Application of cefradine as bovine enterovirus inhibitor

An enterovirus, cefradine technology, applied in antiviral agents, medical preparations containing active ingredients, organic active ingredients, etc., can solve problems such as decreased milk production, economic losses in the cattle industry, and even blood in the stool.

Active Publication Date: 2020-01-17
DAIRY CATTLE RES CENT SHANDONG ACADEMY OF AGRI SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

After the virus was first reported by Moll and Davis in 1959, it has been widely found and prevalent in major cattle-raising countries and regions around the world. It has been found in cattle farms in many regions of my country, and it can cause various syndromes in cattle. It is mainly transmitted through the fecal-oral route, and the source of infection is mainly sick cows and asymptomatic virus carriers. Cows infected with bovine enterovirus show mild diarrhea and mild respiratory symptoms, loss of appetite, and even blood in the stool in severe cases, and milk production. The sharp decline has caused severe economic losses to the cattle industry

Method used

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  • Application of cefradine as bovine enterovirus inhibitor
  • Application of cefradine as bovine enterovirus inhibitor
  • Application of cefradine as bovine enterovirus inhibitor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Separation and identification of BEV

[0033] Dilute fresh cow feces with PBS, freeze-thaw repeatedly for 3 times, centrifuge at 5,000 r / min for 5 min, take the supernatant and add penicillin (200IU / mL) and streptomycin (100μg / mL), take 200μL of the supernatant, follow Viral genomic DNA / RNA extraction kit instructions to extract BEV viral genomic RNA, reverse transcription into cDNA, PCR amplification with BEV specific primers, the stool supernatant identified as positive by PCR is filtered and sterilized with a 0.22um filter membrane , Take 1ml of inoculated MDBK monolayer cells, adsorb them at 37°C for 1h, discard them, and add them to DMEM medium containing 2% FBS. 2 Cultivate in a cell incubator, observe the cytopathic condition every 12h, and observe continuously for 4 days. Freeze and thaw the cells with lesions (CPE) three times, collect the cell virus solution, and identify whether the obtained cell virus is a BEV single infection. If it is not a single v...

Embodiment 2

[0035] Example 2 Virus TCID 50 Determination of

[0036] Digest the MDBK cells to 3×10 per well 5 Cell density of cells / mL was inoculated into 96-well cell culture plate, placed in 37℃, 5% CO 2 After culturing into a monolayer of cells in a cell incubator, discard the cell growth medium in the well, and dilute the bovine enterovirus with a 10-fold serial dilution of the virus (the dilution is 10 -1 -10 -10 ) Inoculated in a 96-well plate full of monolayer cells, 100μL per well, placed in 37℃, 5% CO 2 Continue to cultivate in the incubator, observe the CPE of the cells daily, and record the number of cytopathic wells in detail. At the same time, a normal cell control group and a blank control group were set up, with 8 replicates in each group, and the results were determined when the cytopathic changes no longer occurred. The cytopathic hole is the corresponding cell hole of the above cells, and the virus TCID is calculated according to Karber method 50 .

[0037] Table 1 TCID 50 o...

Embodiment 3

[0045] Example 3 Toxicity experiment of cefradine on MDBK cells:

[0046] MDBK cells are susceptible cells to bovine enterovirus. Therefore, firstly, the cytotoxicity of cefradine on MDBK cells was tested, and the specific experimental procedures were as follows:

[0047] (1) Inoculate 100μL cells (MDBK 3×10 4 Pcs / hole).

[0048] (2) After culturing for about 12 hours, proceed to the next step of drug addition analysis. The culture medium was discarded, and 100 μL of 2% FBS DMEM containing different drug concentrations was added to each well, and three parallels were made for each concentration. At the same time control wells: add 100μL of 2% FBS DMEM medium. Zero adjustment hole: do not plate cells.

[0049] (3) At 37℃, 5% CO 2 After culturing for 48 hours under the conditions, operate according to the CCK-8 kit instructions, and measure the OD value at 490nm with a microplate reader.

[0050] (4) 37℃, 5% CO 2 After continuing to culture for 4 hours under the conditions, the absorb...

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Abstract

The invention belongs to the technical field of prevention and treatment of bovine enterovirus, and particularly relates to application of cefradine as a bovine enterovirus inhibitor. The bovine enterovirus can cause gastrointestinal diseases and respiratory symptoms of cattle, and has serious harm to the breeding industry, research on the bovine enterovirus is little at present, and moreover, effective prevention and treatment drugs have are not disclosed yet. The invention provides application of the cephradine in inhibiting and killing the bovine enterovirus. The research shows that the cefradine can inhibit and kill the bovine enterovirus on an in-vitro MDBK cell model, can effectively inhibit invasion and replication of the bovine enterovirus, has low cytotoxicity, can be used as a new class of bovine enterovirus resisting drugs, lays a foundation for prevention and control of the bovine enterovirus and research and development of the drugs, and also lays an experimental foundation and provides a new visual field for development of high-efficiency specific bovine enterovirus resisting drugs.

Description

Technical field [0001] The present disclosure belongs to the technical field of bovine enterovirus prevention and treatment, and specifically relates to the application of cefradine as a bovine enterovirus inhibitor. Background technique [0002] Disclosure of the background information is only intended to increase the understanding of the overall background of the present disclosure, and is not necessarily regarded as an acknowledgement or in any form suggesting that the information constitutes the prior art known to those of ordinary skill in the art. [0003] Bovine enterovirus (BEV) and monkey enterovirus, porcine enterovirus, human Coxsackie virus, poliovirus, human intestinal cytopathic vibriovirus, etc. belong to the Picornavirus family, A member of the genus Virus, the size of the virus particle is 25-30nm, and it is a spherical, non-enveloped single-stranded RNA virus. According to the latest classification of viruses, bovine enteroviruses belong to the enterovirus specie...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/545A61P31/14
CPCA61K31/545A61P31/14
Inventor 程凯慧楚会萌杨宏军任亚初解晓莉张亮于志君孙阳阳朱彤
Owner DAIRY CATTLE RES CENT SHANDONG ACADEMY OF AGRI SCI
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