Food preparation for animals that protects, conveys orally and maintains the functionality of dna mo

专利摘要:
Food preparation for animals that protects, conveys orally and maintains the functionality of dna molecules with interest in production and animal health, as well as the procedure for obtaining them. Dna molecules, in particular dna plasmids, are incorporated into nano-micro-macrocapsules of chitosan, alginate and other components, and are subsequently included within a mass of food ingredients subjected to extrusion, granulation or a combination of both procedures, under conditions that guarantee the integrity of the dna within the feed pellets. The subsequent oral administration of the preparation allows a precise dosage of the amount of dna, protects it during transit through the digestive tract and maintains its viability to the sections of the intestine in which exerts its physiological effect. The plasmids thus expressed express in living animals the genes of interest they carry in their construction for at least 60 days. (Machine-translation by Google Translate, not legally binding)
公开号:ES2641601A1
申请号:ES201600417
申请日:2016-05-09
公开日:2017-11-10
发明作者:Tomás Francisco MARTÍNEZ MOYA；Francisco Javier ALARCÓN LÓPEZ；María Isabel SAÉZ CASADO
申请人:Universidad de Almeria；
IPC主号:

专利说明:

 2 DESCRIPTION Food preparation for animals that protects, orally vehicles and maintains the functionality of DNA molecules with interest in animal production and health, as well as the procedure for obtaining them.   5 Technical sector The present invention is generally framed in the field of agriculture, specifically in animal production and specifically in the aquaculture sector.   Background of the Invention There are several commercial vaccines authorized for use in fish, the vast majority against bacterial diseases, and very few against viral diseases.  In 15 real fish farming situations, the fish are immunized with the use of vaccines by two usual procedures: administration by injection, either intramuscularly or intraperitoneally, or administration by immersion.  On a still experimental level, it is worth mentioning other procedures that will be discussed later, among which the oral administration of vaccines is, today, the most promising.   20 Each method has its advantages and disadvantages with regard to levels of protection, depending on its side effects, and with respect to its practical application and profitability (Gudding et al. , Recent developments in fish vaccinology.  Vet lmmunol lmmunop. , 1999, volume 72, pages 203-212; De las Heras et al. , lmmunogenic and 25 protective effects of an oral DNA vaccine against infectious pancreatic necrosis virus in fish.  Fish Shellfish Immunol. , 2010, volume 28, pages 562-570).    In recent years, pDNA vaccines are booming, mainly because, unlike conventional vaccines (based on protein subunits or in 30 inactivated pathogenic microorganisms), they are capable of inducing very effective immune responses both at the cellular and humoral  They are based on the expression of the antigens encoded by recombinant plasmids (pDNA) in cells of the immunized animal, being able to induce a long-lasting immune response that greatly decreases the need for reinmunizations (Tonheim et al. , What 35 happens to the DNA vaccine in fish A review of current knowledge.  Fish Shellfish lmmunol. , 2008, volume 25, pages 1-18).    In addition, and from a practical point of view, the DNA vaccine is relatively inexpensive and easy to produce.  In fact, there are different vaccine preparations of 40 cDNAs authorized for veterinary use both in the United States and in Canada.  As an important limitation of them, it should be mentioned that they also force their administration by injection, since oral administration implies that the ingested DNA is hydrolyzed in the intestine to small fragments as a result of the digestive process, which involves mechanical, chemical phenomena ( mainly heartburn) and enzymatic (nuclease action) in the gastrointestinal tract.  In these circumstances, neither the immunostimulatory effects nor the vehicle of genes of interest in the pDNA are possible, since in particular the pDNA needs to reach the intestinal absorption zones with its complete sequence, its circular structure, and even its structure. three-dimensional spatial ("supercoiling") to preserve its capacity for expression in tissues.  Therefore, DNA is a very vulnerable molecule in the digestive tract, and hence the poor success of its oral administration as a naked molecule.    3 Limitations of vaccine administration methods in aquaculture The administration of vaccines in aquatic animals gives rise to technical problems that do not occur in terrestrial animals.  Fish can be immunized in three ways, by injection, by immersion or by oral administration of the vaccine (Heppell and 5 Davis, Application of DNA vaccine technology to aquaculture.  Adv Drug Del Rev. , 2000, volume 43, pages 29-43; Adelmann et al. , Development of an oral vaccine for immunization of rainbow trout (Oncorhynchus mykiss) against viral haemorrhagic septicaemia.  Vaccine, 2008, volume 26, pages 837-844).    10 Injection vaccination can be performed intramuscularly (i. m. ), although the intraperitoneal route (i. p. ), despite causing more stress in animals than the i. m. , has been shown to be more effective in immunizing fish.  Before injecting the vaccine, the fish must be anesthetized, and after puncture it is returned to the water.  This procedure is not operative to be applied to fish of less than 5 grams and, therefore, does not serve to immunize the animals in their early stages of development, a period that is precisely of the utmost interest for early prevention against the appearance of diseases.  In lots of fish larger than that weight, the individual puncture of each animal is still an enormously laborious work and of stressful consequences for the animals.   20 On the other hand, injectable vaccines usually include adjuvants to enhance the immune response and prolong its effect, which rarely cause problems related to both local and systemic inflammatory phenomena, capable of ultimately causing even considerable mortality.  Another 25 disadvantages of this route are related to the reduction of post-vaccination intake, as well as the possibility of producing an inadvertent puncture of the intestine.  In addition, the use of needles increases the risk of transmission of pathogens from water to animals and among the fish themselves (Vinitnantharat et al. , Fish vaccines.  Adv Vet Med. , 1999, volume 41, pages 539-550).   30 A good part of the aforementioned problems associated with intraperitoneal injection are solved by the administration of immersion vaccines.  In this case, the fish are sprayed or immersed in a solution containing the concentrated vaccine, being therefore easy to apply and although not as effective as parenteral injection 35 in terms of the immunity it generates, its ease of handling It is the method of choice for small fish, with live weights of less than 5 grams.  Among its limitations, it should be mentioned that its use is restricted to intensive aquaculture, given the environmental risks arising from the release of vaccine preparations to the cultivated water and, consequently, to the environment in which aquaculture production is developed.  Additionally, a significant amount of the substance to be administered is needed, rather than in certain cases, such as viral vaccines.  It makes its practical application impossible.  On the other hand, there is no precise control of the route of entry of the vaccine antigens, which must be produced by absorption through the skin and / or gills, so that this aspect of the dosage control can be qualified 45 as enormously erratic and imprecise.  And finally, in the best case, the immune response is of less magnitude and duration than when the parenteral route is used by injection (Vinitnantharat et al. , Fish vaccines.  Adv Vet Med. , 1999, volume 41, pages 539-550), which is why it may be necessary to revaccinate with some frequency, something that implies continuous manipulation of the fish.   50 If we confine ourselves to the case of pDNA vaccines, their administration by immersion is an unthinkable practice because of its zero viability, not only because of the erratic nature of the aquatic environment, but mainly due to the rapid degradation of the4 cDNA molecule in the aquatic environment itself.  To date, the application of this type of vaccine is necessarily done by intramuscular or intraperitoneal injection.    In any case, and despite the limitations mentioned, to date the efficacy of vaccination by immersion far exceeds that obtained by oral vaccination in fish.  The reason lies in the degradation of oral vaccines that takes place during their transit through the digestive tract, thanks to the existence of gastrointestinal physiological barriers, the most important of which are heartburn and digestive enzymatic activities in both the stomach and in the intestine   10 From the practical point of view in farmed conditions, the possibility of administering oral vaccines in fish is considered as the ideal route, due to its numerous advantages, among which it is possible to mention: i) it is the only method that could be used in aquaculture extensive, in which the individual control of animals is not possible, 15 but if they receive artificial feeding through feed; ii) it does not cause stress in animals, since it exempts them from painful or annoying manipulations; iii) enables mass administration in animals of any size without the need for additional costs; iv) allows for the previous reason to immunize at very early stages.  provided that the animals eat inert food, and v) simulates the natural route of oral entry of a multitude of ichthiopathogenic agents and the consequent natural mechanism of recognition and triggering of the immune response.  As main disadvantages we could mention that: i) it would be necessary to administer large quantities of these substances to obtain an adequate response; ii) is only applicable to fish that eat inert food, and iii) in the current state of technology, the response obtained is not equivalent to that produced by injection or immersion (Horne and Ellis, in "Fish Vaccination", ed.  TO.  AND.  Ellis  Academic Press, London, 1988, pages 55-66).    In any case, the last problem that hinders its use is that exogenous substances are degraded or inactivated in the digestive tract of the fish, and therefore do not exert the desired effect (Ounn et al. , Vaccines in aquaculture: the search for an efficient delivery system.  Aquacult Eng. , 1990, volume 9, pages 23-32).    