WO2005084312A2 - Controle des maladies chez la crevette - Google Patents

Controle des maladies chez la crevette Download PDF

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Publication number
WO2005084312A2
WO2005084312A2 PCT/US2005/006626 US2005006626W WO2005084312A2 WO 2005084312 A2 WO2005084312 A2 WO 2005084312A2 US 2005006626 W US2005006626 W US 2005006626W WO 2005084312 A2 WO2005084312 A2 WO 2005084312A2
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seq
clone
nucleic acid
acid sequence
shrimp
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PCT/US2005/006626
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WO2005084312A3 (fr
WO2005084312A8 (fr
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Arun Kumar Dhar
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San Diego State University Foundation
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43509—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from crustaceans

Definitions

  • the invention pertains to the identification, monitoring, and treatment of infection in crustaceans, particularly shrimp.
  • the compositions and methods use nucleic acids and polypeptides identified by differentially expression in non-infected and infected crustaceans as therapeutics, diagnostics, and screening reagents.
  • white spot disease of shrimp caused by the white spot syndrome virus (WSSN) is considered to be the most important viral disease of cultured shrimp worldwide (Office International de Epizooties, 2002). Since the initial report of WSSN in East Asia during 1992 to 1993 (Inouye et al. 1994), WSSN has spread to much of Asia and the Americas causing catastrophic losses to shrimp farmers (Krishna et al. 1997; Jory and Dixon 1999). The cumulative loss due to WSSN in the Asia since 1992 is estimated to be $4-6 billion. The losses due to WSSN in the Americas have been estimated to $1-2 billion.
  • WSSN virions are ellipsoid to bacilliform in shape, enveloped with a tail-like appendage at one end of the particle.
  • the genome of WSSN contains a circular double- stranded D ⁇ A of -300 kb in length (van Hulten et al. 2001 ; Yang et al. 2001).
  • the WSSN has a morphological similarity with baculovirus, sequence analysis revealed that WSSV shares very little similarity with any known viruses (van Hulten et al, 2001 ; Yang et al. , 2001).
  • WSSV has been placed in a new family, the Nimaviridae, and a new genus, Whispovirus.
  • WSSN infects all commercially important species of penaeid shrimp and a number of other crustaceans, including crabs and crayfish (Flegel 1997). Since the initial report of WSSN in East Asia during 1992 to 1993 (Inouye et al. 1994), a number of WSSV-encoded genes, such as the capsid genes (van Hulten et al. 2000a; van Hulten et al. 2000b; Zhang et al. 2001; Chen et al. 2002; Marks et al. 2003), a ribonucleotide reductase gene (Tsai et al.
  • LGBP a pattern recognition protein
  • proPO prophenoloxidase
  • PRP pattern recognition protein
  • LGBP gene expression is upregulated in WSSV-infected animals, proPO gene expression is downregulated as the WSSV infection progresses, suggesting that WSSV infection regulates the activation and / or activity of the prophenoloxidase cascade in a novel way (Roux et al. , 2002).
  • a syntenin-like protein (TE8) with a post-synaptic density protein (PDZ) domain has been isolated from Penaeus monodon shrimp and was upregulation during WSSV-infection (Bangrak et al., 2002).
  • the shrimp syntenin- like protein may function as an adapter that couples the PDZ-binding protein to cell-to-cell signal transduction during WSSV pathogenesis (Bangrak et al., 2002).
  • Antiviral substances capable of binding to a variety of D ⁇ A and R ⁇ A viruses (Sindbis virus, vaccinia virus, vesicular somatitis virus, mengo virus, banzi virus and poliomyelitis virus) have been isolated from shrimp ⁇ Penaeus setiferus), although the genes representing these proteins have not yet been cloned (Pan et al, 2000).
  • These antiviral proteins probably represent a component of the innate immune response in shrimp (Pan et al, 2000).
  • This invention has identified cellular genes that play a critical role in viral, specifically white spot syndrome virus (WSSV), pathogenesis in shrimp, and viral pathogenesis in invertebrates in general.
  • WSSV white spot syndrome virus
  • Several immune genes in shrimp have been isolated that showed differential expression between healthy and WSSV-infected shrimp.
  • the nucleic acids comprising these genes, or fragments thereof, can used as potential targets for developing therapeutics against white spot disease and other viral, bacterial, and fungal diseases in shrimp.
  • the invention provides methods for screening for compounds for treating or diagnosing WSSV using the nucleic acids of the invention or the polypeptides they encode.
  • the invention provides compositions comprising, or consisting of, sets of differentially expressed genes selected by their differential expression on viral infection as compared to healthy shrimp expression profiles.
  • the viral infection is white spot syndrome virus, Taura syndrome virus, infectious hypodermal and hematopoietic virus, yellowhead virus or baculovirus penae.
  • the genes are selected from expressed sequence tag libraries designed by differential selection of genes that are up- or down-regulated during viral infection.
  • the viral infection is white spot syndrome virus.
  • the genes can the genes described herein, e.g., the nucleic acids as set forth in Table 2 and the appendices.
  • the viral infection is selected from white spot syndrome virus, Tarua syndrome virus, infectious hypodermal and hematopoietic virus, yellowhead virus and baculovirus penae.
  • the genes can be selected from the shrimp expressed sequence tag differential library used for the treatment of disease.
  • the disease can be selected from white spot syndrome virus, Tarua syndrome virus, infectious hypodermal and hematopoietic virus, yellowhead virus and baculovirus penae.
  • the differential library can be produced from shrimp infected with WSSV, or, from shrimp infected with viruses selected from white spot syndrome virus, Tarua syndrome virus, infectious hypodermal and hematopoietic virus, yellowhead virus and baculovirus penae.
  • the invention also provides methods of determining the metabolic or disease state of shrimp using the compositions described herein, including the polypeptides of the invention and the nucleic acids of the invention.
  • the invention provides isolated or recombinant nucleic acids having at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity, when compared and aligned for maximum correspondence, as measured using one any known sequence comparison algorithm, as discussed in detail below, or by visual inspection, to an exemplary
  • the invention provides isolated or recombinant polypeptides having at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 61%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity, when compared and aligned for maximum correspondence, as measured using one any known sequence comparison algorithm, as discussed in detail below, or by visual inspection, to an exemplary polypeptides of the invention, which include all polypeptides described herein, and include all polypeptides encoded by nucleic acids of the invention, which include all nucleic acids sequences
  • the invention provides nucleic acid and polypeptide sequences having substantial identity to an exemplary sequence of the invention over a region of at least about 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 70O, 750, 800, 850, 900, 950, 1000 or more residues.
  • ESTs expressed sequence tags isolated from Penaeid shrimp tissue that specifically correspond to the onset of viral disease.
  • ESTs expressed sequence tags
  • This approach can also be applied to diseases in shrimp caused other viruses, bacteria, fungi, as well as in other aquaculture species including fish and shellfish suffering from fungal, bacterial, and viral diseases.
  • the invention provides a method for treatment of both acute and chronic diseases via delivery of therapeutic recombinant protein(s) and/or nucleic acids.
  • ESTs differentially expressed on viral infection As a biopesticide.
  • the invention provides both a composition that can be used for prevention of disease, a method of prevention of disease, and diagnostic tools for evaluation of the health of the animals affected by disease.
  • FIG. 1 is a graphical representation of the function classes of genes isolated from hepatopancreas cDNA libraries of healthy and WSSV infected shrimp (P. vannamei) that showed similarities with the GenBank database entries;
  • FIG. 2 is a graphical representation of the differential expression of expressed sequence tags (ESTs) in white spot syndrome virus (WSSV) infected shrimp (Penaeus vannamei) compared to healthy shrimp (P. vannamei). Bars above the X-axis indicate up- regulation, and bar below the X-axis indicates down-regulation of the gene in WSSV-infected compared to healthy shrimp; and [0032] FIG.
  • ESTs expressed sequence tags
  • WSSV white spot syndrome virus
  • FIG. 3 is a graphical representation of the quantification of differently expressed immune genes in WSSV infected shrimp (P. vannemei) by SYBR Green real-time PCR. Bars above the axis represent up-regulated genes and bars below the axis represent down-regulated genes.
  • GILT gamma interferon inducible lysosomal thiol reductase
  • Interleukin enhancer-binding factor 3 3) tetraspanin-2
  • NF-kappa ⁇ essential modulator 5) Chitmase; 6) prophenoloxidase-activating proteinase 2; 7) p-selectin; 8) T-cell activation protein; 9) O- sialoglycoprotein endopeptidase; 10) ubiquitin; 11) anti-lipopolysaccharide factor.
  • nucleic acid sequence comprising a nucleic acid sequence of at least 65% identity to a nucleic acid sequence as set forth in as set forth in SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:l 1, SEQ ID NO: 13, SEQ ID NO:15, SEQ ID NO: 17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID O:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ
  • SEQ ID NO: 103 SEQ ID NO 105, SEQ ID NO 107, SEQ ID NO 109, SEQ ID NO:l ll, SEQ ID NO:113, SEQ ID NO 115, SEQ ID NO 117, SEQ ID NO 119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO 125, SEQ ID NO 127, SEQ ID NO 129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO 135, SEQ ID NO 137, SEQ ID NO 139, SEQ ID NO-.141, SEQ ID NO:143, SEQ ID NO 145, SEQ ID NO 147, SEQ ID NO 149, SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO 155, SEQ ID NO 157, SEQ ID NO 159, SEQ ID NO-.161, SEQ ID NO: 163, SEQ ID NO 165, SEQ ID NO 167, SEQ ID NO 169, SEQ ID NO:171, SEQ ID NO:17
  • the nucleic acid sequence has at least 95% identity. In another embodiment, the nucleic acid sequence has 100% identity.
  • the invention also provides the polypeptide encoded by the nucleic acid sequences disclosed herein.
  • a vector comprising the nucleic acid sequence provided herein, and a cell comprising said vector.
  • a feed or feed supplement comprising a recombinant protein or biologically active fragment thereof encoded by a nucleic acid sequence having at least 65% sequence identity to a shrimp gene or fragment thereof, wherein said shrimp gene is up-regulated during viral infection.
  • the shrimp gene is selected from a group of shrimp genes consisting of tetraspanin-2, P-selectin precursor, T-cell activation protein, anti-lipopolysaccharide factor, and heat shock protein STIl.
  • the shrimp gene is in clone PvWl 1 A5, clone PvW4E03, clone PvW5G04, clone PvHBl 1, or clone PvW10D06.
  • the protein or biologically active fragment thereof is encoded by a nucleic acid listed in Appendix A2. The protein can provided within a cell or provided as a disrupted cell.
