WO2020177004A1 - Procédé pour la production recombinante de molécules peptidiques à propriétés immunnomodulatrices et anti-inflammatoires chez les poissons - Google Patents

Procédé pour la production recombinante de molécules peptidiques à propriétés immunnomodulatrices et anti-inflammatoires chez les poissons Download PDF

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WO2020177004A1
WO2020177004A1 PCT/CL2020/050015 CL2020050015W WO2020177004A1 WO 2020177004 A1 WO2020177004 A1 WO 2020177004A1 CL 2020050015 W CL2020050015 W CL 2020050015W WO 2020177004 A1 WO2020177004 A1 WO 2020177004A1
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peptide
concatemer
peptides
expression
piv
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PCT/CL2020/050015
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Spanish (es)
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Jannel Acosta Alba
Jorge Toledo Alonso
Oliberto Sanchez Ramos
Ivan GONZALEZ CHAVARRIA
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Universidad de Concepción
Centro De Biotecnologia Y Biomedicina Spa
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products

Definitions

  • the present invention relates to the technical field of biotechnology, more particularly it relates to a method for the large-scale production of peptides of interest by expressing a concatemer that encodes a polypeptide containing tandem sequences of the peptide of interest, and the cleavage of said polypeptide to obtain a plurality of individual copies of said peptide of interest.
  • the invention is particularly useful for obtaining neuropeptides with immunomodulatory and anti-inflammatory properties for use in the aquaculture industry.
  • Peptides are small molecules of between 2 to 50 amino acids, which have a wide variety of important functions in vivo.
  • PACAP pituitary adenylate cyclase polypeptide PACAP (38 amino acids), the growth hormone releasing hormone GHRH (44 amino acids), vasoactive intestinal peptide PIV (28 amino acids), neuropeptide Y NPY (36 amino acids) and ghrelin (28 amino acids), among others.
  • PACAP the pituitary adenylate cyclase polypeptide PACAP
  • GHRH 44 amino acids
  • vasoactive intestinal peptide PIV 28 amino acids
  • neuropeptide Y NPY 36 amino acids
  • ghrelin 28 amino acids
  • peptides Due to the small size of the peptides, they do not cause serious immune responses and are also rapidly eliminated from the body, so they do not accumulate in specific organs, which minimizes their possible toxic side effects in the body. All these advantages make peptides attractive molecules for the development of therapies and diagnostic methods for the aquaculture industry.
  • peptides can be easily obtained by chemical synthesis or by recombinant DNA technology, compared to protein molecules of higher i size.
  • cost of production and the market price is still high to market products of a peptide nature.
  • patent document WO2013 / 138850 A1 refers to a method for producing recombinant peptides of VSDL, which comprises expressing a fusion polypeptide with concatemic repeats, where the peptide contains at its C-terminal end enzymatic cleavage sites by trypsin .
  • patent document US 6,558,924 B1 refers to a method for producing the insulin C peptide, which comprises expressing in a host cell a multimeric polypeptide comprising multiple copies of said peptide, and cutting the expressed polypeptide to release the copies of the peptide.
  • the polypeptide incorporates cleavage sites between each peptide monomer, which can be a combination of trypsin and carboxypeptidase B, where the cleavage sites begin or end at arginine residues.
  • cleavage sites between each peptide monomer, which can be a combination of trypsin and carboxypeptidase B, where the cleavage sites begin or end at arginine residues.
  • Ghrelin for example, is the endogenous ligand for GHS-R1a, is produced primarily by cells in the stomach, and serves as a potent circulating orexigenic hormone that controls food intake, energy expenditure, adiposity, and secretion. by GH.
  • the functional roles of circulating ghrelin in the immune system and in states of stress and inflammatory injury are currently being explored.
  • ghrelin is a potent anti-inflammatory mediator in mammals both in vitro and in vivo in lymphocytes, monocytes and dendritic cells through the inhibition of oxidative stress, cell apoptosis, cell adhesion and expression. proinflammatory cytokine and promoting IL-10 expression and cell migration.
  • ghrelin may be a promising therapeutic agent in the treatment of various inflammatory conditions and autoimmune diseases and tissue injuries. Furthermore, ghrelin has also been shown to promote lymphocyte development in the primary lymphoid organs (bone marrow and thymus). Despite the fact that the coding sequence for ghrelin has been reported in salmonids, there are no previous studies that characterize the function of ghrelin in the immune system in fish.
