JP4472336B2 - Crustaceans as production systems for therapeutic proteins - Google Patents

Description

Background of the Invention The present invention relates to the production and use of proteins, nucleic acids, viruses and virus-like particles in crustaceans. Such products are produced by conventional methods of livestock or aquaculture and can have significant therapeutic potential in humans as well as animals. These products can be given to humans or animals either directly by consumption of the crustaceans themselves or after purification of the active substances produced by the crustaceans. Crustaceans produce the substance as a result of infection with a virus containing one or more genes for substance production in the crustacean. This method does not change the genetic material of the crustaceans themselves.

2. 2. Description of Related Art Certain human therapeutic proteins are currently expressed directly by directly modifying the genome of the plant, resulting in a protein that the plant has never produced before. Such plants are referred to as genetically modified crops (GMOs). Concerns about the use of GMOs in conventional foods have increased due to the potential for new allergens to be produced from the incorporated genes. Nevertheless, plants provide an excellent and inexpensive embodiment for producing certain human therapeutic proteins.

  Modern agricultural biotechnology has also established a mode of using plants as a production system for certain human therapeutic proteins without directly modifying the genome of the plant itself. In such cases, plants (eg tobacco) are infected by a virus carrying a gene for a human therapeutic protein (eg tobacco mosaic virus; TMV). In such cases, plant expression is modified without directly altering the plant DNA. The human therapeutic protein is then isolated, purified and used for human therapeutic purposes.

  Recombinant microorganisms such as bacteria, yeast and fungi have been utilized to produce human therapeutic proteins. However, such recombinant microorganisms generally do not produce an accurate mimic of proteins made in mammalian cells due to changes in post-translational modifications. Furthermore, such recombinant microorganisms have not been used for agricultural purposes on the premise that the whole organism is ingested. In both plants and microorganisms, recombinant organisms are used simply as factories, and then therapeutic proteins are isolated and purified prior to use.

  Certain recombinant proteins are produced in insect cells using an insect virus expression system (baculovirus). Such proteins are also produced in intact larvae after infection with modified baculoviruses. In either case, the insect cells or larvae are used as a factory for producing the protein of interest, and then the recombinant protein is purified for pharmaceutical purposes. In general, proteins produced by insect cells are mimics that are closer to proteins produced in mammalian cells because the post-translational modifications are more similar, and the method is generally much less expensive than production in mammalian cells It is. However, proteins produced by insect cells or larvae still require many purification steps before they can be used for therapy.

  Certain crustaceans (eg, brine shrimp) are used as feed for various aquaculture harvests (eg, fish or shrimp), as being representative of live feed to an important first larval stage . In many cases, a kind of microalgae of hoonen shrimp is “loaded” and the microalgae are delivered to larvae by consumption of hoonen shrimp. In this way, certain beneficial nutrients can be provided from algae to larvae through feed.

SUMMARY OF THE INVENTION An object of the present invention is to deliver a composition that is an animal feed or feed component comprising a therapeutic protein or peptide, and a therapeutic dose of a bioactive peptide or protein to an animal that consumes such feed. To provide the use of this feed for the purpose.

  In one embodiment, the invention provides a recombinant virus (eg, baculo) that expresses or carries a gene for expressing a protein of therapeutic value (eg, an antibody, or a protein having an immunological response or antibacterial ability). Provided is a feed containing crustaceans (for example, spinach shrimp) infected by a virus. Such feed would provide an oral vaccine for the species to be consumed.

  In another embodiment of the present invention, hoonen shrimp is used as a low-cost, large-scale production system for the target therapeutic protein. Such proteins can be consumed by humans or other animals as part of the total biomass of the shrimp, or the proteins can be extracted and purified to be taken orally, injected, or transdermally or nasally. It can also be provided as a conventional pharmaceutical product by any of the delivery.

  The marine environment is filled with bacteria and viruses that can attack fish and / or shellfish. Infection with such bacteria or viruses can devastate intensive marine farms very quickly. The same is true for terrestrial environments where viral or bacterial infections can dramatically limit farm productivity. Today, one of the major disease control problems in shrimp aquaculture is infection with certain viruses (eg, vitiligo, taura, etc.). Neither antibiotics nor probiotic strategies are effective in this state, and shrimp cannot be vaccinated in a manner similar to that used for fish. Like all crustaceans, shrimp have only an undeveloped immune system and are particularly susceptible to devastation from viral attacks.