Encapsulation as an oral vehicle of bioactive molecules 35 Among the strategies of oral vehicle transport in fish, different methods of spraying or adding by various systems of a wide range of aqueous and oily solutions containing plasmids or other types of vaccine preparations have been described previously prepared feed pellets.  In spite of the existence of numerous patents in this regard, the limitations of the same include the erraticity of the dosage, derived from the lack of guarantee of uniform distribution on the feed (usually small volumes of solution / vaccine emulsion applied on considerable amounts of feed, procedures, in addition, whose practical utility is in question, taking into account that they will be used in a medium as difficult to control as the aquatic associated with commercial fish farms.  And above all, the lack of demonstration of its effectiveness through in vivo tests.    Encapsulation has offered more promising results for oral administration of bioactive molecules, cells, drugs and other substances of interest (Chen et al. , Genipin cross-linked polymeric alginate-chitosan microcapsules for oral delivery: in vitro 50 analysis lnt J Polymer Sci. , 2009, article ID 617184, 16 pages, doi: 10. 1155/2009/617184).    5 The main requirement of these capsules is that they consist of biocompatible components, if possible, food grade, that originate stable capsules with adequate resistance to gastrointestinal conditions, and that have some permeability.  The structure of these capsules consists of three-dimensional polymeric networks of high molecular weight and hydrophilic character, capable of absorbing large amounts of water or biological fluids.  These networks are insoluble due to the presence of chemical crosslinks or physical crosslinks, which provide the network with a defined structure and physical integrity.  Currently, the capsules have numerous applications in the medical and pharmacological industry, as they resemble natural living tissues more than any other class of materials.  Within the polymers 10 most used in encapsulation procedures, alginate, an anionic polysaccharide isolated from brown algae, satisfactorily meets the requirements mentioned above.  The main drawback is that it forms relatively unstable polymeric structures that can lead to destabilization of the capsules (Wang et al. , Preparation of uniform sized chitosan microspheres by membrane 15 emulsification technique and application as a carrier of protein drug.  J Controlled Release, 2005, volume 106, pages 62-75).    However, to avoid this problem alginate capsules can be stabilized with other cationic natural polymers (Anal et al. , Chitosan-alginate multilayer beads for 20 gastric passage and controlled intestinal release of protein.  Drug Dev lnd Pharm. , 2003, volume 29, pages 713-724).    The most widely used is chitosan, which is a copolymer of the amino-polysaccharide type composed of D-glucosamine and N-acetyl-D-glucosamine [oly (N-acetyl-D-glucosamine)].  25 which is obtained by the alkaline deacetylation of chitin, the main structural material in exoskeletons of crustaceans and other arthropod animals.  It is the only natural cationic polysaccharide and is known for its biocompatibility, biodegradability, as well as for its resistance to gastric conditions and its ability to adhere to the intestinal mucosa, the latter characteristic that increases the contact time of the 30 capsules with the mucous membranes and enhances the absorption of molecules through the epithelial cells of the mucosa (Artursson et al. , Effect of chitosan on the permeability of monolayers of intestinal epithelial cells (Caco-2).  Pharm Res. , 1994, volume 11, pages 1358-1361).    35 It also has certain advantages over other polymers when used as a vehicle for oral administration of drugs or substances sensitive to the stomach acid environment (Li et al. , Preparation of alginate coated chitosan microparticles for vaccine delivery.  BMC Biotechnol. , 2008, volume 8, 11 pages, doi: 10. 1186 / 1472-6750-8-89).    40 For all of the above, if linear DNA or pDNA molecules are to be used orally to take advantage of their biological properties, it is essential to develop a preparation that meets the following requirements: 1) that can be administered in such a way that both are avoided stressful management of the 45 animals, such as alterations in routine personnel operations, or changes in the pattern and composition of animal feeding; that is, that it can be administered as usual feed, 2) that the exogenous DNA, and in particular the pDNA, maintain the viability and functionality within the feed from its elaboration until it is administered to the fish in the aquatic environment,6 3) that the preparation does not dilute or lose properties during the time between adding it to the aquatic environment and the animal ingests it, that is, it behaves like the particles of the usual commercial feed, 4) that during transit through the digestive tract, ensure the arrival in the intestine of a sufficient number of linear DNA or pDNA to ensure, on the one hand, a local effect, and on the other, an effective intestinal absorption, and 5) that such absorption results , in the case of genes contained in a plasmid construction, in a subsequent effective expression in animal tissues.   10 The feed thus prepared must include, in addition to the usual food ingredients, particles (nano, micro or macrocapsules) made from non-toxic substances, food grade, easily available in the market and inexpensive.  The most widely authorized ingredients for the preparation of inert capsules 15 are alginate and chitosan.  Alginate is a natural anionic heteropolysaccharide consisting of D-mannuronic acid and L-guluronic acid and which in the presence of calcium can produce gels.  Diva lens ions, such as Ca2 +, bind preferentially to the L-guluronic acid polymer.  As a polymer to form capsules, calcium alginate is very appropriate due to its simplicity, absence of toxicity, biocompatibility and low cost 20 (Sheu and Marshall, Microentrapment of lactobacilli in calcium alginate gels.  J Food Sci. , 1993, volume 54, pages 557-561).    The solubilization of the calcium alginate matrix when calcium ions are sequestered is another advantage of this ingredient.  This property makes it especially valuable for oral administration, since the alkaline pH of the intestine solubilizes the polymer, and allows the release of its contents.  The second substance of interest for this purpose is chitosan, a cationic natural polymer, which can be used alone or in combination with alginate, thereby increasing its stability (Anal et al. , Chitosan-alginate multilayer beads for gastric passage and controlled intestinal release of 30 protein.  Drug Dev Ind Pharm. , 2003, volume 29, pages 713-724).    The success of alginate-chitosan combinations for oral vehicle and protection of exogenous protein in fish has been demonstrated recently (Sáez et al. , Effect of alginate and chitosan encapsulation on the tate of BSA protein delivered orally to gilthead 35 sea bream (Sparus aurata).  Anim Feed Sci Tech. , 2015, volume 210, pages 114-124).    In view of the above, the most appropriate solution would be the inclusion of the pDNA within different encapsulation formats, which in turn, are incorporated into the mass of the feeds that are usually administered to animals, from the points of view. animal welfare, as well as the comfort of administration, and the possibility of repeating administration as many times as necessary.    There have been numerous attempts in this regard, but no procedure is known that is capable of incorporating viable DNA plasmids into the feed mass.  The performance of physical factors such as temperature and pressure during granulation and extrusion procedures end the integrity of the pDNA, and for this reason, practically all the references consulted incorporate the solution of ADNp on the surface of the granules, once that these have already been elaborated.  Some study solves these problems by producing non-food pellets, such as polyethylene glycol (Adelmann et al. , Development of an oral vaccine for immunization of rainbow trout (Oncorhynchus mykiss) against viral haemorrhagic septicaemia.  Vaccine  2008, volume 26, pages 837-844), which although they can be used for laboratory experimentation conditions for administrations7 specific, however, they present other problems, in fish farm, such as the lack of guarantee that the animals ingest them due to their lack of palatability, since they have nothing to do with the usual food, or the more than probable side effects related to its use (Schultze et al. , Concentration-dependent effects of polyethylene glycol 400 on gastrointestinal transit and drug absorption.  Pharm Res.  2003, 5 volume 20, pages 1984-1988).    In any case, the main objection to all these procedures is the lack of evidence of both the persistence of the pDNA and its effectiveness in vivo.    10 Explanation of the invention Food preparation for animals that protects, orally vehicles and maintains the functionality of DNA molecules with interest in animal production and health, as well as the procedure for obtaining them.   The invention consists of a food preparation intended for production animals in general, and fish in particular, which serves as a vehicle for oral administration of linear DNA molecules or DNA plasmids (pDNAs) capable of expression in eukaryotic cells, as well as the procedure for obtaining it.  The preparation is characterized by having as a non-exclusive characteristics a cylindrical or spherical granule format, of uniform morphology and modifiable size, of crumb, sheet, tape among others, to adapt to the different vital stages of the target animals.  The granules can be the result of a process of granulation, or extrusion, or a combination of both, and are characterized by containing within their mass, in addition to the usual components in feed for food purposes, chitosan nanoparticles , or micro or macroparticles made with the same polymer alone or in combination with other polymers, mainly alginate, as well as other molecules with technological interest, such as cyclodextrins, gelatin, polylactic-co-polyglycolic acid (PLGA), starch, carrageenans , guar gum, etc. , and to which different amounts of linear or circular DNA are incorporated during the manufacturing process, in particular, one or more eukaryotic expression vectors, consisting of DNA plasmids that include one or more genes encoding proteins for interest in the field of animal production and / or health, and whose main application is for the purpose of immunization, immunostimulation, or to exert hormonal or other effects, mediated by protein factors.  