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HO-1 and HO-1 inducer for inhibiting PRRS virus (PRRSV) infection as novel blocker

A virus infection, HO-1 technology, applied in antiviral agents, medical preparations containing active ingredients, peptide/protein components, etc., can solve the problem of unclear pathogenic mechanism and immune mechanism

Active Publication Date: 2013-12-25
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because PRRSV has the characteristics of antigenic variability, macrophage, antibody-dependent enhancement (ADE) and persistent infection, it is often mixed with other pathogens in clinical practice, and the pathogenesis and immune mechanism of the disease are still unclear. Clearly, so there are still no effective preventive measures

Method used

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  • HO-1 and HO-1 inducer for inhibiting PRRS virus (PRRSV) infection as novel blocker
  • HO-1 and HO-1 inducer for inhibiting PRRS virus (PRRSV) infection as novel blocker
  • HO-1 and HO-1 inducer for inhibiting PRRS virus (PRRSV) infection as novel blocker

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] The preparation of embodiment 1 CoPP and hemin

[0016] Dissolve CoPP and hemin (both purchased from Sigma, USA) in 0.2 mol / L NaOH, adjust the pH value to 7.4 with 1 mol / L HCL, then dilute with PBS, and filter through a 0.2 μm filter membrane Sterilize, wrap in tinfoil after aliquoting, and store at -80°C for later use.

Embodiment 2

[0017] Example 2 CoPP and hemin treatment of cells infected by PRRSV

[0018] Marc-145 cells and PAM (10 5 cells / ml) were cultured in six-well cell cultures containing DMEM and RPMI-1640 medium (containing penicillin 100 U / ml, streptomycin sulfate 50 μg / ml, gentamicin 50 μg / ml, 10% fetal bovine serum) plate, 37°C, 5% CO 2 Culture in a humidified incubator to a confluence of 70% to 80%, and discard the medium. Insert 0.1MOI of PRRSV into each well, absorb at 4°C for 1 hour (h), incubate at 37°C for 1h, discard unadsorbed virus liquid, and wash once with 1ml PBS / well. Add 2ml of the medium containing CoPP at different concentrations (0μM, 20μM, 40μM, 60μM, 80μM, 100μM) diluted in Example 1 to each well, and place it at 37°C in 5% CO 2 cultured in a humidified incubator until virus infection for 48 hours, and the cell supernatant and cells were collected respectively.

[0019] Marc-145 cells and PAM (10 5 cells / ml) were cultured in six-well cell cultures containing DMEM and ...

Embodiment 3

[0020] Example 3 CoPP and hemin inhibit the replication of PRRSV genome and the expression of nucleocapsid protein

[0021] Real-time fluorescent quantitative PCR detection of N gene expression of HO-1 and PRRSV: After the cells treated in Example 2 were trypsinized, they were collected in a 1.5ml centrifuge tube, centrifuged at 500g for 10min at 4°C, and the supernatant was discarded. Add 1ml TRI zol (purchased from Invitrogen) to each tube, and extract RNA according to the instructions. RNA was reverse-transcribed into cDNA using PrimeScript RT reagent Kit Perfect Real Time kit (purchased from TAKARA). Finally, apply the SYBR GREEN kit (purchased from Roche Company in Switzerland) and the above reverse-transcribed cDNA template and the designed upstream and downstream primers of HO-1 and N genes, and perform real-time PCR on the StepOnePlus Real-Time PCR System of Applied Biosystems. Fluorescent quantitative PCR detection. The reaction conditions were as follows: denaturat...

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Abstract

The invention provides a method for inhibiting PRRSV infection of cells by adopting heme oxygenase 1 (HO-1) for inhibiting the PRRSV infection of the cells and an HO-1 inducer cobalt protoporphyrin (CoPP) and hemin as a blocker. The expression is substantially increased after the infection of highly-pathogenic PRRSV, and the expression is substantially decreased after the infection of classic PRRSV. The expression is substantially increased after a PRRSV-permissive cell African green monkey kidney cell line is infected with the highly-pathogenic PRRSV, and the expression is substantially decreased after the PRRSV-permissive cell African green monkey kidney cell line is infected with classic PRRSV. The CoPP and the hemin are used to induce the HO-1 expression of Marc-145 and pig alveolar macrophage (PAM). Results show that each of the CoPP and the hemin substantially induces the HO-1 expression and inhibits the PRRSV infection of the Marc-145 and the PAM cells.

Description

technical field [0001] The present invention relates to HO-1 and inducers of HO-1 as novel blockers for inhibiting PRRS virus infection. Background technique [0002] Porcine reproductive and respiratory syndrome (PRRS), commonly known as porcine blue ear disease, is an important infectious disease of pigs caused by PRRS virus (PRRSV) infection, which is extremely harmful to the global pig industry. Because PRRSV has the characteristics of antigenic variability, macrophage, antibody-dependent enhancement (ADE) and persistent infection, it is often mixed with other pathogens in clinical practice, and the pathogenesis and immune mechanism of the disease are still unclear. Clearly, so there are still no effective preventive measures. Therefore, there is an urgent need to develop new drugs against PRRS virus infection to effectively prevent the occurrence of PRRS. Contents of the invention [0003] In order to solve the deficiencies of the prior art, the present invention pr...

Claims

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Application Information

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IPC IPC(8): A61K38/44A61K38/41A61K31/409A61P31/14
Inventor 肖书奇周恩民
Owner NORTHWEST A & F UNIV
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