Fucosan sulfate high-affinity binding protein as well as preparation method and application thereof

Abstract

The invention discloses a fucosan sulfate high-affinity binding protein as well as a preparation method and application thereof, and belongs to the technical field of marine bioengineering. The invention provides a Fucoidan specific binding protein SCBP-Fuc, the amino acid sequence of the Fucoidan specific binding protein SCBP-Fuc is as shown in SEQ ID NO: 1, and the nucleotide sequence of a gene for coding the SCBP-Fuc is as shown in SEQ ID NO: 2; the SCBP-Fuc has specific binding capacity and high affinity to Fucoidan, and is suitable for being used as a polysaccharide antibody to replace a binding molecule for a series of application and development. The protein can be widely applied to specific enrichment, detection, separation and purification of polysaccharides, is suitable for developing various detection methods such as ELISA (enzyme-linked immuno sorbent assay) and biosensors, can realize sensitive detection of Fucoidan in complex systems such as food safety and drug research and development, and has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of marine bioengineering, and in particular relates to a fucoidan sulfate high-affinity binding protein and a preparation method and application thereof. Background Art

[0002] Fucoidan sulfate is a polysaccharide containing fucose and sulfate groups. It can be structurally divided into type I and type II. Type I consists of a polymer of α-1,3-linked fucose residues, while type II is primarily composed of alternating α-1,3 and α-1,4-linked fucose residues. Fucoidan is primarily derived from marine algae, such as Ascophyllum nodosum, Laminaria japonica, and Kjellmaniella crassifolia. As a marine drug candidate, fucoidan exhibits antioxidant, anti-tumor, anti-inflammatory, anti-diabetic, anticoagulant, anti-thrombotic, and antiviral activities. Fucoidan also holds significant therapeutic potential in the treatment of liver disease, urinary tract diseases, kidney disease, gastrointestinal maintenance, and surgical procedures. As an oral polysaccharide, studying the in vivo metabolic fate of fucoidan plays an important role in promoting nonclinical pharmacokinetic studies. Currently, the main methods for quantitative detection of fucoidan include liquid chromatography-mass spectrometry (LC-MS), immunology, biological assay, and isotope labeling tracer method. These methods are relatively complex and tedious to operate, take a long time to detect, and rely on large-scale professional equipment and professional knowledge.

[0003] Enzyme-linked immunosorbent assays (ELISAs) play a crucial role in drug metabolism assays due to their high sensitivity and specificity, high throughput, ease of use, and compatibility with complex matrices. These assays rely on the interaction of highly specific antibodies with antigens to quantitatively detect target antigens. By building upon the biological properties of polysaccharides and capturing them with highly specific biomolecules, ELISAs can provide an effective means for highly sensitive prototype detection of polysaccharide drug candidates.

[0004] Polysaccharide antibodies are currently mainly prepared by traditional hybridoma technology and phage display technology. This method has a long preparation cycle, high cost and cannot be produced in large quantities. In addition, due to the properties of polysaccharides such as large molecular weight, poor water solubility and low immunogenicity, it is difficult to obtain highly specific and high-affinity polysaccharide antibodies. In addition, due to the complexity of polysaccharide structure and the polydispersity of molecular weight, not all types of polysaccharides can be used to prepare highly specific antibodies. In addition to polysaccharide antibodies, polysaccharide-specific recognition molecules also include lectins, oligonucleotide aptamers, carbohydrate binding domains, etc. Although these molecules can recognize and bind to polysaccharides, their affinity for polysaccharides is relatively weak and they lack specificity, which limits their application in the targeted enrichment and quantitative analysis of target polysaccharides.

[0005] This shows that it is very difficult to obtain highly specific and high-affinity antibodies or binding proteins for polysaccharides through immunization, which is difficult to apply to the establishment of polysaccharide microdetermination methods, and also limits the research and development of polysaccharide drugs to a certain extent. Summary of the Invention

[0006] The purpose of the present invention is to provide a fucoidan sulfate high-affinity binding protein, and to provide a preparation method and specific application thereof, so as to make up for the deficiencies of the prior art.

[0007] In order to achieve the above object, the specific technical solution adopted by the present invention is:

[0008] A high-affinity fucoidan sulfate binding protein is based on site modification of fucoidan glycoside hydrolase. It is genetically modified and modified using bioinformatics and molecular biology analysis techniques to eliminate its glycosidic bond hydrolysis activity while retaining its polysaccharide binding activity.

