Serotonin-silicon dioxide microsphere compound, preparation method thereof and sialylated glycocalyxin extraction process

Abstract

The invention relates to extraction of sialylated glycocalyxin, in particular to a serotonin-silicon dioxide microsphere compound, a preparation method thereof and a sialylated glycocalyxin extraction process, and aims to solve the problems that in the prior art, when the serotonin is used as a ligand to enrich the sialylated glycocalyxin, the sialylated glycocalyxin is difficult to obtain through rapid separation, and the purity of the sialylated glycocalyxin is low. Meanwhile, when the serotonin-magnetic particle compound is used for enriching the sialylated glycocalyxin, impurities difficult to remove are easily generated, and the method repeatability is poor. According to the extraction process of the sialylated glycocalyxin, a serotonin-silicon dioxide microsphere compound and the sialylated glycocalyxin are combined and then eluted, and the serotonin-silicon dioxide microsphere compound uses silicon dioxide microspheres as a carrier; the serotonin is covalently coupled to the surface of the epoxidized / activated carboxylation / formylation modified silicon dioxide microsphere, the combination and elution time is short, the separation efficiency is high, and the method is suitable for industrial production.

Description

Technical Field

[0001] The invention relates to the extraction of sialylated glycocalyx protein, and in particular to a serotonin-silicon dioxide microsphere complex and a preparation method thereof, and a sialylated glycocalyx protein extraction process. Background Art

[0002] Glycocalyx refers to a layer of structure rich in carbohydrates on the surface of animal cells, which is composed of oligosaccharide chains extending from glycoproteins and glycolipids that constitute the plasma membrane. Glycocalyx is also a general term for the sticky substance secreted by bacteria, epithelial cells or other cells that covers the surface of cells. The original meaning of this term refers to the glycoproteins and glycolipids that cover the surface of epithelial tissue and are secreted by epithelial cells. Glycocalyx has the following functions: 1) Protective effect, providing a buffer for the cell membrane to prevent it from being chemically damaged, allowing the immune system to detect and selectively attack foreign organisms; 2) Prevent cancer, changes in glycocalyx on cancer cells can enable the immune system to detect and attack cancer cells; 3) Prevent inflammation, the glycocalyx layer of the endothelial cell wall prevents white blood cells from adhering in a healthy state. Glycocalyx contains sialic acid (Sia) and galactose (Gal) sugar chain structures, which are usually sialic acid-linked galactose sugar chain structures on proteins. Such proteins are also called sialylated glycocalyx proteins or sialylated glycoproteins. Sialic acid is a general term for a class of nine-carbon monosaccharide compounds with carboxyl groups and their derivatives, which are usually located at the end of the protein sugar chain structure. Protein sialylation modification is one of the important post-translational modifications of proteins. It has a very important influence on the structure and function of proteins and plays an important biological role in organisms, such as recognition between viruses and cells, signal transduction, immune response and cell-to-cell interaction. Sialylation is a type of glycosylation involving the covalent addition of sialic acid to the glycan ends of glycoproteins and glycolipids. This enzymatic process is strictly regulated by sialyltransferases (STs) and sialidases / neuraminidase (NEUs). According to the type of glycosidic bond, sialylation can be divided into three types: α2-3-, α2-6- and α2-8-sialylation. Since sialylated glycocalyx proteins have low abundance in complex samples (such as biological samples such as breast milk, cow milk, goat milk, blood, saliva and urine), the research and application of sialylated glycocalyx proteins must first solve the problem of enrichment (or separation and purification) of sialylated glycocalyx proteins.

[0003] At present, the enrichment methods for sialylated glycocalyx protein mainly include enrichment using specific lectins, hydrophilic chromatography, strong cation exchange chromatography, titanium dioxide chromatography, wood fiber chromatography, and enrichment using 5-hydroxytryptamine (abbreviated as 5-HT) as a ligand. However, these methods all have their own shortcomings, such as the high cost of the method using lectins to enrich, the lectins are easily shed into the product, and the lectins (with certain toxicity) are polluted by themselves; hydrophilic chromatography, strong cation exchange chromatography, titanium dioxide chromatography (the European Commission has listed titanium dioxide as a Class II suspected carcinogen), and wood fiber chromatography are affected by phosphorylated proteins, and the enrichment process is time-consuming and the reaction conditions are harsh. At present, 5-hydroxytryptamine is used as a ligand to enrich sialylated glycocalyx protein, and the main problem is that it is difficult to quickly separate the bound sialylated glycocalyx protein after ligand recognition and binding.

[0004] Patent CN201711206127.9 proposes to provide a serotonin-magnetic particle complex and a method for enriching sialylated glycoproteins. The route provided by this scheme is simple and easy to operate, but there are many problems after industrial scale-up: (1) The operation is not simple and is time-consuming and labor-intensive; (2) The sialylated glycoproteins separated by the directional response of the magnetic particles to the magnetic field are mixed with black iron impurities, which are difficult to further remove and seriously affect the quality and performance of the product; (3) The magnetic particles have poor stability. After the serotonin-magnetic particle complex is reused 3 to 4 times, it is severely degraded and cannot be reused multiple times.

[0005] Silica microspheres are non-porous spherical microspheres with large specific surface area; their surface is hydrophilic, with good dispersibility and low cost. At the same time, due to their good optical and mechanical properties, they can meet the scientific research needs in the application field of inorganic microspheres. Silica microspheres are divided into two categories, those with unmodified surfaces and those with surface modifications such as carboxyl, amino and streptavidin functional groups, which can be widely used in nucleic acid separation, cell separation, immunoassay and other scientific research fields. Cyanogen bromide activation is one of the commonly used activation methods. The activation process mainly generates iminocarbonic acid active groups, which can react with primary amino groups (NH2) to mainly generate isourea derivatives. The matrix activated by cyanogen bromide can bind to ligands under mild conditions, and the amount of bound ligands is large. Many other derivatives can be obtained by further processing the matrix activated by cyanogen bromide. The disadvantage of this method is that the amino group in the isourea derivative generated after the matrix and ligand are coupled by the cyanogen bromide activation method has a pKa of 10.4, so it usually carries a certain positive charge, so that the matrix may have an anion ion exchange effect, which increases nonspecific adsorption and affects the resolution of affinity chromatography. In addition, the binding of the matrix activated by cyanogen bromide to the ligand is not stable enough, especially when it is bound to a small ligand, the ligand may fall off. In addition, cyanogen bromide is highly toxic and volatile, so it is inconvenient to operate.

[0006] At present, the main difficulty in preparing silica microspheres coupled with serotonin is to combine the surface characteristics of silica microspheres, design the modified structure for coupling with serotonin, and separate the sialylated glycocalyx protein rich in galactose and sialic acid structure through serotonin recognition and binding to sialic acid sugar chain structure. In the process of separation and purification of the target, avoid the contamination of the target by exogenous organic substances, and further investigate the reusability of silica microspheres coupled with serotonin. Summary of the invention

[0007] The purpose of the present invention is to solve the problems in the prior art that it is difficult to quickly separate sialylated glycocalyx proteins when serotonin is used as a ligand to enrich sialylated glycocalyx proteins, and that it is easy to generate impurities that are difficult to remove and the method has poor repeatability when serotonin-magnetic microparticle complexes are used to enrich sialylated glycocalyx proteins, and to provide a serotonin-silica microsphere complex and a preparation method thereof, and a sialylated glycocalyx protein extraction process.

[0008] To achieve the above objectives, the technical solutions provided by the present invention are as follows:

[0009] A serotonin-silicon dioxide microsphere complex is special in that the complex is silicon dioxide microspheres with serotonin fixed on the surface.

[0010] Furthermore, the serotonin is fixed on the surface of the silica microspheres through the reaction of its amino group with the epoxy / aldehyde group / activated carboxyl group modified on the silica microspheres.

[0011] Further, it includes silicon atoms bonded to the surface of the silica microspheres and functional groups connected to the silicon atoms;

[0012] The structure of the functional group is:

[0013]

[0014] At the same time, a method for preparing the above-mentioned serotonin-silica microsphere composite is also provided, which is special in that it comprises the following steps:

[0015] Modified silica microspheres are used as carriers for fixing serotonin, and serotonin is covalently coupled to the surface of the carrier; the modified silica microspheres are at least one of epoxidation / activated carboxylation / aldehyde modification silica microspheres.

