Patent Publication Number: US-2018028636-A1

Title: Methods and compositions for emergency post-infection treatment of anthrax

Description:
CROSS REFERENCE TO RELATED APPLICATION 
     This application is a continuation of U.S. patent application Ser. No. 13/922,719, filed on Jun. 20, 2013, which claims the benefit of U.S. provisional application No. 61/663,271, filed Jun. 22, 2012, all of which is incorporated by reference as if fully set forth. 
     The sequence listing electronically filed with this application titled “Sequence Listing,” created on Oct. 19, 2017, and having a file size of 92,962 bytes is incorporated herein by reference as if fully set forth. 
    
    
     FIELD OF INVENTION 
     The invention relates to methods and compositions to produce recombinant proteins in plants for the post-infection treatment or prophylaxis of the anthrax disease to neutralize the action of the anthrax toxin in the infected subjects. In particular the invention provides recombinant proteins, genetic constructs comprising polynucleotides encoding the proteins thereof, vectors and transgenic plants that include the genetic constructs. Methods for producing recombinant proteins, preparing compositions that include recombinant proteins and methods of protecting subjects by administering the compositions are also provided. 
     BACKGROUND 
     Despite the progress in studying mechanisms of anthrax, a zoonotic disease caused by the Gram-positive bacterium  Bacillus anthracis , a problem remains concerning the prevention and/or post-exposure treatment of the infection, especially with bio-terrorism threats. Although conventional vaccines against anthrax exist, induction and maintenance of adequate protection requires multiple immunizations followed by yearly boosters which frequently cause dangerous side effects (Rainy, Young, 2004). These side effects often inhibit the use of preventive vaccines among the human population. 
     SUMMARY 
     An aspect of the invention relates to a recombinant protein. The recombinant protein includes a first protein fused to a second protein. The first protein is a toxin binding ligand. The second protein is a carrier-protein. The carrier-protein is selected from the group consisting of: Fc-IgA, Fc-IgG, Fc-IgM, oleosin, and VP2 coat protein. 
     An aspect of the invention relates to a genetic construct. The genetic construct includes a first polynucleotide and a second polynucleotide. The first polynucleotide encodes a toxin binding ligand. The second polynucleotide encodes a carrier-protein. The carrier-protein is selected from the group consisting of: Fc-IgA, Fc-IgG, Fc-IgM, oleosin, and VP2 coat protein. 
     An aspect of the invention relates to a genetic construct. The genetic construct includes a sequence with at least 90% identity to a reference sequence selected from the group consisting of: SEQ ID NO: 16 (PgA1-3:B1-3), SEQ ID NO: 17 (PgA1-4:B1-4), SEQ ID NO: 18 (PgA1-3:B2-4), SEQ ID NO: 19 (PgA1-4:B2-14SEQ ID NO: 20 (PgA1-3:B3-3), SEQ ID NO:21 (PgA1-4:B3-4), SEQ ID NO: 22 (PgA1-3:B4-2), SEQ ID NO: 23 (PgA1-3:B5-2), SEQ ID NO: 37 (PgA2-4:B1-5), and SEQ ID NO: 38 (PgA1-5:B1-5). 
     An aspect of the invention relates to a transgenic plant. The transgenic plant comprises a genetic construct. The genetic construct includes a first polynucleotide and a second polynucleotide. The first polynucleotide encodes a toxin binding ligand. The second polynucleotide encodes a carrier-protein. The carrier-protein is selected from the group consisting of: Fc-IgA, Fc-IgG, Fc-IgM, oleosin, and VP2 coat protein. 
     An aspect of the invention relates to a method for producing a recombinant protein in a plant. The method includes contacting a plant with a genetic construct. The genetic construct includes a nucleic acid encoding the recombinant protein. The recombinant protein includes a toxin binding ligand fused to a carrier-protein. The carrier-protein is selected from the group consisting of: Fc-IgA, Fc-IgG, Fc-IgM, oleosin, and VP2 coat protein. The method also includes obtaining a plant including the genetic construct and expressing the recombinant protein. 
     An aspect of the invention relates to a method for preparing a composition effective for treating or preventing an anthrax infection in a subject. The method includes providing a recombinant protein produced by any method described herein. 
     An aspect of the invention relates to a method of protecting a subject against anthrax infection. The method includes providing a composition that includes a recombinant protein. The recombinant protein includes a toxin binding ligand fused to a carrier-protein. The carrier-protein is a protein selected from the group consisting of: Fc-IgA, Fc-IgG, Fc-IgM, oleosin, and VP2 coat protein. The composition is effective in preventing or reducing at least one symptom of an anthrax infection in a subject. The method also includes administering the composition to the subject in need thereof. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The following detailed description of the preferred embodiments will be better understood when read in conjunction with the appended drawings. For the purpose of illustration, there are shown in the drawings embodiments which are presently preferred. It is understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown. In the drawings: 
         FIG. 1A  is a schematic drawing illustrating the formation of anthrax toxin complexes and their entry into cells. 
         FIG. 1B  illustrates an infusion of a recombinant protein that includes an anthrax toxin binding ligand and a carrier protein (component A-B) into the bloodstream of the infected human or animal. 
         FIG. 2A  illustrates a comparison of a native antibody with a recombinant protein that includes a toxin binding receptor (TBL) fused to the Fc-region of the immunoglobulin.  FIG. 2B  illustrates possible configurations (quaternary structures) of self-assembling A1-B recombinant proteins. 
         FIG. 3A  illustrates a map of an intermediate plasmid that contains a nucleotide sequence encoding a synthetic gene PgA1B1. 
         FIG. 3B  illustrates a schematic drawing of an expression cassette included in the pBIN vector. 
         FIGS. 4A-4B  illustrate a pBI binary vector that including the TBL-Fc expression cassette ( FIG. 4A ) and steps for production of the recombinant TBL protein in plants ( FIG. 4B ). 
         FIGS. 5A-5F  illustrate in vitro selection of transgenic tobacco plants engineered to express a recombinant TBL protein. 
         FIG. 5A  illustrates formation of transgenic tobacco shoots on the medium supplemented with to 50 mg/L of kanamycin. 
         FIG. 5B  illustrates development of the transgenic tobacco shoots on selection medium. 
         FIG. 5C  illustrates the rooted transgenic tobacco plant grown in a Magenta box. 
         FIG. 5D  illustrates a transgenic tobacco plant grown on in soil. 
         FIG. 5E  illustrates PCR analysis of the transgenic tobacco plants using nptII-specific primers. 
         FIG. 5F  illustrates Western blot analysis of the transgenic tobacco lines using c-myc tag-specific antibodies. 
