Antisense DNA (Deoxyribose Nucleic Acid) sequence for treating and preventing porcine reproductive and respiratory syndrome and application thereof

A respiratory syndrome and DNA sequence technology, applied in recombinant DNA technology, DNA / RNA fragments, gene therapy, etc., can solve problems such as outbreaks, atypical PRRS epidemics, vaccine safety and efficiency, and achieve low toxicity and side effects. The effect of significant curative effect and moderate cost

Active Publication Date: 2014-12-10
YUNNAN AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In view of the serious harm caused by PRRS to the pig industry, there are various deficiencies in the prevention and control measures based on vaccination: for example, the outbreak of the disease caused by attenuated vaccination, and the outbreak of atypical PRRS in pigs inoculated with inactivated vaccines epidemics, etc., raising questions about the safety and efficacy of vaccine use

Method used

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  • Antisense DNA (Deoxyribose Nucleic Acid) sequence for treating and preventing porcine reproductive and respiratory syndrome and application thereof
  • Antisense DNA (Deoxyribose Nucleic Acid) sequence for treating and preventing porcine reproductive and respiratory syndrome and application thereof
  • Antisense DNA (Deoxyribose Nucleic Acid) sequence for treating and preventing porcine reproductive and respiratory syndrome and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Embodiment 1: the design of antisense DNA

[0024] Antisense DNA design strategy: 1) Completely predict the primary secondary structure and secondary secondary structure of mRNA, and these structure predictions mainly follow the minimum free energy, not in the folding region. Find the single-stranded region as much as possible. If the length of the single-stranded region is not enough, both ends can extend to the double-stranded region; 2) The length of the antisense nucleic acid requires 20nt. Generally, the start codon (AUG) or the 20-base sequence near it is selected, or at both ends of the mRNA, or the neck loop structure, as far as possible in the single-stranded region; 3) Try to contain CCAC, TCCC, ACTC, GCCA or CTCT bases, these bases help to enhance the effect of antisense nucleic acid; 4) CG content also helps antisense nucleic acid to play a role, control G+C content between 40% and 60%; 5) Select RNA two The unstable part in the secondary structure is used ...

Embodiment 2

[0034] Example 2: Antisense DNA inhibits viral cytopathic effect

[0035]1. Digest the well-grown Marc-145 cells with 0.25% porcine trypsin, count, then add 1×104 cells / well, 100 μl per well, into a 96-well culture plate, and place the cells in a carbon dioxide incubator Grow to a single layer and set aside. The antisense DNA of each experimental group is within the maximum non-toxic dose range (TM 2000 transfected into Marc-145 cells, replaced with normal medium after 4 hours, cultured in 37°C, 5% CO2 incubator, and continuously cultivated for 60 hours, observed the degree of cell lesion in each experimental group day by day, and compared with the normal cell control group compared with the virus control group.

[0036] 2. The result is as follows figure 2 As shown, a variety of antisense DNA and positive and negative control antisense DNA sequences were transfected into Marc-145 cells inoculated with PRRSV and cultured continuously for 60 hours. It can be seen that YN-2, ...

Embodiment 3

[0037] Embodiment 3: antisense DNA suppresses PPRSV mRNA expression level analysis

[0038] 1. Digest well-grown Marc-145 cells with 0.25% porcine trypsin, count them, and count them according to 1×10 4 Cells were cultured at the density of each well, 100 μl per well, added to a 96-well culture plate, and placed in a carbon dioxide incubator until the cells grew to a monolayer, ready to use. Using 25 TCIDs 50 The YN-1 strain PRRS virus per well was inoculated into the cells, and after the virus was incubated for 1.5h, 32 μM antisense DNA (within the non-toxic dose range) of each experimental group was used Lipofectamine TM 2000 transfected into Marc-145 cells, changed to 5% DMEM complete medium after 4h, 37°C, 5% CO 2 Cells were harvested after continuous culture in the incubator for 60 hours, RNA was extracted, and then reverse-transcribed into cDNA. Finally, real-time fluorescent quantitative PCR was used to detect the effects of different concentrations of antisense DNA o...

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Abstract

The invention provides an antisense DNA (Deoxyribose Nucleic Acid) sequence for treating and preventing PRRS (Porcine Reproductive and Respiratory Syndrome) and an application thereof. The single-chain antisense DNA sequence is 20nt antisense DNA which is designed and synthesized by taking the partial sequence of a PRRSV (Porcine Reproductive And Respiratory Syndrome Virus) gene as a target spot without other chemical modifications. Monkey kidney epithelial Marc-145 cells are transfected by adopting a lipidosome mediated method, so that the proliferation of the PRRSV on the Marc-145 cells can be outstandingly inhibited; and the antisense DNA sequence is detected through a fluorescent quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) method and an indirect immunofluorescence experiment method to have the effects of outstandingly reducing the nucleoprotein gene expression of the PRRSV and inhibiting virus multiplication. Thus, the antisense DNA sequence provided by the invention has a wide application prospect on the treatment and control of infection of the PRRSV and important value on the gene therapy of the PRRS and has the advantages of easiness for source, low cost and great market value.

Description

technical field [0001] The present invention relates to the application in the treatment of porcine reproductive and respiratory syndrome virus infection, especially the antisense DNA sequence and application thereof for the treatment and prevention of porcine reproductive and respiratory syndrome. technical background [0002] Porcine reproductive and respiratory syndrome (Porcine Reproductive and Respiratory Syndrome, PRRS), also known as blue ear disease, is one of the important infectious diseases that cause huge economic losses to the pig industry worldwide. Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) has large genetic variability, and PRRSV often becomes the primary pathogen again due to vaccination failure, which brings great difficulties to the prevention and treatment of the disease. Therefore, how to effectively control the prevalence of the disease is a serious challenge and a major issue that researchers and producers are currently facing. Effici...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113A61K48/00A61P31/14
Inventor 尹革芬毕峻龙李文贵李想郑龙龙杨贵树
Owner YUNNAN AGRICULTURAL UNIVERSITY
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