Cryptocaryon irritans cyst wall separation and purification method, cryptocaryon irritans cyst wall purity identification method and cryptocaryon irritans cyst wall component identification and data set construction method

Abstract

The invention provides a separation and purification method, a purity identification method and a component identification and data set construction method of cryptocaryon irritans cyst walls, and relates to the technical field of cryptocaryon irritans detection. The separation and purification method of the cryptocaryon irritans cyst wall comprises the following steps: treating a cyst by using low-concentration sodium hypochlorite and a cell protective agent (DMSO and sterile seawater) containing 1-3wt.% of PMSF, and separating and purifying the cyst wall by using a hypotonic buffer solution and a differential centrifugation and density gradient centrifugation method; a purified capsule wall with a clean surface and complete structure and components is obtained, and the problem that an existing capsule wall is not pure and complete in separation is solved. According to the method for identifying the purity of the capsule wall, a form and molecule two-dimensional verification standard is provided, and the blank of lack of systematic purity identification and control in the prior art is filled. The capsule wall protein data set constructed in the component identification and data set construction method can be used for quickly retrieving the capsule wall components, and an intelligent platform is provided for subsequent diagnosis and drug target screening.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of stimulating Cryptocaryon irritans detection, in particular to a separation and purification method of stimulating Cryptocaryon irritans cyst wall, a purity identification method, and a component identification and data set construction method. BACKGROUND

[0002] As a specific parasite on the body surface of marine hard fish, Cryptocaryon irritans Brown, 1951 is listed as a class II animal disease in China since 2008. In recent years, the disease has frequently broken out in the southeast coastal areas of China, causing huge economic losses to the marine hard fish breeding industry. The life history of Cryptocaryon irritans includes four stages: trophont, precyst, cyst and larva. The cyst stage is the longest and free-living stage. Due to the hard cyst wall, it shows strong resistance to drugs and harsh environments. Therefore, in-depth study of the composition and structure of the cyst wall is of great significance for effective prevention and control of Cryptocaryon irritans disease, and the separation and purification of the cyst wall is a basic prerequisite for related research. Currently, in the field of separation and purification of Cryptocaryon irritans cyst wall, the conventional method is to collect the empty cyst wall after the detachment of the larva in seawater under a microscope, and then to obtain the cyst wall sample by washing with seawater for multiple times. Although this method can obtain cyst wall with certain purity, it still has many defects. For example, bacteria and larva pollution may be introduced during the operation process, and the larva membrane breaking process and seawater environment may lead to loss of cyst wall components and damage to the structure, affecting the accuracy of subsequent component identification and structure observation of the cyst wall. SUMMARY

[0003] In view of the deficiencies in the prior art, the present application provides a separation and purification method of Cryptocaryon irritans cyst wall, a purity identification method, and a component identification and data set construction method. The structure and components of the Cryptocaryon irritans cyst wall obtained by the separation and purification method are complete and the surface is clean.

[0004] The specific technical solutions of the present application are as follows: In a first aspect, the present application provides a separation and purification method of Cryptocaryon irritans cyst wall, comprising the following steps: S1, pretreatment: sodium hypochlorite solution is used for sterilization treatment of Cryptocaryon irritans cyst, the mass concentration of the sodium hypochlorite solution is 0.1wt.%-0.5wt.%, the sterilization treatment time is 30-60 s, and then the pretreated cyst is obtained by washing with phosphate buffered saline solution; S2, separating: adding benzylsulfonyl fluoride with a mass concentration of 1wt.%-3wt.% into a cell-protecting agent containing dimethyl sulfoxide and sterile seawater to form an incubation solution; adding benzylsulfonyl fluoride with a mass concentration of 1wt.%-3wt.% into a dehydrating agent containing ethanol to obtain a dehydrating solution; after the pretreated capsules in step S1 are incubated in the incubation solution, adding the dehydrating solution to perform dehydration treatment, and then performing ultrasonic disruption in a hypotonic buffer containing tris(hydroxymethyl)aminomethane, magnesium chloride, glycine and benzylsulfonyl fluoride to obtain a capsule wall crude extract; S3, purifying: purifying the capsule wall crude extract in step S2 by differential centrifugation with a centrifugal force of 1000-5000×g and a centrifugal speed of 3000-8000 rpm to obtain a primary purified capsule wall; and performing secondary purification by density gradient centrifugation to obtain a purified capsule wall.

[0005] Further, the mass ratio of dimethyl sulfoxide to sterile seawater in the cell-protecting agent in step S2 is (1-3):(7-9).

[0006] Further, the mass concentration of benzylsulfonyl fluoride in the dehydrating solution in step S2 is 1wt.%-3wt.%, and the mass concentration of ethanol in the dehydrating agent is 75wt.%-80wt.%.

[0007] Further, in the hypotonic buffer in step S2, the concentration of tris(hydroxymethyl)aminomethane is 10-15 mmol / L, the concentration of magnesium chloride is 10-15 mmol / L, the concentration of glycine is 0.05-0.07 mol / L, and the mass concentration of benzylsulfonyl fluoride is 1wt.%-3wt.%.

[0008] Further, the ultrasonic disruption in step S2 is performed under ice water bath conditions by cyclic ultrasonic disruption with a cycle number of 20-30 times, and each ultrasonic disruption is performed by ultrasonic disruption with an ultrasonic energy of 30%-50% for 5-8 s and then stopping for 10-15 s.

