic acid, 3-8 mM magnesium chloride, 0.5-1.5 mM dithiothreitol, 0.1-1.0 mM sodium ethylenediaminetetraacetate, 20-30 mM potassium chloride, pH is 7.1-7.8, and the dosage is 100-300 μL.
[0017] A further technical solution is that in step S1, the flagella shedding treatment comprises adding a flagella shedding solution of Euglena gracilis to the resuspended Euglena gracilis at a volume ratio of 1:4.5-6 under ice bath conditions, and continuously stirring for 1 hour under ice bath conditions.
[0018] The flagella shedding solution of Euglena gracilis contains 20-30 mM sodium acetate, 10-15% (V / V) ethanol, 0.10-0.15% (w / V) sodium ethylenediaminetetraacetate, 5-10% (w / V) sucrose and 30-35 mM calcium chloride.
[0019] A further technical solution is that in step S2, the first differential centrifugation method specifically includes: using a centrifugal force of 100-300g to centrifuge in a 0-4 degree Celsius refrigerated centrifuge for centrifugation 2-4 times, each time for 5-15 minutes, taking the supernatant obtained after each centrifugation for the next centrifugation; using a centrifugal force of 11000-14000g to centrifuge in a 0-4 degree Celsius refrigerated centrifuge for 30-60 minutes to obtain crude extracted slender euglena flagella.
[0020] A further technical solution is that in the step S3, the first centrifugation operation includes centrifuging in a 0-4 degree Celsius refrigerated centrifuge at a centrifugal force of 100-300 g for 1-3 times, each time for 5-15 minutes; in the step S3, the second centrifugation operation includes centrifuging in a 0-4 degree Celsius refrigerated centrifuge at a centrifugal force of 17000-24000 g for 10-30 minutes.
[0021] A further technical solution is that in step S4, the second differential centrifugation method specifically includes: first using 4000-8000g centrifugal force to centrifuge in a 0-4 degree Celsius micro refrigerated centrifuge for 3-10 minutes, recovering the precipitate, which is the axoneme of the flagella of the slender algae, and using 18000-25000g centrifugal force to centrifuge in a 0-4 degree Celsius micro refrigerated centrifuge for 10-20 minutes to recover the precipitate, which is the microfilament of the flagella of the slender algae.
[0022] Compared with the prior art, the technical effects achieved by the present invention include:
[0023] The method for separating flagellar microfilaments and axonemes of gracilis provided by the present invention comprises the following steps: resuspending gracilis with a first buffer solution, adding a flagella shedding solution, performing flagella shedding treatment, and obtaining a mixture of flagella and gracilis without flagella; separating the flagella in the mixture by a first differential centrifugation method, and obtaining crude flagella of gracilis; resuspending the crude flagella of gracilis with a second buffer solution, performing a first centrifugation, and collecting a supernatant; adding a detergent to the supernatant for extraction, performing a second centrifugation, and recovering a precipitate, and obtaining a mixture of axonemes and microfilaments of gracilis flagella; resuspending the mixture of axonemes and microfilaments of gracilis flagella with a third buffer solution, adding a coenzyme for incubation, and separating the axonemes of gracilis flagella and microfilaments of gracilis flagella by a second differential centrifugation method. The present invention optimizes the purification conditions of the flagella of elliptic algae, and is suitable for obtaining complete flagellar components of protozoan elliptic algae that are different from existing bacteria and eukaryotic algae and plants on the basis of small-amount purification. By further separation, samples of elliptic algae flagella axonemes and microfilaments that are more conducive to high-resolution cryo-electron microscopy single-particle high-resolution structural analysis are obtained, which serves as a good sample basis for the structural biology research of flagella in basic biological research, and is expected to solve and supplement the structural information of flagella in protozoa except bacteria and plants, and contribute to the research on microbial flagella in basic life science research. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.
[0025] Figure 1 A schematic flow chart of the method for separating flagellar filaments and axonemes of Euglena gracilis provided by the present invention.
[0026] Figure 2 This is an optical microscope picture of the flagellated Euglena gracilis in Example 1.