[0016] Furthermore, the preparation method of the epoxidized / activated carboxylation / formylation-modified silica microspheres is:

[0017] Under alkaline and anhydrous conditions, an epoxy silanization agent is added to the silica microspheres for reaction to obtain epoxy-modified silica microspheres, which are then stored in anhydrous ethanol;

[0018] Under alkaline and anhydrous conditions, a compound having an amino group and a carboxyl group is added to the epoxidation-modified silica microspheres for reaction and activation to obtain activated carboxyl-modified silica microspheres, which are then stored in anhydrous ethanol;

[0019] Under alkaline and anhydrous conditions, a coupling agent having a dialdehyde group is added to the amino-silane-modified silica microspheres to react to obtain aldehyde-modified silica microspheres, which are then stored in anhydrous ethanol;

[0020] The aminosilane-modified silica microspheres are prepared by reacting (3-aminopropyl)trimethoxysilane with silica microspheres.

[0021] Further, the epoxy silanization agent is 3-glycidylpropyl ether trimethoxy silane or 3-glycidylbutyl ether trimethoxy silane or 3-glycidylpentyl ether trimethoxy silane;

[0022] The compound having amino and carboxyl groups is γ-aminobutyric acid, and the concentration of γ-aminobutyric acid is 0.5-1.5 mol / L;

[0023] The coupling agent is glutaraldehyde, and the concentration of glutaraldehyde is 0.5-1.5 mol / L.

[0024] Furthermore, the preparation method of the epoxidation-modified silica microspheres is specifically as follows:

[0025] 1.1.1. Wash the silica microspheres with anhydrous ethanol;

[0026] 1.1.2. Add 1-5% NaOH solution to the silica microspheres and react for 0.5-2 hours;

[0027] 1.1.3. Pour off the NaOH solution, wash the silica microspheres with ultrapure water, let the silica microspheres settle, separate and discard the supernatant;

[0028] 1.1.4. Drain the residual liquid in the silica microspheres, wash the silica microspheres with anhydrous ethanol, and then disperse them in anhydrous ethanol for storage;

[0029] 1.1.5. Preparation of epoxidized silica microspheres

[0030] Slowly add 3-glycidylpentyl ether trimethoxysilane reagent to the silica microspheres obtained in 1.1.4. and react for 1 to 3 hours; after the reaction is completed, separate the solid and liquid and discard the liquid phase to obtain epoxidized silica microspheres;

[0031] The method for covalently coupling serotonin to the surface of the carrier is specifically as follows: adding serotonin hydrochloride solution and modified silica microspheres to a coupling buffer, mixing them, and reacting them in the dark to obtain modified silica microspheres coupled with serotonin;

[0032] The light-proof reaction time is 2 to 5 hours;

[0033] The coupling buffer is a mixed solution containing 0.5-2 mol / L NaHCO3 and 0.01-0.1 mol / L NaCl, with a pH of 8.0-8.5;

[0034] The particle size of the silica microspheres is 40-60 meshes.

[0035] At the same time, a sialylated glycocalyx protein extraction process is also provided, which utilizes the above-mentioned serotonin-silica microsphere complex, and its special feature is that it includes the following steps:

[0036] 1) adding a mixed solution of production raw materials and binding buffer to a protein extraction device containing a serotonin-silica microsphere complex to allow the sialylated glycocalyx protein therein to bind to the serotonin-silica microsphere complex; the production raw materials are raw materials containing sialylated glycocalyx protein, and the binding buffer is used to promote the binding of sialylated glycocalyx protein to the serotonin-silica microsphere complex;

[0037] 2) washing the 5-HT-silica microsphere complex of the protein extraction device with a binding buffer to discharge the unbound protein solution;

[0038] 3) adding an elution solution to a protein extraction device, slowly stirring for elution, collecting the elution solution, and performing protein quantification to obtain a sialylated glycocalyx protein solution.

[0039] Furthermore, the method further comprises step 4), wherein the regeneration of the serotonin-silica microsphere complex is specifically as follows:

[0040] The 5-HT-silica microsphere complex was washed with twice the volume of 1-10 mol / L urea and then with 0.9% saline;

[0041] Alternatively, wash three times with 500 mL of sodium dodecyl sulfate solution and then with 500 mL of purified water;

[0042] After cleaning, it can be used again, or 20% ethanol can be added to it and stored at room temperature.

[0043] Furthermore, in step 1), the serotonin-silica microsphere complex is subjected to a sealing treatment;

[0044] The enclosed 5-HT-silica microsphere complex is 5-HT covalently coupled to at least one of epoxidized / activated carboxylation / aldehyde-modified silica microspheres;

[0045] The production raw material is pasteurized milk.

[0046] The binding buffer is one of sodium phosphate buffer, HEPES buffer, Tris-HCl buffer and sodium acetate-acetate buffer;

[0047] The ratio of the pasteurized milk to the binding buffer is 1:8 to 3:4;

[0048] The binding time is 1-6 hours;

[0049] In step 3), the elution solution is a mixture of 0.1-1.5 mol / L NaHCO3 and 0.5-2.5 mol / L NaCl, or a mixture of Na3PO4 and NaCl, or a coupling buffer;

[0050] The amount of the elution solution added is 80-90% of the volume of the extraction device;

[0051] The elution time is 0.5-3 hours.

[0052] If the binding time is too short, the target protein will not bind fully to the serotonin-silica microsphere complex; if the elution time is too short, the target protein and the complex will not be completely separated, both of which will affect the yield. If the binding and elution time are too long, not only may protein degradation occur, but the process time will also be extended. The selected binding time is the result of comprehensive efficiency and yield considerations.

[0053] Furthermore, in step 1), the serotonin-silica microsphere complex is serotonin covalently coupled to epoxidation-modified silica microspheres;

[0054] The epoxidation-modified silica microspheres are prepared by reacting 3-glycidylpentyl ether trimethoxysilane and silica microspheres;

[0055] The ratio of pasteurized milk to binding buffer was 3:8;

[0056] The binding time is 4 hours;

[0057] The binding buffer is a sodium phosphate buffer with a pH of 6.0±0.5 and a concentration of 0.02 mol / L;

[0058] In step 3), the elution solution is a mixture of 0.1 mol / L NaHCO3 and 0.5 mol / L NaCl;

[0059] The amount of the elution solution added is 80% of the volume of the extraction device;

[0060] The elution time was 1 hour.

[0061] Compared with the prior art, the present invention has the following beneficial effects:

[0062] 1. The present invention uses silica microspheres as carriers for fixing serotonin, and establishes a sialylated glycocalyx protein extraction process with simple operation and good stability through the specific recognition of sialic acid by the serotonin-silica microsphere complex. The silica microspheres have good hydrophilicity, good dispersibility and low cost, which is conducive to industrial production.

[0063] 2. In the preparation process of the serotonin-silica microsphere complex in the present invention, there is no special requirement for the reaction temperature, and the room temperature of 25°C is sufficient. However, the reaction time will be greatly prolonged after industrial scale-up to meet the condition of a larger volume of reaction liquid reaching the specified reaction temperature, thereby reducing production efficiency. Therefore, the present invention is suitable for production after industrial scale-up.

[0064] 3. The 5-HT silica microsphere complex of the present invention can be regenerated and reused at least 20 times to extract and prepare sialylated glycocalyx protein rich in galactose and sialic acid sugar chain structures from complex protein samples (such as milk).