  • PIV Vasoactive Intestinal Peptide
  • porcine intestinal extracts as a vasodilator and was later identified as a multifunctional neuropeptide in the central and peripheral nervous system. Recently, this pleiotropic neuropeptide has been shown to play a key role in maintaining neuroendocrine-immune communication. Some of the peptides are released from the central nervous system through the hypothalamic-pituitary axis as hormones or pro-hormones and reach the lymphoid organs through the circulation. Lymphocytes are the major source of PIV in lymphoid organs, which express and secrete PIV upon activation by various stimuli.
  • PIVergic nerve fibers in central (thymus) and peripheral lymphoid organs (spleen, lymph nodes, and mucosa-associated lymphoid tissue).
  • central (thymus) and peripheral lymphoid organs spleen, lymph nodes, and mucosa-associated lymphoid tissue.
  • PIV has been shown to be a potent anti-inflammatory factor that acts by regulating the production of both anti-inflammatory and pro-inflammatory mediators, and has been identified as a potential candidate in the treatment of inflammatory and autoimmune disorders in mammals.
  • the physiological roles of PIV in teleosts have been described. PIV is distributed in the brain of teleosts and affects the release of growth hormone, gonadotropin and prolactin from cultured teleost pituitary cells in vitro.
  • the present invention refers to a method for the production of multiple copies of a recombinant peptide for the production of peptides with immunomodulatory and anti-inflammatory properties in fish, said method comprising the steps of: a) designing and synthesizing a nucleotide sequence that encodes a concatemer comprising a plurality of copies linked by the Asn-Gly sequence of a peptide;
  • the induction of concatemer expression is carried out with isopropyl ⁇ -D-1 -thiogalactopyranoside (IPTG) at a concentration between 0.1 and 1 mM.
  • IPTG isopropyl ⁇ -D-1 -thiogalactopyranoside
  • the bacteria are lysed with a French press.
  • One of the advantages of the proposed method is that the step of disintegration of the inclusion bodies can be carried out in conjunction with the cleavage of the concatemers using hydroxylamine between 25 and 50% (w / v).
  • the recombinant peptide is purified by consecutive washing and centrifugation steps, thus avoiding the use of chromatographic steps for purification.
  • the nucleotide sequence that encodes a concatemer of the peptide is a sequence that is selected from the nucleotide sequences that encode for Vasoactive Intestinal Peptide (IVP) and for ghrelin from Salmo salar, which are cloned in the expression vector pET22b in a preferred embodiment.
  • IVP Vasoactive Intestinal Peptide
  • This expression vector pET22b is used to transform, in a particularly preferred embodiment, the E. coli strain BL21 (DE3), to obtain recombinant PIV or ghrelin monomers from Salmo salar.
  • the peptides thus obtained can be used for the preparation of a veterinary composition to stimulate the immune system in Salmo salar, to stimulate its growth, to combine them in vaccine formulations to enhance their effect, and / or for the treatment of an inflammatory disease. in said fish.
  • FIG. 1 shows a 15% polyacrylamide gel electrophoresis of the soluble and insoluble fractions that are obtained after lysis of BL21 (DE3) cells transformed with the vector pET22b-Peptide 6X and which were induced to express the concatemer of peptides with 0.4 mM IPTG.
  • FIG. 2 shows a 15% polyacrylamide gel electrophoresis of the purification and quantification of the inclusion bodies where the peptide concatemers are contained.
  • FIG. 3 shows a 17% polyacrylamide gel electrophoresis of the hydroxylamine cleavage of the peptide concatemers and the obtaining of individual copies of the peptide of interest.
  • FIG. 4 shows the effect of the Vasoactive Intestinal Peptide (PIV) and ghrelin from Salmo salar obtained by recombinant route.
  • PIV Vasoactive Intestinal Peptide
  • the present invention relates to a method for the recombinant production of peptide molecules of interest on a large scale in bacteria.
  • the method of the invention is particularly useful for obtaining peptides in fish with immunomodulatory and anti-inflammatory properties.
  • This method makes it possible to increase the productive yields of this type of molecules, compared to the recombinant production and purification methods that currently exist in the state of the art.
  • one of the main advantages of the method of the present invention is to obtain peptide molecules with a high degree of purity, without the need to involve chromatographic steps in the process.
  • This simple and low-cost method allows it to be used for the production of peptides that are particularly useful for the aquaculture industry, where the costs of biotechnological products are usually high, so that this method has a positive impact on the production costs of this industry.