Said DNA remains structurally and fully functional during the industrial processes mentioned for the preparation of the granules of the preparation, thanks to the protection provided by the nano, micro or macroparticles previously prepared, and by the characteristics of the extrusion and granulation process applied.  This invention avoids the destruction of DNA due to the pressure and high temperatures involved in the feed manufacturing process.  The preparation thus formed allows precise dosing of the DNA, in particular of the pDNA, compared to other forms of oral or immersion administration, is stable in the aquatic environment, and keeps the DNA fully functional, which it protects during its passage through the digestive transit of 45 animals, favoring their subsequent intestinal release and subsequent physiological effect, either in the intestinal lumen, or after incorporation into the animal's internal environment, allowing the expression of genes of interest in animal tissues for at least 60 days after ingestion.  The administration of the preparation is oral, and since its format is identical to the usual food of the animals, it avoids stressful handling of the same 50, as well as alterations of the routine operations of the farms, and of the fish farms in particular.    As a result of all of the above, the product:8 1) allows precise oral dosing of linear DNA or pDNA compared to other forms of oral or immersion administration, since it is distributed homogeneously in the mass of the feed, 2) avoids the stressful handling associated with the application by injection 5 intramuscularly or intraperitoneally, 3) it is stable in the aquatic environment for a time similar to commercial feed for aquaculture, 10 4) it maintains the DNA structure and function of the ADNp, previously encapsulated, protecting them both during the industrial manufacturing stages of the product, such as during its subsequent transit through the digestive tract of animals.    5) once it reaches the intestine, it allows the release of DNA, being able to exert its physiological action locally, or be absorbed through the intestinal mucosa, 6) the pDNA absorbed into the internal environment of the animal migrates and can be detected in different tissues, such as liver and muscle, 20 7) the genes carried by the pDNA included in the product are expressed in the different animal tissues for at least 60 days after ingestion.    In the particular case of the use of plasmid expression vectors in eukaryotes it has been shown as a very effective strategy especially for the vaccination of 25 fish parenterally, since they are capable of triggering a potent immune response of both humoral and cellular type.  But the structure and functionality of the pDNA molecule is very sensitive to the physiological conditions existing in the gastrointestinal tract of animals, in particular to heartburn and nuclease enzymes of intestinal secretions, so that its direct use orally 30 It is totally ineffective, and as a consequence, administration is limited to the parenteral route by injection, either intramuscularly or intraperitoneally.    Undoubtedly it is in fish where this oral administration is especially interesting, given that the administration of injectable preparations is relatively simple in terrestrial species, and becomes part of the usual vaccination routines in livestock farms.  On the contrary, in aquaculture the individual injection of juvenile forms of fish is not feasible, for the reasons stated above, and this motivates the need to develop effective, reliable, and feasible forms of oral administration in farmed conditions.   40 Among the mechanisms that are probably involved in the survival of the pDNA in the preparation presented, it is worth mentioning, in addition to encapsulation, that the protection granted by the feed granules themselves is due to the fact that the food components act as an acid barrier stomach and digestive enzymes both gastric and intestinal.  Thus, the components of the feed, rich in protein, have buffer capacity by themselves, so that the acidic stomach pH is largely mitigated by the food.  In fact, the stomach acidifying capacity of aquaculture fish clearly depends on whether or not the stomach contains food.  In other words, for the survival of the exogenous cDNA in the digestive tube 50 it is very important that the administration of the plasmids be carried out in the food itself, and not by procedures that differ in time from the feeding patterns.  Therefore, the present invention, containing the pDNA in9 feed particles seem to have protective effect of ADNp precisely because of the combination of these phenomena.    The proposed feed allows oral administration of the capsules (nanocapsules, microcapsules or macrocapsules) containing pDNA, plasmids that remain viable 5 and protected within the feed without special preservation conditions other than those of the feed, while its composition , beyond the addition of protected cDNA in inert capsules, it is that of any feed for aquaculture, and therefore, designed to be stable in the aquatic environment.  Because of its formulation, it is also attractive to animals, which ingest it voluntarily, under the usual conditions of exploitation, without the need for stressful handling of any kind.  The invention is compatible with the usual large-scale operation in all types of fish farms, both in tanks and in open sea cages.  In this way, the ADNp is protected during its transit through the fish's digestive tract.    15 The preparation is formulated in two stages: Stage l.  Preparation of nano, micro and macrocapsules with ADNp.    Stage II  Preparation of feed containing capsules with ADNp 20 Stage l.  Preparation of nano, micro and macrocapsules with ADNp.    In the first one, the previously extracted DNA plasmids are mixed according to the protocol mentioned in the section "Application examples.  Example 1 "with an adequate volume of the substance or substances to be produced by the capsules (comprising as main components chitosan and / or alginate, but which may also include, gelatin, polylactic-co-polyglycolic acid (PLGA), or other components or mixtures thereof, non-exclusive) in varying proportions (typically 0.2 to 4% w / v for alginate, and 0.01 to 3% w / v for chitosan), as well as a variable amount of other components, such as mono-, di-or oligosaccharides, such as cyclodextrins (typically between 0.05 and 3% w / v), or other food substances for thickener or emulsifying purposes (such as , agar, starch, carrageenans, guar gum, etc. ).  The particles that are made from these ingredients can be designed with a wide range of sizes, and thus, we speak 35 of nanocapsules (identifiable with scanning electron microscopy, and diameters between 10 and 1000 nm), of microcapsules (with diameters between 1 µm and 1 mm), and of macrocapsules (between 1 and 10 mm).  The choice of capsule size will depend on the format of the feed particles in which they will be included later, but all of them allow the introduction of the pDNA into its interior, due to its small size.   40 The microcapsules and macrocapsules can be made in a simple manner by forcing the previous mixture of polymers and cDNAs to be passed dropwise through a needle of varying diameter depending on the desired size, over a solution of CaCI2 (typically 0.5 to 5 % w / v) in such a way that, when the drops or micro drops fall into the fluid under continuous agitation, the gelation of the same takes place, forming spherical and solid capsules.  The process can be automated through the use of an encapsulating device, which by means of peristaltic pumps takes the solution to gel, containing the nano or microparticles with the plasmid inside, of a container, and forces it to pass through nozzles of different sizes depending on the desired diameter, and the drops or microdrops thus formed will have to fall on the above-mentioned CaCl2 solution for gelation.  On the other hand, nanocapsules are made following the procedure described by Kumar et al. , Potential use of chitosan nanoparticles for oral delivery of DNA vaccine in Asian sea bass (Lates10 cacarifer) te protect you from Vibrio (Listonella) anguillarum.  Fish Shellfish Immunol. , 2008, volume 25, pages 47-56) with some modifications, starting from equal volumes (for example, 1 mL) of 0.5% sodium sulfate and 0.5% chitosan (w / v), at that the tenth part (for example, 200 µL) of a plasmid pCMVβ solution of 0 is added. 2 mg pDNA / mL.  The mixture is homogenized in a vortex under intense stirring for 5 30 seconds, and allowed to stand for 30 min at room temperature.  After this time, the suspension is stirred again in the same manner described.  Finally, the nanoparticles are recovered by centrifugation for 3 min at 12. 000 g    All the particle formats containing cDNA described can be used directly after obtaining it to make the feed, or they can be kept refrigerated for periods beyond two weeks without any impairment in its functionality.  However, its main advantage consists in the possibility of lyophilizing the capsules, obtaining a powder or a fine granulate capable of keeping the ADNp totally viable for unlimited periods, which can be reserved until the moment when it is necessary to add it to the dough from which the feed will be made.    Stage II  Preparation of the feed containing the capsules with ADNp 20 The second stage that culminates in obtaining the feed will consist in the preparation itself of a mixture of finely ground flours with the usual food ingredients, together with the authorized additives and the vitamin and mineral supplements that precise each species to which it is intended.  This mixture will then be subjected to either a granulation process, or a two-phase process, one of extrusion followed by another of granulation.  In any case, the nanocapsules or microcapsules that previously contain the cDNA of interest and which have been prepared according to the protocol described above will be added to the food ingredient mixture.    In the case of granulation, the procedure will be carried out without the need for 30 special precautions, since the temperature at the exit of the matrix that determines the size of the feed granules is not likely to rise above 90 -95 ° C, and taking into account that the protection provided by the encapsulation process allows DNA to conserve its structure, and in the case of the particular ADNp, not to lose its supercoiled structure at those temperatures.  In fact, the impossibility of raising the temperature is one of the limitations of granulation when preparing fish feed, which requires physical-chemical characteristics and a transformation of the special ingredients because of their digestive physiology and characteristics of the aquatic environment.  