ES2334083A1 - Method for the identification of antibacterial and antiviral polypeptides obtained from mytilus edulis (Machine-translation by Google Translate, not legally binding) - Google Patents
Method for the identification of antibacterial and antiviral polypeptides obtained from mytilus edulis (Machine-translation by Google Translate, not legally binding) Download PDFInfo
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- ES2334083A1 ES2334083A1 ES200703165A ES200703165A ES2334083A1 ES 2334083 A1 ES2334083 A1 ES 2334083A1 ES 200703165 A ES200703165 A ES 200703165A ES 200703165 A ES200703165 A ES 200703165A ES 2334083 A1 ES2334083 A1 ES 2334083A1
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- polypeptide
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- polypeptides
- mussels
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Abstract
Description
Método para la identificación de polipéptidos antibacterianos y antivirales obtenidos de Mytilus edulis.Method for the identification of antibacterial and antiviral polypeptides obtained from Mytilus edulis .
La invención se relaciona con un método para la identificación de polinucleótidos que codifican las formas precursoras de polipéptidos de las familias de las miticinas y de las mitilinas, así como a las miticinas y mitilinas identificadas con dicho método y a su uso como agentes antibacterianos y antivirales.The invention relates to a method for the identification of polynucleotides encoding the forms precursors of polypeptides of the families of miticins and of the mitilins, as well as the identified miticins and mitilins with said method and its use as antibacterial agents and antivirals
El cultivo de moluscos bivalvos supone una de las mayores producciones de la acuicultura en la Unión Europea llegando a constituir más del 70% de la producción mundial. Concretamente, el cultivo del mejillón (Mytilus galloprovincialis y Mytilus edulis, fundamentalmente) supera las 500.000 toneladas de producción cada año (593.000 toneladas en 2003 según datos de la FAO). A pesar de la elevada producción de estos moluscos, y de la existencia de enfermedades que afectan gravemente a su producción, con las importantes pérdidas económicas que ello conlleva, poco se conoce de cómo responden estos organismos a la enfermedad. Los bivalvos, al igual que el resto de los invertebrados, carecen de un sistema inmune adaptativo específico y sólo poseen un sistema inmune innato que actúa independientemente de la naturaleza del estímulo, lo que imposibilita el uso de vacunas al carecer de memoria inmunológica. Con estas limitaciones, el control de las enfermedades se basaba fundamentalmente en el diagnóstico del agente patógeno y en el buen manejo de los stocks.The cultivation of bivalve molluscs is one of the largest productions of aquaculture in the European Union, reaching more than 70% of world production. Specifically, the mussel culture ( Mytilus galloprovincialis and Mytilus edulis , fundamentally) exceeds 500,000 tons of production every year (593,000 tons in 2003 according to FAO data). Despite the high production of these mollusks, and the existence of diseases that seriously affect their production, with the significant economic losses that entails, little is known about how these organisms respond to the disease. Bivalves, like the rest of the invertebrates, lack a specific adaptive immune system and only have an innate immune system that acts independently of the nature of the stimulus, which makes it impossible to use vaccines because they lack immunological memory. With these limitations, disease control was based primarily on the diagnosis of the pathogen and the proper management of stocks.
Recientemente se han descrito en mejillón una serie de pequeños péptidos con actividad antimicrobiana, capaces de reducir el crecimiento de varios microorganismos analizados. La patente estadounidense US6911524 y Mitta,G. et al. (Eur. J. Biochem., 265:71-78) describen dos péptidos aislados de la hemolinfa de Mytilus galloprovincialis, denominados miticina A y B, que muestran actividad bactericida, fungicida y antiparasitaria frente a ciertos patógenos. Charlet, M. et al. (J Biol. Chem., 1996, 271:21808-21813) han descrito dos polipéptidos aislados de la hemolinfa de Mytilus edulis denominados mitilinas A y B que muestran actividad bactericida. Mitta, G. et al (J. Biol. Chem., 2000, 275:12954-12962) han descrito tres nuevas isoformas de las mitilinas denominadas mitilinas C, D y G1 que también muestran actividad bactericida y fungicida.Recently, a series of small peptides with antimicrobial activity, capable of reducing the growth of several microorganisms analyzed, have been described in mussels. US Patent US6911524 and Mitta, G. et al . (Eur. J. Biochem., 265: 71-78) describe two peptides isolated from the hemolymph of Mytilus galloprovincialis , called miticin A and B, which show bactericidal, fungicidal and antiparasitic activity against certain pathogens. Charlet, M. et al. (J Biol. Chem., 1996, 271: 21808-21813) have described two polypeptides isolated from the hemolymph of Mytilus edulis called mitilins A and B that show bactericidal activity. Mitta, G. et al (J. Biol. Chem., 2000, 275: 12954-12962) have described three new isoforms of mitilins called mitilins C, D and G1 that also show bactericidal and fungicidal activity.
Sin embargo, la puesta en práctica de dichos péptidos como agentes para el control de patógenos requiere de disponer de cantidades suficientes de ellos. Esto sólo es posible mediante la expresión de los péptidos de forma recombinante en células heterólogas, ya que la purificación usando métodos convencionales a partir de los extractos de hemolinfa sólo produce cantidades muy limitadas de los mismos. Con el fin de poner a punto sistemas para la expresión recombinante de estos péptidos, es necesario disponer de la correspondiente secuencia de ADNc. La forma habitual para obtener la secuencia de ADNc para este tipo de péptidos consiste en realizar un screening de una biblioteca de ADNc obtenida de hemolinfa mediante PCR usando cebadores degenerados obtenidos a partir de la secuencia de aminoácidos obtenida de la proteína purificada para así amplificar el ADNc. Sin embargo, dado que estos péptidos se sintetizan en forma de precursores para después convertirse en péptidos maduros mediante su procesamiento post-traduccional, la amplificación del ADNc usando cebadores obtenidos a partir de la secuencia del péptido maduro permite obtener únicamente un ADNc parcial que codifica la secuencia del péptido maduro pero que no se corresponde a la secuencia nucleotídica completa que codifica para el precursor de la proteína. En estos casos, es necesaria una segunda ronda de screening usando el ADNc parcial obtenido en el primer screening como sonda para identificar mediante hibridación en una segunda ronda de screening el ADNc completo de las miticinas y mitilinas (véase Mitta,G. et al., 1999, Eur. J. Biochem., 265:71-78).However, the implementation of said peptides as agents for the control of pathogens requires having sufficient amounts of them. This is only possible by expressing the peptides recombinantly in heterologous cells, since purification using conventional methods from hemolymph extracts only produces very limited amounts thereof. In order to develop systems for the recombinant expression of these peptides, it is necessary to have the corresponding cDNA sequence. The usual way to obtain the cDNA sequence for this type of peptides is to perform a screening of a cDNA library obtained from hemolymph by PCR using degenerated primers obtained from the amino acid sequence obtained from the purified protein in order to amplify the cDNA . However, since these peptides are synthesized as precursors and then become mature peptides by post-translational processing, amplification of the cDNA using primers obtained from the mature peptide sequence allows only a partial cDNA encoding the mature peptide sequence but that does not correspond to the complete nucleotide sequence encoding the protein precursor. In these cases, a second round of screening is needed using the partial cDNA obtained in the first screening as a probe to identify by hybridization in a second round of screening the complete cDNA of the miticins and mitilins (see Mitta, G. et al ., 1999, Eur. J. Biochem., 265: 71-78).
Así, existe una necesidad en el arte de métodos más sencillos que permitan la obtención de ADNcs que codifiquen las secuencias completas de los precursores de las mitilinas y miticinas con el fin de simplificar el proceso global de obtención por via recombinante.Thus, there is a need in the art of methods simpler that allow obtaining cDNAs that encode the complete sequences of the precursors of mitilins and Mythines in order to simplify the overall procurement process recombinantly
Los estudios realizados hasta la fecha con estas formas ya patentadas revelan la existencia de actividad frente a bacterias Gram positivas fundamentalmente, sin apreciarse especificidad de patógeno. Por tanto, es necesario trabajar en el desarrollo de nuevos compuestos alternativos y eficaces, que reduzcan al mínimo posible los problemas antes expuestos, y que puedan ser incluidos dentro de estrategias integradas para el control de las enfermedades durante el cultivo de estos moluscos.The studies carried out to date with these already patented forms reveal the existence of activity against Gram positive bacteria fundamentally, without appreciating pathogen specificity. Therefore, it is necessary to work on the development of new alternative and effective compounds, which minimize the problems described above, and that can be included in integrated strategies for disease control during the cultivation of these mollusks
En un primer aspecto, la invención se relaciona con un método para la identificación de un ADNc que codifica el precursor de una miticina o una mitilina que comprendeIn a first aspect, the invention relates to with a method for the identification of a cDNA encoding the precursor of a miticin or a mitilin comprising
- (i) (i)
- poner en contacto un mejillón un agente inmunogénico,put an agent in contact with a mussel immunogenic,
- (ii) (ii)
- preparar librerías de ADNc de mejillones tratados con el agente inmunogénicoprepare cDNA libraries of treated mussels with the immunogenic agent
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- (iii) (iii)
- efectuar una sustracción de la librería obtenida de los mejillones tratados con una preparación de ácidos nucleicos de mejillones controles ysubtract the library obtained from mussels treated with a nucleic acid preparation of mussels controls and
- (iv) (iv)
- aislar los ADNcs específicos de mejillones tratados con el agente inmunogénico.isolate specific cDNAs from treated mussels with the immunogenic agent.
En otro aspecto, la invención se relaciona con un ADNc identificado mediante un método de la invención.In another aspect, the invention relates to a cDNA identified by a method of the invention.
En otro aspecto, la invención se relaciona con una construcción génica que comprende un ácido nucleico de la invención.In another aspect, the invention relates to a gene construct comprising a nucleic acid of the invention.
En otro aspecto, la invención se relaciona con un vector de expresión que comprende un ácido nucleico o una construcción génica de la invención que está operativamente acoplado con una secuencia que regula la expresión de dicho polinucleótido o de dicha construcción génica.In another aspect, the invention relates to an expression vector comprising a nucleic acid or a gene construct of the invention that is operatively coupled with a sequence that regulates the expression of said polynucleotide or said gene construct.
En otro aspecto, la invención se relaciona con una célula hospedadora que contiene una construcción génica o un vector de acuerdo a la invención.In another aspect, the invention relates to a host cell that contains a gene construct or a vector according to the invention.
En otro aspecto, la invención se relaciona con un polipéptido codificado por un ADNc de la invención.In another aspect, the invention relates to a polypeptide encoded by a cDNA of the invention.
