A fluorescently labeled polysaccharide and a method for fluorescently labeling a polysaccharide

Abstract

The present invention relates to a method for fluorescent labeling of polysaccharides, and specifically relates to a method for fluorescently labeling polysaccharides and fluorescently labeled polysaccharides. Fluorescein isothiocyanate with a histidine tag is provided; the hemiacetal hydroxyl group of fucoidan and the hydroxyl group of tyramine are subjected to condensation dehydration to form an acetal to obtain fucoidan-tyramine, and the amino group in fucoidan-tyramine and the carboxyl group in the fluorescein isothiocyanate with a histidine tag are subjected to an amidation reaction to obtain a crude product; the crude product is successively subjected to impurity removal by a Sephadex G-100 column, purification by a nickel column, and desalting by a Sephadex G-100 column to obtain the product. The fluorescently labeled polysaccharide prepared by the present invention can specifically react with a nickel column, so that the fluorescently labeled polysaccharide can be further separated and purified by the nickel column, thereby improving the fluorescent labeling rate of fucoidan.

Description

Technical Field

[0001] The present invention relates to a method for fluorescently labeling polysaccharides, and specifically relates to a method for fluorescently labeling polysaccharides and a fluorescently labeled polysaccharide. Background Art

[0002] Disclosing the information of this background art section is only intended to enhance the overall understanding of the present invention, and it is not necessarily regarded as an admission or an indication in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Polysaccharides are indispensable biological macromolecules in life science. They lack chromogenic groups and fluorescent groups that are easy to detect. It is necessary to modify polysaccharides with fluorescent labeling substances so that polysaccharides carry fluorescent groups, thereby realizing qualitative and quantitative research on polysaccharides through fluorescence detection, and thus realizing the research on the action mechanism of polysaccharides. Currently, fluorescein isothiocyanate (FITC) is a commonly used fluorescent labeling substance. Fucoidan is a complex water-soluble sulfated polysaccharide, which is derived from brown algae cell walls and some marine invertebrate tissues. It is usually composed of L-fucose and sulfate groups, and also contains a certain proportion of other monosaccharide components, including uronic acid, galactose, xylose, mannose, rhamnose, glucose, arabinose and xylose. The traditional method for labeling fucoidan with FITC is as follows: First, introduce amino groups into fucoidan, and then use the amino groups to carry out a coupling reaction with FITC to produce a fucoidan-FITC complex.

[0004] However, the inventors have found through research that the problem with the traditional method for labeling fucoidan with FITC is that it is difficult to separate the labeled and unlabeled fucoidan in the reaction solution, which reduces the labeling rate of fucoidan. Summary of the Invention

[0005] In view of the above problems, in order to improve the labeling rate of fucoidan, the object of the present invention is to provide a method for fluorescently labeling polysaccharides and a fluorescently labeled polysaccharide. The fluorescently labeled polysaccharide prepared by the present invention can specifically react with a nickel column, so that the fluorescently labeled polysaccharide can be further separated and purified by the nickel column, thereby improving the fluorescent labeling rate of fucoidan.

[0006] To achieve the above object, the technical solution of the present invention is as follows:

[0007] On the one hand, a fluorescently labeled polysaccharide is composed of fluorescein isothiocyanate with a histidine tag and fucoidan connected by a chemical bond. The fluorescein isothiocyanate with a histidine tag is composed of a polypeptide formed by histidine and fluorescein isothiocyanate connected by a chemical bond, and the fluorescein isothiocyanate with a histidine tag has a carboxyl group.

[0008] On the other hand, a method for fluorescently labeling polysaccharides provides fluorescein isothiocyanate with a histidine tag, which is composed of a polypeptide formed by histidine and fluorescein isothiocyanate connected by a chemical bond, and the fluorescein isothiocyanate with a histidine tag has a carboxyl group;

[0009] The hemiacetal hydroxyl group of fucoidan and the hydroxyl group of tyramine are subjected to condensation dehydration to form an acetal to obtain fucoidan-tyramine. The amino group in fucoidan-tyramine and the carboxyl group in the fluorescein isothiocyanate with a histidine tag are subjected to an amidation reaction to obtain a crude product. The crude product is successively subjected to impurity removal by a Sephadex G-100 column, purification by a nickel column, and desalting by a Sephadex G-100 column to obtain the product.

