WO2020177004A1 - Method for recombinant production of peptide molecules with immunomodulatory and anti-inflammatory properties in fish - Google Patents

Method for recombinant production of peptide molecules with immunomodulatory and anti-inflammatory properties in fish 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)
French (fr)
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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

The present invention is related to the technical field of biotechnology; more specifically, the invention relates to a method for the large-scale production of peptides of interest by expression of a concatemer that codes for a polypeptide containing tandem sequences from the peptide of interest, and the excision of said polypeptide to obtain a plurality of individual copies of said peptide of interest. The invention is particularly useful for the production of peptides with immunomodulatory and anti-inflammatory properties for use in the fish-farming industry.

Description

PROCESO BIOTECNOLÓGICO PARA LA PRODUCCIÓN RECOMBINANTE A GRAN ESCALA DE MOLÉCULAS PEPTÍDICAS CON PROPIEDADES INMUNOMODULADORAS Y ANTIINFLAMATORIAS PARA USO EN LA INDUSTRIA BIOTECHNOLOGICAL PROCESS FOR THE RECOMBINANT LARGE-SCALE PRODUCTION OF PEPTIDIC MOLECULES WITH IMMUNOMODULATING AND ANTI-INFLAMMATORY PROPERTIES FOR USE IN INDUSTRY
ACUÍCOLA AQUACULTURE
Sector Técnico Technical Sector
La presente invención se relaciona con el campo técnico de la biotecnología, más particularmente se refiere a un método para la producción a gran escala de péptidos de interés mediante la expresión de un concatémero que codifica un polipéptido que contiene secuencias en tándem del péptido de interés, y la escisión de dicho polipéptido para la obtención de una pluralidad de copias individuales de dicho péptido de interés. La invención es particularmente útil para la obtención de neuropéptidos con propiedades inmunomoduladoras y antiinflamantorias para uso en la industria acuícola. 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.
Técnica Anterior Previous Technique
Los péptidos son moléculas pequeñas de entre 2 a 50 aminoácidos, que tienen una gran diversidad de importantes funciones in vivo. En peces, existen de manera natural numerosos péptidos que cumplen funciones clave en el crecimiento, inmunidad y reproducción de éstos como, por ejemplo, el polipéptido del adenilato ciclasa de la pituitaria PACAP (38 aminoácidos), la hormona liberadora de la hormona de crecimiento GHRH (44 aminoácidos), el péptido intestinal vasoactivo PIV (28 aminoácidos), el neuropéptido Y NPY (36 aminoácidos) y la grelina (28 aminoácidos), entre otros. También existen péptidos antimicrobianos que actúan directamente sobre los patógenos y péptidos inmunomoduladores que aumentan la respuesta inmune de los peces, los que permiten mejorar la resistencia a enfermedades y aumentan la eficacia de las vacunas cuando estos se agregan a dichas formulaciones. Peptides are small molecules of between 2 to 50 amino acids, which have a wide variety of important functions in vivo. In fish, there are naturally numerous peptides that play key roles in their growth, immunity and reproduction, such as, for example, the 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. There are also antimicrobial peptides that act directly on pathogens and immunomodulatory peptides that increase the immune response of fish, which improve resistance to diseases and increase the effectiveness of vaccines when these are added to said formulations.
Debido al pequeño tamaño de los péptidos, éstos no causan respuestas inmunes serias y además se eliminan rápidamente del organismo, por lo que no se acumulan en órganos específicos, lo que minimiza sus posibles efectos secundarios tóxicos en el organismo. Todas estas ventajas hacen que los péptidos sean moléculas atractivas para el desarrollo de terapias y métodos de diagnósticos para la industria acuícola. 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.
Actualmente, los péptidos se pueden obtener fácilmente por síntesis química o mediante tecnología del ADN recombinante, comparado con moléculas proteicas de mayor i tamaño. Sin embargo, el costo de producción y el precio de mercado todavía es alto para comercializar productos de naturaleza peptídica. Currently, peptides can be easily obtained by chemical synthesis or by recombinant DNA technology, compared to protein molecules of higher i size. However, the cost of production and the market price is still high to market products of a peptide nature.
Existen además desafíos técnicos que solucionar como la estabilidad de los péptidos recombinantes, rendimientos durante los procesos de expresión y purificación de los péptidos, y el escalamiento industrial del proceso productivo. There are also technical challenges to be solved, such as the stability of recombinant peptides, yields during the expression and purification processes of peptides, and the industrial scale-up of the production process.
En el estado de la técnica existen propuestas de métodos para la producción de péptidos recombinantes. Por ejemplo, el documento de patente WO2013/138850 A1 se refiere a un método para producir péptidos recombinantes de VSDL, que comprende expresar un polipéptido de fusión con repeticiones concateméricas, donde el péptido contiene en su extremo C-terminal sitios de corte enzimático por tripsina. Por otra parte, el documento de patente US 6,558,924 B1 se refiere a un método para producir el péptido C de insulina, que comprende expresar en una célula hospedera un polipéptido multimérico que comprende múltiples copias de dicho péptido, y cortar el polipéptido expresado para liberar las copias del péptido. El polipéptido incorpora sitios de corte entre cada monómero peptídico, el cual puede ser una combinación entre tripsina y carboxipeptidasa B, donde los sitios de corte comienzan o terminan en residuos de arginina. Sin embargo, todos estos documentos utilizan métodos enzimáticos de corte del polipéptido, lo cual aumenta significativamente el costo de la producción de dichos péptidos. In the state of the art there are proposals for methods for the production of recombinant peptides. For example, the 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 . On the other hand, 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. However, all of these documents use enzymatic methods of cutting the polypeptide, which significantly increases the cost of the production of said peptides.