[0009] A fucoidan sulfate high-affinity binding protein SCBP-Fuc, whose amino acid sequence is shown in SEQ ID NO: 1.

[0010] Furthermore, the SCBP-Fuc is based on the α-1,4-fucoidan endonuclease MfFcnA4 in the glycoside hydrolase family 107 (GH107), and its histidine (His) at position 249 is mutated to glutamine (Q) to obtain the mutant H294Q, which is SCBP-Fuc; the mutant H294Q eliminates its glycoside hydrolysis activity and can be regarded as a specific binding protein of Fucoidan, which specifically recognizes α-1,4 glycosidic bonds; the Fucoidan specific binding protein (Specific Carbohydrate Binding Protein-Fucoidan) SCBP-Fuc was prepared by cloning and expression.

[0011] Of course, key amino acid residues in SCBP-Fuc can be modified by site-specific mutation (e.g., to lysine, arginine, tyrosine, or other natural or unnatural amino acids) to retain or enhance their function while still maintaining high-affinity binding to Fucoidan. Therefore, such mutants also fall within the scope of protection of the present invention.

[0012] Furthermore, the nucleotide sequence of the gene encoding the α-1,4-fucoidan endonuclease mutant H294Q is shown in SEQ ID NO: 2. Of course, the gene encoding the above-mentioned Fucoidan-specific binding protein SCBP-Fuc includes not only the gene with the nucleotide sequence of SEQ ID NO: 2, but also all genes that can be translated into SEQ ID NO: 1.

[0013] Specifically, a method for preparing a fucoidan sulfate high-affinity binding protein SCBP-Fuc comprises the following steps:

[0014] (1) First, molecular docking software was used to perform semi-flexible docking between α-1,4-fucoidan endonuclease and fucoidan characteristic oligosaccharides to analyze the key sites of their catalytic hydrolysis;

[0015] (2) Combined with sequence conservation and protein domain analysis, mutants without hydrolysis activity but retaining high binding capacity were screened and identified;

[0016] (3) Using site-directed mutagenesis technology, the α-1,4-fucoidan endonuclease gene was used as a template to construct the H294Q mutant. Based on the results, the α-1,4-fucoidan endonuclease was mutated at its amino acid position 294. The nucleotide sequence corresponding to the Fucoidan-specific binding protein SCBP-Fuc is shown in SEQ ID NO: 2.

[0017] (4) Based on the clear gene sequence of SCBP-Fuc, a large amount of recombinant protein was produced through heterologous recombination cloning, expression and purification.

[0018] Furthermore, in the step (4), the expression system is selected as the E. coli system; a double-tag protein purification strategy is adopted, a Strep tag is added to its C-terminus, and a 6×His tag is retained at its N-terminus; the gene sequence of SEQ ID NO: 2 is optimized and the whole gene is synthesized according to the E. coli expression system, pET-28a is selected as the expression vector, and a recombinant expression

[0019] vector; the recombinant vector was transformed into Escherichia coli expression engineering bacteria (BL21) for expression, and a large amount of SCBP-Fuc was obtained through positive transformant screening, strain culture, protein expression and purification.

[0020] The fucoidan sulfate high-affinity binding protein SCBP-Fuc has broad practical application value in biological detection and engineering technology scenarios such as fucoidan detection, affinity purification, targeted enrichment, column chromatography capture, biosensor construction, sample pretreatment and drug delivery.

[0021] The invention relates to an application of the fucoidan sulfate high affinity binding protein SCBP-Fuc in the detection of fucoidan sulfate, especially in the micro determination of fucoidan sulfate.

[0022] A kit for establishing a fucoidan microassay based on the fucoidan-specific binding protein SCBP-Fuc comprises a capture protein: SCBP-Fuc having an amino acid sequence as shown in SEQ ID NO: 1, and a detection protein: SCBP-Fuc-His with a His tag at the C-terminus.

[0023] Furthermore, the kit also includes skimmed milk powder blocking solution, enzyme-labeled secondary antibody solution, substrate color development solution, and reaction termination solution; wherein SCBP-Fuc is coated on a microplate.