[0010] The beneficial effects of the present invention are as follows:

[0011] In the present invention, a FITC containing a polypeptide with histidine is designed to fluorescently label fucoidan, making it carry a histidine tag, which can specifically react with a nickel column. When the subsequent reaction solution passes through the nickel column, the labeled and unlabeled fucoidan can be better separated, thereby improving the labeling rate of fucoidan. The cytotoxicity detection by the MTT method shows that the fluorescently labeled polysaccharide provided by the present invention is non-toxic to cells and can be used for the subsequent research on the action mechanism of fucoidan. Description of the Drawings

[0012] The specification drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0013] Figure 1 It is a graph showing the detection results of fluorescence and sugar content in the effluent of a Sephadex G-100 column in an embodiment of the present invention. A is the fluorescence absorption intensity of the effluent at different times after FITC-fucoidan passes through the Sephadex G-100 column, B is the sugar content of the effluent at different times after FITC-fucoidan passes through the Sephadex G-100 column detected by the phenol-sulfuric acid method, and C is the fluorescence absorption intensity of the effluent at different times after k-FITC passes through the Sephadex G-100 column;

[0014] Figure 2 It is the purification of labeled fucoidan by a nickel column in an embodiment of the present invention. A is the fluorescence absorption value of FITC-fucoidan at different retention times, B is the sugar content of labeled fucoidan at different retention times detected by the phenol-sulfuric acid method, C is the fluorescence absorption value of k-FITC at different retention times, and D is the fluorescence absorption value of imidazole at different concentrations;

[0015] Figure 3For the detection of fluorescence and sugar content in the eluate of FITC-fucoidan through a Sephadex G-100 column in the embodiments of the present invention, A is the fluorescence detection of the eluate, and B is the detection of sugar content by the phenol-sulfuric acid method;

[0016] Figure 4 It is the result diagram of detecting the toxicity of FITC-fucoidan by the MTT method in the embodiments of the present invention. ns represents no significant difference, and n = 3. Detailed implementation manners

[0017] It should be noted that the following detailed descriptions are all exemplary and are intended to provide further explanations of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0018] It should be noted that the terms used herein are only for describing specific implementation manners and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless otherwise clearly specified in the context, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0019] In view of the low labeling rate of fucoidan by existing fluorescence labeling methods, the present invention proposes a method for fluorescently labeling polysaccharides and a method for fluorescently labeling polysaccharides.

[0020] A typical embodiment of the present invention provides a fluorescently labeled polysaccharide, which is composed of fluorescein isothiocyanate with a histidine tag and fucoidan connected by a chemical bond. The fluorescein isothiocyanate with a histidine tag is composed of a polypeptide formed by histidine and fluorescein isothiocyanate connected by a chemical bond, and the fluorescein isothiocyanate with a histidine tag has a carboxyl group.

[0021] In some embodiments, the number of histidines in the polypeptide is 4 to 8.

[0022] In some embodiments, the chemical structural formula of the polypeptide formed by histidine in the fluorescein isothiocyanate with a histidine tag is as follows:

[0023]

[0024] Among them, n = 4 to 8.

[0025] Preferably, the number of histidines n in the fluorescein isothiocyanate with a histidine tag is 6.

[0026] In some embodiments, fucoidan is linked by forming an amide bond between a modified amino group and a carboxyl group in the fluorescein isothiocyanate with a histidine tag.

[0027] Specifically, fucoidan is modified with an amino group by reacting with tyramine.

[0028] In some embodiments, the molar ratio of fucoidan to fluorescein isothiocyanate with a histidine tag is 1:2.0 - 2.5.