Debido principalmente a la alta demanda que presentan los salmónidos en la actualidad, la industria acuícola se ha visto obligada a desarrollar condiciones de cultivo intensivo a altas densidades de población para alcanzar los altos niveles de crecimiento requeridos por la industria y el mercado. Estas condiciones de cultivo favorecen la presencia de factores de estrés a los que se ven sometidos los peces. Tales factores de estrés pueden desencadenar una cadena de respuestas moleculares, que a su vez pueden suprimir el sistema inmune y aumentar la susceptibilidad de los peces a las enfermedades, disminuir la tolerancia al agua de mar, inhibir el apetito, afectar el crecimiento y/o supervivencia de los peces. Estas condiciones tienen entre otras consecuencias la disminución de la productividad del cultivo y conlleva habitualmente a cuantiosas pérdidas económicas. Por tanto, la optimización de las estrategias de cultivo que involucren la utilización de productos biotecnológicos que potencien el crecimiento, acortando el ciclo productivo, estimulen el sistema inmune del pez y por tanto la resistencia a las enfermedades, así como que mejoren el apetito durante los períodos con un mayor nivel de estrés y contribuyan a mejorar el proceso de esmoltificación es un aspecto clave para la sostenibilidad de este sector. Existen de manera natural en los peces numerosos neuropéptidos con funciones claves en el crecimiento, la inmunidad, la reproducción como por ejemplos los péptidos PACAP, GHRH, PIV, NPY, GnRH, grelina, entre otros y péptidos antimicrobianos e inmunomoduladores que actúan directamente sobre los patógenos o aumentan la respuesta inmune en los animales, aumentando la resistencia a enfermedades y mejorando la eficacia de las vacunas. Due mainly to the high demand for salmonids today, the aquaculture industry has been forced to develop intensive farming conditions at high population densities to achieve the high growth levels required by the industry and the market. These culture conditions favor the presence of stress factors to which the fish are subjected. Such stressors can trigger a chain of molecular responses, which in turn can suppress the immune system and increase fish susceptibility to disease, decrease tolerance to seawater, inhibit appetite, affect growth and / or fish survival. These conditions have, among other consequences, a decrease in the productivity of the crop and usually leads to large economic losses. Therefore, the optimization of culture strategies that involve the use of biotechnological products that enhance growth, shorten the production cycle, stimulate the fish's immune system and therefore resistance to disease, as well as improve appetite during periods with a higher level of stress and contribute to improving the smoltification process is a key aspect for the sustainability of this sector. There are naturally in fish numerous neuropeptides with key functions in growth, immunity, reproduction, such as the peptides PACAP, GHRH, PIV, NPY, GnRH, ghrelin, among others, and antimicrobial and immunomodulatory peptides that act directly on the pathogens or increase the immune response in animals, increasing resistance to diseases and improving the efficacy of vaccines.
La grelina, por ejemplo, es el ligando endógeno para GHS-R1 a, se produce principalmente por las células en el estómago y sirve como una potente hormona orexigénica circulante que controla la ingesta de alimentos, el gasto de energía, la adiposidad y la secreción de GH. Actualmente se han estado explorando los roles funcionales de la grelina circulante en el sistema inmune y en estados de estrés y lesión inflamatoria. Varios informes de la última década han descrito que la grelina es un potente mediador antiinflamatorio en mamíferos tanto in vitro como in vivo en linfocitos, monocitos y células dendríticas a través de la inhibición del estrés oxidativo, la apoptosis celular, la adhesión celular y la expresión proinflamatoria de citoquinas y la promoción de la expresión de IL-10 y migración celular. Con base en estas actividades, la grelina puede ser un agente terapéutico prometedor en el tratamiento de varios estados inflamatorios y enfermedades autoinmunes y lesiones tisulares. Además, la grelina también ha demostrado promover el desarrollo de linfocitos en los órganos linfoides primarios (médula ósea y timo). A pesar de que la secuencia codificante para grelina ha sido reportada en salmónidos no existen estudios previos que caractericen la función de la grelina en el sistema inmune en peces. 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. Several reports from the last decade have described that 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. Based on these activities, 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.
El PIV (Péptido Intestinal Vasoactivo) es un péptido de 28 aminoácidos que se aisló originalmente de extractos intestinales porcinos como un vasodilatador y fue identificado más tarde como un neuropéptido multifuncional en el sistema nervioso central y periférico. Recientemente, se ha demostrado que este neuropéptido pleiotrópico desempeña un papel clave en el mantenimiento de la comunicación neuroendocrina- inmune. Algunos de los péptidos se liberan desde el sistema nervioso central a través del eje hipotálamo-hipófisis como hormonas o pro-hormonas y llegan a los órganos linfoides a través de la circulación. Los linfocitos son la principal fuente de PIV en los órganos linfoides, que expresan y secretan PIV tras la activación mediante diversos estímulos. Usando inmunohistoquímica, varios investigadores han demostrado la presencia de fibras nerviosas PIVérgicas en órganos linfoides centrales (timo) y periféricos (bazo, nodulos linfáticos y tejido linfoide asociado a la mucosa). Durante la última década, PIV ha demostrado ser un potente factor antiinflamatorio que actúa regulando la producción de mediadores tanto antiinflamatorios como proinflamatorios, y ha sido identificado como un candidato potencial en el tratamiento de trastornos inflamatorios y autoinmunes en mamíferos. Se han descrito los roles fisiológicos de PIV en teleósteos. PIV se distribuye en el cerebro de los teleósteos y afectan la liberación de la hormona del crecimiento, la gonadotropina y la prolactina a partir de células cultivadas de pituitaria teleósteas in vitro. Sin embargo, las funciones de PIV en el sistema inmune de los teleósteos y más específicamente su papel en la respuesta inflamatoria aún no se han dilucidado. Además, a pesar de que la secuencia codificante para PIV ha sido reportada en varias especies de peces, no existen reportes de caracterización de dicho gen en Salmo salar u Oncorhynchus mykiss. PIV (Vasoactive Intestinal Peptide) is a 28 amino acid peptide that was originally isolated from 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. Using immunohistochemistry, several investigators have demonstrated the presence of PIVergic nerve fibers in central (thymus) and peripheral lymphoid organs (spleen, lymph nodes, and mucosa-associated lymphoid tissue). Over the past decade, 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. However, the functions of PIV in the teleost immune system and more specifically its role in the inflammatory response have not yet been elucidated. Furthermore, although the coding sequence for PIV has been reported in several species of fish, there are no reports of characterization of this gene in Salmo salar or Oncorhynchus mykiss.
En consecuencia, todos los desafíos que afronta la producción a gran escala de estas moléculas peptídicas, son aún más relevantes para productos biotecnológicos destinados a la industria acuícola, ya que un aspecto clave en esta industria es el aumento de los rendimientos productivos con bajos costos de producción. En consecuencia, se requiere una nueva estrategia que permita optimizar el proceso de producción y escalamiento de moléculas peptídicas para su uso en esta industria. Consequently, all the challenges faced by the large-scale production of these peptide molecules are even more relevant for biotechnological products destined for the aquaculture industry, since a key aspect in this industry is the increase of productive yields with low costs of production. Consequently, a new strategy is required to optimize the process of production and scaling of peptide molecules for use in this industry.
