Adaptor for specifically distinguishing different glycosylation forms of ApoC-III, screening method and application thereof

Abstract

The application discloses an aptamer for specifically distinguishing different glycosylation forms of ApoC-III, and a screening method and application thereof. The aptamer is specifically combined with non-glycosylated ApoC-III0, ApoC-III1, ApoC-III2, ApoC-III3 and total ApoC-III, and the nucleic acid sequence of the aptamer comprises any one or a combination of at least two of sequences shown in SEQ ID NO:1-SEQ ID NO:5. The aptamer has high affinity and strong specificity, can accurately distinguish the glycosylation spectrum of ApoC-III, and can be used for preparing a high-precision diagnostic kit for hypertriglyceridemia, atherosclerotic cardiovascular diseases and the like.

Description

Technical Field

[0001] This invention belongs to the fields of molecular biology, clinical diagnostics, and drug targeting technology. It relates to an aptamer that specifically distinguishes different glycosylation forms of ApoC-III, its screening method, and its application. Specifically, it relates to a set of nucleic acid aptamers that can specifically bind to apolipoprotein C-III and its four major glycosylation forms, a screening method for the aptamers, and their applications in disease diagnosis, prognosis, and targeted therapy. Background Technology

[0002] Apolipoprotein C-III is a key protein regulating lipid metabolism, and its plasma levels are closely related to the risk of hypertriglyceridemia, atherosclerotic cardiovascular disease, pancreatitis, and metabolic diseases. ApoC-III exists in the human body primarily in four glycosylated forms: non-glycosylated ApoC-III0, monosialotyl-ApoC-III1, disialotyl-ApoC-III2, and trisialotyl-ApoC-III3. Numerous studies have shown that the proportion of different glycosylation forms (glycoform profile) is a more precise disease biomarker than the total ApoC-III concentration. For example, an elevated proportion of ApoC-III2 is significantly associated with the severity of coronary heart disease.

[0003] However, there is currently a lack of molecular recognition tools capable of accurately, rapidly, and economically distinguishing these four highly similar glycosylation forms. Traditional antibodies struggle to differentiate protein variants that differ only in the number of sialic acids at a single O-glycosylation site (threonine 74). Aptamers, as "chemical antibodies," offer advantages such as ease of synthesis, simple modification, good stability, and potentially high specificity, making them an ideal solution to this problem. However, existing technologies lack a screening scheme capable of simultaneously obtaining five aptamers that target total ApoC-III and its four glycoforms independently.

[0004] Furthermore, obtaining high-purity single ApoC-III glycoform protein is a prerequisite and a major technical bottleneck for specific screening. Commercially available recombinant ApoC-III proteins are usually heterogeneously glycosylated or lack glycosylation, while isolating single glycoforms from plasma is a cumbersome process with extremely low yields.

[0005] Therefore, developing a method that can controllably prepare specific glycoproteins and screen for highly specific, cross-reactive aptamers based on them has significant innovative value and clinical significance. Summary of the Invention

[0006] To address the shortcomings of existing technologies and practical needs, this invention provides an aptamer that specifically distinguishes different glycosylation forms of ApoC-III, along with its screening method and applications. The aptamer of this invention exhibits high affinity (KD value of 0.11-2.77 nM) and strong specificity (cross-reactivity <10%), enabling precise differentiation of the glycoform profile of ApoC-III. It can be used to prepare high-precision diagnostic kits for hypertriglyceridemia, atherosclerotic cardiovascular diseases, etc., or as a therapeutic drug targeting specific glycoforms, showing broad clinical application prospects.