En otro aspecto, la invención se relaciona con un método para la obtención de un polipéptido de la invención que comprende transformar una célula con un vector de la invención y recuperar el polipéptido del interior celular o del medio de cultivo.In another aspect, the invention relates to a method for obtaining a polypeptide of the invention that comprises transforming a cell with a vector of the invention and recover the polypeptide from inside the cell or from the medium of culture.
En otro aspecto, la invención se relaciona con un método para la obtención de un polipéptido de la invención que comprende inyectar intramuscularmente en un mejillón un agente inmunogénico y recuperar posteriormente de la hemolinfa uno o varios de los polipéptidos.In another aspect, the invention relates to a method for obtaining a polypeptide of the invention that comprises intramuscularly injecting an agent into a mussel immunogenic and subsequently recover from the hemolymph one or several of the polypeptides.
En otro aspecto, la invención se relaciona con un anticuerpo que reconoce específicamente el polipéptido de la invención.In another aspect, the invention relates to an antibody that specifically recognizes the polypeptide of the invention.
En otro aspecto, la invención se relaciona con una composición farmacéutica que comprende al menos un polipéptido de la invención y un carrier farmacéuticamente aceptable.In another aspect, the invention relates to a pharmaceutical composition comprising at least one polypeptide of the invention and a pharmaceutically acceptable carrier.
En otro aspecto, la invención se relaciona con un polipéptido de la invención o con una composición farmacéutica de la invención para su uso en medicina.In another aspect, the invention relates to a polypeptide of the invention or with a pharmaceutical composition of the invention for use in medicine.
En otro aspecto, la invención se relaciona con el uso de un polipéptido de la invención o de una composición de la invención para la preparación de un medicamento destinado a tratar una enfermedad infecciosa.In another aspect, the invention relates to the use of a polypeptide of the invention or a composition of the invention for the preparation of a medicament intended to be treated an infectious disease
Los autores de la presente invención han conseguido desarrollar un método para la identificación de mitilinas y miticinas que permite obtener en un solo paso la secuencia completa del ADNc que codifica los precursores de dichos péptidos, facilitando así la posterior obtención de dichos péptidos de forma recombinante. Así, en un primer aspecto, la invención se relaciona con un método para la identificación de miticinas y mitilinas (de aquí en adelante "el método de la invención") que comprende las etapas deThe authors of the present invention have managed to develop a method for identifying mitilinas and miticinas that allows to obtain in a single step the complete sequence of the cDNA encoding the precursors of said peptides, thus facilitating the subsequent obtaining of said peptides recombinantly Thus, in a first aspect, the invention is relates to a method for the identification of miticins and mitilins (hereinafter "the method of the invention") which understand the stages of
- (i) (i)
- inyectar intramuscularmente en un mejillón un agente inmunogénico,injecting an agent intramuscularly into a mussel immunogenic,
- (ii) (ii)
- preparar librerías de ADNc de mejillones tratados con el agente inmunogénicoprepare cDNA libraries of treated mussels with the immunogenic agent
- (iii) (iii)
- efectuar una sustracción de la librería de cADN obtenida de los mejillones tratados con una población de ácidos nucleicos obtenida a partir de mejillones controles ysubtract from the cDNA library obtained from mussels treated with a population of acids nuclei obtained from control mussels and
- (iv) (iv)
- aislar los ADNc específicos de mejillones tratados con el agente inmunogénico.isolate specific cDNAs from treated mussels with the immunogenic agent.
Por "miticina" se entiende, en el contexto de la presente invención, una proteína que comprende una de las secuencias definidas en SEQ ID NO:1 a 16 o una variante funcionalmente equivalente de las mismas.By "miticina" is understood, in the context of the present invention, a protein comprising one of the sequences defined in SEQ ID NO: 1 to 16 or a variant functionally equivalent thereof.
Por "mitilina" se entiende, en el contexto de la presente invención, una proteína que comprende una de las secuencias definidas en SEQ ID NO:17 a 19 o una variante funcionalmente equivalente de las mismas.By "mitilina" is understood, in the context of the present invention, a protein comprising one of the sequences defined in SEQ ID NO: 17 to 19 or a variant functionally equivalent thereof.
Por "variante funcionalmente equivalente" se entiende, en el contexto de la presente invención, toda proteína que se puede obtener a partir de las mitilinas o miticinas anteriormente indicadas mediante la sustitución, deleción o inserción de uno o mas aminoácidos y que mantiene sustancialmente la función de la proteína original. Las determinación de la función de las miticinas/mitilinas se puede llevar a cabo usando métodos convencionales ampliamente conocidos para el experto en la materia, tales como los ensayos de actividad antimicrobiana, antifúngica y antiparasitaria descritos en US6911524 para las miticinas o los ensayos de actividad antimicrobiana descritos en Charlet, M. et al. (J. Biol. Chem., 1996, 271:21808-21813) para las mitilinas.By "functionally equivalent variant" is meant, in the context of the present invention, any protein that can be obtained from the aforementioned mitilins or miticins by substitution, deletion or insertion of one or more amino acids and which substantially maintains the function of the original protein. The determination of the role of miticins / mitilins can be carried out using conventional methods widely known to those skilled in the art, such as the antimicrobial, antifungal and antiparasitic activity assays described in US6911524 for miticins or antimicrobial activity assays. described in Charlet, M. et al . (J. Biol. Chem., 1996, 271: 21808-21813) for mitilins.
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Por "precursor de una mitilina o miticina" se entiende toda proteína cuya secuencia comprende la secuencia de la mitilina o miticina y una o más regiones adicionales que mantienen la proteína en su forma inactiva y que se eliminan mediante procesamiento proteolítico de forma post-traduccional para generar la forma madura y activa de las miticinas/mitilinas.By "precursor of a mitilin or miticin" means any protein whose sequence comprises the sequence of mitilin or miticin and one or more additional regions that they keep the protein in its inactive form and they are removed by proteolytic processing so post-translational to generate the mature form and active miticins / mitilins.
En el primer paso del método de la invención, se procede a la administración en un mejillón de un agente innmunogénuico. Preferiblemente, el agente inmunogénico se selecciona del grupo de un cóctel de bacterias muertas, poli I:C y una mezcla de ambos.In the first step of the method of the invention, proceed to the administration in a mussel of an agent Inmunogenic Preferably, the immunogenic agent is select from the group of a dead bacteria cocktail, poly I: C and A mixture of both.
En el segundo paso del método de la invención,
se prepara una librería de ADNc a partir de los mejillones que se
han tratado con el agente inmunogénico. Preferiblemente, la
librería de ADNc se prepara a partir de los hemocitos. El proceso de
obtención de una librería de ADNc de una célula, órgano u organismo
de interés comienza por la preparación de ARNm de la célula en la
que se expresen las miticinas/mitilinas, como por ejemplo, los
hemocitos o la hemolinfa. El mRNA se puede aislar usando métodos
convencionales tales como mediante la extracción usando
fenol/clorofomo, fenol/SDS o guanidina, en cuyo caso es posible la
purificación previa del ARN mediante en centrifugación en gradiente
de cloruso de cesio, gradiente os descritos en Sambrook, J. et
al (Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y.) (vease capítulo
7)) y Ausubel,F (Current Protocols in Molecular Biology) (Units
4.1-4.4). Una vez que se ha aislado el ARN total
del tejido de mejillón, es preferible obtener la fracción
poliA^{+}, que contiene los ARNm. Típicamente, el aislamiento de
los ARN poliA+ se efectúa mediante purificación por afinidad usando
matrices conjugadas con oligo(dT), que permite retener los
ARN mediante su hibridación con las colas de poladenina de los
ARNm. La purificación de ARN poliA+ en matrices de oligodT se puede
llevar a cabo en columna o en suspensión. En el primer caso, la
matriz de oligo(DT) se empaqueta en una columna
cromatográfica sobre la cual se añade el ARN celular total, de forma
que el ARNm queda retenido mientras que el resto de ARN no poliA+
eluye de la columna. EL ARNm se eluye posteriormente lavando la
columna con una solución que permite romper la interacción entre el
ARN poliA+ y las matrices de oligodT, como por ejemplo una mezcla
de EDTA 2 mM y SDS al 0.1%. En caso de que la purificación del ARN
poliA+ se lleve a cabo en suspensión, el procedimiento es
esencialmente idéntico al método en columna con la diferencia de la
separación del ARN no poliA+ que no se ha unido a la matriz de
oligo(dT) del ARNm poliA+ se consigue mediante centrifugación
de las partículas de oligo(dT) lo que resulta en la
sedimentación de las partículas que contienen el ARNm poliA+ unido
mientras que el ARN no poliA+ permanece en el sobrenadante. La
elución del ARNm poliA+ se lleva a cabo mediante lavado de las
partículas con una solución del tipo EDTA 2 mM y SDS al 0.1%. El
ARNm es posteriormente precipitado en presencia de 2.5 volúmenes de
etanol para eliminar el SDS procedente de la solución de
elución.In the second step of the method of the invention, a cDNA library is prepared from mussels that have been treated with the immunogenic agent. Preferably, the cDNA library is prepared from hemocytes. The process of obtaining a cDNA library from a cell, organ or organism of interest begins by preparing mRNA from the cell in which the miticins / mitilins are expressed, such as hemocytes or hemolymph. The mRNA can be isolated using conventional methods such as by extraction using phenol / chlorofome, phenol / SDS or guanidine, in which case it is possible to pre-purify the RNA by gradient centrifugation of cesium chlorine, gradient described in Sambrook, J. et al (Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) (see Chapter 7)) and Ausubel, F (Current Protocols in Molecular Biology) (Units 4.1-4.4) . Once the total RNA has been isolated from the mussel tissue, it is preferable to obtain the polyA + fraction, which contains the mRNAs. Typically, the isolation of polyA + RNAs is effected by affinity purification using conjugated matrices with oligo (dT), which allows the RNAs to be retained by hybridization with the mothdenine tails of the mRNAs. Purification of polyA + RNA in oligodT matrices can be carried out in a column or in suspension. In the first case, the oligo matrix (DT) is packed in a chromatographic column on which the total cellular RNA is added, so that the mRNA is retained while the rest of the non-polyA + RNA elutes from the column. The mRNA is subsequently eluted by washing the column with a solution that allows to break the interaction between polyA + RNA and oligodT matrices, such as a mixture of 2 mM EDTA and 0.1% SDS. In case the purification of the polyA + RNA is carried out in suspension, the procedure is essentially identical to the column method with the difference of the separation of the non-polyA + RNA that has not bound to the oligo matrix (dT) of the mRNA. polyA + is achieved by centrifugation of the oligo particles (dT) resulting in sedimentation of the particles containing the bound polyA + mRNA while the non-polyA + RNA remains in the supernatant. Elution of the polyA + mRNA is carried out by washing the particles with a 2 mM EDTA solution and 0.1% SDS. The mRNA is subsequently precipitated in the presence of 2.5 volumes of ethanol to remove the SDS from the solution of
elution
Una vez que se dispone de una preparación purificada de ARNm, este se pone en contacto con un cebador que puede ser un oligonucleótido que incorpora una región de poli(dT) en su extremo 3' o una mezcla de cebadores de secuencia aleatoria en presencia de una transcriptasa inversa carente de actividad RNase H (por ejemplo, las transcriptasas reversas del virus de la leucemia murina de Moloney, del virus de la mieloblastosis aviar, del virus de la inmunodeficiencia humana, del virus de la inmunodeficiencia en simios, del virus respiratorio sincitial, del virus espumoso humano, del virus de la leucemia bovina, del virus linfotrófico T humano tipo I, o la transcriptasa reversa R2 de Bómbix mori). Si se usa un cebador que contiene una región de poli(dT), el ADNc comienza a formarse por el extremo 3' del ARNm que corresponde a la cola de poliadeninas. Por el contrario, si se usa una mezcla de cebadores de secuencia aleatoria, se obtendrán moléculas de ADNc correspondientes a todas las regiones del ARNm, por lo que esta es la técnica de elección en caso de que se deseen obtener poblaciones de ADNc enriquecidas en los extremos 5' de los ARNm correspondientes.Once a preparation is available purified from mRNA, it contacts a primer that it can be an oligonucleotide that incorporates a region of poly (dT) at its 3 'end or a mixture of primers random sequence in the presence of a reverse transcriptase lacking RNase H activity (for example, transcriptases reversals of the Moloney murine leukemia virus, of the avian myeloblastosis, of the human immunodeficiency virus, of simian immunodeficiency virus, respiratory virus Syncytial, human foamy virus, leukemia virus bovine, human T lymphotrophic virus type I, or transcriptase reverse R2 of Bómbix mori). If a primer containing a poly (dT) region, the cDNA begins to form by the 3 'end of the mRNA corresponding to the polyadenin tail. By on the contrary, if a mixture of sequence primers is used randomized, cDNA molecules corresponding to all mRNA regions, so this is the technique of choice in If you wish to obtain populations of cDNAs enriched in the 5 'ends of the corresponding mRNAs.