Sumario de la invención Summary of the invention
La presente invención se refiere a un método para la producción de múltiples copias de un péptido recombinante para la producción de péptidos con propiedades inmunomoduladoras y antiinflamatorias en peces dicho método que comprende los pasos de: a) diseñar y sintetizar una secuencia nucleotídica que codifica un concatémero que comprende una pluralidad de copias unidas por la secuencia Asn-Gly de un péptido; 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;
b) clonar dicha secuencia nucleotídica en un vector de expresión; b) cloning said nucleotide sequence into an expression vector;
c) transformar una bacteria con dicho vector de expresión; c) transforming a bacterium with said expression vector;
d) inducir la expresión del concatémero durante el cultivo de la bacteria; d) inducing the expression of the concatemer during the culture of the bacterium;
e) lisar la bacteria para obtener los cuerpos de inclusión a partir de esta; e) lysing the bacteria to obtain inclusion bodies from it;
f) disgregar los cuerpos de inclusión para obtener el concatémero; y f) disintegrating the inclusion bodies to obtain the concatemer; and
g) escindir el concatémero con un agente químico, para obtener una pluralidad de copias individuales de dicho péptido; y g) cleaving the concatemer with a chemical agent, to obtain a plurality of individual copies of said peptide; and
h) purificar dicho péptido recombinante. En una modalidad preferida del método de la invención la inducción de la expresión del concatémero se realiza con isopropil^-D-1 -tiogalactopiranósido (IPTG) a una concentración entre 0,1 y 1 mM. h) purifying said recombinant peptide. In a preferred embodiment of the method of the invention, the induction of concatemer expression is carried out with isopropyl ^ -D-1 -thiogalactopyranoside (IPTG) at a concentration between 0.1 and 1 mM.
En otra modalidad preferida de la invención las bacterias se lisan con una prensa francesa. In another preferred embodiment of the invention the bacteria are lysed with a French press.
Una de las ventajas del método propuesto es que el paso de disgregación de los cuerpos de inclusión se puede llevar a cabo de manera conjunta con la escisión de los concatémeros utilizando para ello hidroxilamina entre el 25 y el 50% (p/v). 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).
En el método de la presente invención el péptido recombinante se purifica mediante pasos consecutivos de lavado y centrifugación, evitándose así el uso de pasos cromatográficos para la purificación. In the method of the present invention, the recombinant peptide is purified by consecutive washing and centrifugation steps, thus avoiding the use of chromatographic steps for purification.
En una realización preferida de la presente invención, la secuencia nucleotídica que codifica un concatémero del péptido es una secuencia que se selecciona de las secuencias nucleotídicas que codifican para el Péptido Intestinal Vasoactivo (PIV) y para la grelina de Salmo salar, las cuales se clonan en el vector de expresión pET22b en una modalidad preferida. In a preferred embodiment of the present invention, 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.
Este vector de expresión pET22b se utiliza para transformar, en una modalidad particularmente preferida, la cepa de E. coli BL21 (DE3), para obtener monómeros de PIV o grelina recombinantes de Salmo salar. 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.
Los péptidos así obtenidos pueden utilizarse para la preparación de una composición veterinaria para estimular el sistema inmune en Salmo salar, para estimular su crecimiento, para combinarlos en formulaciones de vacunas para potenciar el efecto de estas, y/o para el tratamiento de una enfermedad inflamatoria en dicho pez. 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.
Breve descripción de las figuras Brief description of the figures
La FIG. 1 muestra una electroforesis en gel de poliacrilamida al 15% de las fracciones solubles e insolubles que se obtienen luego de la lisis de las células BL21 (DE3) transformadas con el vector pET22b-Péptido 6X y a las cuales se les indujo la expresión del concatémero de péptidos con IPTG 0,4 mM. 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.
La FIG. 2 muestra una electroforesis en gel de poliacrilamida al 15% de la purificación y cuantificación de los cuerpos de inclusión donde están contenidos los concatémeros de péptidos. La FIG. 3 muestra una electroforesis en gel de poliacrilamida al 17% de la escisión con hidroxilamina de los concatémeros de péptidos y la obtención de las copias individuales del péptido de interés. 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.
La FIG. 4 muestra el efecto del Péptido Intestinal Vasoactivo (PIV) y la grelina de Salmo salar obtenidos por vía recombinante. FIG. 4 shows the effect of the Vasoactive Intestinal Peptide (PIV) and ghrelin from Salmo salar obtained by recombinant route.
Divulgación detallada de la invención Detailed disclosure of the invention
La presente invención se refiere a un método para la producción recombinante de moléculas peptídicas de interés a gran escala en bacterias. Particularmente, el método de la invención es particularmente útil para la obtención de péptidos en peces con propiedades inmunomoduladoras y antiinflamantorias. Este método permite aumentar los rendimientos productivos de este tipo de moléculas, comparado con los métodos de producción recombinante y purificación que actualmente existen en el estado de la técnica. Además, una de las principales ventajas del método de la presente invención, es la obtención de las moléculas peptídicas con un alto grado de pureza, sin necesidad de involucrar pasos cromatográficos en el proceso. The present invention relates to a method for the recombinant production of peptide molecules of interest on a large scale in bacteria. In particular, 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. Furthermore, 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.
Este método sencillo y de bajo costo, permite utilizarlo para la producción de péptidos particularmente útiles para la industria acuícola, donde los costos de los productos biotecnológicos suelen ser altos, de manera que este método incide de manera positiva en los costos productivos de esta industria. 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.
Todos los términos técnicos y científicos utilizados para describir la presente invención tienen el mismo significado entendido para una persona con conocimientos básicos en el campo técnico en cuestión. No obstante, para definir con más claridad el alcance de la invención, a continuación, se incluye una lista de la terminología utilizada en esta descripción y su significado. All technical and scientific terms used to describe the present invention have the same meaning understood by a person with basic knowledge in the technical field in question. However, to more clearly define the scope of the invention, the following is a list of the terminology used in this description and their meaning.
Se debe entender por el término “concatémero”, una secuencia nucleotídica que contiene una pluralidad de copias de una misma secuencia nucleotídica dispuestas en serie o tándem. En el caso particular de la presente invención, el concatémero es una secuencia nucleotídica sintética. The term "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.
