WO2021159650A1 - 一种特异性识别β-乳球蛋白的核酸适配体及其应用 - Google Patents

一种特异性识别β-乳球蛋白的核酸适配体及其应用 Download PDF

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WO2021159650A1
WO2021159650A1 PCT/CN2020/097954 CN2020097954W WO2021159650A1 WO 2021159650 A1 WO2021159650 A1 WO 2021159650A1 CN 2020097954 W CN2020097954 W CN 2020097954W WO 2021159650 A1 WO2021159650 A1 WO 2021159650A1
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lactoglobulin
nucleic acid
acid aptamer
aptamer
sequence
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French (fr)
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段诺
吴世嘉
齐硕
王周平
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江南大学
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/16—Aptamers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4713—Plasma globulins, lactoglobulin

Definitions

  • the invention belongs to the field of biology and medicine, and particularly relates to a nucleic acid aptamer that specifically recognizes ⁇ -lactoglobulin and its application.
  • Food allergy is an adverse reaction of some people to certain foods (including IgE-mediated and non-IgE-mediated immune reactions). Food allergic reactions will not only seriously affect the quality of life of patients, but also cause malnutrition and health problems such as asthma, gastrointestinal and skin inflammation. In severe cases, anaphylactic shock can be life-threatening.
  • Cow milk and dairy products have gradually become an important part of human healthy diet because they contain essential amino acids and rich minerals. But at the same time as one of the eight categories of allergic foods designated by the World Health Organization (WHO) and the United Nations Food and Agriculture Organization (FAO), because cow milk and dairy products contain a variety of allergenic proteins ( ⁇ s1-casein, ⁇ -milk white) Protein and ⁇ -lactoglobulin, etc.) seriously affect the health of some people.
  • WHO World Health Organization
  • FEO Food and Agriculture Organization
  • Epidemiological surveys have shown that up to 8% of infants and 1%-2% of adults are allergic to cow’s milk. The immune function is getting lower and lower, and the incidence of milk allergy in infants and young children is increasing year by year.
  • ⁇ -lactoglobulin accounts for about 10% of milk protein content and about 50% of total whey protein. Human milk does not contain ⁇ -lactoglobulin, and about 82% of patients with IgE-mediated milk allergy The patient is allergic to ⁇ -lactoglobulin, so ⁇ -lactoglobulin is considered to be the main allergen component in milk. Highly sensitive detection of the allergen ⁇ -lactoglobulin in milk and dairy products is of great significance to provide necessary dietary guidance for allergic groups.
  • Nucleic acid aptamer is a new type of single-stranded oligonucleotide that can specifically bind to the target substance by screening artificially synthesized random nucleotide libraries through the exponential enrichment of ligand evolution technology (SELEX).
  • "Antibody” aptamer can form a specific three-dimensional conformation (hairpin, pseudoknot, convex ring and G-quadruplex, etc.), through intermolecular interactions such as spatial structure matching, hydrogen bonding, van der Waals force and electrostatic interaction It can bind to the target substance with high affinity.
  • the English literature of Mohammed Zourob et al. provided a nucleic acid aptamer that binds to ⁇ -lactoglobulin and used it to construct an electrochemical detection scheme.
  • the document uses a target immobilization method to immobilize ⁇ -lactoglobulin on agarose microspheres to screen for nucleic acid aptamers enriched with ⁇ -lactoglobulin.
  • the detection part adsorbs the nucleic acid aptamer on the graphene oxide.
  • the nucleic acid aptamer specifically binds to the graphene oxide and falls off from the graphene oxide, causing changes in electrochemical signals.
  • the disadvantage of this scheme is that the aptamer Kd value is large and the specificity of the aptamer has not been studied. Therefore, there are too many interference factors in the actual sample detection, and the detection limit is high. In addition, the aptamer and ⁇ -lactoglobulin The mechanism of binding is unclear.
  • the purpose of the present invention is to provide a nucleic acid aptamer that specifically recognizes ⁇ -lactoglobulin and its application.
  • the nucleic acid aptamer can specifically bind to the allergen ⁇ -lactoglobulin in milk and dairy products, and provides a new type of recognition molecule for the high-sensitivity and low-cost detection of allergen ⁇ -lactoglobulin, thereby solving the current allergen ⁇ -In the process of lactoglobulin detection, the high cost of relying on chromatographic equipment, large application limitations, and the defects of relying on antibody stability and poor repeatability, and for the detection of allergen ⁇ -lactoglobulin in milk and dairy products
  • the development of the program provides a new tool.
  • the first objective of the present invention is to provide a nucleic acid aptamer that specifically recognizes ⁇ -lactoglobulin
  • the sequence of the nucleic acid aptamer is the sequence shown in SEQ ID NO. 1; or the sequence shown in SEQ ID NO.
  • the sequence shown in 1 has more than 60% homology and can specifically recognize the sequence of ⁇ -lactoglobulin; or the sequence derived from the sequence shown in SEQ ID NO. 1 can specifically recognize the sequence of ⁇ -lactoglobulin.
  • the nucleotide sequence of the nucleic acid aptamer is modified; the modification method includes any one of phosphorylation, methylation, amination, sulfhydrylation, phosphorothioate modification, and sugar ring side chain modification. kind or more.
  • nucleotide sequence of the nucleic acid aptamer is connected to any one of a fluorescent label, a radioactive substance, a therapeutic substance, biotin, digoxigenin, a nano luminescent material, a small peptide, an siRNA, or an enzyme label. kind or more.
  • SEQ The sequence shown in ID NO.1 has intermolecular interactions with ⁇ -lactoglobulin, among which C-45/T-46 and ARG-40, G-56 and SER-36, C-57 and ASN-63, G-58 and ASN-63/TRP-61, G-76 and SER-30 form hydrogen bonds.
  • the second objective of the present invention is to provide a kit including the above-mentioned nucleic acid aptamer.
