Method for extracting foot vesicles of perna viridis

Abstract

The invention provides an extraction method of perna viridis foot vesicles, which comprises the following steps: S1, respectively sampling tissues of a near-end region and a far-end region of a foot, and grinding to obtain a foot tissue homogenate; s2, removing tissue fragments and other impurities by adopting a multi-stage filtration mode to obtain a foot vesicle crude extract; and S3, further removing organelles and other impurities by adopting a multi-time centrifugation mode to obtain purified near-end region vesicles and far-end region vesicles of the feet. According to the method, crude extraction of the vesicles is carried out in manners of multiple times of filtration, low-speed centrifugation, filter membrane filtration and the like, and the vesicles can be obtained under the conditions of less centrifugation times, rotation speed and time, so that the extraction process of the vesicles is simplified, and the activity of biomacromolecules in the vesicles is maintained to the maximum extent; the method can be used in the scientific research fields of byssus formation mechanism research, byssus biomimetic material research and development and the like.

Description

Technical Field

[0001] The present invention relates to the field of marine biotechnology, and in particular to a method for extracting foot vesicles of green mussel. Background Art

[0002] Byssal threads play an important role in the attachment, aggregation, and reproduction of bivalves (McCartney, 2021), but their self-assembly mechanism has not yet been clearly elucidated. This is mainly because byssal thread self-assembly occurs in the confined space of the foot ventral groove, which cannot be directly observed. Currently, direct observation can only be achieved by synthesizing byssal thread proteins in vitro. Byssal thread proteins are characterized by a wide variety, low conservation, and diverse functions (Qin et al., 2016; Li et al., 2017; Liu, 2022). Therefore, the annotation and prediction of byssal thread proteins have progressed slowly.

[0003] Direct proteomic sequencing of the byssus fibers cannot reveal the complete byssus protein family. However, using foot tissue or foot glands for omics sequencing (DeMartini et al., 2017) presents the problem of interference from other tissues, making it difficult to accurately identify the proteins involved in byssus self-assembly. Byssus proteins are stored in a liquid phase within the foot gland, the foot gland. Upon encountering a suitable substrate, bivalves transport the byssus proteins into vesicles located in the foot ventral groove. The vesicles rupture, releasing the byssus proteins. Under the influence of pH and other factors, the byssus proteins rapidly self-assemble within minutes, forming a solid byssus with a distinct hierarchical structure (Yu et al., 2011; Priemel et al., 2017 & 2020). To identify the byssus proteins present in these vesicles, isolation and extraction of foot gland vesicles is urgently needed. Summary of the Invention

[0004] In view of this, the object of the present invention is to provide a method for extracting foot vesicles of the green mussel, which helps to reduce the vesicle extraction time and maximize the activity of byssus protein and other biomacromolecules in the vesicles.

[0005] The technical solution adopted by the present invention to achieve the technical purpose is:

[0006] A method for extracting vesicles from the foot of the green mussel comprises the following steps:

[0007] S1. Sample the proximal and distal regions of the foot and grind them to obtain foot tissue homogenate;

[0008] S2. Using multi-stage filtration to remove tissue debris and other impurities, obtaining a crude extract of foot vesicles;

[0009] S3. Multiple centrifugation steps are used to further remove organelles and other impurities to obtain purified vesicles from the proximal and distal regions of the foot.

[0010] Furthermore, in step S1, the tissues of the proximal and distal regions of the foot are sampled separately, including: removing the epidermis and part of the muscle tissue of the foot of the green mussel, dividing the foot tissue into two regions, the proximal and distal regions, rinsing them with double-distilled water, and storing them in different centrifuge tubes for later use.

[0011] Furthermore, the foot tissue is divided into two regions, proximal and distal, according to the location of the foot glands that secrete byssal threads and adhesion discs.

[0012] Furthermore, in step S1, the grinding liquid used for grinding the foot tissue contains 0.2 M, pH 5.5 citrate-phosphate buffer, 0.4 M sucrose, 0.15 M EDTA and 0.005 M ascorbic acid.