  • a feed or feed supplement comprising a nucleic acid sequence of at least 65% sequence identity to a shrimp gene or biologically active portion thereof in a set of genes up-regulated by infection with a virus.
  • the nucleic acid sequence can be a nucleic acid sequence listed in Appendix A2.
  • the shrimp gene is selected from a group of shrimp genes consisting of tetraspanin-2, P-selectin precursor, T-cell activation protein, anti- lipopolysaccharide factor, and heat shock protein STIl.
  • the shrimp gene is contained in clone Pv l 1 A5, clone PvW4E03, clone PvW5G04, clone PvHBl 1, or clone PvWIODO ⁇ .
  • the nucleic acid sequence can be contained or provided within a cell or provided as a disrupted cell. In some embodiments, the nucleic acid sequence is produced synthetically.
  • a feed or feed supplement comprising a recombinant protein or biologically active fragment thereof encoded by a nucleic acid sequence with at least 65% or sequence identity to a nucleic acid sequence found in the set of shrimp genes down-regulated with viral infection.
  • the protein is gamma-interferon inducible lysosomal thiol reductase precursor, interleukin enhancer-binding factor 3, NF-kappaB essential modulator, chitinase, prophenoloxidase-activating proteinase 2, O-sialoglycoprotein endopeptidase, ubiquitin, or lysozyme.
  • the protein is encoded by a nucleic acid sequence selected from the group of shrimp nucleic acid sequence contained in clonePvHlA02, clone PvW8B06, clone PvW8E09, clone PvW9El l, clone PvW4F07, clone PvHC06, clone PvW04C06, and clone PvW10F4.
  • the protein or biologically active fragment thereof is encoded by a nucleic acid sequence listed in Appendix Al .
  • the protein can be provided within a cell or provided as a disrupted cell.
  • a feed or feed supplement comprising a nucleic acid sequence with at least 65% sequence identity to a shrimp gene or biologically active fragment thereof, wherein said nucleic acid sequence encodes a shrimp gene down-regulated at or during viral infection.
  • the nucleic acid sequence can be one listed in Appendix A.
  • the shrimp gene is gamma-interferon inducible lysosomal thiol reductase precursor, interleulcin enhancer-binding factor 3, NF-kappaB essential modulator, chitinase, prophenoloxidase- activating proteinase 2, O-sialoglycoprotein endopeptidase, ubiquitin, and lysozyme.
  • the shrimp nucleic acid sequence is contained in clonePvHl A02, clone PvW8B06, clone PvW8E09, clone PvW9Ell, clone PvW4F07, clone PvHC06, clone PvW04C06, and clone PvW10F4.
  • the nucleic acid sequence can be provided within a cell or provided as a disrupted cell. In some embodiments, the nucleic acid sequence is produced synthetically.
  • a therapeutic comprising a recombinant protein or biologically active portion thereof having at least 65% sequence identity to a shrimp protein encoded by a shrimp gene up-regulated during viral infection.
  • the shrimp gene is a nucleic acid sequence listed in Appendix A2.
  • the shrimp gene can be tetraspanin-2, P-selectin precursor, T-cell activation protein, anti-lipopolysaccharide factor, and heat shock protein STIl .
  • the shrimp gene is contained in clone PvWl 1 A5, clone PvW4E03, clone PvW5G04, clone PvHBl 1, and clone PvW10D06.
  • the protein or biologically active portion thereof can be delivered orally, by immersion, by injection, or any suitable method.
  • a therapeutic comprising a nucleic acid sequence of at least 65% or greater sequence identity to a shrimp gene or biologically active fragment thereof that is up- regulated by infection with a virus.
  • the shrimp gene can be selected from those listed in Appendix A2.
  • the shrimp gene is tetraspanin-2, P-selectin precursor, T- cell activation protein, anti-lipopolysaccharide factor, and heat shock protein STIl.
  • the shrimp gene is contained in clone PvWl 1 A5, clone PvW4E03, clone PV 5G04, clone PvHBl 1, and clone PvW10DO6.
  • the nucleic acid sequence can be delivered orally, by immersion, by injection, or any suitable method that permits expression.
  • a therapeutic comprising a recombinant protein or biologically active fragment thereof encoded by a nucleic acid sequence with at least 65% sequence identity to a shrimp protein down-regulated by infection with a virus.
  • the protein or biologically active fragement thereof can be encoded by a nucleic acid sequence listed in Appendix Al .
  • the protein or biologically active fragment thereof is gamma-interferon inducible lysosomal thiol reductase precursor, interleukin enhancer-binding factor 3, NF- kappaB essential modulator, chitinase, prophenoloxidase-activating proteinase 2, O- sialoglycoprotein endopeptidase, ubiquitin, or lysozyme.
  • the protein or biologically active fragment thereof is contained in clonePvHl A02, clone PvW8B06, clone PvW8E09, clone PVW9E11, clone PVW4F07, clone PvHC06, clone PvW04C06, or clone PV T0F4.
  • the protein or biologically active portion thereof can be delivered orally, by immersion, by injection, or any suitable method.
  • a therapeutic comprising a nucleic acid sequence of at least 65% sequence identity to a shrimp gene or faction thereof down-regulated by infection with a virus.
  • the nucleic acid sequence can be one listed in Appendix Al .
  • the nucleic acid sequence is gamma-interferon inducible lysosomal thiol reductase precursor, interleukin enhancer-binding factor 3, NF-kappaB essential modulator, chitinase, prophenoloxidase-activating proteinase 2, O-sialoglycoprotein endopeptidase, ubiquitin, and lysozyme.
  • the nucleic acid sequence is contained in clone PvHlA02, clone PvW8B06, clone PVW8E09, clone PvW9El l, clone PvW4F07, clone PvHC06, clone PvW04C06, or clone PvW10F4.
  • the nucleic acid sequence can be delivered orally, by immersion, by injection, or any suitable method that permits expression.
  • a screening method for identifying a substrate of a protein that is up- regulated following infection by a pathogen comprising: (a) providing a polypeptide of the invention; or a polypeptide encoded by a nucleic acid of the invention; (b) providing a test substrate; and (c) contacting the polypeptide of step (a) with the test substrate of step (b) and detecting a decrease in the amount of substrate or an increase in the amount of reaction product, wherein a decrease in the amount of the substrate or an increase in the amount of a reaction product identifies the test substrate as the polypeptide substrate.
  • the protein can be encoded by a nucleic acid sequence selected from Appendix A2.
  • the protein is tetraspanin-2, P-selectin precursor, T-cell activation protein, anti-lipopolysaccharide factor, or heat shock protein STIl .
  • the protein is encoded by the nucleic acid seqeunce contained in clone PvWl 1 A5, clone PvW4E03, clone PvW5G04, clone PvHBl 1, or clone PvW10D06.
  • a screening method cfor identifying a substrate of a protein that is down-regulated following infection by a pathogen comprising: (a) providing a polypeptide of the invention; or a polypeptide encoded by a nucleic acid of the invention; (b) providing a test substrate; and (c) contacting the polypeptide of step (a) with the test substrate of step (b) and detecting a decrease in the amount of substrate or an increase in the amount of reaction product, wherein a decrease in the amount of the substrate or an increase in the amount of a reaction product identifies the test substrate as the polypeptide substrate.
  • the protein can be encoded by a nucleic acid sequenced selected from Appendix Al .
  • the protein is garnma-interferon inducible lysosomal thiol reductase precursor, interleukin enhancer-binding factor 3, NF-kappaB essential modulator, chitinase, prophenoloxidase-activating proteinase 2, O-sialoglycoprotein endopeptidase, ubiquitin, or lysozyme.
  • the protein is encoded by the nucleic acid sequence contained in clonePvHl A02, clone PvW8B06, clone PvW8E09, clone PvW9El 1, clone PvW4F07, clone PvHC06, clone PvW04C06, or clone PvW10F4.
  • hererin is a diagnostic kit for evaluation of infection in crustaceans in aquaculture and food processing comprising at least one differentially expressed gene as a nucleic acid or recombinant polypeptide, wherein the gene can be used to identify a crustacean as being infected or non-infected with the pathogen of interest; and optionally instructions for use of the kit.
  • the gene can be one selected from Appendix Al or Appendix A2.
  • Provided herein is a method for treating or preventing infection by a pathogen in an aquatic animal, comprising providing at least one differentially expressed gene as a nucleic acid or recombinant polypeptide, wherein the gene is up-regulated with infection by the pathogen.
  • the gene can be one selected from Appendix A2.
  • the gene is tetraspanin-2, P-selectin precursor, T-cell activation protein, anti-lipopolysaccharide factor, or heat shock protein STIl.
  • the gene is contained in clone PvWl 1A5, clone PvW4E03, clone PvW5G04, clone PvHBl 1, or clone PvW10D06.
  • a method for treating or preventing infection by a pathogen in an aquatic animal comprising providing at least one differentially expressed gene as a nucleic acid or recombinant polypeptide, wherein the gene is down-regulated with infection by the pathogen.
  • the gene can be one selected from Appendix Al .
  • the gene is gamma-interferon inducible lysosomal thiol reductase precursor, interleukin enhancer-binding factor 3, NF-kappaB essential modulator, chitinase, prophenoloxidase-activating proteinase 2, O-sialoglycoprotein endopeptidase, ubiquitin, or lysozyme.
  • the gene is the nucleic acid sequence contained clonePvHl A02, clone PvW8B06, clone PvW8E09, clone PvW9El 1, clone PvW4F07, clone PvHC06, clone PvW04C06, or clone PvW10F4.
  • the aquatic animal is a crustacean, preferably a shrimp.
  • an inhibitory RNA molecule comprising a nucleic acid sequence comprising a nucleic acid sequence or fragment thereof, or complementary sequence, of the nucleic acid sequence listed in Appendix Al or A2.
  • the inhibitory RNA molecule can be used in a method of preventing or treating infection of an aquatic animal comprising administering the inhibitory RNA molecule to the animal.
  • the inhibitory RNA molecule can be used in a feed or feed supplement.
  • the inhibitory RNA molecule can be provided in a cell or as a disrupted cell.
  • the cell can be a bacterial, yeast, insect, fish, crustacean, or mammalian cell.
  • Provided herein is a method of identifying shrimp lines that are resistant to viral, bacterial, or fungal diseases, comprising identifying at least one differentially expressed gene as a nucleic acid or protein, wherein the gene comprises a nucleic acid sequence provided in Appendix Al or Appendix A2.