         FIGS. 6A-6E  illustrate in vitro selection of transgenic  Echinaceia angustifolia  plants engineered to express a recombinant TBL protein. 
         FIG. 6A  illustrates formation of the transgenic  Echinaceia angustifolia  shoot on the medium supplemented with to 50 mg/L of kanamycin. 
         FIG. 6B  illustrates mass-propagation of the transgenic  Echinaceia angustifolia  plants. 
         FIG. 6C  illustrates the rooted transgenic  Echinaceia angustifolia  plant grown in a Magenta box. 
         FIG. 6D  illustrates a transgenic  Echinacea angustifolia  plant growing on the selection medium supplemented with kanamycin. 
         FIG. 6E  illustrates Western blot analysis of the transgenic  Echinacea angustifolia  using c-myc tag-specific antibodies. 
         FIGS. 7A-7C  illustrate in vitro selection of transgenic  Kalanchoe pinnata  plants engineered to express a recombinant TBL protein 
         FIG. 7A  illustrates formation of the transgenic  Kalanchoe pinnata  shoot on the medium supplemented with to 50 mg/L of kanamycin. 
         FIG. 7B  illustrates propagation of the transgenic transgenic  Kalanchoe pinnata  shoots on the kanamycin selection medium. 
         FIG. 7C  shows rooting of  Kalanchoe pinnata  shoots on the kanamycin selection medium. 
         FIG. 8  illustrates steps of the method for producing and administering plant-derived compositions effective to prevent anthrax infection in a subject. 
         FIGS. 9A-9C  illustrates analysis of the PgA1-B1 proteins produced in plants. 
         FIG. 9A  illustrates Western blot analysis of transgenic plants expressing PgA1-B1 proteins. 
         FIG. 9B  illustrates Western blot analysis of a total and soluble protein extracted from transgenic tobacco plants. 
         FIG. 9C  illustrates extraction and purification of the plant PgA1-B1 recombinant protein from tobacco leaf tissue. 
         FIGS. 10A-10B  illustrates the ability of the recombinant protein PgA1-B1 to protect cells against the anthrax toxin. 
         FIG. 10A  illustrates an analysis of the PA binding by the recombinant protein PgA1-B1. 
         FIG. 10B  illustrates neutralization of the lethal toxin activity by the recombinant protein PgA1-B1. 
     
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     Certain terminology is used in the following description for convenience only and is not limiting. The words “right,” “left,” “top,” and “bottom” designate directions in the drawings to which reference is made. The words “a” and “one,” as used in the claims and in the corresponding portions of the specification, are defined as including one or more of the referenced item unless specifically stated otherwise. This terminology includes the words above specifically mentioned, derivatives thereof, and words of similar import. The phrase “at least one” followed by a list of two or more items, such as “A, B, or C,” means any individual one of A, B or C as well as any combination thereof. 
     The onset of acute symptoms of the anthrax disease occurs due to the effects of bacterial toxin consisting of non-lethal components: protective antigen (PA) combined with lethal factor (LF) or edema factor (EF) ( FIG. 1A ; see Young, Collier, 2007). PA 83  binds to cellular receptors where it is cleaved by a furin protease to PA 63 , which assembles into a heptameric pore. The pore can binds up to three units of LF, EF or both. Endocytosys of this structure leads to the entry of LF and/or EF into cytosol, where each factor causes its toxic effects. Neutralizing anthrax toxin activity could provide time for antibacterial agents or the immune system to clear up infection. Therefore, early post-infection treatment of anthrax infection with effective antitoxins that can block the action of toxin in vivo is important (Rainy, Young, 2004). 
     Embodiments herein provide technologies to express the chemically active recombinant anti-anthrax antitoxin in plants.  FIG. 1B  illustrates an infusion of recombinant proteins (Component A-B) into the bloodstream of the infected human or animal. Component A-B may be capable of binding PA 83  protein and preventing it from binding to the anthrax toxin receptor on the cell surface, thus protecting cells from translocation of other toxin component, EF or LF, into the cells. Production of active recombinant antitoxin protein in plants may be easily scaled up. The potential to produce large quantities of antitoxin protein may be useful for preindustrial and industrial scale production, during threats of bioterrorism and continuous outbreaks of anthrax infections. The plant-derived compositions may also be produced at a lower cost compared to traditional antibodies. An advantage of plant-derived antitoxin compositions is that these compositions are free of mammalian pathogens. 
     In an embodiment, a recombinant protein is provided. The recombinant protein may include a first protein fused to a second protein. The first protein may be a toxin binding ligand (referred to herein as “component A”). The toxin binding ligand may be capable of binding anthrax toxin with high affinity. 
     The toxin binding ligand may be a human or animal anthrax receptor (ATR) protein. The toxin binding ligand may be a Capillary Morphogenesis Protein 2 (CMG-2; component A1). The toxin binding ligand may be a soluble domain of the CMG-2. The toxin binding ligand may be a soluble domain of another ATR protein. The toxin binding ligand may be a soluble domain of another anthrax toxin-binding polypeptide. 
     The toxin binding ligand may be a polypeptide capable of high affinity binding to a protective antigen (PA) region necessary for PA interaction with a lethal factor (LF) or an edema factor (EF) components of the anthrax toxin. The toxin binding ligand may be a protective antigen binding domain of a lethal factor (PA-LF; component A2). 
     In an embodiment, the human capillary morphogenesis protein 2 may include, consist essentially of, or consist of a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to a reference sequence of: SEQ ID NO: 24 (A1-CMG2) or SEQ ID NO: 60 (PgA1-5). 
     In an embodiment, the PA-LFn protein may include, consist essentially of, or consist of a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to a reference sequence of: SEQ ID NO: 25 (A2/PA-LF). 
     In an embodiment, the toxin binding ligand alone may be capable of protecting a subject against anthrax toxin. 
     The second protein may be a carrier protein (referred to herein as “component B”). The carrier-protein may be capable of improving production, stability, solubility, extraction, secondary structure or other characteristics of the recombinant protein. The carrier-protein may be capable of improving translocation of the recombinant protein through biological membranes and its delivery into the bloodstream of an infected subject. The carrier protein may be capable of simplifying purification of the recombinant protein. 
