Production method, preparation and administration route of irisin

Abstract

The invention relates to a method for producing irisin. The method comprises the following steps: providing an expression vector containing a nucleotide sequence for coding the irisin; inserting the expression vector into at least one first bacterium belonging to the Agrobacterium genus, thereby obtaining a first Agrobacterium comprising the expression vector; treating at least one plant material with the first agrobacterium comprising the expression vector, thereby obtaining a treated plant material; incubating the treated plant material so that it expresses the irisin; and extracting the irisin. The invention also relates to irisin contained in liposomes, their use as a medicament and route of administration, as well as a synthetic gene comprising a sequence encoding irisin and additional elements.

Description

Technical Field

[0001] The present invention relates to a method for producing irisin, in particular recombinant irisin, in an organism of plant origin, and also relates to a preparation of irisin and a route of administration thereof. Background Art

[0002] Irisin is a molecule produced by muscle during physical exercise and is a myokine released upon cleavage of the fibronectin type III domain-containing membrane protein (FNDC5). Initially, its ability to induce transdifferentiation of white adipocytes to brown adipocytes was described, but subsequent studies have highlighted broader effects of irisin on other tissues and organs. Many of these effects have important clinical implications: the ability of low-dose irisin to prevent and treat osteoporosis and muscle atrophy in the musculoskeletal system; its role in regulating energy metabolism by reducing insulin resistance; and its ability to protect memory and slow cognitive decline in neurodegenerative diseases such as Alzheimer's disease.

[0003] As a result, irisin is emerging as an important molecular key to metabolic diseases and other diseases that can be improved after physical exercise.

[0004] The translation of the above results, obtained mainly in mouse models, to humans could lead to the development of drugs that would represent a highly innovative therapeutic strategy for mimicking exercise with multiple applications in medicine. However, further preclinical research steps, especially those aimed at regulating clinical prodromal symptoms to human clinical trials, involve experimental studies in large animal models and therefore require large amounts of protein that must be produced in accordance with good manufacturing practices ("GMP").

[0005] Such development first requires large-scale, cost-effective production of molecules that are as safe as possible for patients.

[0006] Recombinant DNA technology has made it possible to produce irisin in Escherichia coli and Chinese hamster ovary cells ("CHO" cells). However, there are many disadvantages to expressing recombinant proteins in these systems, including low yields, complex protocols for large-scale application, the potential presence of immunogenic endotoxins, and extremely high costs. These disadvantages, combined with the high cost, make it particularly complicated to develop a potential irisin-based drug, the use of which could affect a large number of patients with a variety of diseases.

[0007] Therefore, it is necessary to provide a method for producing irisin that can obtain a large amount of irisin simply, quickly and at low cost.

[0008] In addition to the technical issues associated with irisin production, there are a number of issues associated with irisin administration. For example, in its natural form, irisin may not be administered orally because proteases present in various parts of the gastrointestinal tract would digest it at the gastric and / or intestinal level, which would limit compliance for patients who might receive chronic irisin therapy.

[0009] Purpose of the Invention

[0010] Therefore, an object of the present invention is to provide a method for producing irisin that can easily and quickly obtain a large amount of irisin.

[0011] A further object of the present invention is to provide a method for producing irisin with high yield and low cost.

[0012] Another object of the present invention is to provide a formulation of irisin that improves the pharmacodynamic and pharmacokinetic properties of the molecule and increases patient compliance in human trials.

[0013] Another object of the present invention is to provide an irisin preparation that can be safely, conveniently and quickly administered.

[0014] Another object of the present invention is to provide a synthetic gene capable of achieving high-level expression of irisin. Summary of the invention

[0015] The above objects and other objects are achieved by the content of the present invention, that is, a method for producing irisin, in particular a method for producing recombinant irisin, which comprises the following steps:

[0016] a) providing at least one expression vector, preferably at least one plasmid, comprising a nucleotide sequence encoding irisin;

[0017] b) inserting the expression vector into at least one bacterium of the genus Agrobacterium, thereby obtaining a bacterium of the genus Agrobacterium containing the expression vector;

[0018] c) treating at least one plant material with the bacterium comprising the expression vector, thereby obtaining a treated plant material;

[0019] d) cultivating the treated plant material to express the irisin;

[0020] e) extracting the irisin from the plant material.

[0021] In fact, it has been observed that organisms of plant origin are able to express irisin, in particular recombinant irisin, for example in a non-glycosylated or lightly glycosylated form, but at levels significantly higher than those achievable with known technologies.

[0022] In addition, plants have several advantages that make them attractive for producing recombinant proteins, especially for pharmacological purposes. For example, they can be easily grown on a large scale in small greenhouses and do not contain immunogenic bacterial endotoxins, which are a major challenge in the purification of proteins produced in E. coli.

[0023] Furthermore, compared to traditional expression systems such as microbial fermentation and mammalian cell culture, plant production systems are low-cost, easy to implement on an industrial scale, and found to be free of pathogens that are harmful to human health.

[0024] Herein, the term "recombinant irisin" refers to irisin obtained by transcription and translation of a recombinant DNA fragment inserted into a host organism.

[0025] As used herein, the term "recombinant DNA" refers to a DNA sequence artificially obtained by combining genetic material from (possibly) different sources, such as a plasmid containing a gene of interest.

[0026] In an embodiment of the present invention, irisin is in a non-glycosylated form or a lightly glycosylated form.

[0027] The production of non-glycosylated or lightly glycosylated irisin has several advantages over the production of glycosylated irisin (i.e., compared to the production of irisin where the majority of the molecule is in the glycosylated form). In fact, surprisingly, it has been found that the production of non-glycosylated or lightly glycosylated irisin has significantly higher yields than the production of glycosylated irisin (i.e., compared to the production of irisin where the majority of the molecule is in the glycosylated form). In particular, western blot and ELISA assays enable observation of how methods suitable for the production of glycosylated irisin (i.e., compared to the production of irisin where the majority of the molecule is in the glycosylated form) fail to produce irisin in significant quantities.

[0028] For example, production of non-glycosylated irisin can be achieved by using a gene sequence that does not include a signal peptide coding sequence capable of directing irisin to the endoplasmic reticulum.

[0029] For example, production of lightly glycosylated irisin can be achieved by using a gene sequence including a sequence encoding a KDEL tag, which allows irisin to be maintained in the endoplasmic reticulum.

[0030] The nucleotide sequence encoding irisin is currently commercially available in the form of a plasmid, preferably a pUC57 plasmid (eg, from GenScript, Piscataway, NJ) comprising the nucleotide sequence.

[0031] The irisin encoding sequence can be isolated from the original plasmid by using appropriate restriction enzymes, such as BamHI and XmaI restriction enzymes.