  The present invention provides a solution to this problem by providing a nutritional control method using target animal feed as a vector for delivering antiviral antibodies or fragments thereof directly to shrimp. These “designer feeds” are those that deliver therapeutic doses of antibodies directly to the shrimp intestine. This approach is known as “passive immunity” because the antibody stays outside the host organism and only prevents viral entry through the intestinal wall. The present invention contemplates the use of transgenic multicellular organisms that deliver antibodies to the intestinal tract of a target animal by consumption of the transgenic multicellular organisms (plants, animals, insects, etc.). Alternatively, the feed source itself can be infected by a host-specific virus designed to produce the antibody of interest or fragment thereof in a multicellular organism that can be given to the target animal. Alternatively, the feed material may deliver a portion of the virus (eg, a coat protein) or fragment thereof to actively immunize shrimp, other shellfish or fish or terrestrial animals that consume the feed.

FIG. 1 shows Pacific white shrimp (Penaeus vannamei) orally infected with baculovirus (AcNPV-eGFP) modified to express GFP as a fusion protein. When observed 72 hours after infection, GFP-labeled recombinant baculovirus was localized in the hepatopancreas region and in the craniothoracic region of the shrimp.
Detailed Description of Embodiments The present invention is advantageous compared to microbial sources in the production and delivery of antimicrobial compounds, antibodies or therapeutic proteins within crustacean sources. Aseptic fermentation is important for pharmaceutical GMP compliance, while lower purity sources are no problem for food or feed utilization. Crustaceans are common elements in any food chain of aquatic or terrestrial species (including aquaculture or agriculture). One of the major problems with bacterial production of human proteins is that the recombinant proteins produced by microorganisms are ineffective due to incorrect post-translational modifications. Eukaryotic processes (eg, by crustacean species) provide post-translational modifications that may be more “natural” compared to recombinant products from bacteria.

  Antibodies or antibody fragments against the desired target, such as vitiligo virus or taura virus, can be obtained by screening normal immunity and selection of monoclonal antibody-producing hybridomas, or by screening viral or bacterial expression libraries of immunoglobulin genes and gene fragments Can be adjusted. See "Current Protocols in Immunology", edited by Collagen et al., Wiley-Interscience, 1991 and periodic augmentation. Nucleic acid sequences encoding the binding sites of selected antibodies can be obtained using standard methods (edited by Ausubel et al., “Current Protocols in Molecular Biology”, Wiley-Interscience 1987, and periodic augmentation). The antibody can be expressed from a recombinant plant, recombinant animal or recombinant insect, or can be cloned into a virus that infects the desired feed material.

  There are a number of well-known bactericidal and bacteriostatic peptides that inhibit microbial growth, including but not limited to cecropins, peneadins, bactenecins, callinectins, mycinin (Myticin), tachyplesin, clavanin, misgulin, misuro, pleurosidin, parasin, histones, acidic proteins and lysozymes. According to the present invention, these peptides are produced in crustacean hosts using recombinant methods well known to those skilled in the art and can be provided as feed ingredients that confer resistance or resistance to invasion in some cases. Crustaceans are food for many aquaculture species, and the present invention contemplates recombinant production of therapeutic proteins in the diet of juvenile fish (eg, semi-bred catfish) and larval fish in natural or farm farms Yes. Thus, the host organisms that constitute part of the food chain in larval, juvenile and adult feeding in aquaculture and the same life chain in the feed of terrestrial animals (eg, pigs, chickens and cows) Within the spirit of the invention.

  The edible material may be any material that is ingested. One embodiment of the invention is genetically modified such that the crustacean directly produces exogenous peptides and / or antibodies or antibody fragments, and the modified crustacean is ingested.

  Certain post-harvest processing may be required for the preparation of the materials used. The present invention contemplates standard (known) processing for converting crustacean material into food. Such standard processing includes homogenization followed by extrusion into pellets of various sizes depending on the application object (eg, larvae, juveniles and adults). Other modes of preparation also include spray drying, fluid bed drying, or providing the material as a liquid suspension. Alternatively, crustaceans expressing the therapeutic protein can be grown and harvested and then the fraction containing the therapeutic protein can be isolated from the crustacean. In some cases, additional processing steps known to those skilled in the art may be applied to further purify the therapeutic protein.

The invention contemplated herein is described in the following examples, but the utility is not limited to the examples provided.
Example 1. Production of viral antigens in hoonen shrimp.