La transcriptasa reversa extiende el cebador o los cebadores usando como molde la secuencia del ARNm creando una molécula de ADN de cadena sencilla cuya secuencia viene dictada por la secuencia del ARNm que se ha usado como molde y al cual permanece unida formando un híbrido ARNm/ADNc. La siguiente etapa consiste en la eliminación de la cadena de ARNm del híbrido con la simultánea síntesis de la segunda cadena de ADNc. Para ello, el híbrido ARNm/ADNc se pone en contacto con una RNasa H, con la ADN polimerasa de E. coli y con la ADN ligasa de E. coli. De esta forma, la RNasa H introduce cortes en la cadena de ARNm que son reparados por la ADN polimerasa de E. coli a la vez que, gracias a su actividad exonucleasa 3'-5', elimina los restos de ARNm y los reemplaza por una cadena nueva de ADNc, que son ligados a los fragmentos contiguos por medio de la actividad de la ADN ligasa, formándose así el ADN de doble cadena o ADNc. El ADNc resultante es tratado para eliminar nucleótidos sobrantes en los extremos 5' y 3'. Alternativamente, si la síntesis de la primera cadena de ADNc se lleva a cabo usando una transcriptasa reversa que posee actividad RNasa H, el producto resultante no es un híbrido ADNc/ARNm sino una cadena sencilla de ADNc.Reverse transcriptase extends the primer or primers using the mRNA sequence as a template by creating a single stranded DNA molecule whose sequence is dictated by the mRNA sequence that has been used as a template and to which it remains attached forming a mRNA / cDNA hybrid . The next stage consists in the elimination of the mRNA chain from the hybrid with the simultaneous synthesis of the second cDNA chain. To do this, the hybrid mRNA / cDNA is contacted with an RNase H, with DNA polymerase and E. coli DNA ligase E. coli. In this way, RNase H introduces sections in the mRNA chain that are repaired by E. coli DNA polymerase while, thanks to its 3'-5 'exonuclease activity, it eliminates the mRNA residues and replaces them with a new cDNA chain, which are linked to adjacent fragments through the activity of DNA ligase, thus forming double stranded DNA or cDNA. The resulting cDNA is treated to remove excess nucleotides at the 5 'and 3' ends. Alternatively, if the synthesis of the first cDNA chain is carried out using a reverse transcriptase that possesses RNase H activity, the resulting product is not a cDNA / mRNA hybrid but a simple cDNA chain.
Una vez que se ha obtenido una población de ADNc que representa los ARNm que aparecen en células de organismos que se han estimulado con un agente inmunogénico, es necesario incorporar dicha población en un vector de clonación para así poder amplificar la población de ADNc. El experto en la materia apreciará que existen múltiples alternativas para dicho propósito. Preferiblemente, la población de ADNc una nucleasa y el fragmento Klenow de la ADN polimerasa de E. coli para así eliminar nucleótidos no apareados en los extremos 5' y 3' de la secuencia y obtener moléculas de extremos romos. Posteriormente, las moléculas de ADNc de extremos romos son tratadas con una ADN metilasa (por ejemplo la metilasa EcoRI) en presencia de un donante de grupos metilo (normalmente S-adenosil metionina). A continuación, las moléculas de ADNc de extremos romos y metiladas se pueden ligar a adaptadores o enlazadores (linkers) que contienen sitios diana para la enzima de restricción cuya metilasa se utilizó en la etapa anterior (por ejemplo la endonuclease de restricción EcoRI). Una vez que las moléculas de ADNc se han ligado a los adaptadores, se digieren con la enzima de restricción correspondiente y se ponen en contacto con un vector de clonación linearizado mediante digestión con la misma enzima en presencia de una ADN ligasa (por ejemplo, la ADN ligasa del fago T4, la ADN ligasa de Taq o la ADN ligasa de E. coli) para dar lugar a la recircularización de los vectores conteniendo los insertos de ADNc en su interior. La población de moléculas de ADNc ligadas a los adaptadores puede ser fraccionadas mediante cromatografía de exclusión en gel con el fin de seleccionar aquellos insertos de mayor peso molecular.Once a population of cDNAs representing mRNAs that appear in cells of organisms that have been stimulated with an immunogenic agent has been obtained, it is necessary to incorporate said population into a cloning vector in order to amplify the cDNA population. The person skilled in the art will appreciate that there are multiple alternatives for this purpose. Preferably, the population of cDNA a nuclease and the Klenow fragment of E. coli DNA polymerase so as to eliminate unpaired nucleotides at the 5 'and 3' ends of the sequence and obtain blunt end molecules. Subsequently, blunt-end cDNA molecules are treated with a DNA methylase (for example EcoRI methylase) in the presence of a donor of methyl groups (usually S-adenosyl methionine). Next, blunt and methylated cDNA molecules can be ligated to adapters or linkers containing target sites for the restriction enzyme whose methylase was used in the previous step (for example, the EcoRI restriction endonuclease). Once the cDNA molecules have been ligated to the adapters, they are digested with the corresponding restriction enzyme and contacted with a linearized cloning vector by digestion with the same enzyme in the presence of a DNA ligase (e.g., the Phage T4 ligase DNA, Taq ligase DNA or E. coli DNA ligase) to result in the recircularization of the vectors containing the cDNA inserts therein. The population of cDNA molecules bound to the adapters can be fractionated by gel exclusion chromatography in order to select those inserts of greater molecular weight.
En el tercer paso del método de la invención, una vez que se dispone de una genoteca de ADNc correspondiente a la población de ARNm presente en una célula en una condición determinada, se efectúa una "sustracción" de la genoteca obtenida de células estimuladas (población (+)) con una población de ácidos nucleicos obtenidos de las mismas células en ausencia de dicho estímulo (población (-)) de forma que se eliminen de la genoteca aquellas secuencias cuya expresión ocurre indistintamente en presencia y en ausencia de dicha condición. Para ello la población de ácidos nucleicos usada para efectuar la sustracción puede ser una preparación de ARNm aislada de mejillones controles o, alternativamente, una preparación de ADNc obtenida a partir de dicho mARN.In the third step of the method of the invention, once a cDNA library corresponding to the mRNA population present in a cell in a condition determined, a "subtraction" of the library is made obtained from stimulated cells (population (+)) with a population of nucleic acids obtained from the same cells in the absence of said stimulus (population (-)) so that they are removed from the library those sequences whose expression occurs interchangeably in the presence and absence of said condition. To do this the population of nucleic acids used to effect subtraction it can be an mRNA preparation isolated from control mussels or, alternatively, a cDNA preparation obtained from said mRNA.
En el caso de que se disponga de una población de ADNc de cada una de las poblaciones celulares, se pueden usar métodos ampliamente conocidos para el experto en la materia tales como los métodos descritos en Ausubel, F.M, Current Protocols in Molecular Biology, Unidad 25B.1). En caso de que se disponga de una preparación de ADNc de la muestra en la que se encuentran los genes de expresión diferencial y una preparación de ARNm de la muestra en la que no se expresan dichos genes, también es posible llevar a cabo una substracción mediante métodos conocidos para el experto en la materia como por ejemplo (i) hibridación del ARNm al ADNc desnaturalizado y separación de los híbridos ADNc/ARNm en base a su capacidad de unión a columnas de hidroxiapatito a 68ºC, (ii) hibridación de ARNm a una genoteca de ADNc expresada en fagemidos de cadena sencilla y (iii) hibridación de ARNm a ADNc y separación de los híbridos mediante unión a oligo(dT)-latex.In the event that a population is available cDNA from each of the cell populations, can be used methods widely known to the person skilled in the art such as the methods described in Ausubel, F.M, Current Protocols in Molecular Biology, Unit 25B.1). In case of having a cDNA preparation of the sample in which the genes are found of differential expression and a mRNA preparation of the sample in which these genes are not expressed, it is also possible to lead to perform a subtraction by methods known to the expert in matter such as (i) hybridization of mRNA to cDNA denatured and separation of cDNA / mRNA hybrids based on their Hydroxyapatite column binding capacity at 68 ° C, (ii) mRNA hybridization to a cDNA library expressed in fagemids single chain and (iii) mRNA hybridization to cDNA and separation of hybrids by binding to oligo (dT) -latex.