[0013] Furthermore, in step S1, the grinding method includes: placing the centrifuge tubes storing the proximal and distal foot tissues respectively into liquid nitrogen for pre-cooling, and then transferring them into a grinding tube for cryogenic grinding for 5 minutes.

[0014] Furthermore, in the grinding tube, the foot tissue in the proximal area of each tube is preferably from about 6-8 green mussels, and the foot tissue in the distal area of each tube is preferably from about 3-4 green mussels, and 5 mL of grinding liquid and 6 grinding beads are added to the tube.

[0015] Furthermore, in step S2, a multi-stage filtration method is used to remove tissue fragments and other impurities, including: filtering with a 0.5 mm stainless steel mesh to remove residual tissue fragments and collect the filtrate; then filtering with a 100 μm cell strainer to collect the filtrate; finally, filtering with a 40 μm cell strainer, and the collected filtrate is the crude extract of the foot vesicles.

[0016] Furthermore, in step S3, multiple centrifugations are used to further remove organelles and other impurities, including: centrifuging the crude extract of the foot vesicles at 1000×g and 4°C for 10 minutes, aspirating the supernatant, and discarding the precipitate; then centrifuging at 10,000×g and 4°C for 10 minutes, removing the supernatant, and the precipitate is the purified foot proximal region vesicles and distal region vesicles.

[0017] Furthermore, the above method also includes: S4. Using light microscopy, transmission electron microscopy and proteome sequencing to test the vesicle extraction effect.

[0018] Furthermore, in step S4, the purified vesicles are resuspended in a grinding solution, mixed by pipetting, and then one drop is added to a glass slide. The vesicles are stained with Sirius red, and the red granular substances observed under an optical microscope are vesicles.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The green mussel vesicle extraction method provided by the present invention is reported for the first time. Vesicles can be obtained under conditions of fewer centrifugation times, rotation speeds and time. Compared with other bivalve vesicle extraction methods, it not only simplifies the vesicle extraction process and greatly reduces the time for vesicle separation and extraction, but also can maximize the protection of the activity of byssus protein and other biological macromolecules in the vesicles. The extracted vesicles have complete morphology, and the byssus protein has high biological activity. It can be used in related fields such as studying the byssus self-assembly mechanism and the development of biomimetic materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Shown are the results of Sirius red staining of the proximal region (A) and distal region (B) of the foot of the green mussel.

[0022] Figure 2 Shown are transmission electron microscopy observations of vesicles in the proximal (A) and distal (B) regions of the foot of the green mussel.

[0023] Figure 3 Showing the KEGG enrichment results of the proteomes of the proximal region (A) and distal region vesicles (B) of the green mussel foot. DETAILED DESCRIPTION

[0024] In order to more clearly illustrate the present invention, the present invention is further described in detail below in conjunction with embodiments and with reference to the accompanying drawings. It should be understood by those skilled in the art that the following specific description is illustrative rather than restrictive and should not be used to limit the scope of protection of the present invention.

[0025] Unless otherwise specified, the experimental methods used in the examples of the present invention are all conventional methods.

[0026] Unless otherwise specified, the materials, reagents, etc. used in the examples of the present invention can be obtained from commercial sources.

[0027] The green mussels of the present invention are all from the vegetable market in Haikou City.