Se debe entender por el término“secuencia de nucleótidos o secuencia nucleotídica”, una doble hebra de ADN, o una hebra simple de ADN, natural o sintética, o productos de la transcripción de dicho ADN (por ejemplo, moléculas de ARN). A su vez,“secuencia de nucleótidos que codifica un péptido de interés”, se debe entender como una secuencia de nucleótidos que transcribe una molécula de ARN funcional, o que codifica un péptido funcional de interés para la presente invención. Se debe entender, que la presente invención no se relaciona a secuencias de nucleótidos genómicos en su estado natural, si no que se refiere a secuencias de nucleótidos en un estado aislado, o purificado, o parcialmente purificado, o recombinante, obtenida mediante cualquier método de ingeniería genética conocido en el estado de la técnica. The term "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). In turn, "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. It should be understood that 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.
Se debe entender en el contexto de la presente invención por el término“secuencia de péptidos o secuencia peptídica o secuencia de aminoácidos o secuencia aminoacídica” una secuencia de aminoácidos pequeña de hasta 50 aminoácidos, natural o sintética, o productos de la traducción de ARN. In the context of the present invention the term "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.
Se debe entender por el término“recombinante” cualquier secuencia nucleotídica (ADN, ARN) o aminoacídica modificada mediante cualquier método de ingeniería genética conocido en el estado de la técnica, que genera como resultado una nueva secuencia nucleotídica o aminoacídica distinta a la que se encuentra en la naturaleza. The term "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.
Se debe entender por el término “cuerpos de inclusión”, agregados proteicos o peptídicos intracelulares que se forman comúnmente en bacterias recombinantes. The term "inclusion bodies" is to be understood as intracellular protein or peptide aggregates that are commonly formed in recombinant bacteria.
Se debe entender por la expresión“disgregar los cuerpos de inclusión” en el contexto de la presente invención, la solubilización de los agregados proteicos o peptídicos que se forman durante el proceso de producción recombinante en E. coli. In the context of the present invention, 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.
Se debe entender por el término“precipitado de ruptura” en el contexto de la presente invención, a la fracción insoluble que se forma como resultado del proceso de ruptura celular y posterior centrifugación. In the context of the present invention, the term "rupture precipitate" should be understood as the insoluble fraction that is formed as a result of the process of cellular rupture and subsequent centrifugation.
Se debe entender por el término“sobrenadante de ruptura” en el contexto de la presente invención, a la fracción soluble que se forma como resultado del proceso de ruptura celular y posterior centrifugación. The term "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.
Se debe entender por el término“debris celular”, restos orgánicos que se producen luego de una lisis celular. The term "cell debris" should be understood as organic remains that are produced after cell lysis.
Se debe entender por el término“transformar un microorganismo”, un proceso por el cual se incorpora una secuencia nucleotídica exógena a una célula hospedera, que en este caso y para el contexto de la presente invención, corresponde a una célula procariota. The term "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.
El método de la presente invención comprende producir moléculas peptídicas a gran escala comenzando con el diseño y síntesis de una secuencia nucleotídica que codifica un concatémero que comprende una pluralidad de copias de un péptido de interés. En el caso particular de la presente invención, dicho péptido de interés es un neuropéptido que tiene propiedades inmunomoduladoras y antiinflamantorias en peces tal como el PIV y la grelina. La producción de estos péptidos de interés en forma de concatémeros mejora la estabilidad de dichos péptidos, las cuales son menos susceptibles a la degradación proteolítica durante el proceso de producción en el microorganismo hospedero. 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. In the particular case of the present invention, 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.
El concatémero puede contener entre 6 y 8 copias del péptido de interés, sin limitarse a dicha cantidad mencionada. Preferentemente, el concatémero contiene 6 copias en tándem del péptido de interés. Cada secuencia nucleotídica individual que codifica al péptido de interés se encuentra separada por una secuencia nucleotídica que codifica los aminoácidos Asp-Gly. De esa forma, se puede inducir la escisión del concatémero con el compuesto inorgánico hidroxilamina para liberar las copias individuales de los péptidos de interés. Esto le otorga una gran ventaja pues el presente método no utiliza técnicas enzimáticas para el corte del concatémero, lo que reduce los costos de producción de péptidos para la industria acuícola. The concatemer can contain between 6 and 8 copies of the peptide of interest, without being limited to said quantity. Preferably, 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. In this way, 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.
La secuencia nucleotídica diseñada que contiene el concatémero del péptido de interés, se clona en un vector de expresión para su posterior expresión en un microorganismo adecuado. La secuencia nucleotídica que contiene el concatémero del péptido de interés puede clonarse en cualquier vector del sistema de expresión pET. Dicho vector de expresión es preferentemente pET22b, el cual contiene un promotor T7, un operador lac y sitios de múltiple clonación útiles para la inserción del concatémero. En una modalidad preferida, el microorganismo preferido para la expresión del concatémero es procarionte, preferentemente E. coli, más preferentemente la cepa BL21 (DE3), aunque puede utilizarse cualquier cepa de E. coli del sistema de expresión pET. 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. In a preferred embodiment, 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.
La inducción de la expresión del concatémero se realiza preferentemente con isopropil- b-D-l -tiogalactopiranósido (IPTG) a una concentración entre 0,1 y 1 mM, entre 4 y 18 horas de cultivo y a una temperatura entre 30 y 37eC. 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.
Luego de incubar el cultivo con IPTG durante el periodo de tiempo establecido, el cultivo se centrifuga a una velocidad entre 6000 y 8000 g durante 5 a 15 minutos a una temperatura entre 2 y 6eC para obtener un precipitado de microorganismos. Posteriormente, el precipitado de microorganismos se resuspende en una disolución de lisis apropiada, utilizando medios físicos como, por ejemplo, una prensa francesa para lograr la lisis celular. El debris obtenido luego de la lisis se centrifuga a una velocidad entre 8000 y 10000 g durante 25 a 40 minutos a una temperatura entre 2 y 6eC para separar el sobrenadante de ruptura del precipitado de ruptura donde están contenidos los cuerpos de inclusión, y luego se lava el precipitado nuevamente con la disolución de lisis y se centrifuga nuevamente a una velocidad entre 8000 y 10000 g durante 25 a 40 minutos, a una temperatura entre 2 y 6eC para obtener los cuerpos de inclusión. El precipitado que contiene los cuerpos de inclusión se resuspende nuevamente en la disolución de solubilización y esta suspensión se mantiene en agitación durante 6 a 12 horas a una temperatura entre 21 y 25eC. Luego, se centrifuga a una velocidad entre 8000 y 10000 g durante 15 a 30 minutos a una temperatura entre 2 y 6eC, para obtener así los cuerpos de inclusión solubilizados. 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.