These problems are solved in the commercial feed industry with the extrusion process, much more aggressive in terms of physical effects on the 40 components.  Therefore, if it is necessary for the feed to be extruded, which is common in aquaculture feed, it must be taken into account that much higher temperatures can be reached (up to 120-140ºC).  In this case, it is possible to continue including the nano or micro / macrocapsules in the feed mass, but they will have to be incorporated once the extrusion process is finished, and then subject the mixture to a subsequent granulation stage.  For this, it is necessary that the mixture of ingredients conditioned and forced to transit through the extruder screw, before incorporating the DNA: a) does not impersonate the matrix or the cutter at the end of the extruder screw, avoiding 50 ace! that acquires the final format of the granule, pellet or sheet, but that it is freely allowed to obtain an amorphous mass of extruded ingredients,11 b) for this, water vapor must be added during the passage through the screw in sufficient quantity so that the mass of ingredients maintains a high humidity (above 50%) at the extruder outlet, c) this extruded mass is It will then be introduced into a mixer that guarantees homogeneous kneading, during which the dough temperature will be checked, allowing it to cool down below about 90-95ºC, but preventing the temperature from falling below about 45-50ºC.  Within this temperature range, the suspension of the capsules with plasmids may be added without any risk of DNA alteration, but sufficient fluidity will be maintained so that the mixing of the capsules with the 10 feed ingredients is homogeneous, and thus allows the subsequent passage of the dough through the granulating machine with the necessary fluidity.  For this, the humidity should be maintained above 50%, adding hot water at the desired temperature if necessary.    D) the described mixture, previously extruded and already containing the encapsulated and homogeneously distributed DNA, will then be subjected to a granulation process equal to that described above, as a second stage after extrusion of the food components.    20 The final preparation obtained by this procedure allows the exogenous DNA to be orally vehicular, which will continue to retain its structure and functionality, something that represents a special interest for its use in aquaculture fish and has the following advantages over its injected or immersion administration as well as against other preparations proposed for oral administration: 25 1) the double protection provided by the combination of encapsulation techniques, together with the feed processing conditions that have been used, either granulation, or extrusion / granulation, they allow the incorporation into the mass of the feed of DNA molecules, and in particular of ADNp, without these being destroyed or inactivated during the manufacturing process, something that does occur when DNA is added naked.    2) the feed thus formed guarantees the precise dosage of the DNA to the animals that consume it, since it can be incorporated into the mass thereof and conveniently homogenized as much DNA as necessary during the preparation of the mixture, distributing it throughout the mass of the food granule, and not only as an external layer added a posteriori on the feed granules, something common in the inventions for this purpose proposed so far.    3) Once manufactured, the feed maintains the viability of the DNA for long periods of storage, since this once encapsulated molecule is stable under the low humidity conditions of the feed, below 10%.    4) the extrusion / granulation procedures used for the preparation of the invention are specifically tailored to the needs of aquaculture fish, so as to guarantee not only the persistence of the DNA inside the feed granules in the aquatic environment during Enough time for fish to ingest it, but also the properties of a commercial extruded feed.   50 5) when the granules described herein are thrown into the aquatic environment, there is no loss due to "rinsing" of the DNA, unlike what happens when it is subsequently incorporated on the surface of the feed granules, normally12 as an oily film that adheres to them.  The proposed invention solves the serious problem of uncertainty about the amount of DNA that the fish actually ingests when it is applied as a cover layer on the feed.  Said quantity may be calculated by multiplying the average daily feed intake by the known concentration of DNA per unit weight of the feed.   5 6) the feed containing the capsules loaded with DNA, during its transit through the digestive tract, is capable of transporting said DNA to the later sections of the intestine, overcoming the physiological barriers of the fish capable of destroying it if it is administered naked.   10 7) the orally protected pDNA administered inside the feed granules described herein is absorbed in the intestine, and distributed by the different tissues of the fish, so that it is possible to detect and identify its sequences in intestinal, hepatic and muscular.   15 8) Protected and vehicularly transmitted pDNA with the feed described here, and which is subsequently absorbed through the intestine and distributed by animal tissues remains viable and fully functional after this transit, and for this reason it is possible to detect and quantify the activity of the genes of interest inserted in the plasmids in the tissues of the animals that ingested them for at least 60 days post-ingestion.    The proposed invention meets the requirement of novelty, since it has not been disseminated by written or oral description, nor has it been used beyond the experimental scope in the laboratory, before applying for the patent.   25 The inventive activity represents a new contribution on the state of the art from a double point of view: 1.   DNA vaccines have shown their potential in immunization not only on an experimental scale, but also in some commercial preparations.  They represent a medium-term line of research of great interest, although in the case of fish its main limitation is the need for injected administration.  Despite the interest in oral administration, there is to date no preparation capable of achieving efficient vehicle transport in a food preparation that allows its commercial use in real fish farm conditions.    2.   The inclusion in the polymeric vehicle whose characteristics have been described in the corresponding sections solves the double technological problem that limits the practical use of DNA plasmids orally in feed: i) the destruction of them due to high temperatures and pressures that take place during the manufacture of the feed, and ii) the acidic and enzymatic hydrolysis that the naked plasmids undergo during their transit through the digestive tract of the animals, which prevents that sufficient quantity of the same arrives for their intestinal absorption.  These weaknesses completely prevent the use of plasmids of DNA orally on a commercial scale if they are not transported and protected with the proposed invention.    We understand that the proposed invention is susceptible to industrial exploitation, considering that the cost / benefit of its exploitation is favorable for the following 50 reasons: 1.   The inclusion of gene sequences encoding proteins of immunological, hormonal or other interest in specific areas within plasmids to ensure13 its subsequent expression in animal cells is a relatively simple procedure, available to any laboratory with a basic command of molecular biology techniques.  On the other hand, the cloning and subsequent multiplication of these plasmids of interest within bacteria is a routine and cheap procedure, which allows to obtain industrial quantities thereof.  From this point of view, this process can be developed on an industrial scale without special difficulty.    2.   The manufacture of the capsules does not require an excessively expensive equipment, since a wide variety of machinery is available on the market at reasonable prices for all the industrial scales that are required.  The reason 10 lies in the multitude of applications, especially pharmacological, that use various forms of encapsulation, and that is why the industrial production of the proposed invention is perfectly feasible, and in fact it is a reality in many fields of research and Commercial product development.    15 3.   The substances used in the manufacture of the capsules are ingredients authorized for food use, easily accessible and cheap.    Four.   The DNA encapsulation yields are excellent (above 95%), and the maintenance of their integrity within the capsules is also very high, 20 such that almost all of the pDNA obtained after purification can end up in the capsules. , with hardly any losses.    5.   The ease of regulating both the diameter (from nanocapsules of micrometer fractions, to macrocapsules of several millimeters) and the DNA content of the capsules 25 makes the procedure extremely versatile.  Thus, manufacturing can be adapted to specific needs related to the size and species of fish of interest, and can be incorporated into feed particles of very different formats (sizes from 0.2 mm to several cm), always using the same machinery, no need for parallel or multiplied industrial lines.   30 6.   The subsequent elaboration of the feeds in which the capsules with the DNA will be included does not require a different machinery from the usual one in the factories of feed, since the formulated ones, aside from including the DNA, can be the habitual ones of the feeds commercial, including as macro-ingredients to 35 materials of animal and vegetable origin authorized for animal feed, in adequate proportions for gara: 1tizar the protein and energy content necessary for each species and productive stage, conveniently milled and screened, and subjected to high pressures and temperatures that guarantee high digestibility, inactivation of antinutritive factors, as well as adequate persistence in the aquatic environment.    Brief description of the drawings To complement the description that is being made and in order to help a better understanding of the characteristics of the invention, a set of figures in which, for illustrative purposes, is attached as an integral part of said description and not limiting, the following has been represented: Figure 1.  Details of the PCR products obtained that confirmed the presence of the 50 plasmid pCMVβ in the colonies of E.  coli DH5aα transformed.  The size (base pairs) of the amplified fragments is indicated.  M: 1 kb marker (100-12. 000 bp).    14 Figure 2.  Electrophoretic separation in 0.8% agarose gels of purified pDNA from transformed E. cultures.  