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siRNA segment and application thereof used for curing and/or preventing porcine reproductive and respiratory syndrome

A technology for respiratory syndrome and pig breeding, applied in the direction of DNA/RNA fragments, applications, gene therapy, etc., can solve problems such as inability to prevent re-infection, difficulty in obtaining vaccine results, and infection of pigs

Active Publication Date: 2010-06-23
INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there are inactivated vaccines and attenuated vaccines for preventing PRRSV, the use of these vaccines can only provide partial protection, only make the body no longer have clinical symptoms, but cannot prevent reinfection
Moreover, the attenuated vaccine strain may also have a strong virulence phenomenon in the natural state, and may be transmitted to the fetus through the placenta, resulting in continuous infection of the infected pigs, making it difficult for some conventional vaccines to achieve the desired effect

Method used

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  • siRNA segment and application thereof used for curing and/or preventing porcine reproductive and respiratory syndrome
  • siRNA segment and application thereof used for curing and/or preventing porcine reproductive and respiratory syndrome
  • siRNA segment and application thereof used for curing and/or preventing porcine reproductive and respiratory syndrome

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 : Design of RNAi sequences

[0028] siRNA design strategy: 1) Search for the ideal siRNA sequence from 50-100 nucleotides downstream of the start codon AUG of the target gene, the closer to the 3' end of the target gene, the better the gene silencing effect; 2) siRNA The sequence is preferably AA(Nn)UU (N represents any base; n is the number of bases, between 19 and 29 nt), and NA(Nn)UU and NA(Nn)NN sequences are also acceptable. 3) It has a balanced base content (that is, the G / C content is between 30% and 70%). According to the genome sequence of PRRSV S1 strain (GenBank accession number: AF090173), two pairs of siRNA fragments were designed, and their sequences are:

[0029] M-229 (SEQ ID NO.1):

[0030] 5'-GCAGUAGUUGCACUCCUUU-3'

[0031] 3'-CGUCAUCAACGUGAGGAAA-5';

[0032] M-379 (SEQ ID NO.2):

[0033] 5'-GCAAAUGAUAACCACGCAU-3'

[0034] 3'-CGUUUACUAUUGGUGCGUA-5'

[0035] M-167 (SEQ ID NO.3):

[0036] 5'-CCUUCGGGUACAUGACUUU-3'

[0037] 3'-GGAAGCC...

Embodiment 2

[0048] Example 2 : siRNA transfection of Marc145 cell line and PRRSV infection

[0049] Experimental materials: Marc145 cells, American PRRSV S1 strain (MOI=0.01), American PRRSV polyclonal antibody.

[0050] 1. Transfection: Marc145 cells were incubated with RPMI 1640 medium (GIBCO) containing 10% fetal bovine serum at 37°C and 5% CO 2 cultivated under conditions. Cell transfection was performed using liposome Hiperfect (Qiagen), and the operation method could be completely in accordance with the manufacturer's instructions. For transfection, Marc145 cells (6-well plate) were transfected with 1 μg siRNA.

[0051] 2. PRRSV infection: 24 hours after transfection, PRRSV cytotoxicity (MOI=0.1) was added to each culture plate, and the culture was continued for 24 hours.

Embodiment 3

[0052] Example 3 : Detection of siRNA interference effect

[0053] I. Real-time quantitative PCR (Real-time RCR) analysis:

[0054] 1. Extraction of total RNA

[0055] Total cellular RNA was extracted using Trizol reagent (Invitrogen), and the steps were referred to the instructions of Invitrogen Company.

[0056] 2. Real-time quantitative PCR analysis

[0057] 1) Primer and probe design

[0058] The GAPDH gene of the baboon (Papio anubis) and the beta-actin gene of the long-tailed monkey (C.aethiops) were used as positive controls for the real-time quantitative PCR reaction, and the sequences of primers and probes were designed as shown in Table 1.

[0059] Table 1 GAPDH gene, beta-actin gene and PRRSV-M gene primer and probe sequence

[0060]

[0061] 2) Reagents and instruments

[0062] Reagents: TaqMan (TaqMan) general reagents for real-time quantitative PCR (Shanghai Gemma Biotechnology Co., Ltd.);

[0063] Instrument: FTC-2000A real-time quantitative PCR instr...

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Abstract

The invention provides a siRNA segment and application thereof used for curing and / or preventing Porcine Reproductive and Respiratory Syndrome. Both the siRNA segment and a carrier comprising the siRNA segment play the protection role on the Marc145 clones inflected by PRRSV; the siRNA segment is discovered to be capable of reducing the mRNA level of PRRSV-M protein by about 30 percent-50 percent through real-time quantitative PCR and immunoblot assay; therefore, the siRNA segment and the carrier comprising the siRNA segment of the invention can be used for preparing drugs used for curing and / or preventing Porcine Reproductive and Respiratory Syndrome, and have important values on curing Porcine Reproductive and Respiratory Syndrome with genes.

Description

technical field [0001] The present invention relates to an siRNA fragment and its application, in particular to an siRNA fragment for treating and / or preventing porcine reproductive and respiratory syndrome and its application. Background technique [0002] RNA interference (RNA interference, RNAi) is sequence-specific gene silencing mediated by double-stranded RNA. In February 1998, Fire at the Carnegie Institute of Washington and Mello at the University of Massachusetts Cancer Center proposed this concept for the first time in the study of Caenorhabditis elegans. The phenomenon of quelling found in Neurospora belongs to the mechanism of post-transcriptional gene silencing. This mechanism has been confirmed to exist in almost all eukaryotic cells such as fungi, Arabidopsis, trypanosomes, hydra, planarians, and zebrafish, and even a similar mechanism exists in Escherichia coli. After discovering that this mechanism also exists in mammalian cells, RNAi, as a rapid, effectiv...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/867C12N15/861C12N15/86C12N15/63A61K31/713A61K48/00A61P31/14
Inventor 何宏轩周凯赵宝华
Owner INST OF ZOOLOGY CHINESE ACAD OF SCI
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