  • a method of screening for a therapeutic that modulates infectious disease in an aquatic animal comprising identifying a compound which modulates the expression of the gene as a nucleic acid sequence or protein in an infected animal, wherein the gene comprises a nucleic acid sequence listed in Appendix Al or Appendix A2, whereby the compound is identified as a modulator of infectious disease when said modulation results in the ameriolation or prevention of one or more symptoms caused by the infection.
  • a microsatellite marker comprising at least one nucleic acid sequence listed in Appendix Al or Appendix A2.
  • a microarry comprising at least one nucleic acid sequence or biologically active fragment thereof listed in Appendix Al or Appendix A2.
  • a biopesticide comprising at least one nucleic acid sequence or biologically active fragment thereof listed in Appendix Al or Appendix A2.
  • a method for developing a biopesticide employing at least one nucleic acid sequence or biologically active fragment thereof listed in Appendix Al or Appendix A2.
  • a transgenic plant expressing a protein or biologically active fragment thereof encoded by a nucleic acid sequence disclosed in Appendix Al or Appendix A2.
  • aquaculture refers to the cultivation of aquatic organisms under controlled conditions.
  • An "aquatic organism” is an organism grown in water, either fresh- or saltwater.
  • Aquatic organisms include, but are not limited to, fish, e.g., bass, striped bass, tilapia, catfish, sea bream, rainbow trout, zebrafish, red drum, and carp; crustaceans, e.g., penaeid shrimp, brine shrimp, freshwater shrimp, and Artemia; and rotifers.
  • a "coding sequence” is an in-frame sequence of codons that (in view of the genetic code) correspond to or encode a protein or peptide sequence. Two coding sequences show similarity or homology to each other if the sequences or their complementary sequences encode the same or similar amino acids.
  • An "EST” or “expressed sequence tag” is a piece of nucleic acid that is found under specific conditions and is derived from a cDNA library. A unique DNA sequence derived from a cDNA library (therefore from a sequence which has been transcribed in some tissue or at some stage of development). The EST can be mapped, by a combination of genetic mapping procedures, to a unique locus in the genome and serves to identify that genetic locus.
  • a "feed” refers to a preparation providing nutritional value to an aquatic animals including, but not limited to fish, shrimp, lobsters, crawfish, ollusks, sponges, and jellyfish.
  • a “feed additive” is any substance added to feed, regardless of nutritional or therapeutic value.
  • the term “shrimp” refers to any of the group of crustaceans that are commonly cultured for aquaculture or captured in the wild fisheries. The term “shrimp” includes shrimp eggs, shrimp larvae, shrimp post-larvae and adult shrimp. The term “shrimp” and “prawn” will be used interchangeably throughout the specification.
  • antibody includes a peptide or polypeptide derived from, modeled after or substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, capable of specifically binding an antigen or epitope, see, e.g. FUNDAMENTAL IMMUNOLOGY, Third Edition, W.E.
  • antibody includes antigen-binding portions, i.e., "antigen binding sites," (e.g., fragments, subsequences, complementarity determining regions (CDRs)) that retain capacity to bind antigen, including (i) a Fab fragment, a monovalent fragment consisting of the VL, NH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (
  • array or “microarray” or “biochip” or “chip” as used herein is a plurality of target elements, each target element comprising a defined amount of one or more polypeptides (including antibodies) or nucleic acids immobilized onto a defined area of a substrate surface, as discussed in further detail, below.
  • expression cassette refers to a nucleotide sequence which is capable of affecting expression of a structural gene (i.e., a protein coding sequence, such as an recombinant protein encoded by a nucleic acid of the invention) in a host compatible with such sequences.
  • Expression cassettes include at least a promoter operably linked with the polypeptide coding sequence; and, optionally, with other sequences, e.g., transcription termination signals. Additional factors necessary or helpful in effecting expression may also be used, e.g., enhancers. Thus, expression cassettes also include plasmids, expression vectors, recombinant viruses, any form of recombinant "naked DNA” vector, and the like.
  • "Operably linked” as used herein refers to a functional relationship between two or more nucleic acid (e.g., DNA) segments. Typically, it refers to the functional relationship of transcriptional regulatory sequence to a transcribed sequence.
  • a promoter is operably linked to a coding sequence, such as a nucleic acid of the invention, if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell or other expression system.
  • promoter transcriptional regulatory sequences that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are c/s-acting.
  • some transcriptional regulatory sequences, such as enhancers need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance.
  • a "vector" comprises a nucleic acid which can infect, transfect, transiently or permanently transduce a cell.
  • a vector can be a naked nucleic acid, or a nucleic acid complexed with protein or lipid.
  • the vector optionally comprises viral or bacterial nucleic acids and/or proteins, and/or membranes (e.g., a cell membrane, a viral lipid envelope, etc.).
  • Vectors include, but are not limited to replicons (e.g., RNA replicons, bacteriophages) to which fragments of DNA may be attached and become replicated.
  • Vectors thus include, but are not limited to RNA, autonomous self-replicating circular or linear DNA or RNA (e.g., plasmids, viruses, and the like, see, e.g., U.S. Patent No.
  • promoter includes all sequences capable of driving transcription of a coding sequence in a cell, e.g., a plant cell.
  • promoters used in the constructs of the invention include cw-acting transcriptional control elements and regulatory sequences that are involved in regulating or modulating the timing and/or rate of transcription of a gene.
  • a promoter can be a cts-acting transcriptional control element, including an enhancer, a promoter, a transcription terminator, an origin of replication, a chromosomal integration sequence, 5' and 3' untranslated regions, or an intronic sequence, which are involved in transcriptional regulation.
  • These cis-acting sequences typically interact with proteins or other biomolecules to carry out (turn on/off, regulate, modulate, etc.) transcription.
  • “Constitutive” promoters are those that drive expression continuously under most environmental conditions and states of development or cell differentiation, "inducible” or “regulatable” promoters direct expression of the nucleic acid of the invention under the influence of environmental conditions or developmental conditions. Examples of environmental conditions that may affect transcription by inducible promoters include anaerobic conditions, elevated temperature, drought, or the presence of light. [0072] "Plasmids” can be commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accord with published procedures. Equivalent plasmids to those described herein are known in the art and will be apparent to the ordinarily skilled artisan.
  • gene includes a nucleic acid sequence comprising a segment of DNA involved in producing a transcription product (e.g., a message), which in turn is translated to produce a polypeptide chain, or regulates gene transcription, reproduction or stability.
  • Genes can include regions preceding and following the coding region, such as leader and trailer, promoters and enhancers, as well as, where applicable, intervening sequences (introns) between individual coding segments (exons).
  • nucleic acid or “nucleic acid sequence” includes oligonucleotide, nucleotide, polynucleotide, or to a fragment of any of these, to DNA or RNA (e.g., mRNA, rRNA, tRNA) of genomic or synthetic origin which may be single-stranded or double-stranded and may represent a sense or antisense strand, to peptide nucleic acid (PNA), or to any DNA- like or RNA-like material, natural or synthetic in origin, including, e.g., iRNA, ribonucleoproteins (e.g., iRNPs).
  • DNA or RNA e.g., mRNA, rRNA, tRNA
  • PNA peptide nucleic acid
  • DNA- like or RNA-like material natural or synthetic in origin, including, e.g., iRNA, ribonucleoproteins (e.g., iRNPs).
  • nucleic acids i.e., oligonucleotides, containing known analogues of natural nucleotides.
  • the term also encompasses nucleic-acidlike structures with synthetic backbones, see e.g., Mata (1997) Toxicol. Appl. PbLarmacol. 144:189-197; Strauss-Soukup (1997) Biochemistry 36:8692-8698; Straussense Nucleic Acid Drug Dev 6:153-156.
  • polypeptide and protein include amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain modified amino acids other than the 20 gene-encoded amino acids.
  • polypeptide also includes peptides and polypeptide fragments, motifs and the like. The term also includes glycosylated polypeptides.
  • the peptides and polypeptides of the invention also include all "mimetic” and “peptidomimetic” forms, as described in further detail, below.
  • biologically active fragment thereof thereof refers to a fragment of the protein or nucleic acid that retains at least one biological activity of the whole sequence.
  • the fragment retains all of the relevant biological activities of the protein or nucleic acid sequence.
  • the biologically active fragment of a protein is one that protects the organism growing in aquaculture from one or more deleterious effects of a pathogen, preferably a virus.
  • isolated includes a material removed from its original environment, e.g., the natural environment if it is naturally occurring.
  • a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated.
  • Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.
  • an isolated material or composition can also be a "purified" composition, i.e., it does not require absolute purity; rather, it is intended as a relative definition.
  • Individual nucleic acids obtained from a library can be conventionally purified to electrophoretic homogeneity.
  • the invention provides nucleic acids which have been purified from genomic DNA or from other sequences in a library or other environment by at least one, two, three, four, five or more orders of magnitude.
  • nucleic acids can include nucleic acids adjacent to a "backbone” nucleic acid to which it is not adjacent in its natural environment.
  • nucleic acids represent 5% or more of the number of nucleic acid inserts in a population of nucleic acid "backbone molecules.”
  • Backbone molecules include nucleic acids such as expression vectors, self-replicating nucleic acids, viruses, integrating nucleic acids, and other vectors or nucleic acids used to maintain or manipulate a nucleic acid insert of interest.
  • the (isolated, recombinant, enriched) nucleic acids represent 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 61%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more of the number of nucleic acid inserts in the population of recombinant backbone molecules.
  • Recombinant polypeptides or proteins refer to polypeptides or proteins produced by recombinant DNA techniques; e.g., produced from cells transformed by an exogenous DNA construct encoding the desired polypeptide or protein.
  • synthetic polypeptides or protein are those prepared by chemical synthesis, as described in further detail, below. [0079]
  • a promoter sequence can be "operably linked to" a coding sequence when RNA polymerase which initiates transcription at the promoter will transcribe the coding sequence into mRNA, as discussed further, below.
  • Oligonucleotide includes either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically synthesized. Such synthetic oligonucleotides have no 5' phosphate and thus will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide can ligate to a fragment that has not been dephosphorylated.
  • nucleic acids or polypeptides can refer to two or more sequences that have, e.g., at least about 50%, 51 >, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more nucleotide or amino acid residue (sequence) identity, when compared and aligned for maximum correspondence, as measured using one any known sequence comparison algorithm, as discussed in detail below, or by visual inspection.
  • the invention provides nucleic acid and polypeptide sequences having substantial identity to an exemplary sequence of the invention over a region of at least about 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 or more residues, or a region ranging from between about 50 residues to the full length of the nucleic acid or polypeptide.