coli DH5α.  Note in both cases the greatest electrophoretic migration of the band corresponding to the plasmid in supercoiled state.  M, 1 kb marker (100-2. 000 bp).    5 Figure 3.  Electrophoretic separation in agarose gels from the supernatants and the pDNA contained in the nanocapsules after disintegration with sodium bicarbonate.  The presence of pDNA outside the capsules (supernatant) is not detected.  The encapsulation maintained the functional form of the pDNA (supercoiled state).  M, 1 kb marker (100-12. 000 bp).   10 Figure 4.  Electrophoretic separation in agarose gels from naked pDNA subjected to different pH values (2, 4, 7 and 9) and at different times (0, 10, 20, 30, 45, 60, 90 minutes) in an in vitro system.  1 Kb: molecular size marker (100 - 12. 000 bp).   15 Figure 5.  Electrophoretic separation in agarose gels from the encapsulated cDNA in calcium alginate and subjected to different pH values (2, 4, 7 and 9) and at different times (0, 10, 20, 30, 45, 60, 90 minutes) in a system in vitro    20 Figure 6.  Electrophoretic separation in agarose gels from the pDNA encapsulated in chitosan nanospheres subjected to different pH values (2, 4, 7 and 9) and at different times (0, 10, 20, 30, 60, 90 minutes) in an in vitro system .    Figure 7  (1): Feed granules with cDNA nanocapsules incorporated in the mass 25 before granulation (lot A) and with the same nanocapsules incorporated in their surface after immersion (lot B) observed under natural light, without any difference being appreciated .  (2): The same granules of (1) observed with ultraviolet light (at 312 nm).  Note the fluorescence emission from encapsulated DNA stained with SYBR Green ® throughout the mass of the granules A, but only on the surface in the 30 granules B.  (3): Sheets from the cross section of the previous granules observed under ultraviolet light (λ 312 nm).  Note the fluorescence emission from encapsulated DNA stained with SYBR Green® in the sheet from granule A.  but only on the outer surface of the sheet from granule B.   35 Figure 8.  Electrophoretic separation in agarose gels of the PCR products obtained from the tissue extracts (muscle, intestine and liver) of the control fish (without administration of pDNA) for the pair of primers specified after 7, 15, 30 and 60 post-administration days.   40 Figure 9.  Electrophoretic separation in agarose gels of the PCR products obtained from the tissue extracts (muscle, intestine and liver) of the fish that were administered feed with cDNA nanocapsules for the pair of primers specified after 7, 15 , 30 and 60 days post-administration.   45 Figure 10.  Electrophoretic separation in agarose gels of the PCR products obtained from the tissue extracts (muscle, intestine and liver) of the fish to which the naked pDNA was administered by intramuscular injection to the pair of primers specified after 7, 15, 30 and 60 days post-administration.   50 Figure 11.  13-galactosidase activity (UA / mL) measured in the muscle, liver and gut extracts from gilthead over 60 days.  (A) administration of the oral preparation (I THINK) containing the plasmid pCMVβ.  (B) administration of the same plasmid15 by intramuscular injection (IM).  No β-galactosidase activity was detected in any of the controls. "    Preferred Embodiment of the Invention The embodiment of the invention is described in more detail by the following examples.  The examples of preferred embodiment are oriented exclusively to provide a more complete description of the selected embodiments of the invention and should not be considered as limiting the scope thereof.   When describing the preferred embodiment of the present invention, the indicated specifications can describe it as a sequence of steps to be followed in a very specific order.  However, considering that the preparation of the preparation is not limited to the particular order of the steps to follow established in this document, its obtaining 15 should not be limited to the particular sequence described.  It is clear that any person skilled in the art would appreciate that it is possible to obtain a similar product by varying the sequence of steps, and therefore, the particular order of the steps to be described in the preferred embodiment of the invention should not be construed as a limitation of The claims.  Therefore, the sequence of steps to be followed for the embodiment of the invention can be varied in the order, but it would still remain within the scope of the present invention.    Example 1: Plasmid multiplication protocol and procedure for encapsulation 25 The experimental work has been carried out with a plasmid construction that has been designed to express control genes in eukaryotic organism cells.  The expression vector pCMVβ (GenBank accession number: U02451) has been used.  This plasmid construction is marketed by Clontech Laboratories lnc (Ciontech.  30 2013a), and is a modified version of the preceding plasmid pCMV (MacGregor and Caskey, Construction of plasmids that express E.  coli β-galactosidase in mammalian cells.  Nucleic Acids Res. , 1989, volume 17, pages 2365-2365).    The plasmid contains the human cytomegalovirus (CMV) promoter, the apex SV40 polyadenylation signal, an ampicillin resistance gene as a marker for the selection of plasmids transformed with the plasmid, and a lac-Z control gene which is expressed using the molecular machinery of eukaryotic cells and that codes for the enzyme β-galactosidase.    40 1.  E. transformation  coli DH5α with the model plasmid The plasmid was used to transform E. bacteria separately.  coli DH5α, selecting only the colonies transformed by a LB culture medium (Luria Bertani) that included ampicillin.  From one of the transformed colonies a mass bacterial culture was obtained from which the plasmid DNA was purified using the commercial kit Qiagen Plamid Maxi (Sigma-Aldrich, Madrid, Spain), according to the instructions proposed by the manufacturer.  Once purified, the quality and integrity of the pDNA was analyzed by performing 1% agarose gel electrophoresis, verifying that the proportion of DNA in a supercoiled circular state (greater migration in the gel) 50 was higher than that of the rest of the plasmid species (circular or open, less mobility in the gel).  The identification of the pDNAs in the transformed bacteria and in the purified pDNAs was confirmed by conventional PCR.  The procedure followed in each of these stages is described in more detail below.   16 In order to obtain competent cells, it was necessary to prepare 6 mL of Escherichia coli (E.  coli DH5a) in an inclined tube with LB medium, which was kept under stirring at 37 ° C for 24 h.  After this time, 2 mL of the culture was inoculated into 50 mL of LB medium with 10 mM MgSO4 (45 mL of LB medium and 5 mL MgSO4), and the whole was kept under gentle stirring at 37 ° C until an optical density was reached. (λ 600 nm) of 0.5, using the culture medium itself as blank.  The initial culture was distributed at a rate of 10 mL in sterile 15 mL Falcon tubes that were kept in an ice bath for 10-15 min.  Subsequently, these were centrifuged at 2. 000 rpm and 4 ° C for 10 min, discarding the supernatant.  The cell pellet was then resuspended in 3.2 mL of RF1 reagent (6 g RbCI, 4.95 g MnCl2 * 4H2O, 1.47 g CH3CO2K, 10 0.75 g CaCl2 * 2H2O and 59.5 mL of glycerol in water distilled up to 500 mL), and the whole was adjusted with 0.2 M acetic acid to pH 5.8.  The cell suspension was then incubated for 15 min in an ice bath.  Subsequently, it was centrifuged under the same conditions above, and the precipitate was resuspended in 2 mL RF2 reagent (mixture 2 mL MOPS 0.5 M (3- (n-morpholino) -propanesulfonic acid-4-15 morpholinopropanesulfonic acid), 0.12 g RbCI, 1.1 g CaCI2 * H2O, 11.9 mL glycerol and 100 mL of distilled water, which was adjusted with NaOH until a pH of 6 was reached. 8).  The cell suspension was cooled on ice for 15 min.  Reagents RF1 and RF2 were sterilized by filtration (0.45 µm, Millipore, Madrid, Spain).  Finally, the suspension of competent bacteria was distributed in 100-200 µL aliquots, maintaining cold conditions, and these were stored at -80 ° C until use.    The transformation of E. cells  Competent coli with plasmids was performed in ice bath.  For this, 50 µL of the plasmid sample was suspended in 50 µL of sterile TE buffer (1M Tris-HCI, 0.5 M EDTA at pH 8), and the mixture was kept in an ice bath.  Then 100 µL of the previously thawed competent cell suspension was added.  The mixture was subjected to thermal shock keeping it 15 min on ice, followed by 3 min at 37 ° C and finally another 5 min on ice.    Then 900 µL of LB was added to the mixture, stirring gently for 1 hour at 37 ° C.  After this time, a seeding of the E cells was performed.  coli DH5α in solid LB medium with ampicillin to select bacteria transformed with the plasmid.  At 24 hours, 12 resistant colonies were selected from each of the cell cultures transformed with each pDNA.    35 2.  Confirmation of colonies transformed by PCR The 12 colonies selected for each plasmid were reseeded in petri dish with LB medium supplemented with antibiotic by dividing the plate into 12 equal grids.  The plate was incubated for 24 h at 37 ° C to allow bacterial growth.  A sample with a sterile 1 µL pipette tip was taken from the colony of each grid, and this was placed in a PCR tube next to 10 µL of ultrapure water.    The DNA samples were amplified by PCR (Eppendorf Ibérica, Madrid, Spain) following the standard protocol used in the laboratory.  The conditions of the PCR cycles included an activation step of 5 min, followed by 36 cycles of 1 min at 95 ° C, followed by 2 min 50 ° C and 1 min at 72 ° C, with a final extension step at 72 ° C for 10 min. .  The amplification process was completed at 4 ° C.    To confirm the presence of plasmid pCMVβ (GenBank accession number: 50 U02451) in the selected colonies, three pairs of primers were used that amplify by PCR three specific regions of the same length, whose sequences are indicated in the SEQUENCE LISTING section, and whose denomination has been the following:17 Pair 1, formed by primer F3237, sense primer, with the number SEQ ID NO 1 in the sequence listing attached, and by primer R3918, antisense primer, with number SEQ ID NO 2 in the sequence listing.  This pair generates a 682 base pair PCR product.    5 Pair 2, formed by primer F5714, sense primer, with number SEQ ID NO 3 in the sequence listing, and by primer R6959, antisense primer, with number SEQ ID NO 4 in the sequence listing.  This pair generates a 1346 base pair PCR product.    10 Pair 3, formed by primer F729, sense primer, with the number SEQ ID NO 5 in the sequence listing, and by primer R2089, antisense primer, with number SEQ ID NO 6 in the sequence listing.  