[0009] Further, the differential centrifugation in step S3 is performed by first centrifuging the capsule wall crude extract in step S2 at a centrifugal force of 1000×g and a centrifugal speed of 3000 rpm for 10-20 min, and then centrifuging at a centrifugal force of 5000×g and a centrifugal speed of 8000 rpm for 10-20 min.

[0010] Further, the centrifugal force of the density gradient centrifugation in step S3 is 100000-200000×g, and the centrifugal speed is 28000-40000 rpm.

[0011] Further, the density gradient centrifugation method in step S3 adopts a sucrose solution with a gradient concentration of 20wt.%-60wt.%, and the sucrose solution with a mass concentration of 40wt.% is collected after centrifugation.

[0012] In a second aspect, the application further provides a purity detection method for stimulating Cryptocaryon irritans cyst wall, comprising the following steps: S41, morphological evaluation: first, the morphological characteristics of the purified cyst wall are evaluated by using an optical microscope and a scanning electron microscope; S42, characteristic protein detection: the purified cyst wall is pretreated to obtain cyst wall protein, and then the cyst wall protein is detected by using a larva-specific surface antigen Cl-iAg1 antibody; The purified cyst wall is prepared by the above-mentioned separation and purification method for stimulating Cryptocaryon irritans cyst wall.

[0013] In a third aspect, the application further provides a component identification and data set construction method for stimulating Cryptocaryon irritans cyst wall, comprising the following steps: S51, component identification: the proteins in the purified cyst wall are identified by using liquid chromatography-mass spectrometry technology to obtain an identification result of the components of the stimulating Cryptocaryon irritans cyst wall, and the purified cyst wall is prepared by the above-mentioned separation and purification method for stimulating Cryptocaryon irritans cyst wall; S52, data set construction: the data set is constructed based on the identification result obtained in step S51.

[0014] The positive progress effect of the application is that: The application provides a method for stimulating the separation and purification of Cryptocystidium cyst wall, a purity identification method, and a component identification and dataset construction method. In the pretreatment step, the cyst is quickly treated with low-concentration sodium hypochlorite to effectively remove other parasites and bacteria on the surface of the cyst without damaging the cyst. In the separation step, the cyst is treated with a cell protective agent (DMSO + sterile seawater) containing 1wt.%-3wt.% benzylsulfonyl fluoride to reduce the freezing point and inhibit the formation of ice crystals, while effectively inhibiting the degradation of cyst wall membrane proteins, ensuring the integrity of the subsequent cyst wall proteins. In the separation step, the cyst wall can be effectively separated by using a low-osmotic buffer to treat the cells at low temperature and using low-energy ultrasonic treatment. In the purification step, the cyst wall is separated and purified by differential centrifugation and density gradient centrifugation. Finally, the purified cyst wall with clean surface, complete structure and complete components is obtained, solving the problem of impure and incomplete separation of the existing cyst wall. The cyst wall purity identification method provides a morphological and molecular dual-dimension verification standard, filling the gap in the lack of systematic purity identification and control in the prior art. The cyst wall protein dataset constructed in the component identification and dataset construction method can be used for rapid retrieval of cyst wall components, providing an intelligent platform for subsequent diagnosis and drug target screening. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 Optical microscope image of the purified cyst wall prepared in Example 1.

[0016] Figure 2 Optical microscope image of the purified cyst wall prepared in Example 3.

[0017] Figure 3 Scanning electron microscope image of the purified cyst wall prepared in Example 1.

[0018] Figure 4 Protein detection image of the purified cyst wall prepared in Example 1. DETAILED DESCRIPTION

[0019] In order to make the above-mentioned purposes, features and advantages of the application more obvious and easy to understand, the specific embodiments of the application are described in detail below. It should be noted that the following embodiments are only used to illustrate the implementation method and typical parameters of the application, and do not limit the parameter range described in the application, and reasonable changes derived therefrom are still within the protection scope of the claims of the application.

[0020] It should be noted that the endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0021] Unless otherwise defined, all terms, symbols, and other scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In some instances, terms having a conventional meaning are defined herein for clarification or ease of reference, and such definitions should not be construed as indicating a significant difference from conventional understanding in the art. The technical methods described or referenced herein are generally well understood by those skilled in the art and employed by conventional methods. Unless otherwise stated, the use of commercially available kits, reagents, and instruments shall be performed according to the manufacturer's instructions and parameters.

[0022] The specific technical solution of this invention is as follows: In a first aspect, the present invention provides a method for isolating and purifying Cryptocaryon cysts by stimulating their cyst walls, comprising the following steps: S1. Pretreatment: The cysts of Cryptocaryon irritans were sterilized with sodium hypochlorite solution at a concentration of 0.1 wt.%-0.5 wt.% and a sterilization time of 30-60 s. Then, they were washed with phosphate buffer solution to obtain pretreated cysts. S2, Separation: 1 wt.%-3 wt.% PMSF was added to a cell protectant containing dimethyl sulfoxide (DMSO) and sterile seawater to form an incubation solution; 1 wt.%-3 wt.% PMSF was added to a dehydrating agent containing ethanol to obtain a dehydrated solution; after incubation in the incubation solution, the pretreated capsules from step S1 were added to the dehydrated solution for dehydration, and then sonicated in a hypotonic buffer containing tris(hydroxymethyl)aminomethane (Tris), magnesium chloride, glycine, and PMSF to obtain a crude extract of the capsule walls; S3. Purification: The crude extract of the capsule wall in step S2 is purified by differential centrifugation. The centrifugal force of differential centrifugation is 1000-5000×g and the centrifugation speed is 3000-8000 rpm to obtain the first purified capsule wall. The second purification is carried out by density gradient centrifugation to obtain the purified capsule wall.