[0065] 4. The extraction steps of sialylated glycocalyx protein in the present invention are simple, and only a simple protein extraction device is needed to realize semi-automatic and simple operation of the reaction process, thereby avoiding lengthy separation process and purification process of intermediate compounds in the post-treatment process, simplifying manual operation, thereby saving time and resources and improving yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 This is a reaction principle diagram for preparing epoxidation-modified silica microspheres in Example 1 of the present invention;

[0067] Figure 2 This is a physical picture of the silica microspheres, epoxidation-modified silica microspheres and the 5-HT-silica microsphere composite in Example 1 of the present invention;

[0068] Figure 3This is an infrared spectrum detection diagram of the silica microspheres, epoxidized silica microspheres and epoxidized silica microspheres coupled with serotonin in Example 1 of the present invention; wherein SiO2 represents silica microspheres, SiO2-O represents epoxidized silica microspheres, and SiO2-O-5HT represents epoxidized silica microspheres coupled with serotonin;

[0069] Figure 4 is a schematic diagram of the silver staining results of sialylated glycocalyx protein in Example 1 of the present invention;

[0070] Figure 5 Schematic diagram of the identification result of sialylated glycocalyx protein using a fluorescence scanner in Example 1 of the present invention (lectin MAL-II blot);

[0071] Figure 6 Schematic diagram of the identification result of sialylated glycocalyx protein using a fluorescence scanner in Example 1 of the present invention (lectin SNA blot);

[0072] Figure 7 Schematic diagram of the relationship between the mesh size of silica and the concentration of eluted sialylated glycocalyx protein in Example 1 of the present invention;

[0073] Figure 8 This is a table of eluted sialylated glycocalyx protein concentrations corresponding to different silica mesh sizes and modification types in Example 1 of the present invention;

[0074] Fig. 9 This is a schematic diagram of the concentration of eluted sialylated glycocalyx protein corresponding to different silica mesh sizes and modification types in Example 1 of the present invention;

[0075] Fig.10 Schematic diagram of the effect of the serotonin-silica complex prepared by silanization reagents with different carbon arm lengths on the eluted protein concentration in Example 1 of the present invention;

[0076] Fig.11 Schematic diagram of the effect of binding time on the concentration of eluted sialylated glycocalyx protein in Example 1 of the present invention;

[0077] Fig.12 Schematic diagram of the effect of elution time on the concentration of eluted sialylated glycocalyx protein in Example 1 of the present invention;

[0078] Fig.13 Schematic diagram of the effect of the ratio of milk to binding buffer on the concentration of eluted sialylated glycocalyx protein in Example 1 of the present invention;

[0079] Fig.14 This is a schematic diagram of the regeneration verification of the serotonin-silica microsphere complex in Example 1 of the present invention. DETAILED DESCRIPTION

[0080] The present invention is further described in detail below with reference to the accompanying drawings and specific embodiments:

[0081] 1. Experimental reagents and instruments

[0082] The epoxy silanization reagent was 3-glycidylpropyltrimethoxysilane, 3-glycidylbutyltrimethoxysilane or 3-glycidylpentyltrimethoxysilane; the amino silanization reagent was (3-aminopropyl)trimethoxysilane; γ-aminobutyric acid, glutaraldehyde, NaOH, glacial acetic acid, ethanolamine, and potassium bromide (KBr) were purchased from Sigma-Aldrich, USA; sodium chloride, trisodium phosphate, serotoninamine hydrochloride (pentahydrotryptamine hydrochloride), sodium bicarbonate and other commonly used reagents were domestic analytical grade reagents; silica of different particle sizes (40-60 mesh, 60-80 mesh, 80-100 mesh, 100-120 mesh) was purchased from Qingdao Ocean Chemical Company; ZHWY-2101C constant temperature air oscillator was purchased from Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd.; infrared spectrometer (FT-IR) Nicolet 5700 was purchased from Thermo electron Company, USA. Special iron stand for chromatography column, 2000mL standard-mouth conical flask, 1000mL sand filter G3 suction funnel, 500mL sand filter G5 suction funnel, 1000mL measuring cylinder, 500mL measuring cylinder, 1000mL beaker, 500mL beaker. All solutions used, unless otherwise specified, use ultrapure water as the solvent.

[0083] 2. Experimental process

[0084] 1. Synthesis of 5-HT-SiO2 Microsphere Complex

[0085] 1.1. Preparation of epoxidized silica microspheres

[0086] 1.1.1. Take 125g of silica microspheres of different particle sizes and place them in a 2L conical flask. Add 500mL of anhydrous ethanol to wash the silica microspheres. Specifically, use magnetic stirring for 10 minutes to suspend the silica microspheres in anhydrous ethanol, turn off the magnetic stirrer, let the silica microspheres settle, separate and discard the supernatant. Repeat the washing of the silica microspheres with anhydrous ethanol for 3 to 5 times, each washing for 5 to 10 minutes.

[0087] When cleaning the silica microspheres, use recycled anhydrous ethanol for the first time, and use fresh anhydrous ethanol for the second and third times. The anhydrous ethanol can be recycled for the third time, which takes about 30 minutes.

[0088] The particle sizes of the silica microspheres include 40-60 mesh, 60-80 mesh, 80-100 mesh, and 100-120 mesh. In other embodiments, the cleaning time needs to be adjusted according to the particle size, weight, etc. of the silica microspheres.

[0089] 1.1.2. Prepare a NaOH solution with a mass concentration of 1-5%, pour the NaOH solution into a conical flask, stir with a magnetic stirrer at medium speed for 0.5-2 hours. Put the conical flask into an ultrasonic cleaner and ultrasonicate for 15 minutes at 90% power.

[0090] The NaOH solution is used to expose the hydroxyl groups on the surface of the silica microspheres. In this embodiment, the mass concentration of the NaOH solution is 5%, and the reaction time is 0.5 hours.

[0091] 1.1.3. Pour away the NaOH solution and pour in about 1000 mL of ultrapure water to wash the silica microspheres. Repeat the washing process 3 to 5 times, each time for about 5 to 10 minutes. After washing, allow the silica microspheres to settle, separate and discard the supernatant.

[0092] 1.1.4. Transfer the silica microspheres into a 1000mL sand filter G3 filtration funnel and drain. Add anhydrous ethanol to the sand filter G3 filtration funnel to clean the silica microspheres, and stir with a stirring rod for 5 minutes to allow the anhydrous ethanol and silica microspheres to fully contact, then drain to complete one cleaning. Repeat the cleaning 3 to 5 times to ensure that subsequent reactions are carried out in a water-free environment.

[0093] The cleaned silica microspheres were dispersed in 500-1000 mL of anhydrous ethanol and transferred into a 2 L conical flask for storage.

[0094] 1.1.5. Preparation of epoxidized silica microspheres

[0095] Place the conical flask in 1.1.4 on a stirrer, adjust the stirrer speed to 800-1000 rpm, slowly add 50 mL of epoxy silanization reagent, and continue the reaction at room temperature 25°C for 3 hours; after the reaction is completed, transfer the reaction system to a conical flask, discard the liquid phase after solid-liquid separation, and obtain epoxidized modified silica microspheres, wash them with 500-1000 mL of anhydrous ethanol for 3-5 times, and store them in 500-1000 mL of anhydrous ethanol.

[0096] The epoxy silanization reagent can be 3-glycidyl propyl ether trimethoxy silane, 3-glycidyl butyl ether trimethoxy silane, or 3-glycidyl pentyl ether trimethoxy silane. Repeat step 1.1 for 3 times to prepare three different epoxy-modified silica microspheres using different epoxy silanization reagents. The reaction principle of the preparation using 3-glycidyl pentyl ether trimethoxy silane is as follows: Figure 1 shown.

[0097] 1.2. Preparation of activated carboxyl-modified silica microspheres

[0098] 1.2.1. Place the epoxidized modified silica microspheres dispersed in anhydrous ethanol on a stirrer, adjust the stirrer speed to 800-1000 rpm, slowly add 50-300 mL of 0.5-1.5 mol / L γ-aminobutyric acid, and continue the reaction for 3 hours;

[0099] In this example, the volume of γ-aminobutyric acid is 50 mL and the concentration is 1.5 mol / L;

[0100] 1.2.2. The silica microspheres after the reaction were washed with 500 mL of anhydrous ethanol for 3 to 5 times, and then 50 to 300 mL of 0.5 to 1.5 mol / L N-hydroxysuccinimide (NHS) and 50 to 300 mL of 0.5 to 1.5 mol / L dicyclohexylcarbodiimide (DCC) were added to react for 1 to 5 hours to activate the silica microspheres after the reaction in step 1.2.1; after the reaction, the reaction system was transferred to a conical flask, and the liquid phase was discarded after separation to obtain activated carboxyl-modified silica microspheres, which were washed with 500 mL of anhydrous ethanol for 3 to 5 times and then stored in 500 to 1000 mL of anhydrous ethanol.