  • the third objective of the present invention is to propose the application of the aforementioned nucleic acid aptamer in detecting ⁇ -lactoglobulin, and the method for drawing the standard curve for detecting ⁇ -lactoglobulin of the nucleic acid aptamer is as follows:
  • nucleic acid aptamer and its complementary short chain are mixed uniformly in a 1:1 ratio of the amount of substance, and then denatured;
  • beacon issuing molecules are dissolved in the binding buffer for denaturation
  • Enzyme digestion cycle amplification mix the recognition probe, beacon hairpin molecule and exonuclease III and incubate them in different concentrations of ⁇ -lactoglobulin standard solution for 2 to 3 hours; use a fluorescence spectrometer Fluorescence measurement is performed at an excitation wavelength of 575nm and an emission wavelength of 606nm;
  • the beacon hairpin molecule is denatured at 95°C for 10 minutes, and then the temperature is gradually lowered to 37°C for 3 hours.
  • the incubation time is 2 h
  • the incubation system is 400 ⁇ L
  • the concentration of the beacon hairpin molecule is 400 nM
  • the enzyme activity of the exonuclease III is 0.25 U/ ⁇ L.
  • the molar ratio of the identification probe to the beacon issuing molecule is 1:2.
  • the present invention screens ⁇ -lactoglobulin aptamers through the library immobilization method, which effectively overcomes the steric hindrance effect of traditional methods and The defect of conformational change caused by target fixation.
  • the present invention provides a nucleic acid aptamer that specifically recognizes the allergen ⁇ -lactoglobulin in milk and dairy products. It has been verified that the aptamer can bind to the allergen ⁇ with a dissociation constant below 100 nM. -Lactoglobulin (as shown in Table 1), and can distinguish between proteins ( ⁇ -lactalbumin, casein) and structural analogs (IgG, bovine serum albumin), with good affinity and specificity.
  • the 5'end or the 3'end of the nucleic acid aptamer can be labeled with FAM, FITC and biotin, which can realize the rapid qualitative and quantitative detection of allergen ⁇ -lactoglobulin, with low cost and high specificity And the advantages of wide application range. It can be said that the nucleic acid aptamer that specifically recognizes the allergen ⁇ -lactoglobulin in milk and dairy products proposed by the present invention is very useful as an allergen recognition molecule in the fields of food processing, food management, and nutrition planning. A tool.
  • the nucleic acid aptamer contains a typical stem-loop structure in the secondary structure of the nucleotide sequence, which indicates that the aptamer has good stability.
  • the binding of aptamer and target depends on the secondary structure of the aptamer, and the stem-loop structure is one of them.
  • the formation of the stem-loop structure is the structural basis for the affinity of aptamers.
  • the present invention constructs the three-dimensional conformation of the nucleic acid aptamer, simulates the interaction between the nucleic acid aptamer and the allergen ⁇ -lactoglobulin through molecular docking technology, and explores the binding mode of the nucleic acid aptamer and ⁇ -lactoglobulin It explains the inherent reason for the high affinity of the allergen ⁇ -lactoglobulin aptamer, and can provide better theoretical guidance for the subsequent optimization of the allergen ⁇ -lactoglobulin aptamer.
  • Figure 1 is a graph showing the change of fluorescence enrichment rate during the screening of nucleic acid aptamers in Example 2.
  • Figure 2 is the calorimetric curve of the nucleic acid aptamer Lg-18 in Example 3.
  • Figure 3 is the binding saturation curve of the nucleic acid aptamer Lg-18 in Example 3 under the fluorescence method.
  • Fig. 4 is a diagram showing the specific binding of the nucleic acid aptamer Lg-18 in Example 3 to the target.
  • Fig. 5 is a standard curve of the biosensor constructed by the nucleic acid aptamer Lg-18 in Example 4 for the detection of allergen ⁇ -lactoglobulin.
  • FIG. 6 is a schematic diagram of the secondary structure prediction of the nucleic acid aptamer Lg-18 in Example 5.
  • Fig. 7 is a schematic diagram of the docking of nucleic acid aptamer Lg-18 and ⁇ -lactoglobulin molecule in Example 6.
  • Fig. 8 is a map of the binding site of part A in Fig. 7.
  • a single-stranded oligonucleotide library with a length of 80 nt and a library capacity of up to 1012-1024 was constructed, consisting of a random sequence region of 40 nt in the middle (determining the richness of the library) and a fixed sequence region of 20 nt at both ends (necessary for primer binding) Sequence) composition.
  • the sequence is: 5’-AGCAGCACAGAGGTCAGATG-40 random
  • the base -CCTATGCGTGCTACCGTGAA-3’ was synthesized by Dalian Bao Biological Co., Ltd.
  • Biotinylated complementary short chain 5’- Biotin- AGCACGCATAGG-3’
  • the primers were synthesized by Shenggong Bioengineering (Shanghai) Co., Ltd.
  • the single-stranded oligonucleotide library and primers were prepared with TE buffer into a 100 ⁇ M stock solution and stored at -20°C for later use.
  • the single-stranded oligonucleotide library is complemented with the biotinylated complementary short chain, and then it is immobilized on the streptavidin-ylated magnetic beads and incubated with the allergen ⁇ -lactoglobulin, because the single-stranded Nucleic acid interacts with ⁇ -lactoglobulin and falls off from the magnetic beads, and is released into the solution.
  • the supernatant is used as a template for the secondary library for PCR amplification, and then the single strand is prepared by enzyme digestion and alcohol precipitation.
  • the secondary library is used for the next round of screening. Specific steps are as follows:
  • the single-stranded oligonucleotide library is complementary to the biotinylated complementary strand: the ssDNA library and the biotinylated complementary short strand are mixed in a ratio of 1:1.5 (the amount of the single-stranded oligonucleotide library used in the first round of screening is 1nmol , The amount of material in the secondary library used in the remaining rounds is 100pmol), denatured at 95°C for 10min, and then slowly cooled to 37°C for 3h until the two are fully complementary.
  • Fixation of single-stranded oligonucleotide library react the above complementary mixture with a certain amount of streptavidin magnetic beads (the mass ratio of nucleic acid library to magnetic beads is 1:400) at 37°C, 200rpm for 2h, and use The strong binding effect of biotin and streptavidin fixes the single-stranded oligonucleotide library on the magnetic beads.
