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Nucleic acid aptamer for specifically recognizing bovine pregnancy-related glycoprotein 4 and application thereof

A nucleic acid aptamer and glycoprotein technology, which is applied in biochemical equipment and methods, material inspection products, instruments, etc., can solve the problems of no research reports on glycoprotein nucleic acid aptamers related to livestock pregnancy, and achieve good affinity and specificity, easy chemical modification, and low-cost effects

Active Publication Date: 2020-08-07
XINJIANG ACADEMY OF AGRI & RECLAMATION SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there are no research reports on nucleic acid aptamers of pregnancy-associated glycoproteins in livestock

Method used

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  • Nucleic acid aptamer for specifically recognizing bovine pregnancy-related glycoprotein 4 and application thereof
  • Nucleic acid aptamer for specifically recognizing bovine pregnancy-related glycoprotein 4 and application thereof
  • Nucleic acid aptamer for specifically recognizing bovine pregnancy-related glycoprotein 4 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Screening of bPAG4 nucleic acid aptamers

[0033] 1. Synthetic random single-stranded DNA (ssDNA) library and primers:

[0034] Random single-stranded DNA (ssDNA) library:

[0035] 5'-CTACGGTGCCTTGAAGTGAC-N36-CATAGCAGGTCACTTCCAGG-3', wherein, N36 represents 36 random nucleotides, and the library was synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.;

[0036] Upstream primer: 5'-FAM-CTACGGTGCCTTGAAGTGAC-3',

[0037] Downstream primer: 5'- 20A-spacer18-CCTGGAAGTGACCTGCTATG-3',

[0038] Among them, in the downstream primers, 20A represents a polyA tail composed of 20 adenosine (A), and Spacer 18 represents an 18-atom hexaethylene glycol interarm. The above primers were synthesized by Nanjing GenScript Biotechnology Co., Ltd.

[0039] Use DPBS buffer (NaCl: 8 g / L, KCl: 0.2 g / L, Na2HPO4: 1.15 g / L, KH2PH4: 0.2 g / L; PH 7.4) and stored at -20°C for later use.

[0040] 2. Magnetic beads-bPAG4 protein (MB-bPAG4) coupling

[0041] Take 50 μL of carboxylate...

Embodiment 2

[0053] Embodiment 2, the affinity analysis of nucleic acid aptamer

[0054] Several nucleic acid aptamers synthesized in Example 1 were taken, and each nucleic acid aptamer was prepared into a series of gradient concentrations (0nM, 100 nM, 200 nM, 400 nM, 800 nM, 1600 nM, 3200 nM, 6400 nM in 10 mM PBS nM) aptamer solution; follow step 4 to analyze the binding of each nucleic acid aptamer to MB-bPAG4 in turn, use GraphPad Prism7.0 software to fit the binding curve, and calculate the dissociation constant Kd value of each nucleic acid aptamer . image 3 It is the saturated binding curve of the nucleic acid aptamer shown in SEQ ID No.1 and the target protein bPAG4, the aptamer has high affinity with the bPAG4 protein, and the dissociation constant is 11.7 nM. The secondary structure prediction analysis of the aptamer sequence shown in SEQ ID No. 1 was carried out using online Mfold, and the results are shown in Figure 4 .

Embodiment 3

[0055] Embodiment 3: the specificity analysis of nucleic acid aptamer

[0056] Prepare MB-bPAG1, MB-bPAG4, MB-bPAG9, MB-BSA and MB-OVA respectively according to step 2, and dilute the nucleic acid aptamer sequence shown in SEQ ID No.1 to a concentration of 0.5 μM with 10mM PBS, and follow the steps 4 Analyze the binding of the nucleic acid aptamer to each protein-coupled magnetic bead, and the magnetic bead not connected to any protein is used as a negative control. See the test results Figure 5 , the nucleic acid aptamer shown in SEQ ID No. 1 preferentially binds to the target protein bPAG4, and has a higher binding ability with similar family proteins, but does not bind with other BSA and OVA proteins.

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Abstract

The invention provides a nucleic acid aptamer for specifically combining bovine pregnancy-related glycoprotein 4. The sequence of the nucleic acid aptamer comprises any one of SEQ ID No. 1 sequences.The nucleic acid aptamer provided by the invention can also be various similar sequences with higher homology or derivatives obtained from the sequences provided by the invention. The invention also provides an application of the aptamer. The nucleic acid aptamer disclosed by the invention is obtained by screening through a magnetic bead-SELEX technology; the aptamer can be combined with bPAG4 target protein, can also be selectively combined with bPAG4 like protein, is not specifically combined with other proteins, can be used for capturing the bPAG4 protein from a complex system, and is suitable for detection, separation and purification of the bPAG4 protein and quick diagnosis of bovine early pregnancy.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to a nucleic acid aptamer that specifically recognizes bovine pregnancy-associated glycoprotein 4 (bPAG4), and in particular to the use of the nucleic acid aptamer in the detection of pregnancy-associated glycoprotein (bPAG) and livestock Application in early pregnancy diagnosis. Background technique [0002] Bovine pregnancy-associated glycoproteins (bPAG) belong to the aspartic protease family, and have more than 50% identical amino acid sequences with pepsin, cathepsin D, and cathepsin E. There are many types of bPAG, including at least 22 kinds of bPAG proteins, which are expressed by placental trophoblast cells (Xie et al., 1994; Hughes et al., 2000), enter the maternal blood after embryo implantation, and express and secrete in time and space throughout pregnancy specificity (Green et al., 2000; Wooding et al., 2005; Telugu et al., 2009), and is often used as a marker for...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115G01N33/68G01N33/53
CPCC12N15/115C12N2310/16G01N33/5308G01N33/68G01N2800/36
Inventor 刘长彬卢春霞
Owner XINJIANG ACADEMY OF AGRI & RECLAMATION SCI
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