Infectious pancreatic necrosis virus (IPNV) is an example of a virus that can cause high mortality in juvenile trout and juvenile salmon. In these animals, it is best to vaccinate as soon as possible. Therefore, it can be advantageous to deliver oral vaccines at an early juvenile stage. The IPNV genome contains two segments, the larger segment containing the genes for VP2, VP3 and VP4 virus coat proteins. Full-length cDNAs containing one or more virus coat protein genes and transfection markers (eg, green fluorescent protein-GFP) are prepared using conventional molecular biological techniques. This fragment is then ligated to a cloning site downstream of the P10 promoter region on a baculovirus transformation vector such as pAcUW21. Recombinant baculovirus containing both virus coat protein gene and GFP gene is used for transfection into hoonen shrimp. Infection and expression of the desired product (eg VP2) is monitored by the appearance of green fluorescence within the spinach shrimp. If the expression is satisfactory, the shrimp can be harvested and fed to juvenile fish. Ingestion of VP2-expressing hoonen shrimp by young fish will provide an inexpensive and effective oral vaccination method.
Example 2 Production of antibodies against vitiligo virus (WSV) in hoonen shrimp.
WSV contains three major viral coat proteins, and all three proteins can be used to prepare antibody fragments (Fabs), the genes for these Fabs are recombinant phage and E. coli. The antibody fragments (Fabs) can be prepared by expression in Coli. These Fabs genes can be prepared and isolated by techniques known to those skilled in the art. The gene is then spliced into a baculovirus vector and used to infect hoon prawns.

Starting with 1 to 5 mg of antigen (each of three viral proteins), two balb / c mice are immunized, the spleen is removed, and the spleen cells are isolated. Messenger RNA is extracted from splenocytes and purified on an oligo dT column. A cDNA library is generated and amplified by PCR prior to gel analysis. Bands are isolated from the gel and Vh and V chains are precipitated with ethanol. After assembly and fill-in reaction, a second amplification may be required before purification and isolation of purified ScFv. This DNA is then digested with Sfi and Not1 and ligated into a plasmid such as pCANTAB5E. The E. coli is then transformed, the recombinant phage library rescued, and screened three times to select antigen positive recombinant phage antibodies. The ScFv gene encoding the most specific Fab is then isolated from the plasmid and ligated into a baculovirus expression system. Such viruses may contain expression markers for transfection such as green fluorescent protein (GFP). Hoonen shrimp can then be infected with baculovirus and the degree of infection can be easily monitored by the intensity of the green color. In such cases, the infected shrimp also produce antibodies against WSV. Passive immunity is provided by using biomass as a feed additive and introducing antibodies or antibody fragments into animals.
Example 3 FIG. Expression of bactericidal or bacteriostatic proteins in spinach shrimp.

Bactericidal or bacteriostatic proteins are selected for the specific application. Preferred examples include penaeidin proteins for pathogenicity control in shrimp. Penaidins are one of a family of antimicrobial peptides isolated from crustaceans (eg, Penaeus shrimp). Antibacterial peptides can also be obtained from insects and antlers, including but not limited to cecropins, peneazines, bactenecins, carinectins, mytisines, tachypressins, clavanins, misgrins, preurocidins, paracin , Histones, acidic proteins and lysozymes. The gene for the selected protein or peptide can be isolated from either the original source or the source of amplification or can be made synthetically. The gene is spliced into a baculovirus vector that can also contain a green fluorescent protein (GFP) gene. The virus is then used to transfect hoonen shrimp and the production of bactericidal or bacteriostatic proteins is monitored by the expression intensity of GFP. Once the shrimp that produces the desired product at the appropriate level is isolated, it is added to the fish or shrimp diet. In this way, the bacteriostatic or bactericidal protein is delivered directly to the intestinal tract of the animal in the form of scallops themselves. Whole hounen shrimp can be used as feed or feed ingredients. Alternatively, crustaceans can be homogenized and extruded into pellets suitable for feed. Baculovirus vectors can be inactivated by high temperature prior to use as feed or by other methods well known to those skilled in the art.
Example 4 Expression of human insulin in hoonen shrimp.