     The carrier-protein may be an Fc (fragment crystallizable) region of an antibody capable of interacting with cell surface receptors called Fc receptors.  FIG. 2A  illustrates a comparison of a native antibody (left) with a recombinant protein that includes the toxin binding receptor fused to Fc-region of an immunoglobulin. The Fc-region may be but is not limited to an IgG isotype (Component B1), an IgA isotype (Component B2), or an IgM isotype (component B3). The Fc region of an antibody may be a single isotype. The Fc region may be a combination of any of IgA, IgG, or IgM isotypes. The Fc-region of an antibody may be combined with elements of several immunoglobulin isotypes that possess properties or have the natural ability of transport across the intestinal epithelium or penetrate higher cell- and mucose surfaces. The Fc-regions of recombinant proteins may be capable of assembling into quaternary structures consisting of several similar units. For example, the toxin binding ligand fused to an Fc-region of a human or an animal antibody under native conditions may be capable of forming dimers with another Fc molecule through the formation of disulfide bonds ( FIG. 2B ). The assembly of the individual Fc-molecules (monomers) into quaternary structures consisting of several similar units (dimers or multimers) may be facilitated by a helper element (referred to herein as “component C”). The helper element may facilitate self-assembly of the Fc-molecules into quaternary structures after bringing them into close proximity with each other, particularly after delivery into a human or a non-human animal organism. The helper element may provide a higher stability in a bloodstream, binding avidity for the anthrax toxin due to its multivalency and ability to induce host immune response to the bound anthrax toxin. The helper element may be an IgJ antibody. The helper element may be a VP1 coat protein of the JC virus.  FIG. 2B  illustrates possible configurations of self-assembling A1-B recombinant proteins, where the Fc fragment (component B) originates from an antibody (Ab) of different isotypes. This figure shows that the isotype IgG (B1) may form a bivalent structure (left). The isotype IgA (B2; middle) and the isotype IgM (B3; right) may be capable of binding multiple units together in the presence of “J,” a helper element, assisting in self-assembly of separate antibody molecules into dimmer or multimer structures. 
     The carrier-protein may be of virus origin. The carrier-protein may originate from a polyoma or a papiloma virus proteins capable of self-assembly into quaternary structures resembling virus like particles. The carrier protein may be a virus structural protein. The carrier protein may be a virus coat protein. The carrier protein may be a VP2 coat protein of the JC virus. 
     The carrier-protein may be capable of targeting itself as well as another covalently fused protein to plant cell oil bodies thus providing accumulation of the target protein in the plant lipid fraction, where it can be easily extracted with cheap available technologies. Particularly, the carrier-protein may be, but is not limited to, a plant oleosin. The oleosin may be capable of targeting the recombinant protein into a lipid fraction of plant cells and accumulating the recombinant protein in an outer surface of plant cell oil bodies, thus providing easy and cheap extraction of the protein from plant biomass (McLean et al., 2012,  Transgenic Res , Mar. 2, Epub). 
     The carrier-protein may be a protein capable of changing solubility under specific temperature condition, thus providing cheap and easy extraction using recently developed technologies available on market. The carrier-protein may be a thermo-stable protein or a thermo-labile protein. The carrier-protein may be a protein from thermophilic bacteria. The carrier-protein from the termophilic bacteria may be glucuronidase or lichenase B (U.S. Pat. No. 8,173,408, incorporated herein by reference as if fully set forth). The carrier-protein may be an Elastin-Like Polypeptides capable of undergoing a reversible, inverse phase transition and providing technical simplicity, low cost, ease of scale-up of extraction using “inverse transition cycling” technique (Hassouneh et al., 2010,  Curr Protocol Protein Sci  6:6.11; Meyer, Chilkoti, 1999,  Nat Rev. Immunol  5:905-916, all of which are incorporated by reference as if fully set forth). The carrier protein may be any other protein. 
     In an embodiment, the carrier-protein may include, consist essentially of, or consist of a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to a reference sequence selected from the group consisting of: SEQ ID NO: 26 (B1), SEQ ID NO: 27 (B2), SEQ ID NO: 28 (B3), SEQ ID NO: 29 (B4) and SEQ ID NO: 30 (B5). 
     In an embodiment, the helper element may include, consist essentially of, or consist of a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to a reference sequence selected from the group consisting of: SEQ NO 35 (C1-protein) and SEQ ID NO: 36 (C-protein). 
     The first protein or the second protein may be linked to a targeting peptide. The first protein or the second protein may be linked to a peptide tag for detection or purification. The detection or purification tag may be chosen from, but is not limited to, Poly-Arg, Poly-His, FLAG, Strep-tag II, c-myc, S-, HAT-, 3xFLAG, Calmodulin-binding peptide, Cellulose-binding domain, SBP, Chitin-binding domain, Glutation S-transferase, Maltose-binding protein, and Elastin-like peptide. 
     In an embodiment, the first protein may be fused to the second protein, or the purification tag using a flexible linker. The flexible linker may be a self-cleavable peptide. The flexible linker may be a peptide that is a site for cleaving with a specific protease, available in a composition or naturally present in mammal blood, providing, when necessary, release of the soluble toxin binding ligand from the carrier-protein or the tag during process of extraction or upon delivery of the recombinant protein into a bloodstream of a subject. 
     In an embodiment, the recombinant protein may include, consists essentially of, or consists of a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to a reference sequence of SEQ ID NO: 31 (PgA1-1:B1-1). 
     In an embodiment, a genetic construct comprising a first polynucleotide and a second polynucleotide is provided. The first polynucleotide may encode a toxin binding ligand. The first polynucleotide may encode a CMG-2 protein. The first polynucleotide may encode a PA-LF protein. 
     The first polynucleotide may include, consist essentially of, or consist of a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to a reference sequence selected from the group consisting of: SEQ ID NO: 1(PgA1-1), SEQ ID NO: 2 (PgA1-2), SEQ ID NO: 47 (PgA1-3), SEQ ID NO: 49 (PgA1-4) and SEQ ID NO: 58 (PgA1-5). The first polynucleotide may include, consist essentially of, or consist of a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to a reference sequence selected from the group consisting of: SEQ ID NO: 3 (PgA2-1), SEQ ID NO: 4(PgA2-2), SEQ ID NO: 5 (A2-3/PA-LF) and SEQ ID NO: 57 (PgA2-4). 
     The first polynucleotide may include a sequence capable of hybridizing under conditions of one of low, moderate, or high stringency to a reference sequence selected from the group consisting of: SEQ ID NO: 1(PgA1-1), SEQ ID NO: 2 (PgA1-2), SEQ ID NO: 47 (PgA1-3), SEQ ID NO: 49 (PgA1-4) and SEQ ID NO: 58 (PgA1-5). The first polynucleotide may include a sequence capable of hybridizing under conditions of one of low, moderate, or high stringency to a reference sequence selected from the group consisting of: SEQ ID NO: 3 (PgA2-1), SEQ ID NO: 4(PgA2-2), SEQ ID NO: 5 (A2-3/PA-LF) and SEQ ID NO: 57 (PgA2-4). 
