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Aptamer realizing specific binding with newcastle disease virus as well as screening method and applications of aptamer

A nucleic acid aptamer, Newcastle disease virus technology, applied in the field of molecular biology, can solve the problems affecting the efficiency of screening and ligand affinity, and achieve the effects of shortening incubation time, reducing differences, and improving efficiency

Active Publication Date: 2017-08-18
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Mix the screening library and target molecules in a certain ratio, and select appropriate conditions (medium, temperature, buffer system, time, etc. for incubation). Although this step is simple, different conditions will affect the efficiency of screening and the affinity of the ligand.

Method used

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  • Aptamer realizing specific binding with newcastle disease virus as well as screening method and applications of aptamer
  • Aptamer realizing specific binding with newcastle disease virus as well as screening method and applications of aptamer
  • Aptamer realizing specific binding with newcastle disease virus as well as screening method and applications of aptamer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1. In vitro screening of Newcastle disease virus nucleic acid aptamers

[0047] S1. Construct a random nucleic acid library with a synthetic length of 81 nt, the sequence of which is shown in SEQ ID NO.1, 5'-CCGGAATTCCTAATACGACTC–(N)40–TATTGAAAACGCGGCCGCGG-3'; where the two ends are 20 and 21 bases respectively The sequence is fixed, and the middle 40 bases are random sequences.

[0048] The sequences of the upstream primer F and the downstream primer R synthesized for PCR amplification are:

[0049] Upstream primer F: 5'-CCGGAATTCCTAATACGACTC-3', as shown in SEQ ID NO.2;

[0050] Downstream primer R: 5'-CCGCGGCCGCGTTTTCAATA-3', as shown in SEQ ID NO.3;

[0051] Biotin-modified upstream primer Biotin-F: 5'-Biotin-CCGGAATTCCTAATACGACTC-3', as shown in SEQ ID NO.4.

[0052] S2. Incubation and separation of ssDNA library and HN protein, add 35.5 μg ssDNA library to 100 μL binding buffer (50 mM Tris-HCl, 25 mM NaCl, 5 mM MgCl2, 10 mM DTT (dithiothreitol), pH 7.5)...

Embodiment 2

[0063] Example 2. Enzyme-linked immunosorbent assay (ELISA)

[0064] The nucleic acid aptamers obtained by screening were sent to Sangon Bioengineering (Shanghai) Co., Ltd. for synthesis, modified with biotin, and tested for specificity by ELISA method. Different samples were selected, the coating volume was 2 μL / well, and a blank control group was set up at the same time. Coat overnight at 4°C, block with 1% BSA at 37°C, add different biotin-labeled aptamers to the wells for incubation, then add horseradish peroxidase-labeled avidin for incubation, add substrate The substance was used for color development, the color development was terminated, and the OD was measured with a microplate reader 450 value.

[0065] image 3 The ELISA detection of the aptamer B53 screened for SELEX, Figure A shows the affinity of the aptamer to different strains of Newcastle disease virus F48E9, GM and La Sota. like image 3 As shown, the results show that the aptamer B53 has affinity to New...

Embodiment 3

[0066] Embodiment 3. Dot-blot test (Dot-blot)

[0067] Spot 2 μL of HN protein (1mg / mL), GM strain purified virus, AIV, IBV, SPF chick embryo allantoic fluid onto the NC membrane, and set up a blank control group without dripping the solution; after the NC membrane is dry, Add 3% BSA blocking solution to block at 37°C for 2 hours, wash the membrane with PBST solution; add 5 pmol / μL biotin-labeled ssDNA to the NC membrane, incubate at 37°C for 2 hours, then wash the membrane with PBST solution ; Add the NC membrane to the 1:5000 diluted Streptavidin-HRP solution, incubate at 37°C for 1 hour, then wash the membrane with PBST solution for 3 minutes, each time for 3 minutes; stop the color development after 10 minutes of DAB display, take out the membrane, Observe the color development of the membrane.

[0068] The results of aptamer specificity analysis were as follows: Figure 4 as shown, Figure 4 A represents the results of the aptamer against different strains of Newcastle...

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Abstract

The invention discloses an aptamer realizing specific binding with the newcastle disease virus as well as a screening method and applications of the aptamer. The aptamer is specific aptamer B53 obtained through screening by adopting the SELEX technology. Specifically, the random single stranded DNA library and primers are constructed and synthesized in vitro, the newcastle disease HN protein and the newcastle disease virus GM strain realizing prokaryotic expression are adopted as target molecules, after screening, PCR amplification, positive screening and inverse screening, the screened single stranded DNA library realizing specific binding with the newcastle disease virus GM strain is subjected to clone sequencing, and thus the specific aptamer B53 is obtained. The aptamer B53 has the capacity of inhibiting virus replication, virus blood clotting activity and plaque formation, can specifically recognize the newcastle disease virus HN protein and the newcastle disease virus GM strain, has no reaction to avian influenza virus, infectious bursal disease virus and SPF chick embryo allantoic fluid, and can be used for detecting the newcastle disease virus.

Description

technical field [0001] The invention belongs to the field of molecular biology, and more specifically relates to a nucleic acid aptamer specifically binding to Newcastle disease virus, a screening method and application thereof. Background technique [0002] Newcastle Disease (ND) is a highly contagious and lethal severe infectious disease caused by Newcastle Disease Virus (NDV). The main damages to the respiratory tract, digestive tract and central nervous system are caused by poultry infection. feature. NDV belongs to the Paramyxoviridae family. It is a single-strand, non-segmented, and enveloped negative-strand RNA virus. The genome structure is 3′-NP-P-M-F-HN-L-5′, encoding 6 structural proteins. Currently, only a serotype. The disease is distributed worldwide and has caused huge economic losses to the poultry industry. For the poultry industry in most countries, not only the outbreak of Newcastle disease has caused huge economic losses, but also the cost of controlli...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115C12N15/10G01N33/569
CPCC12N15/1048C12N15/115C12N2310/16G01N33/56983C12N2330/31
Inventor 任涛谭阳通谢鹏高潇祎王峰
Owner SOUTH CHINA AGRI UNIV
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