Una vez que se obtienen estos cuerpos de inclusión, se disuelven en disolución de solubilización, a la cual se le agrega hidroxilamina a una concentración entre el 25 y el 50% (p/v), y la mezcla se incuba a 45eC durante entre 12 y 16 horas para producir la escisión de los concatémeros y generar las copias individuales del péptido de interés. Once these inclusion bodies are obtained, they 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.
La solución se centrifuga a una velocidad entre 8000 y 10000 g durante 15 a 30 minutos y a una temperatura entre 2 y 6eC, para obtener un precipitado, el cual se lava al menos 4 veces con PBS 1 X para obtener los péptidos con un alto grado de pureza. 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.
Los siguientes ejemplos están destinados a ilustrar la invención y sus modalidades preferidas, pero en ninguna circunstancia deberán considerarse para restringir el alcance de la invención, que estará definido por el tenor de las reivindicaciones que aquí se adjuntan. The following examples are intended to illustrate the invention and its preferred embodiments, but should under no circumstances be construed to restrict the scope of the invention, which will be defined by the wording of the claims appended herein.
Ejemplos de Aplicación Application Examples
Ejemplo 1. Diseño y síntesis de una secuencia nucleotídica que codifica el concatémero. Example 1. Design and synthesis of a nucleotide sequence encoding the concatemer.
Los péptidos de interés que se utilizaron en el presente estudio fueron dos péptidos con propiedades inmunomoduladoras y antiinflamantorias obtenidos de Salmo salar: el Péptido Intestinal Vasoactivo PIV, cuya secuencia aminoacídica es: HSDAIFTDNYSRFRKQMAVKKYLNSVLT y la grelina cuya secuencia aminoacídica es: GSSFLSPSQKPQVRQGKGKPPRV 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
Para la obtención recombinante de estos péptidos a gran escala, se utilizaron las siguientes secuencias nucleotídicas; Para el PIV: For the recombinant obtaining of these peptides on a large scale, the following nucleotide sequences were used; For the PIV:
C AT ATGCACT CAG AT GCCATTTT CACAG ACAACT ACAGT CGCTT CCGCAAA CAG AT GGCTGT AAAGAAAT AT CT G AACT CGGTT CT G ACAAACGGCCACT CA G AT GCCATTTT CACAGACAACT ACAGT CGCTT CCGCAAACAG AT GGCT GT A AAG AAAT AT CT G AACT CGGTT CT G ACAAACGGCCACT CAG AT GCCATTTT C ACAG ACAACT ACAGT CGCTT CCGCAAACAGAT GGCT GT AAAGAAAT AT CT G AACT CGGTT CT G ACAAACGGCCACT CAG AT GCCATTTT CACAGACAACT AC AGT CGCTT CCGCAAACAGAT GGCT GT AAAGAAAT AT CT G AACT CGGTT CT G ACAAACGGCCACT CAG AT GCCATTTT CACAGACAACT ACAGT CGCTT CCGC AAACAG AT GGCT GT AAAGAAAT AT CT G AACT CGGTT CT GACAAACGGCCAC T CAG AT GCCATTTT CACAGACAACT ACAGT CGCTT CCGCAAACAGAT GGCT GT AAAGAAAT AT CT G A ACT CGGTT CT G AC AT G ACTCG AG C AT ATGCACT CAG AT GCCATTTT CACAG ACAACT ACAGT CGCTT CCGCAAA CAG AT GGCTGT AAAGAAAT AT CT G AACT CGGTT CT G ACAAACGGCCACT CA G AT GCCATTTT CACAGACAACT ACAGT CGCTT CCGCAA GACT ACC ACCC AT CGAGACT CTGAGACT CTAGACT CGAGACT ACAACT ACAGT CGCTT CCGCAAACAGAT GGCT GT AAAGAAAT AT CT G AACT CGGTT CT G ACAAACGGCCACT CAG AT GCCATTTT CACAGACAACT AC AGT CGCTT CCGCAAACAGAT GGCT GT AAAGAAAT AT CT G AACT CGGTT CT G ACA CAACTTGACCT CGGTT CT G ACA CAACTT GAGAC CTAGACCT AACT GAGAC ATGACCT AACT GTAGACCTA CGGTT CT GACAAACGGCCAC T CAG AT GCCATTTT CACAGACAACT ACAGT CGCTT CCGCAAACAGAT GGCT GT AAAGAAAT AT CT GA ACT CGGTT CT G AC AT G ACTCG AG
Las secuencias marcadas en negritas y subrayadas corresponden a los sitios de reconocimiento de las enzimas de restricción Ndel y Xhol utilizadas para la clonación en el vector de expresión pET22b. The 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.
Para la Grelina:
Figure imgf000011_0001
For the Ghrelin:
Figure imgf000011_0001
GGGT AAAGGG AAGCCCCCT CG AGTT AACGGCGGGT CCAGCTT CCT CAGC CCCT CCCAG AAACCACAGGT AAG ACAGGGT AAAGGG AAGCCCCCT CG AGT T AACGGCGGGT CCAGCTT CCT CAGCCCCT CCCAG AAACCACAGGT AAG AC AGGGT AAAGGG AAGCCCCCT CG AGTT AACGGCGGGT CCAGCTT CCT CAG CCCCT CCCAG AAACCACAGGT AAG ACAGGGT AAAGGG AAGCCCCCT CG A GTT AACGGCGGGT CCAGCTT CCT CAGCCCCT CCCAG AAACCACAGGT AAG ACAGGGT AAAGGG AAGCCCCCT CG AGTT AACGGCGGGT CCAGCTT CCT CA GCCCCT CCCAG AAACCACAGGT AAG ACAGGGT AAAGGG AAGCCCCCT CG AGTTT G AAAGCTT GGGT AAAGGG AAGCCCCCT CG AGTT AACGGCGGGT CCAGCTT CCT CAGC CCCT CCCAG AAACCACAGGT AAG ACAGGGT AAAGGG AAGCCCCCT CG AGT T AACGGCGGGT CCAGCTT CCT CAGCCCCT CCCAG AAACCACAGGT AAG AC AGGGT AAAGGG AAGCCCCCT CG AGTT AACGGCGGGT CCAGCTT CCT CAG CCCCT CCCAG AAACCACAGGT AAG ACAGGGT AAAGGG AAGCCCCCT CG A GTT AACGGCGGGT CCAGCTT CCT CAGCCCCT CCCAG AAACCACAGGT AAG ACAGGGT AAAGGG AAGCCCCCT CG AGTT AACGGCGGGT CCAGCTT CCT CA GCCCCT CCCAG AAACCACAGGT AAG ACAGGGT AAAGGG AAGCCCCCT CG AGTTT G AAAGCTT
Las secuencias marcadas en negritas y subrayadas corresponden a los sitios de reconocimiento de las enzimas de restricción Ndel y Hindlll utilizadas para la clonación en el vector de expresión pET22b. The 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.
