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CN-117701572-B - Nucleic acid aptamer of lipoprotein-associated phospholipase A2 and application thereof

CN117701572BCN 117701572 BCN117701572 BCN 117701572BCN-117701572-B

Abstract

The invention relates to a nucleic acid aptamer of lipoprotein-related phospholipase A2, which is at least one of B76-2, B76-4 and B76-5, and has sequences shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 respectively, wherein the lipoprotein-related phospholipase A2 aptamer provided by the invention fills the blank in the field of lipoprotein-related phospholipase A2 aptamers and has higher affinity and specificity. Wherein, 3 aptamers with good affinity and specificity to the lipoprotein-associated phospholipase A2 are screened and verified, and the aptamers can be prepared into molecular probes, used as detection reagents and the like to be applied to a method for detecting the lipoprotein-associated phospholipase A2 or a corresponding kit, so that the accuracy is improved. Can be used as recognition molecules to develop a novel Lp-PLA2 biosensing method, and has great application potential in the aspect of early warning of malignant events of cardiovascular diseases.

Inventors

  • ZHANG SHENGXING
  • NIU HUIMIN
  • QIU SHUQIAN
  • WANG SHUILIANG
  • CHEN LI

Assignees

  • 中国人民解放军联勤保障部队第九〇〇医院

Dates

Publication Date
20260505
Application Date
20231120

Claims (7)

  1. 1. A nucleic acid aptamer of lipoprotein-related phospholipase A2 is characterized in that the nucleic acid aptamer is at least one of B76-2, B76-4 and B76-5, and the sequences of the nucleic acid aptamer are respectively as follows: B76-2：5'- TACCA GTGCG ATGCT CAGGG GGGGG TGGGT GGGGT CACGT CCGGA TGTGT GTCGT GTGCT GAGCA TCGGT AATGA C -3'; B76-4：5'- TACCA GTGCG ATGCT CAGGG GTGGG TGGGT GGGGG AGGAT GGGGG GTGGC CTGAC GTGCT GAGCA TCGGT AATGA C -3' B76-5：5'- TACCA GTGCG ATGCT CAGGG GTGGG GCGGG TGGGG GAGGG GGCGG AATGG TATGT GTGCT GAGCA TCGGT AATGA C -3'.
  2. 2. The nucleic acid aptamer of claim 1, wherein the spatial structure of the nucleic acid aptamer B76-2, B76-4, B76-5 at 25℃and 100mM Na + ,10uM Mg 2+ is as follows: 。
  3. 3. The nucleic acid aptamer of claim 1, wherein the 5 'or 3' end of at least one of the nucleic acid aptamers B76-2, B76-4, B76-5 is chemically modified with a fluorophore, an amino group, biotin, digoxin, or polyethylene glycol.
  4. 4. Use of a nucleic acid aptamer of lipoprotein-associated phospholipase A2 according to any of claims 1-3 for the preparation of a reagent, kit or sensor for detecting lipoprotein-associated phospholipase A2.
  5. 5. Use of a nucleic acid aptamer of lipoprotein-associated phospholipase A2 according to any of claims 1-3 for the preparation of a probe for detecting lipoprotein-associated phospholipase A2 molecules.
  6. 6. A kit for specifically recognizing lipoprotein-associated phospholipase A2, which comprises the nucleic acid aptamer of any of claims 1-3.
  7. 7. A molecular probe comprising the nucleic acid aptamer according to any one of claims 1 to 3.

Description

Nucleic acid aptamer of lipoprotein-associated phospholipase A2 and application thereof Technical Field The invention relates to the technical field of biology, in particular to a nucleic acid aptamer of atherosclerosis marker lipoprotein-related phospholipase A2 and application thereof. Background Lipoprotein-associated phospholipase A2 (LpPLA 2) is a 50kD phospholipase produced and secreted by many immune cells, such as megaphaga, mononuclear, mast, and T lymphocytes, also known as platelet-activating factor acetylhydrolase. LpPLA2 is responsible for hydrolyzing oxidized phospholipids on LDL surfaces, producing proinflammatory byproducts that mimic platelet activating factors, such as lysophosphatidylcholine and oxidized non-esterified fatty acids, and attract monocytes to this region, activating leukocytes and stimulating the production of other inflammatory cytokines (such as IL-6 and TNF- α) to mediate inflammatory responses. LpPLA2 promotes apoptosis and necrosis of macrophages in plaque by attracting smooth muscle cells into the intima, further promoting atherosclerosis. In conclusion, lpPLA2 directly participates in vascular inflammatory reaction which leads to atherosclerosis, quickens vulnerable plaque rupture, further forms thrombus and triggers cardiovascular and cerebrovascular malignant events, and is a novel independent marker aiming at vascular endothelial inflammatory reaction. LpPLA2 is used as a vascular strong specificity factor, can be used as a dynamic monitoring index of vascular inflammation and atherosclerosis, and high level LpPLA2 indicates that atheromatous plaques are easier to break, and can evaluate and early warn the risk of cardiovascular and cerebrovascular embolism diseases. In addition, lpPLA has low biological variability and is less influenced by other factors and indexes, compared with the traditional cardiovascular risk prediction index, the method can directly reflect the inflammation degree of the vascular intima, has higher specificity and has higher relevance with plaque instability. LpPLA2 is the only blood test index approved by the FDA for risk assessment of atherosclerosis-related coronary heart disease, ischemic stroke and various thrombotic diseases. The current methods for detecting the enzyme quality are an enzyme-linked immunosorbent assay (ELISA) method, an immunopotentiation turbidimetry method and a chemiluminescence method. The method for detecting the activity is a continuous detection method of an automatic biochemical analyzer. However, the ELISA method has a complicated flow, and requires a professional technician to operate to avoid the operation error as much as possible, and the required detection time is about 2 hours, and the lppla2 activity detection flow is more required to be performed by a laboratory staff and can be realized by a large instrument. Meanwhile, two monoclonal antibodies and biological enzyme materials required by the kit are complex to prepare, the activity of the antibodies and the enzyme is easily affected by temperature, the cost is high, and in China, the development of the kit for the two methods is still lacking, so that the actual detection requirements of patients suffering from cardiovascular and cerebrovascular diseases can not be met. Therefore, the novel LpPLA detection kit is developed to be used for early warning of cardiovascular malignant events, and has wide application prospects in home monitoring and clinical examination. Nucleic acid Aptamer (Aptamer) refers to a nucleic acid sequence with targeting effect selected by exponential enrichment of ligand systematic evolution technique (SELEX). The three-dimensional structure can be folded into a specific three-dimensional structure, and can be combined with targets including proteins, small molecules, viruses, bacteria, cells and the like through the actions of hydrogen bonding, electrostatic interaction, hydrophobic action, aromatic ring accumulation, van der Waals force and the like, thereby playing an important role in disease diagnosis, targeted drug delivery, environmental monitoring and food safety analysis. Thus if a nucleic acid aptamer with higher affinity to LpPLA and high specificity could be found, it would be helpful to achieve high sensitivity and high specificity detection of LpPLA 2. Disclosure of Invention In view of the fact that the existing method can only realize detection under laboratory conditions, and requires high time, reagent and labor costs, the invention provides an aptamer of lipoprotein-associated phospholipase A2 and application thereof, wherein the aptamer has extremely strong affinity and specificity with lipoprotein-associated phospholipase A2, and can be used for efficiently detecting lipoprotein-associated phospholipase A2. Correspondingly, the invention also provides application of the nucleic acid aptamer of the lipoprotein-related phospholipase A2 as a probe for detecting the lipoprotein-related phospholipase A2 in a