Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Sheep pregnancy-related glycoprotein aptamer and application thereof

A nucleic acid aptamer and glycoprotein technology, applied in biochemical equipment and methods, material inspection products, instruments, etc., to achieve the effects of low production cost, reduced production cost and detection cost, good affinity and specificity

Active Publication Date: 2021-01-26
XINJIANG ACADEMY OF AGRI & RECLAMATION SCI
View PDF3 Cites 2 Cited by
  • 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Sheep pregnancy-related glycoprotein aptamer and application thereof
  • Sheep pregnancy-related glycoprotein aptamer and application thereof
  • Sheep pregnancy-related glycoprotein aptamer and application thereof

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 stop the reaction, and the microplate reader was used at 450 nm (OD 450nm ) to measure its ab...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention provides a sheep pregnancy-related glycoprotein (ovPAG) aptamer. The sequence of the aptamer is shown as SEQ ID No. 1, and the aptamer can also be various similar sequences with relatively high homology or derivatives obtained from the sequences. The invention also provides application of the aptamer. The aptamer is obtained by screening through a magnetic bead-SELEX technology, hashigh affinity with sheep PAG protein, and can be used for separation, enrichment and detection of the PAG protein and rapid diagnosis of early pregnancy of 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
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
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com