Otros métodos adecuados para el aislamiento de genes expresados de forma diferencial incluyen el muestreo aleatorio entre dos o mas poblaciones celulares en el que los clones se seleccionan al azar de una biblioteca de ADBc), hibridación diferencial (en el que sondas obtenidas de los ARNms de los dos poblaciones celulares que se comparan y se usan para realizar un screening de una biblioteca de ADNc y para aislar aquellos clones que hibridan a una sonda pero no a otra y presentación diferencial (differential display) en el que cebadores parcialmente aleatorios se usan para amplificar un subconjunto de ARNms que se expresan en una célula determinada, los fragmentos obtenidos se separan en un gen de acrilamida y las bandas obtenidas de distintas muestras se comparan.Other suitable methods for the isolation of differentially expressed genes include sampling randomized between two or more cell populations in which the clones are randomly selected from an ADBc library), differential hybridization (in which probes obtained from mRNAs from the two cell populations that are compared and used to perform a screening of a cDNA library and to isolate those clones that hybridize to one probe but not another and differential display in which primers partially randomized are used to amplify a subset of MRNAs that are expressed in a given cell, the fragments obtained are separated into an acrylamide gene and the bands obtained of different samples are compared.
En una forma preferida de realización, la sustracción se lleva a cabo mediante la técnica de hibridación mediante supresión de substracción (suppression substractive hybridization o SSH). En este método se parte de ADNc de cadena doble de ambas poblaciones, los ADNc se digieren con endonucleasas de restricción que tienen un diana de corte de 4 pares de bases. A continuación, los fragmentos resultantes se ligan a dos conjuntos diferentes de adaptadores (a1/a2 y b1/b2). Los ADNcs se amplifican con cebadores adecuados (a1 o b1) para dar lugar a las poblaciones de ADN A0 y B0. A continuación, se llevan a cabo dos conjuntos de sustracciones (A0-B0 y B0-A0), lo que permite obtener los genes expresados diferencialmente en la población A y los genes expresados diferencialmente en la población B. En cada caso, los ADNcs (+) y (-) se marcan con una pequeña cantidad de [^{32}P] dCTP y el ADNc de la población (-) se marca con biotina durante la síntesis, bien mediante la incorporación de biotina-11-dUTP durante la reacción de PCR, bien mediante biotinilación del producto de PCR o bien mediante el uso de cebadores biotinilados. Los ADNcs (+) y (-) se mezclan en una relación 1:20, se desnaturalizan y se realinean. Los híbridos (-)/(-) y (+)/(-) se eliminan mediante tratamiento con estreptavidina y extracción con fenol. El resultado es un enriquecimiento en secuencias que aparecen con mayor frecuencia en la población (+). Opcionalmente, se pueden llevar a cabo rondas adicionales de sustración. Una vez que las reacciones de sustracción se han completado, los ADNcs se donan en los vectores apropiados. Preferiblemente, la substracción se lleva a cabo usando kits comerciales tales como el kit PCR-Select cDNA subtraction kit (Clontech).In a preferred embodiment, the subtraction is carried out by hybridization technique by subtraction suppression hybridization or SSH). This method starts with cDNA chain double of both populations, cDNAs are digested with endonucleases of restriction that have a cutting target of 4 base pairs. TO then the resulting fragments are linked to two sets different from adapters (a1 / a2 and b1 / b2). CDNAs are amplified with suitable primers (a1 or b1) to give rise to populations of DNA A0 and B0. Next, two sets of subtractions (A0-B0 and B0-A0), what which allows to obtain differentially expressed genes in the population A and genes differentially expressed in the population B. In each case, the cDNAs (+) and (-) are marked with a small amount of [32 P] dCTP and the population cDNA (-) is labeled with biotin during synthesis, either by incorporating biotin-11-dUTP during the reaction PCR, either by biotinylation of the PCR product or through the use of biotinylated primers. The cDNAs (+) and (-) are they mix in a 1:20 ratio, they are denatured and realigned. The hybrids (-) / (-) and (+) / (-) are eliminated by treatment with Streptavidin and phenol extraction. The result is a enrichment in sequences that appear most frequently in the population (+). Optionally, rounds can be carried out Additional subtraction. Once the reactions of subtraction have been completed, cDNAs are donated in vectors appropriate. Preferably, subtraction is carried out using commercial kits such as the PCR-Select cDNA kit subtraction kit (Clontech).
Usando el método de la invención, es posible la identificación de los ADNcs correspondientes a los ARNms que se inducen en hemocitos de mejillón cuando este es tratado con una composición inmunogénica. Así, en otro aspecto, la invención se refiere a un ADNc que se ha aislado usando el método de la invención. Preferiblemente, los ADNc aislados usando el método de la invención codifican para las formas precursores de mitilinas y miticinas.Using the method of the invention, it is possible to identification of the cDNAs corresponding to the mRNAs that are induce in mussel hemocytes when this is treated with a immunogenic composition Thus, in another aspect, the invention is refers to a cDNA that has been isolated using the method of invention. Preferably, cDNAs isolated using the method of the invention encodes for the precursor forms of mitilins and Mythines
En otro aspecto, la invención se relaciona con una construcción génica que comprende, además del ácido nucleico de la invención, elementos que regulan la expresión de dicho gen. Dichos elementos reguladores incluyen promotores y potenciadores. Los promotores se encuentran típicamente posicionados en posición 5' con respecto al sitio de iniciación de la transcripción o traducción. Los potenciadores son capaces de influenciar la expresión de genes cuando se encuentran en posición 5'o 3 'con respecto al ADNc o cuando se encuentran formando parte de un intrón. Secuencias reguladoras incluyen, además de promotores, secuencias que facilitan la traducción, señales de procesamiento para los intrones, codones de terminación, secuencias señales, sitios internos de unión al ribosoma (IRES) y señales de poliadenilación.In another aspect, the invention relates to a gene construct comprising, in addition to the nucleic acid of the invention, elements that regulate the expression of said gene. Such regulatory elements include promoters and enhancers. Promoters are typically positioned in position 5 'with respect to the transcription initiation site or translation. Enhancers are able to influence the gene expression when they are in position 5'or 3 'with regarding the cDNA or when they are part of a intron Regulatory sequences include, in addition to promoters, sequences that facilitate translation, processing signals for introns, termination codons, signal sequences, internal ribosome binding sites (IRES) and signals from polyadenylation
En otro aspecto, la invención se relaciona con un vector de expresión que comprende un ácido nucleico de la invención o con una construcción génica de la invención que está operativamente acoplado con una secuencia que regula la expresión de dicho polinucleótido o de dicha construcción génica. El experto en la materia advertirá que el tipo de vector adecuado para la expresión de los ácidos nucleicos y construcciones génicas de la invención dependerá del organismo en el que se desee expresar el ADNc de la invención.In another aspect, the invention relates to an expression vector comprising a nucleic acid of the invention or with a gene construct of the invention that is operatively coupled with a sequence that regulates the expression of said polynucleotide or said gene construct. The expert in matter will warn that the right type of vector for the nucleic acid expression and gene constructs of the invention will depend on the organism in which it is desired to express the CDNA of the invention.
Así, para la expresión en organismos procariotas, la invención contempla el uso de vectores del tipo de pUC18, pUC19, Bluescript y sus derivados, mp18, mp19, pBR322, pMB9, CoIE1, pCR1, RP4, fagos y vectores "shuttle" tales como pSA3 and pAT28. Para la expresión en levaduras, vectores del tipo de plásmidos de 2 micras, plásmidos de integración, vectores YEP, plásmidos centroméricos y similares. Para la expresión en baculovirus, la invención contempla el uso de vectores de la serie pAC y de la serie pVL. Para la expresión en plantas, la invención contempla el uso de vectores del tipo en los que la expresión de los polinucleótidos de la invención puede estar dirigida por promotores virales del tipo de los promotores 35S y 19S del CaMV, que pueden ser usados opcionalmente en combinación con la secuencia omega de TMV. Alternativamente, se puede usar el promotor del gen que codifica para la subunidad pequeña de RUBISCO, el promotor del gen de la nopalina sintetasa o promotores regulados por estrés térmico y similares.Thus, for expression in organisms prokaryotes, the invention contemplates the use of vectors of the type of pUC18, pUC19, Bluescript and its derivatives, mp18, mp19, pBR322, pMB9, CoIE1, pCR1, RP4, phage and shuttle vectors such as pSA3 and pAT28. For expression in yeasts, vectors of the type of 2 micron plasmids, integration plasmids, YEP vectors, centromeric plasmids and the like. For the expression in baculovirus, the invention contemplates the use of series vectors pAC and pVL series. For expression in plants, the invention contemplates the use of vectors of the type in which the expression of the polynucleotides of the invention may be directed by viral promoters of the CaMV 35S and 19S promoter type, which can be optionally used in combination with the sequence TMV omega. Alternatively, the gene promoter can be used. coding for the small subunit of RUBISCO, the promoter of nopaline synthetase gene or stress regulated promoters Thermal and similar.
En otro aspecto, la invención se relaciona con una célula o un organismo hospedador que contiene una construcción génica de la invención o un vector tal que definido en la invención. Cualquier tipo de organismo hoispedadro conocido para el experto en la materia puede ser usado en la presente invención, tales como una cepa bacteriana (Escherichia coli, Bacillus subtilis y similares), una cepa de levadura (Saccharomyces cerevisiae, Pichia pastoris, Kluyveromyces lactis, Hansenula polymorpha y similares), una planta transgénica (dicotiledoneas o monocotildoneas), una célula de insecto, por ejemplo, baculovirus, una célula de mamífero (células COS, CHO, C127, HeLa y similares) y un transgénico no humano (por ejemplo, un ratón, una vaca, una cabra, un conejo, un cerdo, etc.).In another aspect, the invention relates to a cell or host organism that contains a gene construct of the invention or a vector such as defined in the invention. Any type of hoispedadro organism known to the person skilled in the art can be used in the present invention, such as a bacterial strain ( Escherichia coli , Bacillus subtilis and the like), a yeast strain ( Saccharomyces cerevisiae , Pichia pastoris , Kluyveromyces lactis , Hansenula polymorpha and the like), a transgenic plant (dicotiledoneas or monocotyldoneas), an insect cell, for example, baculovirus, a mammalian cell (COS, CHO, C127, HeLa and the like cells) and a non-human transgenic (for example, a mouse, a cow, a goat, a rabbit, a pig, etc.).