[0027] Figure 3 This is an optical microscope picture of the gracilis after the flagella were removed in Example 1. It can be seen from the picture that the gracilis has lost its flagella.
[0028] Figure 4 for Figure 3 Magnified image of .
[0029] Figure 5 This is a picture of the axoneme of the slender euglena flagellum obtained in Example 1.
[0030] Figure 6 for Figure 5 Magnified image of .
[0031] Figure 7 This is a picture of the microfilaments of the slender euglena flagella obtained in Example 1.
[0032] Figure 8 for Figure 7 Magnified image of .
[0033] Fig. 9 This is an optical microscope picture of the flagellated Euglena gracilis in Example 2.
[0034] Fig.10 This is an optical microscope picture of the gracilis after the flagella were removed in Example 2. It can be seen from the picture that the gracilis has lost its flagella.
[0035] Fig.11 This is the sample obtained by density gradient centrifugation in Example 2.
[0036] Fig.12 This is a microscope picture of the sample with 0-40% sucrose layer in Example 2.
[0037] Fig.13 This is a microscope picture of a sample of a 40-60% sucrose layer in Example 2.
[0038] Fig.14 for Fig.13 Magnified image of . DETAILED DESCRIPTION
[0039] The technical solutions in the embodiments will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Similar component numbers in the drawings represent similar components. Obviously, the embodiments described below are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0040] It should be understood that when used in this specification and the appended claims, the terms "include" and "comprises" indicate the presence of described features, integers, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or combinations thereof.
[0041] It should also be understood that the terms used in this description of the embodiments of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the embodiments of the present invention. As used in the description of the embodiments of the present invention and the appended claims, unless the context clearly indicates otherwise, the singular forms of "a", "an" and "the" are intended to include plural forms.
[0042] Example 1
[0043] The embodiment of the present invention provides a method for separating flagellar filaments and axonemes of Euglena gracilis, comprising the following steps:
[0044] 3 L of the culture of Euglena gracilis was recovered by centrifugation at 500 g for 20 minutes. The precipitate obtained was Euglena gracilis. Under optical microscope observation, Euglena gracilis with flagella can be seen, such as Figure 2 .
[0045] The above-mentioned Euglena gracilis was resuspended in 37.5 mL of a solution containing 25 mM sodium acetate, pH 7.0, and 195 mL of a flagella shedding solution of Euglena gracilis containing 25 mM sodium acetate, 12% (V / V) ethanol, 0.12% (w / V) sodium ethylenediaminetetraacetic acid, 7% (w / V) sucrose and 32 mM calcium chloride, pH 7.0, was added on ice, and stirred in an ice bath for 1 hour to perform flagella shedding treatment, thereby obtaining a mixture of flagella and Euglena gracilis without flagella. After the flagella shedding treatment, the Euglena gracilis without flagella was observed under an optical microscope, as shown in FIG. Figure 3 , Figure 4 .
[0046] The mixture was centrifuged three times in a refrigerated centrifuge at 150 g for 10 minutes each time at 4 degrees Celsius. The supernatant obtained after each centrifugation was centrifuged in the next step. The precipitate obtained from each centrifugation was recovered, and the combined precipitate was the flagella of Euglena gracilis removed. The supernatant obtained from the last step of centrifugation was packaged and centrifuged in a refrigerated centrifuge at 12150 g for 45 minutes at 4 degrees Celsius to obtain the crude flagella of Euglena gracilis.
[0047] The crudely extracted flagella of Euglena gracilis are resuspended with 500uL of a buffer solution containing 40mM tris(hydroxymethyl)aminomethane and pH 8.0, and the flagella are centrifuged at 150g for 10 minutes in a micro refrigerated centrifuge at 4 degrees Celsius, centrifuged twice, and the supernatant is collected to remove the contamination of the flagella of Euglena gracilis, thereby obtaining a flagella suspension of Euglena gracilis; 1% NP-40 detergent is added to the purified flagella suspension of Euglena gracilis, and the flagella are extracted for 1 hour, and then the flagella are centrifuged at 20000g for 15 minutes in a micro refrigerated centrifuge at 4 degrees Celsius, and the precipitate is recovered, and the precipitate is a mixture of axonemes and microfilaments of Euglena gracilis flagella.
