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High-affinity aptamers specifically binding to lactoxins 1 and 4 and their applications

A technology of lactoxin and aptamer, which is applied in the field of biotechnology and biotechnology, and can solve the problems of identification, optimization and application of affinity constants that have not yet been reported.

Active Publication Date: 2018-11-09
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, there are no relevant reports on molecular recognition probes specifically binding to GTX1 and GTX4, high-affinity aptamers specifically binding to GTX1 and GTX4, and the identification, optimization and application of the aptamer's affinity constant.

Method used

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  • High-affinity aptamers specifically binding to lactoxins 1 and 4 and their applications
  • High-affinity aptamers specifically binding to lactoxins 1 and 4 and their applications
  • High-affinity aptamers specifically binding to lactoxins 1 and 4 and their applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1. Construction of random ssDNA library and primers thereof

[0031] 1. Construct a random ssDNA library with a length of 80 nucleotides

[0032] 5′-AGCAGCACAGAGGTCAGATG-N 40 -CCTATGCGTGCTACCGTGAA-3'; wherein, N represents any one of the bases A, T, C, G, N 40 Represents a random sequence of length 40bp.

[0033] 2. Construction of primers

[0034] Upstream primer: 5'-AGCAGCACAGAGGTCAGATG-3', SEQ ID NO: 15

[0035]Downstream primer 1: 5'-TTCACGGTAGCACGCATAGG-3', SEQ ID NO: 16

[0036] Downstream primer 2: 5′-poly(dA20)-Spacer18-TTCACGGTAGCACGCATAGG-3′

Embodiment 2

[0037] Example 2. Screening of GTX1 and GTX4 nucleic acid adaptation

[0038] In order to obtain nucleic acid aptamers with high affinity and high specificity for GTX1 and GTX4, eight rounds of screening were carried out based on graphene oxide’s ability to non-specifically adsorb ssDNA, as shown in figure 1 shown; where the reverse screening was introduced from the sixth round, that is, the ssDNA library was first incubated with the reverse targets (GTX2, GTX3, STX and neoSTX), and then an appropriate amount of graphene oxide was added for co-incubation; after the supernatant was removed by centrifugation, Add free target toxins GTX1 and GTX4 and incubate again; finally, aptamers that specifically bind to GTX1 and GTX4 are recovered. In each round of screening, the recovery rate of aptamers binding to target toxins GTX1 and GTX4 was as follows: figure 2 shown.

[0039] Take 1 nmol of the initial ssDNA library, add 200 μL of binding buffer (20 mM Tris-HCl, 100 mM NaCl, 2 mM...

Embodiment 3

[0048] Example 3. Truncation optimization of GO18 aptamers

[0049] All truncated sequences are shown in Table 2. After the primers at both ends of the aptamer GO18 were truncated, the binding affinity was determined by biomembrane interferometry. affinity. Based on the secondary structure of GO18-T such as image 3 , forming three diameter rings a, b, c. The binding affinity of the aptamer GO18-T-a to the target toxin was significantly increased, while GO18-T-b and GO18-T-c showed no binding. It may be that the radial loop a on the aptamer GO18-T-a is fully exposed after the truncated rings b and c, and binds more tightly to the target molecules GTX1 and GTX4, resulting in a significant increase in the affinity of the two. Therefore, the aptamer GO18-T-d containing only 25 nucleotides was obtained after further truncation.

[0050] Table 2: Aptamer GO18 truncated sequence and its affinity constant Kd value

[0051]

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Abstract

The invention relates to the technical field of biology, and provides an aptamer capable of being specifically combined with gonyautoxin 1 (GTX1) and gonyautoxin 4 (GRX4). The general formula is 5'-AGCAGCACAGAGGTCAGATG-N40-CCTATGCGTGCTACCGTGAA-3', wherein N represents any one of bases A, T, C and G, and N40 represents a random sequence with the length of 40bp. The high-affinity aptamer capable of being specifically combined with GTX1 and GTX4 is obtained by screening on the basis of graphene oxide SELEX technology. The single-chain DNA (deoxyribonucleic acid) adapter can be used for separation and enrichment of trace amounts of GTX1 and GRX4 in the sample, analysis detection of GTX1 and GRX4, preparation of neutral or antagonistic GTX1 and GRX4 drugs, and removal of toxins in a water body.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to a high-affinity aptamer that specifically binds to lactoxins 1 and 4 obtained by screening based on graphene oxide SELEX technology, which is the aptamer for trace amounts of lactoxins 1 and 4 in samples. The separation and enrichment, the rapid detection of lactoxins 1 and 4, the preparation of drugs that neutralize or antagonize lactoxins 1 and 4, and the removal of toxins in water have laid the foundation. Background technique [0002] Paralytic shellfish poisoning is one of the three major worldwide marine biological hazards. Gonyautoxin 1 and 4 (Gonyautoxin1 and Gonyautoxin 4, GTX1 and GTX4) and their analogues Gonyautoxin 2 and 3 (GTX2and GTX3), Saxitoxin (STX) and Neolithiatoxin (neoSTX ) is the most representative neurotoxin among paralytic shellfish toxins. GTX1 and GTX4 can accumulate in fish and shellfish through the aquatic food chain, and humans will cause para...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/115G01N33/53A61K31/7088A61P39/02C02F1/58
CPCC02F1/58C12N15/115C12N2310/16G01N33/53
Inventor 高顺祥郑欣胡波刘德婧孙铭娟焦炳华王梁华
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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