En otro aspecto, la invención se relaciona con un método para la obtención de un polipéptido según la invención que comprende transformar una célula con un vector según la invención y recuperar el polipéptido del interior celular o del medio de cultivo. Las proteínas de la invención se pueden expresar usando una variedad de sistemas de expresión. Normalmente, los péptidos pueden ser recuperados del medio de cultivo puesto que las miticinas y mitilinas contienen una secuencia señal que dirige la expresión de las proteínas al medio extracelular. En la presente invención, se pueden usar células hospedadoras procariotas, más concretamente, bacterias tales como E. coli y B. subtilis transformadas con un plásmido, cósmido o bacteriófago que contiene el gen del precursor de la miticina y/o mitilina, levaduras tales como Saccharomyces y Pichia, transformadas con vectores de expresión en levaduras, células de insecto infectadas con vectores virales capaces de infectar dichas células (baculovirus), células de plantas infectas con vectores virales específicos de plantas (virus del mosaico de la coliflor, virus del mosaico del tabaco) o transformadas con vectores plasmídicos (por ejemplo, el plásmido Ti) o células de mamíferos (por ejemplo células COS, CHO, BHK, 293, 3T3, HeLa, C127, Hep G2 y similares).In another aspect, the invention relates to a method for obtaining a polypeptide according to the invention which comprises transforming a cell with a vector according to the invention and recovering the polypeptide from the cell interior or from the culture medium. The proteins of the invention can be expressed using a variety of expression systems. Normally, the peptides can be recovered from the culture medium since the miticins and mitilins contain a signal sequence that directs the expression of the proteins to the extracellular medium. In the present invention, prokaryotic host cells can be used, more specifically, bacteria such as E. coli and B. subtilis transformed with a plasmid, cosmid or bacteriophage containing the precursor gene of miticin and / or mitilin, yeasts such as Saccharomyces and Pichia, transformed with yeast expression vectors, insect cells infected with viral vectors capable of infecting said cells (baculovirus), infected plant cells with plant-specific viral vectors (cauliflower mosaic virus, mosaic virus tobacco) or transformed with plasmid vectors (for example, the Ti plasmid) or mammalian cells (for example COS, CHO, BHK, 293, 3T3, HeLa, C127, Hep G2 and the like cells).
El experto en la materia advertirá que la invención incluye la expresión de los ADNcs tal y como se han aislado usando el método de la invención pero que también es posible la modificación de dichos ADNcs de forma que la secuencia de la mitilcina y/o mitilina sea ligada a una secuencia heteróloga que sea traducida en el sistema de expresión anteriormente indicado. Dicha secuencia heteróloga puede tener utilidad tanto para la purificación de la proteína como para su identificación si existen anticuerpos específicos para ella. Dichas secuencias heterólogas incluyen aquellas que codifican para la glutation-S-transferasa (GST), la proteína de unión a maltosa (MBP), tioredoxina (Trx), péptido de unión a calmodulina (CBP), hexahistidina, FLAG, c-myc y hemaglutinina. GST, MBP, Trx, CBP y 6-His permiten la purificación de la proteína asociada a ellas usando glutation, maltosa, óxido de fenilarsina, calmodulina o metales inmovilizados, respectivamente. FLAG, c-myc y hemaglutinina permiten la purificación mediante inmunoafinidad usando anticuerpos monoclonales y policlonales específicos que reconocen los distintos epítopos.The subject matter expert will notice that the invention includes the expression of cDNAs as they have been isolated using the method of the invention but that is also possible the modification of said cDNAs so that the sequence of the mitilcin and / or mitilin is linked to a heterologous sequence that be translated into the expression system indicated above. Said heterologous sequence can be useful for both protein purification as for identification if they exist specific antibodies to her. These heterologous sequences include those that code for the glutathione-S-transferase (GST), the maltose binding protein (MBP), thioredoxin (Trx), peptide binding to calmodulin (CBP), hexahistidine, FLAG, c-myc and hemagglutinin. GST, MBP, Trx, CBP and 6-His allow protein purification associated with them using glutathione, maltose, phenylarsine oxide, calmodulin or immobilized metals, respectively. FLAG, c-myc and hemagglutinin allow purification by immunoaffinity using monoclonal antibodies and specific polyclonal that recognize the different epitopes.
Puesto que las miticinas y mitilinas se sintetizan en forma de precursor que contiene, además de la forma madura, un primer péptido en la región N-terminal y un segundo péptido en la región C-terminal que deben ser eliminados durante su maduración intracelular, el experto en la materia advertirá que la secuencia heteróloga para su localización/purificación debe estar situada en la secuencia de la mitilina/miticina adyacentemente a la forma madura del polipéptido, bien asociado a través de su extremo N-terminal como asociado a través de su extremo C-terminal. Alternativamente, las secuencias que codifican para las mitilinas y/o miticinas y las secuencias que codifican para las proteínas o péptidos heterólogos pueden estar separadas por una secuencia heteróloga adicional que codifica para un sitio de reconocimiento para una proteasa, de forma que la secuencia heteróloga pueda ser eliminada tras la purificación de la proteína de fusión.Since the miticins and mitilins are synthesized as a precursor that contains, in addition to the form mature, a first peptide in the N-terminal region and a second peptide in the C-terminal region that they must be eliminated during their intracellular maturation, the expert in the matter will notice that the heterologous sequence for its location / purification must be located in the sequence of the mitilin / miticin adjacent to the mature form of the polypeptide, well associated through its N-terminal end as an associate through its C-terminal end. Alternatively, the sequences coding for the mitilins and / or miticins and the sequences that code for proteins or heterologous peptides can be separated by a sequence additional heterologous coding for a recognition site for a protease, so that the heterologous sequence can be removed after purification of the fusion protein.
Usando el método objeto de la invención, los autores han puesto de manifiesto que, de forma sorprendente, el repertorio de péptidos con actividad antibacteriana presentes en hemolinfa de mejillón es muy superior a lo que se conocía hasta la fecha, puesto que los autores han identificado la existencia de 16 nuevas formas de miticinas y 3 nuevas formas de mitilinas, todas ellas distintas a las descritas previamente. Así, en otro aspecto, la invención se relaciona con un polipéptido seleccionado del grupo de:Using the method object of the invention, the authors have revealed that, surprisingly, the repertoire of peptides with antibacterial activity present in mussel hemolymph is far superior to what was known until the date, since the authors have identified the existence of 16 new forms of miticins and 3 new forms of mitilins, all they are different from those previously described. So, in another aspect, the invention relates to a polypeptide selected from the group from:
- (i) (i)
- un polipéptido que comprende una de las secuencias definidas en SEQ ID NO:1 a 19.a polypeptide comprising one of the sequences defined in SEQ ID NO: 1 to 19.
- (ii) (ii)
- una variante del polipéptido según (i) que muestra una identidad en la secuencia aminoacídica de al menos 90% con los polipéptidos definidos en las secuencias de 1 a 16, ya variant of the polypeptide according to (i) which shows an identity in the amino acid sequence of at least 90% with the polypeptides defined in sequences 1 to 16, and
- (iii) (iii)
- una variante del polipéptido según (ii) que muestra una identidad en la secuencia aminoacídica de al menos 66% con los polipéptidos definidos en las secuencias 17 a 19.a variant of the polypeptide according to (ii) showing an identity in the amino acid sequence of at least 66% with the polypeptides defined in sequences 17 to 19.
Según se entiende en el contexto de la presente invención, una variante de un polipéptido según se define en las secuencias de SEQ ID NO:1 a 19 es todo aquel péptido que se puede obtener a partir de la secuencias 1 a 16 mediante sustitución, inserción o deleción de uno o más amino ácidos. En el caso de las variantes por substitución, la substituciones son preferentemente substituciones conservativas, esto es, los aminoácidos se sustituyen por otros con características similares en cuanto a la propiedades de su cadena lateral. Así, substituciones conservativas incluyen substituciones dentro de los grupos de amino ácidos según la tabla 1.As understood in the context of this invention, a variant of a polypeptide as defined in the sequences of SEQ ID NO: 1 to 19 is all that peptide that can be obtain from sequences 1 to 16 by substitution, insertion or deletion of one or more amino acids. In the case of variants by substitution, the substitutions are preferably conservative substitutions, that is, amino acids are replaced by others with similar characteristics in terms of properties of its side chain. Thus, conservative substitutions include substitutions within the amino acid groups according to table 1.
Adicionalmente, uno ó más de los amino ácidos de las variantes de la invención pueden estar sustituidos por amino ácidos no convencionales naturales o sintéticos como por ejemplo, beta-amino ácidos, ácido 2-aminoadípico, alpha-asparagina, ácido 2-aminobutanoico, ácido 2-aminiocáprico, alpha-glutamina, alpha-metilalanina, ácido 2-aminopimélico, ácido gamma-amino-beta-hidroxibenzenopentanoico, ácido 2-aminosubérico, 2-carboxiazetidina, beta-alanina, ácido beta-aspártico, ácido 3,6 diaminohexanoico, ácido butanoico, ácido 4-amino 4-amino-3-hidroxi butanoico, ácido gamma-amino-beta-hidroxiciclohexanepentanoico, N5-aminocarbonilornitina, 3-sulfoalanina, ácido 2,4 diaminobutanoico, ácido diaminopimélico, ácido 2,3 diaminopropanoico, ácido 2,7 diaminosubérico, S-etiltiocisteina, ácido gamma-glutámico, ácido gamma-carboxiglutámico, ácid pirogultámico, homarginina, homocisteina, homohistinba, homoserina, ácido 2-hidroxiisovalérico, ácid 2-hidroxipentanoico, 5-hidroxilisina, 4-hidroxipriolina, 2-carboxioctahidroindol, 3-carboxiisoquinolina, isovalina, ácido 2-hidroxipropanoico, ácido mercaptoacético, ácid mercaptobutanoico, 4-metil-3-hidroxiprolina, ácido mercaptopropanoico, norlceucina, nortirosina, norvalina, ornitina, penicilamina, 2-fenilglicina, 2-carboxipiperidina, sarcosina, 1-amino-1 -carboxiciclopentano, statin, 3-tienilalanina, epsilon-N-trimetilisina, 3-tiazolialanina, ácido alpha-amino-2,4-dioxopirimidinapropanoico.Additionally, one or more of the amino acids of variants of the invention may be substituted by amino unconventional natural or synthetic acids such as beta-amino acids, acid 2-aminoadipic, alpha-asparagine, 2-aminobutanoic acid, acid 2-aminiocapric, alpha-glutamine, alpha-methylalanine acid 2-aminopimelic acid gamma-amino-beta-hydroxybenzenopentanoic acid, 2-aminosuberic acid, 2-carboxiazetidine, beta-alanine, beta-aspartic acid, 3.6 diaminohexanoic acid, butanoic acid, 4-amino acid 4-amino-3-hydroxy butanoic acid gamma-amino-beta-hydroxycyclohexanepentanoic acid, N5-aminocarbonylornitine, 3-sulfoalanine, 2,4-diaminobutanoic acid, acid diaminopimelic, 2,3 diaminopropanoic acid, 2,7 diaminosuberic, S-ethylthiocysteine, acid gamma-glutamic acid gamma-carboxyglutamic pyrogultamic acid, homarginine, homocysteine, homohistinba, homoserine, acid 2-hydroxyisovaleric acid 2-hydroxypentanoic acid, 5-hydroxylysine, 4-hydroxyproline, 2-carboxyioctahydroindole, 3-carboxyisoquinoline, isovaline, acid 2-hydroxypropanoic acid, mercaptoacetic acid, acid mercaptobutanoic, 4-methyl-3-hydroxyproline, mercaptopropanoic acid, norlceucine, nortyrosine, norvaline, Ornithine, penicillamine, 2-phenylglycine, 2-carboxypiperidine, sarcosine, 1-amino-1-carboxycyclopentane, statin, 3-thienylalanine, epsilon-N-trimethylisin, 3-thiazolialanine acid alpha-amino-2,4-dioxopyrimidinepropanoic.
