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Anti-pig O type foot and mouth disease virus shRNA design and carrier construction method

A technology of foot-and-mouth disease virus and RNA interference, applied in DNA/RNA fragments, DNA preparation, recombinant DNA technology, etc., can solve problems such as poor immunogenicity, narrow antigen spectrum, and difficulty in epidemics of foot-and-mouth disease

Inactive Publication Date: 2013-06-12
SHIHEZI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Commonly used vaccines for FMD include inactivated vaccines and attenuated vaccines. FMD inactivated vaccines have poor immunogenicity and a narrow antigen spectrum; although attenuated vaccines have good immunogenicity, they play an important role in the prevention and control of FMD. However, due to unsafe factors such as virulence reversion of foot-and-mouth disease, incomplete virus inactivation, and live virus escape from vaccine processing factories, the application of this type of vaccine is limited.
At the same time, since FMDV is a highly variable RNA virus serotype, and there is no cross-protection among various types, it is becoming more and more difficult to prevent and control the epidemic of foot-and-mouth disease through immunization

Method used

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  • Anti-pig O type foot and mouth disease virus shRNA design and carrier construction method
  • Anti-pig O type foot and mouth disease virus shRNA design and carrier construction method
  • Anti-pig O type foot and mouth disease virus shRNA design and carrier construction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0101] Embodiment 1, the design of anti-porcine O-type foot-and-mouth disease virus shRNA, such as figure 1 Shown:

[0102] (1) According to the O-type foot-and-mouth disease genome sequence published in GenBank, select VP 1 and the conserved segment in the 3D gene, select a 19bp long nucleic acid sequence. Design the sense strand and antisense strand oligonucleotide sequences respectively containing the target gene siRNA sequence, the GC content of the nucleotide sequence is 40%-60%; there are at least 3 A or T nucleic acids in the 15-19 position Residues can enhance the RNA interference activity; ensure that the 19bp nucleic acid sequence will not form a secondary structure.

[0103] (2) Add the spacer sequence TTCAAGAGA between the sense strand and the antisense strand oligonucleotide sequence to form a hairpin structure, add the RNA polymerase termination sequence TTTTTT at the 3' end of the antisense strand, and then add ECORI and BamHI enzyme cutting site adapter seq...

Embodiment 2

[0105] Example 2 Synthesis of anti-porcine O-type foot-and-mouth disease virus shRNA

[0106] Dissolve the artificially synthesized sense and antisense strand DNA fragments in deionized water to a final concentration of 100 μMol / L, take 10 μl each, and synthesize double strands on a PCR instrument: 95°C for 30 seconds, 72°C for 2 minutes, 37°C for 2 minutes, and 25°C After 2 minutes, store on ice at -20°C for later use. Part of the link results are as follows figure 2 shown.

Embodiment 3

[0107] Example 3 Design and synthesis of anti-porcine O-type foot-and-mouth disease virus shRNA

[0108] pSIREN-FMDV-VP 1 -1

[0109] 5'-GATCCAAATTACTCAGACACTTGCAGTTTCAAGAGAACTGCAAGTGTCTGAGTAATTTTTTTTACGCGTG-3'

[0110] 5'-AATTCACGCGTAAAAAAAAATTACTCAGACACTTGCAGTTCTCTTGAAACTGCAAGTGTCTGAGTAATTG-3'

[0111] pSIREN-FMDV-VP 1 -2

[0112] 5'-GATCCAAAGTTAATGTGTTGGACCTGATTCAAGAGATCAGGTCCAACACATTAACTTTTTTTTTACGCGTG-3'

[0113] 5'-AATTCACGCGTAAAAAAAAGTTAATGTGTTGGACCTGATCTCTTGAATCAGGTCCAACACATTAACTTG-3'

[0114] pSIREN-FMDV-VP 1 -3

[0115] 5'-GATCCAAACAGCTTACCACAAGGAACCTTCAAGAGAGGTTCCTTGTGGTAAGCTGTTTTTTTACGCGTG-3'

[0116] 5'-AATTCACGCGTAAAAAAAAACAGCTTACCACAAGGAACCTCTCTTGAAGGTTCCTTGTGGTAAGCTGTTG-3'

[0117] pSIREN-FMDV-VP 1 -4

[0118] 5'-GATCCAAAGGCAGAAGAACTCTGCCTTTCAAGAGAAGGCAGAGTTCTTTCTGCCTTTTTTTTACGCGTG-3'

[0119] 5'-AATTCACGCGTAAAAAAAAGGCAGAAGAACTCTGCCTTCTCTTGAAAGGCAGAGTTCTTTCTGCCTTG-3'

[0120] pSIREN-FMDV-VP 1 -5

[0121] 5'-GATCCAAAGGCAAC...

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Abstract

The invention relates to an anti-pig O type foot and mouth disease virus shRNA and carrier construction method, which is characterized in that: according to the O type foot and mouth disease virus genome sequence which is published by the GenBank, the protective sections of VP1 and 3D genes are selected, and a section of nucleic acid sequence of 19bp long is selected; and the nucleic acid sequence of 19bp is the specific target gene which only aims at the pig foot and mouth disease virus, does not have more similarity with the other genes, and the DNA sequence which corresponds to the hairpin-shaped shRNA is synthetized according to the characteristics of the carrier. The method adopts the synthesis method and utilizes the genetic engineering technology to construct the plasmid and screenthe excellent interference sections, the plasmid has the capability of restraining the replication of the foot and mouth disease virus, and can obviously restrain the replication of the foot and mouth disease virus (FMDV) at the cell level; and the VP1 and 3D genes in FMDV gene open reading frames are selected according to the RNA interference technological principle as the target points to carryout RNA interference. When the virus RNA is replicated, firstly, a complementary strand is synthesized from the 3' end of the plus strand RNA under the 3D action; and then the negative strand is taken as the template to synthesize the progeny plus strand RNA. The nucleotide sequence and the amino acid sequence of 3Dpol have high conservative property.

Description

technical field [0001] The invention relates to a method for constructing an anti-pig O-type foot-and-mouth disease virus shRNA and a carrier, in particular to a method of utilizing genetic engineering technology, through shRNA design, synthesis, carrier construction, virus attack, RT-PCR and other processes and the use of RNA The interference technology is used to inhibit the replication of porcine O-type foot-and-mouth disease virus, and belongs to the technical field of inhibiting the replication of foot-and-mouth disease virus (FMDV). Background technique [0002] Foot-and-mouth disease (FMD) is an acute, febrile and highly contagious disease caused by foot-and-mouth disease virus (FMDV) that seriously harms cloven-hoofed animals. The disease is widespread in countries all over the world. The disease has become the number one "economic disease" among livestock diseases due to its rapid spread, high incidence and huge economic losses. The first disease of the first cate...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/10C12N15/63
Inventor 陈创夫马铈委乔军赛务加甫哈孜王鹏雁
Owner SHIHEZI UNIVERSITY
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