  • Nucleic acid sequences of the invention can be substantially identical over the entire length of a polypeptide coding region.
  • a "substantially identical" amino acid sequence also can include a sequence that differs from a reference sequence by one or more conservative or non-conservative amino acid substitutions, deletions, or insertions, particularly when such a substitution occurs at a site that is not the active site of the molecule, and provided that the polypeptide essentially retains its functional properties.
  • a conservative amino acid substitution for example, substitutes one amino acid for another of the same class (e.g., substitution of one hydrophobic amino acid, such as isoleucine, valine, leucine, or methionine, for another, or substitution of one polar amino acid for another, such as substitution of arginine for lysine, glutamic acid for aspartic acid or glutamine for asparagine).
  • Hybridization includes the process by which a nucleic acid strand joins with a complementary strand through base pairing. Hybridization reactions can be sensitive and selective so that a particular sequence of interest can be identified even in samples in which it is present at low concentrations.
  • Stringent conditions can be defined by, for example, the concentrations of salt or formamide in the prehybridization and hybridization solutions, or by the hybridization temperature, and are well known in the art.
  • stringency can be increased by reducing the concentration of salt, increasing the concentration of formamide, or raising the hybridization temperature, altering the time of hybridization, as described in detail, below.
  • nucleic acids of the invention are defined by their ability to hybridize under various stringency conditions (e.g., high, medium, and low), as set forth herein.
  • “Variant” includes polynucleotides or polypeptides of the invention modified at one or more base pairs, codons, introns, exons, or amino acid residues (respectively) yet still retain the biological activity of the recombinant polypeptide of the invention.
  • the term "therapeutic” refers to a protein or biologically active fragment thereof, or a nucleic acid sequence encoding the protein or fragment thereof that can heal, or provide a remedial, palliative, or preventive effect on a pathologic process. Therapeutic substances and compounds can be used to treat medical diseases, disorders, conditions, or syndromes.
  • macroalgae refers to algae that form structures easily discemable with the naked eye in at least one life stage. Usually these organisms have secondary vascularization and organs. Examples of different groups containing macroalgae include, but are not limited to, the chlorophyta, rhodophyta, and phaeophyta.
  • microalgae include both prokaryotic and eukaryotic algae that are classed in many different genera. Prokaryotic algae are typically referred to as cyanobacteria or bluegreen algae.
  • Eukaryotic microalgae come from many different genera, some of which overlap with the macroalgae, but can be generally differentiated by their size and lack of defined organs. Microalgae can have specialized cell types. Examples of different groups containing microalgae include, but are not limited to the chlorophyta, rhodophyta, phaeophyta, dinophyta, euglenophyta, cyanophyta, prochlorophyta, and cryptophyta.
  • the term “up-regulated” refers to an increase of mRNA or protein expression in a crustacean infected with a pathogen, preferably a virus, when compared to an uninfected crustacean.
  • the term “down-regulated” refers to a decrease of mRNA or protein expression in a crustacean infected with a pathogen, preferably a virus, when compared to an uninfected crustacean.
  • ESTs nucleic acids, polypeptides, and fragments thereof [0090]
  • a composition consisting of a set of differentially expressed gene selected by their differential expression in viral-infected shrimp relative to non-virally- infected, or healthy shrimp.
  • Expressed sequence tag (EST) analysis is an effective, comprehensive, and relatively straightforward method of examining gene expression. This method is particularly useful for species where no or limited information is available regarding the genome of the species.
  • An EST approach was used to examine gene expression in different tissues (cephalothorax, eyestalks, and pleopod) in black tiger shrimp, P. monodon (Lehnert et al, 1999). The putative identities of many of these ESTs revealed the occurrence of tissue- specific expression that includes novel genes.
  • one aspect of the present invention is a composition of ESTs representing differentially expressed genes and substantially identical nucleic acids in the presence and absence of infection in crustaceans.
  • Any suitable crustacean can be analyzed.
  • the crustacean is a shrimp.
  • the shrimp is a P. vannamei shrimp.
  • Any suitable type of infection can be analyzed.
  • the infection is a viral infection.
  • the virus can be white spot syndrome virus, Taura syndrome virus, infectious hypodermal and hematopoietic virus, yellowhead virus or baculo"virus penae.
  • the virus is a member of the genus Whispovirus.
  • the virus is white spot syndrome virus (WSSV).
  • Differential expression of genes include, but are not limited to genes that are up or down regulated relative to the genes expressed in a non- infected crustacean.
  • differential gene expression is determined using E STs isolated from cDNA libraries of virally-infected shrimp and non-virally infected shrimp. The gene expression can be determined using the hepatopancreas tissue or the hemocyte population.
  • the hepatopancreas tissue is the source of the ESTs. Any suitable numbers of ESTs may be considered.
  • Gene expression can be analyzed at any time during infection, includiag but not limited to time periods representing the onset of infection or the resolving of infection as well as during acute and chronic infections.
  • the EST isolated specifically correspond to the onset of infection, particularly that of viral disease. It is contemplated that gene expression analysis may also be performed if the crustacean has more than one infective entity.
  • the ESTs are isolated from shrimp that non-iaifected or infected with WSSV at the onset of viral infection.
  • the invention provides isolated and recombinant nucleic acids, including ESTs.
  • the invention further provides probes comprising or consisting of nucleic acids, e.g., ESTs, of the invention.
  • the invention provides isolated or recombinant nucleic acids comprising a nucleic acid sequence having at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%o, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequence identity to an exemplary nucleic acid of the
  • the nucleic acid encodes at least one polypeptide having a biologic activity, and the sequence identities are determined by analysis with a sequence comparison algorithm or by a visual inspection.
  • the invention provides nucleic acids for use as probes, inhibitory molecules (e.g., antisense, iRNAs), transcriptional or translational regulation, and the like.
  • nucleic acids of the invention include isolated or recombinant nucleic acids comprising a nucleic acid sequence as set forth in Appendix A, and subsequences thereof, e.g., at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500 or more residues in length, or over the full length of a gene or transcript.
  • Another aspect of the invention is an isolated or recombinant nucleic acid including at least 10 consecutive bases of a nucleic acid sequence of the invention, sequences substantially identical thereto, and the sequences complementary thereto.
  • the invention provides isolated or recombinant nucleic acids comprising a sequence that hybridizes under stringent conditions to a nucleic acid of the invention, e.g., any one of the sequences in Appendix A.
  • the nucleic acid can be at least about 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500 or more residues in length or the full length of the gene or transcript.
  • the stringent conditions include a wash step comprising a wash in 0.1X SSC at a temperature of about 65°C for about 15 minutes.
  • the gene of interest is a lectin gene.
  • Lectin are known to plays a critical role in the innate immunity in vertebrates by activating complement factor after binding to the carbohydrate moieties on the surfaces of viral capsid proteins (Vorup- Jensen et al, 2000).
  • the C-type lectin exhibits CD4-independent binding of the envelope glycoprotein, gpl20, of human immunodefficiency virus (HIV) (Curtis et al, 1992).
  • Human MBL was also shown to bind to the envelope proteins of the influenza A virus, neutralizing the virus by inhibiting the spread of the virus and simultaneously activating the complement cascade (Kase et al, 1999; Malhotra et al., 1994).
  • nucleic acids of the invention can be made, isolated and/or manipulated by, e.g., cloning and expression of cDNA libraries, amplification of message or genomic DNA by PCR, and the like.
  • Techniques for the manipulation of nucleic acids such as, e.g., subcloning, labeling probes (e.g., random-primer labeling using Klenow polymerase, nick translation, amplification), sequencing, hybridization and the like are well described in the scientific and patent literature, see, e.g., Sambrook, ed., MOLECULAR CLONING: A LABORATORY MANUAL (2ND ED.), Vols.
  • the invention provides isolated or recombinant nucleic acids that hybridize under stringent conditions to an exemplary sequence of the invention, or a nucleic acid that encodes a polypeptide of the invention.
  • the stringent conditions can be highly stringent conditions, medium stringent conditions, low stringent conditions, including the high and reduced stringency conditions described herein. In one aspect, it is the stringency of the wash conditions that set forth the conditions which determine whether a nucleic acid is within the scope of the invention, as discussed below.
  • nucleic acids of the invention as defined by their ability to hybridize under stringent conditions can be between about five residues and the full length of nucleic acid of the invention; e.g., they can be at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 90, 100, 150, 200, 250, 300, 350, 00, 50, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, or more, residues in length. Nucleic acids shorter than full length are also included.
  • nucleic acids of the invention are defined by their ability to hybridize under high stringency comprises conditions of about 50% formamide at about 37°C to 42°C. In one aspect, nucleic acids of the invention are defined by their ability to hybridize under reduced stringency comprising conditions in about 35% to 25% formamide at about 30°C to 35°C.
  • nucleic acids of the invention are defined by their ability to hybridize under high stringency comprising conditions at 42°C in 50% formamide, 5X SSPE, 0.3% SDS, and a repetitive sequence blocking nucleic acid, such as cot-1 or salmon sperm DNA (e.g., 200 n/ml sheared and denatured salmon sperm DNA).
  • nucleic acids of the invention are defined by their ability to hybridize under reduced stringency conditions comprising 35% formamide at a reduced temperature of 35°C.
  • the filter may be washed with 6X SSC, 0.5% SDS at 50°C. These conditions are considered to be “moderate” conditions above 25% formamide and "low” conditions below 25% formamide.
  • a specific example of “moderate” hybridization conditions is when the above hybridization is conducted at 30% formamide.
  • a specific example of “low stringency” hybridization conditions is when the above hybridization is conducted at 10% formamide.
  • the temperature range corresponding to a particular level of stringency can be further narrowed by calculating the purine to pyrimidine ratio of the nucleic acid of interest and adjusting the temperature accordingly.
  • Nucleic acids of the invention are also defined by their ability to hybridize under high, medium, and low stringency conditions as set forth in Ausubel and Sambrook. Variations on the above ranges and conditions are well known in the art. Hybridization conditions are discussed further, below.
  • the above procedure may be modified to identify nucleic acids having decreasing levels of homology to the probe sequence.
  • the hybridization temperature may be decreased in increments of 5°C from 68°C to 42°C in a hybridization buffer having a Na + concentration of approximately 1M.
  • the filter may be washed with 2X SSC, 0.5% SDS at the temperature of hybridization.
  • These conditions are considered to be "moderate” conditions above 50°C and "low” conditions below 50°C.
  • a specific example of “moderate” hybridization conditions is when the above hybridization is conducted at 55°C.
