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Proteolysis targeting virus, live vaccine thereof, preparation method of proteolysis targeting virus, preparation method of live vaccine, application of proteolysis targeting virus, and application of preparation method

A proteolytic and viral technology, applied in the biological field

Pending Publication Date: 2021-01-05
司龙龙 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] However, there is currently no report on the use of this technology for virus transformation

Method used

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  • Proteolysis targeting virus, live vaccine thereof, preparation method of proteolysis targeting virus, preparation method of live vaccine, application of proteolysis targeting virus, and application of preparation method
  • Proteolysis targeting virus, live vaccine thereof, preparation method of proteolysis targeting virus, preparation method of live vaccine, application of proteolysis targeting virus, and application of preparation method
  • Proteolysis targeting virus, live vaccine thereof, preparation method of proteolysis targeting virus, preparation method of live vaccine, application of proteolysis targeting virus, and application of preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0155] Example 1 Establishment of stable mammalian cell lines HEK293-TEVp and MDCK-TEVp capable of stably expressing TEVp.

[0156] The mammalian cell line stably expressing TEVp was commissioned by Beijing CorreGene Biotechnology Co., Ltd. (CorreGene).

[0157] (1) Construction of TEVp overexpression lentiviral vector:

[0158] TEVp overexpression lentiviral vector map such as figure 1 shown. The lentiviral vector is puromycin resistant.

[0159] (2) Purification and quantification of lentiviral packaging

[0160] ① Cell inoculation on the 0th day: 293T was inoculated in a 10cm / 15cm culture dish (the inoculum number is determined by the expected amount of virus, 1×10^8 / 15cm culture dish), control the inoculation density, and grow to 80% confluence the next day;

[0161] ②Plasmid transfection on day 1: Use the TEVp overexpression lentiviral vector described in (1), psPAX2 plasmid (this plasmid can be obtained from addgene) and pVSVG plasmid (this plasmid can be obtained fr...

Embodiment 2

[0194] Example 2: Construction of a gene vector for influenza virus WSN comprising a cleavable proteolytic targeting molecule.

[0195] (1) Rescue the acquisition of the plasmid of wild-type influenza virus WSN:

[0196] According to the gene sequence of influenza virus A / WSN / 1933 published by pubmed

[0197] https: / / www.ncbi.nlm.nih.gov / nuccore / ? term=WSN+PB2 ;

[0198] https: / / www.ncbi.nlm.nih.gov / nuccore / ? term=WSN+PB1 ;

[0199] https: / / www.ncbi.nlm.nih.gov / nuccore / ? term=WSN+PA ;

[0200] https: / / www.ncbi.nlm.nih.gov / nuccore / ? term=WSN+HA ;

[0201] https: / / www.ncbi.nlm.nih.gov / nuccore / ? term=WSN+NA ;

[0202] https: / / www.ncbi.nlm.nih.gov / nuccore / ? term=WSN+NP;

[0203] https: / / www.ncbi.nlm.nih.gov / nuccore / ? term=WSN+M ;

[0204] https: / / www.ncbi.nlm.nih.gov / nuccore / ? term=WSN+NS, the genes of each gene segment of the influenza virus are obtained through whole gene synthesis. Then they were connected to pHH21, pCDNA3(neo), and pcAAGGS / MCS ve...

Embodiment 3

[0237] Example 3: Rescue of PROTAC influenza virus modified by site-directed mutagenesis

[0238] According to the normal influenza virus rescue method, the 12 plasmids used for influenza virus rescue were co-transfected into a stable cell line, and the corresponding plasmids among the 12 plasmids were replaced with the plasmids transformed by site-directed mutagenesis in Example 2. Corresponding to each well of the six-well plate, add 0.2 μg of each plasmid. After transfection, observe the pathological changes of the cells, and screen out insertion sites that can rescue the virus and are dependent on TEVp, proteolysis targeting molecules, linkers that can be cleaved by TEVp, and combinations thereof. The screened strains were named according to the protein and the introduced cleavable proteolytic targeting molecule.

[0239] As an example, after introducing the cleavable proteolytic targeting molecule TEVcs+PROTAC-1 on the Ben3 pPolI-WSN-PA plasmid, this plasmid was combin...

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Abstract

The invention provides a proteolysis targeting virus. The virus comprises one or more proteolysis targeting molecules capable of being recognized by a ubiquitin-proteasome system at one or more different protein sites; the virus protein and the proteolysis targeting molecule are connected through one or more connecting chains; and the connecting chains can be selectively cut. The invention also provides a preparation method of the proteolysis targeting virus. Specifically, the invention provides the proteolysis targeting influenza virus and a preparation method thereof. The proteolysis targeting virus and the preparation method thereof provided by the invention can be widely applied to live vaccines, attenuated vaccines, inactivated vaccines, virulent safety models and the like. Compared with the prior art, the proteolysis targeting virus and the preparation method thereof provided by the invention are used for designing the vaccine with different inactivation degrees, and have safe controllability, and the vaccine has good immunogenicity.

Description

technical field [0001] The invention belongs to the field of biotechnology. Specifically, the present invention relates to a proteolytic targeting virus and its use. The invention also provides a preparation method of the virus. Background technique [0002] Influenza is an acute respiratory disease caused by influenza virus, which can infect poultry, mammals and humans seasonally. Influenza viruses can be further divided into three types: A, B, and C, among which type A (also known as type A) has the most frequent outbreaks. Influenza A virus belongs to the Orthomyxoviridae family, and its genome consists of 8 independent single-stranded RNA segments, encoding 10 proteins: hemagglutinin protein (HA); matrix protein (M), which is divided into M1 and M2; Neuraminidase (NA); nucleocapsid protein (NP); nonstructural protein (NS), including NS1 and NEP; and three polymerases, PB1, PB2 and PA. Each protein has an important biological function for the influenza virus. Influen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/44C12N5/10C12N15/85A61K47/64A61K39/145A61P31/16
CPCC12N7/00C12N9/50C12N15/85A61K47/64A61K39/12A61P31/16C12N2760/16121C12N2760/16134A61P31/12C12N9/506A61K2039/5254C12N2770/34022C07K14/005C07K2319/50C12N2760/16122A61P37/04A61K39/145C12N5/0687C12N2740/15043C12N2760/16162C12N2800/107C12Y304/22044
Inventor 司龙龙甄兆裕牛四文
Owner 司龙龙
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