This pair generates a PCR product of 1361 base pairs.    15 The PCR reaction was carried out in three batches of 12 tubes for each pair of primers, each corresponding to one of the colonies of bacteria selected after transformation with the pDNAs.  1 µL of the bacterial suspension (sample of the suspended bacterial colony and ultrapure water) and 14 µL of the premix for PCR was added to the tube.  The final concentration for all PCR components in the 20 reaction volume (25 µL) was 10 nmol of each dNTPs, 4 nmol of each first plasmid specific, 20 ng / µL of genomic DNA extract, 0.5 units of Taq polymerase Amplitaq Go Taq (GoTaqFiexi DNA polymerase, PROMEGA) and 5 µL of Taq buffer pH 8.5 (5X Green GoTaqFiexi Buffer, Biomol, Madrid, Spain) and 2 mM MgCl2.   25 The PCR products were separated into 1% agarose gels [2 g of agarose in 200 mL of TBE buffer (1X) and 5 µL of SYBR GREEN (Biomol. , Madrid, Spain)], and displayed on an ETXF transilluminator (Vilber-Lourmat, Madrid, Spain) using a filter that provides a wavelength of 312 nm.  In general, 7 µL of 30 PCR products were applied per well.    3.  Mass culture of E.  coli and plasmid DNA purification The liquid culture medium used for mass culture of E. colonies.  Transformed coli 35 was 10 g / L of tryptone, 5 g / L of yeast extract and 5 g / L of NaCl (pH 7.2).  This medium was sterilized at 121 ° C for 20 m in.    On the first day, 5 mL of LB (sterile) culture medium, 50 µL of the E suspension was introduced into a flask.  coli (in which the presence of pDNA 40 was previously confirmed by PCR) and 5 µL ampicillin (0.1 g / mL).  The culture was kept under stirring for 24 h at 37 ° C.  On the second day, 5 mL of the culture medium from the previous day was taken, and transferred to 45 ml of LB (sterile) culture medium with 45 µL of ampicillin.  The whole was kept under constant stirring for 24 h at 37 ° C.  On the third day, the culture was scaled to a 1 L Erlenmeyer flask, incorporating the 50 ml of culture medium with the activated bacteria, in 750 ml of LB (sterile) with 750 µL of ampicillin.  After keeping the culture under stirring for 24-48 h at 37 ° C, it was centrifuged at 6. 000 rpm for 10 min in 250 mL bottles, removing the supernatant, and recovering the cell pellet that was frozen at 80 ° C until use.  The culture was continued until a fresh bacterial biomass of approximately 15-18 g was obtained, quantity 50 recommended for subsequent purification of pDNA.    Plasmid purification was performed from bacterial biomass using the Gigaprep Gen Elute HP SelectPiasmid Kit (Sigma-Aldrich, Madrid, Spain) following the18 manufacturer's instructions.  Thus, about 15-18 g of bacterial biomass was started and 50 ml of an aqueous solution containing the plasmid DNA was obtained.  Finally, the solution was distributed in 5 ml aliquots, and once frozen at -80 ° C they were lyophilized.    5 4.  Quantification and integrity of plasmid DNA Each aliquot of lyophilized material was resuspended in 1 ml of ultrapure water to proceed with quantification of pDNA using the commercial DNA Quantitation Kit FluorescenceAssay kit (Sigma-Aldrich, Madrid, Spain).  A standard curve 10 was prepared using a standard commercial DNA solution (Sigma-Aldrich) in the range of 3 to 1. 000 µg / mL prepared in 10 mM Tris-HCL buffer, 1 mM EDTA at pH 7.4.  In the wells of a 96-cell plate for fluorimetry readings, 200 µL of a bis-benzamidine solution (2 µg / ml) prepared in fluorescence buffer (100mM Tris-HCI buffer, 10 mM EDTA, 2M NaCI at pH) were applied 7.4) and 2 µL of the corresponding 15 serial dilutions of the commercial DNA stock.  The readings were made in a fluorimeter (Fluoroskanascent, Thermo, Madrid, Spain) using as excitation and emission wavelengths 355 and 460 nm, respectively.  From the values obtained, the linear equation that relates the values of relative fluorescence units to the concentration of DNA in the problem samples was established.    On the other hand, in addition to confirmation and quantification, the integrity of the purified DNA was also assessed by electrophoretic separation on 1% agarose gel.  The proportion of pDNA in the supercoiled state (preserving its functionality, that is, its ability to transfer and express its information in eukaryotic cells), compared to the open or linear plasmid (inactive), was evaluated based on its electrophoretic migration differences in gels of agarose, as described in Tian et al.  (Chitosan microspheres as candidate plasmid vaccine carrier for oral immunization of Japanese flounder (Paralichthys olivaceus).  Int Imunopharm. , 2008, volume 8, pages 30 900-908).    5.  Plasmid DNA encapsulation methods Two encapsulation procedures were used to obtain particles with different sizes; nanoparticles and micro and macrocapsules.    5. one.  Encapsulation of pDNA in micro and macrocapsules For the formation of micro and macrocapsules, sterile 40 ml 15 ml tubes were introduced: 2 ml of sterile H2O, 3 ml of 3% sodium alginate (previously heated to 40 ° C to reduce its viscosity ) and 500 µL of the aqueous solution with the plasmid pCMVβ (450 mg DNA).  The mixture was then homogenized well and kept in a bath at 40 ° C until use.  This solution was passed through a STARTUP encapsulator equipment (Encapbiosystem INC, Switzerland), coupled to a series of nozzles which allow extrusion of the gelling solution through a hole with a diameter of 200 µm - 5 mm.  The drops formed were dispensed in a gelling solution (3% CaCl2 dissolved in distilled H2O), in which they were kept under stirring for 30 min (220 rpm) in order to complete the gelation process.    50 The production of chitosan-coated capsules was performed as described above, except that in this case the gel solution contained, in addition to calcium chloride, 1 mL of chitosan (Sigma-Aldrich, Madrid, Spain) at 1%.    19 5. 2.  Encapsulation of pDNA in nanoparticles The method is based on the formation of nanocomplexes between the DNA molecules and the chitosan due to electrostatic attractions caused by differences in charges between the two molecules.  They were made following the procedure described by Kumar et 5 al. , Potential use of chitosan nanoparticles for oral delivery of DNA vaccine in Asían sea bass (Lates cacarifer) to protect from Vibrio (Listonella) anguillarum.  Fish Shellfish Immunol. , 2008, volume 25, pages 47-56) with some modifications, starting from 500 µL of 0.5% sodium sulfate plus 500 µL of 0.5% chitosan (w / v) and 100 µL of the plasmid solution pCMVβ containing 0.2 g cDNA / mL.  The mixture was homogenized in a vortex under intense stirring for 30 seconds, and allowed to stand for 30 min at room temperature.  After this time, the vortex suspension was stirred again.  Finally, the nanoparticles were recovered by centrifugation for 3 min at 12. 000 g    15 A sample of the nanoparticles was metallized and visualized by transmission electron microscopy (SEM; Hitachi SV-3500N, Hitachi High-Tech, Japan) in the Electron Microscopy service of the Central Research Services of the UAL.    20 Results 1.  Quantification and integrity of ADNp.    Initially, the presence of plasmid pCMVβ was confirmed in one of the colonies of E.  25 coli DH5α by the appearance of the amplified fragments using the primer pairs, specified above, in the PCR products (Fig.  one).  The concentration of purified pDNA was estimated by reading the fluorimeter after resuspending a lyophilized vial in 1 mL of purified water.  The concentration of pDNA in the vials was estimated from the standard curve performed with commercial DNA, which is between 0.8-0.9 mg pDNA / vial.  The structural integrity of the plasmid, determined by the presence of its supercoiled form, was electrophoretically confirmed (Fig.  2).    2.  Encapsulation of the plasmid DNA sequences The two encapsulation procedures allowed to obtain particles capable of efficiently retaining the pDNA (Fig.  3).  The encapsulation yield was very high (greater than 98%), and no pDNA was detected in the liquid media in which both types of capsules (supernatants) were prepared.  In particular, the nanocapsules were very stable, and the interior pDNA could only be recovered when they were disintegrated with NaHCO3.  It was also found that the encapsulation process did not reduce the proportion of pDNA in the supercoiled state.    Example 2: Evaluation of the stability of capsules and plasmids of DNA 45 under conditions of in vitro gastrointestinal hydrolysis 1.  Stability analysis of bare pDNA versus encapsulation at different pH values 50 Stability of pDNA was analyzed at acidic (2 and 4), neutral (7) and alkaline (9) pH, in order to cover the full range of possible pH values that plasmids could find during their transit through the digestive tract of fish with stomach.   20 In the case of the microcapsules, beakers were used in which 2.5 mL of buffer (0.1 M glycine-HCI for pH 2 and 4; and Tris-HCI for pH 7 and 9) were dispensed.  A sample of naked pDNA, another of material encapsulated in calcium alginate and, a third of cDNA encapsulated in calcium alginate and covered with chitosan were used in the study.  In the case of the naked pDNA, 180 µL of the solution of plasmid pCMVβ was added in each beaker, while in the case of the encapsulated material, 50 capsules were immersed in each of the buffered solutions to each of the values of pH tested, maintaining continuous stirring.  At different times (0, 10, 20, 30, 45, 60, 90 min) a 50 µL sample of the solution containing the naked pDNA was taken, or, where appropriate, three capsules that were placed in eppendorf tubes.  The capsules were disintegrated with 300 µL sodium citrate (0.1 M) and with the help of a pistil.  The stability of the pDNA was analyzed by visualizing the samples after their electrophoretic separation in agarose gels.    In the case of nanoparticles, the method was similar to that described for obtaining 15 capsules.  In brief, the nanoparticle suspension was divided into 12 eppendorf tubes at a rate of 150 µL / tube, and these were centrifuged for 3 min at 12. 000 g  The supernatant was then removed, and 150 µL of the different buffer solutions adjusted to pH 2, 4, 7 and 9 values were dispensed, the whole being initially agitated with a vortex, and subsequently kept under continuous stirring for 20 min.  At different incubation times (0, 10, 20, 30, 60 and 90 min) samples were taken from each tube.  The samples were centrifuged and the supernatant was removed.  To the precipitate 150 µL of 0.4 M sodium bicarbonate was added, and the particles were disintegrated with the help of a pistil.  The samples were kept for 24 hours at 4 ° C to complete the disintegration process.  Finally, the integrity of the 25 pDNA in agarose gel electrophoresis was visualized.    2.  