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Nucleic acid aptamer capable of specifically recognizing beta-lactoglobulin and application of nucleic acid aptamer

A nucleic acid aptamer and lactoglobulin technology, which is applied in the fields of biology and medicine, can solve the problems of large application limitations, poor repeatability, and high cost, and achieve wide application range, good affinity and specificity, and low cost. low effect

Active Publication Date: 2020-05-19
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The nucleic acid aptamer can specifically bind the allergen β-lactoglobulin in milk and dairy products, and provide a new type of recognition molecule for the high-sensitivity and low-cost detection of the allergen β-lactoglobulin, thereby solving the problem of the current allergen β-lactoglobulin. - In the detection process of lactoglobulin, it depends on the high cost of chromatographic instruments, the problems of large application limitations, the stability of antibodies, and the defects of poor repeatability, and it is suitable for the detection of the allergen β-lactoglobulin in milk and dairy products Program development provides a new tool

Method used

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  • Nucleic acid aptamer capable of specifically recognizing beta-lactoglobulin and application of nucleic acid aptamer
  • Nucleic acid aptamer capable of specifically recognizing beta-lactoglobulin and application of nucleic acid aptamer
  • Nucleic acid aptamer capable of specifically recognizing beta-lactoglobulin and application of nucleic acid aptamer

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Experimental program
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Effect test

Embodiment 1

[0040] Design and synthesis of high-capacity single-stranded oligonucleotide library and primers

[0041] A single-stranded oligonucleotide library with a length of 80nt and a library capacity of up to 1012-1024 was constructed, consisting of a random sequence region of 40nt in the middle (determining the richness of the library), and a fixed sequence region of 20nt at both ends (necessary for binding primers) sequence) composition. The sequence is: 5'-AGCAGCACAGAGGTCAGATG-40random base-CCTATGCGTGCTACCGTGAA-3' was synthesized by Dalian Bao Biological Co., Ltd.

[0042] Forward primer: 5'-FAM-AGCAGCACAGAGGTCAGATG-3'

[0043] Reverse primer: 5'-P-TTCACGGTAGCACGCATAGG-3'

[0044] Biotinylated complementary short strand: 5'-Biotin-AGCACGCATAGG-3'

[0045] Primers were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.

[0046] Both the single-stranded oligonucleotide library and primers were made into 100 μM stock solution with TE buffer and stored at -20°C for later us...

Embodiment 2

[0048] Screening of allergen β-lactoglobulin aptamers based on Capture-SELEX technology

[0049] The single-stranded oligonucleotide library was first complemented with biotinylated complementary short strands, then immobilized on streptavidinized magnetic beads, and incubated with the allergen β-lactoglobulin, due to the single-stranded Nucleic acid interacts with β-lactoglobulin to detach from the magnetic beads and release it into the solution. After incubation, the supernatant is used as a template for the secondary library for PCR amplification, and then the single strand is prepared by enzyme digestion, alcohol precipitation, etc. The secondary library was used for the next round of screening. Specific steps are as follows:

[0050] The single-stranded oligonucleotide library is complementary to the biotinylated complementary chain: the ssDNA library and the biotinylated complementary short chain are mixed at a ratio of 1:1.5 (the amount of the single-stranded oligonucl...

Embodiment 3

[0057] Affinity and Specificity Analysis

[0058] (1) Affinity analysis based on isothermal titration calorimetry (ITC)

[0059] Binding analysis was performed on 10 candidate sequences using PEAQ-ITC (Malvern Instruments Ltd., UK). First, inject 300 μL of β-lactoglobulin solution (6 μM) into the sample cell, suck 38 μL of aptamer solution (100 μM) into the sampling needle, and set the program as the initial titration volume of the first drop of the sampling needle is 0.4 μL (avoid the injection at the top of the sampling needle). The sample reacts with the sample in the sample cell to affect the final result), followed by 18 drops of 2μL, with an interval of 150s, and the rotation speed of the injection needle is 750rpm to ensure that each injection is fully reacted. At the same time, the heat of dilution caused by the titration of the buffer into the β-lactoglobulin solution was subtracted. The binding force and thermodynamic parameters in the binding process can be obtain...

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Abstract

The invention belongs to the fields of biology and medicine, and particularly relates to a nucleic acid aptamer capable of specifically recognizing beta-lactoglobulin and application of the nucleic acid aptamer. The nucleic acid aptamer has a sequence shown in SEQ ID NO.1; or a sequence which has 60% of homology or above with the sequence shown in the SEQ ID NO.1 and can specifically recognize thebeta-lactoglobulin; or a sequence which is derived from the sequence shown in the SEQ ID NO.1 and can specifically recognize the beta-lactoglobulin. The nucleic acid aptamer can specifically bind theallergen beta-lactoglobulin in cow milk and milk products, and a new tool is provided for high-sensitivity and low-cost detection of the allergen beta-lactoglobulin.

Description

technical field [0001] The invention belongs to the fields of biology and medicine, and in particular relates to a nucleic acid aptamer specifically recognizing β-lactoglobulin and its application. Background technique [0002] Food allergy is an adverse reaction of some people to certain foods (including IgE-mediated and non-IgE-mediated two types of immune reactions). Food allergies will not only seriously affect the quality of life of patients, but also cause malnutrition and health problems, such as asthma, gastrointestinal tract and skin inflammation, etc. In severe cases, anaphylactic shock may be life-threatening. [0003] Milk and dairy products have gradually become an important part of human's healthy diet because they contain essential amino acids and rich minerals. But at the same time, it is one of the eight categories of allergenic foods designated by the World Health Organization (WHO) and the Food and Agriculture Organization of the United Nations (FAO), bec...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115G01N33/68
CPCC12N15/115G01N33/68C12N2310/16G01N2333/4713G01N33/542G01N33/5308
Inventor 段诺齐硕吴世嘉王周平
Owner JIANGNAN UNIV
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