Application of β-lactoglobulin-specific aptamers

Abstract

The present invention belongs to the field of biomedicine technology and relates to the application of a β-lactoglobulin-specific aptamer. Based on the co-localization phenomenon between the binding epitope of a β-lactoglobulin cyclic aptamer on the surface of β-lactoglobulin and the binding epitope of the antibody IgE on the surface of β-lactoglobulin, the present invention can effectively mask the allergen binding epitope on the surface of β-lactoglobulin. The invention proposes the use of a β-lactoglobulin-specific aptamer to alleviate cow's milk allergic reactions caused by β-lactoglobulin. Cell experiments and animal experiments have verified that the cyclic aptamer can effectively inhibit allergic reactions caused by β-lactoglobulin, and has good application prospects in alleviating cow's milk allergic reactions.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and relates to the application of a beta-lactoglobulin-specific aptamer, in particular to the application of the aptamer in alleviating cow's milk allergic reactions. Background Art

[0002] Cow's milk allergy (CMA) is an immune response to proteins in cow's milk. In recent years, with the rapid development of the global economy and changing lifestyles, the prevalence of breastfeeding has gradually declined. Cow's milk, with its similar nutritional composition to breast milk, has become an important source of nutrition for infants and young children, considered the best supplement or substitute for breast milk. Cow's milk-based infant formula has gradually become a staple of infants' daily diet. However, as one of the eight major allergenic foods identified by the WHO / FAO, cow's milk allergy should not be ignored. Currently, cow's milk allergy has become the most common food allergy in infants and young children, seriously impacting their health. Epidemiological surveys show that the global incidence of CMA in infants and young children is 1.9% to 4.9%, but the incidence in infants and young children aged one year and under is 1.8% to 7.5%. Surveys indicate that the allergen in approximately 82% of cow's milk allergies is β-lactoglobulin, making it considered the primary allergenic protein in cow's milk.

[0003] Milk allergy, a specific immune system disorder, can recur after exposure to foods containing milk allergens. This condition involves multiple factors, including individual genetics and environmental conditions. While antihistamines and corticosteroids can be used to prevent and treat allergic reactions, a complete cure is still not possible. To date, the most effective measure for food allergy sufferers is strict avoidance of the corresponding allergen, severely impacting the health and quality of life of those with milk allergy. Therefore, the development of a novel treatment strategy for milk allergy is an urgent need. Summary of the Invention

[0004] The present invention aims to solve the above problems and provides an application of a β-lactoglobulin-specific aptamer for alleviating cow's milk allergic reactions.

[0005] The first aspect of the present invention provides the use of a β-lactoglobulin-specific aptamer in the preparation of a drug for alleviating a cow's milk allergic reaction. The β-lactoglobulin-specific aptamer is a cyclic covalent aptamer (cyclic aptamer) of β-lactoglobulin, and the cow's milk allergic reaction is a cow's milk allergic reaction caused by β-lactoglobulin.

[0006] Furthermore, the cyclic covalent aptamer for β-lactoglobulin is formed by connecting two aptamer monomers carrying complementary base sequences through base pairing. The core portion of the aptamer monomer is a β-lactoglobulin binding unit, and the sequence of the β-lactoglobulin binding unit is shown in SEQ ID NO: 1.

[0007] Specifically, the cyclic covalent aptamer of β-lactoglobulin comprises two β-lactoglobulin binding units and is prepared by cyclization design of aptamer #T4M8 (SEQ ID NO: 1) and enzymatic reaction.

[0008] Furthermore, the drug may be an inhibitor.

[0009] Furthermore, the cyclic covalent aptamer of β-lactoglobulin is used to mask the antibody IgE binding epitope on the surface of β-lactoglobulin to inhibit antibody IgE binding, thereby inhibiting the sensitization of β-lactoglobulin.

[0010] Specifically, the binding targets of the cyclic covalent aptamer of β-lactoglobulin and β-lactoglobulin are TYR-20\GLN-35\ARG-40\ASN-152\THR-154, and the IgE antibody binding epitopes on the surface of β-lactoglobulin mainly include three regions (epitope I: AA41-60\epitope II: AA102-124\epitope III: AA149-162). Therefore, the cyclic covalent aptamer can effectively mask the allergen binding epitopes on the surface of β-lactoglobulin.