[0032] Before inserting the nucleotide sequence encoding irisin into an expression vector suitable for inserting into plant material, the sequence can be inserted into an intermediate vector (or amplification vector) for amplification, i.e., to generate a large number of copies of the sequence. The intermediate vector containing the sequence encoding irisin is used to transform E. coli cells. The E. coli cells are then cultured, and at the end of cell growth, the intermediate vector containing the sequence encoding irisin is purified.

[0033] Therefore, in an embodiment, the method according to the present invention comprises a preliminary step, i.e., before step a), in which an intermediate vector (or amplification vector) comprising a nucleotide sequence encoding irisin is inserted into E. coli and amplified by culturing the E. coli.

[0034] Preferably, the intermediate vector is a plasmid, more preferably a pGEM-NOS plasmid.

[0035] After the above-mentioned preliminary amplification step, the sequence encoding irisin is inserted into an expression vector, preferably together with a sequence having a plant terminator function, preferably a Nos-ter terminator. A terminator is a sequence capable of terminating gene transcription, allowing the expression vector to be integrated into the plant genome. The Nos-ter terminator is a known terminator in the art. In an embodiment, the sequence encoding irisin is inserted into an expression vector, preferably together with a sequence having a plant terminator function, optionally after amplification in Escherichia coli.

[0036] After amplification in E. coli, the intermediate vector containing the sequence encoding irisin is purified, and the sequence encoding irisin is extracted therefrom, preferably together with a sequence having a plant terminator function (eg, Nos-ter), and then inserted into an expression vector.

[0037] In an embodiment, the expression vector is a plasmid, preferably a pBIΩ plasmid. Once the nucleotide sequence encoding irisin is inserted, the plasmid is called pBIΩ-IRS.

[0038] In an embodiment, the expression vector is a plasmid, preferably a modified pJL-TRBO plasmid.

[0039] Reference herein to a “modified pJL-TRBO plasmid” refers to a pJL-TRBO plasmid in which the restriction sites pBluescriptKS, Pvu I, GFP-R, BstZ17 I, and GFP-F are removed, and the restriction sites Sal I, Mlu I, NheI, Bmt I, Eco53k I, Sac I, Nco I, Nru I, Asc I–BssH II, ApaL I, AsiS I, Swa I, Kas I, Nar I, Sfo I, PluT I, and Avr II are inserted.

[0040] pBluescriptKS, Pvu I, GFP-R, BstZ17 I and GFP-F, as well as restriction sites such as Sal I, Mlu I, Nhe I, BmtI, Eco53k I, Sac I, Nco I, Nru I, Asc I–BssH II, ApaL I, AsiS I, Swa I, Kas I, Nar I, Sfo I, PluT I and Avr II are known in the art.

[0041] Reference to the expression "pJL-TRBO" or "unmodified pJL-TRBO" refers to the original version of the pJL-TRBO plasmid currently commercially available and sold by Addgene (Watertown, MA, USA).

[0042] The modified pJL-TRBO plasmid contains 47 restriction sites, while the unmodified pJL-TRBO plasmid contains 35 restriction sites.

[0043] The modified pJL-TRBO plasmid can be obtained from the unmodified pJL-TRBO plasmid by methods known in the art.

[0044] The modified pJL-TRBO vector can be used to express irisin in plant materials.

[0045] The use of the modified pJL-TRBO plasmid can significantly improve the Agrobacterium-infection efficiency, which is much higher than that of the unmodified pJL-TRBO plasmid. In addition, the use of the modified pJL-TRBO plasmid can achieve a higher level of irisin expression than the unmodified pJL-TRBO plasmid. In addition, the use of the modified pJL-TRBO plasmid can avoid the formation of virus particles during the infection-replication cycle.

[0046] In an embodiment, the expression vector may be selected from a pBIΩ plasmid or a modified pJL-TRBO plasmid.

[0047] In addition, the modified pJL-TRBO plasmid contains the 35S promoter from the cauliflowe mosaic virus (CAMV), which allows high-level expression of proteins in plants, as well as a replication origin site, a kanamycin antibiotic resistance gene, a replication initiation protein (trfA) gene, a 5'-leader sequence of the tobacco mosaic virus (TMV) (called Ω (Omega)), a "left border repeat of T-DNA" region, a "right border repeat of T-DNA" region, and a KS primer sequence for amplifying and sequencing the right end of the gene.

[0048] Once the region between the border repeat regions of the T-DNA (i.e., between the "left border repeat of T-DNA" and the "right border repeat of T-DNA" regions) is identified in Agrobacterium, the region is transferred into plant cells.

[0049] The expression vector can be inserted into at least one bacterium of the genus Agrobacterium by methods known in the art, such as electroporation.

[0050] The plant material may be selected from a plant, or at least a part of a plant, or at least a cell of the plant.

[0051] For example, the plant material can be selected from the following plants: tobacco (preferably Nicotiana benthamiana), hemp (Cannabis sativa), spirulina (Arthrospira platensis), chlorella, Arabidopsis, corn, rice, soybean, rapeseed, alfalfa, sunflower, sorghum, wheat, cotton, peanut, tomato, potato, lettuce and pepper, or at least a part of these plants or at least a cell thereof.

[0052] Preferred plant material is selected from the tobacco plant (preferably Nicotiana benthamiana), or at least a part of such a plant or at least a cell thereof.

[0053] In certain embodiments, for example, plant leaves can be used as plant material of the present invention to express recombinant proteins in Nicotiana benthamiana. Advantageously, using leaves as plant material can eliminate the need for flowering, thereby significantly reducing the potential risk of causing gene diffusion to the environment through the spread of pollen or seeds. In addition, tobacco is a non-food crop, which eliminates the risk of plant-produced recombinant proteins entering the food chain.

[0054] The treatment of the plant material according to step c) of the method can be carried out by immersing the plant material (preferably completely submerging it) in a solution containing one or more bacteria of the genus Agrobacterium and an expression vector and applying a vacuum. In an embodiment, immersing the plant material in a solution containing Agrobacterium (preferably in a desiccator) takes only a few minutes. For example, immersing the plant material in a solution containing Agrobacterium may take 5 to 15 minutes. Preferably, the vacuum is applied and released quickly once about 10 mm Hg is reached.

[0055] Step c) in the method of the present invention can be defined as an agro-infiltration step.

[0056] In an embodiment, step c) of the method of the present invention further comprises treating the plant material with at least one second bacterium of the genus Agrobacterium, wherein the second bacterium comprises an expression vector comprising a gene for preventing silencing of irisin expression in the treated plant material.

[0057] In some embodiments, in step c) of the method of the present invention, the plant material is treated with a mixture comprising a first bacterium and a second bacterium, wherein the first bacterium comprises an expression vector comprising a nucleotide sequence encoding irisin, and the second bacterium comprises a gene for preventing silencing of irisin expression in the treated plant material.