     The second polynucleotide may include, consist essentially of, or consist of a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to a reference sequence selected from the group consisting of: SEQ ID NO:6 (PgB1-1), SEQ ID NO: 7 (PgB2-1), SEQ ID NO: 8 (PgB3-1), SEQ ID NO: 9 (PgB1-2), SEQ ID NO: 10 (PgB2-2), SEQ ID NO: 11 (PgB3-2), SEQ ID NO: 12 (PgB4-1), SEQ ID NO: 13 (PgB5-1), SEQ ID NO: 48 (PgB1-3), SEQ ID NO: 50 (PgB1-4), SEQ ID NO: 51 (PgB2-3), SEQ ID NO: 52 (PgB2-4), SEQ ID NO: 53 (PgB3-3), SEQ ID NO: 54 (PgB3-4), SEQ ID NO: 55 (PgB4-2), and SEQ ID NO: 56 (PgB5-2). 
     The second polynucleotide may include a sequence capable of hybridizing under conditions of one of low, moderate, or high stringency to a reference sequence selected from the group consisting of: SEQ ID NO:6 (PgB1-1), SEQ ID NO: 7 (PgB2-1), SEQ ID NO: 8 (PgB3-1), SEQ ID NO: 9 (PgB1-2), SEQ ID NO: 10 (PgB2-2), SEQ ID NO: 11 (PgB3-2), SEQ ID NO: 12 (PgB4-1), SEQ ID NO: 13 (PgB5-1), SEQ ID NO: 48 (PgB1-3), SEQ ID NO: 50 (PgB1-4), SEQ ID NO: 51 (PgB2-3), SEQ ID NO: 52 (PgB2-4), SEQ ID NO: 53 (PgB3-3), SEQ ID NO: 54 (PgB3-4), SEQ ID NO: 55 (PgB4-2), and SEQ ID NO: 56 (PgB5-2). 
     In an embodiment, the genetic construct may further include a third polynucleotide encoding a helper element. The third polynucleotide may include, consist essentially of, or consist of a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to a reference sequence selected from the group consisting of: SEQ ID NO: 14 (PgC1-1), SEQ ID NO: 15 (PgC2-1), SEQ ID NO: 32 (PgC1-2), SEQ ID NO: 33 (PgC1-3), and SEQ ID NO: 34 (PgC2-2). The third polynucleotide may include a sequence capable of hybridizing under conditions of one of low, moderate, or high stringency to a reference sequence selected from the group consisting of: SEQ ID NO: 14 (PgC1-1), SEQ ID NO: 15 (PgC2-1), SEQ ID NO: 32 (PgC1-2), SEQ ID NO: 33 (PgC1-3), and SEQ ID NO: 34 (PgC2-2). 
     In an embodiment, a genetic construct may include, consist essentially of, or consist of a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% to a reference sequence selected from the group consisting of: SEQ ID NO: 16 (PgA1-3:B1-3), SEQ ID NO: 17 (PgA1-4:B1-4), SEQ ID NO: 18 (PgA1-3:B2-4), SEQ ID NO: 19 (PgA1-4:B2-14SEQ ID NO: 20 (PgA1-3:B3-3), SEQ ID NO:21 (PgA1-4:B3-4), SEQ ID NO: 22 (PgA1-3:B4-2), SEQ ID NO: 23 (PgA1-3:B5-2), SEQ ID NO: 37 (PgA2-4:B1-5), and SEQ ID NO: 38 (PgA1-5:B1-5). The genetic construct may include a sequence capable of hybridizing under conditions of one of low, moderate, or high stringency to a reference sequence selected from the group consisting of: SEQ ID NO: 16 (PgA1-3:B1-3), SEQ ID NO: 17 (PgA1-4:B1-4), SEQ ID NO: 18 (PgA1-3:B2-4), SEQ ID NO: 19 (PgA1-4:B2-14SEQ ID NO: 20 (PgA1-3:B3-3), SEQ ID NO:21 (PgA1-4:B3-4), SEQ ID NO: 22 (PgA1-3:B4-2), SEQ ID NO: 23 (PgA1-3:B5-2), SEQ ID NO: 37 (PgA2-4:B1-5), and SEQ ID NO: 38 (PgA1-5:B1-5). 
     Determining percent identity of two amino acid sequences or two nucleic acid sequences may include aligning and comparing the amino acid residues or nucleotides at corresponding positions in the two sequences. If all positions in two sequences are occupied by identical amino acid residues or nucleotides then the sequences are said to be 100% identical. Percent identity may be measured by the Basic Local Alignment Search Tool (BLAST; Altschul, S. F., Gish, W., Miller, W., Myers, E. W. &amp; Lipman, D. J, 1990 “Basic local alignment search tool.” J. Mol. Biol. 215:403-410, which is incorporated herein by reference as if fully set forth). 
     In an embodiment, the toxin binding ligand may be a derivative of a human or non-human anthrax toxin receptor protein. The toxin binding ligand may be a derivative of a bacterial protein specifically binding PA protein. The toxin binding ligand may be a variant ATR from a human or a non-human animal. The toxin binding ligand may be a variant of  B. anthracis  LF and EF proteins. A variant may include an amino acid sequence with at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to an amino acid sequence of native toxin-binding proteins. 
     Variants may include conservative amino acid substitutions, i.e., substitutions with amino acids having similar properties. Conservative substitutions may be a polar for polar amino acid (Glycine (G), Serine (S), Threonine (T), Tyrosine (Y), Cysteine (C), Asparagine (N) and Glutamine (Q)); a non-polar for non-polar amino acid (Alanine (A), Valine (V), Thyptophan W), Leucine (L), Proline (P), Methionine (M), Phenilalanine (F)); acidic for acidic amino acid Aspartic acid (D), Glutamic acid (E)); basic for basic amino acid (Arginine (R), Histidine (H), Lysine (K)); charged for charged amino acids (Aspartic acid (D), Glutamic acid (E), Histidine (H), Lysine (K) and Arginine (R)); and a hydrophobic for hydrophobic amino acid (Alanine (A), Leucine (L), Isoleucine (I), Valine (V), Proline (P), Phenilalanine (F), Tryptophan (W) and Methionone (M)). Conservative nucleotide substitutions may be made in a nucleic acid sequence by substituting a codon for an amino acid with a different codon for the same amino acid. Variants may include non-conservative substitutions. 
     In an embodiment, fragments of a toxin binding ligand, a carrier protein or a helper element are provided. Fragments may include 100, 150, 200, 300, 400, 600, contiguous amino acids or more, such as 700. 
