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Method for producing mullerian inhibitor substance in plants

A technology for inhibiting substances and plants, applied in biochemical equipment and methods, introduction of foreign genetic material using vectors, chemical instruments and methods, etc., can solve problems such as inapplicability of methods and inability to produce MIS

Inactive Publication Date: 2013-09-25
PHILIP MORRIS PROD SA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

All of these problems are particularly acute in the context of the industrial production of recombinant proteins in plants, where multiple or complex steps may render the method unsuitable
[0005] Current MIS production systems are not capable of producing MIS at levels required for clinical trials or commercial applications

Method used

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  • Method for producing mullerian inhibitor substance in plants
  • Method for producing mullerian inhibitor substance in plants
  • Method for producing mullerian inhibitor substance in plants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0135] Example 1 - Materials and methods

[0136] Cloning and infiltration

[0137] Nucleic acid constructs comprising nucleotide sequences encoding gamma-zein wild-type gene, its fragments and variants were respectively linked to synthetic sequences encoding mature MIS. In the case of using a fragment or variant of γ-zein, the nucleic acid construct further comprises a nucleotide sequence encoding the signal peptide of the native γ-zein at the 5' end, if it is present in the fragment or variant words that exist in the body. For some experiments, a synthetic nucleic acid sequence encoding a linker comprising a protease cleavage site between the gamma-zein coding sequence and the MIS coding sequence was also included in the construct. The coding sequence of mature MIS has been optimized for expression in plants. The nucleic acid construct is cloned into a vector at a site where expression of the nucleic acid construct in tobacco plant cells is driven by the minimal 35S promo...

Embodiment 2

[0152] Example 2 - Expression Levels of MIS-γ-Zein Fusion Proteins

[0153] The gamma-zein-enterokinase-MIS fusion protein construct (gamma-zein-MIS) was introduced into tobacco plants using the Agroinfiltration method. Total protein was extracted and quantified by Western blotting using γ-zein specific antibodies. Control experiments were also carried out using MIS expressed in the absence of γ-zein under the same conditions (MIS). Expression levels from the average of 4 Agroinfiltration events were as follows:

[0154] For γ-zein-MIS, the expression level was between about 0.2 and 0.6 g γ-zein-MIS / kg fresh weight.

[0155] For MIS without γ-zein, the expression level was between about 0.1 and 0.3 g γ-zein-MIS / kg fresh weight.

[0156] Based on these average results, it was concluded that the expression of γ-zein-MIS was about 30 times higher than that in the absence of γ-zein.

Embodiment 3

[0157] Example 3 - Analysis of different non-naturally occurring repeat motifs in gamma zein

[0158] γ-Zein-MIS fusion constructs were made using different non-naturally occurring repeat motifs in γ-zein. The following constructs were tested: γ-zein peptide only (γ-zein); γ-zein with (PPPVAL)n repeat motif; γ-zein with (PPPVEL)n repeat motif Prolamins; gamma-zein with (PPPAPA)n repeat motif and gamma-zein with (PPPEPE)n repeat motif.

[0159] The constructs were separately infiltrated into different tobacco plants using the Agroinfiltration method. Total protein was extracted and quantified by Western blotting using γ-zein specific antibodies. Expression levels from the mean of 2 Agroinfiltration events relative to γ-zein wild type were as follows:

[0160] Constructs tested

[0161] Three of the five constructs tested (γ-zein-Glu, γ-zein-(PPPAPA)n and γ-zein-(PPPAPA)n and γ-zein- Zein-(PPPEPE)n) significantly increased expression levels.

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Abstract

The present invention provides, in one aspect, a method for producing Mullerian Inhibitor Substance in a plant comprising incubating or growing a plant into which has been introduced or infiltrated a nucleic acid construct comprising, consisting or consisting essentially of a nucleic acid sequence encoding a Mullerian Inhibitor Substance fusion protein that comprises a fusion protein partner that induces the formation of a protein body in a plant, preferably, wherein the step of introducing or infiltrating the plant is performed prior to the incubating or growing step.

Description

field of invention [0001] The present invention relates to a method for producing Mullerian Inhibitor Substance (MIS) in plants by accumulating Mullerian Inhibitor Substance (MIS) in protein bodies. Nucleic acid sequences, nucleic acid constructs, vectors, expression vectors, etc. for carrying out the methods are also disclosed. Background technique [0002] Proteins of the TGF-β family mediate important embryogenic and immune functions, including chemotaxis, production of extracellular matrix, regulation of cell growth and differentiation, and development and regulation of the immune system. Thus, if available in sufficient quantities, these molecules can find application in a number of different therapies. Mullerian inhibitory substance (MIS), also known as anti-Müllerian hormone (AMH), is a member of the TGF-beta family. MIS is a 140kDa dimeric glycoprotein hormone. Like other TGF-β proteins, MIS is synthesized as a bulky precursor with a short signal sequence followed...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82
CPCC12N15/8257C07K14/495
Inventor K·奥伊什
Owner PHILIP MORRIS PROD SA
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