[0048] The mixture of the flagellar axial filaments and microfilaments of the above-mentioned Euglena gracilis was resuspended in 200uL of a solution containing 30mM 4-hydroxyethylpiperazineethanesulfonic acid, 5mM magnesium chloride, 1mM dithiothreitol, 0.5mM sodium ethylenediaminetetraacetic acid, 25mM potassium chloride, pH 7.4, and a final concentration of 10mM adenosine triphosphate powder was added to the system for rotation incubation at room temperature for 1 hour. After the incubation, the system was first centrifuged at 6000g in a 4 degree Celsius micro refrigerated centrifuge for 5 minutes to recover the precipitate, which is the axial filament part of the flagella of Euglena gracilis. The precipitate was treated with 60uL of 30mM 4-hydroxyethylpiperazineethanesulfonic acid, 5mM magnesium chloride, 1mM dithiothreitol, 0.5mM sodium ethylenediaminetetraacetic acid, 25mM potassium chloride, pH The supernatant was centrifuged at 20000 g for 15 minutes in a micro refrigerated centrifuge at 4 degrees Celsius to recover the precipitate, which was the flagellar filament part of the slender algae. The precipitate was resuspended with 60uL of a solution of 30mM 4-hydroxyethylpiperazineethanesulfonic acid, 5mM magnesium chloride, 1mM dithiothreitol, 0.5mM sodium ethylenediaminetetraacetic acid, 25mM potassium chloride, pH 7.4 to obtain filaments with a concentration of 6mg / mL.
[0049] The axoneme and microfilaments of the flagella of the slender Euglena separated in this example were prepared into samples with a concentration of 2 mg / mL, and observed by negative staining electron microscopy. Figure 5-Figure 8 It was found that the flagellar axoneme and microfilaments of this embodiment were well separated, the axoneme part was thick and long, and it could be clearly observed that the microfilament part was mainly composed of two different types of microfilaments, thick and thin. The impurity content in the sample was extremely low and the sample purity was high.
[0050] Example 2
[0051] The embodiment of the present invention provides a method for separating flagellar filaments and axonemes of Euglena gracilis, comprising the following steps:
[0052] 3 L of the culture of Euglena gracilis was recovered by centrifugation at 500 g for 20 minutes. The precipitate obtained was Euglena gracilis. Under optical microscope observation, Euglena gracilis with flagella can be seen, such as Fig. 9 .
[0053] The above-mentioned Euglena gracilis was resuspended in 37.5 mL of a solution containing 25 mM sodium acetate, pH 7.0, and 195 mL of a flagella shedding solution of Euglena gracilis containing 25 mM sodium acetate, 12% (V / V) ethanol, 0.12% (w / V) sodium ethylenediaminetetraacetic acid, 7% (w / V) sucrose and 32 mM calcium chloride, pH 7.0, was added on ice, and stirred in an ice bath for 1 hour to perform flagella shedding treatment, thereby obtaining a mixture of flagella and Euglena gracilis without flagella. After the flagella shedding treatment, the Euglena gracilis without flagella was observed under an optical microscope, as shown in FIG. Fig.10 .
[0054] The mixture was centrifuged three times in a refrigerated centrifuge at 150 g for 10 minutes each time at 4 degrees Celsius. The supernatant obtained after each centrifugation was centrifuged in the next step. The precipitate obtained from each centrifugation was recovered, and the combined precipitate was the flagella of Euglena gracilis removed. The supernatant obtained from the last step of centrifugation was packaged and centrifuged in a refrigerated centrifuge at 12150 g for 45 minutes at 4 degrees Celsius to obtain the crude flagella of Euglena gracilis.
