Application of PRKRA gene as target spot in inhibition of peste des petits ruminants virus replication

A kind of PPR, gene technology, applied in application, gene therapy, antiviral agent and other directions, can solve the problem that the pathogenic mechanism has not made a big breakthrough, and achieve the effect of inhibiting replication

Active Publication Date: 2021-09-21
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 has not been a great breakthrough in the study of etiology, pathogenic mechanism, etc.

Method used

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  • Application of PRKRA gene as target spot in inhibition of peste des petits ruminants virus replication
  • Application of PRKRA gene as target spot in inhibition of peste des petits ruminants virus replication
  • Application of PRKRA gene as target spot in inhibition of peste des petits ruminants virus replication

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1 Results of PPRV virus replication after PRKRA gene silencing

[0060] 1. Design of small interfering RNA (siRNA)

[0061] Design the RNA interference target sequence of PRKRA gene as shown in PRKRA siRNA (SEQ ID NO.3-4) and NC siRNA (SEQ ID NO.5-6), the specific sequences are as follows:

[0062] PRKRA-siRNA-F: 5'-CCCAGUUUAUGAAUGUGAATT-3' (SEQ ID NO.3);

[0063] PRKRA-siRNA-R:5'-UUCACAUUCAUAAACUGGGTT-3' (SEQ ID NO.4);

[0064] NC siRNA-F:5'-UUCUCCGAACGUGUCACGUTT-3'(SEQ ID NO.5);

[0065] NC siRNA-R: 5'-ACGUGACACGUUCGGAGAATT-3' (SEQ ID NO. 6).

[0066] 2. Construction of PRKRA gene silencing cell lines:

[0067] (1) Preparation of PRKRA 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 Resuspend 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

[0074] Example 2 PRKRA gene knockout Vero cell line

[0075] 1. Design of sgRNA targeting PRKRA gene

[0076] The Ensemble database was used to query the PRKRA gene sequence, and the first exon segment of PRKRA in the overlapping region of different transcripts in the genome was located for target design.

[0077] According to the design principles of CRISPR / Cas9, log in to the CRISPR online design website http: / / crispor.tefor.net / to design sgRNA, respectively named: PRKRA-sgRNAsp1, PRKRA-sgRNAsp2; add at the 5' end of the forward sequence of the sgRNA fragment CACC sticky end, AAAC sticky end was added at the 5' end of the reverse sequence as sgRNA oligonucleotide (sgRNA1-oligo) targeting PRKRA gene. The sgRNA1-oligo was synthesized by Jinweizhi Biotechnology Co., Ltd., and the detailed sequence is shown in Table 1.

[0078] Table 1 sgRNA oligonucleotides targeting PRKRA gene

[0079]

[0080] Note: The underlined sequence is the added restriction site, and the ununder...

Embodiment 3

[0097] Example 3 Effect of PRKRA Gene Knockout Vero Cell Line on PPRV Replication

[0098] PRKRA gene knockout Vero cells and wild-type Vero cells were used for normal subculture. The cells were plated on 35 mm cell culture dishes and inoculated with PPRV Nigeria 75 / 1 virus. Western blotting and qPCR methods were used to detect PPRV replication, respectively.

[0099] The results of Western blotting are as follows: Figure 7 As shown, the results showed that the expression of N protein of PPRV was basically undetectable in PRKRA knockout Vero cells.

[0100] qPCR test results such as Figure 8 As shown, the mRNA levels of N protein and P protein in PRKRA knockout Vero cells were significantly down-regulated compared with wild-type cells after inoculation with PPRV. Indirect immunofluorescence results such as Figure 9 As shown, the viral capsid protein in wild-type cells increased with the passage of virus infection time, while the expression of PPRV capsid protein was almo...

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Abstract

The invention belongs to the technical field of biological genetic engineering, and particularly relates to application of a PRKRA gene as a target spot in inhibition of peste des petits ruminants virus replication. The invention firstly finds that by inhibiting or silencing the host PRKRA gene, the replication of the peste des petits ruminants virus can be inhibited, and the PRKRA gene can be used as a target spot for preparing the medicine for inhibiting the replication of the peste des petits ruminants virus; secondly, by taking the PRKRA gene as a target spot, the small interfering RNA is designed, and the small interfering RNA can interfere with the replication of the peste des petits ruminants virus and can be used for preparing a medicine for inhibiting the replication of the peste des petits ruminants virus; moreover, the invention provides sgRNA for specifically targeting the PRKRA gene, the sgRNA can specifically target the PRKRA gene, complete knockout of the PRKRA gene is realized by combining with a CRISPR-Cas9 technology, and the obtained monoclonal cell line PRKRA-KOs has a resistance phenotype to PPRV, can significantly inhibit duplication of PPRV in cells, and a research tool and a material are provided for further researching a molecular mechanism of the PRKRA gene for regulating and controlling pathogenic microorganism replication in cells.

Description

technical field [0001] The invention belongs to the technical field of biogenetic engineering, and specifically relates to the application of a PRKRA gene as a target in inhibiting the replication of Peste des petits ruminants virus. Background technique [0002] Peste des petits ruminants virus (PPRV) belongs to the Paramyxoviridae (Paramyxoviridae) genus Morbillivirus. It is a single-strand negative-sense and non-segmented RNA virus. It mainly infects small ruminants such as goats and sheep. animal. PPRV mainly infects lymphoid tissue, digestive tract and respiratory tract epithelial cells. Clinically, it mainly manifests as acute fever, enteritis, bronchopneumonia and abortion of pregnant ewes, causing huge economic losses to the livestock breeding industry in my country and developing countries. Immunization is currently the most effective way to protect against Peste des petits ruminants. Compared with inactivated vaccines, the attenuated vaccines (Nigeria 75 / 1 and Su...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6851C12N15/113C12N15/55C12N5/10A61K48/00A61K31/713A61P31/14C12R1/91
CPCC12Q1/6851C12N15/1131C12N9/22C12N5/0686C07K14/4702A61K31/713A61P31/14C12N2310/20C12N2510/00C12Q2531/113C12Q2563/107C12Q2521/107
Inventor 郑海学陈淑莹朱紫祥杨帆曹伟军齐晓兰唐闻达马昭张向乐刘湘涛
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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