Analysis of the integrity of the ADNp by electrophoresis.    The integrity of the pDNA was checked by 0.8% agarose gel electrophoresis.  30 This percentage of agarose allows a visualization of the different species that make up the pDNA (supercoiled, circular or linear), especially in encapsulated DNA samples.  In brief, 0.4 g of agarose in 50 mL of TBE plus 1.6 µL of SYBR GREEN® (fluorescent substance that specifically binds to the AON molecule) were prepared.  In the first well, a marker (3 µL of milliQ water and 5 µL of the 1 kb marker) was introduced, and in the rest of the wells 5 µL of sample were applied.  All samples were mixed with 2 µL of loading buffer before being applied to the agarose gel.  The electrophoresis were developed at a continuous voltage of 90 V per gel for 45 min in a Labolan Max cuvette (Labolan, Madrid, Spain).  The gels were visualized on an ETXF transilluminator (Vilber-Lourmat, Madrid, Spain) 40 using a 312 nm wavelength filter.    Results 1.  Analysis of the stability of the naked pDNA against encapsulation at different pH values.    one. one.  PH stability analysis of naked pDNA.    Fig.  4 shows the results obtained after incubation of the plasmid in solutions with different pH.  Plasmid pCMVβ was extremely unstable at pH 2, since at 10 min all the pDNA disappeared (supercoiled, circular and linear plasmid).  At pH 4 a progressive decrease of the band corresponding to the supercoiled form was evidenced as the residence time in an acidic environment progressed,21 fact that cancels its functionality.  The neutral (7) and alkaline (9) pH values did not affect the electrophoretic mobility of the pDNA, as verified by observing the fraction corresponding to the supercoiled plasmid, indicating a marked stability of the functional form of the plasmid.    5 1. 2.  Analysis of the pH stability of the pDNA in calcium alginate capsules Fig.  5 shows that encapsulation in calcium alginate prevents degradation of pDNA under acidic conditions that occurs when naked plasmids are used (Fig.  4).  However, incubation of the capsules at pH 2 results in a progressive reduction of 10 the intensity of the plasmid band in the supercoiled state, while observing an increase in that of less electrical mobility (non-functional circular shape).  In contrast, when the capsules were incubated at pH 4, the plasmid was not affected after 90 min incubation.  Incubation of the capsules at pH 7 and 9 did not modify the supercoiled structure of the pDNA.   15 1. 3.  Analysis of the pH stability of the pDNA in chitosan nanoparticles The results indicated that the nanospheres protect plasmids from extreme pH values (Fig.  6).  At pH 2 the protective effect was less, however the two bands of the pDNA could be recovered after 90 min.  In the nanoparticles subjected to pH 4 the two bands were recovered, although their intensity was considerably reduced.  Similarly, it was noted that part of the pDNA added to the wells did not migrate in the agarose gel, a fact that shows the enormous stability of the chitosan-cDNA interaction in this type of nanoparticles.   25 Example 3: Preparation of feed containing pDNA and in vivo evaluation of the same in juveniles of golden sea bream (Sparus aurata) 3. one.  Preparation of the feed and evaluation of the distribution of the pDNA in the granules In order to check whether the amount of pDNA incorporated into the mass of the feed granules by the proposed procedure (described in detail in the corresponding section), is greater than the amount incorporated by means of the procedures based on the application on the surface of the granules of the solution of ADNp, granules of identical composition were elaborated, but following both strategies.   35 A feed mass consisting of fishmeal (60%), degreased soybean meal (20%), and wheat flour (10%) as main components was prepared, in addition to small amounts of fish oil, soybean lecithin , squid flour and vitamin and mineral complex.  To the mass of feed was added volumes of a solution of chitosan nanocapsules containing 1 mg / mL pDNA (the same plasmid pCMVβ used in the previous examples).  The mixture was homogenized in a semi-industrial kneader, and then the dough was introduced into a granulating press (model 14-175, Amandus Kahl Ibérica, S. L. , Madrid) to obtain feed pellets with a diameter of 0.8 cm.  The final concentration of pDNA was 10 45 µg of pDNA per g of feed.    In order to visualize the homogeneity of the distribution of the incorporated pDNA by nanocapsules to the feed mass, a small amount thereof (lot A) was prepared following the same procedure described, but in this case, the nanoencapsulated 50 pDNA was previously stained with SY8R Green®, a fluorescent compound that interacts specifically with the DNA molecule, and emits green coloration when UV light of λ 312 nm falls on it.  Next, the feed granules were made as previously indicated.   22 Another part of the mass of ingredients was not added pDNA during mixing (batch B), but once the feed granules were formed, they were immersed for 5 min in a nanoencapsulated solution of pDNA (1 mg / mL) and previously dyed with SYBR Green®.  The granules were then removed and dried in an oven at 37 ° C in the dark.   5 Next, both batches (A and B) of ultraviolet light of λ 312 nm were struck, in order to observe the possible emission of fluorescence.    3. 2.  Detection of the expression of vehicular genes in pDNA vectors administered orally versus intramuscularly in gilthead specimens (Sparus aurata).  Rehearsal in vivo.    3. 2. one.  Animals and facilities 15 The in vivo studies were carried out at the facilities of the research aquarium of the University of Almeria, an animal experimentation center authorized by the Ministry of Agriculture of the Junta de Andalucía.  60 specimens of sea bream (Sparus aurata) were used, with an average weight of 20 grams.  The animals were acquired from a commercial pre-fat farm, and after the corresponding period of 20 quarantine they were introduced into the experimental tanks.  The fish were fed twice a day, with a usual maintenance ration based on a commercial feed, in a daily amount equivalent to 2% of their live weight (0.4 g per fish per day).  After an acclimatization period of 15 days, the tests were started.    25 3. one. 2.  Detection of transfer of plasmid DNA to animal tissues by PCR.    Administration of plasmids to fish 30 An essential requirement was to ensure that each fish received the same amount of plasmid DNA both orally and parenterally.  Three groups of fish were arranged: i) control, to which no exogenous DNA was administered; ii) intramuscular administration (i. m. ) of naked pDNA, iii) oral administration of feed containing nanoencapsulated pDNA.   35 For this study, 5 fish were taken for each of the experimental lots and sampling times, for a total of 60 animals: 3 experimental lots x 4 times (7, 15, 30 and 60 days) x 5 replicates.    40 Special care was taken regarding dosing, so that each fish was given the same amount of plasmid pCMVβ specifically 40 µg of pDNA.  For intramuscular administration, 100 µL of 0.85% NaCl solution containing 40 µg of pDNA was injected in a single administration.  In the case of the oral route, the experimental feed (containing 100 µg of pDNA per gram) was offered to the fish in an amount equivalent to 2% of the biomass (0.4 g), so that each fish ingested 40 µg of ADNp.    From this experimental administration, the animals received feed with the same composition, but without incorporated pDNA, for a period of 60 days, taking 5 50 fish from each of the experimental batches at 7, 15, 30 and 60 days after the administration of ADNp.     23 Sampling After the fish was slaughtered, extremely scrupulous handling was carried out avoiding cross-contamination between animals that received plasmids and those that did not (controls).  Work was always started first with the control animals.  Samples of dorsal muscle, intestine and liver were extracted from all fish, and were immediately introduced into liquid nitrogen.    DNA extraction from fish tissues 10 The extraction procedure was based on the homogenization of samples of tissues previously sprayed in liquid N, and on the subsequent use of the commercial kit DNeasy Tissue Kit (Quiagen), according to the instructions manufacturer.  The quality of the extracted DNA was determined by electrophoretic separation in 0.8% agarose gels, and its quantification was carried out spectrophotometrically (Nanodrop 2000, Thermo 15 Scientific) by measuring the absorbance ratio at 260 and 280 nm.    Amplification of plasmid DNA fragments by PCR The DNA extracted from the different tissues was used in PCR reactions with specific primers for different areas of plasmid pCMVβ under the conditions described in Application Example 1.    For the identification of plasmid pCMVβ, the PCR products were resolved by 1% agarose electrophoresis using the primer pairs indicated in SEQUENCE LISTING section, with the numbers SEQ ID NO 1 to SEQ ID NO 6.  Simultaneously, the amplification of a fragment of the constitutive gene of gilthead 13-actin was used as a positive PCR control, using a pair of primers designed by our laboratory, whose sequences are indicated in the SEQUENCE LISTING section, and whose designation has been the following: 30 Actin couple, formed by the β-actinF primer, sense primer, with the number SEQ ID NO 7 in the attached sequence listing, and by the β-actinR primer, antisense primer, with the number SEQ ID NO 8 in the sequence listing.  This pair generates a 1325 base pair PCR product.   35 Detection and quantification of lacZ gene expression in muscle tissue The possible expression of the lacZ control gene contained in plasmid pCMVβ, involves the intracellular synthesis of the bacterial enzyme β-galactosidase, whose expression can be measured by an enzymatic assay.  Therefore, the presence of β-galactosidase activity in tissues, if any, would have as its only origin the expression of the bacterial lacZ gene contained in the pCMVβ expression vector in fish cells.  That is, its origin would be exogenous, since it is a bacterial enzyme that is not constitutionally present in fish.   This β-galactosidase activity can be quantified in the homogenates of the different tissues studied according to the procedure described by An et al.  (Expression of bacterial β-galactosidase in animal cells.  Mol Cell Biol. , 1982, volume 2, pages 1628-1632), using o-nitrophenyl-β-D-galactopyranoside (ONPG) as a substrate, and measuring spectrophotometrically at λ 420 nm in microplates the amount of o-nitrophenol released from the substrate by action of the enzyme β-galactosidase.     