[0023] The application provides a method for stimulating the separation and purification of cyst wall of Cryptocaryon irritans. In the pretreatment step, the cysts are rapidly treated with a low-concentration sodium hypochlorite solution with a mass concentration of 0.1 wt.%-0.5 wt.% for 30-60 s, so that bacteria and other parasites attached to the surface of the cysts can be effectively removed, while the structure of the cysts is not damaged, and the risk of microbial contamination in subsequent experiments is effectively reduced. The cyst wall of Cryptocaryon irritans can resist the oxidation of low-concentration sodium hypochlorite due to its unique stress-resistant structure; and most bacteria and free-living parasites in seawater can be killed due to the release of nascent oxygen, strong oxidation and chlorination by sodium hypochlorite. After being washed with phosphate buffered saline (PBS buffer) for several times, clean pretreated cysts can be obtained. In the separation step, the cysts are treated with a cell protection agent (DMSO + sterile seawater) containing 1 wt.%-3 wt.% PMSF in the cell protection stage. DMSO, as a permeable cryoprotectant, can form a hydrogen bond network in the cells by passing through the cell membrane, thereby reducing the freezing point and inhibiting the formation of ice crystals, so as to avoid membrane damage caused by ice crystal expansion during low-temperature treatment (4 DEG C). PMSF, as a serine protease inhibitor, can effectively inhibit the degradation of cyst wall membrane proteins by irreversibly combining with the serine residues of the active sites of proteases, thereby ensuring the integrity of the cyst wall proteins in the subsequent step. In the cell dehydration stage, the cysts are treated with an ethanol solution containing 1 wt.%-3 wt.% PMSF, so that a large amount of water in the cells can be replaced in a short time without damaging the surface membrane proteins, thereby enhancing the effect of subsequent cyst treatment with a low-osmotic solution. Finally, the cyst wall can be separated by using a low-osmotic buffer for low-temperature treatment of the cells and using low-energy ultrasonic treatment, wherein Tris, MgCl2 and glycine can stabilize the pH value of the solution to prevent changes in the molecular structure caused by changes in the acidity and alkalinity. In the purification step, the differential centrifugation method is used to separate the cell contents and impurities with a large mass under the centrifugal force of 1000-5000 x g and the centrifugal speed of 3000-8000 rpm. Thereafter, in combination with density gradient centrifugation, supersonic density gradient centrifugation is formed, and organelles and cyst walls are effectively separated according to the density. Under the combined action of various factors in the above steps, the purified cyst wall with a clean surface, complete structure and complete composition is separated.

[0024] Further, the mass ratio of dimethyl sulfoxide to sterile seawater in the cell protection agent of step S2 is (1-3):(7-9). The sterile seawater provides an ion strength close to the seawater environment, and when DMSO is mixed with sterile seawater at a mass ratio of (1-3):(7-9), the osmotic pressure of the system is maintained in the isotonic range, so that cell shrinkage or swelling can be avoided.

[0025] Further, the mass concentration of benzyl sulfonamide fluoride in the dehydrated solution of step S2 is 1wt.%-3wt.%, and the mass concentration of ethanol in the dehydrating agent is 75wt.%-80wt.%. The ratio of 1wt.%-3wt.% PMSF mass concentration and 75wt.%-80wt.% ethanol mass concentration enhances the dehydration efficiency and structural stability of the capsule wall through synergistic effect.

[0026] Further, in the low-osmotic buffer of step S2, the concentration of Tris is 10-15 mmol / L, the concentration of magnesium chloride is 10-15 mmol / L, the concentration of glycine is 0.05-0.07 mol / L, and the mass concentration of PMSF is 1wt.%-3wt.%. When 1wt.%-3wt.% PMSF and 10-15 mmol / L MgCl2 are used together, metal ion-dependent proteases can be inhibited, and the presence of 0.05-0.07 mol / L glycine can reduce the hydrolytic inactivation of PMSF under alkaline conditions, and the neutral environment maintained by 10-15 mmol / L Tris further optimizes its inhibition efficiency.

[0027] Further, the ultrasonic crushing process of step S2 is as follows: under ice water bath conditions, cyclic ultrasonic is performed, the cycle number of cyclic ultrasonic is 20-30 times, and the process of single ultrasonic is as follows: after ultrasonic for 5-8 s with 30%-50% ultrasonic energy, stop for 10-15 s. The above ultrasonic crushing process is regulated by three factors: low-temperature control activity, intermittent energization, and cumulative membrane rupture. The ice water bath quickly removes the heat generated during ultrasonic process through heat conduction, intermittent ultrasonic allows the ice water bath to fully dissipate heat, and repeated cycles of cumulative membrane rupture reduce the continuous effect of mechanical stress, thereby protecting the activity of the capsule wall protein and achieving efficient dissociation of the capsule structure.