  • concatemer should be understood as a nucleotide sequence that contains a plurality of copies of the same nucleotide sequence arranged in series or tandem. In the particular case of the present invention, the concatemer is a synthetic nucleotide sequence.
  • nucleotide sequence or nucleotide sequence is to be understood as a double strand of DNA, or a single strand of DNA, natural or synthetic, or products of the transcription of said DNA (eg, RNA molecules).
  • nucleotide sequence encoding a peptide of interest should be understood as a nucleotide sequence that transcribes a functional RNA molecule, or that encodes a functional peptide of interest for the present invention.
  • the present invention does not relate to genomic nucleotide sequences in their natural state, but rather refers to nucleotide sequences in an isolated, or purified, or partially purified, or recombinant state, obtained by any method of genetic engineering known in the state of the art.
  • peptide sequence or peptide sequence or amino acid sequence or amino acid sequence is to be understood as a small amino acid sequence of up to 50 amino acids, natural or synthetic, or products of RNA translation.
  • recombinant should be understood as any nucleotide (DNA, RNA) or amino acid sequence modified by any genetic engineering method known in the state of the art, which generates as a result a new nucleotide or amino acid sequence different from the one found In nature.
  • inclusion bodies is to be understood as intracellular protein or peptide aggregates that are commonly formed in recombinant bacteria.
  • the expression “disintegrating the inclusion bodies” should be understood as the solubilization of the protein or peptide aggregates that are formed during the recombinant production process in E. coli.
  • rupture precipitate should be understood as the insoluble fraction that is formed as a result of the process of cellular rupture and subsequent centrifugation.
  • rupture supernatant in the context of the present invention should be understood as the soluble fraction that is formed as a result of the process of cellular rupture and subsequent centrifugation.
  • cell debris should be understood as organic remains that are produced after cell lysis.
  • transforming a microorganism should be understood as a process by which an exogenous nucleotide sequence is incorporated into a host cell, which in this case and for the context of the present invention, corresponds to a prokaryotic cell.
  • the method of the present invention comprises producing peptide molecules on a large scale beginning with the design and synthesis of a nucleotide sequence that encodes a concatemer comprising a plurality of copies of a peptide of interest.
  • said peptide of interest is a neuropeptide that has immunomodulatory and anti-inflammatory properties in fish such as PIV and ghrelin.
  • the production of these peptides of interest in the form of concatemers improves the stability of said peptides, which are less susceptible to proteolytic degradation during the production process in the host microorganism.
  • the concatemer can contain between 6 and 8 copies of the peptide of interest, without being limited to said quantity.
  • the concatemer contains 6 tandem copies of the peptide of interest.
  • Each individual nucleotide sequence encoding the peptide of interest is separated by a nucleotide sequence encoding the amino acids Asp-Gly.
  • cleavage of the concatemer can be induced with the inorganic hydroxylamine compound to release individual copies of the peptides of interest. This gives it a great advantage since the present method does not use enzymatic techniques to cut the concatemer, which reduces the production costs of peptides for the aquaculture industry.
  • the designed nucleotide sequence containing the concatemer of the peptide of interest is cloned into an expression vector for its subsequent expression in a suitable microorganism.
  • the nucleotide sequence containing the concatemer of the peptide of interest can be cloned into any vector of the pET expression system.
  • Said expression vector is preferably pET22b, which contains a T7 promoter, a lac operator, and multiple cloning sites useful for inserting the concatemer.
  • the preferred microorganism for expression of the concatemer is prokaryote, preferably E. coli, more preferably strain BL21 (DE3), although any strain of E. coli of the pET expression system can be used.
  • Induction of expression of the concatemer is preferably performed with BDL -tiogalactopiranósido isopropyl- (IPTG) at a concentration between 0.1 and 1 mM, between 4 and 18 hours of culture and at a temperature between 30 and 37 and C.
  • IPTG BDL -tiogalactopiranósido isopropyl-
  • the culture After incubating the culture with IPTG during the set time, the culture is centrifuged at a speed between 6000 and 8000 g for 5 to 15 minutes at a temperature between 2 and 6 and C to obtain a precipitate of microorganisms. Subsequently, the microorganism precipitate is resuspended in an appropriate lysis solution, using physical means such as, for example, a French press to achieve cell lysis.
  • the debris obtained after lysis is centrifuged at a speed between 8000 and 10000 g for 25 to 40 minutes at a temperature between 2 and 6 and C to separate the supernatant breaking the precipitate of rupture which are contained the inclusion bodies, and then the precipitate is washed again with lysis solution and centrifuged again at a speed between 8000 and 10000 g for 25 to 40 minutes, at a temperature between 2 and 6 and C to obtain the inclusion bodies.