     In an embodiment, fragments of CMG2 or PA-LF proteins are provided. Fragments may include 100, 150, 200, 300, 400, 500 contiguous amino acids or more, such as 580. 
     The functionality of a recombinant protein, variants or fragments thereof, may be determined using any methods. The functionality may include conferring ability to bind the components of the anthrax toxin in a solution as determined by immunodetection methods. The functionality may be assessed using protection of cells growing in vitro. The functionality of a protein, or variants, or fragments thereof, may be assessed based on an ability to protect animals after administering of a recombinant antitoxin following of the infection of animals with the causative agent of anthrax. 
     In an embodiment, polynucleotides are provided having a sequence as set forth in any one of the nucleic acids listed herein or the complement thereof. In an embodiment, polynucleotides having a sequence that hybridizes to a nucleic acid having the sequence of any nucleic acid listed herein or the complement thereof are provided. In an embodiment, the hybridization conditions are low stringency conditions. In an embodiment, the hybridization conditions are moderate stringency conditions. In an embodiment, the hybridization conditions are high stringency conditions. Examples of hybridization protocols and methods for optimization of hybridization protocols are described in the following book: in Green and Sambrook. Molecular Cloning: a Laboratory Manual. 4th ed. Spring Harbor, N.Y: Cold Spring Harbor Laboratory Press, which is incorporated by reference in as if fully set forth. 
     Moderate conditions may be as follows: filters loaded with DNA samples are pretreated for 2-4 hours at 68° C. in a solution containing 6× citrate buffered saline (SSC; Amresco, Inc., Solon, Ohio), 0.5% sodium dodecyl sulfate (SDS; Amresco, Inc., Solon, Ohio), 5× Denhardt&#39;s solution (Amresco, Inc., Solon, Ohio), and denatured salmon sperm (Invitrogen Life Technologies, Inc. Carlsbad, Calif.). Hybridization is carried in the same solution with the following modifications: 0.01 M EDTA (Amresco, Inc., Solon, Ohio), 100 μg/ml salmon sperm DNA, and 5−20×10 6  cpm  32 P-labeled or fluorescently labeled probes. Filters are incubated in hybridization mixture for 16-20 hours and then washed for 15 minutes in a solution containing 2×SSC and 0.1% SDS. The wash solution is replaced for a second wash with a solution containing 0.1×SSC and 0.5% SDS and incubated an additional 2 hours at 20° C. to 29° C. below Tm (melting temperature in ° C.), where: 
         Tm= 81.5+16.61 Log 10 ([Na + ](1.0+0.7[Na + ]))+0.41(%[ G+C ])−(500/ n )− P−F;  
         [Na+]=Molar concentration of sodium ions;   %[G+C]=percent of G+C bases in DNA sequence;   N=length of DNA sequence in bases;   P=a temperature correction for % mismatched base pairs (˜1° C. per 1% mismatch);   F=correction for formamide concentration (=0.63° C. per 1% formamide).
 
Filters are exposed for development in an imager or by autoradiography.
       

     Low stringency conditions refers to hybridization conditions at low temperatures, for example, between 37° C. and 60° C., and the second wash with higher [Na + ] (up to 0.825M) and at a temperature 40° C. to 48° C. below Tm. High stringency refers to hybridization conditions at high temperatures, for example, over 68° C., and the second wash with [Na+]=0.0165 to 0.0330M at a temperature 5° C. to 10° C. below Tm. 
     In an embodiment, polynucleotides having a sequence that has at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity along its length to a contiguous portion of a nucleic acid having any one of the sequences set forth herein or the complements thereof are provided. The contiguous portion may be the entire length of a sequence set forth herein or the complement thereof. 
     In an embodiment, isolated nucleic acids, polynucleotides, or oligonucleotides are provided having a portion of the sequence as set forth in any one of the nucleic acids listed herein or the complement thereof. These isolated nucleic acids, polynucleotides, or oligonucleotides are not limited to but may have a length in the range from 10 to full length, 10 to 1000, 10 to 900, 10 to 800, 10 to 10 to 600, 10 to 500, 10 to 400, 10 to 300, 10 to 200, 10 to 100, 10 to 90, 10 to 80, 10 to 70, 10 to 60, 10 to 50, 10 to 40, 10 to 35, 10 to 30, 10 to 25, 10 to 20, 10 to 15, or 20 to 30 nucleotides or 10, 15, 20 or 25 nucleotides. An isolated nucleic acid, polynucleotide, or oligonucleotide having a length within one of the above ranges may have any specific length within the range recited, endpoints inclusive. The recited length of nucleotides may start at any single position within a reference sequence (i.e., any one of the nucleic acids herein) where enough nucleotides follow the single position to accommodate the recited length. In an embodiment, a hybridization probe or primer is 85 to 100%, 90 to 100%, 91 to 100%, 92 to 100%, 93 to 100%, 94 to 100%, 95 to 100%, 96 to 100%, 97 to 100%, 98 to 100%, 99 to 100%, or 100% complementary to a nucleic acid with the same length as the probe or primer and having a sequence chosen from a length of nucleotides corresponding to the probe or primer length within a portion of a sequence as set forth in any one of the nucleic acids listed herein. In an embodiment, a hybridization probe or primer hybridizes along its length to a corresponding length of a nucleic acid having the sequence as set forth in any one of the nucleic acids listed herein. In an embodiment, the hybridization conditions are low stringency. In an embodiment, the hybridization conditions are moderate stringency. In an embodiment, the hybridization conditions are high stringency. 
     The variants or fragments of the polynucleotides encoding the recombinant proteins may be identified, isolated or synthesized by any known methods. For optimal expression in a host cell, a polynucleotide sequence encoding a first and a second protein may be codon-optimized by adapting the codon usage to that most preferred in host genes. In case the host is a plant, codon usage may be optimized to native plant genes (Itakura et al. 1977 Science 198:1056; Bennetzen et al. 1982 J Mol Chem 257: 3026) using codon usage tables. Codon usage table are publicly available for various plant species (Nakamura et al. 2000 Nucl Acid Res 28: 292). 
     In an embodiment, a genetic construct having a nucleic acid encoding a recombinant protein may be provided in an expression cassette suitable for expression in plant cell, tissues, organs, and/or whole organism. 