[0029] Another embodiment of the present invention provides a method for fluorescently labeling a polysaccharide, providing fluorescein isothiocyanate with a histidine tag, which is composed of a polypeptide formed by histidine and fluorescein isothiocyanate connected by a chemical bond, and the fluorescein isothiocyanate with a histidine tag has a carboxyl group;

[0030] The hemiacetal hydroxyl group of fucoidan and the hydroxyl group of tyramine are subjected to condensation dehydration to generate an acetal to obtain fucoidan-tyramine. The amino group in fucoidan-tyramine and the carboxyl group in the fluorescein isothiocyanate with a histidine tag are subjected to an amidation reaction to obtain a crude product. The crude product is successively subjected to impurity removal by a Sephadex G-100 column, purification by a nickel column, and desalting by a Sephadex G-100 column to obtain the product.

[0031] In some embodiments, the number of histidines in the polypeptide is 4 - 8.

[0032] In some embodiments, the chemical structural formula of the polypeptide formed by histidine in the fluorescein isothiocyanate with a histidine tag is as follows:

[0033]

[0034] Among them, n = 4 - 8.

[0035] Preferably, the number of histidines n in the fluorescein isothiocyanate with a histidine tag is 6. According to the property that histidine can specifically bind to nickel ions, the present invention uses a polypeptide formed by 6 histidines, namely hexahistidine-FITC, which has the strongest binding ability to the nickel column, so as to better separate the labeled and unlabeled fucoidan in the reaction solution, and thus improve the labeling rate of fucoidan.

[0036] In some embodiments, tyramine is added to the fucoidan solution for dissolution, and sodium cyanoborohydride is added for reaction. Specifically, after the reaction, dialysis and drying are carried out. Specifically, fucoidan is added to an alkaline phosphate buffer solution for reaction for 12 to 36 h to obtain a fucoidan solution. Specifically, the pH of the alkaline phosphate buffer solution is 7.3 to 7.5. Specifically, after adding tyramine, the reaction is carried out at 36 to 38 °C for 12 to 36 h. The reaction conditions after adding sodium cyanoborohydride are: the reaction is carried out at 36 to 38 °C for 84 to 108 h.

[0037] In some embodiments, the process of the amidation reaction is as follows: Fluorescein isothiocyanate with a histidine tag is dissolved, N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) are added for activation, then sodium hydroxide or potassium hydroxide is added to adjust the pH, and then fucoidan-tyramine is added for reaction. Specifically, fluorescein isothiocyanate with a histidine tag is dissolved using a phosphate buffer solution. More specifically, the pH of the phosphate buffer solution is 5.8 to 6.2. Specifically, the activation process is stirring at room temperature for 20 to 40 min. Specifically, the pH is adjusted to 7.3 to 7.5 using sodium hydroxide or potassium hydroxide. Specifically, the temperature for adding fucoidan-tyramine for reaction is 36 to 38 °C. Specifically, the time for adding fucoidan-tyramine for reaction is 36 to 60 h.

[0038] In some embodiments, the reaction solution obtained from the amidation reaction is passed through a Sephadex G-100 column, collected and freeze-dried to obtain a first freeze-dried sample. The first freeze-dried sample is redissolved and passed through a nickel column, the reaction solution is collected and freeze-dried to obtain a second freeze-dried sample. The second freeze-dried sample is redissolved and passed through a Sephadex G-100 column, collected and freeze-dried to obtain the product. Specifically, the solvent used for redissolution is ultrapure water.

[0039] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with specific embodiments.

[0040] Example 1

[0041] A method for fluorescent labeling of fucoidan, comprising the following steps:

[0042] (1) Synthesis of fucoidan-tyramine: Dissolve 2 g of fucoidan in 75 mL of 0.2 mol / L phosphate buffer solution with pH = 7.4, place it in a shaker at 37 °C and 100 r / min for 24 h. First, add 2 g of tyramine and stir to completely dissolve it, then add 600 mg of sodium cyanoborohydride, and continue the reaction for 96 h. The reaction solution is centrifuged at 4000 rpm for 10 min, and the supernatant is dialyzed in an 8000 - 14000 dialysis bag for 24 h, and then freeze-dried to obtain the freeze-dried sample of fucoidan-tyramine.