[0101] In this embodiment, the volume of NHS is 50 mL, the concentration is 1.5 mol / L, the volume of DCC is 50 mL, the concentration is 1.5 mol / L; the reaction time is 1 hour;

[0102] Among them, the epoxysilanized silica microspheres prepared by using 3-glycidylpropyltrimethoxysilane are preferred, and the surface of the silica microspheres contains a layer of epoxy groups, which are easy to react with γ-aminobutyric acid to cover the surface of the silica microspheres with carboxyl groups.

[0103] 1.3. Preparation of aldehyde-modified silica microspheres

[0104] 1.3.1. Take 125g of silica microspheres and place them in a 2L Erlenmeyer flask. Wash the silica microspheres three times with anhydrous ethanol. The washing process is the same as that in step 1.1.1.

[0105] 1.3.2. Use sodium hydroxide to react with silica microspheres to expose the hydroxyl groups on their surfaces, wash the silica microspheres obtained by the reaction, disperse them in 500 mL of anhydrous ethanol, and transfer them to a 2 L conical flask for storage. The specific method is the same as steps 1.1.2 to 1.1.4.

[0106] 1.3.3. Preparation of amino-modified silica microspheres

[0107] Place the conical flask on a stirrer, adjust the stirrer speed to 800-1000 rpm, stir for 30-60 minutes, then slowly add 50-100 mL of (3-aminopropyl)trimethoxysilane in sequence, and continue the reaction for 1-3 hours; after the reaction is completed, transfer the reaction system to a conical flask, discard the liquid phase after solid-liquid separation, and obtain amino-modified silica microspheres, wash them with 500-1000 mL of anhydrous ethanol for 3-5 times, and then store them in 500 mL of anhydrous ethanol.

[0108] In this embodiment, after stirring for 30 minutes, 50 mL of (3-aminopropyl)trimethoxysilane was added and the reaction was continued for 3 hours;

[0109] 1.3.4. Preparation of aldehyde-modified silica microspheres

[0110] The amino-modified silica microspheres dispersed in anhydrous ethanol are placed on a stirrer, the stirrer speed is adjusted to 800-1000 rpm, and 50-100 mL of 0.5-1.5 mol / L glutaraldehyde are slowly added in sequence, and the reaction time is 1-3 hours; after the reaction is completed, the reaction system is transferred to a conical flask, and the liquid phase is discarded after solid-liquid separation to obtain aldehyde-modified silica microspheres, which are washed 4 times with 500 mL of anhydrous ethanol and stored in anhydrous ethanol.

[0111] In this example, the volume of glutaraldehyde is 50 mL, the concentration is 1 mol / L, and the reaction time is 3 hours;

[0112] The aminated silica microspheres prepared with (3-aminopropyl)trimethoxysilane have a layer of amino groups on the surface, which can be covalently coupled with a coupling agent to cover the surface of the aminated silica microspheres with aldehyde groups. In this embodiment, glutaraldehyde is preferably used as a coupling agent because glutaraldehyde is stable and relatively cheap.

[0113] 1.4. Preparation of 5-HT-SiO2 Microsphere Complex

[0114] 1.4.1. Preparation of coupling buffer

[0115] The coupling buffer includes 0.5-2 mol / L NaHCO3 and 0.01-0.1 mol / L NaCl;

[0116] In this embodiment, the coupling buffer includes 0.5 mol / L NaHCO3 and 0.1 mol / L NaCl, and the pH is 8.0-8.5. The specific preparation method is: accurately weigh 42 g of NaHCO3 and 146.1 g of NaCl and place them in a 5 L beaker, add 4 L of pure water, stir to fully dissolve them, finally adjust the volume to 5 L, and adjust the pH to 8.0-8.5.

[0117] 1.4.2. Take 125 g of epoxidized / aldehyded / activated carboxyl-modified silica microspheres respectively, mix them in a 2L three-necked flask, wash them 3-5 times with 500-1000 mL coupling buffer, and then disperse them in 500-1000 mL coupling buffer;

[0118] 1.4.3. Weigh 2-5 g of 5-hydroxytryptamine hydrochloride, dissolve it in 500 mL of coupling buffer, and add it to the coupling buffer in step 1.4.3. Stir at room temperature, away from light, and carry out coupling reaction at 150-200 rpm for 2-5 hours. After the reaction is completed, discard the liquid phase to obtain the 5-hydroxytryptamine-silica microsphere complex, and wash it with 500-1000 mL of coupling buffer for 3-5 times, and then discard the liquid phase to obtain the 5-hydroxytryptamine-silica microsphere complex. Then, the 5-hydroxytryptamine-silica microsphere complex is blocked again.

[0119] In this example, 3 g of serotonin hydrochloride was added and the reaction time was 3 hours.

[0120] The blocking of the 5-HT-silica microsphere complex is as follows:

[0121] (1) Preparation of ethanolamine solution

[0122] The concentration of ethanolamine solution is 0.5-2%. In this embodiment, the concentration is 2%, specifically:

[0123] Measure 10 mL of ethanolamine and place it in a beaker. Add pure water and stir to mix thoroughly. Adjust the pH to about 8.0-8.5 with hydrochloric acid and make the volume to 500 mL.

[0124] (2) Closed

[0125] Add 500-1000 mL of ethanolamine solution to the serotonin-silica microsphere complex washed in 1.4.3, and stir at 100-120 rpm for 1-5 hours to achieve the sealing of the serotonin-silica microsphere complex.

[0126] In this embodiment, the amount of ethanolamine used is 500 mL, and the stirring reaction time is 3 h;

[0127] 1.4.4. Detection of the coupling effect of serotonin and modified silica microspheres;

[0128] Specifically, a small amount of 5-HT-silica microsphere complex was placed at 50°C overnight and dried. Then it was ground and mixed evenly with about 200 mg of potassium bromide (KBr) dried overnight, placed in a mold, and 8×10 6 Pa pressure was pressed into transparent thin sheets on a tablet press, and infrared measurement was performed using an infrared spectrometer to detect the coupling effect of serotonin and modified silica microspheres.

[0129] 2. Column Packing of 5-HT-Silica Microsphere Complex

[0130] 2.1. Preparation of binding buffer

[0131] The binding buffer was a sodium phosphate buffer with a pH of 6.0 ± 0.5 and a concentration of 0.01-0.05 mol / L;

[0132] In this embodiment, the binding buffer is a 0.02 mol / L sodium phosphate buffer with a pH of 6.0. The specific preparation method is: weigh 38 g of trisodium phosphate and place it in a beaker, add 5 L of pure water, stir to dissolve it completely, add 15 mL of concentrated hydrochloric acid to mix thoroughly, transfer to a brown bottle, and store away from light for later use.

[0133] In other embodiments of the present invention, the binding buffer may also be a HEPES buffer having a pH of 7.0±0.5 and a concentration of 0.01 to 0.05 mol / L, or a Tris-HCl buffer having a pH of 7.0±0.5 and a concentration of 0.02 to 0.05 mol / L, or a sodium acetate-acetate buffer having a pH of 6.0±0.5 and a concentration of 0.2 to 0.5 mol / L; other buffers that do not react with other substances in the separation and purification process may also be used.

[0134] 2.2. Move the sealed 5-HT-silica microsphere complex into a protein extraction device, let the microspheres slowly sink, and let the ethanolamine solution slowly flow out. The protein extraction device can be a chromatography column or a larger-scale reactor. In this embodiment, a chromatography column is used.

[0135] 2.3. Wash the free ethanolamine in the device with 500-1000 mL of binding buffer.

[0136] 3. Isolation and purification of sialylated glycocalyx proteins from milk

[0137] 3.1. Sample addition

[0138] Fresh pasteurized milk was centrifuged at 5000 rpm and 4°C for 30 minutes, the upper fat layer was removed, 225 mL of the lower layer of liquid was added to the protein extraction device, and a binding buffer was added to make the volume of the mixed solution in the protein extraction device 825 mL. The binding buffer can be used to promote the binding of sialylated glycocalyx protein to the serotonin-silica microsphere complex.

[0139] In other embodiments of the present invention, the samples used as production raw materials can be animal or human milk and its isolates, blood and its isolates (such as pig serum), plant (peanut, soybean, etc.) homogenates and other raw materials containing sialylated glycocalyx protein.

[0140] 3.2. Binding and washing

[0141] Under room temperature conditions, the lower port of the device was closed, and after slow stirring for 4 hours, the lower port clamp was released, and the first effluent was collected and stored at 4°C. The first effluent contained unbound components.