Human insulin is a human therapeutic protein that can be produced in crustaceans. The gene for human insulin (or any other human therapeutic protein) is obtained or synthetically produced and ligated into a baculovirus expression vector. A baculovirus expression vector may also contain a transfection marker such as green (or red) fluorescent protein (GFP or R-FP). Baculoviruses are then used to infect crustacean cultures, such as shrimp. The expression level of the therapeutic protein is determined by the intensity of fluorescence generated by GFP or RFP. The crustacean is harvested at the point of maximum production of therapeutic protein. Brine shrimp can be used directly as a wet paste or can be dried by any suitable method that preserves the integrity of the product (eg, spray drying, extrusion, etc.). Alternatively, brine shrimp can be ground and the protein of interest isolated and purified by conventional biochemical methodologies. The purified protein can be used in oral, nasal, transdermal or injectable delivery forms.
Embodiment 5 FIG. Production from other crustaceans and rotifers.

The processed baculovirus described in any of Examples 1 to 4 can be infected with other crustaceans (eg, crabs, crayfish, lobster, etc.). Such infections may occur at a later stage in the life cycle so that fully mature crustaceans deliver drug products in a form that is much more acceptable as food. Alternatively, certain other crustaceans (longitudinal aunts, crabs, crayfish) may be used as therapeutic protein delivery vectors after the procedures of Examples 1-4. Alternatively, rotifers may be used as therapeutic protein delivery vectors after the procedures of Examples 1-4.
Example 6 Incorporation of therapeutic protein gene into baculovirus and use of infectious material as feed Pacific white shrimp (Penaeus vannamei) was orally transfected with processed baculovirus (AcNPV-cGFP) as a fusion protein GFP was expressed. The Bacmid Bac-to-Bac® baculovirus expression system (Invitrogen) was used for cloning and transfection. A 720 kb fragment containing GFP was fused to the polyhedron ((pPolh) promoter and ligated to pPolh at the XhoI site 3 '. Transfection of Sf9 insect cells with recombinant baculovirus using methods described in the Invitrogen product literature. After 72 hours, plaque formation was visually observed and 70 ml of culture medium was pelleted to 100 g for 5 minutes at 4 ° C. The resulting cell pellet was brought to 4 ° C. until used for subsequent oral transfection. The corresponding supernatant was centrifuged for 2 hours at 27,000 sucrose gradient, 4 ° C., 80,000 g to obtain purified virus, which was used for subsequent oral transfection. Until 4 ° C.

  An air stone for oxygenation was attached to a shrimp isolation chamber consisting of a 3 quart container filled with dechlorinated water with a salinity of 30 ppt. Three 1 gram shrimp were placed in each container and allowed to acclimate overnight.

The following procedure was performed within 30 minutes before feeding the shrimp. First, a pellet matrix was prepared by adding 100 mg of alginic acid (Sigma) to 10 ml of distilled deionized water (ddH ₂ O) in a beaker and heating to 40 ° C. with stirring. After the start of gel formation, 150 mg starch (Sigma) was added. After the solution was mixed for 1 minute, 500 mg of krill meal was added. The heat source was removed while stirring the solution.

An aliquot (500 μl) of the pellet matrix was combined with 5 μl of infected cell pellet or 5 μl of sucrose purified virus and gently mixed with a vortex mixer. The infected pellet matrix was aspirated into a tuberculin syringe followed by a 21 gauge needle. The forming solution was formed by dissolving 5 grams of calcium chloride (JT Baker) and 1 gram of sodium chloride (Research Organics) in 100 ml of ddH ₂ O. While slowly stirring the forming solution, the matrix was squeezed through the needle into this solution to form tubular pellets. Immediately upon extrusion, pellets formed in the solution and a spatula was used to clean the needle between the pellets. The pellet volume was seen as 25-30 μl. The pellet was washed in 10% NaCl and added to a shrimp isolation container. Shrimp consumed the pellets immediately, but gave them until they were full. Each shrimp consumed approximately one pellet.

  72 hours after consumption of the virus-infected matrix, shrimp were placed in a Petri dish and observed on a dark reader (Dark Reader®) transilluminator (Claire Chemical Research). Shrimps expressing GFP showed greenish light (FIG. 1). Uninfected shrimp showed no fluorescence. The GFP-labeled recombinant baculovirus observed at 72 hours was specifically located in the hepatopancreas region of the shrimp craniothoracic region (FIG. 1).

References This specification is best understood with reference to the following references, which are incorporated herein in their entirety.

  Edited by Ausubel et al. (1987 and regular enhancements) “Current Protocols in Immunology”, Wiley-Interscience.