  • Target incubation Incubate the above-mentioned immobilized single-stranded nucleotide library with 50 ⁇ g/mL ⁇ -lactoglobulin in a 300 ⁇ L system at 37°C and 200rpm, and calculate the relative fluorescence enrichment rate of each screening cycle. According to the results of fluorescence monitoring during the screening process, in the sixth and tenth rounds, an anti-screening process is added. Before incubating with the target, coexisting substances ( ⁇ -lactalbumin, casein) and structural analogs (BSA, IgG) are introduced into the system ) To increase the pressure of screening and obtain better affinity and specific sequences.
  • coexisting substances ⁇ -lactalbumin, casein
  • BSA structural analogs
  • PCR amplification and verification After magnetic separation, the supernatant after the target incubation is obtained, which is used as a template for PCR amplification.
  • Amplification system is 1 ⁇ L template, 0.5 ⁇ L Taq DNA polymerase (5U/ ⁇ L), 5 ⁇ L 10X polymerase buffer (containing 20mM MgCl2), 0.5 ⁇ L forward primer and reverse primer, 1 ⁇ L dNTP Mix (5mM), 42 ⁇ L ddH2O.
  • the amplification program was 95°C pre-denaturation for 5 minutes, 95°C denaturation for 30 seconds, 58°C annealing for 30 seconds, 72°C extension for 30 seconds, 10 cycles, and finally 72°C extension for 5 minutes, and 4°C storage.
  • After 8% non-denaturing polyacrylamide gel electrophoresis observe whether the electrophoresis band is single and bright, and whether the band is at the 80bp position to ensure the success of the PCR amplification template.
  • Preparation of secondary library single-stranded oligonucleotide After the amplified product is purified, the antisense strand of the phosphorylated PCR product is specifically digested with lambda exonuclease to prepare single-stranded.
  • the digestion conditions are 350 ⁇ L PCR amplification product, 35 ⁇ L exonuclease buffer, 2 ⁇ L lambda exonuclease, reaction at 37°C, and the digestion process is judged by electrophoresis on 8% denaturing polyacrylamide gel (containing 7M urea). After the reaction is over, The reaction was terminated by inactivating the enzyme at 75°C for 10 min. After phenol/chloroform purification and alcohol precipitation, single-stranded oligonucleotides of the secondary library were obtained.
  • PEAQ-ITC (British Malvern Instruments Co., Ltd.) was used to analyze the associativity of 10 candidate sequences.
  • the sample at the top reacts with the sample in the sample cell and affects the final result), then 18 drops of injection are carried out, each drop is 2 ⁇ L, the interval is 150s, and the rotation speed of the injection needle is 750rpm to ensure that each injection is fully reacted.
  • the heat of dilution caused by the buffer titration into the ⁇ -lactoglobulin solution is deducted.
  • the binding force and thermodynamic parameters in the binding process can be fitted in the PEAQ-ITC analysis software.
  • the buffer used in the isothermal titration calorimetry (ITC) analysis is 10 mM Tris-HCl, pH 7.4.
  • aptamer (Lg-18) has the best binding ability to ⁇ -lactoglobulin.
  • the sequence of the aptamer (Lg-18) is shown below.
  • the ITC analysis result of the aptamer (Lg-18) is shown in Figure 2. It can be seen from Figure 2 that the nucleic acid aptamer Lg-18 has a good binding ability to ⁇ -lactoglobulin.
  • the abscissa is time and the ordinate is thermal power.
  • the peak area between the peak base and the peak tip is the total heat released during each titration.
  • the abscissa is the molar ratio of the titrant to the sample solution, and the ordinate is the total heat generated by the titration.
  • homologous sequences of aptamer Lg-18 (Lg-9, Lg-16) and ⁇ -lactoglobulin also have good binding properties.
  • Lg-9 and Lg-16 are as follows:
  • the aptamer used is the 5'end of the sequence shown in Lg-18, Lg-9, and Lg-16 modified with FAM fluorescent groups or other fluorescent molecules.
  • Recognition probe construction (Lg-18): The nucleic acid aptamer and its complementary short chain are mixed uniformly in a 1:1 ratio of substance, denatured at 95°C for 10 minutes, and then slowly cooled to 37°C for 3 hours until the two are fully complementary.
  • the beacon hairpin molecule was dissolved in the binding buffer (100mM NaCl, pH7.4) at 95°C for 10min, and then the temperature was gradually reduced to 37°C for 3h to ensure that the hairpin structure was fully formed.
  • Enzyme digestion cycle amplification Recognition probe (200nM), beacon issuing molecule (400nM) and exonuclease III (0.25U/ ⁇ L) are mixed and mixed at different concentrations (20 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, 1000 ng/mL, 2000 ng/mL, 5000 ng/mL) ⁇ -lactoglobulin standard solutions were incubated for 2h, and the incubation system was 400 ⁇ L.
  • the binding effect of aptamer and ⁇ -lactoglobulin is stronger than the interaction between the aptamer and the complementary short chain, and the complementary short chain falls off from the aptamer , And then complementary to the 3'end of the beacon hairpin molecule to form a double-stranded region, the beacon hairpin molecule is turned on, and the fluorescence is restored.
  • the exonuclease III in the system acts on the double-stranded region along 3'-5 'Digest the single-stranded oligonucleotide, the complementary short chain can be released for circulation, and the fluorescence signal is further enhanced.
  • F7000 fluorescence spectrometer was used for fluorescence measurement at 575nm excitation wavelength and 606nm emission wavelength.
  • the aptamer sequence was introduced into Mfold, and the secondary structure formation conditions were set (Na+: 100mM; Mg2+: 1mM; 37°C).
  • the formed secondary structure is shown in Figure 6, and the secondary structure contains typical Stem-loop structure.
