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Application of eEF1D protein in preparation of medicine for preventing or treating foot-and-mouth disease virus infection

A virus infection, virus technology, applied in applications, viruses, antiviral agents, etc., can solve problems such as weak effect

Active Publication Date: 2020-10-20
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Previous studies have shown that TANK-binding kinase 1 (TBK1) is the main kinase that phosphorylates IRF3 / 7, and is also a key kinase in the expression of IFN-β mediated by TLR and RLR; TBK1 can not only act as a new E3 ubiquitin ligase , and can effectively degrade viral VP3 protein, thereby significantly inhibiting the protein assembly of Picornaviridae, especially FMDV, and inhibiting the replication of Picornaviridae viruses, but TBK1 can only effectively degrade viral VP3 protein, while other structures protein is weak

Method used

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  • Application of eEF1D protein in preparation of medicine for preventing or treating foot-and-mouth disease virus infection
  • Application of eEF1D protein in preparation of medicine for preventing or treating foot-and-mouth disease virus infection
  • Application of eEF1D protein in preparation of medicine for preventing or treating foot-and-mouth disease virus infection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Changes in eEF1D mRNA and protein expression levels in cells detected after infection with foot-and-mouth disease virus in embodiment 1

[0052] 1. Preparation of PK-15 cell samples infected with FMDV

[0053] will be 6×10 5 Each PK-15 cell was plated in a single well of a 6-well plate, and a total of 3 dishes were plated. When the cells grow to 70%-80% confluence, use liposome reagents to transfect 0, 1, and 2 μg of FLAG-eEF1D plasmid respectively, transfect for 24 hours, inoculate with 1 MOI of foot-and-mouth disease virus, and change to maintenance medium culture after inoculation for 1 hour .

[0054] 2. qPCR detection of eEF1D mRNA expression level

[0055] The cells infected with foot-and-mouth disease virus were harvested at 0, 4, 6, 8, 10 and 12 hours respectively, and washed once with PBS. The RNA extraction process is as follows:

[0056] Add 1mL Trizol reagent, pipette vigorously to completely lyse the cells for 5-15min, and transfer the liquid to a 1.5m...

Embodiment 2

[0068] Example 2 Evaluation of the effect of eEF1D protein inhibiting the replication of foot-and-mouth disease virus

[0069] 1. Construction of FLAG-eEF1D plasmid

[0070] The FLAG-eEF1D plasmid was constructed by Lanzhou Ruibolai Biotechnology Co., Ltd. For information on plasmid construction, see image 3 As shown, the eEF1D gene is synthesized according to the porcine eEF1D gene sequence, and the accession number is: XM_021090459.1.

[0071] 2. Preparation of PK-15 cell samples infected with FMDV

[0072] will be 6×10 5 PK-15 cells were plated in a single well of a 6-well plate, and a total of 3 dishes were plated. When the cells grow to 70%-80% confluence, use liposome reagents to transfect 0, 1, and 2 μg of FLAG-eEF1D plasmid respectively, transfect for 24 hours, inoculate with 1 MOI of foot-and-mouth disease virus, and change to maintenance medium culture after inoculation for 1 hour .

[0073] 3. Detection of viral protein content

[0074] The detection method i...

Embodiment 3

[0092] Example 3 Overexpression of eEF1D lentivirus and construction of cell lines

[0093] The gene sequence of eEF1D was synthesized by Shanghai Gemma Pharmaceutical Technology Co., Ltd.

[0094] The lentiviral vector plasmid was digested with a restriction endonuclease for 2 hours, and then the lentiviral plasmid and the eEF1D gene were ligated. The ligation product was transferred into competent cells, and the successfully constructed lentiviral plasmid was extracted.

[0095] Enzyme digestion system (10μL):

[0096]

[0097] Ligation reaction system (10μL):

[0098]

[0099] After the host cells grew to about 80%, the constructed lentiviral plasmid was packaged according to the instructions of the lentiviral packaging kit to obtain Lenti-eEF1D-puro lentivirus. The lentivirus can promote the overexpression of eEF1D protein, and can be used to prepare vaccines or vaccine compositions for preventing or treating picornaviridae virus infection.

[0100] Infect the ce...

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Abstract

The invention belongs to the technical field of biology, and particularly relates to application of an eEF1D protein in preparation of a medicine for preventing or treating foot-and-mouth disease virus infection. The invention finds that the expression of the eEF1D mRNA is increased and the content of the eEF1D protein is reduced after the foot-and-mouth disease virus infection, namely, the eEF1DmRNA and the eEF1D protein can be used as detection markers for the foot-and-mouth disease virus infection and are used for evaluating whether the foot-and-mouth disease virus infection occurs or not;secondly, the eEF1D protein can remarkably reduce the structural protein VP1 of the foot-and-mouth disease virus, the expression of mRNA of the foot-and-mouth disease virus and the titer of the foot-and-mouth disease virus, remarkably inhibits the replication of the foot-and-mouth disease virus, and can be used for preventing or treating the infection of the foot-and-mouth disease virus; and finally, after knocking down the eEF1D protein, the expression of the foot-and-mouth disease virus structural protein VP1 is promoted, and the expression of the foot-and-mouth disease virus mRNA and the virus titer are improved. Therefore, the eEF1D protein can be knocked down in a production cell strain through a genetic engineering means, and a cell line with better performance than that of the foot-and-mouth disease virus vaccine produced by the existing production cell line is obtained and is used for producing foot-and-mouth disease virus vaccines.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the application of eEF1D protein in the preparation of medicines for preventing or treating foot-and-mouth disease virus infection. Background technique [0002] Picornavirus (RNA) Viridae is a family composed of the smallest group of RNA viruses, mainly including Enterovirus, Rhinovirus, Cardiovirus and Oral Virus, and the Picornaviridae four genera of viruses Structural proteins have a high degree of homology. Among them, foot-and-mouth disease belongs to the genus Aphthovirus of the family Picornaviridae, and is an important disease that infects cloven-hoofed animals caused by foot-and-mouth disease virus. [0003] Foot-and-mouth disease is an acute, febrile and contact infectious disease caused by foot and mouth disease virus (FMDV) infecting artiodactyls (pigs, cattle, sheep, camels, etc.). The nucleic acid type of FMDV is single-stranded positive-sense RNA, includ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12N5/10C12N15/12C12N15/867C12N15/113C07K14/47G01N33/68G01N33/569A61K45/00A61P31/14
CPCA61K45/00A61P31/14C07K14/4702C12N15/113C12N15/86C12N2740/15043C12Q1/701C12Q2600/158C12N2310/20G01N33/56983G01N33/68
Inventor 郑海学刘会胜薛巧朱紫祥杨帆薛钊宁曹伟军刘湘涛
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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