[0028] Further, the process of differential centrifugation of step S3 is as follows: the capsule wall crude extract in step S2 is first centrifuged at 1000xg centrifugal force and 3000 rpm centrifugal speed for 10-20 min, and then centrifuged at 5000xg centrifugal force and 8000 rpm centrifugal speed for 10-20 min. The above differential centrifugation process efficiently removes large particle impurities through the first stage of centrifugation (centrifugation at 1000xg centrifugal force and 3000 rpm centrifugal speed for 10-20 min), and effectively separates the capsule wall from the organelle through the second stage of centrifugation (centrifugation at 8000 rpm centrifugal speed for 10-20 min). This process uses a progressive strategy of "removing large particles first, then enriching the target", which ensures the integrity of the capsule wall structure while efficiently removing impurities.

[0029] Further, the centrifugal force of the density gradient centrifugation in step S3 is 100000-200000xg, and the centrifugal speed is 28000-40000 rpm. When the centrifugal force of the density gradient is 100000-200000xg and the centrifugal speed is 28000-40000 rpm, nanoparticles with a density difference of ≤0.02 g / cm3 can be separated, and the separation precision is high.

[0030] Further, the density gradient centrifugation in step S3 uses a gradient concentration sucrose solution with a mass concentration of 20wt.%-60wt.%, and the sucrose solution with a mass concentration of 40wt.% is collected after centrifugation. The sucrose concentration range of 20wt.%-60wt.% can form a continuous density gradient from low to high, providing a differentiated sedimentation environment for the capsule wall components (such as proteins, polysaccharides, membrane structures, etc.). Particles of different densities will migrate to a position equal to their density in the gradient, achieving accurate physical separation. The density of the capsule wall is usually matched with the density of the 40wt.% sucrose solution (about 1.2-1.3 g / cm3). At this concentration, the target components stop settling due to the equal density with the surrounding medium, forming a clear band, facilitating targeted collection.

[0031] In a second aspect, the present application also provides a purity detection method for stimulating Cryptocaryon irritans capsule wall, comprising the following steps: S41, morphological evaluation: first, the morphological characteristics of the purified capsule wall are evaluated by optical microscope and scanning electron microscope; S42, characteristic protein detection: the purified capsule wall is pretreated to obtain capsule wall protein, and then the capsule wall protein is detected by using the Cl-iAg1 antibody specific to the surface antigen of the larvae; The purified capsule wall is obtained by the above-mentioned separation and purification method for stimulating Cryptocaryon irritans capsule wall.

[0032] The purity detection method for stimulating Cryptocaryon irritans capsule wall provided by the present application first evaluates the morphological characteristics of the purified capsule wall by optical microscope and electron microscope characterization. The optical microscope can evaluate the content removal effect of the capsule, and the scanning electron microscope characterization can provide high-definition pictures of the surface structure of the capsule wall, providing evidence for the surface contamination of the capsule wall. Then, the Cl-iAg1 antibody is used to evaluate the characteristic protein, which further proves the content removal effect of the capsule, and the Cl-iAg1 antibody specific to the surface antigen of the larvae can be used to evaluate the degree of contamination of the larvae at the molecular level.

[0033] In a third aspect, the present application also provides a component identification and data set construction method for stimulating Cryptocaryon irritans capsule wall, comprising the following steps: S51, component identification: using liquid chromatography-mass spectrometry technology to identify the purified capsule wall protein, obtaining the identification result of the capsule wall component of the Myxobolus spiculiformis, and the purified capsule wall is prepared by the separation and purification method of the Myxobolus spiculiformis capsule wall described above; S52, data set construction: based on the identification result obtained in step S51, the data set is constructed.

[0034] The Myxobolus spiculiformis capsule wall component identification and data set construction method provided by the application can quickly retrieve the capsule wall components through the protein ID, gene name, signal pathway and GO analysis content, and provides an intelligent platform for subsequent diagnosis and drug target screening.

[0035] Hereinafter, the technical solutions of the application will be further described in combination with specific examples. All reagents used in the examples can be commercially available or synthesized according to conventional methods, and can be directly used without further treatment. The instruments used in the examples are commercially available.

