Swine-origin miR-c89 capable of resisting PRRSV (Porcine reproductive and respiratory syndrome virus) infection, and application thereof

A mir-c89, porcine-derived technology is applied to porcine-derived miR-c89 against PRRSV infection and its application fields, which can solve the problems of limited protection and no anti-PRRS virus.

Active Publication Date: 2019-04-26
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the characteristics of PRRS virus antigenic variability, macrophage, antibody-dependent enhancement (ADE) and persistent inf...

Method used

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  • Swine-origin miR-c89 capable of resisting PRRSV (Porcine reproductive and respiratory syndrome virus) infection, and application thereof
  • Swine-origin miR-c89 capable of resisting PRRSV (Porcine reproductive and respiratory syndrome virus) infection, and application thereof
  • Swine-origin miR-c89 capable of resisting PRRSV (Porcine reproductive and respiratory syndrome virus) infection, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Effect of miR-c89 on HP-PRRSV replication when first transfecting 50nM miR-c89 mimics in PAMs and then infecting highly pathogenic HP-PRRSV

[0038] (1) miR-c89 mimic transfection and cell inoculation:

[0039] 1) Plating: Isolate porcine alveolar macrophages from the lung tissue of 6-week-old piglets, perform cell counting and plating, and store at 37°C in 5% CO 2 Cultivated in an incubator;

[0040] 2) Transfection: The transfection reagent is Roche's X-tremeGENE siRNA Transfection Reagent, and the transfection is performed according to the operation steps of the transfection reagent. 12 hours after plating, a transfection mixture was prepared (the mock and siRNAtransfection reagent were dissolved in Opti-MEM respectively, and then the two were mixed) and incubated at room temperature for 20 minutes. Take the 24-well plate out of the incubator, wash it with PBS and replace it with Opti-MEM, add the mixture drop by drop, shake and mix well, and culture it in the cell...

Embodiment 2

[0081] Example 2 When PAMs cells were first transfected with 50nM miR-c89 mimic and then infected with low pathogenic N-PRRSV, the effect of miR-c89 on the replication of low pathogenic N-PRRSV

[0082] (1) miR-c89 mimic transfection and cell inoculation:

[0083] Plating, transfection, inoculation (0.1 MOI N-PRRSV CH1a) and collection were the same as in Example 1.

[0084] 2. RT-qPCR detection of relative expression level of PRRSV ORF7 mRNA

[0085] Extraction of RNA in cells, reverse transcription into cDNA and qPCR detection are the same as in Example 1.

[0086] The result is as Figure 5 As shown, when PAMs cells were transfected with 50nM miR-c89 mimics and then infected with N-PRRSV CH1a strain, the relative expression of PRRSV ORF7 decreased by 96.6% and 96.6% respectively at 12hpi and 24hpi compared with the control mimic group. 99.3%.

[0087] 3. RT-qPCR detection and determination of the PRRSV genome copy number in the supernatant sample

[0088] Extract the R...

Embodiment 3

[0094] Example 3 When PAMs cells were infected with highly pathogenic HP-PRRSV and transfected with 50nM miR-c89 mimics, the effect of miR-c89 on the replication of highly pathogenic HP-PRRSV

[0095] (1) Cell inoculation and miR-c89 mimic transfection:

[0096] 1) decking is with embodiment 1;

[0097] 2) Infection: Inoculate the HP-PRRSV GD-HD strain at 0.01 MOI 12 hours after plating, and calculate the required virus solution according to the formula PFU=number of cells×MOI=0.7×TCID50; take out the 24-well plate and wash it with PBS Afterwards, add virus diluent and incubate at 37°C;

[0098] 3) Transfection: The transfection reagent is Roche's X-tremeGENE siRNA Transfection Reagent, and the transfection is carried out according to the operation steps of the transfection reagent; after 1 hour of infection, the transfection mixture is prepared (the mock and siRNA transfection reagent are respectively dissolved in Opti-MEM and then mix the two) and incubate at room temperat...

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Abstract

The invention discloses a swine-origin miR-c89 capable of resisting PRRSV (Porcine reproductive and respiratory syndrome virus) infection. Through RT-qPCR (Real Time-quantitative Polymerase Chain Reaction), TCID50 (Tissue Culture Infectious Dose 50) and western blot methods, miR-c89 derived from swine coding is proved to obviously inhibit the replication and the proliferation of high pathogenicityand low pathogenicity PRRSV for the first time, the micoRNA is hopefully developed into a novel medicine capable of preventing and curing porcine reproductive and respiratory syndrome and used for researching the gene-modified pig capable of resisting the porcine reproductive and respiratory syndrome, and a foundation is laid for the prevention and control of the PRRSV.

Description

technical field [0001] The invention relates to the field of biotechnology, and more specifically relates to a porcine miR-c89 resistant to PRRSV infection and application thereof. Background technique [0002] Porcine reproductive and respiratory syndrome (Porcine reproductive and respiratory syndrome, PRRS) is a viral infection caused by PRRS virus (Porcine reproductive and respiratory syndrome virus, PRRSV), characterized by reproductive impairment in sows and respiratory damage in piglets. sick. The disease first broke out in the United States in 1987, and then spread and became popular all over the world, bringing huge economic losses to the global pig industry. In 2006, a "pig high fever disease" characterized by high fever, high morbidity, and high mortality broke out in pig farms in southern my country. The pathogen that caused the disease was finally confirmed to be a mutated PRRSV, which was named as highly pathogenic Sexual PRRSV (Highly pathogenic PRRSV, HP-PRRS...

Claims

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Application Information

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IPC IPC(8): C12N15/113A61K31/713A61P31/14
CPCA61P31/14A61K31/713C12N15/113C12N2310/14
Inventor 肖书奇李爽闫云欢张晓彬
Owner NORTHWEST A & F UNIV
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