[0058] Advantageously, the use of genes for preventing silencing of irisin expression in treated plant material can inhibit plant cell defense against exogenous genes (in this case, the irisin gene); said fragmentation and silencing are defense mechanisms that plant cells may activate when the production of exogenous transcripts significantly exceeds the transcripts of endogenous genes of the host plant, i.e., in this case, the production of irisin transcripts is significantly increased.

[0059] A gene preferably used to prevent silencing of irisin expression is the P19 gene.

[0060] The P19 gene encodes a protein called "p19 RNA silencing suppressor protein". The p19 protein can specifically bind to double-stranded RNAs of 19 to 21 nucleotides, which function as small interfering RNAs (siRNAs) in the RNA silencing system of plant cells. By sequestering these siRNAs, p19 suppresses the silencing of exogenous RNA.

[0061] A preferred Agrobacterium bacterium is Agrobacterium tumefaciens.

[0062] The preferred nucleotide sequence encoding irisin is:

[0063]

[0064] The nucleotide sequence SEQ.ID.NO.2 includes the restriction site of BamHI enzyme (the first underlined 6 nucleotides) and the restriction site of SmaI enzyme (the last underlined 6 nucleotides). The two stop codons are shown in bold, i.e., the 6 nucleotides immediately before the restriction site of SmaI enzyme.

[0065] In an embodiment, the nucleotide sequence encoding irisin is contained in a gene (particularly a synthetic gene) that, in addition to the sequence encoding irisin, also includes a polynucleotide sequence encoding an N-terminal signal peptide for secretion from alfalfa disulfide isomerase from Medicago sativa; a sequence encoding a thrombin cleavage site, followed by a sequence encoding an 8-histidine residue tail (8-His tag), and a sequence encoding a KDEL terminal tail that allows the protein to be retained in the endoplasmic reticulum. The nucleotide sequence encoding irisin is located between the polynucleotide sequence encoding the N-terminal signal peptide for secretion from alfalfa disulfide isomerase and the sequence encoding the thrombin cleavage site.

[0066] Therefore, in an embodiment, an expression vector comprising a nucleotide sequence encoding irisin may include a gene (particularly a synthetic gene) comprising: a polynucleotide sequence encoding an N-terminal signal peptide for secretion from Medicago truncatula disulfide isomerase; a sequence encoding irisin; a sequence encoding a thrombin cleavage site; a sequence encoding an 8 histidine residue tail (8-His tag); and a sequence encoding a KDEL terminal tail.

[0067] Therefore, another object of the present invention is a gene (i.e. a polynucleotide sequence, in particular a synthetic gene) comprising a polynucleotide sequence encoding an N-terminal signal peptide for secretion of alfalfa disulfide isomerase from alfalfa; a sequence encoding irisin; a sequence encoding a thrombin cleavage site; a sequence encoding an 8-histidine residue tail (8-His tag); and a sequence encoding a KDEL terminal tail, which sequences are fused to each other.

[0068] Using such a synthetic gene, a polypeptide (particularly a synthetic peptide) can be obtained that consists of an N-terminal signal peptide for secretion from disulfide isomerase of Medicago truncatula, the amino acid sequence of the irisin protein, followed by a thrombin cleavage site and an 8-His tag (i.e., a tail of 8 histidine residues), and a KDEL terminal tag (i.e., a tail consisting of lysine, aspartic acid, glutamic acid and leucine residues).

[0069] Advantageously, particularly high levels of irisin expression can be achieved using this synthetic gene.

[0070] The N-terminal signal peptide for secretion of the disulfide isomerase from Medicago truncatula allows the synthesized polypeptide to be directed to the secretory pathway. The N-terminal signal peptide of the disulfide isomerase is introduced 5' to the irisin coding sequence and can be removed by enzymatic treatment, for example, in the same plant cell expressing the polypeptide.

[0071] The thrombin cleavage site is the site where thrombin cleaves, and after cleavage, the 8-His tail and the KDEL tail are removed.

[0072] The 8-His tag was used for the extraction and purification of irisin.

[0073] The KDEL terminal tag (i.e., a tail consisting of lysine, aspartic acid, glutamic acid, and leucine) can keep the protein in the endoplasmic reticulum. Advantageously, the use of the KDEL tail can increase the expression of the recombinant protein by 2 to 10 times. The KDEL tag can be removed by enzymatic treatment, for example, after irisin is extracted from plant material by thrombin, it is removed together with the histidine tail.

[0074] For example, the nucleotide sequence encoding irisin in the present invention is contained in a gene (especially a synthetic gene) having the following sequence:

[0075]

[0076] The polynucleotide sequence SEQ.ID.NO.8 includes the polynucleotide sequence of irisin. The bold part is the sequence encoding the N-terminal signal peptide for secretion of alfalfa disulfide isomerase; the single underlined part is the sequence encoding the thrombin cleavage site; the double underlined part is the sequence encoding the 8-His tag; the bold and single underlined part is the sequence encoding the KDEL tag.

[0077] Preferably, the polypeptide obtained by using the synthetic gene has the following sequence:

[0078]

[0079]

[0080] The amino acid sequence SEQ.ID.NO.9 includes the amino acid sequence of irisin. The bold part is the N-terminal signal peptide sequence for secretion of alfalfa disulfide isomerase; the single underlined part is the thrombin cleavage site; the double underlined part is the 8-His tag; the bold and single underlined part is the KDEL tag.

[0081] In embodiments, when irisin is produced using the synthetic genes of the present invention, irisin may exhibit light glycosylation, i.e., slight glycosylation.

[0082] The expression "lightly glycosylated" refers to partial glycosylation, in which about 10% of the irisin molecules are glycosylated. For example, the term "lightly glycosylated" refers to partial glycosylation, in which the number of glycosylated irisin molecules is less than or equal to 10% of the total number of irisin protein molecules.

[0083] In some embodiments, for example, lightly glycosylated irisin is enzymatically treated to remove glucose groups, thereby yielding non-glycosylated irisin.

[0084] In some embodiments, irisin extracted from plant materials may be partially glycosylated (particularly lightly glycosylated, wherein about 10% or less of the irisin molecules are glycosylated), and the method further comprises a step of removing glycosylation (i.e., removing glucose groups), for example, by enzymatic treatment. The process of removing glycosylation can be performed using techniques known in the art.

[0085] According to step d) of the inventive method, the cultivation time of the treated plant material can vary depending on the plant used, preferably carried out in a greenhouse. For example, if the plant is a tobacco plant, the cultivation time of the treated plant material is 2 to 7 days, preferably 6 to 7 days.