[0007] To achieve this objective, the present invention adopts the following technical solution: In a first aspect, the present invention provides an aptamer that specifically distinguishes different glycosylation forms of ApoC-III, said aptamer comprising any one or a combination of at least two of the following: (1) An aptamer that specifically binds to non-glycosylated ApoC-III0, the nucleic acid sequence of which includes the sequence shown in SEQ ID NO:1; (2) An aptamer that specifically binds to monosialylated ApoC-III1, the nucleic acid sequence of which includes the sequence shown in SEQ ID NO:2; (3) An aptamer that specifically binds to bisialylated ApoC-III2, the nucleic acid sequence of which includes the sequence shown in SEQ ID NO:3; (4) An aptamer that specifically binds to trisialylated ApoC-III3, the nucleic acid sequence of which includes the sequence shown in SEQ ID NO:4; (5) An aptamer that specifically binds to total ApoC-III protein, the nucleic acid sequence of which includes the sequence shown in SEQ ID NO:5.

[0008] The aptamers of this invention have high affinity (KD value of 0.11-2.77 nM) and strong specificity (cross-reactivity <10%), and can accurately distinguish the glycoform profile of ApoC-III. They can be used to prepare high-precision diagnostic kits for hypertriglyceridemia, atherosclerotic cardiovascular diseases, etc., or as therapeutic drugs targeting specific glycoforms, and have broad clinical application prospects.

[0009] The nucleic acid sequences of the aptamers that specifically distinguish different glycosylation forms of ApoC-III described in this invention are shown in Table 1.

[0010] Table 1 In a second aspect, the present invention provides a method for screening the aptamers described in the first aspect, the screening method comprising the following steps: (1) Prepare a single ApoC-III glycoprotein, wherein the single ApoC-III glycoprotein includes any one of ApoC-III0, ApoC-III1, ApoC-III2 or ApoC-III3; (2) The total ApoC-III protein containing ApoC-III0, ApoC-III1, ApoC-III2 and ApoC-III3 was immobilized on a solid support and incubated with a single-stranded DNA library; (3) A competitive step-elution method was used to sequentially elute the binding sequences with different glycoform protein solutions, and the aptamer sub-libraries specifically bound to each glycoform were collected respectively; (4) Multiple rounds of screening and enrichment were performed on the sub-cubes to finally obtain specific aptamers that target different glycoforms.

[0011] Preferably, the preparation of the single ApoC-III glycoform protein in step (1) includes: expressing recombinant ApoC-III protein using a eukaryotic system; preliminarily separating different glycoforms by anion exchange chromatography; further purifying by reversed-phase high-performance liquid chromatography to obtain a single glycoform protein; and identifying the protein glycoform by mass spectrometry.

[0012] In one embodiment of the present invention, the controllable preparation of a single glycosylated ApoC-III protein includes: Recombinant human ApoC-III was expressed using the human HEK293F expression system. Its near-nature glycosylation modification capability was used to obtain a mixture of glycoforms, which were then finely separated by two-step chromatography.

[0013] (a) Expression and capture: A His-tagged human APOC3 gene expression vector was constructed and transfected into HEK293F cells for suspension culture. The supernatant was collected and preliminarily captured and purified using nickel affinity chromatography.

[0014] (b) Glycotype enrichment and separation: Continuous separation is performed by utilizing the differences in charge (different sialic acid number) and hydrophobicity of different glycotypes in the ApoC-III protein mixture.

[0015] Step 1: Anion exchange chromatography. A Source 15Q column was used for gradient elution with a 20 mM Tris-HCl (pH 8.0) buffer containing 0-500 mM NaCl. Since sialic acid is negatively charged, the more highly glycosylated form is retained more strongly on the column, which allows for the preliminary separation of different glycoforms.

[0016] Step 2: Fine separation by reversed-phase high-performance liquid chromatography. The peaks from anion exchange chromatography were collected and further purified using a C4 reversed-phase column with gradient elution in water and acetonitrile containing 0.1% trifluoroacetic acid to obtain four single glycoform proteins (ApoC-III0, ApoC-III1, ApoC-III2, ApoC-III3) and a pool containing all forms of "total ApoC-III" protein.