[0024] A sandwich enzyme-linked immunosorbent assay (ELISA) method for micro-determination of fucoidan is established based on the fucoidan-specific binding protein SCBP-Fuc. The method comprises:

[0025] (1) SCBP-Fuc (without His tag at the C-terminus) protein was selected as the capture protein, SCBP-Fuc-His (with His tag at the C-terminus) was selected as the detection protein, and Antibody-His-HRP was selected as the enzyme-labeled detection antibody;

[0026] (2) SCBP-Fuc was coated on a microplate at 4°C overnight;

[0027] (3) Add skim milk powder blocking solution to each well for blocking;

[0028] (4) Add the Fucoidan sample solution to the sealed microplate, react at 37°C, wash, and pat dry;

[0029] (5) Add SCBP-Fuc-His solution to each well, incubate at 37°C, wash, and pat dry;

[0030] (6) Add enzyme-labeled secondary antibody (1:2000-1:8000) solution to each well, incubate at 37°C for 1 h, wash 8 times, and pat dry on absorbent paper;

[0031] (7) Add H2O2-TMB substrate colorimetric solution to each well and incubate at room temperature;

[0032] (8) Add stop solution to each well to terminate the reaction, read the absorbance of each well with a microplate reader at a wavelength of 450 nm, and compare it with the standard test curve to complete qualitative and quantitative determination.

[0033] Furthermore, the concentration of the SCBP-Fuc capture protein coated on the microplate is 5-10 μg / mL, the working concentration of the detection protein SCBP-Fuc-His is 0.5-1 μg / mL, and the dilution ratio of the Antibody-His-HRP secondary antibody is 1:2000-1:8000.

[0034] Furthermore, the microplate was coated with capture protein overnight and blocked with 1%-5% skim milk powder. The sample was incubated for 1-4 hours, and the detection proteins SCBP-Fuc-His and Antibody-His-HRP were incubated for 1 hour.

[0035] The present invention establishes a sandwich enzyme-linked immunosorbent assay (ELISA) method for micro-determination of fucoidan based on the fucoidan-specific binding protein SCBP-Fuc. PBS is used as the detection matrix, and the detection range is 7.8 ng / mL-250 ng / mL; treated plasma is used as the detection matrix, and the detection range is 50 ng / mL-1000 ng / mL.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] The technical problem to be solved by the present invention is that Fucoidan, as an oral macromolecular active polysaccharide candidate drug, lacks an efficient method for preparing a large amount of Fucoidan with high binding capacity and specific binding to proteins, resulting in certain difficulties in establishing a sensitive and reliable micro-immunoassay method that can be performed in complex systems. As a result, its metabolic distribution in the body has not been clearly explored, which is not conducive to the research and development of Fucoidan marine candidate polysaccharide drugs.

[0038] To address the above problems, the present invention provides a high-affinity Fucoidan-specific binding protein SCBP-Fuc, which has specific binding ability and high affinity for Fucoidan and is suitable for use as a polysaccharide antibody alternative binding molecule for a series of application developments; based on SCBP-Fuc, the enzyme-linked immunosorbent assay (ELISA) method developed by the present invention has high sensitivity and can achieve trace detection of Fucoidan in complex systems, promoting its development in the food and pharmaceutical fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is the molecular docking diagram of α-1,4-fucoidanase and Fucoidan characteristic oligosaccharides.

[0040] Figure 2 It is used to construct expression vectors of endonuclease, SCBP-Fuc and SCBP-Fuc-His.

[0041] Figure 3 This is the electrophoresis diagram of the purification of endonuclease, SCBP-Fuc and SCBP-Fuc-His.

[0042] Figure 4 It is a TLC comparison chart of the degradation activity of the wild type and mutant.

[0043] Figure 5 This is a graph showing the SCBP-Fuc affinity evaluation results.

[0044] Figure 6 : SCBP-Fuc specificity evaluation results.

[0045] Figure 7 It is a standard curve graph.

[0046] Figure 8 This is a diagram showing the matrix adaptability verification results. DETAILED DESCRIPTION

[0047] To further clarify the objectives, technical solutions, and advantages of the embodiments of the present invention, the following will provide a clear and complete description of the technical solutions of the embodiments of the present invention, with reference to the accompanying drawings. Example drawings of the embodiments are shown in the drawings. The embodiments described with reference to the drawings are illustrative only and include only partial examples for the purpose of explaining and understanding the present invention. They are not intended to limit the scope of protection of the present invention.