  “Bac-to-Bac Baculovirus expression systems manual”, Invitrogen Life Technologies, Cat. No. 10359-016.

  Charfee. M.M. Tsu. Y. (Chalfie, M., Tu, Y.), Eukirchen. G. , Word. W. W. And Pusher. D. C. (1994) "Green Fluorescent Protein as a Marker for Gene Expression", Science, 263: 802-805.

  Edited by Collagen et al. (1991 and regular augmentation) “Current Protocols in Immunology”, Wiley-Interscience.

  Inoue. S. , And Tsuji. F. I. (1994) “Aequorea green-fluorescent protein: Expression of the gene and fluorescens characteristics of the recombinant protein”, FEBS Letters, pages 341-7.

  Lewis, David. L. , De Camillis, Mark. A. Brunetti, Craig. R. Halder. G. Kasner, Victoria. A. , Selegue. J. et al. E. Higgs. S. , And Carroll. S. B. (1999) "Ectopic gene expression and homeotropic transformations in artistic recombinant Sindbis viruses", Current Biology 9: 1279-1287.

  Pusher. D. C. Eckenrod. V. K. , Word. W. W. , Pendergast. F. G. And Cormier. M.M. J. et al. (1992), "Primary structure of the Aequorea victoria green-fluorescent protein", Gene, 111: 229-233.

Represents Pacific White Shrimp (Penaeus vannamel) orally transfected with baculovirus (AcNPV-eGFP) designed to express GFP as a fusion protein. The GFP-tagged recombinant baculovirus observed 72 hours after infection is specifically located in the hepatopancreas region of the shrimp craniothoracic region.

Claims (15)

A crustacean host infected with a recombinant infectious baculovirus , wherein the recombinant infectious baculovirus encodes and expresses one or more proteins that are foreign to the crustacean host and the infectious virus The foreign protein is therapeutically effective in a target animal different from the crustacean host and delivered to the target animal, and the genetic material of the crustacean host is modified by infection with the recombinant baculovirus infectious virus. Not a crustacean host . The crustacean host, anostraca, crayfish, lobster, a crab or shrimp, according to claim 1 crustacean host. The virus, including vitiligo virus of claim 1 crustacean host. The crustacean host according to any one of claims 1 to 3, wherein the virus encodes a foreign protein that is an antibacterial compound. The antibacterial compound is selected from cecropins, peneazines, bactenecins, carinectins, mytisines, tachypressins, clavanins, misgrins, preurocidins, paracins, histones, acidic proteins and lysozymes, Crustacean host according to claim 4. Wherein said virus is an antibody, antibody fragments, virus-like particles, and encoding a foreign protein is selected from the group consisting of hormones, crustaceans host according to any one of claims 1-3. The crustacean host according to any one of claims 1 to 3, wherein the protein is an antibody or an antibacterial agent and comes into contact with a disease infectious agent . The protein inhibits the growth or replication of bacteria, according to claim 7 crustaceans host. The crustacean host of claim 8, wherein the bacterium is a Vibrio species. A method of producing a protein or peptide is delivered to the target animal, the protein or peptide is encoded by the nucleic acid to be introduced into a recombinant infectious baculovirus, then the recombinant infectious baculovirus, the target animal is introduced into a different crustaceans host and produced in該甲shells such host, the protein or peptide is foreign to the crustacean host and the infectious virus, the genetic material of the crustacean host the recombinant A method that has not been modified by infection with a baculovirus infectious virus . 11. A method according to claim 10 , wherein the crustacean host is selected from honnen shrimp, crayfish, lobster, crab and shrimp. (A) preparing a cDNA clone of the desired protein ;
(B) by ligating the cDNA clone to a baculovirus transformation vector ;
Producing a recombinant infectious baculovirus ;
(C) said recombinant infectious baculovirus to infect to the crustacean host,
The method of claim 10 , further comprising: It said recombinant infectious baculovirus is a vitiligo virus A method according to any one of claims 10-12. A method for delivering a therapeutic protein to shrimp as a target animal, the method comprising feeding biomass containing a hoon shrimp host infected with a recombinant infectious baculovirus to the shrimp as a target animal, The infectious baculovirus encodes the therapeutic protein that is foreign to the hoonen shrimp host and the infectious virus, and the genetic material of the hoonen shrimp host has been modified by such infection by the recombinant infectious baculovirus No way. The crustacean host according to claim 1, which is contained in an aquaculture diet .

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