[0017] (c) Protein characterization: Each purified protein was identified using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALS). Approximately 1 μg of protein sample was mixed with an equal volume of sinapic acid matrix solution and targeted, and mass spectra were acquired in positive ion linear mode. The glycoform was confirmed by determining the molecular weight of the intact protein (the molecular weight increased by approximately 291 Da for each additional sialic acid molecule).

[0018] Preferably, the single-stranded DNA library in step (2) is an oligonucleotide library containing a random sequence region, the length of which is 30-50 nt, for example 30 nt, 40 nt or 50 nt.

[0019] The 5' primer sequence of the random sequence region is shown in SEQ ID NO:6, and the 3' primer sequence of the random sequence region is shown in SEQ ID NO:7.

[0020] SEQ ID NO: 6: CACGACGTTGTAAAACGAC.

[0021] SEQ ID NO:7: GGTAGCGACTCGATCGTA.

[0022] Preferably, the competitive step elution in step (3) includes: sequentially using ApoC-III0, ApoC-III1, ApoC-III2 and ApoC-III3 protein solutions as competitive eluting agents; collecting the elution buffers from each step to obtain the corresponding aptamer sublibraries of the glycotypes; and eluting to obtain the total ApoC-III specific aptamer sublibrary.

[0023] In this invention, a highly specific aptamer screening method based on "competitive step elution" uses total protein as a fixed target and a single glycoprotein as a competitive eluent to achieve simultaneous "positive enrichment" and "negative screening" of the aptamer library, ultimately obtaining specific sequences.

[0024] In one embodiment of the present invention, the highly specific aptamer screening method based on "competitive step elution" includes the following steps: (a) Screening and Immobilization of Targets: The total ApoC-III protein obtained in "Program 1" was immobilized on sulfosuccinimide activated magnetic beads by amino coupling. For each reaction, approximately 100 pmol of protein and 1 mg of magnetic beads were reacted in 1 mL of coupling buffer (e.g., 0.1 M MES, pH 6.0) for 2 h.

[0025] (b) Initial affinity screening: approximately 1 nmol (~10 14 An initial single-stranded DNA randomized library (5'-CACGACGTTGTAAAACGAC-(N40)-GGTAGCGACTCGATCGTA-3') was dissolved in 500 μL binding buffer (1× PBS, 5 mM MgCl2, 0.1 mg / mL salmon sperm DNA, 0.05% Tween-20) and incubated with magnetic beads immobilized with total ApoC-III at 25°C for 1 h. The sample was washed 5 times with binding buffer to remove unbound sequences.

[0026] (c) Competitive stepwise elution: First round of elution (obtaining the aptamer pool with basic affinity for total protein): Elution was performed using 500 μL of 5 mM EDTA-PBS solution, and the eluent was collected and labeled "Primary Aptamer Pool (P0)". The DNA from this pool was amplified by PCR, purified, and used as the input library for the next round of screening.

[0027] The second round of screening and elution (enrichment of glycotype specificity): After incubating and washing the P0 library with new total ApoC-III magnetic beads, instead of eluting directly with buffer, high concentrations of single glycotype protein solutions were used as competitive eluting agents. Specifically, 200 μL of competitive elution buffer (binding buffer formulation) containing 50 nM purified ApoC-III0 protein was added and incubated at 25°C for 30 min. The eluted sequences, representing the subset with the highest affinity for ApoC-III0 and capable of being effectively competed for by it, were collected as secondary library S0. The remaining magnetic beads were washed with binding buffer and then eluted with 50 nM ApoC-III1 protein solution to collect secondary library S1. This process was repeated sequentially with 50 nM ApoC-III2 and 50 nM ApoC-III3 protein solutions for competitive elution, yielding secondary libraries S2 and S3, respectively. Finally, the sequences still bound to the magnetic beads were eluted with 5 mM EDTA-PBS to obtain secondary library S4. This library contains sequences with extremely high affinity for common epitopes on the total protein and are not easily competed for by a single glycoform, serving as a specific candidate aptamer source for "total ApoC-III".