  • the precipitate containing the inclusion bodies resuspended again in the solubilization solution and this suspension is kept under stirring for 6 to 12 hours at a temperature between 21 and 25 and C. Then, centrifuged at a speed between 8000 and 10000 g for 15 to 30 minutes at a temperature between 2 and 6 and C, to obtain the solubilized inclusion bodies.
  • inclusion bodies are dissolved in solubilization solution, to which is added hydroxylamine at a concentration between 25 and 50% (w / v), and the mixture is incubated at 45 e C during between 12 and 16 hours to produce the cleavage of the concatemers and generate the individual copies of the peptide of interest.
  • the solution is centrifuged at a speed between 8000 and 10000 g for 15 to 30 minutes at a temperature between 2 and 6 and C, to obtain a precipitate, which is washed at the least 4 times with 1X PBS to obtain peptides with high degree of purity.
  • Example 1 Design and synthesis of a nucleotide sequence encoding the concatemer.
  • the peptides of interest used in the present study were two peptides with immunomodulatory and anti-inflammatory properties obtained from Salmo salar: the Vasoactive Intestinal Peptide PIV, whose amino acid sequence is: HSDAIFTDNYSRFRKQMAVKKYLNSVLT and ghrelin whose aminoacid sequence is: GSSQVPGPGPKPSK
  • sequences marked in bold and underlined correspond to the recognition sites of the restriction enzymes Ndel and Xhol used for cloning in the expression vector pET22b.
  • sequences marked in bold and underlined correspond to the recognition sites of the restriction enzymes Ndel and Hindlll used for cloning in the expression vector pET22b.
  • the E. coli strain BL21 (DE3) was transformed with said expression vector and grown in LB solid medium [Luria-Bertani: Tryptone 1% (p / v), Yeast extract 0.5% (w / v), NaCl 171, 1 mM, pH 7.5, bacteriological agar at 1.5% (w / v)] supplemented with 50 pg / mL of ampicillin.
  • LB solid medium Lia-Bertani: Tryptone 1% (p / v), Yeast extract 0.5% (w / v), NaCl 171, 1 mM, pH 7.5, bacteriological agar at 1.5% (w / v)
  • IPTG isopropyl-thio ⁇ -D-galactoside
  • Example 2 Obtaining inclusion bodies and purification of the PIV and Ghrelin peptides.
  • the culture is centrifuged at 8000 g for 10 minutes at 4 ° C.
  • the microorganism precipitate is resuspended in a 1% PBS solution 1 X-Triton x 100 at 1% at a rate of 10g / 100ml, and cell disruption using a French press.
  • the cell debris obtained after breaking centrifuged at a speed of 8000 g for 30 minutes at 4 C and the supernatant to separate the precipitate rupture rupture which are contained the inclusion bodies.
  • the precipitate is then washed with a 1% 1X-Triton x 100 PBS solution, 1M NaCl, at a rate of 10g / 100ml. Centrifuge again at a rate of 8000 g for 25 to 40 minutes at a temperature between 2 and 6 and C to purify and obtain the inclusion bodies.
  • Figure 1 represents a 15% polyacrylamide gel electrophoresis of the soluble and insoluble fractions that are obtained after the disruption of BL21 (DE3) cells transformed with the vector pET22b-Peptide 6X and to which expression was induced. of the peptide concatemer with 0.4 mM IPTG, in which MW is the molecular weight standard; S.rup is the cleavage supernatant, that is, the soluble intracellular fraction and C. Inc is the cleavage precipitate, or fraction corresponding to inclusion bodies.
  • This figure shows a band between 25 and 35 kDa in the fraction of the rupture precipitate, which corresponds to the concatemer of peptides in the form of inclusion bodies.
  • the precipitate is resuspended in a solution of PBS 1 X, Urea 8M, b-Mercapto 10 nM, PMSF 1 mM, pH 8, at a rate of 10g / ml. This suspension obtained is kept stirred overnight at room temperature then centrifuged at a speed of 8000 g for 20 minutes at 4 e C.
  • the precipitate is dissolved at a rate of 1 g / 5 ml of 1X PBS solution, 8M Urea, 10mM b-Mercapto, 0.1-1mM PMSF, pH8; 1 ml HC1 1M Tris, pH 9.5 and 4 ml hydroxylamine (50% solution in water) and incubated at 45 C and overnight. This step allows to generate the individual copies of the peptide of interest.