Estas secuencias se sintetizaron químicamente y se insertaron en el vector de expresión pET22b en los sitios de restricción Ndel/Xhol, para obtener el concatémero de PIV y Ndel/Hindlll para obtener el concatémero de grelina. These sequences were chemically synthesized and inserted into the expression vector pET22b at the restriction sites Ndel / Xhol, to obtain the PIV concatemer and Ndel / Hindlll to obtain the ghrelin concatemer.
Posteriormente la cepa de E. coli BL21 (DE3) se transformó con dicho vector de expresión y se creció en medio sólido LB [Luria-Bertani: Triptona 1 % (p/v), Extracto de levadura 0,5% (p/v), NaCI 171 ,1 mM, pH 7,5, agar bacteriológico al 1 ,5% (p/v)] suplementado con 50 pg/mL de ampicilina. Para la inducción de la expresión de los concatémeros se tomaron colonias aisladas de la cepa E. coli BL21 (DE3) transformadas con las construcciones de interés y se inocularon en cultivos de 300 mi con medio LB líquido suplementado con ampicilina 50 pg/ml (LBA) y se dejaron crecer durante 12 horas. Al cabo de este tiempo se inocularon 50 mi de células crecidas en 1 L de medio LBA y se dejó crecer a 37 eC hasta obtener una D.O. a 600 nm entre 0,1 y 1 . Se indujo la expresión de los concatémeros con isopropil-tio^-D-galactósido (IPTG). Para ello se agrega IPTG al medio de cultivo de los microorganismos a una concentración final de 0,4 mM, y el cultivo se incuba durante 6 horas a 37eC. Subsequently, 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. For the induction of the expression of the concatemers, isolated colonies of the E. coli BL21 (DE3) strain transformed with the constructions of interest were taken and inoculated into 300 ml cultures with liquid LB medium supplemented with 50 pg / ml ampicillin (LBA ) and grown for 12 hours. A the end of this time 50 ml of grown cells were inoculated in 1 L of LBA medium and grown at 37 and C until an OD 600 nm between 0.1 and 1. The expression of the concatemers was induced with isopropyl-thio ^ -D-galactoside (IPTG). For this, IPTG is added to the culture medium of the microorganisms at a final concentration of 0.4 mM, and the culture is incubated for 6 hours at 37 ° C.
Ejemplo 2. Obtención de cuerpos de inclusión y purificación de los péptidos PIV y Grelina. Example 2. Obtaining inclusion bodies and purification of the PIV and Ghrelin peptides.
Transcurridas 6 horas el cultivo se centrifuga a 8000 g por 10 minutos a 4eC. Posteriormente, el precipitado de microorganismos se resuspende en una disolución de PBS 1 X-Tritón x 100 al 1 % a razón de 10g/100ml, y se produce la ruptura de células utilizando una prensa francesa. El debris celular obtenido luego de la ruptura se centrifuga a una velocidad de 8000 g durante 30 minutos a una temperatura de 4eC para separar el sobrenadante de ruptura del precipitado de ruptura donde están contenidos los cuerpos de inclusión. Luego se lava el precipitado con una disolución de PBS 1 X- Tritón x 100 al 1 %, NaCI 1 M, a razón de 10g/100ml. Se centrifuga nuevamente a una velocidad de 8000 g durante 25 a 40 minutos a una temperatura entre 2 y 6eC para purificar y obtener los cuerpos de inclusión. After 6 hours, the culture is centrifuged at 8000 g for 10 minutes at 4 ° C. Subsequently, 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.
La Figura 1 representa una electroforesis en gel de poliacrilamida al 15% de las fracciones soluble e insoluble que se obtienen luego de la ruptura de las células de BL21 (DE3) transformadas con el vector pET22b-Péptido 6X y a las cuales se les indujo la expresión del concatémero de péptidos con IPTG 0,4 mM, en la cual PM es el patrón de pesos moleculares; S.rup es el sobrenadante de ruptura, es decir la fracción intracelular soluble y C. Inc es precipitado de ruptura, o fracción correspondiente a los cuerpos de inclusión. 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.
En esta figura se muestra una banda entre 25 y 35 kDa en la fracción del precipitado de ruptura, que corresponde al concatémero de péptidos en forma de cuerpos de inclusión. 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.
Para la purificación de los concatémetos, se resuspende el precipitado en una disolución PBS 1 X, Urea 8M, b-Mercapto 10 nM, PMSF 1 mM, pH 8, a razón de 10g/ml. Esta suspensión obtenida se mantiene en agitación durante toda la noche a temperatura ambiente, luego, se centrifuga a una velocidad de 8000 g por 20 minutos a 4eC. Para la escisión de los concatémeros el precipitado se disuelve a razón de 1 g/5 mi de disolución de PBS 1 X, Urea 8M, b-Mercapto 10mM, PMSF 0,1 -1 mM, pH8; 1 mi Tris HC1 1 M, pH 9,5 y 4 mi hidroxilamina (solución al 50% en agua) y se incuba a 45eC durante toda la noche. Este paso permite generar las copias individuales del péptido de interés. For the purification of the concatemetes, 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. For cleavage of the concatemers, 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.
En la FIG. 2 se muestra una electroforesis en gel de poliacrilamida al 15% de la purificación y cuantificación de los cuerpos de inclusión donde están contenidos los concatémeros de péptidos, en la cual PM es el Patrón de pesos moleculares; BSA es la Curva de BSA para la cuantificación de las proteínas; Péptido 6X es el concatémero del péptido de interés contenido en los cuerpos de inclusión. In 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.
La mezcla anterior se centrifuga a una velocidad a 8000 g durante 30 minutos a 4eC y luego se lava el precipitado 4 veces con PBS 1 X para obtener los péptidos con un alto grado de pureza. 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.