[0036] Example 1

[0037] The embodiment provides a separation and purification method of the Myxobolus spiculiformis capsule wall, which comprises the following steps: S1, pretreatment: obtaining the Myxobolus spiculiformis capsule precursor just detached from the host, taking the moment of detachment as 0 h, and culturing the capsule precursor in sterile seawater for 12 h; then, centrifuging the cultured capsule, setting the centrifugal force to 300xg and the centrifugation time to 10 min, and removing the supernatant seawater; adding 50 μL of 0.1wt.% sodium hypochlorite solution to the capsule, treating for 30 s, then immediately adding 3 mL of PBS buffer for neutralization, and then centrifuging at 800xg for 3 min; then, repeatedly washing the capsule with sterile PBS buffer for 5 times to obtain the pretreated capsule; S2, separation: in the pretreatment of the capsule in step S1, 3 mL of cell protection solution is added, the mass concentration of PMSF in the cell protection solution is 1wt.%, and the rest is a cell protection agent, the cell protection agent is composed of 10wt.% DMSO and 90wt.% sterile seawater, and the capsule is incubated at 4°C for 10 min; after the incubation is completed, centrifugation is performed at a centrifugal force of 300xg and a temperature of 4°C for 5 min to remove the residual cell protection agent; then 3 mL of dehydration solution is added, the mass concentration of PMSF in the dehydration solution is 1wt.%, and the rest is a dehydration agent, the composition of the dehydration agent is 75wt.% ethanol, and the same is incubated at 4°C for 15 min, and then centrifuged at a centrifugal force of 300xg and a temperature of 4°C for 5 min to remove the ethanol; then 5 mL of hypotonic buffer is added, the hypotonic buffer is composed of 10 mM Tris, 10 mM MgCl2, 0.05 M glycine and 1wt.% PMSF, and incubated at 4°C for 2 h; then the capsule is transferred to an ice water bath environment, and is broken by using an ultrasonic disrupter, the ultrasonic energy is set to 30%, and the ultrasonic operation is 5 s followed by a pause of 10 s, and the cycle operation is 20 times; finally, centrifugation is performed at a centrifugal force of 800xg and a temperature of 4°C for 10 min, and the supernatant is collected to obtain the capsule wall crude extract; S3, purification: the capsule wall crude extract in step S2 is preliminarily purified by differential centrifugation, the crude extract is centrifuged at a centrifugal force of 1000xg and a centrifugal speed of 3000 rpm and a temperature of 4°C for 10 min, and the supernatant is collected; then the collected supernatant is centrifuged at a centrifugal force of 5000xg and a centrifugal speed of 8000 rpm and a temperature of 4°C for 10 min, and the supernatant is collected to obtain the first purified capsule wall; then secondary purification is performed, and the density gradient centrifugation method is used, 20wt.%, 40wt.% and 60wt.% sucrose solutions are respectively configured, and the density gradient is formed in the centrifuge tube in the order of 60wt.%, 40wt.% and 20wt.%; the first purified capsule wall is slowly added to the upper layer of the sucrose solution, the centrifuge force is set to 100000xg and the centrifugal speed is set to 28000 rpm, and the temperature is set to 4°C, and the centrifugation is performed for 1.5 h; after the centrifugation is completed, the sample in the 40wt.% sucrose layer is sucked by using the puncture method; 8 mL of sterile PBS is added to the sucked sample, and centrifugation is performed at a centrifugal force of 100000xg and a centrifugal speed of 28000 rpm and a temperature of 4°C for 1 h, and the precipitate is collected; after the precipitate is washed twice, the purified capsule wall sample is obtained, and the finally obtained purified capsule wall sample is stored in a-80°C refrigerator.

[0038] Example 2 The embodiment provides a method for stimulating the separation and purification of cryptosporidium parvum capsule wall, which comprises the following steps: S1, pretreatment: obtain the Cryptocaryon irritans cyst precursor just dropped from the host, mark the moment when it drops as 0 h, place the cyst precursor in sterile seawater for 5 h; then, centrifuge the cultured cysts, set the centrifugal force to 300 x g, and the centrifugal time to 10 min, remove the supernatant seawater; add 50 μL of 0.3wt.% sodium hypochlorite solution to the cysts, treat for 30 s, then immediately add 3 mL of PBS buffer for neutralization, and then centrifuge at 800 x g for 3 min; then wash the cysts with sterile PBS buffer for 5 times, and obtain the pretreated cysts; S2, separation: in the pretreated cysts of step S1, add 3 mL of cell protection solution, the mass concentration of PMSF in the cell protection solution is 2wt.%, and the rest is a cell protection agent, the cell protection agent is composed of 20wt.% DMSO and 80wt.% sterile seawater, incubate the cysts at 4°C for 10 min; after incubation, centrifuge at 300 x g and 4°C for 5 min to remove residual cell protection agent; then add 3 mL of dehydration solution, the mass concentration of PMSF in the dehydration solution is 2wt.%, and the rest is a dehydration agent, the composition of the dehydration agent is 85wt.% ethanol, also incubate at 4°C for 15 min, and then centrifuge at 300 x g and 4°C for 5 min to remove ethanol; then add 5 mL of hypotonic buffer, the hypotonic buffer is composed of 12 mM Tris, 13 mM MgCl2, 0.06 M glycine, and 2wt.% PMSF, incubate at 4°C for 2 h; then transfer the cysts to an ice water bath environment, use an ultrasonic disrupter for crushing treatment, set the ultrasonic energy to 40%, ultrasonic for 6 s, pause for 12 s, and repeat for 25 times; finally, centrifuge at 800 x g and 4°C for 10 min, collect the supernatant to obtain the cyst wall crude extract; S3, purification: the crude extract of the capsule wall in step S2 is preliminarily purified by differential centrifugation, the crude extract is centrifuged at a centrifugal force of 2000xg, a centrifugal speed of 4250 rpm and a temperature of 4°C for 15 min, and the supernatant is collected; the collected supernatant is centrifuged at a centrifugal force of 5000xg, a centrifugal speed of 8000 rpm and a temperature of 4°C for 15 min, and the supernatant is collected to obtain the first purified capsule wall; then, secondary purification is performed by using ultracentrifugation with density gradient, sucrose solutions with concentrations of 20wt.%, 40wt.% and 60wt.% are respectively configured, and the sucrose solutions are sequentially added to the centrifuge tube in the order of 60wt.%, 40wt.% and 20wt.% to form a density gradient; the first purified capsule wall is slowly added to the upper layer of the sucrose solution, and the centrifuge is set to a centrifugal force of 200000xg, a centrifugal speed of 40000 rpm and a temperature of 4°C, and centrifuged for 1.5 h; after centrifugation, the sample in the 40wt.% sucrose layer is sucked by using the puncture method; 8 mL of sterile PBS is added to the sucked sample, and centrifuged at a centrifugal force of 200000xg, a centrifugal speed of 40000 rpm and a temperature of 4°C for 1 h, and the precipitate is collected; after the precipitate is repeatedly washed twice, the purified capsule wall sample is obtained, and the finally obtained purified capsule wall sample is stored in a-80°C refrigerator.