[0142] The serotonin-silica microsphere complex in the protein extraction device was washed 5 to 6 times with binding buffer until the absorbance of the washed solution at 280 nm was close to zero to confirm that there was no protein in the outflow solution. The liquid in the protein extraction device was drained and the outflow generated during the washing was used as the second outflow and stored at 4°C.

[0143] The purpose of washing is to remove non-specifically bound proteins on the surface of the 5-HT-silica microsphere complex.

[0144] 3.3. Elution

[0145] An elution solution is added to a protein extraction device, the added volume being 80% of the volume of the protein extraction device, the elution solution comprising 0.1-1.5 mol / L NaHCO3 and 0.5-2.5 mol / L NaCl, the pH being 8.0-8.5, and slowly stirred at room temperature for 0.5 hour, and a third effluent is collected, wherein the third effluent contains a target substance, which is a sialylated glycocalyx protein rich in sialic acid and galactose sugar chain structure, and the third effluent is quantified for protein, and stored at 4°C to achieve the extraction of the sialylated glycocalyx protein.

[0146] In this embodiment, the elution solution includes 0.1 mol / L NaHCO3 and 2.0 mol / L NaCl, and the pH is 8.3;

[0147] In other embodiments of the present invention, the elution solution may also be a coupling buffer or a mixed solution of Na3PO4 and NaCl, and the elution is performed by controlling the amount of the elution solution added and the elution time until the OD280 of the third effluent is less than 0.02.

[0148] 4. Regeneration and preservation of 5-HT-silica microsphere complex

[0149] The 5-HT-silica microsphere complex was washed with twice the volume of 1-10 mol / L urea, and then washed with 0.9% saline to remove the urea;

[0150] Alternatively, wash three times with 500 mL of sodium dodecyl sulfate solution and then with 500 mL of purified water.

[0151] After cleaning, it can be used again, or 20% ethanol can be added to it and stored at room temperature.

[0152] 5. Identification of isolated and purified sialylated glycocalyx proteins

[0153] Take 40 μL of the 9-fold diluted milk solution and the third effluent, add 10 μL of SDS-PAGE protein loading buffer, boil in boiling water for 5-8 minutes, and perform vertical SDS-PAGE electrophoresis on 5% concentrated gel and 10% separation gel until bromophenol blue runs out of the boundary. Use silver staining method to develop color, the results are as follows Figure 4 As shown, based on the color band, it is determined whether the target protein band is obtained and whether the obtained protein is sialylated glycocalyx protein.

[0154] 6.Lectin-blot verification of sialylated glycocalyx protein

[0155] 6.1. Take a PVDF membrane and soak it in methanol for 10 to 30 seconds, then quickly transfer it to the transfer buffer solution to balance for 1 minute. The transfer buffer solution is prepared by adding 3.08g Tris, 14.4g glycine, 20mL methanol, and then dilute to 1L with sterile water;

[0156] 6.2. Transfer the separation gel after electrophoresis in step 5 to a PVDF membrane at a constant voltage of 100 V for 80 minutes to transfer the protein on the separation gel to the PVDF membrane. The specific transfer time is adjusted appropriately according to the molecular weight of the protein.

[0157] The transfer device is installed in the following order: negative electrode plate - sponge pad - three-layer filter paper - separation gel - PVDF membrane - three-layer filter paper - sponge pad - positive electrode plate. No bubbles should appear between the layers.

[0158] 6.3. After the transfer, place the PVDF in 1×Carbo free aqueous solution at room temperature for 1 hour;

[0159] 6.4. After blocking, add MAL-Ⅱ and SNA labeled with Cy-5 fluorescent dye, incubate at 4℃ in dark overnight, remove the PVDF membrane, wash it three times in PBST buffer, and detect the fluorescence signal with a fluorescence scanner. The results are as follows: Figure 5 and 6 shown.

[0160] 3. Results and Discussion

[0161] 1. Coupling of 5-HT to Silica Microspheres Modified with Different Chemical Groups

[0162] Serotonin can be fixed on the surface of silica microspheres through the reaction of its amino group with the epoxy / aldehyde / carboxyl group modified on the silica microspheres. The properties of silica microspheres, epoxidation-modified silica microspheres and serotonin-silica microsphere composites were compared. Figure 2As shown in the figure, the silica modified with 5-HT is dark brown and does not fade. At the same time, the silica microspheres (SiO2), epoxidized silica microspheres (SiO2-O) and epoxidized silica microspheres coupled with 5-HT (SiO2-O-5HT) were detected by infrared spectroscopy. The results are as follows Figure 3 As shown, the results show that after epoxidation modification, the -1 The broad peaks on the left and right are the vibrations related to the epoxy group, indicating that the epoxy silanization reagent has successfully modified the silica microspheres with epoxy groups. After the epoxidized microspheres are coupled with 5-HT, the peak at 1350 cm -1 The peaks on the left and right are the CN vibrations on the secondary amine group (-NH-) formed after the ring-opening reaction between the epoxy group and the amino group, indicating that serotonin is coupled to the surface of the silica microspheres through the reaction between the amino group and the epoxy group; at 1100cm -1 The peaks on the left and right are vibrations related to hydroxyl groups.

[0163] Based on the simplicity, cost and extraction efficiency of the epoxy / aldehyde / carboxyl group modified method on silica microspheres, through experimental comparison, it was found that when the epoxy-modified silica microspheres were used to extract sialylated glycocalyx protein, the eluted sialylated glycocalyx protein (hereinafter referred to as eluted protein) had a higher concentration. The results are as follows Figure 8 and 9 As shown, the epoxidation-modified silica microspheres have the highest working efficiency.

[0164] 2. Effect of binding time and elution time on eluted protein concentration

[0165] Controlling other conditions to be the same, adjusting the binding time of milk and binding buffer in step 3.2 to 1, 2, 3, 4, 5, and 6 hours respectively, separating and purifying sialylated glycocalyx protein in milk multiple times at different binding times, obtaining multiple different third effluents, and using BCA protein concentration determination kit to detect the protein content in multiple third effluents, such as Fig.11 As shown, it is concluded that within the range of 1-3 hours, the eluted protein concentration gradually increases, reaching the peak eluted protein concentration at 3 hours, and then the eluted protein concentration no longer increases when the time is increased, but tends to be stable. If the binding time exceeds 6 hours, not only protein degradation may occur, but also the time required for the process will be extended. Therefore, the preferred binding time is 3-6 hours.

[0166] While keeping other conditions the same, the elution time of milk and elution buffer in step 3.3 was adjusted to 0.5, 1, 1.5, 2, 2.5, and 3 hours, respectively. Sialyl glycocalyx protein in milk was separated and purified multiple times at different elution times to obtain multiple different third effluents. The protein content in the multiple third effluents was detected using a BCA protein concentration assay kit, such as Fig.12As shown, it is concluded that within the range of 0.5-1 hour, the eluted protein concentration gradually increases, reaching the peak eluted protein concentration in 1 hour, and then as the time is further increased, the eluted protein concentration no longer increases but tends to be stable, so the preferred elution time is 1-3 hours.

[0167] 3. Optimal ratio of binding buffer to milk volume

[0168] In step 3.1, the volume ratio of milk to binding buffer was 1:8, 1:4, 3:8, 1:2, and 3:4, respectively, so that the volume of the mixed solution was 825 mL. Step 3 was repeated multiple times at different volume ratios to separate and purify sialylated glycocalyx protein in milk to obtain multiple different third effluents. The protein content in the multiple third effluents was detected using a BCA protein concentration assay kit. The results are as follows: Fig.13 As shown, when the volume ratio of milk to binding buffer is 3:8, the content of sialylated glycocalyx protein in the third effluent is the highest. When the milk content is further increased, the final amount of sialylated glycocalyx protein does not increase, but decreases. When the volume ratio is 1:2, the eluted protein concentration is close to the concentration of 3:8. Therefore, the preferred volume ratio of milk to binding buffer is 3:8-1:2, and the optimal ratio is 3:8.

[0169] The binding buffer is preferably a 20 mmol / L sodium phosphate solution at pH 6.0. Under the condition of pH 6.0, the integrity of the sialic acid structure on the protein in the sample can be protected, which is conducive to the mutual binding of sialic acid and serotonin.