[0055] The crudely extracted flagella of Euglena gracilis are resuspended with 500uL of a buffer solution containing 40mM tris(hydroxymethyl)aminomethane and pH 8.0, and the flagella are centrifuged at 150g for 10 minutes in a micro refrigerated centrifuge at 4 degrees Celsius, centrifuged twice, and the supernatant is collected to remove the contamination of the flagella of Euglena gracilis, thereby obtaining a flagella suspension of Euglena gracilis; 1% NP-40 detergent is added to the purified flagella suspension of Euglena gracilis, and the flagella are extracted for 1 hour, and then the flagella are centrifuged at 20000g for 15 minutes in a micro refrigerated centrifuge at 4 degrees Celsius, and the precipitate is recovered, and the precipitate is a mixture of axonemes and microfilaments of Euglena gracilis flagella.
[0056] In this embodiment, sucrose density gradient centrifugation is used to separate the mixture of the flagellar filaments and microfilaments of the slender euglena flagella from the microfilaments and the slender euglena flagella. The specific operation includes: making a sucrose density gradient layer: using a buffer solution containing 40mM tris(hydroxymethyl)aminomethane, 60% (w / V) sucrose, pH 8.0 as the bottom layer, using a buffer solution containing 40mM tris(hydroxymethyl)aminomethane, 40% (w / V) sucrose, pH 8.0 as the middle layer, using a mixed solution containing 40mM tris(hydroxymethyl)aminomethane, pH 8.0 buffer solution to resuspend the mixture of the flagellar filaments and microfilaments of the slender euglena flagella as the upper layer, centrifuging the sucrose density gradient layer at 20000g centrifugal force in a 4 degree Celsius micro refrigerated centrifuge for 30 minutes, and through density gradient centrifugation, it is found that the precipitated sample is mainly concentrated in the 0-40% sucrose layer and the 40-60% sucrose layer, with a clear boundary, as shown in FIG. Fig.11 .
[0057] Samples of the 0-40% sucrose layer and the 40-60% sucrose layer were resuspended in 60uL of a solution of 30mM 4-hydroxyethylpiperazineethanesulfonic acid, 5mM magnesium chloride, 1mM dithiothreitol, 0.5mM sodium ethylenediaminetetraacetate, 25mM potassium chloride, pH 7.4, and then observed by negative staining electron microscopy. Figure 12-14, it can be seen that the samples with 0-40% sucrose layer are mainly flagellar microfilaments, there are more impurity proteins in the samples, and the background is dirty, indicating that the sample purity is insufficient and the concentration is low. The samples with 40-60% sucrose layer do not have obvious complete flagellar axoneme structure, but it can be seen that there are also a large number of microfilaments in the protein sample. Compared with the sample separated in Example 1, the single microtubule structure separated from the flagella 9+2 axoneme structure in Example 2 is also seriously contaminated, and no obvious separation effect can be achieved.
[0058] In Example 2 of the present invention, when separating the mixture of flagellar axonemes and microfilaments of Euglena gracilis, a sucrose density gradient centrifugation method is used. The sample finally separated contains more impurity proteins and the separation effect cannot be achieved.
[0059] In summary, the method for separating the flagellar filaments and axonemes of Euglena gracilis provided by the present invention optimizes the purification conditions of Euglena gracilis flagella, adds ATP to the obtained mixture of axonemes and microfilaments of Euglena gracilis flagella, incubates it, and then performs differential centrifugation, so that the axonemes and microfilaments of the flagella can be effectively and completely separated, and the samples have a sufficiently high purity to meet the research needs.
[0060] In the above embodiments, the description of each embodiment has its own emphasis. For parts that are not described in detail in a certain embodiment, reference can be made to the relevant descriptions of other embodiments.
[0061] The above is a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can easily think of various equivalent modifications or replacements within the technical scope disclosed by the present invention, and these modifications or replacements should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.
Claims
1. A method for separating flagellar filaments and axonemes of Euglena gracilis, characterized in that: The following steps are involved: S1, after resuspending the gracilis with a first buffer solution, adding a flagella shedding solution to perform flagella shedding treatment to obtain a mixture of flagella and gracilis without flagella; S2, separating the flagella in the mixture by a first differential centrifugation method to obtain crude extracted flagella of Euglena gracilis; S3, resuspending the crudely extracted flagella of Euglena gracilis with a second buffer solution, performing a first centrifugation, and collecting the supernatant; adding a detergent to the supernatant for extraction, performing a second centrifugation, and recovering the precipitate to obtain a mixture of axonemes and microfilaments of Euglena gracilis flagella; S4, resuspending the mixture of the axoneme and microfilaments of the flagella of Euglena gracilis with a third buffer solution, adding a coenzyme for incubation, and separating the axoneme and microfilaments of the flagella of Euglena gracilis by a second differential centrifugation method.