[0028] (d) Repeated screening and cloning sequencing: The five secondary libraries S0 to S4 were amplified by PCR to prepare single-stranded DNA. Then, each library was independently subjected to a new round of fixation, binding, and competitive elution (using the same glycoprotein) targeting the specific glycoform protein used in its elution. This process was repeated 8-12 times until the binding signal no longer increased significantly. The libraries from the final round were cloned and sequenced. Representative candidate aptamer sequences were selected from each library through sequence alignment and family analysis.

[0029] Thirdly, the present invention provides a kit for detecting the glycoform profile of ApoC-III, the kit comprising the aptamer described in the first aspect that specifically distinguishes different glycosylation forms of ApoC-III.

[0030] Preferably, the kit for detecting ApoC-III glycotypes further includes a marker or solid support.

[0031] Preferably, the marker includes any one or a combination of at least two of biotin, fluorescent groups, enzymes, or radionuclides.

[0032] Preferably, the solid support comprises any one or a combination of at least two of the following: a chip, a magnetic bead, or a microporous plate.

[0033] Fourthly, the present invention provides the use of the aptamers described in the first aspect that specifically distinguish different glycosylation forms of ApoC-III in the preparation of products for the diagnosis, prognostic assessment or monitoring of diseases associated with changes in the ApoC-III glycotype profile.

[0034] Preferably, the disease includes any one or a combination of at least two of the following: hypertriglyceridemia, atherosclerotic cardiovascular disease, pancreatitis, metabolic syndrome, or liver disease.

[0035] Fifthly, the present invention provides a method for detecting ApoC-III glycoform profiles in biological samples. The method comprises: mixing the aptamer described in the first aspect, which specifically distinguishes different glycosylation forms of ApoC-III, with the biological sample to be tested; detecting the binding signal between the aptamer and the ApoC-III glycoform protein in the biological sample to be tested; and determining the content or proportion of one or more glycoforms among ApoC-III0, ApoC-III1, ApoC-III2, and ApoC-III3 in the biological sample to be tested based on the binding signal.

[0036] Compared with the prior art, the present invention has the following beneficial effects: (1) The protein preparation method provided by the present invention can obtain four high-purity single ApoC-III glycoform proteins in a relatively controllable manner from the recombinant expression system, laying a material basis for the subsequent development of highly specific molecular recognition tools; (2) The “competitive step elution” screening strategy pioneered in this invention utilizes the subtle differences between glycoforms to perform “negative screening”, which can systematically and in parallel enrich aptamers with optimal specificity for five highly similar targets from the same initial library, avoiding the blindness and inefficiency of independent screening with a single glycoform as the target. (3) The aptamers obtained by this invention can accurately distinguish the glycoform profile of ApoC-III and can be used to develop high-precision diagnostic or prognostic monitoring kits for cardiovascular diseases, metabolic syndrome, liver diseases, etc. In addition, aptamers targeting specific pathogenic glycoforms (such as ApoC-III2) can also be used as targeted blocking agents to develop novel therapeutic drugs. Attached Figure Description

[0037] Figure 1 This is a schematic diagram of the pcDNA3.4-ApoCIII-His plasmid. Figure 2 The graph shows the results of the ApoC-III0 correlation analysis. Figure 3 The graph shows the results of the correlation analysis of ApoC-III1. Figure 4 The graph shows the results of the correlation analysis of ApoC-III2. Figure 5 The graph shows the results of the correlation analysis of ApoC-III3. Detailed Implementation

[0038] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0039] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0040] Example 1 Preparation and identification of four ApoC-III glycoproteins.

