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A goat pregnancy-associated glycoprotein nucleic acid aptamer and its application

A technology of nucleic acid aptamers and glycoproteins, applied in instruments, biochemical equipment and methods, analytical materials, etc.

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] Technical problem to be solved: Aiming at the deficiencies of the existing livestock early pregnancy detection technology, the present invention provides a sheep pregnancy-related glycoprotein nucleic acid aptamer and its application

Method used

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  • A goat pregnancy-associated glycoprotein nucleic acid aptamer and its application
  • A goat pregnancy-associated glycoprotein nucleic acid aptamer and its application
  • A goat pregnancy-associated glycoprotein nucleic acid aptamer and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Screening of goat PAG nucleic acid aptamers

[0032] 1. Synthetic random single-stranded DNA (ssDNA) library and primers:

[0033] Random single-stranded DNA (ssDNA) library:

[0034] 5'-CTACGGTGCCTTGAAGTGAC-N36-CATAGCAGGTCACTTCCAGG-3', wherein, N36 represents 36 random nucleotides, and the library was synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.;

[0035] Upstream primer: 5'-FAM-CTACGGTGCCTTGAAGTGAC-3',

[0036] Downstream primer: 5'- 20A-spacer18-CCTGGAAGTGACCTGCTATG-3',

[0037] 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.

[0038] Binding buffer solution (BB: NaCl 136.89mM, KCl 2.68 mM, Na 2 HPO 4 8.09 mM, KH 2 pH 4 1.47 mM, CaCl 2 0.9 mM, MgCl 6H 2 O 0.49 mM; PH 7 .4) were dissolved and stored at -20°C for later use.

[0039] 2. Magne...

Embodiment 2

[0052] Embodiment 2: Nucleic acid aptamer dissociation constant ( K d ) Determination

[0053] Select the nucleic acid aptamer with the highest binding ability in Example 1, and use BB buffer to prepare a series of aptamer solutions with gradient concentrations (0, 100, 200, 400, 800, 1600, 3200, 6400 nM); according to the example In Step 5 of 2, analyze the binding situation of the nucleic acid aptamer to MB-ovPAG7, use GraphPad Prism 7.0 software to fit the binding curve, and calculate the dissociation constant of the nucleic acid aptamer ( K d value). image 3 It is the saturation binding curve of the aptamer shown in SEQ ID No. 1 after truncating part of the X and Y sequences to the target protein ovPAG7, and its dissociation constant reaches 13.6 nM. Online Mfold was used to predict the secondary structure of the aptamer sequence, and the results were as follows: Figure 4 As shown, the aptamer has a typical stem-loop structure.

Embodiment 3

[0054] Embodiment 3: the specificity analysis of nucleic acid aptamer

[0055] The nucleic acid aptamer shown in SEQ ID No. 1 was truncated by 10 bases at both ends, and sent to Shanghai Sangong for synthesis, and the biotin group was labeled at the 5' end. Proteins such as ovPAG7, bPAG6, BSA, and OVA were diluted to 3 μg / mL with 10 mM PBS, then coated on a 96-well plate, and coated overnight at 4°C. Then the plate was washed 3 times with 200 μL of PBST (10 mM PBS, 0.05% Tween-20, pH 7.0), blocked by adding 200 μL of blocking solution (1% BSA) for 2 h, and washed 3 times with PBST. Add 100 μL of biotin-modified aptamer (100 nM), incubate for 20 min, wash 3 times with PBST, and pat dry. Add 100 μL of SA-HRP (0.5 U / mL) to each well, wash with PBST 3-5 times, pat dry, add 100 μL of TMB substrate chromogenic solution, and react for 5-7 min. Finally, 100 μL of stop solution was added to terminate the reaction, and the microplate reader was used at 450 nm (OD 450nm ) to measure i...

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Abstract

The present invention provides a nucleic acid aptamer of ovine pregnancy-associated glycoprotein (ovPAG). Derivatives derived from the sequences of the 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, has high affinity with sheep PAG protein, and can be used for separation, enrichment and detection of PAG protein, and rapid diagnosis of early pregnancy in livestock.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular, to a goat pregnancy-associated glycoprotein nucleic acid aptamer and its derivatives, and in particular to the use of the nucleic acid aptamer and its derivatives in the detection of pregnancy-associated glycoprotein (PAG) and the early stage of livestock Application in pregnancy diagnosis. Background technique [0002] In sheep breeding, the fecundity of ewes is the key to determine the production efficiency of sheep breeding industry. If we can accurately diagnose the early pregnancy of ewes after mating, distinguish between pregnant and non-pregnant ewes as soon as possible, and take timely recombination measures for non-pregnant ewes, it will not only help reduce the empty tire rate and empty tire time, but also improve the quality of ewes. Reproductive efficiency; It also helps to carry out group feeding management and reduce feeding costs. Therefore, early pregnancy tec...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/115G01N33/68G01N33/58G01N33/53
CPCC12N15/115C12N2310/16G01N33/53G01N33/581G01N33/689G01N2333/471
Inventor 刘长彬卢春霞
Owner XINJIANG ACADEMY OF AGRI & RECLAMATION SCI
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