[0048] Example 1: Construction of α-1,4-fucoidanase mutant

[0049] The crystal structure of α-1,4-fucoidan endonuclease was downloaded from the protein database, and the characteristic oligosaccharide of Fucoidan was modeled using CarbohydrateBuilder. Semi-flexible docking was performed using molecular docking software to analyze the docking score and binding mode. On this basis, the binding modes such as hydrogen bonds, hydrophobic interactions, and salt bridges between the characteristic oligosaccharides of Fucoidan and α-1,4-fucoidan endonuclease were visually analyzed to determine the key catalytic residues. The docking results are shown in the figure below. Figure 1 shown.

[0050] Molecular visualization software was used to analyze the three-dimensional structure of the complex, determine the binding pocket amino acids around the key catalytic amino acids, and screen the mutation sites through sequence alignment and structural analysis to make them lose hydrolysis activity but retain substrate binding ability.

[0051] Depend on Figure 1As shown, the characteristic oligosaccharide ligand is precisely embedded in the active pocket of the receptor in an energy-optimal conformation, forming a tight van der Waals interaction with the hydrophobic cavity of the receptor. At the same time, the hydroxyl group of the ligand forms a key hydrogen bond with the side chain amide hydrogen of His294, Sre227 / 272 and Asn230 of the receptor. Among them, histidine (His) at position 294 is a key catalytic amino acid, which is mutated to glutamine (Gln) to destroy its hydrolysis activity while avoiding affecting substrate binding.

[0052] The gene sequence was submitted to Shanghai Bioengineering Co., Ltd. for full gene synthesis. Codon optimization was performed according to the E. coli expression system. pET-28a was selected as the expression vector. A 6×His (HHHHHH) tag was retained at its N-terminus and a Strep (WSHPQFEK) tag was added to its C-terminus. The tag and the target gene were connected with a flexible linker (GGGGS). The α-1,4-fucoidanase expression vector, SCBP-Fuc expression vector, and SCBP-Fuc-His expression vector were constructed respectively. Figure 2 shown.

[0053] Example 2: Heterologous expression of SCBP-Fuc gene in Escherichia coli and its purification

[0054] The α-1,4-fucanase expression vector, SCBP-Fuc expression vector, and SCBP-Fuc-His recombinant plasmids (prepared in Example 1) were extracted according to the instructions of the Jianshi Company's bacterial plasmid extraction kit. The recombinant strains were transformed into E. coli BL21 (DE3) competent cells using the 42°C heat shock method to construct pure recombinant strains. The strains were spread onto LB culture plates (100 μg / mL kanamycin) and cultured at 37°C for 18 hours. Positive monoclonal colonies on the LB resistance plates were picked and inoculated into 10 mL of LB liquid medium (100 μg / mL kanamycin) and cultured overnight at 37°C and 180 rpm.

[0055] The bacterial solution was transferred to 500 mL LB liquid medium at a 1% inoculum volume and cultured at 37°C with shaking until the bacterial solution OD 600 When the concentration was between 0.6 and 0.8, 500 μL IPTG (0.5 mol / L) was added to the bacterial solution and cultured at 20°C and 180 rpm for 18-24 h.

[0056] After collecting the cells by centrifugation, an appropriate amount of lysis buffer was added according to the cell weight and ultrasonically disrupted. The supernatant was collected by high-speed centrifugation. The recombinant protein was purified according to the instructions for use of Strep-Tactin XT Sepharose from Cytiva. The lysis buffer formula is shown in Table 1.

[0057] Table 1 Bacterial lysis solution formula

[0058]

[0059] Refer to the instructions of the ready-to-use BCA protein quantification kit of Yazyme Biotechnology Co., Ltd. to quantify the purified protein components and take an appropriate amount of sample for SDS-PAGE electrophoresis analysis, such as Figure 3 The results are shown in the figure. M represents the protein molecular weight standard. The molecular weight of the purified α-1,4-endofucanase, SCBP-Fuc, and SCBP-Fuc-His sample bands is approximately 75 kDa, consistent with the standard. Grayscale analysis indicates the protein purity is approximately 90%.

[0060] Example 3: Functional verification of glycosidase and high affinity binding protein

[0061] 1. Hydrolytic Activity Assay

[0062] Determination of glycosidase and high-affinity binding protein degradation activity: Take 100 μL of 2 mg / mL Fucoidan polysaccharide (deionized water as solvent), add 20 μL of purified protein liquid, react at 37°C and 600 rpm for 24 hours, boil in water bath for 10 minutes, cool and centrifuge to obtain the supernatant, and observe the polysaccharide degradation by thin layer chromatography (TLC) spot plate.