[0086] According to step e) of the method of the present invention, the extraction of the synthesized irisin from the plant material can be performed using techniques known in the art. For example, the extraction can be performed using a PBS buffer supplemented with a protease inhibitor.

[0087] According to an embodiment of the present invention, irisin does not require a histidine tail ("His-tag") during the purification process. Advantageously, the lack of a His-tag tail can greatly reduce costs. In fact, the His-tag histidine tail must be removed by enzymatic treatment with, for example, enterokinase (EK) before irisin is administered in vivo, which means that the cost is significantly increased compared to methods for producing and purifying irisin without using a His-tag. In addition, it was observed that recombinant irisin with a His-tag at the C-terminal position completely lost important biological functions (e.g., the ability to stimulate osteoblast differentiation), which further confirms the necessity of removing the histidine tail so that irisin can perform important biological functions.

[0088] In embodiments, for example, when irisin is produced using the synthetic gene of the invention, the His-tag tail can be removed by enzymatic treatment with thrombin. Advantageously, the presence of the histidine tail allows for very rapid, efficient, and accurate purification of irisin, for example by immobilized metal ion affinity chromatography (IMAC).

[0089] In an embodiment, the expression vector is a pJL-TRBO plasmid, in which the restriction sites pBluescriptKS, Pvu I, GFP-R, BstZ17 I and GFP-F are removed, and the restriction sites Sal I, Mlu I, Nhe I, Bmt I, Eco53k I, Sac I, Nco I, Nru I, Asc I–BssH II, ApaL I, AsiS I, Swa I, Kas I, Nar I, Sfo I, PluT I, Avr II are inserted, and wherein the expression vector comprises the following genes: a polynucleotide sequence encoding an N-terminal signal peptide for secretion of alfalfa alfalfa disulfide isomerase; a sequence encoding irisin; a sequence encoding a thrombin cleavage site; a sequence encoding an 8-histidine residue tail (8-His tag); and a sequence encoding a KDEL terminal tail.

[0090] Advantageously, use of a modified pJL-TRBO expression vector and inclusion of the irisin encoding sequence in a gene that also includes a polynucleotide sequence encoding an N-terminal signal peptide for secretion from the disulfide isomerase of Medicago truncatula, a sequence encoding a thrombin cleavage site, a sequence encoding an 8 amino acid residue histidine tail (8-His tag), and a sequence encoding a KDEL terminal tail can result in irisin production in yields exceeding 5 mg protein per gram of fresh plant material.

[0091] In an embodiment, the expression vector is a modified pJL-TRBO plasmid, and the nucleotide sequence encoding irisin is contained in a gene (particularly a synthetic gene) having the following sequence:

[0092]

[0093] Another object of the present invention is to include irisin in liposomes. In other words, the present invention relates to liposomes (at least one liposome) containing irisin, preferably liposomes encapsulating irisin.

[0094] In this specification, the term "liposome" refers to a vesicle consisting of at least one lipid bilayer and an aqueous solution core encapsulated in the lipid bilayer. The lipids constituting the liposome bilayer (or the lipids forming the liposome) may include a mixture mainly composed of phospholipid substances, such as phosphatidylcholine and cholesterol. Preferably, irisin is encapsulated in the above-mentioned liposome.

[0095] The irisin contained in the liposome may be non-glycosylated irisin.

[0096] In an embodiment, the irisin contained in the liposomes is produced in plant material, ie obtained according to the method of the invention.

[0097] For example, liposomes containing irisin can be prepared by dissolving soybean lecithin in a solution of methanol, adding irisin to the solution, and then ultrasonicating the solution, and then adding a chitosan solution to the solution.

[0098] In an embodiment, the liposomes comprise lecithin and chitosan, and preferably are nanoparticles comprising lecithin and chitosan.

[0099] Advantageously, liposomes provide an effective way to help active compounds they carry pass through physiological barriers, thereby increasing the bioavailability of these active compounds. Liposomes are able to form vesicles (packets) that encapsulate the active compound (irisin in this case), which are physically isolated from the surrounding environment and can protect the active compound from any chemical or enzymatic degradation processes before reaching the target tissue.

[0100] Therefore, another object of the present invention is to use irisin contained in liposomes as a medicament. In other words, one aspect of the present invention relates to liposomes containing irisin for use as a medicament.

[0101] For example, irisin-containing liposomes (i.e., irisin-containing liposomes) can be used to treat and / or prevent osteoporosis, sarcopenia, energy metabolism disorders, cardiovascular system diseases, neurodegenerative diseases, diabetes, obesity, kidney diseases, and metabolic diseases.

[0102] Advantageously, liposomes are not restricted in their use and can be administered via a variety of routes. In fact, liposomes are a biologically safe system because they are composed of phospholipids, which are natural components of all cell membranes.

[0103] In some embodiments, irisin contained (preferably encapsulated) in liposomes can be administered sublingually and / or subcutaneously and / or intradermally, preferably by using a skin gel; and / or nasally, preferably by using a nasal spray. In other words, the liposomes containing irisin can be administered sublingually and / or subcutaneously and / or intradermally, preferably by using a skin gel; and / or nasally, preferably by using a nasal spray.

[0104] The present invention demonstrates that irisin can be expressed in plant-derived organisms and that the protein has biological activity, the effects of which can overlap with those caused by commercial irisin used at the same concentration. Therefore, expressing irisin in plants such as Nicotiana benthamiana has many advantages over the traditional use of bacteria (prokaryotic cells) or mammalian cells (eukaryotic cells) as recombinant protein production systems. For example, plants can be easily cultivated on a large scale without immunogenic bacterial endotoxins and pathogens. In addition, the cost of using plant materials is significantly lower than known methods, making it possible to produce large amounts of irisin at a lower cost, thereby promoting its use in treatment. In addition, the present invention enables rapid advancement of research in the medical field that requires large amounts of protein.

[0105] By way of illustrative, non-limiting examples described in the following experimental section and in conjunction with Figure 1-6 , the content of the present invention can be better understood. BRIEF DESCRIPTION OF THE DRAWINGS

[0106] - Figure 1 The different steps of the process for preparing an expression vector comprising a nucleotide sequence encoding irisin are shown. After obtaining the pBIΩ-IRS vector, it is inserted into a bacterium of the genus Agrobacterium.

[0107] - Figure 2 Results of quantification of irisin expression by Western blotting are shown.

[0108] - Figure 3 Shown are the results of quantification of irisin expression by ELISA experiments.

[0109] - Figure 4 The results of an in vitro mouse osteoblast MAP kinase phosphorylation experiment are presented to verify the biological effects of irisin contained in Nicotiana benthamiana extract.

[0110] - Figure 5 Results of experiments evaluating the biological activity of irisin expressed and purified from plant cells and contained in liposomes are presented.

[0111] - Figure 6 A map of the modified pJL-TRBO expression vector is shown.