  • a specific example of "low stringency” hybridization conditions is when the above hybridization is conducted at 45°C.
  • the invention also provides nucleic acid probes that can be used, e.g., for identifying nucleic acids encoding a polypeptide with a biologic activity or fragments thereof or for identifying genes.
  • the probe comprises at least 10 consecutive bases of a nucleic acid of the invention.
  • a probe of the invention can be at least about 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150 or about 10 to 50, about 20 to 60 about 30 to 70, consecutive bases of a sequence as set forth in a nucleic acid of the invention.
  • the probes identify a nucleic acid by binding and/or hybridization.
  • the probes can be used in arrays of the invention, see discussion below, including, e.g., capillary arrays.
  • the probes of the invention can also be used to isolate other nucleic acids or polypeptides.
  • the invention provides expression vectors and cloning vehicles comprising nucleic acids of the invention, e.g., sequences encoding the recombinant proteins encoded by the nucleic acids of the invention.
  • Expression vectors and cloning vehicles of the invention can comprise viral particles, baculovirus, phage, plasmids, phagemids, cosmids, fosmids, bacterial artificial chromosomes, viral DNA (e.g., vaccinia, adenovirus, foul pox virus, pseudorabies and derivatives of SV40), PI -based artificial chromosomes, yeast plasmids, yeast artificial chromosomes, and any other vectors specific for specific hosts of interest (such as bacillus, Aspergillus and yeast).
  • the expression vectors and cloning vehicle comprise yeast plasmids.
  • Vectors of the invention can include chromosomal, non- chromosomal and synthetic DNA sequences. Large numbers of suitable vectors are known to those of skill in the art, and are commercially available.
  • the vector is a baculovirus.
  • the baculovirus is Autographica californica nuclear polyhedrosis virus (AcNPV). Vectors and molecular biology supplies, as well as methods for baculovirus expression vector systems, including AcNPV, are readily available from commercial suppliers
  • the expression vector can comprise a promoter, a ribosome binding site for translation initiation and a transcription terminator.
  • the vector may also include appropriate sequences for amplifying expression.
  • Mammalian expression vectors can comprise an origin of replication, any necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking non-transcribed sequences.
  • DNA sequences derived from the SV40 splice and polyadenylation sites may be used to provide the required non-transcribed genetic elements.
  • the expression vectors contain one or more selectable marker genes to permit selection of host cells containing the vector.
  • Vectors for expressing the polypeptide or fragment thereof in eukaryotic cells can also contain enhancers to increase expression levels.
  • Enhancers are cis-acting elements of DNA, usually from about 10 to about 300 bp in length that act on a promoter to increase its transcription.
  • the invention provides cloning vehicles comprising an expression cassette (e.g., a vector) of the invention or a nucleic acid of the invention.
  • the cloning vehicle can be a viral vector, a baculovirus, a plasmid, a phage, a phagemid, a cosmid, a fosmid, a bacteriophage or an artificial chromosome.
  • the viral vector can comprise an adenovirus vector, a retroviral vector or an adeno-associated viral vector.
  • the cloning vehicle can comprise a bacterial artificial chromosome (BAG), a plasmid, a bacteriophage PI -derived vector (PAC), a yeast artificial chromosome (YAC), or a mammalian artificial chromosome (MAC).
  • BAG bacterial artificial chromosome
  • PAC bacteriophage PI -derived vector
  • YAC yeast artificial chromosome
  • MAC mammalian artificial chromosome
  • the invention provides transformed cell comprising a nucleic acid of the invention or an expression cassette (e.g., a vector) of the invention, or a cloning vehicle of the invention.
  • the transformed cell can be a bacterial cell, a mammalian cell, a fungal cell, a yeast cell, an insect cell or a plant cell.
  • recombinant proteins derived from full-length gene or recombinant truncated amino acids derived from the functional domain of the gene identified using the EST differential libraries provided herein are provided herein.
  • the identified ESTs of interest can be used to clone full length cDNAs using methods known in the art. See e.g., U.S. Patent No. 6,265,165.
  • Recombinant polypeptides generated from these nucleic acids can be individually isolated or cloned and tested for a desired activity. Any recombinant expression system can be used, including bacterial, mammalian, yeast, insect or plant cell expression systems.
  • Recombinant truncated proteins of interest are those that retain at least a detectable amount of the desired biologic activity.
  • the invention provides fusion proteins and nucleic acids encoding them.
  • a polypeptide of the invention can be fused to a heterologous peptide or polypeptide, such as N- terminal identification peptides which impart desired characteristics, such as increased stability or simplified purification.
  • Peptides and polypeptides of the invention can also be synthesized and expressed as fusion proteins with one or more additional domains linked thereto for, e.g., producing a more protective peptide, to more readily isolate a recombinantly synthesized peptide, to identify and isolate antibodies and antibody-expressing B cells, and the like.
  • nucleic acids of the invention and nucleic acids encoding the polypeptides of the invention, or modified nucleic acids of the invention can be reproduced by amplification.
  • Amplification can also be used to clone or modify the nucleic acids of the invention.
  • the invention provides amplification primer sequence pairs for amplifying nucleic acids of the invention.
  • One of skill in the art can design amplification primer sequence pairs for any part of or the full length of these sequences.
  • the invention provides methods of producing a recombinant polypeptide comprising the steps of: (a) providing a nucleic acid of the invention operably linked to a promoter; and (b) expressing the nucleic acid of step (a) under conditions that allow expression of the polypeptide, thereby producing a recombinant polypeptide.
  • the method can further comprise transforming a host cell with the nucleic acid of step (a) followed by expressing the nucleic acid of step (a), thereby producing a recombinant polypeptide in a transformed cell.
  • Any suitable host cell can be used for the production of the recombinant protein or biologically active fragment thereof.
  • the invention provides isolated or recombinant antibodies that specifically bind to the recombinant polypeptides of the invention. These antibodies can be used to isolate, identify or quantify the recombinant polypeptides of the invention or related polypeptides. These antibodies can be used to isolate other polypeptides within the scope the invention.
  • the antibodies can be designed to bind to an active site of a recombinant polypeptide. Thus, the invention provides methods of inhibiting polypeptides using the antibodies of the invention.
  • the antibodies can be used in immunoprecipitation, staining, immunoaffinity columns, and the like.
  • nucleic acid sequences encoding for specific antigens can be generated by immunization followed by isolation of polypeptide or nucleic acid, amplification or cloning and immobilization of polypeptide onto an array of the invention.
  • the methods of the invention can be used to modify the structure of an antibody produced by a cell to be modified, e.g., an antibody's affinity can be increased or decreased.
  • the ability to make or modify antibodies can be a phenotype engineered into a cell by the methods of the invention.
  • Antibodies also can be generated in vitro, e.g., using recombinant antibody binding site expressing phage display libraries, in addition to the traditional in vivo methods using animals. See, e.g., Hoogenboom (1997) Trends Biotechnol. 15:62-70; Katz (1997) Annu. Rev. Biophys. Biomol. Struct. 26:27-45. [0124] Polyclonal antibodies generated against the polypeptides of the invention can be obtained by direct injection of the polypeptides into an animal or by administering the polypeptides to a non-human animal. The antibody so obtained will then bind the polypeptide itself.
  • any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique, the trioma technique, the human B-cell hybridoma technique, and the EBV- hybridoma technique (see, e.g., Cole (1985) in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
  • Another aspect of the invention is a method of protecting crustacean from infection using recombinant protein or nucleic acids derived from the full-length gene or recombinant truncated protein or nucleic acids derived from the functional domain of the gene identified using the EST differential libraries of the instant invention.
  • the infection is a viral infection.
  • the viral infection is WSSV.
  • the infection can also be one caused by bacteria, fungi, and the like.
  • the crustacean is a shrimp.
  • the recombinant protein or nucleic acid, or biologically active fragment thereof, can be administered in any suitable manner in a single dose or repeatedly.
  • the recombinant protein or nucleic acid can be administered alone or in combination with other treatment modalities for the infection.
  • a therapeutically effective amount of recombinant polypeptide or biolgically active fragment thereof is an amount that reduces or eliminates at least one symptom of the infection. In one embodiment, a therapeutically effective amount prevents infection or cures established infection.
  • the protein or biologically active fragment thereof can be one that is upregulated or downregulated following infection with a pathogen.
  • the up-regulation can be mediated by any mechanism including, but not limited to increased transcription, increased mRNA stability, decreased mRNA degradation, or increased translation.
  • the down-regulation can be mediated by any mechanism known in the art. Expression can be determined using routine methods.
  • Exemplary methods include those disclosed in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Ausubel et al., John Wiley & Sons, most recent edition). Infection with pathogen is readily determined using routine methods that include but are not limited to bioassays using indicator hosts, monitoring clinical signs, histopathology, dot blot and in situ hybridization using a virus specific probe, and PCR. See, e.g., Dhar et al., J. Clin. Microbiol. 39:2835-45 (2001).
  • the protein or biologically active fragment is one that enhances the innate, non-adaptive immunity of the organism. For example, in shrimp, immunity consists of a humoral and a cellular response.
  • the crustaceans are Pacific white shrimp (Penaeus vannamei) and the baculovirus is Autographa californica nuclear polyhedrosis virus (AcNPV).
  • the crustacean can be infected either by injection or orally by incorporating the virus into the crustacean's food.
  • the baculovirus can be engineered to express green fluorescent protein (GFP) for monitoring infection.
  • GFP green fluorescent protein
  • the therapeutic proteins can inhibit the growth or replication of bacteria (e.g., Vibrio) or viruses (e.g., Taura or White Spot virus).
  • the crustacean can be provided a feed or feed additive which incorporates recombinant protein or nucleic acids identified using the EST differential libraries of the instant invention as a prophylactic or therapeutic treatment for the deleterious effects of the virus on the host.
  • the present invention provides for a composition comprising at least one recombinant protein or nucleic acid, or biologically active fragment thereof, in a food source for the crustacean.
  • the invention provides edible enzyme delivery matrices comprising a polypeptide of the invention, e.g., a polypeptide encoded by the nucleic acid of the invention.
  • the protein or biologically active fragment thereof can be provided in a cell or in a disrupted cell.
  • the bioactive food complex can be preserved without drying as a semi-solid, moist paste, not a liquid, composed of microcapsules or beads, or as moist noodles, pellets, sheets or other forms, or can be stored frozen by employing cryopreservatives. See, e.g., U.S. Patent No. 5,698,246; 4,741,904.
  • the bioactive food complex can be added directly to aquaculture animal containment systems to be eaten by aquatic animals.