     The expression of any one of the first and the second polynucleotide sequences or the third polynucleotide sequence of the genetic construct included the expression cassette may be under control of a promoter, which provides for transcription of the polynucleotide in a plant. The promoter may be a constitutive promoter, or tissue specific, or an inducible promoter. A constitutive promoter may provide transcription of the polynucleotides throughout most cells and tissues of the plant and during many stages of development but not necessarily all stages. An inducible promoter may initiate transcription of the polynucleotide sequences only when exposed to a particular chemical or environmental stimulus. A tissue specific promoter may be capable of initiating transcription in a particular plant tissue. Plant tissue may be, but is not limited to, a stem, leaves, trichomes, anthers, or seeds. Constitutive promoter may be, but is not limited to, the Cauliflower Mosaic Virus (CaMV) 35S promoter, the Cestrum Yellow Leaf Curling Virus promoter (CMP), or the CMP short version (CMPS), the Rubisco small subunit promoter, or the maize ubiquitin promoter. 
     An expression cassette may further include a terminator sequence, which terminates transcription the first and the second polynucleotide sequences or the third polynucleotide sequence and may be included at the 3′ end of a transcriptional unit of the expression cassette. The terminator may be derived from a variety of plant genes. The terminator may be derived from the nopaline synthase or octopine synthase genes of  Agrobacterium tumefaciens.    
     In an embodiment, an expression cassette is provided in a vector. For stable plant transformation, an expression cassette may be included in a T-DNA binary vector or a co-integrate vector. A vector may include multiple cloning sites to facilitate molecular cloning and selection markers to facilitate selection. A selection marker may be, but is not limited to, a neomycin phosphotransferase (npt) gene conferring resistance to kanamycin, a hygromycin phosphotransferase (hpt) gene conferring resistance to hygromycin, and a bar gene conferring resistance to phosphinothricin. 
     For transient expression of the toxin binding ligands, carrier proteins, or helper peptides in a plant, an expression cassette may be included in a viral-based vector. A viral-based vector may be obtained from a virus, which is not infectious for a mammalian object and therefore not requiring elimination of the vector components from the compositions herein. A viral-based vector may be, but is not limited to, a tobacco mosaic virus (TMV)-based vector or a potato virus X (PVX)-based vector. 
     In an embodiment, a transgenic plant is provided. The transgenic plant may include a genetic construct. The genetic construct may include a first polynucleotide and a second polynucleotide. The first polynucleotide may encode a toxin binding ligand. The second polynucleotide may encode a carrier-protein. The carrier-protein may be but is not limited to Fc-IgA, Fc-IgG, Fc-IgM, oleosin, or VP2 coat protein. The toxin binding ligand may be a capillary morphogenesis protein 2. The toxin binding ligand may be a PA-LF protein. The first polynucleotide may encode the capillary morphogenesis protein 2 or a PA-LF protein. The first polynucleotide may include, consist essentially of, or consisting of a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% to a reference sequence selected from the group consisting of: SEQ ID NO: 1(PgA1-1), SEQ ID NO: 2 (PgA1-2), SEQ ID NO: 3 (PgA2-1), SEQ ID NO: 4 (PgA2-2), SEQ ID NO: 5 (A2/PA-LF), SEQ ID NO: 47 (PgA1-3), SEQ ID NO: 49 (PgA1-4), SEQ ID NO: 57 (PgA2-4), and SEQ ID NO: 58 (PgA1-5). The second polynucleotide may include, consists essentially of, or consists of a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% to a reference sequence selected from the group consisting of: SEQ ID NO:6 (PgB1-1), SEQ ID NO: 7 (PgB2-1), SEQ ID NO: 8 (PgB3-1), SEQ ID NO: 9 (PgB1-2), SEQ ID NO: 10 (PgB2-2), SEQ ID NO: 11 (PgB3-2), SEQ ID NO: 12 (PgB4-1), SEQ ID NO: 13 (PgB5-1), SEQ ID NO: 48 (PgB1-3), SEQ ID NO: 50 (PgB1-4), SEQ ID NO: 51 (PgB2-3), SEQ ID NO: 52 (PgB2-4), SEQ ID NO: 53 (PgB3-3), SEQ ID NO: 54 (PgB3-4), SEQ ID NO: 55 (PgB4-2), and SEQ ID NO: 56 (PgB5-2). 
     The transgenic plant may further include a third polynucleotide. The third polynucleotide may encode a helper element. The third polynucleotide may include, consists essentially of, or consists of a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% to a reference sequence selected from the group consisting of: SEQ ID NO: 14 (PgC1-1), SEQ ID NO: 15 (PgC2-1), SEQ ID NO: 32 (PgC1-2), SEQ ID NO: 33 (PgC1-3), and SEQ ID NO: 34 (PgC2-2). 
     The transgenic plant may include a genetic construct that includes, consists essentially of, or consists of a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% to a reference sequence selected from the group consisting of: SEQ ID NO: 16 (PgA1-3:B1-3), SEQ ID NO: 17 (PgA1-4:B1-4), SEQ ID NO: 18 (PgA1-3:B2-4), SEQ ID NO: 19 (PgA1-4:B2-14SEQ ID NO: 20 
     (PgA1-3:B3-3), SEQ ID NO:21 (PgA1-4:B3-4), SEQ ID NO: 22 (PgA1-3:B4-2), SEQ ID NO: 23 (PgA1-3:B5-2), SEQ ID NO: 37 (PgA2-4:B1-5) and SEQ ID NO: 38 (PgA1-5:B 1-5). 
     The transgenic plant may be any plant, or a part of a plant. The part of a plant may be a stem, a leaf, a flower, a seed, or a callus. The transgenic plant may be a progeny, or descendant of a transgenic plant. The transgenic plant may be obtained through crossing of a transgenic plant and non-transgenic plant as long as it retains the genetic construct as described above. The transgenic plant may be a crop cultivated for purposes of obtaining food, feed or plant derived products including carbohydrates, oil, and medicinal ingredients. A crop plant may be selected from group consisting of: tomato, tobacco, pepper, eggplant, lettuce, sunflower, oilseed rape, broccoli, cauliflower and cabbage crops, cucumber, carrot, melon, watermelon, pumpkin, squash, sugar beet, peanut, chard, Swiss chard, soybean, cotton, beans, cassava, potatoes, sweet potato, okra, barley, pearl millet, wheat, rye, buckwheat, sorghum, rice. The transgenic plant may include forage grasses. The transgenic plant may include tree species and fleshy fruit species. The transgenic plants may include grapes, peaches, plums, cherries, strawberries, cranberries, mangos, and bananas. 
     The transgenic plant may be a medicinal plant. A medicinal plant may be a plant thought to have medicinal property and used in herbalism. A medicinal plant may be selected from a group consisting of, but not limited to:  Arthemis nobilis, Calendula officinalis, Caragana sinica, Codonopsis pilosulae, Echinacea angustifolia, Hedyotis diffusa, Houttuynia cordata, Hydrastis canadensis, Kalanchoe pinnata, Lonicera japonica, Morinda offcinalis , and  Oenothera odorata.    