[0063] TLC developing solvent (formic acid: n-butanol: water (V:V:V) = 6:4:1)

[0064] Sugar color developer: Dissolve 4g diphenylamine in 4mL aniline and 200mL ethyl acetate, add 20mL 85% phosphoric acid and stir evenly, then add 2mL concentrated hydrochloric acid and stir to clarify.

[0065] The results are as follows Figure 4 As shown, the wild-type glycosidase clearly generates corresponding sugar spots at the monosaccharide position under the action of the substrate, indicating its good polysaccharide hydrolysis activity. In contrast, the mutant SCBP-Fuc, in which the histidine in the active center is mutated to glutamine, did not produce sugar spots under the same experimental conditions, indicating that it has completely lost the ability to hydrolyze polysaccharide substrates.

[0066] This phenomenon demonstrates that histidine plays an irreplaceable role in enzyme catalysis. Substitution of histidine with glutamine disrupts the proton donor / acceptor function of the catalytic center, weakening its ability to localize and polarize the substrate, leading to a complete loss of enzyme activity. The results further support the crucial catalytic role of this histidine residue in glycosidic bond cleavage reactions and emphasize its central role in maintaining enzyme conformational stability and substrate conversion efficiency.

[0067] 2. Surface Plasmon Resonance Determination of Protein Specificity and Affinity

[0068] Surface Plasmon Resonance (SPR) technology was used to analyze the binding specificity of SCBP-Fuc to different polysaccharides and determine their affinity parameters (Kd value, Ka value, etc.). A 1:1 volume mixture of EDC / NHS (0.2M EDC, 0.05M NHS) was used to activate the CM5 chip surface, and SCBP-Fuc was diluted to 20μg / mL in mobile phase buffer (HEPES pH 7.4). The protein solution was injected onto the chip surface at a flow rate of 10μL / min to achieve an appropriate binding amount. 1M ethanolamine (pH 8.5) was flowed through the chip to remove unbound active groups. Then, 10μg / mL of different polysaccharides (type I / II-Fucoidan) were added in sequence for flow-through binding. After binding, HEPES elution was completed, and the specificity of the binding signal intensity (Response Unit, RU) of different polysaccharides for Fucoidan binding protein was evaluated. The data were fitted using a 1:1 Langmuir binding model. The specific results are shown in the figure. Figure 5 shown.

[0069] like Figure 5 As shown, by analyzing the binding ability of the protein to various polysaccharide substrates, the results showed that there was almost no obvious binding behavior between it and mannose, glucan and chondroitin sulfate, indicating that the protein lacks specific recognition or affinity sites for the above-mentioned polysaccharide structures.

[0070] In contrast, the protein showed a certain binding ability to both type I and type II fucoidans. In particular, when interacting with type II fucoidan, the binding curve showed a typical binding pattern, and there was almost no obvious dissociation process after binding, indicating that the complex was stable. The binding constant (Kd) obtained by kinetic fitting was 2.589×10 -9 M, which is at the nanomolar level, suggests that this protein binds specifically and with high affinity to type II fucoidan. In contrast, although type I fucoidan also exhibits some binding, its dissociation is relatively pronounced, indicating that the binding is relatively unstable and has a lower affinity than type II fucoidan.

[0071] Binding curve characteristics and Kd values suggest that the protein may interact with the highly sulfated fucose residues in fucoidan through electrostatic adsorption and hydrogen bonding via one or more specific positively charged domains. Furthermore, type II fucoidan may possess a spatial conformation or sulfate group distribution that is more suitable for the binding pocket of the protein, further promoting the formation of a stable complex.

[0072] The above experiments show that a mutant of α-1,4-fucanase has mutated the histidine (His) at position 249 of the wild-type α-1,4-fucanase to glutamine (Q). Compared with the wild-type, SCBP-Fuc eliminates its hydrolysis activity against fucoidan while retaining its fucoidan binding ability. SPR analysis shows that the KD value of SCBP-Fuc binding to fucoidan is 2.589×10 -9 M, indicating that a fucoidan microdetermination method can be established based on SCBP-Fuc, showing the good application potential of SCBP-Fuc.

