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A DNAzyme for inhibiting prrsv infection

A technology of PRRSV and deoxyribozyme, applied in the field of biological enzymes, can solve problems in the experimental stage and achieve the effect of inhibiting infection, good effect, and simple genome

Inactive Publication Date: 2015-09-02
SHANGHAI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the structure and function of deoxyribozymes have been preliminarily understood, but most studies are still in the experimental stage

Method used

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  • A DNAzyme for inhibiting prrsv infection
  • A DNAzyme for inhibiting prrsv infection
  • A DNAzyme for inhibiting prrsv infection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Each ASON-Bugle-DNAzyme-PRRSV (respectively M-1, 5'-UTR-1, 3'-UTR-1, ORF1b-1, CD163-1) was synthesized according to the sequence SEQ NO 2-6, provided by Shanghai Jierui Synthesized by Bioengineering Ltd.

Embodiment 2

[0049] cell transfection

[0050] 1) Take a bottle of Marc-145 cells in a good growth state, transfer them to a 24-well plate, and inoculate about 1×10 cells in each well 5 Cells were cultured in complete DMEM medium without antibiotics at 37°C, 5% CO 2 Culture in an incubator, and ensure that the adherent cells in each well grow to 80-85% confluence before transfection.

[0051] 2) ASON-Bugle-PRRSV and transfection reagent dilution

[0052] ASON-Bugle-PRRSV each take 40pmol to 46μL DMEM medium without antibiotics and serum. Take the transfection reagent Lipofectamine TM 2000 (Invitrogen) 2 μL to 48 μL DMEM medium without antibiotics and serum, shake gently to mix, and incubate at room temperature for 5 minutes.

[0053] 3) Mix the 5 diluted ASON-Bugle-DNAzyme-PRRSV and 50 μL transfection reagent in equal volumes, and incubate at room temperature for 20 minutes.

[0054] 4) The culture medium in the 24-well plate was discarded, washed twice with PBS and once with DMEM, an...

Embodiment 3

[0062] Determination of viral TCID by Reed-Muench method 50 (50% tissue culture infectious dose, half of the cell culture infectious dose)

[0063] 1) Spread the plates, trypsinize the Marc-145 cells in a good growth state, count them, and adjust the density to 1×10 5 cells / ml, added to a 96-well cell culture plate, 175 μL / well, and grown into a monolayer in about 24 hours.

[0064] 2) Infect PRRSV-S3 with serum-free DMEM medium in a 96-well cell culture plate, and the cell supernatant (infected cells obtained in Example 2) for 72h is serially diluted 10 times, starting from 10 -1 -10 -10 .

[0065] 3) The diluted virus cell supernatant was inoculated on the Marc-145 monolayer cells, each dilution was inoculated into a vertical row of 8 wells, and each well was inoculated with 100 μL. Normal cells were arranged in two longitudinal rows as controls.

[0066] 4) Observe the cytopathic effect (CPE) of each well on a daily basis and record the results, usually for 5-7 days. ...

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Abstract

The invention discloses a deoxyribozyme for inhibiting porcine reproductive and respiratory syndrome virus (PRRSV) infection. Nucleotide sequence of the deoxyribozyme is represented in SEQ NO.1. The deoxyribozyme can be used for effectively inhibiting PRRSV infection.

Description

technical field [0001] The invention relates to the technical field of biological enzymes, in particular to a deoxyribozyme for inhibiting porcine reproductive and respiratory syndrome virus (PRRSV) infection. Background technique [0002] porcine reproductive and respiratory syndrome [0003] Porcine reproductive and respiratory syndrome (PRRS) is an acute infectious disease caused by PRRS virus (PRRSV) infection. Immunosuppression and persistent infection, etc. The PRRSV genome is a single-stranded positive-sense RNA that is not segmented. The 3' non-coding region (3'-UTR) of the genome has a poly(A) tail sequence, and the 5' non-coding region (5'-UTR) has a cap leader sequence . There are also 9 overlapping open reading frames in the genome. ORF1a and ORF1b mainly encode viral RNA replicase and polymerase; ORF2-7 encode GP2, E, GP3, GP4, GP5, matrix protein M and nucleocapsid protein N respectively . [0004] PRRSV invades host cells mainly by binding to specific rec...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113A61P31/14
Inventor 李红易建中刘成倩
Owner SHANGHAI ACAD OF AGRI SCI