  • Simulation docking Download the three-dimensional conformation of ⁇ -lactoglobulin (PDB: 2Q2M) from the PDB website, and use MGL Tools to perform a series of pre-treatments, including removing water molecules, adding non-polar hydrogen, and charging Kollman Charges. Finally generate the .pdbqt file; import the three-dimensional conformation of the aptamer Lg-18 as the ligand, add charge to it as above, and add non-polar hydrogen; use Autodock vina performs simulated docking to search for the best binding position between the aptamer and ⁇ -lactoglobulin.
  • the docking parameters are as follows:

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Abstract

提供一种特异性识别β-乳球蛋白的核酸适配体，所述核酸适配体的序列为SEQ ID NO.1所示的序列；或与SEQ ID NO.1所示的序列具有60％以上同源性并且能够特异性识别β-乳球蛋白的序列；或由SEQ ID NO.1所示的序列衍生的能够特异性识别β-乳球蛋白的序列；该核酸适配体能特异性结合牛乳及乳制品中过敏原β-乳球蛋白。

Description

一种特异性识别β-乳球蛋白的核酸适配体及其应用 技术领域
本发明属于生物和医药领域，尤其涉及一种特异性识别β-乳球蛋白的核酸适配体及其应用。
 背景技术
食物过敏是部分人群对某些食物产生的不良反应（包括IgE介导和非IgE介导的两大类免疫反应）。食物过敏反应不仅会严重影响患者的生活质量，还能引起患者营养不良而出现健康性问题，如哮喘、胃肠道和皮肤炎症等，严重者会引起过敏性休克危及生命。
牛乳及乳制品因含有人体必需的氨基酸、丰富的矿物质，逐渐成为人类健康饮食的重要组成部分。但同时作为世界卫生组织（WHO）和联合国粮食及农业组织（FAO）划定的八大类易过敏食物之一，因牛乳及乳制品中含有多种致敏蛋白（αs1-酪蛋白、α-乳白蛋白和β-乳球蛋白等）严重影响着的部分人群的身体健康。流行病学调查显示，高达8%的婴幼儿和1%-2%的成人对牛乳过敏，近年来由于生活方式的改变、微生物的暴露、饮食习惯的改变等多种因素，婴幼儿的肠道免疫功能越来越低，婴幼儿牛乳过敏发生率正呈逐年上升的趋势。
β-乳球蛋白约占牛乳蛋白含量的10%，约占乳清蛋白总量的50%，人乳中不含有β-乳球蛋白，且IgE介导的牛乳过敏病人中，约82%的患者对β-乳球蛋白过敏，因此β-乳球蛋白被认为是牛乳中主要的过敏原成分。对牛乳及乳制品中过敏原β-乳球蛋白的高灵敏度检测，为易过敏群体提供必要的饮食指导具有重要的意义。
目前食物中牛乳过敏原分析检测常用的方法：（1）基于色谱分析的高效液相色谱方法（HPLC），但是仪器费用昂贵，使用场景局限性大，成本较高；（2）基于识别特征基因的荧光定PCR (qPCR)、环介导等温扩增（LAMP），然而却存在特异性差、操作复杂的问题；（3）基于抗体特异性识别的酶联免疫方法（ELISA），然而抗体属于蛋白质，稳定性差，灵敏度低，很难以较低的成本高灵敏度分析食物中过敏原。
核酸适配体（aptamer）是通过指数富集配体进化技术（SELEX）从人工合成的随机核苷酸文库中筛选获得能特异性结合靶物质的单链寡核苷酸，作为一种新型的“抗体”，适配体可形成特定的三维构象（发卡、假结、凸环和G-四联体等），通过空间结构的匹配、氢键、范德华力和静电相互作用等分子间相互作用力与靶物质进行高亲和性结合。与抗体相比具有很多优势：（1）人工合成，不依赖动物体内免疫，批次之间的差异小；（2）稳定性好，可长期保存，耐热性强；（3）亲和力高，特异性强，适配体Kd可低至纳摩尔级别，能很好的区分结构类似物；（4）易于修饰，适配体两端标记化学基团（如FAM、ROX、FITC和生物素等）不影响其亲和性，并可拓展其适用范围，被广泛应用于基于适配体识别的检测技术中。
 技术问题
Mohammed Zourob等的英文文献中提供了一条结合β-乳球蛋白的核酸适配体，并利用其构建了电化学的检测方案。所述文献利用靶标固定的方法，将β-乳球蛋白固定在琼脂糖微球上筛选富集了β-乳球蛋白的核酸适配体。检测部分将核酸适配体吸附在氧化石墨烯上，当存在β-乳球蛋白时，因核酸适配体与其特异性结合而使得从氧化石墨烯上脱落引起电化学信号的变化。该方案的缺点是：适配体Kd值较大且并未研究适配体的特异性，因此实际样品检测中干扰因素过多，检出限较高，另外适配体与β-乳球蛋白的结合机理不清楚。
 技术解决方案
为解决上述技术问题，本发明的目的是提供一种特异性识别β-乳球蛋白的核酸适配体及其应用。该核酸适配体能特异性结合牛乳及乳制品中过敏原β-乳球蛋白，为过敏原β-乳球蛋白的高灵敏度、低成本检测提供一种新型的识别分子，从而解决目前过敏原β-乳球蛋白检测过程中依赖色谱仪器的成本高、适用局限性大的问题和依赖抗体的稳定性、重复性欠佳的缺陷，并且为牛乳及乳制品中过敏原β-乳球蛋白的检测方案的开发提供一种新的工具。
本发明的技术方案如下：
本发明的第一个目的是提出一种特异性识别β-乳球蛋白的核酸适配体，所述核酸适配体的序列为SEQ ID NO.1所示的序列；或与SEQ ID NO.1所示的序列具有60%以上同源性并且能够特异性识别β-乳球蛋白的序列；或由SEQ ID NO.1所示的序列衍生的能够特异性识别β-乳球蛋白的序列。
进一步的，所述核酸适配体的核苷酸序列被修饰；所述修饰的方法包括磷酸化、甲基化、氨基化、巯基化、硫代磷酸修饰、糖环侧链修饰中的任一种或多种。
进一步的，所述核酸适配体的核苷酸序列连接有荧光标记物、放射性物质、治疗性物质、生物素、地高辛、纳米发光材料、小肽、siRNA或酶标记物中的任一种或多种。