[0041] 1. Expression and purification: The constructed pcDNA3.1-ApoCIII-His plasmid ( Figure 1HEK293F cells in logarithmic growth phase were transfected, and the supernatant was collected after 5 days of culture. The supernatant was filtered through a 0.45 μm filter and loaded onto a pre-equilibrated Ni-NTA agarose column at a flow rate of 2 mL / min. After washing with buffer containing 20 mM imidazole, the target protein was eluted with buffer containing 250 mM imidazole. The elution peak was collected and dialyzed overnight at 4°C against 20 mM Tris-HCl (pH 8.0) buffer.

[0042] 2. Anion Exchange Chromatography: The dialyzed sample was loaded onto a HiTrap Q HP 1 mL column. Using 20 mM Tris-HCl (pH 8.0) as mobile phase A and 20 mM Tris-HCl (pH 8.0) containing 1 M NaCl as mobile phase B, the following gradient elution was performed: 0–5 CV, 0% B; 5–25 CV, 0%–40% B; 25–30 CV, 40%–100% B. The flow rate was 1 mL / min, and the UV absorbance at 280 nm was monitored. Several incompletely separated peaks appeared within the 15%–25% B gradient range.

[0043] 3. Reversed-phase chromatography for fine purification: Collect each peak component from anion exchange chromatography, lyophilize, and redissolve in 0.1% trifluoroacetic acid aqueous solution. Use a Jupiter C4 column with 0.1% TFA aqueous solution as mobile phase A and acetonitrile containing 0.1% TFA as mobile phase B, performing gradient elution: 0-5 min, 10% B; 5-50 min, 10%-60% B; 50-52 min, 60%-90% B. Flow rate: 1 mL / min. This step successfully separated each mixed peak into single symmetrical peaks, which were collected in the order of elution.

[0044] 4. Mass spectrometry identification: 1 μL of each purified fraction was mixed with an equal volume of sinapic acid matrix and spotted onto the target. The samples were then detected using a MALDI-TOF mass spectrometer in positive ion linear mode. The measured molecular weights were 8764.2 ± 1.5 Da, 9055.8 ± 2.1 Da, 9347.3 ± 1.8 Da, and 9638.5 ± 2.3 Da, respectively. These values ​​highly matched the theoretical molecular weights of ApoC-III0, ApoC-III1, ApoC-III2, and ApoC-III3, confirming the successful acquisition of four single glycoproteins.

[0045] Example 2 "Competitive step-wash" screening of aptamers.

[0046] 1. Screening Preparation (1) Initial library and primers: 1 nmol (~10 14A 77 nt full-length ssDNA library (5'-CACGACGTTGTAAAACGAC-(N40)-GGTAGCGACTCGATCGTA-3') was constructed. The upstream primer was biotin-CACGACGTTGTAAAACGAC (SEQ ID NO:8); the downstream primer was TACGATCGAGTCGCTACC (SEQ ID NO:9).

[0047] (2) Target immobilization: 100 pmol of the total ApoC-III protein mixture (ApoC-III0, ApoC-III1, ApoC-III2, ApoC-III3) prepared in Example 1 was reacted with 1.0 mg of sulfonyl-NHS activated magnetic beads in 1 mL of coupling buffer (0.1 M MES, pH 6.0) at 25°C for 2 h. After blocking with buffer containing 1 M ethanolamine, the mixture was washed 3 times with 1× PBS (pH 7.4), resuspended in 1 mL of binding buffer (1× PBS, 5 mM MgCl2, 0.1 mg / mL salmon sperm DNA, 0.05% Tween-20), and stored at 4°C for later use.

[0048] (3) PCR reaction system: The total volume of each reaction is 50 μL, containing: 1× Phusion high-fidelity buffer, 200 μM dNTPs, 0.5 μM upstream primer, 0.5 μM downstream primer, 10 ng template DNA, and 1 U Phusion DNA polymerase. Cyclic program: 98°C for 30 s; 20 cycles (98°C for 10 s, 56°C for 30 s, 72°C for 30 s); 72°C for 5 min.