En la FIG. 3 se muestra una electroforesis en gel de poliacrilamida al 17% de la escisión con hidroxilamina de los concatémeros de péptidos y la obtención de los monómeros del péptido de interés, en la que PM es el patrón de pesos moleculares y Péptido 1 X Cl : el péptido monómerico obtenido luego del corte con hidroxilamina. In 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.
Ejemplo 3: Determinación del efecto sobre el sistema inmune de los péptidos obtenidos por vía recombinante. Example 3: Determination of the effect on the immune system of peptides obtained by recombinant route.
La actividad biológica de los péptidos producidos utilizando el sistema de expresión recombinante previamente descrito se determinó in vitro mediante el análisis de la expresión de genes relacionados con el sistema inmune. 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.
Las células SHK-1 (células derivadas de riñón anterior de salmón Atlántico, Salmo sa/ar) se incubaron con 20 nM de los péptidos PIV y GRE (Péptido intestinal vasoactivo y Grelina, respectivamente) de Salmo salar obtenidos en los ejemplos 1 y 2. Se determinó la expresión de INFy, I L- 1 b , TNF-D y la quimioquina CCL19 a las 4 y 8 horas post tratamiento. Los valores de expresión relativa se normalizaron con la expresión de ARNm de la proteína de unión poliadelinato 1 (PBP-1 ). 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 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).
Como resultado se obtuvo que todos los péptidos aumentaron significativamente la expresión de INF-g a las 4 horas de tratamientos, mientras que, a las 8 horas, solo el PIV muestra un aumento significativo en la expresiónde esta citoquina. El INFy es una citocina TH1 que es crítica para casi todas las fases de las respuestas inmunes. Al igual que el INFy de mamífero, los INFy de los peces teleósteos median la activación de macrófagos, a través del aumento de la actividad de estallido respiratorio, la producción de óxido nítrico y la fagocitosis, induciendo la expresión de los genes antivirales típicos. El IFNy también induce la apoptosis, especialmente durante la infección viral, e inhibe la proliferación celular. La inmunidad celular de tipo TH1 es esencial en la defensa inmune contra virus y bacterias intracelulares. As a result, it was obtained that all the peptides significantly increased the expression of INF-g at 4 hours of treatments, while, at 8 hours, only the PIV shows a significant increase in the expression of this cytokine. INFy is a T H 1 cytokine that is critical for almost all phases of immune responses. Like mammalian INFy, 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.
Los péptidos no produjeron cambios significativos en la expresión de CCL19 a las 4 horas de tratamiento. Por su parte, la expresión de IL-1 b no mostró diferencias significativas a las 4 y 8 horas de administrados los péptidos. The peptides did not produce significant changes in the expression of CCL19 at 4 hours of treatment. For its part, the expression of IL-1 b did not show significant differences at 4 and 8 hours after the peptides were administered.
A las 8 horas de tratamiento ambos péptidos disminuyeron significativamente la expresión de CC19. La quimiocina CCL19 interviene en la inflamación aguda y el reclutamiento de linfocitos y otras células. En peces, se ha demostrado que CCL19 induce la proliferación celular, la actividad de estallido respiratorio y la quimiotaxis de leucocitos de sangre periférica (PBL), lo que sugiere su capacidad para ejercer funciones inflamatorias y homeostáticas. Teniendo en cuenta esto, se puede sugerir que los péptidos pueden poseer efectos antiiflamatorios en peces. At 8 hours of treatment, both peptides significantly decreased the expression of CC19. CCL19 chemokine is involved in acute inflammation and the recruitment of lymphocytes and other cells. In fish, 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.
En la FIG. 4 se muestra el efecto de los péptidos PIV y GRE obtenidos de manera recombinante como se describió anteriormente sobre la expresión de genes involucrados en la respuesta inmune. Los valores en el gráfico representan la media + DE (n=4). Los datos se analizaron mediante un Test T. * significa p<0,05; ** significan p<0,01 ; *** significan p<0,001 . Las células SHK1 se incubaron con 20nm con cada uno de los péptidos. Se midió la expresión INF-g, CC19, I L-1 b y TNF-a a 4 y 8 horas post tratamiento. Los valores de expresión relativa se normalizaron con la expresión de ARNm de la proteína de unión poliadelinato 1 (PBP-1 ). In 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 values in the graph represent the mean + SD (n = 4). 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).
Ventajas de la solución propuesta Advantages of the proposed solution
Se ha desarrollado un proceso basado en la expresión recombinante de concatémeros de péptidos en bacterias y la obtención de monómeros peptídicos biológicamente activos mediante la escisión de los concatémeros con hidroxilamina. La invención, entre otras, presenta las siguientes ventajas con respecto a la técnica anterior: 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, among others, has the following advantages over the prior art:
1 ) Permite la obtención por vía recombinante de moléculas peptídicas en forma de concatémeros lo que mejora la estabilidad de dichas moléculas las cuales son menos susceptibles a la degradación proteolítica durante el proceso de producción en el organismo hospedero; 2) Permite la producción de péptidos que pueden ser tóxicos para el microorganismo hospedero donde estos se expresan; y 1) It allows the recombinant obtaining of peptide molecules in the form of concatemers, which improves the stability of said molecules, which are less susceptible to proteolytic degradation during the production process in the host organism; 2) It allows the production of peptides that can be toxic to the host microorganism where they are expressed; and
3) Permite obtener los péptidos biológicamente activos y con un alto grado de pureza sin el uso de técnicas cromatográficas para el proceso de purificación. Estos péptidos, en dependencia de su función biológica, pueden ser utilizados en la acuicultura para aumentar el crecimiento, la sobrevida, la calidad de los alevines y mejorar el sistema inmune y la resistencia a patógenos. También pueden ser utilizados bajo determinadas condiciones como moléculas antiiflamatorias en peces. 3) It allows obtaining biologically active peptides with a high degree of purity without the use of chromatographic techniques for the purification process. These peptides, depending on their biological function, can be used in aquaculture to increase growth, survival, quality of the fingerlings and improve the immune system and resistance to pathogens. They can also be used under certain conditions as anti-inflammatory molecules in fish.
El proceso sencillo y de bajo costo desarrollado para la producción de estos péptidos permite utilizarlos en la industria acuícola donde los costos de los productos biotecnológicos que se apliquen deben ser bajos de manera que se pueda incidir de manera positiva en los costos productivos de la industria. The simple and low-cost process developed for the production of these peptides allows them to be used in the aquaculture industry where the costs of the biotechnological products applied must be low so that the production costs of the industry can be positively affected.

