Nucleic acid aptamer specifically recognizing bovine pregnancy-associated glycoprotein 4 and its application

A nucleic acid aptamer and glycoprotein technology, which can be applied in the fields of instruments, biochemical equipment and methods, analytical materials, etc., and can solve problems such as no research reports on livestock pregnancy-related glycoprotein nucleic acid aptamers.

Active Publication Date: 2021-09-10
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 specifically recognizing bovine pregnancy-associated glycoprotein 4 and its application
  • Nucleic acid aptamer specifically recognizing bovine pregnancy-associated glycoprotein 4 and its application
  • Nucleic acid aptamer specifically recognizing bovine pregnancy-associated glycoprotein 4 and its application

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; according to step 4, analyze the binding of each nucleic acid aptamer to MB-bPAG4 in turn, use GraphPadPrism 7.0 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, dilute the nucleic acid aptamer sequence shown in SEQ ID No.1 with 10mM PBS to a concentration of 0.5 μM, 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 specifically binding to bovine pregnancy-associated glycoprotein 4, and the nucleic acid aptamer sequence comprises any one of the sequences of SEQ ID No.1. The nucleic acid aptamers of the present invention can also be various similar sequences with higher homology or derivatives obtained from the sequences of the present invention. The invention also provides the application of the nucleic acid aptamer. The nucleic acid aptamer of the present invention is screened by magnetic bead-SELEX technology. It can not only bind bPAG4 target protein, but also selectively bind bPAG similar proteins without specific binding with other proteins, and can be used to capture from complex systems The bPAG protein is suitable for the detection, separation and purification of bPAG protein and the rapid diagnosis of early pregnancy in cattle.

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 Patents(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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