[0011] Furthermore, the cyclic covalent aptamer of β-lactoglobulin is used to inhibit the degranulation of basophilic leukemia cell line (RBL-2H3) induced by β-lactoglobulin, which can be specifically manifested as reducing the levels of histamine and β-hexosaminidase.

[0012] Furthermore, the cyclic covalent aptamer of β-lactoglobulin is used to inhibit the sensitization response of RBL-2H3 cells induced by β-lactoglobulin, which can be specifically manifested by reducing the level of cytokines mainly including interleukin-4 (IL-4).

[0013] Furthermore, the cyclic covalent aptamer of β-lactoglobulin is used to inhibit β-lactoglobulin-induced allergic reactions in BALB / c mice, which can be specifically manifested by reducing the levels of β-lactoglobulin-specific antibodies, mMCPT-1 protein and IL-4 in mouse serum.

[0014] Furthermore, the sequences of the two aptamer monomers carrying complementary base sequences are shown as SEQ ID NO: 2 and SEQ ID NO: 3, or SEQ ID NO: 4 and SEQ ID NO: 5, or SEQ ID NO: 6 and SEQ ID NO: 7.

[0015] Furthermore, the aptamer monomer is modified with a labeling molecule and / or a chemical functional group.

[0016] Furthermore, the labeling molecules and / or chemical functional groups are selected from one or more of fluorescent groups, isotopes, electrochemical labels, enzyme labels, affinity ligands and thiol groups.

[0017] The second aspect of the present invention provides a drug for allergic reaction to cow's milk, wherein the cow's milk allergic reaction is a cow's milk allergic reaction caused by β-lactoglobulin;

[0018] The drug comprises a β-lactoglobulin-specific aptamer, which is formed by connecting two aptamer monomers carrying complementary base sequences through base pairing. The core portion of the aptamer monomer is a β-lactoglobulin binding unit, and the sequence of the β-lactoglobulin binding unit is shown in SEQ ID NO: 1.

[0019] Furthermore, the sequences of the two aptamer monomers carrying complementary base sequences are shown as SEQ ID NO: 2 and SEQ ID NO: 3, or SEQ ID NO: 4 and SEQ ID NO: 5, or SEQ ID NO: 6 and SEQ ID NO: 7.

[0020] Furthermore, the aptamer monomer is modified with a labeling molecule and / or a chemical functional group, and the labeling molecule and / or chemical functional group is selected from one or more of a fluorescent group, an isotope, an electrochemical marker, an enzyme marker, an affinity ligand and a thiol group.

[0021] Furthermore, the medicine also includes additives.

[0022] Furthermore, the additive is selected from one or more of antioxidants, preservatives, solubilizers, disintegrants, lubricants, colorants, dispersants and surfactants.

[0023] Furthermore, the drug is administered by intravenous injection or intramuscular injection.

[0024] Furthermore, the dosage of the drug is: 2-15 nmol β-lactoglobulin-specific aptamer per 100 mg β-lactoglobulin.

[0025] The technical solution of the present invention has the following advantages over the prior art:

[0026] The present invention is based on the co-localization phenomenon of the binding epitope of the β-lactoglobulin cyclic aptamer on the surface of β-lactoglobulin and the binding epitope of the antibody IgE on the surface of β-lactoglobulin, which can effectively mask the allergen binding epitope on the surface of β-lactoglobulin. It proposes the use of β-lactoglobulin-specific aptamers to alleviate cow's milk allergic reactions;

[0027] Cell experiments and animal experiments have verified that this circular aptamer can effectively inhibit allergic reactions caused by β-lactoglobulin and has good application prospects in alleviating cow's milk allergic reactions. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Schematic diagram of the secondary structure of the two constituent monomers of the cyclic aptamer Cb-#T4M8.

[0029] Figure 2 This is the binding mode of the circular aptamer Cb-#T4M8 to β-lactoglobulin and the target of the aptamer.