[0049] 2. Filtering loop (taking the 4th round as an example) (1) Binding and washing: Take 100 pmol of single-stranded DNA library (third round product) and incubate it with magnetic beads immobilized with total protein in 500 μL binding buffer at 25°C for 60 min with gentle shaking. Place it on a magnetic rack for separation and discard the supernatant. Wash the magnetic beads 5 times with 500 μL of pre-cooled binding buffer.

[0050] (2) Competitive stepwise elution: Add 200 μL of competitive elution buffer containing 20 nM purified ApoC-III0 protein and incubate at 25°C for 30 min. Magnetic separation is performed, and the supernatant is collected and labeled as library S0. The magnetic beads are washed three times with 200 μL of binding buffer. The elution and washing steps are repeated sequentially with competitive elution buffer containing 20 nM ApoC-III1, ApoC-III2, and ApoC-III3 proteins to obtain libraries S1, S2, and S3, respectively. Finally, add 200 μL of 5 mM EDTA-PBS solution to the magnetic beads, incubate at 25°C for 10 min, and collect the final eluent as library S4.

[0051] (3) Amplification and single-strand preparation 100 μL of elution buffer from each library was used for PCR amplification. The PCR products were then bound to 100 μL of streptavidin magnetic beads at 25°C for 15 min. After magnetic separation, the samples were washed twice (2 min each time) with 200 μL of 0.1 M NaOH solution. The supernatants were combined and immediately neutralized with 1 M Tris-HCl (pH 7.4). After purification by ethanol precipitation, the samples were quantified to obtain the single-stranded DNA libraries for the fifth round of screening.

[0052] 3. Cloning and sequencing After 12 rounds of screening, each library was amplified by PCR (without single-strand preparation), and the products were cloned into T-vectors. At least 50 clones were selected for sequencing. ClustalX software was used for multiple sequence alignment to classify the sequences into different families, and 2-3 representative sequences from the main family of each library were selected for synthesis and verification.

[0053] Example 3 Surface plasmon resonance (SPR) affinity determination.

[0054] 1. Chip pretreatment: The Biacore T200 system and Series S sensor chip SA (streptavidin chip) were used. The chip was equilibrated with HBS-EP+ buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% v / v surfactant P20, pH 7.4) at a flow rate of 10 μL / min.

[0055] 2. Aptamer Immobilization: 5' biotin-labeled candidate aptamers (APC-0 to APC-4) purified by HPLC were diluted to 100 nM with HBS-EP+ buffer. They were then injected into different flow cells over 120 s, with a target immobilization amount of 100-150 response units. Non-specific binding was subsequently removed by injection with 1 M NaCl for 60 s.

[0056] 3. Combined with kinetic analysis: The corresponding purified glycoform proteins (ApoC-III0, I, II, III and total protein) were serially diluted with running buffer (concentration gradient: 0 nM, 0.78 nM, 3.13 nM, 12.5 nM, 50 nM). Each concentration was injected at a flow rate of 30 μL / min for 180 s, followed by dissociation for 600 s. Between each analysis, the chip surface was regenerated with 10 mM glycine-HCl (pH 1.5) for 30 s.

[0057] 4. Data Processing: Using Biacore T200 evaluation software, after subtracting the blank channel signal (reference cell), a 1:1 Langmuir binding model was used to globally fit the binding and dissociation curves, and the kinetic parameters were calculated: binding rate constant (ka, unit M). -1 s -1 ), dissociation rate constant (kd, unit s) -1 ) and the equilibrium dissociation constant derived therefrom (KD = kd / ka, unit M).

[0058] The results are shown in Table 2.

[0059] Table 2 The results showed that the KD values ​​of the five preferred aptamers (named APC-0 to APC-4) to their main targets ranged from 0.11 nM to 2.77 nM, with APC-2 and APC-4 having the best KD values, reaching the picomolar level.

[0060] Example 4 Enzyme-linked oligonucleotide adsorption method (ELONA) cross-reactivity test.