  • the bioactive food complex or the primary emulsion of the bioactive food complex can be added to pelleted or extruded aquatic feeds as a top-dress coating or enrobing of the pelleted or extruded aquatic feed.
  • the foodstuffs incorporated into the paniculate fish foods of this invention may be those which are normally used for other particulate fish foods. Frequently fish meal will provide at least 30% of the formulation and often it will provide at least 50% of the formulation.
  • Particulate fish foods are customarily manufactured by three routes, all of which involve some form of extrusion through a die. The methods are normally classified as compressed pelleting (also known as steam pelleting),/extruded pelleting and moist pelleting.
  • the feed of the invention may be adjusted for the requirements of the marine animal being fed as is l ⁇ iown.
  • the feed comprises animal protein, brine shrimp, egg product, betaine, alanine, isoleucine, leucine, serine, valine, glycine, astaxanthin, vitamin A supplement, vitamin B 12 supplement, riboflavin supplement, calcium pantothenate, niacin supplement, vitamin D3 supplement, vitamin E supplement, menadione sodium bisulfite complex, folic acid, biotin, thiamine, pyridoxine hydrochloride, inositol and choline chloride.
  • the particulate feed may also include medicaments.
  • an edible oil is employed.
  • Typical oils include fish oil, peanut oil, olive oil, com oil, sunflowerseed oil, cotton seed oil, soybean oil, rapeseed oil, coconut oil, and palm oil.
  • the oil may provide the omega-3 HUFA (highly unsaturated fatty acid) dietary requirements of marine shrimp and fish by providing EPA (eicosapentaenoic acid) and DHA (docosahexenoic acid).
  • amplified cDNA can be cloned into bacterial expression vector (such as pETlOO Directional TOPO Expression vector, Invitrogen Inc.) and used to transform BL 21 Star (DE3) One Shot Chemically Competent Escherichia coli (Invitrogen, Inc.) following the manufacturer's protocol.
  • Escherichia coli strain BL21 cells carrying IPTG inducible gene can be grown in LB medium containing ampicillin, and subsequently induced with IPTG to elicit expression of recombinant protein.
  • the expression of the recombinant protein will be empirically optimized to obtain maximum induction using routine methods.
  • Bacterial biomass containing cells, which express the shrimp recombinant protein, are then added to shrimp feed in a free or microbound format (in beads composed of alginate and starch in a polymeric form). Attractants can optionally be added to make the feed or beads more palatable to the target species.
  • krill meal is employed as the attractant.
  • RNAi reagents [0136]
  • the invention provides an RNA inhibitory molecule, a so-called "RNAi" molecule, comprising a recombinant protein or nucleic acid sequence of the invention to be administered as a prophylactic or therapeutic regimen for infection.
  • the RNAi molecule comprises a double-stranded RNA (dsRNA) molecule.
  • dsRNA double-stranded RNA
  • the RNAi can inhibit expression of a gene encoding a nucleic acid of the invention.
  • the RNAi is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more duplex nucleotides in length.
  • RNAi can enter a cell and cause the degradation of a single-stranded RNA (ssRNA) of similar or identical sequences, including endogenous mRNAs.
  • ssRNA single-stranded RNA
  • dsRNA double-stranded RNA
  • mRNA from the homologous gene is selectively degraded by a process called RNA interference (RNAi).
  • RNAi RNA interference
  • a possible basic mechanism behind RNAi is the breaking of a double-stranded RNA (dsRNA) matching a specific gene sequence into short pieces called short interfering RNA, which trigger the degradation of mRNA that matches its sequence.
  • the RNAi's of the invention are used in gene-silencing therapeutics, see, e.g., Shuey (2002) Drug Discov. Today 7: 1040-1046.
  • the invention provides methods to selectively degrade RNA using the RNAi's of the invention. The process may be practiced in vitro, ex vivo or in vivo.
  • the RNAi molecules of the invention can be used to generate a loss-of-function mutation in a cell, an organ or an animal. Methods for making and using RNAi molecules for selectively degrade RNA are well known in the art, see, e.g., U.S. Patent No.
  • a library of ESTs as well as cDNAs are provided for use in functional genomic analysis.
  • a variety of apparatus and methodologies can be used to in conjunction with the polypeptides and nucleic acids of the invention, e.g., to screen polypeptides for biologic activity, to screen compounds as potential modulators, e.g., activators or inl ibitors, of an activity of a recombinant polypeptide encoded by a nucleic acid of the invention, for antibodies that bind to a polypeptide of the invention, for nucleic acids that hybridize to a nucleic acid of the invention, to screen for cells expressing a polypeptide of the invention and the like.
  • Nucleic acids or polypeptides of the invention can be immobilized to or applied to an array.
  • Arrays can be used to screen for or monitor libraries of compositions (e.g., small molecules, antibodies, nucleic acids, etc.) for their ability to bind to or modulate the activity of a nucleic acid or a polypeptide of the invention.
  • a monitored parameter is transcript expression of a gene.
  • One or more, or, all the transcripts of a cell can be measured by hybridization of a sample comprising transcripts of the cell, or, nucleic acids representative of or complementary to transcripts of a cell, by hybridization to immobilized nucleic acids on an array, or "biochip.”
  • array By using an “array” of nucleic acids on a microchip, some or all of the transcripts of a cell can be simultaneously quantified.
  • arcays comprising genomic nucleic acid can also be used to determine the genotype of a newly engineered strain made by the methods of the invention.
  • Polypeptide arrays can also be used to simultaneously quantify a plurality of proteins.
  • arrays are generically a plurality of “spots” or “target elements,” each target element comprising a defined amount of one or more biological molecules, e.g., oligonucleotides, immobilized onto a defined area of a substrate surface for specific binding to a sample molecule, e.g., mRNA transcripts.
  • biological molecules e.g., oligonucleotides
  • any known array and/or method of making and using arrays can be inco ⁇ orated in whole or in part, or variations thereof, as described, for example, in U.S. Patent Nos. 6,277,628; 6,277,489; 6,261,776; 6,258,606; 6,054,270; 6,048,695; 6,045,996; 6,022,963; 6,013,440; 5,965,452; 5,959,098; 5,856,174; 5,830,645; 5,770,456; 5,632,957; 5,556,752; 5,143,854; 5,807,522; 5,800,992; 5,744,305; 5,700,637; 5,556,752; 5,434,049; see also, e.g., WO 99/51773; WO 99/09217; WO 97/46313; WO 96/17958; see also, e.g., Johnston (1998) Curr.
  • baculovirus expressing shrimp chitinase gene can be used as a biological agent to infect and kill lepidopteran insects that infect agricultural crops.
  • Baculovimses have been widely used as a biological control agent in agricultural crop pest management. Further effectiveness can be achieved by co-expressing shrimp chitinase gene with the recombinant polypeptide or biologically active fragment thereof, and then spraying this recombinant baculovims on crop fields infected by an insect pest.
  • the invention provides methods for identifying a substrate of a recombinant polypeptide of interest, comprising the following steps: (a) providing a polypeptide of the invention; or a polypeptide encoded by a nucleic acid of the invention; (b) providing a test substrate; and (c) contacting the polypeptide of step (a) with the test substrate of step (b) and detecting a decrease in the amount of substrate or an increase in the amount of reaction product, wherein a decrease in the amount of the substrate or an increase in the amount of a reaction product identifies the test substrate as the polypeptide substrate.
  • the invention also provides methods of determining whether a test compound specifically binds to a polypeptide comprising the following steps: (a) expressing a nucleic acid or a vector comprising the nucleic acid under conditions permissive for translation of the nucleic acid to a polypeptide, wherein the nucleic acid comprises a nucleic acid of the invention, or, providing a polypeptide of the invention; (b) providing a test compound; (c) contacting the polypeptide with the test compound; and (d) determining whether the test compound of step (b) specifically binds to the polypeptide.
  • the invention provides methods for identifying a modulator of a recombinant polypeptide' s activity comprising the following steps: (a) providing a polypeptide of the invention or a polypeptide encoded by a nucleic acid of the invention; (b) providing a test compound; (c) contacting the polypeptide of step (a) with the test compound of step (b) and measuring an activity of the recombinant polypeptide, wherein a change in the recombinant polypeptide activity measured in the presence of the test compound compared to the activity in the absence of the test compound provides a determination that the test compound modulates the recombinant polypeptide activity.
  • the recombinant polypeptide activity can be measured by providing an recombinant polypeptide substrate and detecting a decrease in the amount of the substrate or an increase in the amount of a reaction product, or, an increase in the amount of the substrate or a decrease in the amount of a reaction product.
  • a decrease in the amount of the substrate or an increase in the amount of the reaction product with the test compound as compared to the amount of substrate or reaction product without the test compound identifies the test compound as an activator of recombinant polypeptide activity.
  • An increase in the amount of the substrate or a decrease in the amount of the reaction product with the test compound as compared to the amount of substrate or reaction product without the test compound identifies the test compound as an inhibitor of recombinant polypeptide activity.
  • kits comprising at least one differentially expressed gene as a nucleic acid or recombinant polypeptide, wherein the kit can be used to positively identify a crastacean as being infected or non-infected with the pathogen of interest. Such ldts are also useful for monitoring the course of infection.
  • the kit comprises at least one antibody or antigen binding fragment thereof that binds the polypeptide encoded by the EST of interest.
  • the kit can be in any suitable format, and in some embodiments includes instructions for use.
  • ESTs have been isolated from cDNA libraries of healthy and WSSV-infected shrimp hepatopancreas tissue. Two cDNA libraries were constructed from hepatopancreas tissues of healthy (PvH) and WSSV-infected (PvW) P. vannamei shrimp using SMART cDNA amplification method (BD Bioscience, California) and cloned into a plasmid vector (pAL16, Evrogen, Inc., Moscow, Russia). Randomly selected recombinant clones were sequenced using vector-derived primers (SP6 and T7).
  • SP6 and T7 vector-derived primers
  • PvH clones represented 269 singletons and 124 contigs (each contig contains between 2-20 clones), whereas the PvW clones represented 281 singletons and 211 contigs (each contig contains 2-20 clones).
  • the average GC content of the PvH clones was 0.427 ⁇ 0.005 and for the PvW clones was 0.424 ⁇ 0.0O5.
  • the PvH and PvW unigene sets were categorized into the following nine functional classes following published protocol (Adams et al, 1995): Cell division, cell signaling/ communication, cell structure, cell defense, gene/protein expression, metabolism, ribosomal proteins, unknown genes and no matches (Fig. 1, Table 1).