[0043] (2) Synthesis of fucoidan-tyramine-FITC: Weigh 15 mg of k-FITC and add it to 75 mL of phosphate buffer solution with pH = 6, then add 29 mg of EDC and 43.5 mg of NHS, and stir magnetically at room temperature for 30 min. After adjusting the pH of the solution to 7.4 with sodium hydroxide, add the freeze-dried sample of fucoidan-tyramine, place it in a shaker at 37 °C and 100 r / min for 48 h, and then obtain fucoidan-tyramine-FITC (FITC-fucoidan).

[0044] The structural formula of k-FITC is as follows:

[0045]

[0046] The k-FITC used in this example was synthesized by GeneScript Biotech Corporation (Shanghai) Co., Ltd. after being designed by the laboratory itself. The designed k-FITC sequence is HHHHHHK-FITC, and GeneScript synthesized it according to the conventional polypeptide synthesis method. Among them, HHHHHHK is shown in SEQ ID NO.1.

[0047] (3) Impurity removal by Sephadex G-100 column: Weigh 2 mg of Sephadex G-100 in 40 mL of ultrapure water and leave it overnight at room temperature to obtain the activated Sephadex G-100. Wash it twice with ultrapure water, slowly add it to the XK16 column, connect the AKTA Pure system, press the column at a relatively high flow rate for a certain time, and then change to a flow rate of 1 mL / min to equilibrate for 1 h. Add 1 mL of the FITC-fucoidan reaction solution, set the flow rate to 1 mL / min, collect once every minute, determine the target sample range according to the fluorescence absorption value and sugar content of each tube, and freeze-dry it. The mobile phase is ultrapure water.

[0048] (4) Nickel column purification: Prepare the buffer according to the above ratio. Connect the nickel column to the AKTA Pure system, add Buffer 1, and equilibrate for 30 min at a flow rate of 0.5 mL / min. Dissolve the sample obtained by freeze-drying in (3) with ultrapure water, load 1 mL each time, with a flow rate of 0.1 mL / min during loading, and collect every 0.1 mL; during buffering, the flow rate is 0.5 mL / min, and collect every 0.5 mL; elute with Buffer 1 and 2 in a 0 - 500 mM imidazole concentration gradient, with a flow rate of 0.5 mL / min, and collect every 0.5 mL. Collect the eluate and perform freeze-drying on it.

[0049] (5) Desalting with Sephadex G-100 column: Subsequently, use the Sephadex G-100 column to remove imidazole and salts, and freeze-dry to obtain FITC-fucoidan. The method is the same as (3).

[0050] There are impurities such as unlabeled fucoidan, k-FITC, NHS, and EDC in the FITC-fucoidan reaction solution. The presence of these impurities reduces the purity of FITC-fucoidan and affects its use. Therefore, in order to obtain high-purity fucoidan, it needs to be purified.

[0051] First, use the molecular sieve principle of the Sephadex G-100 column to remove impurities with smaller molecular weights, especially k-FITC. As Figure 1 A, Figure 1 C shows, the fluorescence intensity detection results show that when FITC-fucoidan passes through the Sephadex G-100 column, there is obvious fluorescence absorption from 10 - 14 min, and the sugar content results also show obvious sugar absorption in this range. Combining Figure 1 B, the sugar content results of the effluent of the FITC-fucoidan Sephadex G-100 column at different times detected by the phenol-sulfuric acid method show strong ultraviolet absorption from 10 - 14 min, indicating that 10 - 14 min is the elution time of FITC-fucoidan. At the same time, Figure 1 C shows that the fluorescence intensity detection results of k-FITC show that the elution range of k-FITC is 15 - 19 min, which is significantly different from that of FITC-fucoidan. Therefore, the fraction from 10 - 14 min can be collected for further purification. In summary, the Sephadex G-100 column can effectively remove the unreacted k-FITC in the reaction solution to eliminate its influence in the next purification step.

