Separation and purification method capable of obtaining enterospora hepatopenaei EHP spores with high purity and low empty shell rate
By removing oil particles from shrimp hepatopancreas and combining with Percoll density gradient centrifugation method, the problems of low purification rate and high empty shell rate of EHP spores in the prior art were solved, and high purity and low empty shell rate of EHP spore separation and purification were achieved.
Patent Information
- Application Number
- CN202510070863.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-16
- Publication Date
- 2025-05-27
AI Technical Summary
The prior art is difficult to effectively isolate and purify EHP spores of shrimp liver, especially in the presence of a large number of oil particles in the shrimp liver and pancreas, resulting in a low purification rate and a high empty shell rate.
The shrimp were dissected and the hepatopancreas were peeled off, wet smears were made and positive tissue blocks were selected, and then homogenized under low temperature conditions, and anhydrous ethanol was added for extraction, and the grease particles were removed. Then further purification was performed using Percoll density gradient centrifugation method, and finally high-purity EHP spores were obtained by cell filter and low centrifugal treatment.
The separation and purification of EHP spores with high purity and low altitude shell rate was achieved, which improved the purification yield, reduced oil interference, retained a large number of EHP spores, and reduced the risk of bacterial growth.
Smart Images
Figure CN120041305A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of extraction methods, and particularly relates to a method for separating and purifying spores of Enterocytozoon hepatopenaei (EHP) with high purity and low empty shell rate. Background Art
[0002] Enterocytozoon hepatopenaei (EHP) is an important newly emerging pathogen that has endangered the healthy farming of Litopenaeus vannamei globally in recent years. It has a wide range of prevalence and has been detected in major farming areas such as Shanghai, Jiangsu, Zhejiang, Shandong, Guangdong, Guangxi, and Hainan in China. It is an obligate intracellular parasitic microsporidium that mainly parasitizes in the hepatopancreas of shrimp. After EHP multiplies, a large number of spores are produced in hepatocytes, resulting in slow growth of shrimp and the situation that individuals in the same batch of seedlings have different sizes under the same farming conditions.
[0003] The separation and purification technology of Enterocytozoon hepatopenaei spores is the basis for further studying the biological characteristics such as the morphological structure, life history, pathogenicity, infection route, and genomic information of the pathogen. Common methods for purifying microsporidium spores mainly include sucrose density gradient centrifugation, percoll density gradient centrifugation, potassium sodium tartrate density gradient centrifugation, etc. These methods have good purification effects on spores in muscle samples. However, the spores of Enterocytozoon hepatopenaei mainly exist in the hepatopancreas of shrimp, and the hepatopancreas of shrimp contains a large number of oil droplets, which seriously interfere with the preparation of the crude purified solution during the purification process, resulting in a decrease in the yield of the finally purified spores. Summary of the Invention
[0004] The technical problem to be solved by the present invention is to provide a method for separating and purifying spores of Enterocytozoon hepatopenaei (EHP) with high purity and low empty shell rate.
[0005] The present invention provides a method for separating and purifying spores of Enterocytozoon hepatopenaei (EHP), including:
[0006] (1) Dissect the shrimp and peel off the hepatopancreas, then take the hepatopancreas to make a wet smear, and use an optical microscope to select positive hepatopancreas tissue blocks containing Enterocytozoon hepatopenaei, and discard the hepatopancreas with negative microscopy results;
[0007] (2) Homogenize the positive hepatopancreas under low-temperature conditions to obtain a homogenate;
[0008] (3) At room temperature, mix the homogenate with absolute ethanol, centrifuge and remove the supernatant, then add absolute ethanol to resuspend the precipitate and continue extraction until the precipitate is gray and no longer fades;
[0009] (4) Suspend the final precipitate in step (3) with water, mix well, then filter through a cell filter, centrifuge, and take the turbid supernatant to obtain a crude spore solution;
[0010] (5) At room temperature, prepare a percoll solution from bottom to top, then add the crude spore liquid. After centrifugation, take out the suspended band layer in the top percoll layer;
[0011] (6) Mix the suspended band layer taken out in step (5) with saline, centrifuge, discard the supernatant, enrich the spores, and finally wash and centrifuge to obtain the spores.
[0012] Preferably, in step (1), the dissected shrimp are 20 - 30 shrimp anesthetized and dissected on ice.
[0013] Preferably, in step (2), there are 10 - 20 positive hepatopancreas.
[0014] Preferably, in step (2), homogenize at low temperature, where the low temperature is an ice bath at 0 - 4°C; use a sterilized and aseptically rinsed glass homogenizer for homogenization.
[0015] Preferably, in step (3), the homogenate is mixed with absolute ethanol, where the absolute ethanol is 5 - 8 times the volume of the homogenate; centrifuge at 10000 rmp / min for 3 - 5 min.
[0016] Preferably, in step (4), the cell filter is a 100 μm cell filter; centrifuge at 500 rmp / min for 2 - 3 min.
[0017] In step (4), the precipitate is suspended in water: the precipitate is suspended in 6 - 10 ml of sterilized deionized water.
