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Carbon monoxide release molecule-2 preparation, application of carbon monoxide release molecule-2 preparation in preparing medicines for resisting porcine reproductive and respiratory syndrome viruses and detection method of carbon monoxide release molecule-2 preparation

A technology for respiratory syndrome and carbon monoxide, applied in the field of anti-virus research, can solve the problem that vaccines cannot provide effective and sustainable disease control, and achieve the effect of mature production process, obvious effect, and easy transportation

Inactive Publication Date: 2017-02-22
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Porcine reproductive and respiratory syndrome poses a serious threat to the entire swine industry. Due to the lack of understanding of the molecular mechanism of immune escape of the disease, the current use of vaccines does not provide effective and sustainable disease control, especially for those RNA species. Viruses are powerless; meanwhile, based on the concern and demand for healthy and safe food, the use of animal antibiotics has caused more and more consumers to oppose

Method used

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  • Carbon monoxide release molecule-2 preparation, application of carbon monoxide release molecule-2 preparation in preparing medicines for resisting porcine reproductive and respiratory syndrome viruses and detection method of carbon monoxide release molecule-2 preparation
  • Carbon monoxide release molecule-2 preparation, application of carbon monoxide release molecule-2 preparation in preparing medicines for resisting porcine reproductive and respiratory syndrome viruses and detection method of carbon monoxide release molecule-2 preparation
  • Carbon monoxide release molecule-2 preparation, application of carbon monoxide release molecule-2 preparation in preparing medicines for resisting porcine reproductive and respiratory syndrome viruses and detection method of carbon monoxide release molecule-2 preparation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 qPCR detection of intracellular PRRSV ORF7 gene mRNA relative expression sample collection

[0031] PRRSV was inoculated into 80% confluent Marc-145 cells, incubated at 37°C for 1 hour, the virus solution was discarded, the cells were washed 3 times with 1×PBS, and CORM-2 containing different concentrations (50 μM, 100 μM, 150 μM) and 3% DMEM maintenance medium culture of fetal bovine serum based on 37°C, 5% CO 2 cultured in a humidified incubator; at the same time, the DMEM maintenance medium containing 3% fetal bovine serum without CORM-2 and the maintenance medium containing 150 μM iCORM-2 were used as controls, and the cells and supernatant were collected at 24 hpi.

[0032] Total RNA extraction from cell samples

[0033] (1) per 10 7 Add 1ml Trizol to each cell, pipette and mix until no visible precipitation is observed, and let stand at room temperature for 5 minutes.

[0034] (2) Add chloroform (1 / 5 the volume of Trizol) to the above-mentioned homoge...

Embodiment 2

[0064] Embodiment 2Western blot detects the impact of CORM-2 on PRRSV infection Marc-145 cells

[0065] The protein samples detected by Western blot were from the cell samples collected in Example 1.

[0066] (1) Preparation: Rinse the beaker, electrophoresis apparatus and its accessories with tap water, then rinse with deionized water, and dry them for later use. The glass plate and the comb were rinsed separately, then rinsed with deionized water, and dried for later use.

[0067] (2) Preparation of 12% separating gel First assemble the rubber plate, and find the usage amount and ratio of each component according to the following table according to the required separating gel concentration, as shown in Table 6.

[0068] Table 6 12% separating gel formula

[0069]

[0070] Add ultrapure water, 30% acrylamide, 1.5M Tris-HCl buffer solution, and 10% APS to the beaker in turn, fully exhaust the air in the mixture with an aspirator, then add 10% SDS, and finally add TEMED. ...

Embodiment 3

[0086] Example 3 Different concentrations of CORM-2 treatment Marc-145 cell culture supernatant PRRSV TCID 50 Determination of

[0087] (1) Plate, adjust the density of trypsinized Marc145 cells to 1×10 5 cells / ml, added to 96 empty cell culture plates, 175μl / well about 24h to grow into a monolayer.

[0088] (2) In a 96-well cell culture plate, use serum-free DMEM medium to make serial 10-fold gradient dilutions of the virus liquid (the virus liquid comes from the supernatant collected in Example 1), from 10 -1 ~10 -10 .

[0089] (3) Inoculate the diluted virus onto the Marc145 monolayer cells, inoculate a vertical row of 8 wells for each dilution, inoculate 100 μL in each well, and make two vertical rows of normal cells as controls, at 37°C, 5% CO 2 After incubation for 1 h, replace with 3% FBS+DMEM maintenance solution and place at 37°C, 5% CO 2 cultured in a humidified incubator.

[0090] (4) Observe and record the results day by day, usually 5 to 7 days.

[0091] (5...

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Abstract

The invention discloses a carbon monoxide release molecule-2 preparation, an application of the carbon monoxide release molecule-2 preparation in preparing medicines for resisting porcine reproductive and respiratory syndrome viruses and a detection method of the carbon monoxide release molecule-2 preparation. The carbon monoxide release molecule-2 preparation is Chinese name of CORM-2 (tricarbonyldichlororuthenium (II) dimer). In-vitro experiments prove that the carbon monoxide release molecule-2 has a significant effect of obviously inhibiting replication of the porcine reproductive and respiratory syndrome viruses, therefore a strong support is provided for enhancing prevention and control of the porcine reproductive and respiratory syndrome viruses through drug therapy.

Description

technical field [0001] The invention belongs to the technical field of antiviral research, and in particular relates to a carbon monoxide releasing molecule-2 preparation and its application and detection method in the preparation of anti-porcine reproductive and respiratory syndrome virus drugs. Background technique [0002] Porcine reproductive and respiratory syndrome (PRRS), also known as porcine blue ear disease, is caused by porcine reproductive and respiratory syndrome virus (Porcine repsoductive and respiratory syndrome virus, PRRSV), can cause pregnant women Pig reproductive disorders (abortion, weak fetus, stillbirth, mummified fetus, etc.) and pigs of all ages, especially piglet respiratory symptoms (interstitial pneumonia) and high mortality are the main infectious diseases of pigs. The industry is very dangerous. [0003] Porcine reproductive and respiratory syndrome poses a serious threat to the entire swine industry. Due to the lack of understanding of the mo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K33/24A61P31/14
CPCA61K33/24
Inventor 肖书奇周恩民张昂克
Owner NORTHWEST A & F UNIV
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