[0028] Example

[0029] (1) The foot of the green mussel was cut, and the epidermis and part of the muscle tissue were removed. The foot was then divided into two parts, the proximal region and the distal region, according to the location of the foot gland that secretes the byssal thread and the adhesive disc. The samples were rinsed with double distilled water, dried, and placed in 1.5 mL centrifuge tubes. They were then frozen in liquid nitrogen at -196°C for 5 minutes. The byssal thread is divided into two parts, the byssal thread and the adhesive disc. The byssal thread is formed by the foot gland vesicles in the proximal region of the foot, while the adhesive disc is formed by the foot gland vesicles in the distal region of the foot. Mussels are one of the shellfish species with the most in-depth research on byssal thread formation. The vesicles in the proximal and distal regions of their feet have different protein compositions. The specific proteins of the proximal region vesicles (forming the byssal thread) are mainly foot proteins fp-2 and fp-3, while the specific proteins of the distal region vesicles (forming the adhesive disc) are mainly foot proteins fp-2, fp-3, fp-5, and fp-6, which play different functions in byssal thread formation.

[0030] (2) The frozen samples were transferred to grinding tubes. The proximal foot of each tube was from about 6-8 green mussels, and the distal foot of each tube was from about 3-4 green mussels. 5 mL of grinding solution and 6 2.5 mm stainless steel grinding beads were added to the tube. The grinding solution contained 0.2 M, pH 5.5 citrate-phosphate buffer, 0.4 M sucrose (Standard for GC, ≥99.5% (GC)), 0.15 M EDTA and 0.005 M ascorbic acid. After the foot tissue sample and grinding solution were thoroughly mixed, they were placed in a Tissuelyser-24L grinder, set to 60 Hz, and cryogenically ground for 5 minutes.

[0031] (3) The tissue homogenate was filtered for the first time using a 0.5 mm stainless steel mesh to remove residual tissue debris and collect the filtered supernatant. The homogenate was filtered for the second time using a 100 μm nylon cell strainer and the filtrate was collected. Finally, the homogenate was filtered for the third time using a 40 μm nylon cell strainer and the filtrate was collected, which was the crude extract of the foot vesicles.

[0032] (4) The foot tissue filtrate after three filtrations was centrifuged at 1000×g and 4°C for 10 min, and the supernatant was aspirated into a clean centrifuge tube. The filtrate was centrifuged at 10000×g and 4°C for 10 min, and the supernatant was aspirated. The white precipitate at the bottom of the centrifuge tube was the purified foot vesicles.

[0033] (5) Use 600 μL grinding solution to resuspend the vesicles in the proximal and distal regions of the foot. When resuspending, be careful to avoid the insoluble EDTA in the grinding solution from mixing into the vesicle precipitate. Puff and mix thoroughly for microscopic observation. Add an appropriate amount of vesicle solution to the slide, add an equal volume of Sirius red dye solution, mix thoroughly, and observe the vesicle structure under an ordinary optical microscope. The vesicles of the foot gland can be stained red ( Figure 1), and the extent of vesicle accumulation differed between the proximal and distal regions of the foot.

[0034] (6) Take 30 μL of the foot vesicle suspension, drop the vesicles from different areas of the foot onto the sealing film, place the copper mesh with the membrane on the droplet, let it stand for 5 minutes, and absorb the residual liquid with filter paper. Then rinse with PBS for 3 minutes, fix with 1% glutaraldehyde for 3 minutes, rinse with ddH2O 4 times, each rinse for 1 minute, and stain with phosphotungstic acid solution for 3 minutes. Absorb the residual liquid with filter paper, and dry the copper mesh at room temperature. Before loading the electron microscope, try to ensure that the copper mesh is dry to avoid the influence of water-containing substances on the vacuum of the electron microscope. Observe the vesicle structure of the proximal and distal areas of the foot under the electron microscope ( Figure 2 ), the morphology and size of vesicles vary in different regions of the foot.

