Application of USP30 gene as target spot in inhibition of Seneca valley virus replication

A gene and target technology, applied in the application field of inhibiting the replication of Seneca Valley virus, can solve the problems of unclear pathogenic mechanism, the advent of vaccines or drugs, etc., and achieve the effect of inhibiting replication

Active Publication Date: 2022-03-04
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, because the mechanism of SVA infection and pathogenesis is s

Method used

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  • Application of USP30 gene as target spot in inhibition of Seneca valley virus replication
  • Application of USP30 gene as target spot in inhibition of Seneca valley virus replication
  • Application of USP30 gene as target spot in inhibition of Seneca valley virus replication

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1 Results of SVA virus replication after USP30 gene silencing

[0061] 1. Design of small interfering RNA (siRNA)

[0062] Design the RNA interference target sequence of USP30 gene as shown in USP30 siRNA (SEQ ID NO.3-4) and NC siRNA (SEQ ID NO.5-6), the specific sequences are as follows:

[0063] USP30-siRNA-F: 5'-GGAGUACAAGUCUGAAGAATT-3' (SEQ ID NO.3);

[0064] USP30-siRNA-R: 5'-UUCUUCAGACUUGUACUCCTT-3' (SEQ ID NO.4);

[0065] NC siRNA-F:5'-UUCUCCGAACGUGUCACGUTT-3'(SEQ ID NO.5);

[0066] NC siRNA-R: 5'-ACGUGACACGUUCGGAGAATT-3' (SEQ ID NO. 6).

[0067] 2. Construction of USP30 gene silencing cell line:

[0068] (1) Preparation of USP30 gene silencing siRNA Oligo: send the designed interfering RNA sequence to Shanghai Gemma Company for synthesis to obtain the corresponding siRNA Oligo, and use DEPC H 2 O resuspended 1OD of siRNA to make the final concentration 20 μm. Before dissolving, centrifuge at 10,000rpm for 2min, then slowly open the cap of the tube,...

Embodiment 2

[0075] Example 2 USP30 gene knockout HEK293T cell line

[0076] 1. Design of sgRNA targeting USP30 gene

[0077] The Ensemble database was used to query the USP30 gene sequence, and the first exon segment of USP30 in the overlapping region of different transcripts in the genome was located for target design.

[0078] According to the CRISPR / Cas9 design principle, log in to the CRISPR online design website http: / / crispor.tefor.net / to design sgRNA, respectively named: USP30-sgRNAsp1, USP30-sgRNAsp2; at the 5' end of the forward sequence of the sgRNA fragment A CACC sticky end and an AAAC sticky end were added at the 5' end of the reverse sequence as sgRNA oligonucleotides (sgRNA1-oligo) targeting the USP30 gene. The sgRNA1-oligo was synthesized by Jinweizhi Biotechnology Co., Ltd., and the detailed sequence is shown in Table 1.

[0079] Table 1 sgRNA oligonucleotides targeting USP30 gene

[0080]

[0081] Note: The underlined sequence is the added restriction site, and th...

Embodiment 3

[0097] Example 3 Effect of USP30 Gene Knockout HEK293T Cell Line on SVA Replication

[0098] USP30 gene knockout HEK293T cells and wild-type HEK293T cells were used for normal subculture, and the cells were spread to 35 mm cell culture dishes, inoculated with SVA-eGFP virus, and the SVA replication was detected by Western blotting and qPCR methods, respectively.

[0099] Fluorescent detection results such as Figure 7 As shown, the results showed that the replication of SVA in USP30 knockout HEK293T cells was significantly reduced. The results of Western blotting are as follows: Figure 8 As shown, the results showed that the expression level of VP2 protein of SVA in USP30 knockout HEK293T cells was significantly reduced.

[0100] qPCR test results such as Figure 9 As shown, the mRNA levels of the virus in USP30 knockout HEK293T cells were significantly down-regulated compared with wild-type cells after inoculation with SVA.

[0101] The above results indicated that USP30...

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Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to application of a USP30 gene as a target spot in inhibition of Seneca valley virus replication. The method has the advantages that (1) the host USP30 gene is inhibited or silenced, and the replication of the Seneca valley virus can be inhibited, namely, the USP30 can be used as a target spot for screening drugs for inhibiting the replication of the Seneca valley virus; (2) the invention provides a small interfering RNA (Ribonucleic Acid) for interfering the replication of the Seneca valley virus, and the replication of the Seneca valley virus can be inhibited; (3) the sgRNA specifically targeting the USP30 gene is provided, complete knockout of the USP30 gene is realized by combining a CRISPR-Cas9 technology, the obtained monoclonal cell line USP30-KOs has a resistance phenotype to SVA, replication of the SVA in cells can be remarkably inhibited, and a research tool and a material are provided for further research on a molecular mechanism of the USP30 gene for regulating and controlling pathogenic microorganism replication in the cells.

Description

technical field [0001] The invention belongs to the technical field of biological genetic engineering, and specifically relates to the application of a USP30 gene as a target in inhibiting the replication of Seneca Valley virus. Background technique [0002] Seneca valley virus (SVA) belongs to the Senecavirus genus of the Picornaviridae family and is a single-stranded positive-sense RNA virus that mainly infects pigs. The vesicular disease caused by SVA infection is very similar to the lesions caused by foot-and-mouth disease (FMD), swine vesicular disease (SVD) and vesicular stomatitis (VS). Accompanied by symptoms such as limping, anorexia, lethargy, and fever. How to formulate effective diagnosis and prevention strategies and measures to prevent the continuous prevalence and spread of the disease is an urgent problem to be solved. However, because the mechanism of SVA infection and pathogenesis is still unclear, there is no commercial vaccine or drug available. Theref...

Claims

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Application Information

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IPC IPC(8): C12Q1/6851G01N33/573C12N15/113C12N15/85C12N5/10C12N15/55A61K45/00A61K31/713A61K31/7088A61K48/00A61P31/14
CPCC12Q1/6851G01N33/573C12N15/1137C12N15/85C12N9/22C12N9/14A61K45/00A61K31/713A61K31/7088A61K48/005A61P31/14C12Y304/19012C12N2310/20G01N2333/948C12Q2531/113C12Q2563/107
Inventor 郑海学朱紫祥赵振翔齐晓兰杨帆曹伟军张向乐陈淑莹刘湘涛
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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