进一步的，所述SEQ ID NO.1所示的序列与β-乳球蛋白存在分子间相互作用力，其中C-45/T-46与ARG-40、G-56与SER-36、C-57与ASN-63、G-58与ASN-63/TRP-61、G-76与SER-30之间形成氢键。
本发明的第二个目的是提出一种试剂盒，包括上述核酸适配体。
本发明的第三个目的是提出上述核酸适配体在检测β-乳球蛋白中的应用，所述核酸适配体检测β-乳球蛋白的标准曲线的绘制方法如下：
（1）识别探针的构建：核酸适配体与其互补短链以物质的量1：1比例混合均匀，然后进行变性；
（2）信标发卡分子的构建：信标发卡分子溶解在结合缓冲液中进行变性；
（3）酶切循环扩增：将识别探针、信标发卡分子和核酸外切酶 III混合均匀后分别于不同浓度的β-乳球蛋白标准品溶液共孵育2~3 h；利用荧光光谱仪在575nm的激发波长和606nm发射波长下进行荧光测定；
（4）检测标准曲线的绘制：根据不同浓度β-乳球蛋白下荧光信号的强度变化，以β-乳球蛋白浓度的对数值为横坐标，以荧光强度为纵坐标，绘制基于核酸适配体检测β-乳球蛋白的标准曲线。
进一步的，所述步骤（2）中，信标发卡分子95℃变性10min，然后梯度降温至37℃持续3h。
进一步的，所述步骤（3）中，孵育时间为2h，孵育体系为400µL；所述信标发卡分子的浓度为400nM，所述核酸外切酶 III的酶活为0.25U/µL。
进一步的，所述步骤（3）中，所述识别探针与所述信标发卡分子的摩尔比为1:2。
 有益效果
（1）与以往筛选蛋白质的核酸适配体采用靶标固定技术相比，本发明通过文库固定的方法筛选到了β-乳球蛋白的适配体，有效的克服了传统方法的空间位阻效应以及靶标固定引起的构象的改变缺陷。
（2）本发明提供了一种特异性识别牛乳及乳制品中过敏原β-乳球蛋白核酸适配体，经验证，所述适配体可以以100nM以下的解离常数结合到过敏原β-乳球蛋白（如表1所示），且能很好区分蛋白（α-乳白蛋白、酪蛋白）和结构类似物（IgG、牛血清蛋白），具有较好的亲和性和特异性。
（3）所述核酸适配体的5’端或者3’端均可标记FAM、FITC和生物素，可实现过敏原β-乳球蛋白定性和定量的快速检测，具有成本低，特异性高和适用范围广等优点。可以说，本发明提出的特异性识别牛乳及乳制品中过敏原β-乳球蛋白的核酸适配体作为一种过敏原识别分子在食品加工、食品管理及营养规划等领域中是非常有用的一个工具。
（4）所述的核酸适配体，所在核苷酸序列的二级结构中含有典型的茎环结构，这说明适配体具有较好的稳定性。适配体与靶标结合中依赖适配体的二级结构，茎环结构就是其中一种。茎环结构的形成是适配体呈现亲和性的结构基础。
（5）本发明构建了核酸适配体的三维构象，通过分子对接技术模拟核酸适配体与过敏原β-乳球蛋白的相互作用，探究核酸适配体与β-乳球蛋白的结合模式，解释了过敏原β-乳球蛋白的核酸适配体高亲和性的内在原因，可为后续过敏原β-乳球蛋白适配体的优化提供较好的理论指导。
 附图说明
图1是实施例2中核酸适配体筛选过程中荧光富集率的变化图。
图2是实施例3中核酸适配体Lg-18的量热曲线。
图3是实施例3中核酸适配体Lg-18在荧光方法下的结合饱和曲线。
图4是实施例3中核酸适配体Lg-18特异性结合靶标的表征图。
图5是实施例4中核酸适配体Lg-18构建的生物传感器用于过敏原β-乳球蛋白检测的标准曲线。
图6是实施例5中核酸适配体Lg-18的二级结构预测示意图。
图7是实施例6中核酸适配体Lg-18与β-乳球蛋白分子对接示意图。
图8是图7中局部A的结合位点图。
 本发明的实施方式
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。
实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1
高容量单链寡核苷酸文库和引物的设计与合成
构建了长度为80nt，库容量高达1012-1024单链寡核苷酸文库，由中间为40nt的随机序列区（决定文库的丰富度），两端为20nt的固定序列区（结合引物所必须的序列）组成。序列为：5’-AGCAGCACAGAGGTCAGATG-40 random base -CCTATGCGTGCTACCGTGAA-3’由大连宝生物有限公司负责合成。
正向引物：5’-FAM-AGCAGCACAGAGGTCAGATG-3’
反向引物：5’-P-TTCACGGTAGCACGCATAGG-3’
生物素化的互补短链：5’- Biotin- AGCACGCATAGG-3’
引物由生工生物工程（上海）股份有限公司合成。
单链寡核苷酸文库和引物均用TE缓冲液配制成100µM的贮存液存于-20℃备用。
实施例2
基于Capture-SELEX技术的过敏原β-乳球蛋白适配体的筛选
首先将单链寡核苷酸文库与生物素化的互补短链进行互补，然后将其固定在链霉亲和素化的磁珠上，与过敏原β-乳球蛋白共孵育，因单链核酸与β-乳球蛋白的相互作用而从磁珠上脱落，释放到溶液中，孵育后上清液作为次级文库的模板进行PCR扩增，后经酶切制备单链，醇沉等获得次级文库用于下一轮的筛选。具体步骤如下：
单链寡核苷酸文库与生物素化互补链互补：ssDNA文库和生物素化互补短链以1：1.5的比例混合（第一轮筛选所用单链寡核苷酸文库的物质的量为1nmol，其余轮数所用次级文库的物质的量为100pmol），95℃变性10min，然后缓慢降温至37℃持续3h至两者充分互补。
单链寡核苷酸文库的固定：将上述互补混合物与一定量的链霉亲和素的磁珠（核酸文库与磁珠的质量比为1：400）在37℃、200rpm下反应2h，利用生物素与链霉亲和素的强结合作用将单链寡核苷酸文库固定在磁珠上。
靶标孵育：将上述固定化的单链核苷酸文库与50µg/mL β-乳球蛋白在300µL体系下进行孵育，孵育条件为37℃、200rpm，计算每一筛选循环的相对荧光富集率。根据筛选过程中荧光监视的结果，在第六轮和第十轮增加反筛过程，与靶标孵育之前，在体系里引入共存物质（α-乳白蛋白、酪蛋白）和结构类似物（BSA、IgG）以增加筛选的压力，获得更好亲和性和特异性的序列。