Adicionalmente, la invención contempla variantes de los péptidos de la invención en los que uno o mas aminoácidos han sufrido modificaciones en su cadena lateral. Ejemplos de modificaciones de cadena lateral contempladas en la presente invención incluyen modificaciones de grupos amino tales como alquilación, amidinación, acilación, carbomoilación, trinitrobencilación, piridoxilación, modificaciones del grupo guanidino de los restos de arginina consistentes en la formación de condensados heterocíclicos; modificaciones de los grupos carboxilo mediante amidación, modificaciones de tirosinas mediante metoxilación, modificación del animllo imidazólico de la histidina mediante alquilación o N-carboxietilación, modificaciones de la prolina mediante hidrixliación en posición 4. Alternativamente, la invención contempla variantes de los péptidos de la invención mediante glicosilación, es decir, la adición de grupos de glicano bien en la cadena lateral serine y/o treonina (O-glicosliación) o en la cadena lateral asparagina y/o glutamina (N-glicosilación). Los glicanos que pueden incorporarse a los polipéptidos de la invención incluyen un número variable de unidades glucidicas (mono-, di-, tri, tetrasacáridos y sucesivos). Los monosacáridos que forman en glicano incluyen D-alosa, D-altrosa, D-glucosa, D-manosa, D-gulosa, D-idosa, D-galactosa, D-talosa, D-galactosamina, D-glucosamina, D-N-acetylgucosamina, D-N-acetylgalatosamina, D-fucosa o D-arabinosa.Additionally, the invention contemplates variants of the peptides of the invention in which one or more amino acids They have undergone modifications in their side chain. Examples of side chain modifications contemplated herein invention include modifications of amino groups such as alkylation, amidination, acylation, carbomoylation, trinitrobenzylation, pyridoxylation, group modifications guanidino of arginine residues consisting of the formation of heterocyclic condensates; modifications of carboxyl groups by amidation, tyrosine modifications by methoxylation, modification of the imidazole histidine pellet by alkylation or N-carboxyethylation, Proline modifications by hydrolyzing in position 4. Alternatively, the invention contemplates variants of the peptides. of the invention by glycosylation, that is, the addition of glycan groups well in the serine and / or threonine side chain (O-glycosylation) or asparagine side chain and / or glutamine (N-glycosylation). The glycans that may be incorporated into the polypeptides of the invention include a variable number of glycidic units (mono-, di-, tri, tetrasaccharides and successive). The monosaccharides that form in glycan include D-alosa, D-altrosa, D-glucose, D-mannose, D-gulose, D-idosa, D-galactose, D-talose, D-galactosamine, D-glucosamine, D-N-acetylgucosamine, D-N-acetylgalatosamine, D-fucose or D-arabinose.
Alternativamente, la invención contempla variantes de los polipéptidos de la invención en los que se incluyen los estereoisómeros D de al menos uno de los aminoácidos que constituyen la cadena peptídico para dar así lugar a los isómeros retro-inverersos.Alternatively, the invention contemplates variants of the polypeptides of the invention in which include D stereoisomers of at least one of the amino acids which constitute the peptide chain to give rise to retro-invented isomers.
En otra forma de realización, la invención contempla peptidomiméticos de los polipéptidos de la invención, es decir, variantes en las que uno o más de los enlaces peptídicos ha sido reemplazado por un tipo alternativo de enlace covalente. Dichos peptidomiméticos se caracterizan por mostrar una mayor estabilidad al ser más resistentes a proteasas. Modificaciones del esqueleto peptídico incluyen la sustitución o la inserción en los elementos del enlace peptídico (-NH-, -CH-, -CO-) de grupos tales como -O-, -S-, -CH2 en lugar de -NH-, -N-, -C-alquil p -BH- en lugar de -CHR y -CS-, -CH2-, -SOn-, -P=O(OH)- o -B(OH)- en lugar de -CO-. Adicionalmente, es posible aumentar la estabilidad de los péptidos de la invención usando grupos que bloquen el extremo N-terminal tales como t-butiloxicarbonil, acetil, succinil, metoxisuccinil, suberil, adipil, dansil, benciloxicarbonil, fluorenilmetoxicarbonil, metoxiadipil, metoxiadipil, metoxisuberil y 2,3-dinitrofenil. Alternativa p simultáneamente, es posible modificar el extremo C-terminal de los péptidos mediante amidación.In another embodiment, the invention contemplates peptidomimetics of the polypeptides of the invention, is that is, variants in which one or more of the peptide bonds has been replaced by an alternative type of covalent bond. Sayings peptidomimetics are characterized by showing greater stability being more resistant to proteases. Skeleton Modifications peptide include replacement or insertion into the elements of the peptide bond (-NH-, -CH-, -CO-) of groups such as -O-, -S-, -CH2 instead of -NH-, -N-, -C-alkyl p -BH- in instead of -CHR and -CS-, -CH2-, -SOn-, -P = O (OH) - or -B (OH) - instead of -CO-. Additionally, it is possible increase the stability of the peptides of the invention using groups that block the N-terminal end such as t-butyloxycarbonyl, acetyl, succinyl, methoxysuccinil, suberil, adipil, dansyl, benzyloxycarbonyl, fluorenylmethoxycarbonyl, methoxyadipil, methoxyadipil, methoxyseril and 2,3-dinitrophenyl. Alternative p simultaneously, it is possible to modify the C-terminal end of the peptides by amidation.
La determinación del grado de identidad entre las variantes y los polipéptidos definidos en las secuencias 1 a 19 se lleva a cabo usando métodos y algoritmos informáticos ampliamente conocidos para el experto en la materia. Preferentemente, la identidad entre dos secuencias de amino ácidos se determina usando el algoritmo BLASTP (BLASTManual, Altschul, S., et al, NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al., J. Mol. Biol. 21 5: 403-410 (1990). Preferiblemente, los polipéptidos objeto de la invención muestran una identidad de secuencia con los polipéptidos definidos en las secuencias de SEQ ID NO:1 a 19 de al menos 50%, al menos 60%, al menos 70%, al menos 80%, al menos 90%, al menos 91%, al menos 92%, al menos 93%, al menos 94%, al menos 95%, al menos 96%, al menos 97%, al menos 98% o al menos 99%.The determination of the degree of identity between the variants and the polypeptides defined in sequences 1 to 19 is carried out using computer methods and algorithms widely known to those skilled in the art. Preferably, the identity between two amino acid sequences is determined using the BLASTP algorithm (BLASTM Annual, Altschul, S., et al , NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al ., J. Mol. Biol 21 5: 403-410 (1990) Preferably, the polypeptides object of the invention show a sequence identity with the polypeptides defined in the sequences of SEQ ID NO: 1 to 19 of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%.
En otro aspecto, la invención se relaciona con un método para la obtención de un polipéptido de la invención que comprende transformar una célula con un vector según de la invención y recuperar el polipéptido del interior celular o del medio de cultivo. Células y métodos adecuados para la expresión de los polipéptidos de la invención son aquellas que se han definido anteriormente.In another aspect, the invention relates to a method for obtaining a polypeptide of the invention that comprises transforming a cell with a vector according to the invention and recover the polypeptide from inside the cell or from the culture medium. Cells and methods suitable for the expression of the polypeptides of the invention are those that have been defined previously.
En otro aspecto, la invención se relaciona con un método para la obtención de un polipéptido de la invención que comprende inyectar intramuscularmente en un mejillón un agente inmunogénico y recuperar posteriormente de la hemolinfa uno o varios de los polipéptidos. En una forma preferida de realización, el agente inmunogénico que se usa está seleccionado del grupo de un cóctel de bacterias muertas, poli I:C y una mezcla de ambos.In another aspect, the invention relates to a method for obtaining a polypeptide of the invention that comprises intramuscularly injecting an agent into a mussel immunogenic and subsequently recover from the hemolymph one or several of the polypeptides. In a preferred embodiment, the immunogenic agent that is used is selected from the group of a Dead bacteria cocktail, poly I: C and a mixture of both.
En otro aspecto la invención se relaciona con un anticuerpo que reconoce específicamente al menos un polipéptido de la invención. Anticuerpos contemplados en el contexto de la presente invención incluyen antisueros policlonales, moléculas de IgG purificadas, sobrenadantes o líquido ascítico que contiene anticuerpos monoclonales, fragmentos Fv, Fab, Fab' y F(ab')_{2}, ScFvdiabodies, triabodies, tetrabodies anticuerpos humanizados.In another aspect the invention relates to a antibody that specifically recognizes at least one polypeptide of the invention. Antibodies contemplated in the context of the Present invention include polyclonal antisera, molecules of Purified IgG, supernatants or ascites fluid containing monoclonal antibodies, Fv, Fab, Fab 'and fragments F (ab ') 2, ScFvdiabodies, triabodies, tetrabodies humanized antibodies
En otro aspecto, la invención se relaciona con composiciones farmaceúticas que comprenden al menos uno de los polipéptidos de la invención o su forma madura acompañado de un excipiente farmacéuticamente aceptable. Para uso en medicina, los compuestos y combinaciones de compuestos de la invención pueden ser formulados conjuntamente con un excipiente que es aceptable desde el punto de vista farmacéutico. Excipientes preferidos para su uso en la presente invención incluyen azúcares, almidones, celulosas, gomas y proteínas. En una realización particular, la composición farmacéutica de la invención se formulará en una forma farmacéutica de administración sólida (p.ej., comprimidos, cápsulas, grageas, gránulos, supositorios, etc.) o líquida (p.ej., soluciones, suspensiones, emulsiones, etc.). En otra realización particular, las composiciones farmacéuticas de la invención pueden ser administradas por cualquier ruta, incluyendo, sin ser limitante, oral, intravenosa, intramuscular, intrarterial, intramedular, intratecal, intraventricular, transdérmica, subcutana, intraperitoneal, intranasal, entérica, tópica, sublingual o rectal. Una revisión de las distintas formas de administración de principios activos, de los excipientes a utilizar y de sus procedimientos de fabricación puede encontrarse en el Tratado de Farmacia Galénica, C. Faulí i Trillo, Luzán 5, S.A. de Ediciones, 1993.In another aspect, the invention relates to pharmaceutical compositions comprising at least one of the polypeptides of the invention or its mature form accompanied by a pharmaceutically acceptable excipient. For use in medicine, compounds and combinations of compounds of the invention may be formulated together with an excipient that is acceptable from The pharmaceutical point of view. Preferred excipients for use in the present invention include sugars, starches, celluloses, Gums and proteins. In a particular embodiment, the composition Pharmaceutical of the invention will be formulated in a pharmaceutical form solid administration (eg, tablets, capsules, dragees, granules, suppositories, etc.) or liquid (e.g., solutions, suspensions, emulsions, etc.). In another particular embodiment, the pharmaceutical compositions of the invention may be managed by any route, including, but not limited to, oral, intravenous, intramuscular, intrarterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteric, topical, sublingual or rectal. A review of the different forms of administration of active ingredients, of the excipients to be used and their manufacturing procedures can be found in the Treaty of Farmacia Galénica, C. Faulí i Trillo, Luzán 5, S.A. of Editions, 1993
En otra realización particular, cuando el principio activo comprende un polinucleótido de la invención, la composición farmacéutica de la invención puede formularse en forma de una composición destinada para su empleo en terapia génica; a modo ilustrativo, no limitativo, en este caso, la composición farmacéutica de la invención puede contener un vector, viral o no viral, que comprende un polinucleótido de la invención o una construcción génica de la invención. A modo ilustrativo, no limitativo, dichos vectores pueden ser vectores virales, por ejemplo, basados en retrovirus, adenovirus, etc., o no virales tales como los complejos ADN-liposoma, ADN-polímero, ADN-polímero-liposoma, etc. [véase "Nonviral Vectors for Gene Therapy", editado por Huang, Hung y Wagner, Academic Press (1999)]. Dichos vectores, que contienen un polinucleótido o una construcción génica de la invención pueden ser administrados directamente al cuerpo humano o animal por métodos convencionales.In another particular embodiment, when the active ingredient comprises a polynucleotide of the invention, the Pharmaceutical composition of the invention can be formulated in the form of a composition intended for use in gene therapy; to illustrative, not limiting, in this case, the composition Pharmaceutical of the invention may contain a vector, viral or not. viral, comprising a polynucleotide of the invention or a gene construct of the invention. By way of illustration, no limiting, said vectors may be viral vectors, by for example, based on retroviruses, adenoviruses, etc., or non-viral such as DNA-liposome complexes, DNA-polymer, DNA-polymer-liposome, etc. [see "Nonviral Vectors for Gene Therapy", edited by Huang, Hung and Wagner, Academic Press (1999)]. These vectors, which contain a polynucleotide or a gene construct of the invention can be administered directly to the human or animal body by methods conventional.