[0052] In addition to the above-mentioned small molecule impurities in the reaction solution, there are also some impurities such as unlabeled fucoidan and fucoidan-tyramine. It is difficult to remove these macromolecular impurities only using a Sephadex G-100 column. In addition, due to the similar molecular weights of labeled and unlabeled fucoidan, it is very difficult to separate the two using a dextran gel chromatography column. Therefore, in this example, a new FITC, namely k-FITC, was designed to solve this problem. k-FITC is based on FITC and a polypeptide containing histidine is added. After it binds to fucoidan, it can not only make fucoidan fluoresce, but also introduce histidine groups. Subsequently, using the property that histidine can specifically bind to nickel and can be eluted by imidazole, the unlabeled fucoidan is removed. Therefore, FITC-fucoidan with histidine labeling can bind to it, thereby removing the remaining impurities.

[0053] As Figure 2 shown in A, 1 - 5 mL is the loading time range of FITC-fucoidan. The fluorescence intensity detection results show that there is obvious fluorescence absorption within this range; 6 - 14 mL is the buffer time. Within this range, the column is equilibrated with buffer to remove polysaccharides or other substances that do not specifically bind to the nickel column. As shown by the fluorescence intensity in Figure 2 A and the sugar content detection results in Figure 2 B, there is no obvious fluorescence absorption and sugar absorption within this range; 15 - 30 mL is the elution time. At this time, FITC-fucoidan bound to the nickel column is eluted with a gradient concentration of 0 - 500 mM imidazole. As shown by the fluorescence intensity in Figure 2 A and the sugar content detection results in Figure 2 B, there is obvious fluorescence absorption and a relatively high sugar content in the eluate within the range of 15 - 25 mL. As shown in Figure 2 C, and the presence of imidazole does not affect the fluorescence intensity and sugar content detection, as shown in Figure 2 D. The above results indicate that after using buffer to eliminate the interference of the sample, there is still high fluorescence intensity absorption and relatively high sugar content in the imidazole eluate, indicating that the fucoidan labeling is successful. Therefore, the eluate within this range is collected, freeze-dried, and then the imidazole and sodium chloride are removed using a Sephadex G-100 column, as shown in Figure 3 . Subsequently, the obtained liquid is freeze-dried to obtain high-purity FITC-fucoidan.

[0054] Example 2

[0055] The content of fucoidan was detected by the phenol-sulfuric acid method. The specific steps are as follows:

[0056] (1) Reagent preparation

[0057] 1) Preparation of fucose standard solution: Dissolve 40 mg of fucose in 200 mL of pure water to prepare a fucose standard solution with a concentration of 200 μg / mL for standby.

[0058] 2) Preparation of 80% phenol solution: Weigh 80 g of phenol and add 20 g of ultrapure water to dissolve it. Store it in the dark in a refrigerator at 4°C. Add ultrapure water to freshly prepare 6% phenol before each use.

[0059] (2) Detection method

[0060] 1) Preparation of fucose standard curve: Aliquot 0, 2, 4, 6, 8, 10, 12, 14, 16, and 18 mL of the fucose standard solution, add pure water to make up to 25 mL, and shake well; Take 1 mL of fucose solutions with different concentrations into stoppered test tubes for standby.

[0061] 2) Take 1 mL of solution from different collection tubes into stoppered test tubes, and successively add 1 mL of 6% phenol and 5 mL of concentrated sulfuric acid, shake well, and let stand at room temperature for 20 min.

[0062] 3) Detect with an enzyme-labeled instrument at 490 nm. The abscissa is the fucose concentration, and the ordinate is the OD value to make a standard curve.

[0063] (3) Detection of the fluorescence content of fucoidan

[0064] 1) Preparation of k-FITC standard solution: Weigh 5 mg of k-FITC in 25 mL of pure water to prepare a k-FITC standard solution with a concentration of 200 μg / mL for standby.

[0065] 2) Preparation of k-FITC standard curve: Take 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, and 0.9 mL of the k-FITC standard solution into stoppered test tubes, add pure water to make up to 1 mL, shake well, and store in the dark for standby.