[0030] Figure 3 The IC of the cyclic aptamer Cb-#T4M8 inhibiting IgE and β-lactoglobulin was determined by indirect competitive enzyme-linked immunosorbent assay. 50 curve.

[0031] Figure 4 The histamine release levels of RBL-2H3 cells treated with β-lactoglobulin and β-lactoglobulin\Cb-#T4M8 complex.

[0032] Figure 5 The release level of β-hexosaminidase in RBL-2H3 cells treated with β-lactoglobulin and β-lactoglobulin\Cb-#T4M8 complex.

[0033] Figure 6 The IL-4 release levels of RBL-2H3 cells treated with β-lactoglobulin and β-lactoglobulin\Cb-#T4M8 complex.

[0034] Figure 7 To score the allergic symptoms in the β-lactoglobulin-induced BALB / c mouse allergy model.

[0035] Figure 8 is the level of β-lactoglobulin-specific antibodies in the serum of BABL / c mice.

[0036] Figure 9 is the concentration of mMCPT-1 protein in the serum of BALB / c mice induced by β-lactoglobulin.

[0037] Figure 10 is the concentration of IL-4 in the serum of BALB / c mice induced by β-lactoglobulin. DETAILED DESCRIPTION

[0038] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0039] Example 1 Preparation of cyclic aptamer Cb-#T4M8

[0040] 1.1 Synthesis of aptamer monomers

[0041] The aptamer monomers were synthesized by Shanghai Sangon Biotechnology Service Co., Ltd. and the sequences of 5' end biotin label were specifically 5' phosphorylated Cb-#T4M8-1 (SEQ ID NO: 2) and Cb-#T4M8-2 (SEQ ID NO: 3). Figure 1 As shown, Cb-#T4M8-1 and Cb-#T4M8-2 were obtained by adding a "lock chain" type toe structure to the tail end of aptamer #T4M8 (SEQ ID NO: 1). SEQ ID NO: 4 and SEQ ID NO: 5, as well as SEQ ID NO: 6 and SEQ ID NO: 7 were also obtained by circularization design based on aptamer #T4M8. The specific sequences are shown in Table 1.

[0042] Table 1 Aptamer sequences

[0043]

[0044] 1.2. Preparation of cyclic aptamer Cb-#T4M8

[0045] Cb-#T4M8-1 and Cb-#T4M8-2 were first dissolved in T4 DNA Ligase buffer at a concentration of 3 μM, then heated at 95°C for 5 minutes and rapidly cooled to 16°C to form circular aptamers with two gaps. The gapped aptamers were then incubated with T4 DNA ligase at 16°C for 12 hours to form the circular aptamer Cb-#T4M8. Finally, the solution was incubated at 75°C for 10 minutes to denature the ligase, and the DNA sample extracted by phenol-chloroform extraction and ethanol precipitation yielded the circular aptamer Cb-#T4M8.

[0046] Example 2 Analysis of the Target Sites of the Circular Aptamer Cb-#T4M8 and β-lactoglobulin

[0047] 2.1 Preparation of aptamer three-dimensional conformation

[0048] The Vienna format of the aptamer Cb-#T4M8 sequence was generated by the Mfold online tool, and then the optimal three-dimensional conformation of the sequence corresponding to the Vienna format was generated using RNAcomposer software.

[0049] 2.2 Simulation docking

[0050] The 3D conformation of β-lactoglobulin (PDB: 1GX9) was downloaded from the PDB website and pre-processed using MGLTools, including removing water molecules, adding nonpolar hydrogens, and adding Kollman charges. Finally, a .pdbqt file was generated. The 3D conformation of the aptamer Cb-#T4M8 was imported as a ligand, and charges and nonpolar hydrogens were added as described above. Autodock vina was used to simulate docking and search for the optimal binding site between the aptamer and β-lactoglobulin. Pymol was then used to analyze the target site, such as Figure 2 As shown in the figure, the binding targets of aptamer Cb-#T4M8 are TYR-20\GLN-35\ARG-40\ASN-152\THR-154, while the IgE antibody binding epitopes on the surface of β-lactoglobulin mainly include three regions (epitope I: AA41-60\epitope II: AA102-124\epitope III: AA149-162). Based on the coverage of the aptamer on the surface of β-lactoglobulin, it can be judged that the aptamer Cb-#T4M8 can effectively mask the antibody IgE binding epitopes on the surface of β-lactoglobulin.

