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Modified norovirus VP1 proteins and VLPS comprising modified norovirus VP1 proteins

A virus and protein technology, applied in antiviral immunoglobulin, cells and viruses modified by introducing foreign genetic material, etc., can solve the problem of human norovirus inability to grow, lack of VLP and stable cell culture of live attenuated norovirus Systems, deteriorating development of an effective norovirus vaccine, etc.

Pending Publication Date: 2020-09-29
MEDICAGO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Along with the difficulty of rapidly evolving and genetically diversifying norovirus strains, other challenges have exacerbated the development of an effective norovirus vaccine
For example, human noroviruses were unable to grow in cell culture until recently, and even now, robust cell culture systems for VLPs and live attenuated noroviruses are lacking
[0009] Another challenge in vaccine development is that immunity to norovirus infection is strain- and genotype-specific with minimal cross-immunity against other genomes

Method used

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  • Modified norovirus VP1 proteins and VLPS comprising modified norovirus VP1 proteins
  • Modified norovirus VP1 proteins and VLPS comprising modified norovirus VP1 proteins
  • Modified norovirus VP1 proteins and VLPS comprising modified norovirus VP1 proteins

Examples

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example 1

[0276] Example 1: Norovirus VP1 constructs

[0277] Candidate sequences for VP1 and VP2 are available in Genbank (see Figure 2A and 2B ). Non-limiting examples of these sequences are:

[0278] Hu / GI.2 / Leuven / 2003 / BEL (GI.2; SEQ ID NO: 4; Figure 13A );

[0279] Hu / GI.3 / S29 / 2008 / Lilla Edet / Sweden (GI.3; SEQ ID NO: 6; Figure 14A );

[0280] Hu / GI.5 / Siklos / Hun5407 / 2013 / HUN (GI.5; SEQ ID NO: 12; Figure 15A );

[0281] Hu / GI.7 / USA / 2014 / GA5043 (GI.7, SEQ ID NO: 101, Figure 16A )

[0282] Hu / GII.1 / Ascension208 / 2010 / USA (GII.1; SEQ ID NO: 13; Figure 16C );

[0283] Hu / GII.2 / CGMH47 / 2011 / TW (GII.2; SEQ ID NO: 14; Figure 17A );

[0284] Hu / GII.3 / Jingzhou / 2013402 / C HN (GII.3; SEQ ID NO: 15; Figure 18A );

[0285] Hu / GII.4 / Sydney / NSW0514 / 2012 / AU (GII.4; SEQ ID NO: 16; Figure 19A );

[0286] US96 / GII.4 / Dresden174 / 1997 / DE_AY741811 (GII.4; SEQ ID NO: 27; Figure 19C );

[0287] FH02 / GII.4 / FarmingtonHills / 2002 / US_AY502023 (GII.4; SEQ ID NO: 28; Figure 19D );

[0...

example 2

[0314] Example 2: Method

[0315] Agrobacterium tumefaciens transfection

[0316] Using the method described by D'Aoust et al., 2008 (Plant Biotech.J. 6:930-40) by using native Norovirus VP1, native Norovirus VP2 or Norovirus VP1 mutant protein Expression vectors were electroporated to transfect Agrobacterium tumefaciens strain AGL1. The transfected Agrobacterium was cultured in YEB medium supplemented with 10 mM 2-(N-morpholino)ethanesulfonic acid (MES), 20 μM acetosyringone, 50 μg / ml kanamycin and 25 μg / ml carbenicillin (pH5.6) to an OD between 0.6 and 1.6 600 . The Agrobacterium suspension was centrifuged before use and resuspended in infiltration medium (10 mM MgCl 2 and 10mM MES pH 5.6).

[0317] Preparation of plant biomass, inoculum, and Agrobacterium infiltrate

[0318]N. benthamiana plants were grown from seed in shallow boxes filled with a commercial peat moss substrate. Plants were grown in a greenhouse under a 16 / 8 photoperiod and a temperature regime ...

example 3

[0330] Example 3: Production of VP1 protein and VLPs in plants

[0331] As described in Example 2, N. benthamiana leaves were vacuum infiltrated with Agrobacterium tumefaciens comprising expression vectors encoding wild-type norovirus VP1 or mutant norovirus VP1 constructs to allow expression of the VP1 sequence and targeting the VP1 protein and / or VLP generation to check the blades. After 9 days post-infiltration (DPI), total crude protein extracts prepared from leaf homogenates were separated by SDS-PAGE and stained with Coomassie (produced by VP1), or using discontinuous protein as described in Example 2 above. Iodixanol density gradient separation (VLP generation). Fractions from density gradients were examined using Coomassie-stained SDS-PAGE. Norovirus VP1 protein appears in a band at approximately 55 to 60 kDa. The appearance of VP1 protein within the density gradient fraction indicated a fraction commensurate with VLPs during density gradient centrifugation. The ...

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Abstract

Nucleic acids encoding modified norovirus VP1 proteins, and VLPs comprising one or more of the modified norovirus VP1 proteins are provided. Methods for modified norovirus VP1 protein, and norovirus VLP, production in plants, portions of the plant or a plant cell, are also described.

Description

technical field [0001] The present invention relates to a modified norovirus (norovirus) VP1 protein, a VLP comprising the modified norovirus VP1 protein and a production method thereof. Background technique [0002] The global burden of disease caused by norovirus infection is substantial, being associated with an estimated 20% of all diarrheal cases worldwide and killing more than 200,000 people annually. Noroviruses are the leading cause of outbreaks of foodborne illness in North America and the causative agent of most hospital-acquired outbreaks among older adults. Norovirus strains are also considered a major cause of pediatric gastrointestinal disease worldwide. [0003] Noroviruses comprise one of several genera in the family Caliciviridae. The human norovirus genome is a single-stranded positive-sense RNA molecule encoding three open reading frames (ORFs) capped at its 5' end by the VPg protein. ORF1 encodes six nonstructural viral proteins, including VPg, RNA-dep...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/40C07K14/08C12N15/82C12N5/10C07K16/10A01H5/00A61K39/12A61P31/14A61P37/04
CPCA61K39/12C07K16/10C12P21/02C12N15/8258A61P31/14A61P37/04A61K2039/5258C12N2770/16034C07K14/05C12N2770/16023C12N2770/16022C07K14/005C12N7/00
Inventor 皮尔-奥利弗·拉沃伊马克-安德鲁·德奥斯特
Owner MEDICAGO INC