  • PvW library contained a significantly higher number of cell defense genes compared to the PvH library (19.4% vs. 8.1%), whereas the PvH library contained a significantly higher number of metabolic genes (37.5% vs. 25.6%) (Fig.l).
  • Table 2 Summary of immune genes isolated from healthy and WSSV-infected shrimp hepatopancreas cD ⁇ A libraries.
  • Example 2 Confirmation of differential expression of shrimp ESTs. ⁇ 0150] As a proof of principle, the differential expressions of three shrimp ESTs were confirmed by examining their mRNA expression in healthy and WSSV-infected shrimp by real-time RT-PCR assay. These genes include a ⁇ interferon inducible lysosomal thiol reductase (GILT) gene, a lectin gene named P-selectin, and a chitinase gene.
  • GILT ⁇ interferon inducible lysosomal thiol reductase
  • primers were designed using Primer Express software (Applied Biosystem, Inc.). The list of primers is given below (Table 6). Table 6. List of primers used for measuring the expression of immune genes in healthy and white spot syndrome virus (WSSV) infected shrimp (Penaeus vannamei) by real-time RT-PCR.
  • WSSV white spot syndrome virus
  • the real-time RT-PCR assay showed that shrimp ⁇ interferon inducible lysosomal thiol reductase (GILT) , P-selectin, and the chitinase genes showed differential expression between healthy and WSSV-infected shrimp.
  • the mRNA expression of ⁇ interferon inducible lysosomal thiol reductase (GILT) gene was down-regulated, where as the expression of chitinase and P-selectin was up-regulated in WSSV-infected shrimp.
  • the real-time PCR assay was performed using 5 healthy and 5 WSSV-infected laboratory challenged shrimp. The summary of the real-time RT-PCR assay for each of the three genes is provided below (Tables 7-9).
  • HI to H5 represents healthy animals
  • Wl to W5 represents WSSV-infected animals
  • CT represents threshold PCR cycle number
  • ⁇ CT represents the normalized Ct value. Normalized Ct value is obtained by subtracting the CT value of RT-PCR internal control gene, shrimp elongation factor- 1 alpha, from the CT value of the target gene. ⁇ CT obtained by subtracting the ⁇ CT of healthy from the ⁇ CT value for the WSSV-infected sample. Fold changes indicates the changes in target gene expression in the WSSV-infected compared to the healthy animals.
  • HI to H5 represents healthy animals
  • Wl to W5 represents WSSV-infected animals
  • CT represents threshold PCR cycle number
  • ⁇ CT represents the normalized Ct value. Normalized Ct value is obtained by subtracting the CT value of RT-PCR internal control gene, shrimp elongation factor- 1 alpha, from the CT value of the target gene. ⁇ CT obtained by subtracting the ⁇ CT of healthy from the ⁇ CT value for the WSSV-infected sample. Fold changes indicates the changes in target gene expression in the WSSV-infected compared to the healthy animals.
  • HI to H5 represents healthy animals
  • Wl to W5 represents WSSV-infected animals
  • CT represents threshold PCR cycle number
  • ⁇ CT represents the normalized Ct value.
  • Nonnalized Ct value is obtained by subtracting the CT value of RT-PCR internal control gene, shrimp elongation factor- 1 alpha, from the CT value of the target gene.
  • ⁇ CT obtained by subtracting the ⁇ CT of healthy from the ⁇ CT value for the WSSV-infected sample. Fold changes indicates the changes in target gene expression in the WSSV-infected compared to the healthy animals.
  • Example 3 Cloning of shrimp ESTs.
  • primers will be designed using primer express software (Applied Biosystems, Inc.). Primers will also be designed to amplify a truncated version of the gene (representing the functional domain only). Amplified cDNA will be cloned into bacterial expression vector (such as pETlOO Directional TOPO Expression vector, Invitrogen Inc.) and used to transform BL 21 Star (DE3) One Shot Chemically Competent Escherichia coli (Invitrogen, Inc.) following manufacturer's protocol.
  • bacterial expression vector such as pETlOO Directional TOPO Expression vector, Invitrogen Inc.
  • Example 4 Bacterial expression of shrimp recombinant protein and formulation into feed.
  • the expression of the recombinant protein will be either constitutive or under an induction system (e.g., induced by IPTG) and empirically optimized to obtain maximum protein expression.
  • Escherichia coli strain BL21 cells carrying IPTG inducible gene will be grown in LB medium containing ampicillin, and then will be induced with IPTG for the expression of recombinant protein.
  • Bacterial biomass containing cells, which express the shrimp recombinant protein will be added to shrimp feed in a free or microbound format (e.g., in beads composed of alginate and starch in a polymeric form). Attractants are added to make the feed or beads more palatable to the target species (in the case of shrimp, krill meal would be a good attractant).
  • Example 5 Method for protection of shrimp from WSSV infection.
  • Shrimp are fed either a control diet or a diet containing bacterial biomass expressing recombinant protein (Example 4). Animals are challenged with WSSV, and their survivability in response to viral infection will be measured. The WSSV load in the control and treatment samples is measured by real-time PCR following published protocol (Dhar et al 2001). The mRNA expression of the target gene will be measured in the treated and control animals using real-time RT-PCR to determine the difference in expression in two treatment groups following the published method (Dhar et al. 2003). Protection from viral challenge will be determined by an increased survival versus a control not fed the diet containing the WSSV recombinant protein.
  • Example 6 DNA method for protection of shrimp from WSSV infection.
  • Shrimp are immersed in a medium containing nucleic acid encapsulated in a liposome.
  • the DNA will be designed to express proteins that are upregulated during infection by WSSV as detemiined from the differential EST libraries.
  • the DNA is then absorbed through the gills or through gut lining and will then expressing the proteins in the tissues that absorb the DNA and thus providing protection against WSSV.
  • RNAi method for protection of shrimp from WSSV infection A portion of a gene or genes identified using a differential expression library from shrimp (healthy versus WSSV-infected) as described in the specification are used to model a small interfering RNA as described by Timmons et al (2001). This DNA is then fed to the shrimp via incorporation in a feed or feed supplement. As shown for C. elegans in previous research (Timmons et al. 2001), this can interfere with expression of specific genes. Genes that are up-regulated in the EST differential library that have no defense function maybe important to viral infection and will be down regulated using this RNAi and this will suppress or inhibit WSSV infection.
  • Example 8 Diagnostic for WSSV infection.
  • the differentially expressed genes that are tied directly to WSSV infection, or general viral infection, will be used in a multiplexed assay to determine the metabolic health of a cultured shrimp population. Compared to WSSV titer determination, which tell the relative amount of virus in the population, this diagnostic will determine the effect of the infection on the metabolic health of the population (progress of the disease compared to the growth of the pathogen).
  • Metabolic genes will be chosen from the EST library that are most influenced in the early, mid, and late stage infection.
  • Defense genes will be chosen from the EST libraries that are the best indicators of early, mid, and late stage infection.
  • ESTs isolated from the healthy and WSSV-infected shrimp can be used for developing therapeutics for not only WSSV but also for other viral diseases such as Taura syndrome disease caused by the Taura syndrome virus (TSV).
  • Taura disease caused by the TSV, has caused catastrophic losses in the Western Hemisphere, and more recently in Taiwan (Dhar et al, 2004).
  • the cDNA libraries described in Example 1 contained immune genes such as the low-density lipoprotein receptor (LDLr) gene (Table 2) that could be for developing therapeutics against the TSV.
  • LDLr low-density lipoprotein receptor
  • Hepatitis C virus has been shown to complex with LDL and interacts ⁇ vith the LDL receptor (Wunschmann et al, 2000).
  • Type 2 rhinovirus was shown to up-regulate LDL receptor expression on human tracheal epithelial cells (Suzuki et al, 2001) and binding of the HlV-transactivator protein is mediated by LDL-related protein (Liu et al, 2000).
  • Primers will be designed to amplify the full-length open reading frame (ORF) of shrimp LDLr using primer express software (Applied Biosystems, Inc.). Primers will also be designed to amplify a truncated version of the gene (representing the functional domain only).
  • Amplified cDNA will be cloned into bacterial expression vector (such as pETlOO Directional TOPO Expression vector, Invitrogen Inc.) and used to transform BL 21 Star (DE3) One Shot Chemically Competent Escherichia coli (Invitrogen, Inc.) following manufacturer's protocol.
  • Escherichia coli strain BL21 cells carrying IPTG inducible gene will be grown in LB medium containing ampicillin, and then will be induced with IPTG for the expression of recombinant protein. The expression of the recombinant protein will be empirically optimized to obtain maximum induction.
  • Bacterial biomass containing cells, which express the shrimp recombinant protein, will be added to shrimp feed in a free or microbound format (in beads composed of alginate and starch in a polymeric form). Attractants are added to make the feed or beads more palatable to the target species (in the case of shrimp, krill meal would be a good attractant).
  • Animals will be challenged with TSV, and their survivability in response to viral infection will be measured. The TSV load in the control and treatment samples is measured by real-time PCR following published protocol (Dhar et al. 2002).
  • the mRNA expression of the target gene will be measured in the treated and control animals using real-time RT-PCR to determine the difference in expression in two treatment groups following published method (Dhar et al. 2003). Protection from viral challenge will be determined by an increased survival versus a control not fed the diet containing the TSV recombinant protein.
  • Example 10 Use of recombinant lectins for controlling viral and bacterial diseases of shrimp and other aquaculture species.
  • the differentially expressed genes in shrimp included a number of lectin genes (see Table 2, Gene ID# PvH09A06, PvH13C0 ⁇ 4, and PvW04E03. Primers will be designed to amplify the full-length open reading frame (ORF) of shrimp lectins using primer express software (Applied Biosystems, Inc.). Primers will also be designed to amplify a truncated version of the gene (representing the functional domain only).
  • Amplified cDNA will be cloned into bacterial expression vector (such as pETlOO Directional TOPO Expression vector, Invitrogen Inc.) and used to transform BL 21 Star (DE3) One Shot Chemically Competent Escherichia coli (Invitrogen, Inc.) following manufacturer's protocol.
  • Escherichia coli strain BL21 cells carrying IPTG inducible gene will be grown in LB medium containing ampicillin, and then will be induced with IPTG for fJhe expression of recombinant protein. The expression of the recombinant protein will be empirically optimized to obtain maximum induction.
  • Bacterial biomass containing cells, whicr ⁇ express the shrimp recombinant protein will be added to shrimp feed in a free or microbound format (in beads composed of alginate and starch in a polymeric form). Attractants are added to make the feed or beads more palatable to the target species (in the case of shrimp, krill meal would be a good attractant).