     The transgenic plant may be edible or medicinal plants. Edible or medicinal plants may not contain health-threatening components. Edible or medicinal plants may not require any special purification and may be used similar to conventional biologically active dietary supplements produced from plants. 
     In an embodiment, a method for producing a recombinant protein in a plant is provided. The method may include steps of contacting a plant with a genetic construct. The genetic construct may include a nucleic acid encoding the recombinant protein. The recombinant protein may include a toxin binding ligand fused to a carrier protein. The carrier protein may be but is not limited to Fc-IgA, Fc-IgG, Fc-IgM, oleosin, or VP2 coat protein. The method may also include obtaining a plant that includes the genetic construct and expressing the recombinant protein. 
     In an embodiment of the method, the toxin binding ligand may be a capillary morphogenesis protein 2. The toxin binding ligand may be a PA-LF protein. 
     The step of contacting may include contacting with a vectors providing for stable transformation of a plant. The step of contacting may include contacting with a vector providing for transient expression in a plant. The vector may include a first polynucleotide encoding a toxin binding ligand and a second polynucleotide sequence encoding a carrier protein. A method may further include contacting a plant with another vector that includes a third polynucleotide encoding a helper element. 
     In an embodiment, the step of contacting may include contacting with the nucleic acid that may include a first polynucleotide encoding a toxin binding ligand. The nucleic acid may also include a second polynucleotide encoding a carrier protein. The first polynucleotide may include, consists essentially of, consists of a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% to a reference sequence selected from the group consisting of: SEQ ID NO: 1(PgA1-1), SEQ ID NO: 2 (PgA1-2), SEQ ID NO: 47 (PgA1-3), SEQ ID NO: 49 (PgA1-4) and SEQ ID NO: 58 (PgA1-5), SEQ ID NO: 3 (PgA2-1), SEQ ID NO: 4(PgA2-2), SEQ ID NO: 5 (A2-3 /PA-LF), and SEQ ID NO: 57 (PgA2-4). The second polynucleotide may include, consists essentially of, consists of a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% to a reference sequence selected from the group consisting of: SEQ ID NO:6 (PgB1-1), SEQ ID NO: 7 (PgB2-1), SEQ ID NO: 8 (PgB3-1), SEQ ID NO: 9 (PgB1-2), SEQ ID NO: 10 (PgB2-2), SEQ ID NO: 11 (PgB3-2), SEQ ID NO: 12 (PgB4-1), SEQ ID NO: 13 (PgB5-1), SEQ ID NO: 48 (PgB1-3), SEQ ID NO: 50 (PgB1-4), SEQ ID NO: 51 (PgB2-3), SEQ ID NO: 52 (PgB2-4), SEQ ID NO: 53 (PgB3-3), SEQ ID NO: 54 (PgB3-4), SEQ ID NO: 55 (PgB4-2), and SEQ ID NO: 56 (PgB5-2). 
     In an embodiment of the method, the nucleic acid may further include a third polynucleotide encoding a helper element. The third polynucleotide may include, consists essentially of, or consists of a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% to a reference sequence selected from the group consisting of: SEQ ID NO: 14 (PgC1-1), SEQ ID NO: 15 (PgC2-1), SEQ ID NO: 32 (PgC1-2), SEQ ID NO: 33 (PgC1-3), and SEQ ID NO: 34 (PgC2-2). 
     In an embodiment of the method, the step of contacting may include contacting with a genetic construct that may include, consists essentially of, or consists of a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% to a reference sequence selected from the group consisting of: SEQ ID NO: 16 (PgA1-3:B1-3), SEQ ID NO: 17 (PgA1-4:B1-4), SEQ ID NO: 18 (PgA1-3:B2-4), SEQ ID NO: 19 (PgA1-4:B2-14SEQ ID NO: 20 (PgA1-3:B3-3), SEQ ID NO:21 (PgA1-4:B3-4), SEQ ID NO: 22 (PgA1-3:B4-2), SEQ ID NO: 23 (PgA1-3:B5-2), SEQ ID NO: 37 (PgA2-4:B1-5), and SEQ ID NO: 38 (PgA1-5:B1-5). 
     In an embodiment of the method, the recombinant protein including the toxin binding ligand and a carrier-protein that may be capable of protecting a subject against anthrax toxin. The helper element may help to establish secondary structures from the molecules of the recombinant protein consisting of the toxin binding ligand and the carrier protein. 
     The plant may be created by  Agrobacterium -mediated transformation using a vector that includes a nucleic acid encoding the recombinant protein herein. The transgenic plant may be created by other methods for modifying plants. The transgenic plant may be created by direct uptake of plasmid DNA. The transformed plant may be stably transformed. The stably transformed plant may incorporate the genetic construct into the genome of the plant. 
     The plant may be transformed with a viral vector for transient expression of a recombinant protein in a plant. The viral vector may be delivered to a plant by any method. For reference, see U.S. patents (U.S. Pat. Nos. 5,889,190; 5,889,191; 5,316,931; 5,589,367; 7,667,092; 7,670,801; 7,763,458; 8,093,458; and 8,003,381), all of which are incorporated by reference herein as if fully set forth. Viral vectors may be T-DNA vectors. Plants may be infiltrated with a diluted  Agrobacterium  suspension carrying T-DNAs encoding viral replicons. The resulting plants may have a high copy number of RNA molecules that encode a recombinant protein. A recombinant protein may be produced in a transgenic plant in a short period of time. A recombinant protein may be produced in the transgenic plant in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days after transformation. The transgenic plants may have a high copy number of RNA molecules that encode recombinant proteins. Recombinant proteins may be produced in plants rapidly and in increasing volumes of biomass containing recombinant proteins that may not require changes in growing conditions. Features of transient expression system may include non-integration of external genes into plant genome, and thus reducing or eliminating the risk of releasing transgene into environment through pollen, seeds, or other routes. Further, no intact and replication-competent virus may be produced, thus, reducing or eliminating the risk of virus mediated spreading of the recombinant genes. Protein production may be performed in closed indoor settings. 
     In an embodiment, the method may include obtaining a plant expressing the recombinant antitoxin binding protein. The method may include also obtaining a plant expressing the helper peptide. The method may further include crossing the plant expressing the recombinant protein with the plant expressing the helper element. 
     An embodiment of any of the method may further include breeding the transgenic plant and obtaining its progeny, or its descendant. The progeny or the descendant may include the genetic construct. 
     In an embodiment, any of the method further may include obtaining a seed of the transgenic plant. The seed may include the genetic construct that includes the recombinant protein. 