24 Results Evaluation of the incorporation of pDNA into the feed granules In Figure 7, two types of feed granules can be observed, one of them with 5 nanocapsules of pDNA incorporated in the dough before granulation (A) and the other type , with the same nanocapsules incorporated in its surface after immersion of the previously manufactured granule in a solution of said nanocapsules (B).  Under natural light (1) it is not possible to appreciate any difference.  However, once subjected to ultraviolet light (2), fluorescence emission from encapsulated cDNA stained with SYBR Green® can be observed throughout the mass of the granules A, but only on the surface in the granules B.    When thin sheets are obtained from said granules (3), by cross-sections, and observed under ultraviolet light (λ 312 nm), fluorescence emission from encapsulated pDNA held with SYBR Green® on the sheet can be checked A, but only on the outer surface of the sheet B.    PCR amplification of pDNA fragments present in fish tissues 20 The results of Fig.  8 show that in the control fish, which did not receive pDNA in any way, no fragments of the plasmid pCMVβ were detected in any of the tissues studied in view of the absence of PCR products when specific primers were used.  On the contrary, PCR products were obtained from the amplification of the DNA of the muscle, liver and intestine homogenates when the first pair of primers was used (F3237 / R3918, 682 bp, SEQ ID NO 1 and SEQ ID NO 2) at all sampling times (7, 15, 30 and 60 days) post-ingestion, when the pDNA was administered orally through the feed with nanocapsules (Fig.  9).    30 These results demonstrate that there has been a transfer of plasmid pCMVβ DNA when it is administered to fish encapsulated in chitosan nanoparticles and within the mass of the feed to the different tissues of the fish.  Another important fact is that this transfer has been extended in time up to at least 60 days.  This persistence of plasmids over time is within the range observed in previous studies, such as that of Tian et al.  (The formulation and immunization of oral poly (DL-Iactide-co-glycolide) microcapsules containing a plasmid vaccine against lymphocystis disease virus in Japanese flounder (Paralichthys olivaceus).  lnt lmmunopharm. , 2008, volume 8, pages 900-908) with alginate microcapsules and Tian et al.  (Chitosan microspheres as candidate plasmid vaccine carrier for oral immunization 40 of Japanese flounder (Paralichthys olivaceus).  Vet Immunol Immunopat. , 2008, volume 126, pages 220-229) with chitosan microcapsules, where a transfer of pDNA to different tissues (gills, intestine, spleen and kidney) of Japanese sole (Paralichthys olivaceus) was verified by RT-PCR at 10 and 90 days after oral administration.   45 When pDNA was administered without encapsulation and parenterally (IM, Fig.  10), only PCR products (682 bp) are observed for the primer pair F3237 / R3918 (SEQ ID NO 1 and SEQ ID NO 2, respectively) in the muscle at all sampling times (7, 15, 30 and 60 days).  On the contrary, no fragments of pDNA were observed either in the intestine or in the liver.    This result seems in line with the possible path through the anatomy of the pDNA fish during the process of plasmid migration from the injection site.25 to the rest of the tissues.  Thus, it seems reasonable that in the tissue closest to the injection site the amount of residual pDNA is greater than in tissues as distant as the intestinal mucosa and liver.    Quantification in muscle tissue of the β-galactosidase activity resulting from the expression of the lacZ gene contained in the plasmid pCMVβ administered orally and parenterally.    In Fig.  11 shows the measured β-galactosidase activity of the muscle, intestine and liver homogenates of both the control gilthead and the two groups at which 10 pDNA (feed and IM) were administered, at different sampling times (7, 15, 30 and 60 days).    The results indicate that there was a greater β-galactosidase activity in the muscle, intestine and liver extracts when the pDNA was administered orally by feed.  Activity values were higher in intestine and liver compared to muscle.  With respect to the administration of pDNA in free form and by parenteral route (IM), it was lower than that observed using the oral route, and the highest levels of activity in the muscle were detected.  The β-galactosidase activity in the control fish was nil.  This result is in line with the detection of 20 specific fragments of pDNA in the different tissues by PCR in the fish to which the plasmid was administered by any of the routes, while these sequences were not detected in the control fish.    In these application examples it has been possible to detect the expression of the exogenous enzyme 25β-galactosidase after oral or intramuscular administration of the control gene that codes for this enzyme.  This expression is long term, up to 60 days after its administration.  These results of persistence in the muscle coincide with those of Verri et al.  (Assessment of DNA vaccine potential for gilthead sea bream (Sparus aurata) by intramuscular injection of a reporter gene.  Fish Shellfish Immunol. , 2003, volume 15, 30 pages 283-295), who also observed transfer phenomena of up to 60 days when this same expression vector was injected intramuscularly also in gilthead.  In other studies in fish, it has been found that the total duration of expression of exogenous proteins encoded by genes transported in pDNA in tissues varies from less than 1 week (Rahman and Maclean, Fish transgene expression by 35 direct injection into fish muscle.  Mol Mar Biol Biotechnol. , 1992, volume 1, pages 286-289) over 115 days (Anderson et al. , Gene expression in rainbow trout (Oncorhynchus mykiss) following intramuscular injection of DNA Mol Mar Biol Biotechnol. , 1996, volume 5, pages 1 05-113) depending on the species and the gene considered, always using the intramuscular route.   40 Accordingly, expression of the control gene contained in pCMVβ has been demonstrated for β-galactosidase activity after oral and parenteral administration in gilthead.  On the other hand, these results propose the vehiculation and protection of encapsulated cDNA and included within the mass of the feed for oral administration, being able to be an adequate tool for the protection of the same against gastrointestinal barriers, ensuring its subsequent release in the target organs  Therefore, these results indicate the viability of this protection as a vehicle for DNA vaccines in fish.   fifty 

权利要求:
Claims (1)
[1]
26 CLAIMS 1. Vaccine or immunostimulating feed comprising circular or non-circular DNA molecules, either of natural or recombinant origin. 2. Product according to claim 1, wherein the DNA molecule is a DNA plasmid encapsulated in particles with a size between 10 nm and 10 mm. 3. Product according to claims 1 and 2 in which the particles can be nanoparticles of 10 to 1000 nm, microparticles of 1 µm to 1 mm, or macroparticles of 1 10 to 10 mm, of semi-solid consistency, homogeneous content, and stable in aqueous solutions. 4. Product according to claims 2 and 3 in which the particles are composed of DNA molecules copolymerized with alginate in concentrations between 0.2 and 4%, chitosan in concentrations between 0.01 and 3%, in addition to the following compounds: gelatin or polylactic-co-polyglycolic acid, cyclodextrins, agar, carrageenans, starch, guar gum, and combinations thereof. 5. I think according to the preceding claims, which includes in addition to the DNA trapped in 20 particles, other food ingredients authorized for use in animals. 6. Product according to the preceding claim where the feed is in the form of flour. pellet or spherical, cylindrical, crumb, sheet or tape. 7. Process for the preparation of the semisolid particles described in claims 2 to 4 comprising the following steps: a) mixing a DNA solution with the polymer solutions for food use described in claim 4 in a proportion of 1 : 5 to 1:10 by volume, b) formation of the spherical particles from the mixture obtained in step a). 8. Process according to claim 7, wherein step b) consists of intense stirring, from which nanoparticles are obtained. Process according to claim 7, wherein step b) consists of spraying on a gelling solution of CaCl2 in constant stirring at a concentration between 0.5 and 5%, from which microparticles are obtained. 10. Process according to claim 7, wherein step b) consists of dripping onto a gelling solution of CaCl2 under constant stirring at a concentration between 0.5 and 5%, from which macroparticles are obtained. 11 Process for obtaining the feed of claim 1 comprising the following steps: a) the homogeneous mixture of the ingredients authorized for animal feed. b) conditioning the mixture by adding hot water or steam. c) mechanical stirring of the conditioned mass,D) the addition to the conditioned mass of the particles containing the DNA according to claims 2 to 4. e) the simple granulation of the mixture obtaining a granulated feed with a diameter between 0.2 and 10 mm that contains a load between 10 and 100 µg DNA per g of feed. Process according to claim 11, in which between steps c) and d) an extrusion step of the conditioned mixture can be added consisting of: a) passing the mixture through a high pressure extruder screw and temperature, from which a wet mass of extruded food ingredients is obtained. b) adding hot water or steam to said extruded mixture to maintain a humidity above 50%, and a temperature between 45 and 95 ° C. 13. Process to obtain the product according to claim 1 whereby at the end of step e) of claim 11 a step is added to obtain the final formats of the feed described in claim 6 and which may or may not be followed by additional steps of spheronization, drying or lyophilization. twenty

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引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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ES550597A0|1985-12-31|1987-05-16|Valderas Arconada Julio|PROCEDURE FOR OBTAINING A COMPLETE FOOD FOR ANIMALS|

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ES2390428A1|2011-04-15|2012-11-13|Universidad De Almería|Prepared probiotic bacteria for oral management of cultivated fish based on encapsulation in hydrogels of alginate. |

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ES201600417A|ES2641601B1|2016-05-09|2016-05-09|Food preparation for animals that protects, orally vehicles and maintains the functionality of DNA molecules with interest in animal production and health, as well as the procedure for obtaining them|ES201600417A| ES2641601B1|2016-05-09|2016-05-09|Food preparation for animals that protects, orally vehicles and maintains the functionality of DNA molecules with interest in animal production and health, as well as the procedure for obtaining them|