2. The method for separating flagellar filaments and axonemes of Euglena gracilis according to claim 1, characterized in that: The detergent includes at least one of NP-40, TritonX-100 and DDM.
3. The method for separating flagellar filaments and axonemes of Euglena gracilis according to claim 1, characterized in that: In the step S3, the concentration of the added detergent is 0.1-2 wt %, and the extraction is carried out at 0-4 degrees Celsius for 0.5-1.5 hours.
4. The method for separating flagellar filaments and axonemes of Euglena gracilis according to claim 1, characterized in that: In the step S4, the coenzyme added is adenosine triphosphate powder, the final concentration is 8 to 15 mM, and the incubation is carried out at room temperature for 0.5 to 1.5 hours.
5. The method for separating flagellar filaments and axonemes of Euglena gracilis according to any one of claims 1 to 4, characterized in that: Before the step S1, the method further includes: taking 3 to 4 L of the culture of Euglena gracilis that has been cultured for at least 5 days, centrifuging the culture, and obtaining Euglena gracilis.
6. The method for separating flagellar filaments and axonemes of Euglena gracilis according to claim 5, characterized in that: The first buffer solution is a sodium acetate solution with a concentration of 20-30 mM, a pH of 6.5-7.5, and a dosage of 30-50 mL; the second buffer solution is a tris(hydroxymethyl)aminomethane solution with a concentration of 30-50 mM, a pH of 7.5-8.5, and a dosage of 300-800 μL.
7. The method for separating flagellar filaments and axonemes of Euglena gracilis according to claim 5, characterized in that: The third buffer solution contains 25-30 mM 4-hydroxyethylpiperazineethanesulfonic acid, 3-8 mM magnesium chloride, 0.5-1.5 mM dithiothreitol, 0.1-1.0 mM sodium ethylenediaminetetraacetate, 20-30 mM potassium chloride, has a pH of 7.1-7.8, and is used in an amount of 100-300 μL.
8. The method for separating flagellar filaments and axonemes of Euglena gracilis according to claim 1, characterized in that: In the step S2, the first differential centrifugation method specifically includes: using a centrifugal force of 100-300g to centrifuge in a 0-4 degree Celsius refrigerated centrifuge for 2-4 times, each time for 5-15 minutes, and taking the supernatant obtained after each centrifugation for the next centrifugation; using a centrifugal force of 11000-14000g to centrifuge the supernatant obtained for the last time in a 0-4 degree Celsius refrigerated centrifuge for 30-60 minutes to obtain crude extracted gracilis flagella.
9. The method for separating flagellar filaments and axonemes of Euglena gracilis according to claim 1, characterized in that: In the step S3, the first centrifugation operation includes centrifuging in a 0-4 degree Celsius refrigerated centrifuge at a centrifugal force of 100-300 g for 1-3 times, each time for 5-15 minutes; in the step S3, the second centrifugation operation includes centrifuging in a 0-4 degree Celsius refrigerated centrifuge at a centrifugal force of 17000-24000 g for 10-30 minutes.
10. The method for separating flagellar filaments and axonemes of Euglena gracilis according to claim 1, characterized in that: In the step S4, the second differential centrifugation method specifically includes: first using 4000-8000g centrifugal force at 0-4 degrees Celsius micro refrigerated centrifuge for centrifugation for 3-10 minutes, recovering the precipitate, which is the axoneme of the flagella of the slender algae, and using 18000-25000g centrifugal force at 0-4 degrees Celsius micro refrigerated centrifuge for centrifugation for 10-20 minutes to recover the precipitate, which is the microfilament of the flagella of the slender algae.