[0039] Example 3

[0040] The present embodiment provides a method for separating and purifying Cryptocaryon irritans capsule wall, which comprises the following steps: S1, pretreatment: obtain the Cryptocaryon irritans capsule precursor just detached from the host, and take the detachment time as 0 h; culture the capsule precursor in sterile seawater for 4 h; then, centrifuge the cultured capsule, set the centrifugal force to 300xg and the centrifugal time to 10 min, and remove the supernatant seawater; add 50 μL of 0.5wt.% sodium hypochlorite solution to the capsule, treat for 30 s, immediately add 3 mL of sterile phosphate buffered saline (PBS buffer) for neutralization, and then centrifuge at a centrifugal force of 800xg for 3 min; then, repeat the washing of the capsule with sterile PBS buffer for 5 times to obtain the pretreated capsule; S2, separation: in the pretreatment of the capsule in step S1, 3 mL of cell protection solution is added, the mass concentration of PMSF in the cell protection solution is 3wt.%, and the rest is a cell protection agent, the cell protection agent is composed of 30wt.% DMSO and 70wt.% sterile seawater, and the capsule is incubated at 4℃ for 10 min; after the incubation is completed, centrifugation is performed at 300xg and 4℃ for 5 min to remove the residual cell protection agent; then 3 mL of dehydration solution is added, the mass concentration of PMSF in the dehydration solution is 3wt.%, and the rest is a dehydration agent, the composition of the dehydration agent is 75wt.% ethanol, and the same is incubated at 4℃ for 15 min, and then centrifuged at 300xg and 4℃ for 5 min to remove the ethanol; then 5 mL of hypotonic buffer is added, the hypotonic buffer is composed of 15 mM Tris, 15 mM MgCl2, 0.07 M glycine and 1 g / L PMSF, and incubated at 4℃ for 2 h; then the capsule is transferred to an ice water bath environment, and an ultrasonic disrupter is used for crushing treatment, the ultrasonic energy is set to 50%, and the ultrasonic treatment is 8 s, then paused for 15 s, and the cycle operation is 30 times; finally, centrifugation is performed at 800xg and 4℃ for 10 min, and the supernatant is collected to obtain the capsule wall crude extract; S3, purification: first, the capsule wall crude extract in step S2 is preliminarily purified by differential centrifugation, the crude extract is centrifuged at 1000xg, 3000 rpm and 4℃ for 20 min, and the supernatant is collected; then the collected supernatant is centrifuged at 5000xg, 8000 rpm and 4℃ for 20 min, and the supernatant is collected to obtain the first purified capsule wall; then secondary purification is carried out, and the ultracentrifugation method is used, 20wt.%, 40wt.% and 60wt.% sucrose solutions are prepared respectively, and the density gradient is formed in the centrifuge tube in the order of 60wt.%, 40wt.% and 20wt.%; the first purified capsule wall is slowly added to the upper layer of the sucrose solution, the centrifuge force is set to 200000xg and the centrifugal speed is set to 40000 rpm, and the temperature is set to 4℃, and the centrifugation is carried out for 1.5 h; after centrifugation, the sample in the 40% sucrose layer is sucked by puncture method; 8 mL of sterile PBS is added to the sucked sample, and centrifugation is carried out at 100000xg, 40000 rpm and 4℃ for 1 h, and the precipitate is collected; after the precipitate is washed twice, the purified capsule wall sample is obtained, and the finally obtained purified capsule wall sample is stored in a-80℃ refrigerator.

[0041] Example 4

[0042] The embodiment provides a method for detecting the purity of cryptosporidium parvum capsule wall, comprising the following steps: S41, morphological evaluation: Optical microscope detection: the purified cyst wall sample obtained in step S3 was placed on a glass slide and observed under an optical microscope in differential interference phase contrast (DIC) mode, with an ocular magnification of 20x, an objective lens magnification of 20x, and a total magnification of 400x; 10 observation regions were randomly selected within the field of view, and the number and morphological characteristics of the larva residues in each region were recorded; Scanning electron microscope detection: the purified cyst wall sample obtained in step S3 was fixed on conductive glue, dehydrated by gradient ethanol (30wt.%→50wt.%→70wt.%→90wt.%→100wt.% for 15 min at each level) and critical point dried to obtain the sample to be tested, with an accelerating voltage (EHT) of the scanning electron microscope set to 10 kV, a working distance (WD) set to 4.3 mm, and a magnification set to 900x; SEM images of at least 5 different regions were collected to observe the surface texture, pore distribution, and contaminant adhesion of the cyst wall; S42, characteristic protein detection: Protein extraction and quantification: 1x10³ purified cyst walls were taken and 200 μL of SDT lysis buffer (containing 10wt.% SDS, 50 mM Tris-HCl at pH 8.0, and 1 mM DTT) was added, along with 2 μL of 100 mM PMSF and 0.1wt.% Triton X-100; a tissue homogenizer was used for crushing at 4°C, with a power of 30%, a sonication time of 5 s, an interval of 10 s, and 20 cycles of homogenization crushing; the homogenized sample was placed in a 100°C boiling water bath for 10 min, quickly cooled in an ice bath, and centrifuged at 10000xg at 4°C for 10 min, and the supernatant was obtained to obtain the cyst wall protein; the Bradford method was used with bovine serum albumin (BSA) as the standard, and the absorbance was measured at 595 nm to determine the cyst wall protein concentration; Dot Blot detection: 2 μL of each sample, including negative control (PBS), positive control (encysted protein after exflagellation, 50 ng / μL) and test sample (encysted wall protein, 50 ng / μL), were uniformly spotted onto designated areas of a polyvinylidene fluoride (PVDF) membrane and dried at room temperature for 30 min; then the PVDF membrane was immersed in blocking solution, which was tris(hydroxymethyl) aminomethane-hydrochloric acid buffer solution (containing Tween-20) containing 15wt.% skim milk powder (TBST for short), and incubated at 37 ℃ for 2 h with shaking; the surface of the PVDF membrane was quickly washed with TBST buffer solution for 3 times, 10 min each time; then, the primary antibody solution was used for incubation at room temperature for 1 h, the mass ratio of Cl-iAg1 antibody specific to larval surface antigen in the primary antibody solution to the blocking solution was 1:500; then, the PVDF membrane was washed twice with TBST and washed for 3 times, 5 min each time; then, the secondary antibody solution was used for incubation at room temperature for 1 h, the secondary antibody solution was labeled with horseradish peroxidase (HRP), and the mass ratio of Cl-iAg1 antibody specific to larval surface antigen in the secondary antibody solution to the blocking solution was 1:1000; then, the PVDF membrane was washed twice with TBST and washed for 3 times, 5 min each time; finally, the residual liquid on the PVDF membrane was absorbed with filter paper, luminescent solution was added to uniformly cover the membrane, incubated at room temperature for 1 min, washed twice with TBST, and then exposed using a chemiluminescence instrument.