Alternativamente, dichos vectores pueden ser utilizados para transformar, transfectar o infectar células, por ejemplo, células de mamíferos, incluido el hombre, ex vivo, y, posteriormente implantarlas en el cuerpo humano o animal para obtener el efecto terapéutico deseado. Para su administración al cuerpo humano o animal dichas células se formularán en un medio adecuado que no afecte adversamente a la viabilidad de dichas células.Alternatively, said vectors may be used to transform, transfect or infect cells, by example, mammalian cells, including man, ex vivo, and, subsequently implant them in the human or animal body to Obtain the desired therapeutic effect. For administration to human or animal body said cells will be formulated in a medium adequate that does not adversely affect the viability of said cells.
En otro aspecto, la invención se relaciona con el uso de un polipéptido de la invención o de un polinucleótido de la invención para la preparación de un medicamento destinado a tratar una enfermedad infecciosa. Aunque los polipéptidos de la invención se han demostrado útiles para el tratamiento de infecciones bacterianas mediadas por E. coli y de infecciones víricas mediadas por el virus que cause la necrosis pancreática infecciosa, la presente invención contempla el uso de los polipéptidos y polinucleótidos de la invención para el tratamiento de todo tipo de enfermedades infecciosas de origen bacteriano, vírico, fúngico o parasitario. Así, la invención contempla el uso de los polipéptidos y polinucleótidos para el tratamiento de enfermedades causadas por bacterias tales como menigitus, carbunco, apenditis, disenteria, bacteremia, peste, lepra, borreliosis, botulismo, bronquitis, brucelosis, peste bubónica, cerebritos, cervicitis, cólera, conjuntivitis, cistitis, dermatitis, diarrea, encefalitis, endocarditis, fiebre entérica, enteritis, enterocolitis, epididimitos, erisipela, tuberculosis, forúnculos, gangrena, gastritis, gastroenteritis, otitis, glomerulonefritis, impetigo, laringitis, tetano, mastitis, meningitis, meningoencefalitis, listeriosis, nocardiosis, oftalmitis, osteomielitis, otitis media, pancreatitis, parotiditis, neumonía, listeremia, prostatis, sepsis puerperal, abscesos cutaneos, pielonefritis, fiebre reumática, rinitis, romboencefalitis, salmonelosis, escarlatina, sepsis, shigelosis, sífilis, peritonitis, sinusitis, traqueobronquitis, fiebres de Malta, tifus, fiebres tifoideas, ulcera y uretritis.In another aspect, the invention relates to the use of a polypeptide of the invention or a polynucleotide of the invention for the preparation of a medicament for treating an infectious disease. Although the polypeptides of the invention have proven useful for the treatment of bacterial infections mediated by E. coli and viral infections mediated by the virus causing infectious pancreatic necrosis, the present invention contemplates the use of the polypeptides and polynucleotides of the invention. for the treatment of all types of infectious diseases of bacterial, viral, fungal or parasitic origin. Thus, the invention contemplates the use of polypeptides and polynucleotides for the treatment of diseases caused by bacteria such as menigitus, anthrax, appenditis, dysentery, bacteremia, plague, leprosy, borreliosis, botulism, bronchitis, brucellosis, bubonic plague, cerebrites, cervicitis , cholera, conjunctivitis, cystitis, dermatitis, diarrhea, encephalitis, endocarditis, enteric fever, enteritis, enterocolitis, epididymits, erysipelas, tuberculosis, boils, gangrene, gastritis, gastroenteritis, otitis, glomerulonephritis, impetigo, laryngitis, tetanus, mastitis, meningitis, meningoencephalitis, listeriosis, nocardiosis, ophthalmitis, osteomyelitis, otitis media, pancreatitis, mumps, pneumonia, listeremia, prostatis, puerperal sepsis, cutaneous abscesses, pyelonephritis, rheumatic fever, rhinitis, rhomboencephalitis, salmonellosis, scarlet fever, sepsis, syphilis, perisitis sinusitis, tracheobronchitis, Malta fevers, typhoid, typhoid fevers, ulcer and urethritis
En otra forma de realización, la invención contempla el uso de los polipéptidos y polinucleótidos para el tratamiento de enfermedades causadas por hongos tales como aspergilosis, blastomicosis, candidiasis, cromoblastomicosis, criptococsis, dermatomicosis, dermatofitosis, histoplasmosis, mucormicosis, micetoma, queratitis micótica, oculomicosis, otomicosis, paracoccidiomicosis, rinosporidiosis, esporotricosis, tiña, dermatitis seborreica, eccema numular, liquen simple, pitiriasis rosada de Gubert y zigomicosis.In another embodiment, the invention contemplates the use of polypeptides and polynucleotides for the treatment of fungal diseases such as aspergillosis, blastomycosis, candidiasis, chromoblastomycosis, cryptococcosis, dermatomycosis, dermatophytosis, histoplasmosis, mucormycosis, mycetoma, fungal keratitis, oculomycosis, otomycosis, paracoccidiomycosis, rhinosporidiosis, sporotrichosis, ringworm, seborrheic dermatitis, number eczema, lichen simplex, Pityriasis rosea of Gubert and zygomycosis.
En otra forma de realización, la invención contempla el uso de los polipéptidos y polinucleótidos para el tratamiento de enfermedades causadas por protozoos tales como tripanosomiasis africana, tripanosomiais americana, amebiasis, disenteria amebiana, enfermedad de Chagas, coccidiosis, criptoporidiosis, leishmaniasis cutanea, leishmaniais visceral, ciclosporiasis, entamebiasis, giardiasis, isosporiasis, malaria, microsporidosis, neumocistosis, sarcosporidiosis, enfermedad del sueño, toxoplasmosis, tricomoniasis, tripanosomiasis.In another embodiment, the invention contemplates the use of polypeptides and polynucleotides for the treatment of diseases caused by protozoa such as African trypanosomiasis, American trypanosomiais, amebiasis, amoebic dysentery, Chagas disease, coccidiosis, cryptoporidiosis, cutaneous leishmaniasis, visceral leishmaniais, cyclosporiasis, entamebiasis, giardiasis, isosporiasis, malaria, microsporidosis, pneumocystosis, sarcosporidiosis, disease of the sleep, toxoplasmosis, trichomoniasis, trypanosomiasis.
En otra forma de realización, la invención contempla el uso de los polipéptidos y polinucleótidos para el tratamiento de enfermedades de origen viral tales como SIDA, enfermedad respiratoria aguda, fiebre hemorrágica de Ebola, fiebre hemorrágica de Lassa, fiebre hemorrágica de Marburgo, fiebre hemorrágica argentina, fiebre hemorrágica boliviana, encefalomielitis, gripe, gripe aviaria, fiebre aftosa, herpes genital, herpes cutaneo, herpes zoster, herpangina, varicela, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, enfermedad de Hodgkin, linfoma de Burkitt, sarcoma de Kaposi, viruela, síndrome respiratorio severo y agudo (SARS), paperas. Dengue, fiebre amarilla, encefalomielitis, sarampión, resfriado común, conjuntivitis, gastroenteritis, cáncer cervical, linfoma, orquitis, rubéola, encefalitis de San Luis, mononucleosis, menigoencefalitis, laringitis, Molluscum contagiosum, peste porcina, infeccione spor hantavirus, coriomeningitis linfocítica, verruga, polimielitis, rabia, enfermedad de Gerstmann-Straussler-Scheinker, enfermedad de Boma, difteria, encefalitis causada por Arbovirus, fiebre de Mayaro, eritema infeccioso, encefalitis LaCrosse, polimielitis, encefalitis del lóbulo temporal, leucoendefalitis multifocal progresiva, enfermedad respiratoria febril aguda, enfermedad de Bornholm y parotitis.In another embodiment, the invention contemplates the use of polypeptides and polynucleotides for the treatment of diseases of viral origin such as AIDS, acute respiratory disease, Ebola hemorrhagic fever, fever hemorrhagic fever, Marburg hemorrhagic fever, fever Argentine hemorrhagic fever, Bolivian hemorrhagic fever, Encephalomyelitis, flu, bird flu, foot and mouth disease, herpes genital, herpes cutaneous, herpes zoster, herpangina, chickenpox, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, Hodgkin's disease, Burkitt lymphoma, Kaposi's sarcoma, smallpox, severe and acute respiratory syndrome (SARS), mumps. Dengue, yellow fever, encephalomyelitis, measles, cold common, conjunctivitis, gastroenteritis, cervical cancer, lymphoma, orchitis, rubella, St. Louis encephalitis, mononucleosis, menigoencephalitis, laryngitis, Molluscum contagiosum, swine fever, infect spor hantavirus, lymphocytic choriomeningitis, wart, Polio, rabies disease Gerstmann-Straussler-Scheinker, Boma disease, diphtheria, arbovirus encephalitis, Mayaro fever, infectious erythema, LaCrosse encephalitis, Polio, temporal lobe encephalitis, leukoendephalitis progressive multifocal, acute febrile respiratory disease, Bornholm disease and parotitis.