PCR扩增及验证：磁分离后获得靶标孵育后的上清液，以此为模板进行PCR扩增。扩增体系为1µL模板，0.5µL Taq DNA聚合酶(5U/µL)，5µL 10Ⅹ聚合酶缓冲液(含有20mM MgCl2)，0.5µL正向引物和反向引物，1µL dNTP Mix（5mM），42µL ddH2O。扩增程序为95℃预变性5min，95℃变性30s，58℃退火30s，72℃延伸30s，循环10次，最后72℃延伸5min，4℃保存。经8%非变性聚丙烯酰胺凝胶电泳验证，观察电泳条带是否单一明亮，条带是否在80bp位置，确保PCR扩增模板的成功。
制备次级文库单链寡核苷酸：扩增产物经纯化后，利用λ外切酶特异性消化PCR产物磷酸化的反义链来制备单链。消化条件为350µL PCR扩增产物，35µL外切酶缓冲液，2µL λ外切酶，37℃反应，根据8%变性聚丙烯酰胺凝胶（含7M尿素）电泳判断酶切进程，待反应结束，75℃灭酶10min终止反应。后经苯酚/氯仿纯化和醇沉，获得次级文库单链寡核苷酸。
克隆测序分析：根据筛选过程中荧光富集率（如图1所示），将荧光富集率为42.77%，相对饱和的第十三轮作为筛选的终点，利用无标记的正向引物和反向引物对其进行PCR扩增，然后送至通用生物有限公司进行TA克隆测序，获得40条80nt±4nt序列。后经过DNAMAN软件进行同源性分析和Mfold在线工具进行二级结构分析，将40条序列分为7个家族，根据二级结构自由能及同源性从每个家族挑选1-2序列作为候选适配体序列进行后续分析。
实施例3
亲和性和特异性分析
（1）基于等温滴定量热技术（ITC）的亲和性分析
使用PEAQ-ITC（英国马尔文仪器有限公司）对10条候补序列进行结合性分析。首先样品池中注入300µL β-乳球蛋白溶液（6µM），进样针里吸入38µL适配体溶液（100µM），程序设置为初始第一滴进样针滴定体积为0.4µL（避免进样针顶部的样品与样品池的样品反应而影响最后的结果），随后进行18滴进样，每滴2µL，间隔150s，进样针的旋转速度为750rpm，确保每次进样充分反应。同时扣除缓冲液滴定至β-乳球蛋白溶液中而带来的稀释热。结合过程中的结合力和热力学参数可以在PEAQ-ITC分析软件中拟合而得到。
所述等温滴定量热技术（ITC）分析中所用到的缓冲液为10mM Tris-HCl，pH 7.4。
结果发现适配体（Lg-18）对β-乳球蛋白具有最优的结合能力，适配体（Lg-18）的序列如下所示。
Lg-18（SEQ ID NO.1）：
AGCAGCACAGAGGTCAGATGTTCGGCCTTTGCGTTAACGAACTTCTAGCTATGCGGCGTACCTATGCGTGCTACCGTGAA
适配体（Lg-18）的ITC分析结果如图2所示，从图2可以看出核酸适配体Lg-18对β-乳球蛋白具有较好的结合能力。
图2上图中，横坐标为时间，纵坐标为热功率。峰底与峰尖之间的峰面积为每次滴定是释放的总热量。
图2下图中，横坐标为滴定物与样品溶液的摩尔比，纵坐标为滴定产生的总热量。
另外，适配体Lg-18的同源性序列（Lg-9、Lg-16）与β-乳球蛋白也具有较好的结合性能。
Lg-9、Lg-16的序列如下所示：
Lg-9（SEQ ID NO.2）：
AGCAGCACAGAGGTCAGATGGTTTTGTTGGGTCTGCTACGGACTTTGTTGACCTTTGCTCCCTATGCGTGCTACCGTGAA
Lg-16（SEQ ID NO.3）：
AGCAGCACAGAGGTCAGATGTCGTTTGTGGCTGTCAATTGGTGTGTTTACCTGTTTTGGCCTATGCGTGCTACCGTGAA
（2）基于磁分离适配体识别的亲和性及特异性分析
所用适配体为Lg-18、Lg-9、Lg-16所示序列的5’端进行FAM荧光基团或其他荧光分子修饰。
不同浓度（10nM、25nM、50nM、75nM、100nM、150 nM、200 nM、300 nM）的适配体与生物素化的互补短链以物质的量1：1.5混合，95℃变性10min，然后缓慢降温至37℃持续3h至两者充分互补；将上述互补混合物与一定量的链霉亲和素的磁珠（适配体与磁珠的质量比为1：400）在37℃、200rpm下反应2h，利用生物素与链霉亲和素的强结合作用将适配体固定在磁珠上；上述固定化的磁珠与50µg/mL β-乳球蛋白在300µL体系下进行孵育，孵育条件为37℃、200rpm，磁分离后，上清液在485nm激发波长和522nm发射波长下进行荧光测定。通过GraphPad prism 5.0 软件将不同适配体浓度下的相对荧光强度进行非线性拟合获得并计算适配体的解离常数Kd值（见表1）。图3是Lg-18的结合饱和曲线。从表1可以看出其Kd值最小，为65.00±27.42nM，表明适配体Lg-18具有最优的结合性能，适配体Lg-18的同源性序列（Lg-9、Lg-16）与β-乳球蛋白也有较好的结合性能。
利用同样的方法评价上述适配体（Lg-18）的特异性，将200nM适配体固定在磁珠上分别于50 µg/mL共存物质（α-乳白蛋白、酪蛋白）和结构类似物（BSA、IgG）进行孵育，经磁分离后，分别测定上清液中荧光强度，然后评价适配体的特异性。结果如图4所示，说明适配体（Lg-18）具有较好的特异性。
表1
序列名称 解离常数Kd值（nM）
Lg-9 71.85±16.06
Lg-16 65.85±15.16
Lg-18 65.00±27.42
实施例4
基于核酸适配体识别的β-乳球蛋白灵敏检测分析
识别探针的构建（Lg-18）：核酸适配体与其互补短链以物质的量1：1比例混合均匀，95℃变性10min，然后缓慢降温至37℃持续3h至两者充分互补。
信标发卡分子的构建：信标发卡分子溶解在结合缓冲液中(100mM NaCl，pH7.4) 95℃变性10min，然后梯度降温至37℃持续3h确保充分形成发卡结构。
信标发卡分子：5’-ROX-ACCTCTGTATCGACATTCACAGAGGT-BHQ2（标记在T碱基上）-CAGATGTT-3’
酶切循环扩增：识别探针（200nM）、信标发卡分子（400nM）和核酸外切酶 III（0.25U/µL）混合均匀后分别于不同浓度（20 ng/mL、50 ng/mL、100 ng/mL、200 ng/mL、500 ng/mL、1000 ng/mL、2000 ng/mL、5000 ng/mL）的β-乳球蛋白标准品溶液共孵育2h，孵育体系为400µL。当体系中存在待检测物质β-乳球蛋白时，因为核酸适配体与β-乳球蛋白的结合作用强于适配体与互补短链的相互作用，互补短链从适配体上脱落，进而与信标发卡分子3’端互补形成双链区域，信标发卡分子打开，荧光得到恢复，同时体系中的核酸外切酶 III作用于双链区域，沿信标发卡分子的3’-5’消化单链寡核苷酸，互补短链得以释放循环，荧光信号进一步增强。利用F7000荧光光谱仪在575nm的激发波长和606nm发射波长下进行荧光测定。