[0043] Example 5

[0044] The embodiment provides a purity detection method for stimulating the cyst wall of Cryptocaryon irritans, which is different from the method in Example 4 in that the characteristic protein detection method in step S42 is replaced by Elisa instead of Dot Blot, and the specific steps are as follows: The capsule wall protein and the capsule total protein are diluted to 20 ng / μL with a carbonate buffer (50 mM Na2CO3, pH 9.6), 50 μL of the protein is added to each well of an enzyme-labeled plate, and incubated at 37 ℃ for 1 h, the liquid in the well is poured out, washed with TBST buffer for 3 times, each time for 3 min of shaking, and the liquid in the well is patted dry; 100 μL of a primary antibody solution is added to each well, and incubated at 37 ℃ for 1 h in the dark, the mass ratio of the larva-specific surface antigen Cl-iAg1 antibody in the primary antibody solution to the blocking solution is 1:500, and the blocking solution is a TBST buffer containing 5wt.% skimmed milk powder; the well liquid is washed with TBST buffer for 3 times, each time for 3 min of shaking, and the well liquid is patted dry; then 100 μL of a secondary antibody solution is added to each well, and incubated at 37 ℃ for 1 h in the dark, the secondary antibody solution is labeled with HRP, and the mass ratio of the larva-specific surface antigen Cl-iAg1 antibody in the secondary antibody solution to the blocking solution is 1:1000; the well liquid is washed with TBST for 5 times, each time for 3 min of shaking, to ensure that the non-specific binding antibodies are completely removed; 50 μL of fresh 3,3',5,5'-tetramethylbenzidine (TMB) color developing agent is added to each well, and incubated at room temperature for 20 min in the dark, 50 μL of a stop reagent (2 M sulfuric acid) is added to each well, and the optical density (OD) value is read at 450 nm.

[0045] Example 6

[0046] The embodiment provides a method for stimulating cryptosporidium parvum cyst wall component identification and data set construction, comprising the following steps: S51, component identification: the purified capsule wall protein is identified by using a liquid chromatography-mass spectrometry technique, and identification results of the cryptosporidium parvum cyst wall component are obtained, and the purified capsule wall is prepared by the separation and purification method of the cryptosporidium parvum cyst wall in the embodiment 1; S52, data set construction: a development framework is built by using a Visual Studio and a Node.js environment, and a full-stack architecture is realized by using a JavaScript language; project dependencies are built by using Maven, a Spring Boot framework is introduced to build a backend service, and front-end requests and data interaction are processed by using a Spring MVC component; meanwhile, a Node.js npm package management tool is integrated to manage front-end resources, and bidirectional communication between Java and Node.js is realized by using a Java Native Interface; a JavaFXWebView component is used to render an HTML5 page, and a custom UI component library written in JavaScript is used to realize a search interface layout, and the search interface layout comprises a capsule wall protein information search bar and a result visualization area.

[0047] The purified capsule wall obtained by separation and purification in the embodiment is characterized and tested, and the results are as follows: Figure 1 The optical microscope image of the purified cyst wall obtained by the separation and purification in Example 1 is shown in the figure. As can be seen from the figure, the purified cyst wall obtained by the separation and purification method of the Cryptocaryon irritans cyst wall in Example 1 has no residues inside, and the surface of the cyst wall is clean and the structure is complete.

[0048] Figure 2 The optical microscope image of the purified cyst wall obtained by the separation and purification in Example 3 is shown in the figure. As can be seen from the figure, the purified cyst wall obtained by the separation and purification method of the Cryptocaryon irritans cyst wall in Example 3 has no residues inside, and the surface of the cyst wall is clean and the structure is complete.

[0049] Figure 3 The scanning electron microscope image of the purified cyst wall obtained in Example 1 is shown in the figure. As can be seen from the figure, the surface of the purified cyst wall obtained by the separation and purification method of the Cryptocaryon irritans cyst wall in Example 1 is very clean, and the structure is very complete.