[0073] Example 4: Establishment of micro-Fucoidan enzyme-linked immunosorbent assay

[0074] Sandwich ELISA standard curves for SCBP-Fuc and SCBP-Fuc-His were prepared using Example 2. Phosphate buffer (PBS, 0.1 mol / L, pH 7.4) was used as the diluent and reaction buffer for all samples; SCBP-Fuc was used as the capture protein, SCBP-Fuc-His was used as the detection protein, Anti-His-tag-HRP was used as the enzyme-labeled secondary antibody, skim milk powder was used as the blocking agent, and PBST (0.05% Tween) was used as the washing solution. The specific steps were as follows:

[0075] 1. Optimization of detection process conditions

[0076] (1) 1-10 μg / mL SCBP-Fuc was coated on a 96-well microplate at 4°C overnight. Before blocking, the plate was washed three times and patted dry on absorbent paper.

[0077] (2) Add 200 μL of 1-5% skim milk powder blocking solution to each well, block at 37°C for 1 h, wash five times with PBST washing solution, and pat dry on absorbent paper;

[0078] (3) Add 100 μL of Fucoidan sample solution of different concentrations to the sealed microplate, react at 37°C for 2 h, wash 5 times, and pat dry on absorbent paper;

[0079] (4) Add 100 μL of SCBP-Fuc-His (0.5-5 μg / mL) to each well, incubate at 37°C for 1 h, wash three times, and pat dry on absorbent paper;

[0080] (5) Add 100 μL of enzyme-labeled secondary antibody solution with different dilution ratios (1:2000-1:8000) to each well, incubate at 37°C for 1 h, wash 8 times, and pat dry on absorbent paper;

[0081] (6) Add 100 μL of H2O2-TMB substrate colorimetric solution to each well and incubate at room temperature for 5-15 min;

[0082] (7) 50 μL of 2 mol / L sulfuric acid solution was added to each well to terminate the reaction. The absorbance of each well was read using a microplate reader at a wavelength of 450 nm. The absorbance ratio of the sample data to the blank data (Signal / Noise, S / N) was used as the optimization indicator. The specific optimization results are shown in Table 2-5.

[0083] Table 2 Results of optimizing capture protein and detection protein concentrations by checkerboard titration

[0084]

[0085] The working concentrations of the capture and detection proteins were optimized using a checkerboard titration method. Results showed that a capture antibody concentration of 5 μg / mL and a detection antibody concentration of 0.8 μg / mL achieved the highest signal intensity and lowest nonspecific background, achieving the best signal-to-noise ratio (S / N), providing optimal reaction conditions for subsequent detection systems. The concentration combination thus determined was used to establish a standard curve and for subsequent sample testing experiments.

[0086] Table 3 Blocking agent types and optimization results of blocking time

[0087]

[0088] The blocking agent and blocking concentration of the detection system were optimized. The results showed that using skimmed milk powder as a blocking agent had the best signal-to-noise ratio (S / N), and its concentration was optimal at 3%. The blocking combination determined in this way was used to establish a standard curve and subsequent sample detection experiments.

[0089] Table 4 Sample incubation time optimization results

[0090]

[0091] The sample incubation time of the detection system was optimized. The results showed that at a standard concentration of 100 ng / mL, incubation for 3 hours had the best signal-to-noise ratio (S / N). The blocking combination determined in this way was used to establish the standard curve and subsequent sample detection experiments.

[0092] Table 5 Optimization results of protein incubation time

[0093]

[0094] The incubation time for the detection protein was optimized, and the results showed that at a standard concentration of 250 ng / mL, an incubation of 0.5 hours had the best signal-to-noise ratio (S / N). The blocking combination determined in this way was used to establish the standard curve and subsequent sample detection experiments.

[0095] Table 6 Optimization results of secondary antibody dilution multiple and incubation time

[0096]

[0097] To achieve optimal detection sensitivity and specificity, the secondary antibody dilution factor and incubation time were systematically optimized. The results showed that signal intensity gradually decreased with increasing secondary antibody dilution, but background levels increased at higher concentrations. Comprehensive analysis of the signal-to-noise (S / N) ratio determined that a secondary antibody dilution of 1:5000 produced the strongest signal and lowest background, demonstrating optimal detection performance.

[0098] In addition, the secondary antibody incubation time also has a significant impact on the test results. Comparisons under different incubation times (e.g., 30, 45, and 60 minutes) found that the signal intensity reached its peak after 1 hour of incubation. Extending the incubation time did not significantly improve the detection effect, but may lead to an increase in background. Therefore, the secondary antibody dilution factor of 1:5000 and the incubation time of 1 hour were ultimately determined as the optimal detection conditions.