[0112] Experimental Section

[0113] Example 1 - Preparation of an expression vector containing a nucleotide sequence encoding irisin and its insertion into Agrobacterium

[0114] The experimental work was primarily conducted using Nicotiana benthamiana plants; however, the method is reproducible in other types of plant organisms, such as hemp (Cannabis sativa), Spirulina, Chlorella, Arabidopsis, corn, rice, soybean, canola, alfalfa, sunflower, sorghum, wheat, cotton, peanut, tomato, potato, lettuce, and pepper.

[0115] The nucleotide sequence encoding the protein corresponding to the irisin portion is optimized to adapt to the codons of the plant and engineered to allow the construct to be expressed in plant tissues. Based on the amino acid sequence of irisin (112 amino acids, SEQ.ID.NO.1), a nucleotide sequence (SEQ.ID.NO.2) for expression in Nicotiana benthamiana was synthesized and optimized. The nucleotide sequence SEQ.ID.NO.2 includes a restriction site for the BamHI enzyme (the first 6 underlined nucleotides) and a restriction site for the SmaI enzyme (the last 6 underlined nucleotides). The nucleotide sequence SEQ.ID.NO.2 further includes two stop codons (the 6 nucleotides immediately before the restriction site for the SmaI enzyme).

[0116] All synthetic genes were generated using Optimum-Gene TM The algorithm (GenScript, Piscataway, NJ, USA) was optimized to accommodate the expression codons in Nicotiana benthamiana.

[0117] Amino acid sequence of natural irisin

[0118] DSPSAPVNVTVRHLKANSAVVSWDVLEDEVVIGFAISQQKKDVRMLRFIQEVNTTTRSCALWDLEEDTEYIVHVQAISIQGQSPASEPVLFKTPREAEKMASKNKDEVTMKE(SEQ.ID.NO.1)

[0119] Polynucleotide sequence of irisin expressed in Nicotiana benthamiana:

[0120] GGATCCGATTCTCCTTCAGCTCCAGTTAATGTTACAGTTAGACATCTTAAGGCTAATTCTGCTGTTGTTTCATGGGATGTTTTGGAAGATGAGGTTGTTATTGGTTTTGCTATCTCCAACAGAAGAAAGATGTTAGAATGCTTAGGTTCATCCAAGAAGTTAACACTACAACTAGGTCTTGT GCTCTTTGGGATTTGGAAGAGGATACAGAGTACATCGTTCATGTTCAGGCTATCTCAATCCAAGGACAGTCTCCTGCTTCAGAACCAGTTTTGTTTAAAACTCCTAGGGAGGCTGAGAAAATGGCAAGTAAAAACAAGGATGAGGTGACAATGAAAGAGTGATAGCCCGGG (SEQ.ID.NO.2)

[0121] The amino acid sequence encoded by the above polynucleotide sequence:

[0122] DSPSAPVNVTVRHLKANSAVVSWDVLEDEVVIGFAISQQKKDVRMLRFIQEVNTTTRSCALWDLEEDTEYIVHVQAISIQGQSPASEPVLFKTPREAEKMASKNKDEVTMKE(SEQ.ID.NO.3)

[0123] It can be observed that the amino acid sequence of the recombinant irisin produced according to the present invention (SEQ.ID.NO.3) is identical to the amino acid sequence of natural irisin (SEQ.ID.NO.1).

[0124] Schematic diagram of cloning the synthetic gene encoding irisin in the pBIΩ expression vector Figure 1As shown, the synthetic gene encoding irisin has the sequence of SEQ.ID.NO.2. The construct comprising the nucleotide sequence encoding irisin is cloned by GeneScript and inserted into the pUC57 plasmid by using two restriction enzyme sites (BamHI site at the 5' end and XmaI site at the 3' end). From the pUC57-IRS plasmid containing the synthetic gene encoding irisin (i.e., sequence SEQ.ID.NO.2), the insert fragment (IRS, corresponding to the sequence encoding irisin) is purified by cutting with BamHI and XmaI restriction enzymes. Then, the gene is cloned into an intermediate vector. This step uses a binary vector that can be replicated in Escherichia coli and Agrobacterium tumefaciens. The step in Escherichia coli is to improve transcription and amplification efficiency. The step of using an intermediate vector before moving to the vector to be transferred to Agrobacterium is also due to the lack of useful restriction enzyme sites in the final vector. Therefore, the commercial E. coli vector pGEM-NOS was used as an intermediate vector, and the plant terminator Nos-ter was cloned therein; this is necessary for the integration of the final vector in plants, ie for the integration of the recombinant DNA in the plant genome.

[0125] In the next step, the final construct was purified from E. coli (by using a currently commercially available kit) and electroporated into Agrobacterium LBA4404 (i.e., Agrobacterium tumefaciens LBA4404 strain) by using a pBIΩ-IRS expression vector containing a transcription enhancer (CaMV35S promoter, i.e., a viral promoter) at the 5' position to further increase the transcription level.

[0126] In the same vector (pBIΩ), the P19 gene was also cloned, which is essential for suppressing the silencing of the irisin gene. The vector containing the P19 gene is transferred into Agrobacterium, but is transferred separately from the vector containing the irisin gene. This process is necessary to suppress the fragmentation and silencing of plant cells to exogenous genes (i.e., irisin genes). In the presence of overproduced exogenous transcripts, that is, the production amount of exogenous transcripts is much greater than the transcripts produced by the host plant endogenous genes (i.e., the transcripts derived from the irisin gene in this embodiment), plant cells may activate this defense mechanism. Therefore, the Agrobacterium LBA4404 strain culture carrying the binary vector containing the irisin gene and the enhancer, and the Agrobacterium culture carrying the binary vector containing the P19 gene and the enhancer were cultivated separately, and the two cultures were mixed before Agrobacterium infiltration. Finally, the Agrobacterium tumefaciens LBA4404 culture containing these two constructs was used to carry out the Agrobacterium infiltration of plants.