[0051] Example 3: Analysis of the Allergenicity of the Circular Aptamer Cb-#T4M8\β-Lactoglobulin Complex

[0052] The effect of the binding of aptamer Cb-#T4M8 to β-lactoglobulin on β-lactoglobulin sensitization was evaluated by indirect competitive enzyme-linked immunosorbent assay. First, 200 μL of β-lactoglobulin solution (10 μg / mL) was added to the microplate and coated overnight at 4°C; the plate was washed, and 300 μL of protein-free blocking solution was added to block for 2 hours at 37°C and 150 rpm; different concentrations of Cb-#T4M8\β-lactoglobulin complex were mixed with equal volumes of serum from patients with milk allergy and incubated for 1 hour, then added to the enzyme-labeled plate and incubated for 1 hour, and finally HRP-labeled goat anti-human IgE was added and incubated for 1 hour, the plate was washed, 100 μL of TMB-H2O2 solution was added to each well, color was developed for 15 minutes, and then 50 μL of 2M sulfuric acid was added to stop the color development, and the absorbance at 450 nm was measured, and the IC was plotted based on this. 50 Inhibition curve, such as Figure 3 As shown, the allergenicity of Cb-#T4M8\β-lactoglobulin complex was greatly reduced, IC 50 The value was 9.84 μg / mL, while the IC50 The value was 1.035 μg / mL, indicating that the aptamer Cb-#T4M8 greatly reduced the allergenicity of β-lactoglobulin.

[0053] Example 4: Circular aptamer Cb-#T4M8 inhibits β-lactoglobulin-induced degranulation in RBL-2H3 cells

[0054] Cell degranulation is a typical manifestation of allergic reactions. Therefore, the effect of the circular aptamer Cb-#T4M8 on β-lactoglobulin-induced RBL-2H3 cell degranulation was evaluated. First, 2×10 6 RBL-2H3 cells were cultured with 60 μM β-lactoglobulin or β-lactoglobulin\aptamer complex, and cultured in a cell culture incubator for 12 h. The cell culture supernatant was then collected and the level of cell degranulation marker (histamine) in the supernatant was measured. Figure 4 As shown in Figure 2, the histamine level in the β-lactoglobulin\aptamer complex-treated group was significantly lower than that in the β-lactoglobulin-treated group. Figure 5 As shown, the levels of β-hexosaminidase, a marker of cell degranulation, in the cell culture supernatant showed that the β-lactoglobulin\aptamer complex (β-lactoglobulin\Cb-#T4M8)-treated group released significantly less β-hexosaminidase than the β-lactoglobulin-treated group. This suggests that the binding of the aptamer Cb-#T4M8 significantly reduced the allergenicity of β-lactoglobulin.

[0055] Example 5 Cytokine Analysis of the Inhibition of β-lactoglobulin-Induced Sensitization Response of RBL-2H3 Cells by Circular Aptamer Cb-#T4M8

[0056] When RBL-2H3 cells undergo sensitization response, they release a series of cytokines, especially IL-4 (interleukin-4). First, 2×10 6 RBL-2H3 cells were cultured with 60 μM β-lactoglobulin or β-lactoglobulin\aptamer complex, and cultured in a cell culture incubator for 12 h. The cell culture supernatant was then collected and the IL-4 level in the supernatant was determined. Figure 6 As shown in the figure, the IL-4 level in the β-lactoglobulin\aptamer complex-treated group was significantly lower than that in the β-lactoglobulin-treated group, indicating that the circular aptamer Cb-#T4M8 can effectively inhibit the allergenicity of β-lactoglobulin.

