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Construction of RIPLET knockout cell line and application of RIPLET knockout cell line as picornaviridae virus vaccine production cell line

A small ribonucleic acid and cell line technology, applied in the field of genetic engineering, can solve the problems of useless commercial vaccine products, severe prevention and control situation, acute death of newborn piglets, etc., to improve production and antigen expression, knock out High efficiency and effect of promoting replication

Pending Publication Date: 2022-02-18
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

Seneca virus (Senecavirus A, SVA) is a virus of the same family as FMDV, and its infection of pigs can cause clinical symptoms very similar to foot-and-mouth disease, and can cause acute death of newborn piglets, which is very harmful to the pig industry and has not been used so far. The commercialized vaccine products for the prevention and control of the disease make the situation of the prevention and control of the disease extremely severe

Method used

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  • Construction of RIPLET knockout cell line and application of RIPLET knockout cell line as picornaviridae virus vaccine production cell line
  • Construction of RIPLET knockout cell line and application of RIPLET knockout cell line as picornaviridae virus vaccine production cell line
  • Construction of RIPLET knockout cell line and application of RIPLET knockout cell line as picornaviridae virus vaccine production cell line

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 Design of sgRNA targeting RIPLET gene

[0053] Use the NCBI database to query the RIPLET gene sequence, find the whole genome of the golden hamster (GenBank accession number: NW_004801729), locate the first exon segment of RIPLET in the overlapping region of different transcripts in the genome, and use it for target design.

[0054] According to the CRISPR / Cas9 design principle, log in to the CRISPR online design website http: / / crispr-era.stanford.edu / index.jsp to design sgRNA, select 2 pairs of 20bp sgRNA fragments according to the score, and name them respectively: RIPLET-sgRNAsp1( RIPLET-sgRNA1-F, shown in SEQ ID NO.3; RIPLET-sgRNA1-R, shown in SEQ ID NO.4), RIPLET-sgRNAsp2 (RIPLET-sgRNA2-F, shown in SEQ ID NO.5; RIPLET-sgRNA2-R, SEQ ID NO.6); Add CACC cohesive end at the 5' end of the forward sequence of the sgRNA fragment, and add AAAC cohesive end at the 5' end of the reverse sequence, as the sgRNA oligonucleotide (sgRNA1-oligonucleotide) targeting the R...

Embodiment 2

[0058] Example 2 Construction of sgRNA recombinant plasmid PX459-sgRNA

[0059] Obtaining double-stranded sgRNA-oligo: dilute the sgRNA-oligo synthesized in Example 1 to 10 μmol / L, and prepare a total of 10 μL reaction system: upstream primer 4.5 μL; downstream primer, 4.5 μL; 10×TaqBuffer, 1 μL. Reaction program: 99°C, 10min; 16°C, store; anneal the upstream and downstream primers to form a double-stranded sgRNA-oligo.

[0060] Digestion of PX459 vector plasmid: Digest the PX459 vector with BBSI restriction endonuclease, and prepare a total of 50 μl digestion system as follows: PX459 vector, 5 μL; BBSI, 2 μL; 10×Buffer, 5 μL; ddH 2 O, 38 μL. Place at 37°C for 3h for enzyme digestion. Afterwards, nucleic acid electrophoresis was performed, and the linearized fragment of the PX459 vector containing cohesive ends was purified and recovered using the DNA purification and recovery kit from Promega.

[0061] Construction of PX459-sgRNA recombinant plasmid: Ligate the purified an...

Embodiment 3

[0063] Example 3 Cell Transfection

[0064] Resuscitate BHK21 cells in T25 cell flasks before transfection, and culture them in DMEM medium containing 10% FBS and 1% double antibody. In the cell six-well plate, when the degree of cell confluence reached 70% to 80%, 2 μg each of the recombinant plasmids (PX459-RIPLET-sgRNA1 and PX459-RIPLET-sgRNA2) successfully constructed in Example 2 were mixed with Lipofectamine3000, 6 μL (according to Ratio 1 μg: 1.5 μL) was added to 50 μL of Opti-MEM, and the two were mixed after standing still for 5 minutes. The liposome-plasmid DNA mixture was allowed to stand for 15 minutes and then directly added to the cell culture medium. Return the cells to 37°C, 5% CO 2 Incubate for 24 hours in the incubator. Selection was performed with puromycin antibiotic for 72 hours.

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Abstract

The invention belongs to the field of gene engineering, and particularly relates to construction of an RIPLET knockout cell line and application of the RIPLET knockout cell line as a picornaviridae virus vaccine production cell line. Firstly, it is found that inhibition of RIPLET gene expression in host cells can promote replication of picornaviridae viruses, especially foot and mouth disease viruses and Seneca viruses. Secondly, the invention provides sgRNA specifically targeting RIPLET. The sgRNA can specifically target the RIPLET gene, the knockout of the RIPLET gene is realized by combining a CRISPR-Cas9 technology, and an obtained monoclonal cell line can significantly promote the replication of picornaviridae viruses, especially foot-and-mouth disease viruses and Seneca viruses. The production quantity and the antigen expression quantity of picornaviridae virus vaccines are improved, and the cell line can be used as a picornaviridae virus vaccine production cell line and has a wide application prospect.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to the construction of a RIPLET knockout cell line and its application as a picornaviridae virus vaccine production cell line. Background technique [0002] Picornaviridae is a family consisting of the smallest group of RNA viruses, mainly including enterovirus, rhinovirus, cardiovirus and aphth virus, including Seneca virus and foot-and-mouth disease virus. Foot-and-mouth disease belongs to the genus Aphthus, and is an important disease that infects cloven-hoofed animals caused by foot-and-mouth disease virus. Genus, composed of structural proteins VP1-VP4. Seneca virus (Senecavirus A, SVA) is a virus of the same family as FMDV, and its infection of pigs can cause clinical symptoms very similar to foot-and-mouth disease, and can cause acute death of newborn piglets, which is very harmful to the pig industry and has not been used so far. Commercialized vaccine produc...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/55C12N5/10A61K39/125A61K39/135A61P31/14
CPCC12N15/1137C12N9/22A61K39/12A61P31/14C12N2310/20C12N2770/32134C12N2770/32152C12N2770/32034C12N2770/32052
Inventor 郑海学朱紫祥张向乐曹伟军杨帆唐闻达李攀齐晓兰顾峰幸陈文哲张克山刘湘涛
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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