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Ribonucleic acid interference (RNAi) for inhibiting replication of hog cholera virus and preparation method of RNAi

A technology of swine fever virus and lentivirus, which is applied in the preparation of RNAi and the field of RNAi that inhibits the replication of swine fever virus

Inactive Publication Date: 2012-09-12
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still many questions about RNAi to be resolved, such as: the time and target site of RNAi; the interferon response in mammals; the exact mechanism of RNAi; off-target effects, etc.

Method used

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  • Ribonucleic acid interference (RNAi) for inhibiting replication of hog cholera virus and preparation method of RNAi
  • Ribonucleic acid interference (RNAi) for inhibiting replication of hog cholera virus and preparation method of RNAi
  • Ribonucleic acid interference (RNAi) for inhibiting replication of hog cholera virus and preparation method of RNAi

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] 1. Generation of ds oligo expressing shRNA

[0055] With the help of the online design software (BLOCK-iT) of Invitrogen Company of the United States TM RNAi Designer), determine the DNA insertion sequence corresponding to the specific shRNA fragments 115, 207 and 303 required for the pEN / U6 vector, and send it to Yubao Bioengineering (Dalian) Co., Ltd. for synthesis and annealing to generate ds oligo. The insertion sequence is as follows:

[0056] Npro115: 5’→3’

[0057] SEQ ID NO: 1: T CACCGCATGCCCAAGACACACCTTACGA

[0058] ATAAGGTGTGTCTTGGGCATGC

[0059] SEQ ID NO: 2: B CACCGCATGCCCAAGACACACCTTACGA

[0060] ATAAGGTGTGTCTTGGGCATGC

[0061] Npro207: 5’→3’

[0062] SEQ ID NO: 3: TCACCGCCCACTATTAGGCTAGTATACGA

[0063] ATATACTAGCCTAATAGTGGGC

[0064] SEQ ID NO: 4: BAAAAGCCCACTATTAGGCTAGTATATTCG

[0065] TATACTAGCCTAATAGTGGGC

[0066] NS5B303: 5’→3’

[0067] SEQ ID NO: 5: T CACCGGGAGAAATACAACCACAATCCGAA

[0068] GATTGTGGTTGTATTTCTCCC

[0069] SEQ ID NO: 6: B A...

Embodiment 2

[0105] 1-6 steps are the same as embodiment 1

[0106] 7. Preparation of sample 2

[0107] Pig whole blood infected with classical swine fever virus was collected, centrifuged at 12,000 rpm and 4°C for 30 minutes, and the supernatant was filtered through a 0.22um filter membrane and then inoculated with PK-15 cells. For details of the inoculation method, see 7 in Example 1.

[0108] 8. Indirect immunofluorescence detection of the ability of shRNA to inhibit the expression of swine fever whole blood virus

[0109] In order to verify that the positive PK-15 cell clones expressing shRNA inhibited the packaging ability of classical swine fever virus, the positive PK-15 cell clones screened were inoculated with the CFSV cytotoxicity cultured in step 7. After culturing for 24 hours, discard the culture medium, wash the cells twice with PBS buffer (pH7.6), 1.5 minutes each time, add pre-cooled 80% acetone after washing, put them in a -20°C refrigerator for 25 minutes, and discard th...

Embodiment 3

[0113] 1-7 steps are the same as embodiment 2

[0114] 8. Real-time RT-PCR verifies the inhibitory effect test of shRNA on the replication of swine fever whole blood virus, the method is the same as that of Example 1.

[0115] 9. Results

[0116] Compared with pU6-shRNA-CON and non-transgenic normal cells, the relative copy numbers of pU6-shRNA-115, pU6-shRNA-207, and pU6-shRNA-303 viral genes were lower, indicating that the three interference groups all showed strong ability to inhibit viral infection. see attached Figure 9 .

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Abstract

The invention discloses ribonucleic acid interference (RNAi) for inhibiting the replication of hog cholera virus and a preparation method of RNAi. The RNAi comprises a small interfering RNA (siRNA) sequence. The preparation method comprises the steps of constructing short hairpin RNA (shRNA) slow virus expression vector, preparing replication-defective slow virus, infecting slow virus pig kidney (PK)-15 cells (pig kidney cells) by using the slow virus and the like. The invention also discloses a method for verifying the effect of inhibiting the hog cholera virus replication effect. The siRNA sequence has the obvious effect of inhibiting the replication of the hog cholera virus on sensitive cells. According to the invention, the exploration of RNA interference on in vitro and vivo replication of hog cholera virus is carried out, a slow-virtue-mediated stably-integrated RNA interfering technology for special conserved gene segments of a targeted hog cholera virus genome is constructed, and transgenic animals with the siRNA of the targeted hog cholera virus are hopeful to construct. According to the invention, necessary experimental data is accumulated for gene function research of RNAi applied to the hog cholera virtue and prevention and treatment of hog cholera, and early-stage preparation is provided for disease resistance breeding of animals.

Description

technical field [0001] The invention relates to a method for inhibiting the replication of classical swine fever virus, specifically an RNAi for inhibiting the replication of classical swine fever virus composed of three pairs of shRNA sequences, and the invention also includes a preparation method of RNAi. Background technique [0002] Classical swine fever (CSF) is one of the infectious diseases that seriously endanger the swine industry worldwide. In order to distinguish it from African swine fever, Europeans call it "Classical swine fever (CSF)". The pathogen is classical swine fever virus (CSFV). CSFV is an RNA virus belonging to the family Flaviridae, a member of the genus Pestivirus, and the same genus as Bovine viral diarrhea virus (BVDV) and sheep border disease virus (Border disease virus, BDV) They are closely related in antigenicity and structure. CSFV is an enveloped, positive-strand RNA (ribonucleic acid) virus that is infectious. The CSFV genome encodes 4 ...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/10C12N15/867C12N7/04C12N5/07
Inventor 刘湘涛陈妍田宏吴锦艳尚佑军尹双辉王光祥靳野张克山杨顺利刘永杰
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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