[0018] Preferably, in step (5), preparing the percoll solution from bottom to top means preparing percoll solutions of 100%, 75%, 50% and 30% of equal volume from bottom to top.
[0019] Preferably, in step (5), centrifuge at 12000 rmp / min for 20 - 40 min; taking out the suspended band layer in the top percoll layer means taking out the grayish-white suspended band layer in the 100% percoll layer.
[0020] Preferably, in step (6), 1 ml of the spore liquid of the suspended band layer taken out is added to a 1.5 ml conical centrifuge tube, and 0.5 ml of saline is added and mixed; the centrifugation is all at 12000 rmp / min for 3 - 5 min.
[0021] The present invention provides spores extracted by the said method.
[0022] Except for step 1-2, other conditions are at room temperature in the present invention. In step (2), the grease is removed, which solves the problem that the spores adhered by the grease are difficult to settle during the subsequent centrifugation process and reduces its influence on the subsequent percoll purification. In step (4), a large amount of homogenate substances are retained, mainly composed of cells and tissue residues, and a large number of EHP spores are hidden in the cells and tissue residues. These cells and tissue residues are prone to form precipitates during the high centrifugal force process. This method selects a low centrifugal force (500 rmp / min) to remove a small amount of large tissue cell residues while retaining a large number of small-sized tissue cell residues, thereby retaining a large number of EHP spores. This greatly increases the yield of EHP spores after subsequent Percoll purification.
[0023] After ethanol treatment, the phenomenon of empty spore generation due to the germination of EHP spores in steps 4-6 is reduced, thereby reducing the empty spore rate. This operation can obtain a large number of solid spores and fully retain the nucleic acid of the spores.
[0024] After ethanol treatment, the bacteria that may originally exist in the hepatopancreas can be inactivated; and the pure spore solution extracted by the conventional method is prone to bacterial growth during storage at 4°C. This may be because its pure spore solution contains a small amount of nutrients such as grease, providing conditions for bacterial reproduction. The pure spore solution extracted by this method is not prone to bacterial growth even after long-term storage at 4°C, which is convenient for subsequent experiments.
[0025] In step (6), a 1.5 ml centrifuge tube is selected to enrich the spores because under the conditions of the same centrifuge, the same rotation speed, and the same time, the number of spores obtained: 1.5 ml centrifuge tube > 2 ml centrifuge tube > 10 ml centrifuge tube.
[0026] Beneficial effects
[0027] The present invention has the advantages of high yield, high purity, and low empty shell rate, etc. It is applicable to tissues with high fat content and muscle tissues, can avoid the loss of spores caused by repeated washing, and can also avoid the bacterial reproduction during the purification operation due to temperature or time length, which affects the subsequent whole genome extraction. Description of the Drawings
[0028] Figure 1 (a) shows that the spores purified by Example 1 have high purity; (b) shows the spores purified by the conventional method, which contain impurities such as bacteria.
[0029] Figure 2 The purified spores obtained by Example 1 include hollow spores and solid spores. Detailed Embodiments
[0030] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.
[0031] Example 1
[0032] (1) 20-30 shrimps were anesthetized and dissected on ice. The hepatopancreas was peeled off and the outer connective tissue sheath as well as the stomach and intestine connected to it were discarded. A small amount of hepatopancreas from each shrimp was taken to make a wet smear. The wet smear was examined under a 40× or 100× objective lens under an ordinary optical microscope. The positive hepatopancreas tissue blocks containing shrimp hepatoenterocystis were selected and the negative hepatopancreas was discarded.
[0033] (2) Place the positive hepatopancreas (about 10-20) into a 10 ml glass homogenizer that has been sterilized and rinsed with sterile water, and homogenize thoroughly at low temperature (0-4°C ice bath) until no tissue blocks are visible to the naked eye and the homogenate is dark red;
[0034] (3) At room temperature, transfer the red homogenate to a centrifuge tube, add 5-8 times the volume of anhydrous ethanol to extract the fat, let it stand for 10 minutes, invert it several times to mix, centrifuge it at 10,000 rpm for 5 minutes, remove the orange-red supernatant, continue to add an equal amount of anhydrous ethanol, resuspend the precipitate, and continue to extract 2-3 times until the precipitate turns gray and no longer fades;
[0035] (4) The precipitate was suspended in 6-10 ml of sterile deionized water and mixed thoroughly by pipetting (40-50 times). After filtering through a 100 μm cell filter, the filtrate was centrifuged at 500 rpm for 3 min. The small amount of precipitate at the bottom was discarded and the turbid supernatant was taken and mixed thoroughly by pipetting again. This turbid liquid was the crude EHP spore liquid, which retained a large number of EHP spores.