[0099] 2. Standard Curve Establishment and Method Validation

[0100] The Fucoidan standard was diluted with PBST buffer to different concentrations, with the concentration range set at 7.8-5000 ng / mL. PBST was also used as a negative control. The sandwich ELISA method established above was used for detection, and the absorbance (OD) values corresponding to each concentration were read using a microplate reader. Data analysis showed that the standard curve fit well within the standard concentration range of 7.8-250 ng / mL, with a linear regression correlation coefficient (R2) > 0.99, indicating that the method has a good linear relationship in the PBST matrix. The standard curve was fitted using a four-parameter logistic regression model, and the equation is as follows:

[0101] (A=2.43288; B=-1.44297; C=22.05093; D=0.05506)

[0102] To further verify the accuracy and repeatability of the linear range, at least 6 concentration levels within the linear interval were selected, and standard curves of 6 independent batches were constructed at different time points. The results showed that the linear relationship between batches was consistent and the variability was small, indicating that the method has good reproducibility and stability.

[0103] 3. Sample processing and matrix adaptability verification:

[0104] Before testing, human plasma samples were pretreated by centrifugation to remove insoluble impurities to prevent interference. The plasma samples were then diluted fivefold and enzymatically digested with Pronase to effectively reduce matrix effects and improve assay sensitivity and specificity. The treated plasma was used as a diluent, and calibration curves were prepared for Fucoidan standards of varying concentrations spiked into the treated plasma matrix. The samples were then analyzed using the same assay method.

[0105] The results are as follows Figure 8 As shown in the figure, with treated plasma as the matrix, fucoidan can still be well quantified in the concentration range of 50-5000 ng / mL. The standard curve was fitted using a four-parameter logistic regression model with good fitting results (R2>0.99). The equation of the standard curve is as follows:

[0106] (A=1.95763; B=-0.52591; C=2084.71242; D=0.00461)

[0107] This shows that this method has strong adaptability and reliable detection ability in actual sample matrices, providing technical support for the accurate quantification of Fucoidan in subsequent plasma samples.

[0108] 4. Specificity

[0109] After protein coating and blocking, each polysaccharide was diluted to a final concentration of 5 μg / mL in phosphate-buffered saline (PBS, 0.1 mol / L, pH 7.4) for use as a standard for detection. Subsequently, 100 μL of the recombinant SCBP-Fuc-His protein prepared in Example 2 was added to each well for binding. The binding reaction was incubated at 37°C and 600 rpm to ensure sufficient interaction between the protein and polysaccharide.

[0110] After the binding was completed, enzyme-labeled secondary antibody (1:5000) was added for detection, and the color was developed by TMB color reaction system. After the reaction was terminated, the absorbance value of each well was measured using a microplate reader (OD 450nm ).like Figure 7 As shown, SCBP-Fuc-His exhibited excellent specific recognition for different types of fucoidan, while no positive signals were detected for non-binding polysaccharides such as dextran, mannose, and chondroitin sulfate. These results further validated the highly selective binding properties of SCBP-Fuc-His for fucoidan, demonstrating its excellent specificity and ability to discriminate between different structural types of fucoidan molecules.

[0111] 5. Repeatability Experiment

[0112] Inter-batch repeatability and intra-batch repeatability experiments were performed separately. The inter-batch repeatability was performed using antibodies processed from different batches and different ELISA plates, while the intra-batch repeatability was performed using antibodies processed from the same batch and the same ELISA plate. At the same time, a negative control group was set up with three replicates per group. The specific results are shown in Tables 7 and 8.

[0113] Table 7 Inter-batch repeatability experiment

[0114]

[0115] Table 8 Intra-batch repeatability experiment

[0116]

[0117] The inter-batch and intra-batch coefficients of variation of the sandwich ELISA method for detecting Fucoidan established in this experiment were less than 10%, indicating that this method has good reproducibility when performing inter-batch and intra-batch detection.

[0118] The specific embodiments described above further illustrate the purpose, technical solutions and beneficial effects disclosed in the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A fucoidan sulfate high affinity binding protein, characterized in that: The binding protein is based on site modification of Fucoidan glycoside hydrolase, and is genetically modified and modified using bioinformatics and molecular biology analysis techniques to eliminate the activity of hydrolyzing glycosidic bonds while retaining the activity of binding to polysaccharides.

2. A fucoidan sulfate high affinity binding protein SCBP-Fuc, characterized in that: The amino acid sequence of the SCBP-Fuc is shown in SEQ ID NO:

1.