检测标准曲线的绘制：根据不同浓度β-乳球蛋白下荧光信号的强度变化，以β-乳球蛋白浓度的对数值为横坐标，以荧光强度为纵坐标，绘制基于适配体识别的β-乳球蛋白灵敏检测的标准曲线，如图5。标准曲线：Y=1324.85X-1669.47，R2=0.9954。检测线性范围：20ng/mL -5µg/mL，检出限(LOD)：1.02ng/mL。
实施例5
利用Mfold在线工具分析所述适配体（Lg-18）的二级结构
将所述适配体序列导入Mfold中，设置二级结构的形成条件（Na+：100mM；Mg2+：1mM；37℃），所形成的二级结构如图6所示，二级结构中含有典型的茎环结构。
实施例6
分子对接模拟适配体Lg-18与β-乳球蛋白的相互作用
适配体三维构象的准备：适配体Lg-18序列的Vienna格式由Mfold在线工具生成，然后利用RNAcomposer 软件生成Vienna格式对应序列的最优三维构象。。
模拟对接：从PDB网站下载β-乳球蛋白的三维构象（PDB：2Q2M），利用MGL Tools对其进行一系列前处理，包括去除水分子，加非极性氢，加电荷Kollman Charges。最后生成.pdbqt文件；导入适配体Lg-18的三维构象作为配体，同上对其进行添加电荷，添加非极性氢；采用Autodock vina 对其进行模拟对接，搜寻适配体与β-乳球蛋白的最佳结合位置。对接参数如下：
receptor = lacg.pdbqt
ligand = apt.pdbqt
center_x = 41.408
center_y = 54.25
center_z = 25.132
size_x = 125
size_y = 125
size_z = 125
out = vina.pdbqt
对接结果分析：经Autodock vina对接后，可初步确定适配体Lg-18与β-乳球蛋白的结合位置，结合能在-17kcal/mol左右的结合模式具有多种，选择其中一种两者之间存在多种氢键的模式利用pymol软件对其进行结合位点分析。结果如图7、图8所示，核酸适配体Lg-18与β-乳球蛋白之间存在分子间相互作用力，C-45/T-46与ARG-40、G-56与SER-36、C-57与ASN-63、G-58与ASN-63/TRP-61、G-76与SER-30之间可形成氢键，可确定为其主要结合区域。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。
 序列表自由内容
 SEQUENCE LISTING
<110>  江南大学
<120>  一种特异性识别β-乳球蛋白的核酸适配体及其应用
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<170>  PatentIn version 3.3
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agcagcacag aggtcagatg ttcggccttt gcgttaacga acttctagct atgcggcgta     60
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agcagcacag aggtcagatg gttttgttgg gtctgctacg gactttgttg acctttgctc     60
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Claims (9)

  1. 一种特异性识别β-乳球蛋白的核酸适配体，其特征在于，所述核酸适配体的序列为SEQ ID NO.1所示的序列；或与SEQ ID NO.1所示的序列具有60%以上同源性并且能够特异性识别β-乳球蛋白的序列；或由SEQ ID NO.1所示的序列衍生的能够特异性识别β-乳球蛋白的序列。
  2. 根据权利要求1所述的特异性识别β-乳球蛋白的核酸适配体，其特征在于，所述核酸适配体的核苷酸序列被修饰；所述修饰的方法包括磷酸化、甲基化、氨基化、巯基化、硫代磷酸修饰、糖环侧链修饰中的任一种或多种。
  3. 根据权利要求1所述的特异性识别β-乳球蛋白的核酸适配体，其特征在于，所述核酸适配体的核苷酸序列连接有荧光标记物、放射性物质、治疗性物质、生物素、地高辛、纳米发光材料、小肽、siRNA或酶标记物中的任一种或多种。
  4. 根据权利要求1所述的特异性识别β-乳球蛋白的核酸适配体，其特征在于，所述SEQ ID NO.1所示的序列与β-乳球蛋白存在分子间相互作用力，其中C-45/T-46与ARG-40、G-56与SER-36、C-57与ASN-63、G-58与ASN-63/TRP-61、G-76与SER-30之间形成氢键。
  5. 一种试剂盒，其特征在于，包括权利要求1-4任一项所述的核酸适配体。
  6. 权利要求1-4任一项所述的核酸适配体在检测β-乳球蛋白中的应用，其特征在于，核酸适配体检测β-乳球蛋白的标准曲线的绘制方法如下：
    （1）识别探针的构建：核酸适配体与其互补短链以物质的量1：1比例混合均匀，然后进行变性；
    （2）信标发卡分子的构建：信标发卡分子溶解在结合缓冲液中进行变性；
    （3）酶切循环扩增：将识别探针、信标发卡分子和核酸外切酶 III混合均匀后分别于不同浓度的β-乳球蛋白标准品溶液共孵育2~3 h；利用荧光光谱仪在575nm的激发波长和606nm发射波长下进行荧光测定；
    （4）检测标准曲线的绘制：根据不同浓度β-乳球蛋白下荧光信号的强度变化，以β-乳球蛋白浓度的对数值为横坐标，以荧光强度为纵坐标，绘制基于核酸适配体检测β-乳球蛋白的标准曲线。
  7. 根据权利要求6所述的应用，其特征在于，所述步骤（2）中，信标发卡分子95℃变性10min，然后梯度降温至37℃持续3h。
  8. 根据权利要求6所述的应用，其特征在于，所述步骤（3）中，孵育时间为2h，孵育体系为400µL；所述信标发卡分子的浓度为400nM，所述核酸外切酶 III的酶活为0.25U/µL。