[0061] 1. Coating: Dilute the five glycoproteins and bovine serum albumin to 2 μg / mL with coating buffer, add 100 μL to each well of a 96-well plate, and incubate overnight at 4°C.

[0062] 2. Blocking: Discard the coating solution, add 200 μL of PBST buffer containing 3% BSA to each well, and block at 37°C for 2 h.

[0063] 3. Aptamer binding: Dilute each 5' biotin-labeled candidate aptamer to 10 nM with binding buffer. Add 100 μL of aptamer solution to each well and incubate at 37°C for 1 h.

[0064] 4. Washing: Wash the wells 6 times with PBST buffer.

[0065] 5. Signal detection: Add 100 μL of streptavidin-horseradish peroxidase solution diluted 1:5000 to each well and incubate at 37°C for 30 min. After washing, add 100 μL of TMB substrate solution for color development and incubate for 15 min. Finally, add 50 μL of 1N H2SO4 to terminate the reaction. Measure the absorbance at 450 nm using a microplate reader.

[0066] 6. Data processing: The average absorbance of each aptamer to its main target is set to 100%, and the percentage of the binding signal of the aptamer to other targets relative to this value is calculated.

[0067] The results are shown in Table 3.

[0068] Table 3 The results showed that APC-0 to APC-3 had extremely high specificity for their respective targets, while APC-4 could bind to all glycoforms.

[0069] Example 5 Validation using actual clinical samples.

[0070] 1. Sample preparation: 30 clinical plasma samples (including healthy controls and patients with cardiovascular disease) that had been pre-analyzed by MALDI-TOF mass spectrometry and whose absolute concentrations of each glucose form were known were selected.

[0071] 2. Aptamer microarray detection: Aptamers APC-0 to APC-3 are spotted onto the chip according to the designed pattern. Clinical plasma samples are diluted, hybridized with the chip, and then washed.

[0072] 3. Signal Reading and Analysis: The fluorescence intensity at each point was read using a chip scanner. The relative percentage of the corresponding glycoform in the sample was calculated based on the signal values ​​at each aptamer point using the standard curve method.

[0073] 4. Correlation Analysis: The percentages of each glycoform measured by the aptamer chip method were compared with the percentages of the gold standard measured by mass spectrometry. The Pearson correlation coefficient (r) and the coefficient of determination (r²) were calculated. 2 ), the result is as follows Figures 2-5 As shown, the detection results of all glycoform proportions showed a highly significant positive correlation between the aptamer chip method and the gold standard mass spectrometry method (p<0.0001), and the coefficient of determination r was also very high. 2 All values ​​were greater than 0.66, with the highest concordance observed for the key pathological glycoform ApoC-III2 (r). 2 =0.992). This demonstrates that the aptamer of the present invention has the potential to replace complex mass spectrometry methods for precise glycosylation analysis of clinical samples.

[0074] In summary, the aptamers (APC-0 to APC-4) screened by the method of this invention possess high affinity, high specificity, and high accuracy. The KD values ​​for all targets range from 0.11 nM to 2.77 nM, meeting the requirements for high-sensitivity detection. The cross-reactivity of each glycoform-specific aptamer (APC-0 to APC-3) to its main target is generally less than 10%, while the universal aptamer APC-4 can bind to all glycoforms in a balanced manner. In clinical sample validation, the detection results of aptamers ApoC-III0 to ApoC-III2 are highly correlated with the mass spectrometry gold standard method (R > 0.8), and can be used for accurate ApoC-III glycoform profiling analysis.

[0075] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. An aptamer that specifically distinguishes different glycosylation forms of ApoC-III, characterized in that, The aptamer includes any one or a combination of at least two of the following: (1) An aptamer that specifically binds to non-glycosylated ApoC-III0, the nucleic acid sequence of which includes the sequence shown in SEQ ID NO:1; (2) An aptamer that specifically binds to monosialylated ApoC-III1, the nucleic acid sequence of which includes the sequence shown in SEQ ID NO:2; (3) An aptamer that specifically binds to bisialylated ApoC-III2, the nucleic acid sequence of which includes the sequence shown in SEQ ID NO:3; (4) An aptamer that specifically binds to trisialylated ApoC-III3, the nucleic acid sequence of which includes the sequence shown in SEQ ID NO:4; (5) An aptamer that specifically binds to total ApoC-III protein, the nucleic acid sequence of which includes the sequence shown in SEQ ID NO:

5.