[0127] Example 2 - Using Root cancer Agrobacterium infiltration of Nicotiana benthamiana leaves with Agrobacterium tumefaciens LBA4404

[0128] Transient expression in plant leaves was obtained by vacuum infiltration. This expression is defined as "transient" because it only affects infected leaves. Cultivate Agrobacterium tumefaciens (LBA4404) clones carrying the above-mentioned construct respectively, and the bacteria are resuspended by centrifugal sedimentation at 4000g and infiltration buffer (10mM MES, 10mM MgSO4, pH 5.8). Use the Agrobacterium suspension carrying different carriers respectively, or mix them together, so that the final optical density (OD600) of each construct reaches 0.5. Infiltration treatment is carried out to the hydroponic Nicotiana benthamiana plants (being in the 6-7 leaf stage) of six weeks old by immersing each plant completely in the solution containing Agrobacterium in the desiccator. Apply vacuum to about 10mm Hg, then release quickly. Observe and confirm the effect of infiltration by observing that the infiltration area is translucent. Then the plant is placed in a greenhouse, and different and predetermined time after infiltration, i.e., leaf sampling is carried out 2 to 7 days (2-7DPI) after infiltration. Leaves of the same age (usually the 4th and 5th leaves from the bottom, i.e., the “intermediate leaf” position) from three plants were collected and stored in liquid nitrogen (N 2 ). In the same experiment, plants infiltrated with the same vector lacking the gene encoding irisin were cultured and used as negative controls according to the same experimental method. To verify the expression and subsequent purification of irisin, 40 g of leaves infiltrated with tumefaciens of all ages were collected, immediately frozen with liquid nitrogen and stored at -80°C. The samples were then extracted in PBS buffer supplemented with protease inhibitors, and irisin expression was tested and quantified by Western blot and ELISA analysis.

[0129] Example 3 - Quantification of irisin expression by Western blot and ELISA

[0130] Leaf tissue (100 mg) was ground in liquid nitrogen and homogenized in 500 μl of phosphate-buffered saline (PBS) at pH 7.2 containing a protease inhibitor cocktail (Complete™; Roche, Mannheim, Germany). After centrifugation at 20,000 g for 30 min at 4 °C, the supernatant was collected and lysed using DC TM The total content of soluble proteins was quantified by protein assay (Bio-Rad, California, USA). Leaves of plants at the same position (4th and 5th leaves from the bottom) were collected at 2, 3, 4, 5, 6 and 7 days after infiltration (DPI) and expression was detected by Western blot analysis using anti-FNDC5 antibody (Abcam-ab131390). In the same experiment, plants infiltrated with the same vector agrobacterium lacking the gene encoding irisin were cultured according to the same experimental method and used as negative control.

[0131] Western blot analysis of leaf extracts under reducing conditions showed a band with a molecular weight of 12 kD corresponding to irisin, as shown by the band produced by the recombinant protein produced in E. coli, which is currently commercially available and used as a positive control (Adipogen-AG-40B-0103-C010) ( Figure 2 ). The same band did not appear in the negative control.

[0132] The maximum expression of irisin, indicated by the intensity of the 12 kDa band, was detected at day 7 after infiltration ( Figure 2 ).

[0133] An ELISA test (AG-45A-0046YEK-KI01, Adipogen) was performed to quantify the irisin present in the leaf extract. The Phoenix Pharmaceutical ELISA kit is designed for the determination of irisin concentration based on the principle of competitive enzyme immunoassay. The 96-well plates of the kit are pre-coated with recombinant irisin. The irisin-specific polyclonal antibody competes with the known concentrations of recombinant irisin added in the standard curve and the irisin in the unknown concentration of the leaf extract samples in the irisin-coated plates. The final color intensity is inversely proportional to the irisin content in the standard irisin solution or leaf extract. This is due to the competition between the irisin in the standard curve or leaf extract and the irisin molecules present in the 96-well plate coating for binding to the primary antibody. The standard curve was prepared by interpolating the optical density points measured at various known concentrations of the standard. The concentration of irisin in the leaf extract (mean 2.563 μg / ml ± 0.55SD) was determined by extrapolation based on the standard curve ( Figure 3 ).

[0134] In addition, the average level was 0.5 mg / 100 g fresh leaf tissue, accounting for about 0.6% of the total soluble protein content.

[0135] Example 4 - In vitro biological effects of irisin contained in Nicotiana benthamiana extract on mouse osteoblasts

[0136] In order to prove that irisin expressed and purified from plant cells is biologically active, the effects of the extracts were evaluated in vitro. In particular, given that irisin is able to activate the MAP kinase ERK in skeletal progenitor cells osteoblasts, we conducted experiments by stimulating osteoblasts for 5, 10 and 20 minutes with extracts from Nicotiana benthamiana leaves infiltrated with pBIΩ-IRS containing 100 ng / ml irisin or with the corresponding negative control (C-) pBIΩ Agrobacterium without the gene sequence encoding irisin synthesis SEQ.ID.NO.2 ( Figure 4After the stimulation, osteoblasts were lysed using a lysis buffer consisting of 50 mM Tris-HCl (pH 8.0), 150 mM HCl, 5 mM EDTA, 1% NP40, and 1 mM phenylmethylsulfonyl fluoride (PMSF). Protein concentration was determined by using DC TM Protein quantification kit (Bio-Rad, California, USA) was used for determination. 20 μg of cell protein was subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a nitrocellulose membrane (Millipore, Massachusetts, USA). The blot was incubated overnight at 4°C and anti-phosphorylated ERK (pERK) primary antibody (Santa Cruz Biotechnology) and anti-total ERK (tERK) (Santa Cruz Biotechnology) were used. The membrane was then incubated for two hours at room temperature and a secondary antibody labeled with IRDye (680 / 800CW) (LI-COR Biosciences) was used. For immunodetection, the Odyssey infrared imaging system (LI-COR Corp., Lincoln, Nebraska) was used. All data were normalized to tERK and calculated as the fold change (i.e., the fold amplification of the signal) relative to the zero time point (t0).

[0137] Our results showed that a 5-minute stimulation with extracts of Nicotiana benthamiana leaves infiltrated with pBIΩ-IRS Agrobacterium activated the phosphorylation of MAP kinase ERK in osteoblasts. Phosphorylation of ERK remained active for 10 minutes and then stopped 20 minutes after stimulation. In contrast, extracts of Nicotiana benthamiana leaves infiltrated with the negative control (C-) pBIΩ Agrobacterium that does not contain the sequence of SEQ.ID.NO.2 encoding the irisin synthesis gene did not stimulate the phosphorylation of MAP kinase ERK ( Figure 4 ). The data obtained from this bioactivity test showed that irisin produced in N. benthamiana leaves was able to effectively and directly activate intracellular signals triggered in bone-forming cells, as previously observed with irisin produced in E. coli but not in plant material.

[0138] Example 5 - Preparation of lecithin / chitosan nanoparticles (NLC) incorporating liposomal irisin and its application in mice Biological tests of bone cell lines

[0139] To prepare liposomal irisin, a solution of soybean lecithin in methanol was used, to which irisin powder at different concentrations was added. The resulting mixture was sonicated to facilitate the encapsulation of irisin in the liposomes.

[0140] In parallel, a chitosan solution was prepared, which facilitates the transmucosal penetration of substances, and the organic lecithin / irisin solution was added to this solution.

[0141] In this way, irisin-incorporated liposomal NLC nanoparticles (NLC-I) were prepared.

