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Nucleic acid aptamer for specifically recognizing bovine pregnancy-associated glycoproteins and application of nucleic acid aptamer

A nucleic acid aptamer and glycoprotein technology, which is applied in the fields of biochemical equipment and methods, material testing products, instruments, etc., can solve the problems such as no research reports on livestock pregnancy-related glycoprotein nucleic acid aptamers, etc., and achieve good affinity. and specificity, short production cycle, easy chemical modification effect

Active Publication Date: 2020-06-26
XINJIANG ACADEMY OF AGRI & RECLAMATION SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

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-associated glycoproteins and application of nucleic acid aptamer
  • Nucleic acid aptamer for specifically recognizing bovine pregnancy-associated glycoproteins and application of nucleic acid aptamer
  • Nucleic acid aptamer for specifically recognizing bovine pregnancy-associated glycoproteins and application of nucleic acid aptamer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Screening of bPAG nucleic acid aptamers

[0031] 1. Synthetic random single-stranded DNA (ssDNA) library and primers:

[0032] Random single-stranded DNA (ssDNA) library:

[0033] 5'-CTACGGTGCCTTGAAGTGAC-N36-CATAGCAGGTCACTTCCAGG-3', wherein, N36 represents 36 random nucleotides, and the library was synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.;

[0034] Upstream primer: 5'-FAM-CTACGGTGCCTTGAAGTGAC-3',

[0035] Downstream primer: 5'- 20A-spacer18-CCTGGAAGTGACCTGCTATG-3',

[0036] 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.

[0037] The random single-stranded DNA library, 5' end primer, and 3' end primer were respectively washed with PBS buffer (PBS, NaCl: 8 g / L, KCl: 0.2 g / L, NaCl: 0.2 g / L, Na 2 HPO 4 : 1.15 g / L, KH 2 pH 4 : 0.2 g / L; PH 7 .4) dis...

Embodiment 2

[0049] Embodiment 2: SPR detects the dissociation constant of nucleic acid aptamer and bPAG9 protein

[0050] Take several pieces of ssDNA synthesized in Example 1, that is, nucleic acid aptamers, and each nucleic acid aptamer is prepared into a series of gradient concentration aptamer solutions with PBS; couple bPAG9 protein on the chip according to step 4, and then mix different concentrations of The aptamer solution is injected into the chip coupled with bPAG9 sequentially, and the affinity of each nucleic acid aptamer and bPAG9 protein is detected, and it is found that the nucleic acid aptamer of the sequence shown in SEQ ID No.1 and SEQ ID No.2 has the same affinity with the bPAG9 protein High affinity, the dissociation constants obtained by SPR detection are all in nM level (see image 3 ); wherein, sequence 1 is 1.04 nM for the dissociation constant of the nucleic acid aptamer of the sequence shown in SEQ ID No.1 to the bPAG9 protein, and sequence 2 is the nucleic acid ...

Embodiment 3

[0051] Embodiment 3: SPR detects the binding specificity of nucleic acid aptamer and bPAG protein

[0052] Dilute the four similar proteins of bPAG4, bPAG6, bPAG9 and bPAG16, as well as BSA and OVA proteins with sodium acetate solution (pH5.0) to a concentration of 50 μg / mL, and couple the proteins on different channels of the chip according to step 4. The nucleic acid aptamer sequences shown in SEQ ID No. 1 and SEQ ID No. 2 were all prepared in PBS to a concentration of 500 nM, and the binding of each nucleic acid aptamer to each protein was determined according to step 4. Test results such as Figure 5 , the nucleic acid aptamers shown in SEQ IDNo.1 and SEQ IDNo.2 can all be combined with b-PAG 4, b-PAG 6, b-PAG 9, b-PAG 16 in the bPAG protein family, but not with BSA Binds to OVA protein.

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Abstract

The invention discloses a nucleic acid aptamer for specifically recognizing bovine pregnancy-associated glycoproteins (bPAG) and application of the nucleic acid aptamer. The sequence of the nucleic acid aptamer is a DNA (deoxyribonucleic acid) fragment shown in a sequence SEQ ID No.1 or a DNA fragment shown in a sequence SEQ ID No.2. The invention further discloses a nucleic acid aptamer derivative. The nucleic acid aptamer derivative which has functions identical to those of the nucleic acid aptamer is obtained through framework modification or basic group transformation on sequences of the nucleic acid aptamer. The nucleic acid aptamer disclosed by the invention is obtained through screening of a magnetic bead-SELEX (systematic evolution of ligands by exponential enrichment) technique, can be specifically combined with a bPAG protein family, is not specifically combined with other proteins, is easy to synthesize and modify, can be used for capturing the bPAGs from complex systems, isapplicable to detection and separation purification of the bPAGs and rapid diagnosis of early pregnancy of livestock, and has wide application prospects.

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-related glycoproteins, and in particular to the application of the nucleic acid aptamer in the detection of pregnancy-related glycoproteins and the diagnosis of early pregnancy in livestock. 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 early pregnancy ...

Claims

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Application Information

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