2. A method for screening aptamers according to claim 1, characterized in that, The screening method includes the following steps: (1) Prepare a single ApoC-III glycoprotein, wherein the single ApoC-III glycoprotein includes any one of ApoC-III0, ApoC-III1, ApoC-III2 or ApoC-III3; (2) The total ApoC-III protein containing ApoC-III0, ApoC-III1, ApoC-III2 and ApoC-III3 was immobilized on a solid support and incubated with a single-stranded DNA library; (3) A competitive step-elution method was used to sequentially elute the binding sequences with different glycoform protein solutions, and the aptamer sub-libraries specifically bound to each glycoform were collected respectively; (4) Multiple rounds of screening and enrichment were performed on the sub-cubes to finally obtain specific aptamers that target different glycoforms.

3. The method according to claim 2, characterized in that, The preparation of the single ApoC-III glycoform protein in step (1) includes: expressing recombinant ApoC-III protein using a eukaryotic system; preliminarily separating different glycoforms by anion exchange chromatography; further purifying by reversed-phase high-performance liquid chromatography to obtain a single glycoform protein; and identifying the protein glycoform by mass spectrometry.

4. The method according to claim 2 or 3, characterized in that, The single-stranded DNA library mentioned in step (2) is an oligonucleotide library containing a random sequence region, the length of which is 30-50 nt; The 5' primer sequence of the random sequence region is shown in SEQ ID NO:6, and the 3' primer sequence of the random sequence region is shown in SEQ ID NO:

7.

5. The method according to any one of claims 2-4, characterized in that, The competitive step elution in step (3) includes: sequentially using ApoC-III0, ApoC-III1, ApoC-III2 and ApoC-III3 protein solutions as competitive eluting agents; collecting the elution buffers from each step to obtain the corresponding aptamer sublibraries of the glycotypes; and eluting to obtain the total ApoC-III specific aptamer sublibrary.

6. A kit for detecting ApoC-III glycotypes, characterized in that, The kit for detecting ApoC-III glycotypes includes the aptamer described in claim 1 that specifically distinguishes different glycosylation forms of ApoC-III.

7. The reagent kit according to claim 6, characterized in that, The kit for detecting ApoC-III glycotype profiles also includes a marker or solid support; Preferably, the marker includes any one or a combination of at least two of biotin, fluorescent groups, enzymes, or radionuclides; Preferably, the solid support comprises any one or a combination of at least two of the following: a chip, a magnetic bead, or a microporous plate.

8. The use of the aptamer of claim 1, which specifically distinguishes different glycosylation forms of ApoC-III, in the preparation of products for the diagnosis, prognostic assessment, or monitoring of diseases associated with changes in the ApoC-III glycotype profile.

9. The application according to claim 8, characterized in that, The disease includes any one or a combination of at least two of the following: hypertriglyceridemia, atherosclerotic cardiovascular disease, pancreatitis, metabolic syndrome, or liver disease.

10. A method for detecting ApoC-III glycoform profiles in biological samples, characterized in that, The method for detecting the ApoC-III glycoform profile in a biological sample includes: mixing the aptamer described in claim 1, which specifically distinguishes different glycosylation forms of ApoC-III, with the biological sample to be tested; detecting the binding signal between the aptamer and the ApoC-III glycoform protein in the biological sample to be tested; and determining the content or proportion of one or more glycoforms among ApoC-III0, ApoC-III1, ApoC-III2, and ApoC-III3 in the biological sample to be tested based on the binding signal.

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