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Aptamer of grass carp reovirus and derivative, screening method and application of aptamer

A nucleic acid aptamer, reovirus technology, applied in the field of molecular biology, can solve the problems of not being widely used, difficult screening process, etc.

Inactive Publication Date: 2017-08-11
PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, nucleic acid aptamers have not been widely used after more than 20 years of development. , the screening process is very difficult. To obtain aptamers for a certain target, it often requires continuous exploration and creation to obtain nucleic acid aptamer sequences with good application value.

Method used

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  • Aptamer of grass carp reovirus and derivative, screening method and application of aptamer
  • Aptamer of grass carp reovirus and derivative, screening method and application of aptamer
  • Aptamer of grass carp reovirus and derivative, screening method and application of aptamer

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Experimental program
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Effect test

Embodiment 1

[0065] Embodiment 1, the screening of the nucleic acid aptamer of grass carp reovirus

[0066] Use exponential enrichment ligand system evolution technology (SELEX technology) to screen the nucleic acid aptamers of grass carp reovirus, the SELEX technology schematic diagram can be found in figure 1 , the specific implementation method is as follows:

[0067] (1) Virus purification

[0068] GCRV was inoculated on FHM cells and cultured at 28°C until about 80% of the lesions appeared. After harvesting, it was frozen and thawed three times at –80°C, and the cell debris was removed by gradient centrifugation: 3000rpm / min, 15min; 5000rpm / min, 15min; 8000rpm / min, 20min; 10000rpm / min, 30min, to obtain GCRV virus liquid. At the same time, uninfected FHM cells were used as a control for the reverse screening of the screening process, and were also subjected to repeated freeze-thaw and gradient centrifugation under the same conditions to obtain FHM cell liquid, which was stored at –80...

Embodiment 2

[0090] Example 2, Spatial Structure Prediction of Nucleic Aptamers

[0091] Predict the space structure of the 10 nucleic acid aptamers whose sequences were screened out above and whose frequency of repetition was determined: according to the energy principle of the MFOLD software, the minimum energy structure was selected as the most stable structure of the aptamer.

[0092] The result is as figure 2 As shown, all aptamers can form a stem-loop structure, which forms a stem-loop from about thirteen oligonucleotides to twenty-seven oligonucleotides, which may be the aptamer binding The key site of the target and play a role.

Embodiment 3

[0093] Example 3, the effect of nucleic acid aptamers on the viability of FHM cells

[0094] The FHM cells were seeded in a 96-well plate. When the cells grew to 80%, the aptamer FT1 was added at a concentration of 0 μM to 10 μM, cultured at 28°C for 48 hours, 20 μl / well of MTT solution was added, and cultured at 28°C for 4 hours. The ELISA plate reader reads at 490nm.

[0095] Experimental results such as image 3 As shown, after the nucleic acid aptamer FT1 was added to the cells at a concentration of 0uM to 10uM, the cell viability did not change significantly, indicating that the nucleic acid aptamer would not produce toxic side effects on the host cells, which is beneficial to the later preparation of drugs and the application of detection reagents.

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PUM

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Abstract

The invention discloses aptamer of a grass carp reovirus (GCRV) and a derivative, screening method and application of the aptamer. The aptamer is an ssDNA library aiming at the GCRV and is obtained through an SELEX method, and the nucleotide sequence of the aptamer is as shown in optional one of SEQ ID No. 1-10. Experiments show that the aptamer has no evident influence on cell viability, can form a stem-ring secondary structure and can even inhibit GCRV infection, and the aptamer FT6 and the aptamer FT7 have the optimal antivirus effect. The aptamer has promising application prospect in the preparation of medicine for treating viral diseases and the preparation of grass carp reovirus molecular probes, detecting reagents or targeting media.

Description

technical field [0001] The invention belongs to the field of molecular biology, and more specifically relates to a nucleic acid aptamer of grass carp reovirus and its derivatives, screening method and application. Background technique [0002] Grass carp reovirus (GCRV) is recognized as the most virulent strain among aquatic reoviruses, causing grass carp hemorrhagic disease and causing massive fish deaths, with a mortality rate as high as 85%. So far, once the fish body is infected by GCRV, there is no effective antiviral treatment. Although vaccines can effectively prevent the disease, in recent years, virus variants have continuously emerged, and the use of a single vaccine has been limited. Therefore, the prevention of these diseases remains a great challenge in grass carp farming. [0003] Nucleic acid aptamer is a DNA or RNA oligonucleotide that can specifically bind to a target; due to its small molecular weight, chemical synthesis, good stability, non-toxicity, and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115A61K31/7088A61P31/14C12N15/10
CPCA61K31/7088C12N15/1048C12N15/115C12N2310/16C12N2330/31
Inventor 梁红茹李宁求付小哲刘礼辉林强黄志斌黄子君郭慧芝
Owner PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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