[0066] 3) Take 200 μL of solution from different collection tubes into a completely black 96-well plate, and measure the fluorescence intensity at an excitation wavelength of 485 / 20 and an emission wavelength of 528 / 20. Make a standard curve with the k-FITC concentration as the abscissa and the fluorescence absorption value as the ordinate.

[0067] (4) Detection of the labeling rate of fucoidan

[0068] 1) Weigh 1 mg of the FITC-fucoidan lyophilized sample and dissolve it in 10 mL of pure water to a concentration of 100 μg / mL. Aliquot 0.2 mL, 0.4 mL, and 0.8 mL into stoppered test tubes, and add pure water to make up to 1 mL.

[0069] 2) Take 200 μL of the solution from different tubes into a 96-well black plate, and measure the fluorescence intensity at an excitation wavelength of 485 / 20 and an emission wavelength of 528 / 20.

[0070] 3) Calculate the mass of k-FITC in the solution according to the k-FITC standard curve and the fluorescence intensity of FITC-fucoidan, and calculate the fluorescence labeling rate (R L ) according to the following formula: R L = n F / n0, where n F is the molar concentration of k-FITC, and n0 is the molar concentration of FITC-fucoidan.

[0071] Table 1 Detection results of fluorescence labeling rate

[0072]

[0073] Note: A standard curve was plotted with the k-FITC concentration on the x-axis and the fluorescence intensity on the y-axis. The standard curve formula is y = 8662.9x + 8372 (R 2 = 0.9995)

[0074] As shown in Table 1, a standard curve was drawn using k-FITC. The molar concentration of FITC-fucoidan was calculated by detecting the fluorescence intensity of FITC-fucoidan at different concentrations, and the fluorescence labeling rate of fucoidan was obtained by comparing it with the molar concentration of FITC-fucoidan. The average molar labeling rate of FITC-fucoidan was 2.456, indicating that 1 mol of fucoidan can react with 2.456 mol of k-FITC.

[0075] Example 3

[0076] The cytotoxicity of labeled fucoidan on macrophages was detected by the MTT method, and the specific steps were as follows:

[0077] (1) Dilute the FITC-fucoidan solution with DMEM high-glucose medium containing 15% fetal bovine serum to make its concentrations 25, 50, 100, 200, 400, and 800 μg / mL respectively. Macrophages in the logarithmic growth phase were inoculated into a 96-well plate at a cell density of 100 μL per well and 3×10 4 cells / mL, and cultured in an incubator at 5% CO2 and 37°C.

[0078] (2) After 24 h, discard the medium and replace it with DMEM medium containing the above different concentrations of fucoidan. Six parallel wells were set in each group and cultured for another 48 h.

[0079] (3) After the incubation, add 10 μL of MTT reagent to each well in the dark. Incubate at 37 °C for 4 h, then discard the supernatant. Add 100 μL of DMSO solution to each well and then gently vortex for 10 min to mix evenly.

[0080] (4) Detection: Detect at 490 nm using an enzyme-linked immunosorbent assay (ELISA) reader. Calculate the cell viability based on the measured absorbance value.

[0081] The effect of FITC-fucoidan on the proliferation activity of macrophages is as Figure 4 shown. Fucoidan at 25 - 800 μg / mL had no significant effect on the proliferation activity of macrophages within 24 h, indicating that fucoidan after labeling was also non-toxic to macrophages.

[0082] In summary, using k-FITC to label fucoidan can improve the fluorescence labeling rate of fucoidan. The average molar labeling rate of FITC-fucoidan is 2.456, indicating that 1 mol of fucoidan can react with 2.456 mol of k-FITC. Subsequently, the cytotoxicity of FITC-fucoidan was detected by the MTT method. The results showed that fucoidan at 25 - 800 μg / mL had no significant effect on the proliferation activity of macrophages within 24 h, indicating that fucoidan after labeling was non-toxic to macrophages.