[0036] (5) At room temperature, equal volumes of 100%, 75%, 50% and 30% Percoll solutions were layered from bottom to top, and then the above turbid suspension was added. After centrifugation at 12000 rpm for 40 min, the gray-white suspension band layer in the 100% Percoll layer was removed and placed in a new centrifuge tube;
[0037] (6) Add 1 ml of the spore solution taken from the 100% percoll layer to a 1.5 ml conical centrifuge tube, add 0.5 ml of sterile saline, mix well, centrifuge at 12,000 rmp / min for 5 min, discard the supernatant, and perform multiple enrichments of spores in this centrifuge tube. Finally, wash the total precipitate once with 1.5 ml of sterile saline, centrifuge at 12,000 rmp / min for 5 min to completely remove the percoll solution, and finally suspend it in sterile water and store it for later use according to different application requirements.
[0038] 1 Yield:
[0039] In step (6), finally, suspend the precipitate with 1 ml of sterile water to prepare a spore suspension. Use the visual counting method based on a hemocytometer to count the number of spores in 1 ml of the suspension. The formula is D is the dilution factor. The amount of spores obtained this time is 1.6×10 9 spores / ml.
[0040] 2 Judgment of purity:
[0041] Select cover glasses and slides with high cleanliness and no impurity contamination. After staining the purified spores with crystal violet solution, prepare a wet smear of the spores and observe under an oil immersion microscope at 100× to check whether there are particulate matters such as bacteria and other impurities in the purified spores, and evaluate the purity of the spores.
[0042] 3 Statistics of empty shell rate:
[0043] Observe empty spores and solid spores under an oil immersion microscope at 100×. As shown in the figure below, for empty spores, only an outer layer remains (white arrow), while solid spores have a light green refractive property (black arrow). Randomly select different fields of view, count about 100 spores (including solid spores and empty spores), and count the number of empty spores among them. Calculation method of empty shell rate: Number of empty shell spores / Total number of spores.
[0044] Empty shell rate = 4 / 129 = 3.1%
[0045] Comparative Example 1
[0046] Conventional method: Randomly select shrimps suffering from EHP, then aseptically dissect and grind the hepatopancreas. The tissue homogenate is subjected to differential centrifugation (remove impurities at 2000 rpm for 5 min and take the supernatant, take the white precipitate at 12,000 rpm for 20 min), and then subjected to percoll density gradient centrifugation (30%, 60%, 90% percoll) to obtain purified EHP spores.
[0047] Conventional method empty shell rate = 17 / 136 = 12.5%
Claims
1. A method for separating and purifying shrimp hepatoenterozoan EHP spores, comprising: (1) Shrimp were dissected and the hepatopancreas was removed. Then, wet smears were prepared from the hepatopancreas. The positive hepatopancreas tissue blocks containing shrimp hepatoenterostomycetes were selected under an optical microscope, and the negative hepatopancreas was discarded; (2) homogenizing the positive hepatopancreas under low temperature conditions to obtain a homogenate; (3) At room temperature, the homogenate and anhydrous ethanol are mixed, centrifuged and the supernatant is removed, and then anhydrous ethanol is added to resuspend the precipitate and continue extraction until the precipitate turns gray and no longer fades; (4) suspending the final precipitate of step (3) with water, mixing it, filtering it through a cell filter, centrifuging it, taking the turbid supernatant, and obtaining a crude spore liquid; (5) At room temperature, a Percoll solution is prepared from bottom to top, and then the spore crude solution is added. After centrifugation, the suspended band layer in the top Percoll layer is removed; (6) The suspended band layer taken out in step (5) and the saline solution are mixed, centrifuged, the supernatant is discarded, the spores are enriched, and finally washed and centrifuged to obtain the spores.
2. The method according to claim 1, characterized in that: In the step (1), the shrimps are dissected by anesthetizing 20-30 shrimps on ice.
3. The method according to claim 1, characterized in that: In the step (2), there are 10-20 positive hepatopancreas.
4. The method according to claim 1, characterized in that: In the step (2), the homogenization is carried out at a low temperature, wherein the low temperature is an ice bath at 0-4°C; the homogenization is carried out using a glass homogenizer that has been sterilized and rinsed with sterile water.
5. The method according to claim 1, characterized in that: In the step (3), the homogenate is mixed with anhydrous ethanol, wherein the volume of anhydrous ethanol is 5-8 times of the volume of the homogenate; and the centrifugation is performed at 10000 rpm for 3-5 minutes.
6. The method according to claim 1, characterized in that: The cell filter in step (4) is a 100 μm cell filter; the centrifugation is performed at 500 rpm for 2-3 minutes.
7. The method according to claim 1, characterized in that: In the step (5), the percoll solution is prepared from bottom to top by paving equal volumes of 100%, 75%, 50% and 30% percoll solutions from bottom to top.
8. The method according to claim 1, characterized in that: The centrifugation in step (5) is performed at 12000 rpm for 20-40 min; and the removal of the suspended band layer in the uppermost percoll layer is performed by removing the gray-white suspended band layer in the 100% percoll layer.
9. The method according to claim 1, characterized in that: 1 ml of the spore solution of the suspended strip layer taken out in step (6) was added to a 1.5 ml conical centrifuge tube, and 0.5 ml of saline was added and mixed; the centrifugation was performed at 12000 rpm for 3-5 min.
10. Spores extracted by the method of claim 1.