[0142] After verification of the encapsulation efficiency, the filter-sterilized NLC-I was used for biological testing with a final irisin concentration of 100 ng / ml.

[0143] Specifically, mouse osteoblast cell line cultures were stimulated with liposomal irisin and non-encapsulated irisin (as a control) at the same concentration for 8 hours.

[0144] Next, cell extracts were prepared and used to analyze the gene levels of two transcription factors: ATF4, which regulates osteoblast formation, and TFAM, which regulates mitochondrial biogenesis; both of which are known to be regulated by non-encapsulated (non-liposomal) irisin.

[0145] The results showed that the mRNA levels of two transcription factors, ATF4 and TFAM, increased in osteoblasts treated with liposomal irisin, and were comparable to those obtained with non-liposomal irisin stimulation.

[0146] Example 6 - Effect of irisin contained in Nicotiana benthamiana extract and encapsulated in liposomes on mouse muscle cells In vitro biological effects

[0147] To demonstrate that irisin expressed and purified from plant cells is also biologically active when contained in liposomes, the gene expression of mitochondrial transcription factor A (Tfam), a transcription factor regulated by irisin and playing a key role in mitochondrial biogenesis, was evaluated in mouse muscle cells. More specifically, muscle cells were treated for 8 hours with liposomes containing extracts of Nicotiana benthamiana leaves infiltrated with pBIΩ-IRS Agrobacterium or with liposomes containing the corresponding negative control (C-) pBIΩ without the gene encoding irisin (sequence SEQ.ID.NO.2).

[0148] RNA was extracted from muscle cells using the RNeasy Mini kit (Qiagen, Hilden, Germany) by spin columns according to the manufacturer's instructions. Reverse transcription was performed on a MyCycler thermal cycler (Bio-Rad, Hercules, California, USA) using iScript reverse transcription Supermix (Bio-Rad, Hercules, California, USA) according to the manufacturer's instructions. Quantitative real-time polymerase chain reaction (qPCR) was performed using SsoFast EvaGreen Supermix (Bio-Rad, Hercules, California, USA) and CFX96 real-time thermal cycler (Bio-Rad, Hercules, California, USA) with 40 cycles (denaturation, 95°C, 5 seconds; annealing / extension, 60°C, 10 seconds) after an initial enzyme activation step of 30 seconds at 95°C. Primers of interest were identified using Primer-BLAST. Gapdh was selected as a housekeeping gene because it was stably expressed in all samples. The primer sequences used are as follows:

[0149] Gapdh

[0150] Forward primer: acaccagtagactccacgaca (SEQ.ID NO.4)

[0151] Reverse primer: acggcaaattcaacggcacag (SEQ.ID NO.5)

[0152] Tfam

[0153] Forward primer: taggcaccgtattgcgtgag (SEQ.ID NO.6)

[0154] Reverse primer: cagacaagactgatagacgaggg (SEQ.ID NO.7).

[0155] All data were normalized to Gapdh and calculated as fold change (ie, fold signal amplification) relative to the negative control (C-)pBIΩ.

[0156] like Figure 5As shown, the results showed that the use of liposomes containing N. benthamiana leaf extract infiltrated with pBIΩ-IRS Agrobacterium could effectively increase the expression of Tfam mRNA in muscle cells after 8 hours of stimulation compared with liposomes containing N. benthamiana leaf extract infiltrated with negative control (C-) pBIΩ Agrobacterium without the gene encoding irisin. The data obtained from this bioactivity test showed that irisin produced by N. benthamiana leaves and encapsulated in liposomes effectively upregulated mitochondrial generation in muscle cells, which is completely consistent with irisin produced in E. coli instead of in plant material.

[0157] Example 7 - Modified pJL-TRBO expression vector and synthetic gene containing irisin sequence and additional elements

[0158] A synthetic gene was designed that included the sequence encoding irisin and fused to other elements capable of expressing the protein in plants. The resulting cDNA encodes a polypeptide consisting of: I) an N-terminal signal peptide for secretion from Medicago sativa disulfide isomerase, II) the irisin protein, III) a thrombin cleavage site followed by IV) an 8-His tag and V) a KDEL terminal tag that allows the protein to be retained in the endoplasmic reticulum. The overall sequence of the synthetic gene and its encoded protein is shown below (sequences SEQ ID NO.8 and SEQ ID NO.9, respectively)

[0159]

[0160] The polynucleotide sequence SEQ.ID.NO.8 includes the polynucleotide sequence of irisin. The bold part is the sequence encoding the N-terminal signal peptide for secretion of alfalfa disulfide isomerase; the single underlined part is the sequence encoding the thrombin cleavage site; the double underlined part is the sequence encoding the 8-His tag; the bold and single underlined part is the sequence encoding the KDEL tag.

[0161]

[0162] The amino acid sequence SEQ.ID.NO.9 includes the amino acid sequence of irisin. The bold part is the N-terminal signal peptide for secretion of alfalfa disulfide isomerase; the single underlined part is the thrombin cleavage site; the double underlined part is the 8-His tag; the bold and single underlined part is the KDEL tag.

[0163] The synthetic gene was optimized for expression in Nicotiana benthamiana and cloned into a modified version of the pJL-TRBO plasmid vector (ie, cloned into a modified pJL-TLBO plasmid).

[0164] The modified pJL-TRBO plasmid is a pJL-TRBO plasmid in which the restriction sites pBluescriptKS, Pvu I, GFP-R, BstZ17 I, and GFP-F are removed, and the restriction sites Sal I, Mlu I, Nhe I, Bmt I, Eco53k I, Sac I, Nco I, Nru I, Asc I–BssH II, ApaL I, AsiS I, Swa I, Kas I, NarI, Sfo I, PluT I, Avr II are inserted.

[0165] The modified pJL-TRBO plasmid was obtained by techniques known in the art from the original pJL TRBO plasmid, which is currently commercially available and sold by Addgene (Watertown, MA, USA).

[0166] The modified pJL-TRBO plasmid is 10659 base pairs in length.

[0167] Figure 6 The modified pJL-TRBO plasmid is shown.

[0168] like Figure 6 As shown, the modified pJL-TRBO plasmid contains a 35S promoter from CAMV (cauliflower mosaic virus) capable of expressing proteins at high levels in plants, a replication origin site, a kanamycin antibiotic resistance gene, a replication initiation protein (trfA) gene, a 5'-leader sequence of tobacco mosaic virus (TMV) (called Ωa), a "left border repeat of T-DNA" region, a "right border repeat of T-DNA" region, and a KS primer sequence for amplifying and sequencing the right end of the gene.

[0169] Once recognized by the plasmid in Agrobacterium, the region between the T-DNA border repeats is transferred into the plant cell.

