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Anti-PCVD shRNA and design-synthesis method and application thereof

A technology of porcine circovirus and virus, applied in antiviral agents, DNA/RNA fragments, DNA preparation, etc.

Inactive Publication Date: 2010-03-03
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is currently no effective vaccine to prevent PCV-2 infection

Method used

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  • Anti-PCVD shRNA and design-synthesis method and application thereof
  • Anti-PCVD shRNA and design-synthesis method and application thereof
  • Anti-PCVD shRNA and design-synthesis method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0197] Embodiment 1, the selection of anti-porcine circovirus disease shRNA

[0198] 1. According to the genome sequence of PCV-2 type virus, take Rep gene and Cap gene as target gene, select a section of 19bp long nucleic acid sequence. The sequence should not be in the 5' and 3' untranslated regions, and 75 bp after the start codon, because these regions are rich in regulatory proteins, which may affect the effect of RNA interference.

[0199] 2. Calculate the GC content of the 19bp nucleic acid sequence, and the GC content must be between 40% and 60%. The GC content is optimal around 45%.

[0200] 3. There are at least 3 A or T nucleic acid residues in the 15-19 position, which can enhance the RNA interference activity.

[0201] 4. Check the 19bp nucleic acid sequence to ensure that it will not form a secondary structure.

[0202] 5. The 19bp nucleic acid sequence that simultaneously meets the three conditions of step 2, step 3 and step 4 is used as a candidate nucleic a...

Embodiment 2

[0203] Embodiment 2, the design and synthesis of anti-porcine circovirus disease shRNA

[0204] According to the RNA interference vector we used, RNAi-Ready pSIREN-RetroQ-ZsGreen Vector, the designed shRNA sequence was annealed (95°C / 30sec, 72°C / 2min, 37°C / 2min, 25°C / 2min) to form a sticky The double strand at the end can be directly ligated to the linearized RNAi-Ready pSIREN-RetroQ-ZsGreen vector (known vector).

Embodiment 3

[0205] Embodiment 3, the anti-porcine circovirus shRNA of design and synthesis

[0206] PSIREN-REP-1:

[0207] 5'-GATCCGTACGGGAGCTTCCAATCTCTTCAAGAGAGAGATTGGAAGCTCCCGTATTTTTTACGCGTG-3'

[0208] 3'-GCATGCCCTCGAAGGTTAGAGAAGTTTCTCTCTCTAACCTTCGAGGGCATAAAAAATGCGCACTTAA-5'

[0209] PSIREN-REP-2:

[0210] 5'-GATCCAGTGAGCGGGAAAATGCAGTTCAAGAGACTGCATTTTCCCGCTCACTTTTTTTACGCGTG-3'

[0211] 3'-GTCACTCGCCCTTTTACGTCAAGTTCTCTGACGTAAAAGGGCGAGTGAAAAAAAATGCGCACTTAA-5'

[0212] PSIREN-REP-3:

[0213] 5'-GATCCGTGGTACTCCTCAACTGCTGTTCAAGAGACAGCAGTTGAGGAGTACCATTTTTTACGCGTG-3'

[0214] 3'-GCACCATGAGGAGTTGACGACAAGTTCTCTGTCGTCAACTCCTCATGGTAAAAAATGCGCACTTAA-5'

[0215] PSIREN-REP-4:

[0216] 5'-GATCCAATGCAGAAGCGTGATTGGTTCAAGAGACCAATCACGCTTCTGCATTTTTTTACGCGTG-3'

[0217] 3'-GTTACGTCTTCGCACTAACCAAGTTCTCTGGTTAGTGCGAAGACGTAAAAAAAATGCGCACTTAA-5'

[0218] PSIREN-REP-5:

[0219] 5'-GATCCAAGCAAATGGGCTGCTAATTTCAAGAGAATTAGCAGCCCATTTGCTTTTTTTTACGCGTG-3'

[0220]3'-GTTCGTTTACCCGACGATTAAAGTTCTCTTAATCGTCGGGT...

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Abstract

The invention discloses a method for designing and synthesizing anti-PCV shRNA. The method comprises the steps of: 1) selecting a section of nucleotide sequence with length of 19bp according to genomesequence of PCV-2 and by taking Rep gene or Cap gene as target gene; 2) selecting a nucleotide sequence as a candidate nucleotide sequence, which meets the following three conditions: GC content of the nucleotide sequence is between 40 percent and 60 percent; at least three A or T nucleic-acid residues exist among locus from 15 to 10; and secondary structure is ensured not to be generated by 19bpnucleotide sequence after inspection; and 3) comparing the obtained candidate nucleotide sequence with PCV genome so as to determine whether the candidate nucleotide sequence only specially aims at the target gene. The anti-PCV shRNA provided by the invention can play the anti-virus role by restricting reproduction of PCV in cells.

Description

technical field [0001] The invention relates to the key technology and method of using RNA interference technology to resist porcine circovirus disease. Background technique [0002] RNA interference (RNA interference, RNAi) refers to the phenomenon of efficient and specific degradation of homologous mRNA induced by double-stranded RNA (double-stranded RNA, dsRNA), which is highly conserved during evolution. Since the use of RNAi technology can specifically knock out or shut down the expression of specific genes, this technology has been widely used in the field of gene therapy for exploring gene functions and infectious diseases and malignant tumors. [0003] RNAi was first discovered in Caenorhabditis elegans (C.elegans) by Fire et al. They found that injecting dsRNA into nematodes could inhibit the expression of sequence homologous genes, and confirmed that this inhibition mainly acts after transcription, so it is also called RNAi It is post-transcriptional gene silencin...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12N15/11A61K48/00A61P31/20
Inventor 周继勇缪云根杨华军胡嘉彪曹晶晶周芳
Owner ZHEJIANG UNIV
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