[0170] 4. Verification of the enrichment efficiency of different 5-HT-silica microsphere complexes and different elution solutions

[0171] The protein extraction device was filled with a serotonin-silica microsphere complex prepared with 3-glycidylpentyltrimethoxysilane, and other conditions were kept consistent. Step 3 was performed using elution solution A (0.1 mol / L NaHCO3 and 0.5 mol / L NaCl, pH = 8.3) and elution solution B (20 mmol / L Na3PO4 and 0.5 mol / L NaCl, pH = 6.0) to obtain the corresponding third effluent.

[0172] The third effluent was detected by SDS-PAGE and silver staining. The results showed that the protein amount in the third effluent after elution with elution solution A was higher than that in the third effluent after elution with elution solution B. Since the binding between serotonin and sialic acid sialyl glycocalyx protein is not related to the pH value in the system but is related to the ionic strength, it is speculated that the high concentration of salt solution can more effectively dissociate the binding between serotonin and sialylated glycocalyx protein, so it can be eluted more effectively.

[0173] Under the same elution conditions, the 5-hydroxytryptamine-silica microsphere complex prepared by 3-glycidylpropyltrimethoxysilane, 3-glycidylbutyltrimethoxysilane and 3-glycidylpentyltrimethoxysilane was compared, and it was found that the 5-hydroxytryptamine-silica microsphere complex prepared by 3-glycidylpentyltrimethoxysilane had the highest elution protein concentration. Fig.10 As shown, it is shown that the binding and elution effects of this serotonin-silica microsphere complex are better than the other two.

[0174] Since the binding and elution efficiencies of complexes with different carbon arm lengths are different, e.g. Figure 7 As shown, 5-HT-silica microsphere complexes were prepared using silica microspheres with particle sizes of 40-60 mesh, 60-80 mesh, 80-100 mesh, and 100-120 mesh, respectively, and separated and purified. When the particle size was 40-60 mesh, the eluted protein concentration was the highest; as shown Figure 8 As shown, the epoxidized silica microspheres prepared with 3-glycidylpentyltrimethoxysilane and having a particle size of 40-60 mesh had the highest eluted protein concentration, proving that the separation and purification efficiency was the best.

[0175] 5. Identification of Sialyl Glycocalyx Proteins

[0176] Silver staining results Figure 4 As shown in the figure, the three bands of protein silver staining are around 30KDa, 25KDa and 22KDa respectively. Lectin MAL-Ⅱ and SNA can recognize sialic acid with α2-3 and α2-6 connected to the sugar chain terminal of sialic acid sialylated glycocalyx protein respectively. The protein sample extracted by serotonin-silica microsphere complex was incubated with fluorescently labeled MAL-Ⅱ and SNA on PVDF membrane and scanned with a fluorescence scanner. The scanning results of Lectin-blot-MAL-Ⅱ are shown in the figure. Figure 5 As shown in Figure 2, there are two protein bands (molecular weight 30 and 25 KDa) that bind to MAL-Ⅱ, indicating that the proteins at these two bands contain sialic acid linked by α2-3. Figure 6 As shown, it does not show whether the protein at 22 KDa contains sialic acid linked to α2-6. This may be due to the low content of proteins of this molecular weight in the entire extracted protein sample and the sensitivity of the detection method, so no detection results are shown.

[0177] 6. Regeneration of 5-HT-SiO2 Microsphere Complex

[0178] Wash the 5-HT-silica microsphere complex with 1-10 mol / L urea twice the volume of the 5-HT-silica microsphere complex to remove the protein adsorbed on the 5-HT-silica microsphere complex, and then wash it three times with 0.9% saline before continuing to use. Fig.14 As shown, it has been verified that the serotonin-silica microsphere complex modified with 3-glycidylpentyl ether trimethoxysilane can be used continuously for more than 20 times, is suitable for industrial production, and has low raw material loss.

[0179] Embodiment 2

[0180] In this embodiment, the silica microspheres are activated carboxyl-modified silica microspheres, and the preparation method of the serotonin-silica microsphere complex and the sialylated glycocalyx protein extraction process include the following steps:

[0181] 1. Synthesis of 5-HT-SiO2 Microsphere Complex

[0182] 1.1. Preparation of epoxidized silica microspheres

[0183] 1.1.1. Take 125 g of 40-60 mesh silica microspheres and place them in a 2L conical flask. Add 500 mL of anhydrous ethanol to wash the silica microspheres three times, each time for 5 minutes.

[0184] 1.1.2. Pour 1% NaOH solution into a conical flask and stir at medium speed with magnetic force for 2 hours.

[0185] 1.1.3. Pour away the NaOH solution and pour in about 1000 mL of ultrapure water to wash the silica microspheres. Repeat the washing three times, each time for about 10 minutes. After washing, allow the silica microspheres to settle, separate and discard the supernatant.

[0186] 1.1.4. Transfer the silica microspheres into a 1000mL sand filter G3 filtration funnel and drain; wash the silica microspheres three times with anhydrous ethanol in the sand filter G3 filtration funnel. Disperse the washed silica microspheres in 1000mL anhydrous ethanol and transfer to a 2L conical flask for storage.

[0187] 1.1.5. Preparation of epoxidized silica microspheres

[0188] Place the conical flask in 1.1.4 on a stirrer, adjust the stirrer speed to 800-1000rpm, slowly add 100mL of 3-glycidylpentyl ether trimethoxysilane reagent, and continue the reaction at room temperature 25°C for 1 hour; after the reaction is completed, transfer the reaction system to a conical flask, discard the liquid phase after solid-liquid separation, and obtain epoxidized modified silica microspheres, wash them with 500mL of anhydrous ethanol three times, and store them in 500mL of anhydrous ethanol. 1.2. Preparation of activated carboxyl-modified silica microspheres

[0189] 1.2.1. Add 300 mL of 0.5 mol / L γ-aminobutyric acid to the epoxidized silica microspheres and stir for 5 hours;

[0190] 1.2.2. The silica microspheres after the reaction were washed with 500 mL of anhydrous ethanol for 3 times, and then 300 mL of 1.0 mol / L N-hydroxysuccinimide (NHS) and 300 mL of 1.0 mol / L dicyclohexylcarbodiimide (DCC) were added to react for 5 hours; after the reaction, the reaction system was transferred to a conical flask, and the liquid phase was discarded after separation to obtain activated carboxyl-modified silica microspheres, which were washed with 500 mL of anhydrous ethanol for 4 times and then stored in 500 mL of anhydrous ethanol.

[0191] 1.3. Preparation of 5-HT-SiO2 Microsphere Complex

[0192] 1.3.1. Preparation of coupling buffer

[0193] The coupling buffer includes 2 mol / L NaHCO3 and 0.01 mol / L NaCl, and the pH is 8.0-8.5.

[0194] 1.3.2. Take 125 g of activated carboxyl-modified silica microspheres and place them in a 2L three-necked flask. Wash them five times with 500 mL of coupling buffer and then disperse them in 500 mL of coupling buffer.

[0195] 1.3.3. Weigh 2 g of serotonin hydrochloride, dissolve it with 500 mL of coupling buffer, and add it to the coupling buffer of step 1.4.3., protect it from light at room temperature, stir it at 150-200 rpm for coupling reaction, and the reaction time is 2 hours. After the reaction is completed, the liquid phase is discarded to obtain the serotonin-silica microsphere complex, and after washing it with 500 mL of coupling buffer 5 times, the liquid phase is discarded to obtain the serotonin-silica microsphere complex. Then the serotonin-silica microsphere complex is blocked again, and the coupling effect of the obtained serotonin-silica microsphere complex can be detected.

[0196] The blocking of the 5-HT-silica microsphere complex is as follows:

[0197] (1) Prepare 0.5% ethanolamine solution

[0198] Measure 2.5 mL of ethanolamine and place it in a beaker. Add pure water and stir to mix thoroughly. Adjust the pH to about 8.0-8.5 with hydrochloric acid and make the volume to 500 mL.

[0199] (2) Closed

[0200] Add 1000 mL of ethanolamine solution to the 5-HT-silica microsphere complex washed in 1.4.3 above, and stir at 100-120 rpm for 1 hour to achieve the blocking of the 5-HT-silica microsphere complex. 2. Column Packing of 5-HT-Silica Microsphere Complex

[0201] 2.1. Preparation of binding buffer

[0202] The binding buffer was 0.01 mol / L sodium phosphate buffer with a pH of 6.0±0.5.