[0050] Figure 4 The protein detection diagram of the purified cyst wall obtained in Example 1 is shown in the figure. As can be seen from the figure, after the separation and purification of the Cryptocaryon irritans cyst wall in Example 1, the content of the surface antigen was identified, and compared with the cyst after the removal of the package, the content of Cl-iAg1 in the cyst wall after the separation and purification was reduced by 24%, while the content of the internal reference actin (β-ACtin) was unchanged.

[0051] Table 1 is the composition of the cyst wall identified in Example 6. As can be seen from the table, the cyst wall protein identified in Example 6 contains various Cryptocaryon irritans specific antigens and various cytoskeletal proteins, which can be used for drug target screening.

[0052] Table 1 is the composition of the cyst wall identified in Example 6.

[0053] Finally, it should be pointed out that the above embodiments are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or make equivalent replacement to part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for stimulating the isolation and purification of Cryptocaryon irritans cyst wall, characterized by, The method comprises the following steps: S1, pretreatment: the cysts of Cryptocaryon irritans are subjected to sterilization treatment by using a sodium hypochlorite solution with a mass concentration of 0.1wt.%-0.5wt.% for 30-60 s, and then washed with a phosphate buffered saline solution to obtain pretreated cysts; S2, separation: a cell protective agent containing dimethyl sulfoxide and sterile seawater is added with benzylsulfonyl fluoride with a mass concentration of 1wt.%-3wt.% to form an incubation solution; a dehydrating agent containing ethanol is added with benzylsulfonyl fluoride with a mass concentration of 1wt.%-3wt.% to obtain a dehydrating solution; after the pretreated cysts in step S1 are incubated in the incubation solution, the dehydrating solution is added for dehydration treatment, and then ultrasonic disruption is performed in a hypotonic buffer containing tris(hydroxymethyl)aminomethane, magnesium chloride, glycine and benzylsulfonyl fluoride to obtain a crude wall extract of cysts; S3, purification: the crude wall extract of cysts in step S2 is purified by using a differential centrifugation method with a centrifugal force of 1000-5000×g and a centrifugal speed of 3000-8000 rpm to obtain a primary purified cyst wall; a density gradient centrifugation method is used for secondary purification to obtain a purified cyst wall.

2. The separation and purification method according to claim 1, characterized by, The mass ratio of dimethyl sulfoxide to sterile seawater in the cell protective agent in step S2 is (1-3):(7-9).

3. The separation and purification method according to claim 1, characterized by, The mass concentration of benzylsulfonyl fluoride in the dehydrating solution in step S2 is 1wt.%-3wt.%, and the mass concentration of ethanol in the dehydrating agent is 75wt.%-80wt.%.

4. The separation and purification method according to claim 1, characterized by, In the hypotonic buffer in step S2, the concentration of tris(hydroxymethyl)aminomethane is 10-15 mmol / L, the concentration of magnesium chloride is 10-15 mmol / L, the concentration of glycine is 0.05-0.07 mol / L, and the mass concentration of benzylsulfonyl fluoride is 1wt.%-3wt.%.

5. The separation and purification method according to claim 1, characterized by, The ultrasonic disruption in step S2 is performed under ice water bath conditions by using cyclic ultrasonic waves with a cycle number of 20-30 times, and each cycle is performed by using ultrasonic waves with an energy of 30%-50% for 5-8 s and then stopped for 10-15 s.

6. The separation and purification method according to claim 1, characterized by, The differential centrifugation method in step S3 is performed by first centrifuging the crude wall extract of cysts in step S2 at a centrifugal force of 1000×g and a centrifugal speed of 3000 rpm for 10-20 min, and then centrifuging at a centrifugal force of 5000×g and a centrifugal speed of 8000 rpm for 10-20 min.

7. The separation and purification method according to claim 1, characterized by, The centrifugal force of the density gradient centrifugation method in step S3 is 100000-200000×g, and the centrifugal speed is 28000-40000 rpm.

8. The separation and purification method according to claim 1, characterized by, The density gradient centrifugation method in step S3 uses a gradient concentration sucrose solution with a mass concentration of 20wt.%-60wt.%, and the sucrose solution with a mass concentration of 40wt.% is collected after centrifugation.

9. A method for stimulating purity detection of Cryptocaryon irritans cyst wall, characterized in that, The method comprises the following steps: S41, morphological evaluation: the morphological characteristics of the purified cyst wall are evaluated using an optical microscope and a scanning electron microscope; S42, characteristic protein detection: the purified cyst wall is pretreated to obtain cyst wall proteins, and the cyst wall proteins are detected using a larva-specific surface antigen Cl-iAg1 antibody; The purified cyst wall is obtained by the separation and purification method of the cyst wall of the Cryptocaryon irritans according to any one of claims 1-8.

10. A method for identifying the components of the cyst wall of Cryptocaryon worms and constructing a dataset, characterized in that, The method comprises the following steps: S51, component identification: the proteins in the purified cyst wall are identified using liquid chromatography-mass spectrometry to obtain an identification result of the components of the Cryptocaryon irritans cyst wall, wherein the purified cyst wall is obtained by the separation and purification method of the cyst wall of the Cryptocaryon irritans according to any one of claims 1-8; S52, data set construction: a data set is constructed based on the identification result obtained in step S51.

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