[0083] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for fluorescently labeling a polysaccharide, characterized in that Provided is fluorescein isothiocyanate with a histidine tag, which is composed of a polypeptide formed by histidine and fluorescein isothiocyanate connected by a chemical bond, and the fluorescein isothiocyanate with a histidine tag has a carboxyl group; The hemiacetal hydroxyl group of fucoidan and the hydroxyl group of tyramine are subjected to condensation dehydration to form an acetal to obtain fucoidan-tyramine. The amino group in fucoidan-tyramine and the carboxyl group in the fluorescein isothiocyanate with a histidine tag are subjected to an amidation reaction to obtain a crude product. The crude product is successively subjected to impurity removal by a Sephadex G-100 column, purification by a nickel column, and desalting by a Sephadex G-100 column to obtain the product; The process of the amidation reaction is as follows: Dissolve the fluorescein isothiocyanate with a histidine tag, add N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride for activation, then add sodium hydroxide or potassium hydroxide to adjust the pH, and then add fucoidan-tyramine for reaction; The fluorescein isothiocyanate with a histidine tag is dissolved with a phosphate buffer solution; The pH of the phosphate buffer solution is 5.8-6.2; The activation process is to stir at room temperature for 20-40 min; The pH is adjusted to 7.3-7.5 with sodium hydroxide or potassium hydroxide; The temperature for adding fucoidan-tyramine for reaction is 36-38 °C; The time for adding fucoidan-tyramine for reaction is 36-60 h; The reaction solution obtained from the amidation reaction is passed through a Sephadex G-100 column, collected and freeze-dried to obtain a first freeze-dried sample. The first freeze-dried sample is redissolved and passed through a nickel column, the reaction solution is collected and freeze-dried to obtain a second freeze-dried sample. The second freeze-dried sample is redissolved and passed through a Sephadex G-100 column, collected and freeze-dried to obtain the product; The solvent used for redissolution is ultrapure water; During the impurity removal process of the Sephadex G-100 column, the elution time of FITC-fucoidan is 10-14 min, and the elution time of k-FITC is 15-19 min; During the purification process of the nickel column, the sample loading volume is 1-5 mL, the flow rate during sample loading is 0.1 mL / min, the buffer volume is 6-14 mL, the flow rate during buffering is 0.5 mL / min, the elution volume is 15-30 mL, and the flow rate during elution is 0.5 mL / min; The chemical structural formula of the polypeptide formed by histidine in the fluorescein isothiocyanate with a histidine tag is as follows: Among them, n = 4-8.

2. The method for fluorescently labeling a polysaccharide according to claim 1, wherein In the chemical structural formula of the polypeptide formed by histidine in the fluorescein isothiocyanate with a histidine tag, n = 6.

3. The method for fluorescently labeling a polysaccharide according to claim 1, wherein Add tyramine to dissolve in the fucoidan solution, add sodium cyanoborohydride and then react; After the reaction, dialyze and dry.

4. The method for fluorescently labeling a polysaccharide according to claim 3, wherein Add fucoidan to an alkaline phosphate buffer solution and react for 12-36 h to obtain a fucoidan solution.

5. The method for fluorescently labeling a polysaccharide according to claim 4, characterized in that, The pH of the alkaline phosphate buffer solution is 7.3-7.

5.

6. The method for fluorescently labeling a polysaccharide according to claim 3, wherein React at 36-38 °C for 12-36 h after adding tyramine; The reaction conditions after adding sodium cyanoborohydride are: React at 36-38 °C for 84-108 h.

7. A fluorescently labeled polysaccharide prepared by the method according to any one of claims 1-6, characterized in that, It is composed of fluorescein isothiocyanate with a histidine tag and fucoidan connected by a chemical bond. The fluorescein isothiocyanate with a histidine tag is composed of a polypeptide formed by histidine and fluorescein isothiocyanate connected by a chemical bond, and the fluorescein isothiocyanate with a histidine tag has a carboxyl group.

8. The fluorescently labeled polysaccharide according to claim 7, wherein The molar ratio of fucoidan to fluorescein isothiocyanate with a histidine tag is 1:2.0 - 2.5.

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