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Rabies virus like particle production in plants

A technology of rabies virus and rabies, which is applied in the direction of plant products, viruses, and viral peptides, and can solve the problems of reduced yields and other issues

Inactive Publication Date: 2014-08-20
MEDICAGO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In contrast, infectious particles produced in rabies virus mutants lacking the M protein were predominantly cell-associated, and the yield of cell-free infectious virus was reduced by as much as 500,000-fold

Method used

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  • Rabies virus like particle production in plants
  • Rabies virus like particle production in plants
  • Rabies virus like particle production in plants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0133] Example 1: Assembly of expression cassettes with rabies protein

[0134] A-X35S / CPMV-HT / Rabies M / NOS (Construct No. 1066)

[0135]The sequence encoding the M protein from the ERA strain of rabies virus was cloned into the 2X35S / CPMV-HT / NOS expression system of the plasmid containing the Plasto_pro / P19 / Plasto_ter expression cassette using a PCR-based approach as follows. Using a synthetic M gene (corresponding to nt2496-3104 from Genbank accession number FJ913470) ( Figure 4C , SEQ ID NO: 3) as template, using primer IF-RabM-S3.c ( Figure 4A , SEQ ID NO: 1) and IF-RabM-S1-4.r ( Figure 4B , SEQ ID NO: 2) amplifies a fragment comprising the entire M protein coding sequence. PCR products were cloned into the 2X35S / CPMV-HT / NOS expression system using the In-Fusion cloning system (Clontech, Mountain View, CA). Construct No. 1191 was digested with SacII and StuI restriction enzymes ( Figure 4D ) and use the linearized plasmid in an In-Fusion assembly reaction. Cons...

Embodiment 2

[0142] Example 2: Preparation of Plant Biomass, Inoculum and Agrobacterium Infiltration

[0143] As used herein, the terms "biomass" and "plant matter" are meant to reflect any matter derived from plants. Biomass or plant matter may include whole plants, tissues, cells, or any fraction thereof. Additionally, biomass or plant matter can include intracellular plant components, extracellular plant components, bodily fluid or solid extracts of plants, or combinations thereof. Additionally, biomass or plant matter can include plants, plant cells from plant leaves, stems, fruits, roots, or combinations thereof, tissues, liquid extracts, or combinations thereof. A part of a plant may comprise plant matter or biomass.

[0144] N. benthamiana was grown from seed in flats filled with a commercially available peat moss substrate. Plants were grown in a greenhouse with a 16 / 8 photoperiod and a temperature regime of 25°C day / 20°C night. Three weeks after sowing, individual plantlets we...

Embodiment 3

[0155] Example 3: Evaluation of rabies G protein expression and co-expression strategies

[0156] N. benthamiana plants were agro-infiltrated with AGL1 / 1071 (with or without AGL1 / 1066) or AGL1 / 1091 (with or without AGL1 / 1086) inoculum at different concentrations, for 1071-infiltrated plants and 1071-infiltrated plants Leaves were harvested after 5 days post infiltration (DPI) for +1066 infiltrated plants or after 3 to 4 DPI for 1091 infiltrated plants and 1091+1086 infiltrated plants. Western blot analysis of leaf protein extracts from transformed plants showed that the BeYDV element was required to achieve detectable levels of G protein accumulation ( figure 1 Comparison of 1071-infiltrated plants with 1091-infiltrated plants). For plants infiltrated with AGL1 / 1091 co-expressed with or without the M protein (AGL1 / 1086), the maximum level of accumulation was reached at 3 DPI ( figure 1 ).

[0157] Biochemical extraction methods were able to release the rabies G protein from...

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Abstract

A method of producing a virus like particle (VLP) in a plant is provided. The method comprises introducing a first nucleic acid into the plant, or portion of the plant. The first nucleic acid comprising a first regulatory region active in the plant operatively linked to a nucleotide sequence encoding a native rabies virus structural protein. The nucleotide sequence may further comprise one or more than one amplification element. Optionally, a second nucleic acid might be introduced into the plant, or portion of the plant. The second nucleic acid comprising a second regulatory region active in the plant and operatively linked to a nucleotide sequence encoding a matrix protein, for example but not limited to a rabies matrix protein. The plant or portion of the plant is incubated under conditions that permit the expression of the nucleic acids, thereby producing the VLP.

Description

technical field [0001] The present invention relates to the production of native viral proteins in plants. More specifically, the present invention also relates to the production in plants of virus-like particles comprising native rabies virus structural proteins. Background technique [0002] Vaccination provides protection against disease caused by an infectious agent by inducing a subject to mount a defense prior to infection. Routinely, this is accomplished by using a live attenuated or whole inactivated form of the infectious agent as the immunogen. To avoid the dangers of using whole viruses (eg dead or attenuated viruses) as vaccines, recombinant viral proteins (eg subunits) are used as vaccines. Both peptide and subunit vaccines suffer from a number of potential limitations. Subunit vaccines can exhibit poor immunogenicity (due to incorrect folding), poor antigen presentation, or differences in carbohydrate and lipid composition. The main problem is that it is di...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01A01H5/00A61K39/205A61P31/14C07K14/145C07K16/10C12N15/00C12N15/47C12N15/82C12N7/00
CPCA61K2039/55505A61K2039/5258A61K39/205A61K2039/517C12N15/8258C12N2750/12043C07K14/005C12N2760/20123C12N2760/20134A61K39/12A61P31/14A61P31/16A61P37/04C12N7/00C12N15/8257
Inventor 马克-安德烈·德奥斯特皮埃尔-奥列弗·拉瓦路易斯-菲利普·韦齐纳马农·科图雷
Owner MEDICAGO INC
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