Claims

REIVINDICACIONES
1 . Un método para la producción de múltiples copias de un péptido recombinante para la producción de péptidos con propiedades inmunomoduladoras y antiinflamantorias en peces, CARACTERIZADO porque comprende los pasos de: one . 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, CHARACTERIZED because it comprises the steps of:
a. diseñar y sintetizar una secuencia nucleotídica que codifica un concatémero que comprende una pluralidad de copias unidas por la secuencia Asn-Gly de un péptido; to. designing and synthesizing a nucleotide sequence encoding a concatemer comprising a plurality of copies linked by the Asn-Gly sequence of a peptide;
b. clonar dicha secuencia nucleotídica en un vector de expresión; c. transformar una bacteria con dicho vector de expresión; b. cloning said nucleotide sequence into an expression vector; c. transforming a bacterium with said expression vector;
d. inducir la expresión del concatémero durante el cultivo de la bacteria; e. lisar la bacteria para obtener los cuerpos de inclusión a partir de esta; f. disgregar los cuerpos de inclusión para obtener el concatémero; y g. escindir el concatémero con un agente químico, para obtener una pluralidad de copias individuales de dicho péptido; y d. induce the expression of the concatemer during the culture of the bacterium; and. lyse the bacteria to obtain inclusion bodies from it; F. disaggregate the inclusion bodies to obtain the concatemer; and g. cleaving the concatemer with a chemical agent, to obtain a plurality of individual copies of said peptide; and
h. purificar dicho péptido recombinante. h. purify said recombinant peptide.
2. El método de acuerdo con la reivindicación 1 , CARACTERIZADO porque la inducción de la expresión del concatémero se realiza con isopropil^-D-1 - tiogalactopiranósido (IPTG) a una concentración entre 0,1 y 1 mM. The method according to claim 1, CHARACTERIZED in that the induction of the expression of the concatemer is carried out with isopropyl ^ -D-1-thiogalactopyranoside (IPTG) at a concentration between 0.1 and 1 mM.
3. El método de acuerdo con la reivindicación 1 , CARACTERIZADO porque las bacterias se lisan con una prensa francesa. 3. The method according to claim 1, CHARACTERIZED in that the bacteria are lysed with a French press.
4. El método de acuerdo con la reivindicación 1 , CARACTERIZADO porque los cuerpos de inclusión se disgregan junto con la escisión de los concatémeros utilizando hidroxilamina. 4. The method according to claim 1, CHARACTERIZED in that the inclusion bodies disintegrate together with the cleavage of the concatemers using hydroxylamine.
5. El método de acuerdo con las reivindicaciones 4, CARACTERIZADO porque la hidroxilamina se utiliza entre el 25 y el 50% (p/v). 5. The method according to claims 4, CHARACTERIZED in that the hydroxylamine is used between 25 and 50% (w / v).
6. El método de acuerdo con la reivindicación 1 , CARACTERIZADO porque el péptido recombinante se purifica mediante pasos consecutivos de lavado y centrifugación. 6. The method according to claim 1, CHARACTERIZED in that the recombinant peptide is purified by consecutive washing and centrifugation steps.
7. El método de acuerdo con cualquiera de las reivindicaciones 1 a 6, CARACTERIZADO porque la secuencia nucleotídica que codifica un concatémero del péptido es una secuencia que se selecciona de las secuencias nucleotídicas que codifican para el Péptido Intestinal Vasoactivo (PIV) y para la grelina de Salmo salar, las cuales se clonan en un vector de expresión. 7. The method according to any of claims 1 to 6, CHARACTERIZED in that 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 (PIV) and for ghrelin of Salmo salar, which are cloned into an expression vector.
8. El método de acuerdo con las reivindicaciones 7, CARACTERIZADO porque las secuencias nucleotídicas que codifican para el Péptido Intestinal Vasoactivo (PIV) y/o para la grelina de Salmo salar se clonan en el vector de expresión pET22b. 8. The method according to claims 7, CHARACTERIZED in that the nucleotide sequences that encode for Vasoactive Intestinal Peptide (PIV) and / or for ghrelin from Salmo salar are cloned in the expression vector pET22b.
9. El método de acuerdo con la reivindicación 8, CARACTERIZADO porque el vector pET22b se utiliza para transformar una bacteria. 9. The method according to claim 8, CHARACTERIZED in that the vector pET22b is used to transform a bacterium.
10. El método de acuerdo con la reivindicación 9, CARACTERIZADO porque la bacteria que se transforma con vector pET22b es la cepa de E. coli BL21 (DE3). 10. The method according to claim 9, CHARACTERIZED in that the bacterium that is transformed with vector pET22b is the E. coli strain BL21 (DE3).
1 1. El método de acuerdo con la reivindicación 6, CARACTERIZADO porque dicho péptido recombinante es el PIV o la grelina de Salmo salar. 1 1. The method according to claim 6, CHARACTERIZED in that said recombinant peptide is PIV or ghrelin from Salmo salar.
12. Uso de un péptido recombinante obtenido por el método de cualquiera de las reivindicaciones 1 a la 1 1 , CARACTERIZADO porque es útil para la preparación de una composición veterinaria para estimular el sistema inmune en Salmo salar. 12. Use of a recombinant peptide obtained by the method of any of claims 1 to 1 1, CHARACTERIZED because it is useful for the preparation of a veterinary composition to stimulate the immune system in Salmo salar.
13. Uso de un péptido recombinante obtenido por el método de cualquiera de las reivindicaciones 1 a la 1 1 , CARACTERIZADO porque es útil para la preparación de una composición veterinaria para tratar una enfermedad inflamatoria en Salmo salar. 13. Use of a recombinant peptide obtained by the method of any of claims 1 to 1 1, CHARACTERIZED because it is useful for the preparation of a veterinary composition for treating an inflammatory disease in Salmo salar.
14. Uso de acuerdo con las reivindicaciones 12 a 14, CARACTERIZADO porque dicho péptido recombinante es el PIV o la grelina de Salmo salar. 14. Use according to claims 12 to 14, CHARACTERIZED in that said recombinant peptide is PIV or ghrelin from Salmo salar.
PCT/CL2020/050015 2019-03-05 2020-03-04 Method for recombinant production of peptide molecules with immunomodulatory and anti-inflammatory properties in fish WO2020177004A1 (en)

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