[0203] 2.2. Move the sealed 5-HT-silica microsphere complex into the chromatography column, allowing the microspheres to slowly sink and the ethanolamine solution to slowly flow out.

[0204] 2.3. Wash the free ethanolamine in the column with 500-1000 mL of binding buffer.

[0205] 3. Isolation and purification of sialylated glycocalyx proteins from milk

[0206] 3.1. Sample addition

[0207] Fresh pasteurized milk was centrifuged at 5000 rpm and 4°C for 30 minutes, the upper fat layer was removed, 92 mL of the lower layer of liquid was added to the protein extraction device, and binding buffer was added to make the volume of the mixed solution in the protein extraction device 825 mL.

[0208] 3.2. Binding and washing

[0209] At room temperature, close the lower port of the device, slowly stir for 3 hours, then release the lower port clamp, collect the first effluent, and store it at 4°C. The first effluent contains unbound components.

[0210] The serotonin-silica microsphere complex in the protein extraction device was washed 5 to 6 times with binding buffer until the absorbance of the washed solution at 280 nm was close to zero to confirm that there was no protein in the outflow solution. The liquid in the protein extraction device was drained and the outflow generated during the washing was used as the second outflow and stored at 4°C.

[0211] 3.3. Elution

[0212] Add an elution solution to the protein extraction device, the added volume is 90% of the volume of the protein extraction device, the elution solution includes 0.5 mol / L NaHCO3 and 2.5 mol / L NaCl, the pH is 8.0-8.5, slowly stir at room temperature for 3 hours, collect the third effluent, the third effluent contains the target substance, which is sialylated glycocalyx protein rich in sialic acid and galactose sugar chain structure, and quantify the protein in the third effluent, and store it at 4°C to achieve the extraction of sialylated glycocalyx protein.

[0213] Embodiment 3

[0214] In this embodiment, the silica microspheres are aldehyde-modified silica microspheres, and the preparation method of the serotonin-silica microsphere complex and the sialylated glycocalyx protein extraction process include the following steps:

[0215] 1. Synthesis of 5-HT-SiO2 Microsphere Complex

[0216] 1.1. Preparation of aldehyde-modified silica microspheres

[0217] 1.1.1. Take 125 g of 60-80 mesh silica microspheres and place them in a 2L conical flask. Add 500 mL of anhydrous ethanol to wash the silica microspheres 5 times, each washing for 8 minutes.

[0218] 1.1.2. Use sodium hydroxide with a mass concentration of 4% to react with the silica microspheres to expose the hydroxyl groups on their surfaces. Wash the silica microspheres obtained by the reaction, disperse them in 500 mL of anhydrous ethanol, and transfer them into a 2 L conical flask for storage.

[0219] The specific steps are the same as step 1.3.2 in Example 1.

[0220] 1.1.3. Preparation of amino-modified silica microspheres

[0221] The conical flask was placed on a stirrer with the stirrer speed adjusted to 800-1000 rpm. After 40 minutes, 70 mL of (3-aminopropyl)trimethoxysilane was slowly added in sequence and the reaction was continued for 1.5 hours. After the reaction was completed, the reaction system was transferred to a conical flask, and the liquid phase was discarded after solid-liquid separation to obtain amino-modified silica microspheres, which were washed 5 times with 500 mL of anhydrous ethanol and stored in 500 mL of anhydrous ethanol.

[0222] 1.1.4. Preparation of aldehyde-modified silica microspheres

[0223] The amino-modified silica microspheres dispersed in anhydrous ethanol were placed on a stirrer, the stirrer speed was adjusted to 800-1000 rpm, and 80 mL of 0.5 mol / L glutaraldehyde was slowly added in sequence, and the reaction time was 1 hour; after the reaction was completed, the reaction system was transferred to a conical flask, and the liquid phase was discarded after solid-liquid separation to obtain aldehyde-modified silica microspheres, which were washed with 500-1000 mL of anhydrous ethanol for 3-5 times and then stored in anhydrous ethanol.

[0224] 1.2. Preparation of 5-HT-SiO2 Microsphere Complex

[0225] 1.2.1. Preparation of coupling buffer

[0226] The coupling buffer includes 1.5 mol / L NaHCO3 and 0.05 mol / L NaCl, and the pH is 8.0-8.5.

[0227] 1.2.2. Take 125 g of aldehyde-modified silica microspheres and place them in a 2L three-necked flask. Wash them 3-5 times with 800 mL of coupling buffer and then disperse them in 1000 mL of coupling buffer.

[0228] 1.2.3. Weigh 5 g of serotonin hydrochloride, dissolve it in 1000 mL of coupling buffer, add it to the coupling buffer of step 1.2.2., protect it from light at room temperature, stir it at 150-200 rpm for coupling reaction, and the reaction time is 5 hours. After the reaction is completed, discard the liquid phase to obtain the serotonin-silica microsphere complex, and wash it 3 times with 800 mL of coupling buffer, then discard the liquid phase to obtain the serotonin-silica microsphere complex. Then the serotonin-silica microsphere complex is blocked again, and the coupling effect of the obtained serotonin-silica microsphere complex can be detected.

[0229] The blocking of the 5-HT-silica microsphere complex is as follows:

[0230] (1) Prepare 1.2% ethanolamine solution

[0231] Measure 6 mL of ethanolamine and place it in a beaker. Add pure water and stir to mix thoroughly. Adjust the pH to about 8.0-8.5 with hydrochloric acid and make the volume to 500 mL.

[0232] (2) Closed

[0233] Add 500 mL of ethanolamine solution to the serotonin-silica microsphere complex washed in 1.4.3 above, and stir at 100-120 rpm for 5 hours to achieve the sealing of the serotonin-silica microsphere complex.

[0234] 2. Column Packing of 5-HT-Silica Microsphere Complex

[0235] 2.1. Preparation of binding buffer

[0236] The binding buffer was 0.03 mol / L sodium phosphate buffer with a pH of 6.0 ± 0.5.

[0237] 2.2. Move the sealed 5-HT-silica microsphere complex into the protein extraction device, allow the microspheres to slowly sink, and allow the ethanolamine solution to slowly flow out.

[0238] 2.3. Wash the free ethanolamine in the device with 500-1000 mL of binding buffer.

[0239] 3. Isolation and purification of sialylated glycocalyx proteins from milk

[0240] 3.1. Sample addition

[0241] Fresh pasteurized milk was centrifuged at 5000 rpm and 4°C for 30 minutes, the upper fat layer was removed, 225 mL of the lower layer of liquid was added to the protein extraction device, and binding buffer was added to make the volume of the mixed solution in the protein extraction device 525 mL.

[0242] 3.2. Binding and washing

[0243] At room temperature, close the lower port of the device, slowly stir for 6 hours, then release the lower port clamp, collect the first effluent, and store it at 4°C. The first effluent contains unbound components.

[0244] The serotonin-silica microsphere complex in the protein extraction device was washed 5 to 6 times with binding buffer until the absorbance of the washed solution at 280 nm was close to zero to confirm that there was no protein in the outflow solution. The liquid in the protein extraction device was drained and the outflow generated during the washing was used as the second outflow and stored at 4°C.

[0245] 3.3. Elution

[0246] Add elution solution to the protein extraction device, the added volume is 85% of the volume of the protein extraction device, the elution solution includes 1.5mol / L NaHCO3 and 0.5mol / L NaCl, the pH is 8.0-8.5, slowly stir at room temperature for 0.5 hour, collect the third effluent, the third effluent contains the target substance which is sialylated glycocalyx protein rich in sialic acid and galactose sugar chain structure, the third effluent is quantified for protein, and stored at 4°C to achieve the extraction of sialylated glycocalyx protein.

[0247] Embodiment 4

[0248] In this embodiment, the silica microspheres are epoxidized silica microspheres, and the preparation method of the serotonin-silica microsphere complex and the sialylated glycocalyx protein extraction process include the following steps:

[0249] 1. Synthesis of 5-HT-SiO2 Microsphere Complex

[0250] 1.1. Preparation of epoxidized silica microspheres

[0251] 1.1.1. Take 125 g of 40-60 mesh silica microspheres and place them in a 2L conical flask. Add 500 mL of anhydrous ethanol to wash the silica microspheres 4 times, each washing for 6 minutes.

