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Cholesterol hydroxylase CH25H and application thereof

A cholesterol and hydroxylase technology, applied in the field of biotechnology and chemistry, can solve the problems of no approved safe antiviral therapy, no cholesterol-25-hydroxylase anti-enterovirus, etc.

Active Publication Date: 2017-11-24
INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there is no specific vaccine against EV68, and there is no approved safe antiviral therapy that can be used in clinical treatment. There is an urgent need to develop specific and effective preventive and therapeutic drugs to curb the harm of the virus
But there is no report of cholesterol-25-hydroxylase against enterovirus, especially EV71

Method used

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  • Cholesterol hydroxylase CH25H and application thereof
  • Cholesterol hydroxylase CH25H and application thereof
  • Cholesterol hydroxylase CH25H and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] Example 1. Changes in CH25H transcription levels after virus infection

[0091] RD cells (human rhabdomyosarcoma cells, purchased from ATCC, USA) were infected with 1 MOI (multiplicity of infection) EV71 virus (Beijing strain BJ / CHN / 2008), and the cells were harvested at 4h, 8h, and 24h after infection, and extracted Cellular total RNA was subjected to fluorescent quantitative PCR after reverse transcription to detect the mRNA transcription level of CH25H. The experiment was repeated 3 times, and the experimental results were averaged.

[0092] The specific method is as follows:

[0093] 1.1 Amplification of EV71 virus

[0094] Take 30 μl of EV71 virus and 10% FBS (fetal bovine serum, purchased from American Hyclone Company, product number 30396) in DMEM medium (purchased from American Gibco Company, product number C11965500) mixed and inoculated on T75 (growth area 75cm 2 ) in RD cells with 80%-90% abundance (cell fraction per unit area, that is, the extended densit...

Embodiment 2

[0118] The impact of embodiment 2.CH25H overexpression on EV71 virus replication

[0119] RD cells were transfected with different doses of CH25H expression vectors. After 24 hours of transfection, 1 MOI of EV71 virus was added, and the cells were harvested after 24 hours of continuous culture. Total RNA was extracted from the cells. After reverse transcription, fluorescent quantitative PCR was performed to detect the mRNA transcription level of EV71. The experiment was repeated 3 times, and the experimental results were averaged.

[0120] 2.1 Amplification of EV71 virus

[0121] With embodiment 1.1.

[0122] 2.2 Transfection of RD cells with CH25H expression vector

[0123] 1.5×10 RD cells per well 6 Cells were seeded in a 24-well plate, and the next day when the cells grew to 60%-70% abundance (the extension density of the cells in the culture plate reached 60-70% of the bottom area of ​​the culture plate), they could be transfected, before transfection Change to serum-f...

Embodiment 3

[0136] Example 3.CH25H inhibits the replication of EV71 virus

[0137] RD cells were transfected with different doses of CH25H expression vectors. After 24 hours of transfection, 1 MOI of EV71 virus was added, and the cells were cultured for 24 hours to harvest the cells. The structural proteins of EV71 virus were detected by Western blotting. The experiment was repeated 3 times, and the experimental results were averaged.

[0138] The specific method is as follows:

[0139] 3.1 Amplification of EV71 virus

[0140] With embodiment 1.1.

[0141] 3.2 Transfection of RD cells with CH25H expression vector

[0142] With embodiment 2.2.

[0143] 3.3 Infection of transfected RD cells with EV71 virus

[0144] With embodiment 2.3.

[0145] 3.4 Western blot detection of EV71 virus structural proteins

[0146] β-actin (β-actin) was used as an internal reference for Western blot detection:

[0147] (1) The cells harvested in each group of the above steps were processed as follows:...

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Abstract

The invention provides application of cholesterol-25-hydroxylase, nucleotide sequence for encoding cholesterol-25-hydroxylase or a matter for inducing the cholesterol-25-hydroxylase expression in preparing a drug for preventing and / or treating diseases and / or symptoms caused by enterovirus infection, a dosing method and a preparation suitable for the drug, other active ingredients contained in the drug and a combined medicine of the drug and other drugs. The invention also provides a method for achieving a non-therapeutic purpose for restraining enterovirus in the in vitro cells by utilizing a cholesterol-25-hydroxylase expression vector and the application of at least one of the cholesterol-25-hydroxylase, the nucleotide sequence for encoding cholesterol-25-hydroxylase or the matter for inducing the cholesterol-25-hydroxylase expression in achievement of the non-therapeutic purpose for restraining enterovirus in the in vitro cells.

Description

technical field [0001] The invention belongs to the field of biotechnology and chemistry. Specifically, the invention relates to cholesterol-25-hydroxylase, a nucleic acid sequence encoding cholesterol-25-hydroxylase and substances for inducing the expression of cholesterol-25-hydroxylase for the preparation of Use of anti-enteroviral drugs. Background technique [0002] The genus Enterovirus belongs to Picornaviridae, a class of single-stranded positive-sense RNA viruses with similar biological properties, without envelope, and with a genome length of about 7.2-8.4kb. Enteroviruses above the respiratory tract, throat and intestinal tract are the portals of invasion, and first proliferate in the local mucosa, pharynx, tonsil and other lymphoid tissues and intestinal collection lymph nodes, and then release into the blood, forming the first viremia and spreading to the local area. The target tissue with receptors will proliferate again, causing the second viremia and clinica...

Claims

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Application Information

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IPC IPC(8): A61K38/44A61K48/00A61K45/00A61P31/14
CPCA61K38/44A61K45/00A61K48/005C12Y114/99038
Inventor 王健伟雷晓波肖霞张珍珍
Owner INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
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