  • Animals will be challenged with WSSV, TSV, yellohead virus (YHV), and infectious hypodermal and hematopoietic virus (IHHNV) and bacterial pathogens such, as Vibrio sp. including V. penaeicida, an important bacterial pathogen of shrimp. The survivability of shrimp after viral and bacterial challenge will be recorded.
  • the viral load (WSSV, TSV, YHV and IHHNV) will be measured by real-time PCR following published protocol (Dhar et al, 2001, 2002).
  • the mRNA expression of the lectin gene in the virus and bacterial-challenged animals will be niea-sured in the treated and control animals using realtime RT-PCR to determine the difference in expression in two treatment groups following published method (Dhar et al. 2003). Protection from viral challenge will be determined by an increased survival versus a control not fed the diet containing recombinant lectin.
  • homology modeling the tertiary structure of shrimp lectin genes will be determined.
  • Example 11 Method for protection of shrimp and other aquaculture species from bacterial diseases using recombinant anti-lipopolysaccharide protein.
  • Analysis of shrimp EST sequence revealed the presence of an anti- lipopolysaccharide (Anti-LPS) gene (see Table 2 EST ID #PvHBl 1).
  • Anti-LPS gene encodes for small a basic protein that binds and neutralizes LPS, and thus possesses a strong antibacterial effect on the growth of Gram-negative bacteria (Iwanaga et al., 1992; Ried et al., 1996).
  • Primers will be designed to amplify the full-length open reading frame (ORF) of shrimp anti-LPS gene using primer express software (Applied Biosystems, Inc.).
  • Primers will also be designed to amplify a truncated version of the gene (representing the functional domain only).
  • Amplified cDNA will be cloned into bacterial expression vector (such as pETlOO Directional TOPO Expression vector, Invitrogen Inc.) and used to transform BL 21 Star (DE3) One Shot Chemically Competent Escherichia coli (Invitrogen, Inc.) following manufacturer's protocol.
  • Escherichia coli strain BL21 cells carrying IPTG inducible gene will be grown in LB medium containing ampicillin, and then will be induced with IPTG for the expression of recombinant protein. The expression of the recombinant protein will be empirically optimized to obtain maximum induction.
  • Anti-LPS recombinant protein will be purified from the recombinant cells and its Gram-negative bacterial neutralizing activity will be assayed against a number of Gram-negative bacterial pathogens of shrimp, fish and terrestrial agricultural species e. g. Vibrio penaeicida, Vibrio anguillarium, Virio parahemolyticus, Lactococcus garviae, Pasteur ella piscicida. [0167] Bacterial biomass containing cells, which express the shrimp recombinant anti-LPS protein, will be added to shrimp feed in a free or microbound format (in beads composed of alginate and starch in a polymeric form).
  • Attractants are added to make the feed or beads more palatable to the target species (in the case of shrimp, krill meal would be a good attractant).
  • Shrimp will be challenges with the bacterial pathogen and mortality will be compared to control treatment where the animals will be fed pellet without containing any anti- LPS recombinant protein.
  • Example 12 Confirmation of differential expression of shrimp ESTs [0168] As a proof of principle, the differential expression of nine more ESTs in healthy and WSSV-infected slirimp were evaluated by real-time RT-PCR assay. These include Interleukin enhancer-binding factor 3, Tetraspanin-2, NF-kappaB essential modulator, Prophenoloxidase- activating proteinase 2, T-cell activation protein, O-sialoglycoprotein endopeptidase, Ubiquitin, Anti-lipopolysaccharide Factor, Lysozyme 2, and Heat-shock protein (Table 10 and 11; Fig. 3).
  • Interleukin enhancer-binding factor 3 Tetraspanin-2
  • NF-kappaB essential modulator Prophenoloxidase- activating proteinase 2
  • T-cell activation protein T-cell activation protein
  • O-sialoglycoprotein endopeptidase Ubiquitin
  • Taura syndrome (TS) disease caused by the Taura syndrome virus (TSV) pose a major threat to shrimp mariculture in both hemispheres.
  • TSV Taura syndrome virus
  • SPR. TSV-resistant
  • FG Fast Growth
  • a cDNA clone from a healthy Penaeus vannamei (clone ID#PvH04G08) showed similarity with the low-density lipoprotein receptor (LDLr) gene of human, mouse, Drosophila and Caenorhabditis elegans.
  • LDLr low-density lipoprotein receptor
  • the LDLr gene is a member of an evolutionarily conserved family of multifunctional receptors that binds to rhino viruses (Family Picornaviridae) and a variety of ligands. Upon binding to the ligands, LDLr transports the macro olecules through receptor- mediated endocytosis.
  • the LDLr gene expression was measured in both healthy and TSV-infected (acute and chronically infected) shrimp by real-time RT-PCR. LDLr mRNA expression was almost 4-fold higher in the healthy TSV-resistant SPR shrimp compared to the healthy FG TSV-susceptible line. In the SPR animals, LDLr expression increased upon TSV challenge (3.3 to 6.6-fold higher expression depending on the TSV load). In the FG TSV- susceptible acute phase animals, there was no increase in the LDLr expression. However, in the FG TSV-susceptible chronic phase animals, LDLr expression was 5-fold lower compared to the healthy control animals. These data indicate that LDLr expression is differentially modulated in the TSV-resistant and susceptible animals.
  • LDLr gene could be used as a potential target for developing therapeutics against Taura syndrome disease in shrimp.
  • SPR animals showed higher expression of LDLr compared to the TSV-susceptible line making LDLr gene as a candidate genetic marker for marker-assisted selection in developing TSV-resistant line of shrimp.
  • Table 10 List of primers used for measuring the expression of immune genes in healthy and white spot syndrome virus (WSSN) infected shrimp (Penaeus vannamei) by real-time RT-PCR.
  • WSSN white spot syndrome virus
  • Penaeus vannamei infected shrimp
  • Interleukin enhancer-binding faetor 3 7.093 ⁇ 0.392 7.120 ⁇ 0.491 -0.027 -1.0 PvWllA5
  • T-cell activation protein 12.587 ⁇ 0.280 11.925 ⁇ 0.375 +0.662 +1.6 PvHC06
  • Lysozyme 2 2.883 ⁇ 0.390 3.157 ⁇ -0.048 -0.274 -1.2 PvW10D06
  • Heat-shock protein 9.230 ⁇ 0.550 8.470 ⁇ 0.002 +Q.757 +1.7
  • VP35 nucleocapsid protein
  • Tsai MF et al. (2000b) Identification and characterization of a shrimp white spot syndrome virus (WSSV) gene that encodes a novel chimeric polypeptide of cellular-type thymidine kinase and thymidylate kinase. Virology 277:100-110.
  • WSSV shrimp white spot syndrome virus

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Abstract

L'invention concerne une composition et des méthodes d'utilisation de gènes exprimés de façon différentielle chez les crustacés, en particulier la crevette. L'invention concerne également des gènes jouant un rôle particulièrement important dans la capacité des crevettes à combattre les maladies virales, ainsi que leur sélection, leur amplification et leur utilisation à des fins utiles. L'invention concerne en outre des compositions utilisées dans l'alimentation, la thérapie, le diagnostic et la recherche. L'invention concerne, de plus, des méthodes visant à réduire l'effet d'une infection virale, en particulier celui du virus du syndrome du point blanc. L'invention concerne enfin les utilisations des gènes décrits dans la surveillance et le diagnostic d'une maladie virale.
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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009040136A1 (fr) * 2007-09-28 2009-04-02 Ghent University Procédés et compositions utilisables pour le traitement d'organismes aquatiques
JP2014143949A (ja) * 2013-01-29 2014-08-14 Univ Of Miyazaki マルチプレックス遺伝子解析によるエビ類の健康診断法
US8822427B2 (en) 2010-10-27 2014-09-02 Harrisvaccines Methods and compositions to protect aquatic invertebrates from disease
US8828961B2 (en) 2010-10-27 2014-09-09 Harrisvaccines Methods and compositions to protect aquatic invertebrates from disease
CN106699861A (zh) * 2016-11-22 2017-05-24 中国科学院海洋研究所 一种环状的合成多肽h及其应用和抗菌制剂
WO2018075971A1 (fr) * 2016-10-20 2018-04-26 Fred Hutchinson Cancer Research Center Systèmes et procédés de détection de cellules ou d'adn disséminés ou circulants
US10004797B2 (en) 2010-10-27 2018-06-26 Harrisvaccines, Inc. Method of rapidly producing improved vaccines for animals
US10329581B2 (en) 2015-12-18 2019-06-25 Dow Agrosciences Llc Ribosomal protein L40 (RPL40) nucleic acid molecules that confer resistance to coleopteran and hemipteran pests

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109232727B (zh) * 2018-09-05 2020-09-08 内蒙古科技大学 一种图书馆用的绿色无污染抗菌肽及其制备与应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE EST [Online] 16 January 2004 Database accession no. (CK572715) *
DATABASE GENBANK [Online] 02 November 1999 Database accession no. (AJ250830) *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009040136A1 (fr) * 2007-09-28 2009-04-02 Ghent University Procédés et compositions utilisables pour le traitement d'organismes aquatiques
US8822427B2 (en) 2010-10-27 2014-09-02 Harrisvaccines Methods and compositions to protect aquatic invertebrates from disease
US8828961B2 (en) 2010-10-27 2014-09-09 Harrisvaccines Methods and compositions to protect aquatic invertebrates from disease
US9650634B2 (en) 2010-10-27 2017-05-16 Harrisvaccines, Inc. Methods and compositions to protect aquatic invertebrates from disease
US10004797B2 (en) 2010-10-27 2018-06-26 Harrisvaccines, Inc. Method of rapidly producing improved vaccines for animals
JP2014143949A (ja) * 2013-01-29 2014-08-14 Univ Of Miyazaki マルチプレックス遺伝子解析によるエビ類の健康診断法
US10329581B2 (en) 2015-12-18 2019-06-25 Dow Agrosciences Llc Ribosomal protein L40 (RPL40) nucleic acid molecules that confer resistance to coleopteran and hemipteran pests
WO2018075971A1 (fr) * 2016-10-20 2018-04-26 Fred Hutchinson Cancer Research Center Systèmes et procédés de détection de cellules ou d'adn disséminés ou circulants
CN106699861A (zh) * 2016-11-22 2017-05-24 中国科学院海洋研究所 一种环状的合成多肽h及其应用和抗菌制剂
CN106699861B (zh) * 2016-11-22 2020-06-26 中国科学院海洋研究所 一种环状的合成多肽h及其应用和抗菌制剂

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