[0252] 1.1.2. Use 3% sodium hydroxide to react with silica microspheres to expose the hydroxyl groups on their surfaces. Wash the silica microspheres obtained by the reaction, disperse them in 500 mL of anhydrous ethanol, and store them in a 2 L conical flask.

[0253] The specific steps are the same as steps 1.1.2-1.1.4 in Example 2;

[0254] 1.1.3. Preparation of epoxidized silica microspheres

[0255] Place the conical flask in 1.1.2 on a stirrer, adjust the stirrer speed to 800-1000 rpm, slowly add 80 mL of 3-glycidylpentyl ether trimethoxysilane reagent, and continue the reaction at room temperature 25°C for 2 hours; after the reaction is completed, transfer the reaction system to a conical flask, discard the liquid phase after solid-liquid separation, and obtain epoxidized modified silica microspheres, wash them with 500-1000 mL of anhydrous ethanol for 3-5 times, and then store them in anhydrous ethanol.

[0256] 1.2 Preparation of 5-HT-SiO2 Microsphere Complex

[0257] The epoxidation-modified silica microspheres prepared in step 1.1 are coupled with serotonin, and the specific steps are the same as step 1.3 of Example 2;

[0258] The serotonin-silica microsphere complex prepared in step 1.2 was loaded into a column and used to separate and purify sialylated glycocalyx protein in milk. The specific operation steps were the same as step 2 and step 3 of Example 2.

Claims

1. A serotonin-silica microsphere complex, characterized in that: The complex is a silica microsphere with serotonin fixed on the surface.

2. The serotonin-silica microsphere complex according to claim 1, characterized in that: The serotonin is fixed on the surface of the silica microspheres through the reaction of the amino groups of the serotonin with the epoxy / aldehyde / activated carboxyl groups modified on the silica microspheres.

3. The serotonin-silica microsphere complex according to claim 2, characterized in that: It includes silicon atoms bonded to the surface of silica microspheres and functional groups connected to the silicon atoms; The structure of the functional group is:

4. A method for preparing the serotonin-silica microsphere complex according to any one of claims 1 to 3, characterized in that: The following steps are involved: Modified silica microspheres are used as carriers for fixing serotonin, and serotonin is covalently coupled to the surface of the carrier; the modified silica microspheres are at least one of epoxidation / activated carboxylation / aldehyde modification silica microspheres.

5. The method for preparing the serotonin-silica microsphere complex according to claim 4, characterized in that: The preparation method of the epoxidized / activated carboxylation / formylation-modified silica microspheres is as follows: Under alkaline and anhydrous conditions, an epoxy silanization agent is added to the silica microspheres for reaction to obtain epoxy-modified silica microspheres, which are then stored in anhydrous ethanol; Under alkaline and anhydrous conditions, a compound having an amino group and a carboxyl group is added to the epoxidation-modified silica microspheres for reaction and activation to obtain activated carboxyl-modified silica microspheres, which are then stored in anhydrous ethanol; Under alkaline and anhydrous conditions, a coupling agent having a dialdehyde group is added to the amino-silane-modified silica microspheres to react to obtain aldehyde-modified silica microspheres, which are then stored in anhydrous ethanol; The aminosilane-modified silica microspheres are prepared by reacting (3-aminopropyl)trimethoxysilane with silica microspheres.

6. The method for preparing the serotonin-silica microsphere complex according to claim 5, characterized in that: The epoxy silanization agent is 3-glycidyl propyl ether trimethoxy silane or 3-glycidyl butyl ether trimethoxy silane or 3-glycidyl pentyl ether trimethoxy silane; The compound having amino and carboxyl groups is γ-aminobutyric acid, and the concentration of γ-aminobutyric acid is 0.5-1.5 mol / L; The coupling agent is glutaraldehyde, and the concentration of glutaraldehyde is 0.5-1.5 mol / L.

7. The method for preparing the serotonin-silica microsphere complex according to claim 6, characterized in that: The preparation method of the epoxidation-modified silica microspheres is specifically as follows: 1.1.

1. Wash the silica microspheres with anhydrous ethanol; 1.1.

2. Add 1-5% NaOH solution to the silica microspheres and react for 0.5-2 hours; 1.1.

3. Pour off the NaOH solution, wash the silica microspheres with ultrapure water, let the silica microspheres settle, separate and discard the supernatant; 1.1.

4. Drain the residual liquid in the silica microspheres, wash the silica microspheres with anhydrous ethanol, and then disperse them in anhydrous ethanol for storage; 1.1.

5. Preparation of epoxidized silica microspheres Slowly add 3-glycidylpentyl ether trimethoxysilane reagent to the silica microspheres obtained in 1.1.

4. and react for 1 to 3 hours; after the reaction is completed, separate the solid and liquid and discard the liquid phase to obtain epoxidized silica microspheres; The method for covalently coupling serotonin to the surface of the carrier is specifically as follows: adding serotonin hydrochloride solution and modified silica microspheres to a coupling buffer, mixing them, and reacting them in the dark to obtain modified silica microspheres coupled with serotonin; The light-proof reaction time is 2 to 5 hours; The coupling buffer is a mixed solution containing 0.5-2 mol / L NaHCO3 and 0.01-0.1 mol / L NaCl, with a pH of 8.0-8.5; The particle size of the silica microspheres is 40-60 meshes.

8. A process for extracting sialylated glycocalyx protein, using the serotonin-silica microsphere complex according to any one of claims 1 to 3, characterized in that: The following steps are involved: 1) adding a mixed solution of production raw materials and binding buffer to a protein extraction device containing a serotonin-silica microsphere complex to allow the sialylated glycocalyx protein therein to bind to the serotonin-silica microsphere complex; the production raw materials are raw materials containing sialylated glycocalyx protein, and the binding buffer is used to promote the binding of sialylated glycocalyx protein to the serotonin-silica microsphere complex; 2) washing the 5-HT-silica microsphere complex of the protein extraction device with a binding buffer to discharge the unbound protein solution; 3) adding an elution solution to a protein extraction device, slowly stirring for elution, collecting the elution solution, and performing protein quantification to obtain a sialylated glycocalyx protein solution.

9. The sialylated glycocalyx protein extraction process according to claim 8, characterized in that: The step 4) is also included, and the regeneration of the serotonin-silica microsphere complex is specifically as follows: The 5-HT-silica microsphere complex was washed with twice the volume of 1-10 mol / L urea and then with 0.9% saline; Alternatively, wash three times with 500 mL of sodium dodecyl sulfate solution and then with 500 mL of purified water; After cleaning, it can be used again, or 20% ethanol can be added to it and stored at room temperature.

10. The process for extracting sialylated glycocalyx protein according to claim 8 or 9, characterized in that: In step 1), the 5-HT-silica microsphere complex is subjected to a sealing treatment; The enclosed 5-HT-silica microsphere complex is 5-HT covalently coupled to at least one of epoxidized / activated carboxylation / aldehyde-modified silica microspheres; The production raw material is pasteurized milk. The binding buffer is one of sodium phosphate buffer, HEPES buffer, Tris-HCl buffer and sodium acetate-acetate buffer; The ratio of the pasteurized milk to the binding buffer is 1:8 to 3:4; The binding time is 1-6 hours; In step 3), the elution solution is a mixture of 0.1-1.5 mol / L NaHCO3 and 0.5-2.5 mol / L NaCl, or a mixture of Na3PO4 and NaCl, or a coupling buffer; The amount of the elution solution added is 80-90% of the volume of the extraction device; The elution time is 0.5-3 hours.

11. The process for extracting sialylated glycocalyx protein according to claim 10, characterized in that: In step 1), the serotonin-silica microsphere complex is serotonin covalently coupled to epoxidation-modified silica microspheres; The epoxidation-modified silica microspheres are prepared by reacting 3-glycidylpentyl ether trimethoxysilane and silica microspheres; The ratio of pasteurized milk to binding buffer was 3:8; The binding time is 4 hours; The binding buffer is a sodium phosphate buffer with a pH of 6.0±0.5 and a concentration of 0.02 mol / L; In step 3), the elution solution is a mixture of 0.1 mol / L NaHCO3 and 0.5 mol / L NaCl; The amount of the elution solution added is 80% of the volume of the extraction device; The elution time was 1 hour.

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