[0057] Example 6: Cyclic aptamer Cb-#T4M8 inhibits β-lactoglobulin-induced allergic reactions in BALB / c mice

[0058] In order to further evaluate the inhibitory effect of the cyclic aptamer Cb-#T4M8 on β-lactoglobulin sensitization, in vivo experiments were conducted on mice to establish a β-lactoglobulin-induced mouse milk allergy model. First, 3-4 week-old BALB / c mice were adaptively raised in the animal room for about a week. Then, each mouse was gavaged with 10 mg of β-lactoglobulin and 10 μg of cholera toxin for 5 consecutive times, one week apart, to put the mice in a sensitized state. After one week, 50 mg of β-lactoglobulin was continuously gavaged every 3 days to stimulate allergic reactions in the mice. 1 hour before each stimulation, 3 nmol of the cyclic aptamer Cb-#T4M8 was injected into the tail vein of each mouse. After stimulating the mice to be allergic, each mouse was scored and evaluated, and the β-lactoglobulin-specific antibodies in the mouse serum were measured. The results are shown as follows. Figure 7 、 Figure 8 As shown, the mice injected with the aptamer through the tail vein had lower allergy symptom scores, and the level of β-lactoglobulin-specific antibody IgE in the serum was significantly lower than that in the positive control group. These results indicate that the β-lactoglobulin-specific aptamer Cb-#T4M8 can effectively bind to β-lactoglobulin, effectively inhibiting the binding of antibody IgE to the target, thereby inhibiting the bovine milk allergy reaction induced by β-lactoglobulin. In addition, the mMCPT-1 protein, an important marker for allergic reactions, was measured to have a significantly lower level of mMCPT-1 protein in the serum of mice injected with the aptamer through the tail vein than that of mice in the positive control group ( Figure 9 In addition, compared with the positive control group, the IL-4 level in the serum of mice injected with the aptamer via the tail vein decreased significantly ( Figure 10 ), indicating that the cyclic aptamer Cb-#T4M8 can also effectively bind to β-lactoglobulin in mice, thereby effectively inhibiting β-lactoglobulin-induced allergic reactions in vivo.

[0059] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. Use of a β-lactoglobulin-specific aptamer in the preparation of a drug for alleviating bovine milk allergic reaction, wherein the β-lactoglobulin-specific aptamer is a cyclic covalent aptamer of β-lactoglobulin, and the bovine milk allergic reaction is a bovine milk allergic reaction caused by β-lactoglobulin; the cyclic covalent aptamer of β-lactoglobulin is formed by connecting two aptamer monomers carrying complementary base sequences through base pairing, and the sequences of the two aptamer monomers carrying complementary base sequences are shown in SEQ ID NO: 2 and SEQ ID NO:

3.

2. The use according to claim 1, characterized in that The aptamer monomer is modified with a labeling molecule and / or a chemical functional group.

3. The use according to claim 2, characterized in that The labeling molecules and / or chemical functional groups are selected from one or more of fluorescent groups, isotopes, electrochemical labels, enzyme labels, affinity ligands and sulfhydryl groups.

4. A drug for allergic reaction to cow's milk, characterized in that: The cow's milk allergic reaction is a cow's milk allergic reaction caused by β-lactoglobulin; The drug comprises a β-lactoglobulin-specific aptamer, which is formed by connecting two aptamer monomers carrying complementary base sequences through base pairing. The sequences of the two aptamer monomers carrying complementary base sequences are shown in SEQ ID NO: 2 and SEQ ID NO:

3.

5. The drug for allergic reaction to cow's milk according to claim 4, characterized in that: The aptamer monomer is modified with a labeling molecule and / or a chemical functional group, and the labeling molecule and / or chemical functional group is selected from one or more of a fluorescent group, an isotope, an electrochemical label, an enzyme label, an affinity ligand and a thiol group.

6. The drug for allergic reaction to cow's milk according to claim 4, characterized in that: Also includes additives.

7. The drug for allergic reaction to cow's milk according to claim 6, characterized in that: The additive is selected from one or more of an antioxidant, a preservative, a solubilizer, a disintegrant, a lubricant, a colorant, a dispersant and a surfactant.

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