La invención se ilustra a continuación mediante los siguientes ejemplos que han de ser considerados como meramente ilustrativos y no limitativos del alcance de la invención.The invention is illustrated below by the following examples to be considered as merely illustrative and not limiting the scope of the invention.
Para el estudio se partió de un stock de mejillones de las Rías Bajas gallegas, a los que se estimuló mediante inyección intramuscular con un cóctel de bacterias muertas o polyI:C. A las 24 horas se les extrajo la hemolinfa, se separaron por centrifugación suero y células (hemocitos), y se extrajo RNA del pellet de hemocitos siguiendo los protocolos habituales para esta técnica. Con un pool de hemolinfa de 50 mejillones por cada tratamiento, se llevaron a cabo dos sustracciones de librerías génicas, entre animales tratados con bacteria o poly I:C y los animales control (a los que se les había inyectado agua de mar). Para ello, se sintetizó el cDNA de cada pool de hemocitos (muestras de infección con bacteria, muestras de infección con poly I:C y muestras control, sin infección) mediante el kit de síntesis de cDNA de Clontech SMART PCR cDNA Síntesis kit, que permite la amplificación completa de eDNA a partir de transcriptos de mRNA. Posteriormente se realizó una sustracción de librerías génicas mediante la técnica SSH llevada a cabo con el kit PCR-Select eDNA Subtraction kit (Clontech). Mediante esta técnica se identificaron genes expresados o sobre expresados en tejidos infectados, en comparación con los controles no infectados. Las secuencias obtenidas expresadas diferencialmente se amplificaron mediante PCR , se ligaron en vectores de ligación con el kit TOPO TA cloning (Invitrogen) y se trasformaron en bacterias E. coli competentes. Las colonias seleccionadas a partir de la transformación se amplificaron por PCR y se secuenciaron en secuneciador automático DNA sequencer ABI 3730. Las secuencias obtenidas se analizaron y compararon con genes conocidos, con ayuda del BLAST. Una vez caracterizadas las secuencias, se realizaron estudios de expresión mediante PCR en tiempo real " Real Time SYBER Green PCR Assay".For the study, a stock of mussels from the Galician Rias, was started, which was stimulated by intramuscular injection with a cocktail of dead bacteria or polyI: C. After 24 hours the hemolymph was removed, serum and cells (hemocytes) were centrifuged, and RNA was extracted from the hemocyte pellet following the usual protocols for this technique. With a hemolymph pool of 50 mussels for each treatment, two gene library subtractions were carried out, between animals treated with bacteria or poly I: C and the control animals (to which seawater had been injected). For this, the cDNA of each pool of hemocytes (bacterial infection samples, poly I: C infection samples and control samples, without infection) was synthesized by the Clontech SMART PCR cDNA Synthesis kit cDNA synthesis kit, which It allows the complete amplification of eDNA from mRNA transcripts. Subsequently a gene library subtraction was performed using the SSH technique carried out with the PCR-Select eDNA Subtraction kit (Clontech). Through this technique, genes expressed or over-expressed in infected tissues were identified, compared to uninfected controls. Differentially expressed sequences obtained were amplified by PCR, ligated into ligation vectors with the TOPO TA cloning kit (Invitrogen) and transformed into competent E. coli bacteria. The colonies selected from the transformation were amplified by PCR and sequenced in automatic sequencer DNA sequencer ABI 3730. The sequences obtained were analyzed and compared with known genes, with the help of BLAST. Once the sequences were characterized, expression studies were performed by real-time PCR "Real Time SYBER Green PCR Assay".
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Se ha determinado la actividad antimicrobiana de una combinación de miticinas y miticinas contra el virus de la necrosis pancreática infecciosa (IPNV, birnavirus) y la bacteria Escherichia Coli mediante las técnicas usuales de diluciones seriadas resultando en una disminución significativa del título viral y del crecimiento bacteriano.The antimicrobial activity of a combination of miticins and miticins against infectious pancreatic necrosis virus (IPNV, birnavirus) and Escherichia Coli bacteria has been determined by the usual serial dilution techniques resulting in a significant decrease in viral titer and bacterial growth. .
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<110> CONSEJO SUPERIOR DE INVESTIGACIONES CIENTÍFICAS<110> SUPERIOR INVESTIGATION COUNCIL SCIENTISTS
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<120> MÉTODO PARA LA IDENTIFICACIÓN DE POLIPÉPTIDOS ANTIBACTERIANOS Y ANTIVIRALES OBTENIDOS DE MYTILUS EDULIS <120> METHOD FOR THE IDENTIFICATION OF ANTIBACTERIAL AND ANTIVIRAL POLYPEPTIDES OBTAINED FROM MYTILUS EDULIS
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<170> PatentIn version 3.4<170> PatentIn version 3.4
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<213> Mitylus edulis <213> Mitylus edulis
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<213> Mytilus edulis <213> Mytilus edulis
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<212> PRT<212> PRT
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Claims (17)
- (i) (i)
- poner en contacto un mejillón un agente inmunogénico,put an agent in contact with a mussel immunogenic,
- (ii) (ii)
- preparar librerías de ADNc de mejillones tratados con el agente inmunogénicoprepare cDNA libraries of treated mussels with the immunogenic agent
- (iii) (iii)
- efectuar una sustracción de la librería obtenida de los mejillones tratados con una preparación de ácidos nucleicos de mejillones controles ysubtract the library obtained from mussels treated with a nucleic acid preparation of mussels controls and
- (iv) (iv)
- aislar los ADNcs específicos de mejillones tratados con el agente inmunogénico.isolate specific cDNAs from treated mussels with the immunogenic agent.
- (i) (i)
- un polipéptido que comprende una de las secuencias definidas en SEQ ID NO:1 a 19,a polypeptide comprising one of the sequences defined in SEQ ID NO: 1 to 19,
- (ii) (ii)
- una variante del polipéptido según (i) que muestra una identidad en la secuencia aminoacídica de al menos 90% con los polipéptidos definidos en las secuencias de 1 a 16, ya variant of the polypeptide according to (i) which shows an identity in the amino acid sequence of at least 90% with the polypeptides defined in sequences 1 to 16, and
- (iii) (iii)
- una variante del polipéptido según (ii) que muestra una identidad en la secuencia aminoacídica de al menos 66% con los polipéptidos definidos en las secuencias 17 a 19.a variant of the polypeptide according to (ii) showing an identity in the amino acid sequence of at least 66% with the polypeptides defined in sequences 17 to 19.
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ES2763349A1 (en) * | 2018-11-28 | 2020-05-28 | Consejo Superior Investigacion | Mythicin peptide and its use in cell regeneration (Machine-translation by Google Translate, not legally binding) |
CN111606988A (en) * | 2020-06-05 | 2020-09-01 | 深圳大学 | Pernalins, signal peptide of antibacterial peptide, encoding gene of antibacterial peptide and application |
Citations (1)
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FR2796072A1 (en) * | 1999-07-08 | 2001-01-12 | Centre Nat Rech Scient | MOLLUSC ANTI-MICROBIAL PEPTIDES |
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Non-Patent Citations (6)
Title |
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DATABASE UNIPROT [Online] 01 noviembre 1999 (01.11.1999) "{}Mytilin B and MGD2, two antimicrobial peptides of marine mussels: gene structure and expression analysis."{} Mytilin B antimicrobial peptide. Retrieved from EBI. Accesion N$^{o}$: Q9Y0B1 * |
DATABASE UNIPROT [Online] 01 noviembre 1999 (01.11.1999) "Mytilin B and MGD2, two antimicrobial peptides of marine mussels: gene structure and expression analysis." Mytilin B antimicrobial peptide. Retrieved from EBI. Accesion Nº: Q9Y0B1. * |
DATABASE UNIPROT [Online] 11 septiembre 2007 (11.09.2009) "{}Sequence variability of Myticins identified in haemocytes from mussels stimulated with Vibrio and poly I:C suggests ancient host-pathogen interactions."{} Myticin C; Precursor. Retrieved from EBI. Accesion N$^{o}$: A7DWW0; A7DWX8; A7DWX1; A7DWX7; A7DWS4;A7DWX3; A7DWX5; A7DWU9; A7DWW2; A7DWX0; A7DWX6; A7DWY0; A7DWW9;A7DWX4; A7DWX2. * |
DATABASE UNIPROT [Online] 11 septiembre 2007 (11.09.2009) "Sequence variability of Myticins identified in haemocytes from mussels stimulated with Vibrio and poly I:C suggests ancient host-pathogen interactions." Myticin C; Precursor. Retrieved from EBI. Accesion Nº: A7DWW0; A7DWX8; A7DWX1; A7DWX7; A7DWS4;A7DWX3; A7DWX5; A7DWU9; A7DWW2; A7DWX0; A7DWX6; A7DWY0; A7DWW9;A7DWX4; A7DWX2. * |
DATABASE UNIPROT [Online] 23 octubre 2007 (23.10.2007) "{}Gene of the antimicrobial peptide myticin B from the Mediterranean mussel, Mytilus galloprovincialis."{} Myticin B; Precursor. Retrieved from EBI. Accesion N$^{o}$: A7XP66. * |
DATABASE UNIPROT [Online] 23 octubre 2007 (23.10.2007) "Gene of the antimicrobial peptide myticin B from the Mediterranean mussel, Mytilus galloprovincialis." Myticin B; Precursor. Retrieved from EBI. Accesion Nº: A7XP66. * |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
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ES2763349A1 (en) * | 2018-11-28 | 2020-05-28 | Consejo Superior Investigacion | Mythicin peptide and its use in cell regeneration (Machine-translation by Google Translate, not legally binding) |
WO2020109642A1 (en) * | 2018-11-28 | 2020-06-04 | Consejo Superior De Investigaciones Científicas | Myticin peptide and its use in cell regeneration |
CN111606988A (en) * | 2020-06-05 | 2020-09-01 | 深圳大学 | Pernalins, signal peptide of antibacterial peptide, encoding gene of antibacterial peptide and application |
CN111606988B (en) * | 2020-06-05 | 2021-12-24 | 深圳大学 | Pernalins, signal peptide of antibacterial peptide, encoding gene of antibacterial peptide and application |
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