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Modified CPMV enhancer elements

A purpose and promoter technology, applied in the direction of angiosperms/flowering plants, allergic diseases, peptide sources, etc., can solve the problems of virus biological protection concerns, limit the size of inserted sequences, etc.

Active Publication Date: 2017-02-22
MEDICAGO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Despite success, the use of full-length viral vectors limits the size of the insert and movement between plants raises concerns about the biocontainment of the virus

Method used

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  • Modified CPMV enhancer elements
  • Modified CPMV enhancer elements
  • Modified CPMV enhancer elements

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0236] Example 1: 2X35S / CPMV-HT / PDISP / H3 Victoria / NOS (Construct No. 1391)

[0237] The sequence encoding H3 from influenza virus A / Victoria / 361 / 2011 (PDISP / H3 Victoria) in which the native signal peptide has been replaced by the signal peptide of the alfalfa protein disulfide isomerase was cloned into 2X35S using the following PCR-based method - CPMV-HT-NOS expression body. Using primers IF-PDI.S1+3c (Figure 5A, SEQ ID NO:18) and IF-H3V36111.s1-4r (Figure 5B, SEQ ID NO:19), using the PDISP / H3 Victoria sequence (Figure 5C, SEQ ID NO:20) was used as a template to amplify the fragment containing the coding sequence of PDISP / H3 Victoria. PCR products were cloned into the 2X35S / CPMV-HT / NOS expression system using the In-Fusion cloning system (Clontech, Mountain View, CA). Construct number 1191 was digested with SacII and StuI restriction enzymes ( Figure 5D and 5E , SEQ ID NO:21), and the linearized plasmid was used in the In-Fusion assembly reaction. Construct number 1191...

Embodiment 2

[0238] Example 2: 2X35S / CPMV-HT+ / PDISP / H3 Victoria / NOS (Construct No. 1819)

[0239] The sequence encoding H3 from influenza virus A / Victoria / 361 / 2011 (PDISP / H3 Victoria) in which the native signal peptide has been replaced by the signal peptide of the alfalfa protein disulfide isomerase was cloned into 2X35S using the following PCR-based method - CPMV-HT+ / NOS expressor. Using primers IF(SacII)-Kozac_PDI.c (Figure 6A, SEQ ID NO:24) and IF-H3V36111.s1-4r (Figure 5B, SEQ ID NO:19), using the PDISP / H3 Victoria sequence (Figure 8C, SEQ ID NO:19) IDNO:20) was used as a template to amplify a fragment of the PDISP / H3 Victoria 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 number 2181 (Figure 6B) was digested with SacII and StuI restriction enzymes, and the linearized plasmid was used in an In-Fusion assembly reaction. Construct number 2181 is a recipient plasmid d...

Embodiment 3

[0240] Example 3 SacII restriction site and PDISP / H3 in 2X35S / CPMV HT+ / NOS expression system Sequence changes between Victoria's ATGs (construct numbers 1952 to 1959)

[0241] Utilizing the same PCR-based approach as construct number 1819 (see Example 2), with a modified forward primer and keeping all other steps the same, eight SacII-restricted cells containing the 2X35S / CPMV HT+ / NOS expression system were created. Constructs with sequence changes between sites and the ATG of PDISP / H3Victoria. Variants HT1* to HT8* were amplified using the primers listed in Figures 7A-7H to create construct numbers 1952 to 1959, respectively:

[0242] IF-HT1*(-Mprot)-PDI.c (FIG. 7A, SEQ ID NO:27),

[0243] IF-HT2*(-Mprot)-PDI.c (FIG. 7B, SEQ ID NO:28),

[0244] IF-HT3*(-Mprot)-PDI.c (FIG. 7C, SEQ ID NO:29)

[0245] IF-HT4*(-Mprot)-PDI.c (FIG. 7D, SEQ ID NO:30)

[0246] IF-HT5*(-Mprot)-PDI.c (FIG. 7E, SEQ ID NO:31)

[0247] IF-HT6*(-Mprot)-PDI.c (FIG. 7F, SEQ ID NO:32)

[0248] IF-HT...

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PUM

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Abstract

An expression enhancer comprising, in series, a CPMV 5'UTR nucleotide sequence comprising nucleotides 1-160 of SEQ ID NO:1, or comprising a nucleotide sequence comprising from about 80% to 100% sequence similarity with SEQ ID NO:1, and a stuffer fragment is provided. The stuffer fragment comprises a nucleotide sequence encoding an incomplete M protein and one or more kozak sequence active in a plant. Plants and plant matter comprising the expression enhancer and methods using the expression enhancer are also described.

Description

[0001] field of invention [0002] The present invention relates to the expression of target protein in plants. The present invention also provides methods and compositions for producing a protein of interest in plants. [0003] Background of the invention [0004] Plants have great potential as production systems for recombinant proteins. One approach to producing foreign proteins in plants is to generate stable transgenic plant lines. However, this method is time-consuming and labor-intensive. An alternative to transgenic plants is the use of plant virus-based expression vectors. Plant virus-based vectors allow rapid, high-level, transient expression of proteins in plants. [0005] One approach to achieving high-level transient expression of foreign proteins in plants involves the use of RNA plant virus-based vectors, including the Cowpea mosaic virus group, such as Cowpea mosaic virus (CPMV; see, for example, WO2007 / 135480; WO2009 / 087391; US ​​2010 / 0287670, Sainsbury F...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H5/00C12N15/113C12N15/13
CPCC12N15/8216A61P31/16A61P37/04C07K14/11
Inventor 马克-安德烈·蒂奥斯特皮埃尔-奥利维耶·拉瓦伊
Owner MEDICAGO INC
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