3. The fucoidan sulfate high affinity binding protein SCBP-Fuc according to claim 2, wherein: SCBP-Fuc is based on the α-1,4-fucosan endonuclease MfFcnA4 in the glycoside hydrolase family 107. The histidine at position 249 is mutated to glutamine to obtain the mutant H294Q, which is SCBP-Fuc.

4. A method for preparing a fucoidan sulfate high affinity binding protein SCBP-Fuc, characterized in that: The preparation method specifically comprises the following steps: (1) First, molecular docking software was used to perform semi-flexible docking of α-1,4-fucoidanase and fucoidan characteristic oligosaccharides to analyze the key sites of their catalytic hydrolysis; (2) Combined with sequence conservation and protein domain analysis, mutants with no hydrolysis activity but high binding capacity were screened and identified; (3) Using site-directed mutagenesis technology, the α-1,4-fucoidan endonuclease gene was used as a template to construct the H294Q mutant. Based on the results, the α-1,4-fucoidan endonuclease was mutated at its amino acid position 294. The nucleotide sequence corresponding to the Fucoidan-specific binding protein SCBP-Fuc is shown in SEQ ID NO:

2. (4) Based on the clear gene sequence of SCBP-Fuc, a large amount of recombinant protein was produced through heterologous recombination cloning, expression and purification.

5. The preparation method according to claim 4, wherein In the step (4), the E. coli system is selected as the expression system; a double-tag protein purification strategy is adopted, a Strep tag is added to its N-terminus, and a 6×His tag is added to its C-terminus; the gene sequence is optimized and the whole gene is synthesized according to the E. coli expression system, pET-28a is selected as the expression vector, and a recombinant expression vector is constructed; the recombinant vector is transformed into E. coli expression engineering bacteria for expression, and a large amount of SCBP-Fuc is obtained through positive transformant screening, strain culture, protein expression and purification.

6. The preparation method according to claim 4, wherein In the step (4), the clear gene sequence of the SCBP-Fuc is shown as SEQ ID NO:

2.

7. Application of the high-affinity fucoidan sulfate binding protein SCBP-Fuc according to claim 1 in bioassay and engineering technology scenarios of fucoidan detection, affinity purification, targeted enrichment, column chromatography capture, biosensor construction, sample pretreatment, and drug delivery.

8. A kit for establishing a micro-determination of Fucoidan based on the high affinity fucoidan sulfate binding protein SCBP-Fuc, characterized in that: The kit includes a capture protein: SCBP-Fuc with an amino acid sequence as shown in SEQ ID NO: 1, and a detection protein: SCBP-Fuc-His with a His tag at the C-terminus.

9. A sandwich enzyme-linked immunosorbent assay for micro-determination of fucoidan based on the high-affinity fucoidan sulfate binding protein SCBP-Fuc, characterized in that: The method includes: (1) Select the protein without the His tag SCBP-Fuc at the C-terminus as the capture protein, the protein with the His tag SCBP-Fuc-His at the C-terminus as the detection protein, and Antibody-His-HRP as the enzyme-labeled antibody; (2) Coat SCBP-Fuc on a microplate overnight at 4°C; (3) Add skim milk powder blocking solution to each well for blocking; (4) Add the Fucoidan test sample solution into the sealed microplate, react at 37°C, wash, and pat dry; (5) Add SCBP-Fuc-His solution to each well, incubate at 37°C, wash, and pat dry; (6) Add enzyme-labeled secondary antibody solution to each well, incubate at 37°C, wash, and pat dry; (7) Add H2O2-TMB substrate colorimetric solution to each well and incubate at room temperature; (8) Add stop solution to each well to terminate the reaction, read the absorbance of each well with a microplate reader at a wavelength of 450 nm, and compare it with the standard detection curve to complete qualitative and quantitative determination.

10. The sandwich enzyme-linked immunosorbent assay according to claim 9, wherein The concentration of the SCBP-Fuc capture protein coated on the microplate is 5-10 μg / mL, the working concentration of the detection protein SCBP-Fuc is 0.5-1 μg / mL, and the dilution ratio of the Antibody-His-HRP secondary antibody is 1:2000-1:8000; the microplate is coated with the capture protein overnight and blocked with 1%-5% skim milk powder. The sample incubation time is 1-4 hours, and the detection protein SCBP-Fuc-His and Antibody-His-HRP are both incubated for 1 hour.

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