[0170] Especially in Figure 6 In the present invention, the term "minimal CaMV 35S promoter" refers to the 35S promoter of CAMV (cauliflower mosaic virus) capable of high-level protein expression in plants; the term "OriV" refers to the replication initiation site; the term "KanR" refers to "kanamycin resistance"; the term "trfA" refers to "replication initiation protein"; the term "TMVΩ" refers to the 5' leader sequence (called Ω) of tobacco mosaic virus (TMV); the term "LB T-DNA repeat" refers to the term "left border repeat of T-DNA", and the term "RB T-DNA repeat" refers to the "right border repeat of T-DNA"; the term "KS primer" refers to the primer used to amplify and sequence the right end of a gene.

[0171] The final genetic construct was used to transform electrocompetent cells of Agrobacterium tumefaciens LBA4404 and generate glycerol stocks of transformed bacteria.

[0172] Subsequently, N. benthamiana plants were agroinfiltrated with large cultures of the engineered A. tumefaciens cells, resulting in transient expression of the protein of interest (i.e., irisin) in the plant leaves.

[0173] The presence of the His-tag enabled purification of the protein by IMAC (immobilized metal affinity chromatography), and subsequent thrombin treatment ensured the removal of the tag from purified irisin.

[0174] At the end of the test, irisin expression levels reached or exceeded 5 mg protein per gram of fresh leaf tissue.

Claims

1. A method for producing irisin, comprising the following steps: a) providing at least one expression vector, wherein the expression vector comprises a nucleotide sequence encoding the irisin; b) inserting the expression vector into at least one first bacterium of the genus Agrobacterium, thereby obtaining a first bacterium of the genus Agrobacterium comprising the expression vector; c) treating at least one plant material with said first bacterium comprising said expression vector, thereby obtaining a treated plant material; d) cultivating the treated plant material to express the irisin; e) extracting the irisin from the treated plant material.

2. The method according to claim 1, further comprising, before step a), a step of inserting the amplification vector comprising the nucleotide sequence encoding irisin into Escherichia coli and amplifying by culturing the Escherichia coli.

3. The method of claim 1, wherein the irisin is non-glycosylated irisin.

4. The method of claim 1, wherein the irisin is partially glycosylated, and wherein the method further comprises a step of removing the glycosylation to obtain non-glycosylated irisin.

5. The method of claim 1, wherein the plant material is selected from a plant or at least a part of the plant or at least one cell of the plant.

6. The method of claim 5, wherein the plant material is selected from the group consisting of tobacco (preferably Nicotiana benthamiana), hemp, spirulina, chlorella, Arabidopsis, corn, rice, soybean, rapeseed, alfalfa, sunflower, sorghum, wheat, cotton, peanut, tomato, potato, lettuce and pepper, or at least a part of the plant or at least one cell of the plant.

7. The method of claim 1, wherein said step c) further comprises the step of treating said at least one plant material with at least one second bacterium of the genus Agrobacterium, said second bacterium comprising an expression vector comprising a gene suitable for preventing silencing of expression of irisin in said treated plant material.

8. The method of claim 7, wherein: In step c), the at least one plant material is treated with a mixture, the mixture comprising the first bacterium and the second bacterium, the first bacterium comprising the expression vector comprising a nucleotide sequence encoding the irisin, and the second bacterium comprising a gene suitable for preventing silencing of the expression of the iris protein in the treated plant material.

9. The method of claim 1, wherein the bacterium of the genus Agrobacterium is Agrobacterium tumefaciens.

10. The method according to claim 7, wherein the gene suitable for preventing silencing of irisin expression is the P19 gene.

11. The method of claim 1, wherein the expression vector is a pJL-TRBO plasmid, in which the restriction sites pBluescriptKS, Pvu I, GFP-R, BstZ17 I and GFP-F are removed, and the restriction sites Sal I, Mlu I, NheI, Bmt I, Eco53k I, Sac I, Nco I, Nru I, Asc I–BssH II, ApaL I, AsiS I, Swa I, Kas I, Nar I, Sfo I, PluT I, Avr II are inserted.

12. The method of claim 1, wherein the nucleotide sequence encoding irisin is contained in a synthetic gene, which, in addition to the sequence encoding irisin, further comprises a polynucleotide sequence encoding an N-terminal signal peptide for secretion from alfalfa alfalfa disulfide isomerase; a sequence encoding a thrombin cleavage site, followed by a sequence encoding an 8-histidine residue tail and a sequence encoding a KDEL terminal tail, wherein the nucleotide sequence encoding irisin is located between the polynucleotide sequence encoding the N-terminal signal peptide for secretion from alfalfa alfalfa disulfide isomerase and the sequence encoding the thrombin cleavage site.

13. The method of claim 1, wherein the nucleotide sequence encoding irisin is: GGATCCGATTCTCCTTCAGCTCCAGTTAATGTTACAGTTAGACATCTTAAGGCTAATTCTGCTGTTGTTTCATGGGATGTTTTGGAAGATGAGGTTGTTATTGGTTTTGCTATCTCTCAACAGAAGAAAGATGTTAGAATGCTTAGGTTCATCCAAGAAGTTAACACTACAACTAGGTCTTGTGCTCTTTGGGATTTGGAAGAGGATACAGAGTACATCGTTCATGTTCAGGCTATCTCAATCCAAGGACAGTCTCCTGCTTCAGAACCAGTTTTGTTTAAAACTCCTAGGGAGGCTGAGAAAATGGCAAGTAAAAACAAGGATGAGGTGACAATGAAAGAGTGATAGCCCGGG (SEQ. ID. NO. 2).

14. Irisin contained in liposomes.

15. The irisin contained in liposomes according to claim 14, wherein the irisin is non-glycosylated irisin. 16 . The irisin contained in liposomes according to claim 14 , wherein the irisin is encapsulated in the liposomes.

17. The irisin contained in liposomes, wherein the liposomes contain lecithin and chitosan.

18. Irisin contained in liposomes is used as a drug.

19. Irisin contained in liposomes as claimed in claim 18, for use in treating and / or preventing one or more diseases selected from osteoporosis, sarcopenia, energy metabolism disorders, cardiovascular system diseases, neurodegenerative diseases, diabetes, obesity, kidney diseases and metabolic diseases.

20. Irisin for use according to claim 18 or 19, wherein the irisin contained in the liposomes is administered via the sublingual and / or subcutaneous and / or intradermal route through a skin gel, and / or via the nasal route through a nasal spray.

21. A synthetic gene for expressing irisin in plants, comprising: a polynucleotide sequence encoding an N-terminal signal peptide for secretion from Medicago truncatula disulfide isomerase; a sequence encoding irisin; a sequence encoding a thrombin cleavage site; a sequence encoding an 8 histidine residue tail; and a sequence encoding a KDEL terminal tail.

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