[0035] (7) The vesicles from different regions of the foot were subjected to proteomic research by protein extraction, enzyme digestion, liquid chromatography-mass spectrometry tandem analysis, bioinformatics analysis and other methods. Based on the raw files obtained by mass spectrometry detection, a green mussel foot silk protein annotation database was constructed and a database search was performed; based on the results of the database search, common functional annotations were performed on the identified proteins, including GO, KEGG, Protein domain, COG / KOG annotations. The 4D Fast DIA proteome sequencing results showed that the vesicles isolated from the proximal region of the foot (the region forming the foot silk thread) contained 1266 proteins such as foot proteins Pvfp-2 and Pvfp-3, and the vesicles isolated from the distal region of the foot (the region forming the adhesion disc) contained 2092 proteins such as foot proteins Pvfp-2, Pvfp-3, Pvfp-5 and Pvfp-6. Finally, the identified proteins were enriched using the Fisher's exact test method ( Figure 3 ), the KEGG pathways enriched in vesicles in the proximal and distal regions of the foot were basically the same, with only map00630 Glyoxylate and dicarboxylate metabolism (specific to vesicles in the proximal region of the foot) and map03060 Protein export (specific to vesicles in the distal region of the foot) belonging to specific pathways.

[0036] Obviously, the above embodiments of the present invention are merely examples to more clearly illustrate the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made based on the above description. It is impossible to enumerate all implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the scope of protection of the present invention.

Claims

1. A method for extracting foot vesicles of green mussel, comprising the following steps: S1. Sample the proximal and distal regions of the foot and grind them to obtain foot tissue homogenate; S2. Using multi-stage filtration to remove tissue debris and other impurities, obtaining a crude extract of foot vesicles; S3. Multiple centrifugation steps are used to further remove organelles and other impurities to obtain purified vesicles from the proximal and distal regions of the foot.

2. The extraction method according to claim 1, wherein In step S1, the proximal and distal regions of the foot are sampled separately, including: removing the epidermis and part of the muscle tissue of the foot of the green mussel, dividing the foot tissue into two regions, the proximal region and the distal region, rinsing them with double-distilled water, and storing them in different centrifuge tubes for later use.

3. The extraction method according to claim 2, characterized in that According to the location of the foot glands that secrete byssal threads and adhesion discs, the foot tissue is divided into two regions: proximal and distal.

4. The extraction method according to claim 1, wherein In step S1 , the grinding solution used to grind the foot tissue contains 0.2 M pH 5.5 citrate-phosphate buffer, 0.4 M sucrose, 0.15 M EDTA, and 0.005 M ascorbic acid.

5. The extraction method according to claim 2, characterized in that In step S1, the grinding method includes: placing centrifuge tubes storing the proximal and distal foot tissues respectively into liquid nitrogen for pre-cooling, and then transferring them into grinding tubes for cryogenic grinding for 5 minutes.

6. The extraction method according to claim 5, characterized in that In the grinding tube, the foot tissue in the proximal area of each tube comes from 6-8 green mussels, and the foot tissue in the distal area of each tube comes from 3-4 green mussels. 5 mL of grinding liquid and 6 grinding beads are added to the tube.

7. The extraction method according to claim 1, characterized in that In step S2, a multi-stage filtration method is used to remove tissue fragments and other impurities, including: filtering with a 0.5 mm stainless steel mesh to remove residual tissue fragments and collect the filtrate; then filtering with a 100 μm cell strainer to collect the filtrate; finally, filtering with a 40 μm cell strainer, and the collected filtrate is the crude extract of the foot vesicles.

8. The extraction method according to claim 1, characterized in that In step S3, multiple centrifugations are used to further remove organelles and other impurities, including: centrifuging the crude foot vesicle extract at 1000×g and 4°C for 10 minutes, aspirating the supernatant, and discarding the precipitate; then centrifuging at 10,000×g and 4°C for 10 minutes, removing the supernatant, and the precipitate is the purified foot proximal region vesicles and distal region vesicles.

9. The extraction method according to any one of claims 1 to 8, characterized in that Also includes: S4. The vesicle extraction effect was verified by light microscopy, transmission electron microscopy and proteome sequencing.

10. The extraction method according to claim 9, characterized in that In step S4, the purified vesicles are resuspended in a grinding solution, mixed by pipetting, and then one drop is added to a glass slide. The vesicles are stained with Sirius red. Red granular substances observed under an optical microscope are vesicles.

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