  9. 根据权利要求6所述的应用，其特征在于，所述步骤（3）中，所述识别探针与所述信标发卡分子的摩尔比为1:2。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114075565A (zh) * 2022-01-19 2022-02-22 中国农业大学 一种用于检测β-乳球蛋白的双功能G-四链体变构生物传感器

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111172166B (zh) * 2020-02-15 2021-10-15 江南大学 一种特异性识别β-乳球蛋白的核酸适配体及其应用
CN114317543B (zh) * 2021-11-23 2024-01-26 河北农业大学 可特异性识别α-乳白蛋白的核酸适配体及其应用
CN116355908B (zh) * 2023-05-30 2023-08-15 中国农业大学 一种用于检测β-乳球蛋白的磁性适配体生物传感器

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106645050A (zh) * 2016-10-13 2017-05-10 南京大学 一种用于检测乳铁蛋白含量的适配体及其应用
CN107607722A (zh) * 2017-07-21 2018-01-19 南京医科大学 一种定量检测奶粉中β‑乳球蛋白的方法
WO2018089391A1 (en) * 2016-11-08 2018-05-17 Dots Technology Corp. Allergen detection agents and assays
CN109507423A (zh) * 2018-12-27 2019-03-22 南京祥中生物科技有限公司 一种基于适配体探针同时检测α-乳白蛋白、β-乳球蛋白和乳铁蛋白含量的方法
CN111172166A (zh) * 2020-02-15 2020-05-19 江南大学 一种特异性识别β-乳球蛋白的核酸适配体及其应用

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100254901A1 (en) * 2005-07-28 2010-10-07 Smith Cassandra L Compositions comprising nucleic acid aptamers
CN103898119B (zh) * 2014-03-24 2016-08-31 湖南大学 一种多西紫杉醇的核酸适配体、核酸适配体衍生物及其用途
CN105886512B (zh) * 2016-03-07 2020-06-09 江南大学 一组高特异识别盐酸克伦特罗，沙丁胺醇和莱克多巴胺的寡核苷酸适配子
CN107064485B (zh) * 2017-01-13 2020-05-19 南京大学 十面体纳米银探针在利用表面等离子共振成像技术筛选核酸适配体中的应用
CN106834295B (zh) * 2017-03-21 2020-04-24 江南大学 一种特异识别细菌脂多糖的广谱核酸适配体及其定向筛选方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106645050A (zh) * 2016-10-13 2017-05-10 南京大学 一种用于检测乳铁蛋白含量的适配体及其应用
WO2018089391A1 (en) * 2016-11-08 2018-05-17 Dots Technology Corp. Allergen detection agents and assays
CN107607722A (zh) * 2017-07-21 2018-01-19 南京医科大学 一种定量检测奶粉中β‑乳球蛋白的方法
CN109507423A (zh) * 2018-12-27 2019-03-22 南京祥中生物科技有限公司 一种基于适配体探针同时检测α-乳白蛋白、β-乳球蛋白和乳铁蛋白含量的方法
CN111172166A (zh) * 2020-02-15 2020-05-19 江南大学 一种特异性识别β-乳球蛋白的核酸适配体及其应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HE, SHENGFA, HONG-BING CHEN, CAI-YUN LONG, LI-JUN ZOU, GONG-HUA HU, LIANG XIONG, YAN-FANG GAO: "Research progress on the detection methods of cow's milk allergen β-lactoglobulin", JOURNAL OF FOOD SAFETY & QUALITY, vol. 10, no. 7, 1 April 2019 (2019-04-01), pages 1763 - 1769, XP055835530 *
YU FANG, LI HUI, SUN WEI, ZHAO YAJU, XU DANKE, HE FUCHU: "Selection of aptamers against Lactoferrin based on silver enhanced and fluorescence-activated cell sorting", TALANTA, ELSEVIER, AMSTERDAM, NL, vol. 193, 1 February 2019 (2019-02-01), NL, pages 110 - 117, XP055835534, ISSN: 0039-9140, DOI: 10.1016/j.talanta.2018.09.063 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114075565A (zh) * 2022-01-19 2022-02-22 中国农业大学 一种用于检测β-乳球蛋白的双功能G-四链体变构生物传感器

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