  • FIG. 2 shows a 15% polyacrylamide gel electrophoresis of the purification and quantification of the inclusion bodies where the peptide concatemers are contained, in which MW is the molecular weight standard; BSA is the BSA Curve for protein quantification; 6X Peptide is the concatemer of the peptide of interest contained in the inclusion bodies.
  • the above mixture was centrifuged at a speed at 8000 g for 30 minutes at 4 C and then the precipitate was washed 4 times with 1X PBS to obtain peptides with a high degree of purity.
  • FIG. 3 shows a 17% polyacrylamide gel electrophoresis of the cleavage with hydroxylamine of the peptide concatemers and the obtaining of the monomers of the peptide of interest, in which PM is the molecular weight standard and Peptide 1 X Cl: the monomeric peptide obtained after cutting with hydroxylamine.
  • Example 3 Determination of the effect on the immune system of peptides obtained by recombinant route.
  • the biological activity of the peptides produced using the previously described recombinant expression system was determined in vitro by analyzing the expression of genes related to the immune system.
  • SHK-1 cells (cells derived from the anterior kidney of Atlantic salmon, Salmo sa / ar) were incubated with 20 nM of PIV and GRE (vasoactive intestinal peptide and Ghrelin, respectively) peptides from Salmo salar obtained in examples 1 and 2
  • PIV and GRE vasoactive intestinal peptide and Ghrelin, respectively
  • peptides from Salmo salar obtained in examples 1 and 2
  • the expression of INFy, I L-1b, TNF-D and the chemokine CCL19 was determined at 4 and 8 hours after treatment. Relative expression values were normalized to mRNA expression of polyadelenate binding protein 1 (PBP-1).
  • INFy is a T H 1 cytokine that is critical for almost all phases of immune responses.
  • teleost fish INFy mediate activation of macrophages, through increased respiratory burst activity, nitric oxide production, and phagocytosis, inducing the expression of typical antiviral genes.
  • IFNy also induces apoptosis, especially during viral infection, and inhibits cell proliferation.
  • T H 1 cell immunity is essential in immune defense against intracellular viruses and bacteria.
  • the peptides did not produce significant changes in the expression of CCL19 at 4 hours of treatment.
  • the expression of IL-1 b did not show significant differences at 4 and 8 hours after the peptides were administered.
  • CCL19 chemokine is involved in acute inflammation and the recruitment of lymphocytes and other cells.
  • CCL19 has been shown to induce cell proliferation, respiratory burst activity, and peripheral blood leukocyte (PBL) chemotaxis, suggesting its ability to exert inflammatory and homeostatic functions. Taking this into account, it can be suggested that the peptides may possess anti-inflammatory effects in fish.
  • FIG. 4 shows the effect of PIV and GRE peptides obtained recombinantly as described above on the expression of genes involved in the immune response.
  • the data were analyzed using a T Test. * Means p ⁇ 0.05; ** mean p ⁇ 0.01; *** mean p ⁇ 0.001.
  • SHK1 cells were incubated with 20nm with each of the peptides.
  • INF-g, CC19, I L-1 b and TNF-a expression was measured at 4 and 8 hours post treatment. Relative expression values were normalized to mRNA expression of polyadelenate binding protein 1 (PBP-1).
  • PBP-1 polyadelenate binding protein 1
  • a process has been developed based on the recombinant expression of peptide concatemers in bacteria and the obtaining of biologically active peptide monomers through the cleavage of the concatemers with hydroxylamine.
  • the invention has the following advantages over the prior art:

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Abstract

La présente invention concerne le domaine technique de la biotechnologie, plus particulièrement elle concerne un procédé pour la protection à grande échelle de peptides d'intérêt par l'expression d'un concatémère qui code pour un polypeptide qui contient des séquences dudit polypeptide pour l'obtention d'une pluralité de copies individuelles dudit peptide d'intérêt. L'invention est particulièrement utile pour l'obtention de peptides à propriétés immunnomodulatrices et anti-inflammatoires destinés à être utilisés dans l'industrie aquacole.
PCT/CL2020/050015 2019-03-05 2020-03-04 Procédé pour la production recombinante de molécules peptidiques à propriétés immunnomodulatrices et anti-inflammatoires chez les poissons WO2020177004A1 (fr)

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WO2000034312A1 (fr) * 1998-12-10 2000-06-15 Samyang Genex Corporation Procede de purification de peptides antimicrobiens

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