     The method may further include isolating and purifying the recombinant protein from the plant. 
     In an embodiment a method for preparing a composition effective for treating or preventing an anthrax infection in a subject is also provided. The method may include providing a recombinant protein produced by any methods described herein. The therapeutic composition may include a toxin binding ligand (component A) alone. The therapeutic composition may include a toxin binding ligand fused to a carrier-protein (components A and B). The therapeutic composition may include a mixture of two or more recombinant antitoxins of different types related to different component B parts. The therapeutic composition may include any composition described herein and a helper element (component C). The plant-derived therapeutic compositions may neutralize, delay, or attenuate the fatal action of the anthrax toxin in a subject. 
     In an embodiment, a plant-derived therapeutic composition may include active agents. Active agents may include at least one of recombinant antitoxin proteins produced in plants. The therapeutic composition may be therapeutically effective. Therapeutic efficacy may depend on effective amounts of active agents and time of administering necessary to achieve the desired result. 
     In an embodiment, the therapeutic compositions in a “therapeutically effective amount”, i.e., the amount sufficient to protect against accumulation of active deadly toxin in serum, or disappearance of disease symptoms in a subject. Disappearance of disease symptoms may be assessed by decrease of internalization of active LF or EF components of the anthrax toxin by living cells in the subject&#39;s body or by increase of a surviving time of the subject after contact with pathogen. The plant-derived compositions may be administered using any amount and any route of administration effective for a protective action. 
     The exact dosage may be chosen by the physician based on a variety of factors and in view of individual patients. Dosage and administration may be adjusted to provide sufficient levels of the active agent or agents or to maintain the desired effect. For example, factors which may be taken into account include time of potential infection, the type and amount of infection agent and severity of a disease; weight of the patient; availability of other means for treatment or prophylaxis. 
     Therapeutic efficacy and toxicity of active agents in a composition may be determined by standard pharmaceutical procedures, for example, by determining the therapeutically effective dose of the agent in 50% of the population (ED 50 ) and the lethal dose to 50% of the population (LD 50 ) with or without therapeutic agent in cells cultured in vitro or experimental animals. Plant-derived therapeutic compositions may be evaluated based on the dose ratio of toxic to therapeutic effects (LD 50 /ED 50 ), called the therapeutic index, the largest value of which may be used for assessment. The data obtained from and animal studies may be used in formulating a dosage for human use. 
     The therapeutic dose according to currently accepted norm in animal models of anthrax infection may be at least 50 microgram (50 μg) of antitoxin/dose/animal. As plant-based recombinant antitoxin may be readily produced and inexpensively engineered and designed and stored, lesser or greater doses for large animals may be economically feasible. 
     In an embodiment, the method may also include providing the therapeutic composition that includes a pharmaceutically acceptable carrier. The “pharmaceutically acceptable carrier” refers to solvents, diluents, preservatives, dispersion or suspension aids, isotonic agents, thickening or emulsifying agents, solid binders, and lubricants, appropriate for the particular dosage form. The pharmaceutically acceptable carrier may be any known carrier that may be used in formulating pharmaceutical compositions and knows techniques for the preparation thereof. See  Remington&#39;s Pharmaceutical Sciences  Ed. by Gennaro, Mack Publishing, Easton, Pa., 1995. The pharmaceutically acceptable carriers may include, but are not limited to Ringer&#39;s solution, isotonic saline, starches, potato starch, sugars, glucose, powdered tragacant, malt, gelatin, talc, cellulose and its derivatives, ethyl cellulose, sodium carboxymethyl cellulose, cellulose acetate excipients, cocoa butter, suppository waxes, agar, alginic acid, oils, cottonseed oil, peanut oil, safflower oil, sesame oil, olive oil, soybean oil, corn oil, glycols, propylene glycol, esters, ethyl laurate, ethyl oleate, buffering agents, aluminum hydroxide, magnesium hydroxide, phosphate buffer solutions, pyrogen-free water, ethyl alcohol, other non-toxic compatible lubricants, sodium lauryl sulfate, magnesium stearate, coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents. Pharmaceutically acceptable carriers may also include preservatives and antioxidants. 
     In an embodiment, the method includes providing an adjuvant. The adjuvant may be any adjuvant. As used herein, the term “adjuvant” refers to a pharmacological or immunological agent which when administered with an antigen nonspecifically enhances the recipient&#39;s response to that antigen. The adjuvant may be but is not limited to Alum, oil-in-water nannoemulsion (MF59™), the glycolipid monophosphoryl lipid A (MPL®), virus-like particles (VLP), the cholera toxin B subunit (CTB), montanides ISA51 and ISA720, saponines Quil-A, ISCOM and QS-21, syntax adjuvant formulation (SAF), muramyl dipeptides (MDP), immunostimulatory oligonucleotides, TLR ligands,  Escherichia coli  heat-labile exotoxin, or lipid-based adjuvants (Vajdy et al., 2004 Imm and Cell Biol 82:617; Schroder et al., 1999 Vaccine 17:2096, all of which are incorporated by reference herein as if fully set forth). 
     In an embodiment, a method of protecting a subject against anthrax infection is provided. The method may include providing a composition that includes a recombinant protein. The recombinant protein may include a toxin binding ligand fused to a carrier-protein. The carrier protein may be a protein selected from the group consisting of: Fc-IgA, Fc-IgG, Fc-IgM, oleosin, and VP2 coat protein. The composition may effective in preventing or reducing at least one symptom of an anthrax infection in a subject. The method may also include administering the composition to the subject in need thereof. 
     In an embodiment of the method, the toxin binding ligand may be a capillary morphogenesis protein 2. The toxin binding ligand may be a PA-LF protein. The toxin binding ligand may include, consist essentially of, or consist of a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to a reference sequence selected from the group consisting of: SEQ ID NO: 24 (A1-CMG2), SEQ ID NO: 25 (A2/PA-LF) and SEQ ID NO: 60 (PgA1-5). 
     In an embodiment of the method, the carrier-protein may include, consist essentially of, or consist of a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to a reference sequence selected from the group consisting of: SEQ ID NO: 26 (B1), SEQ ID NO: 27 (B2), SEQ ID NO: 28 (B3), SEQ ID NO: 29 (B4) and SEQ ID NO: 30 (B5). 
     In an embodiment of the method, the recombinant protein may include, consist essentially of, or consist of a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to a reference sequence of SEQ ID NO: 31 (PgA1-1:B1-1). 
     In an embodiment, the subject may be a mammal. The mammal may be but is not limited to an agricultural animal, an equine, a high value zoo animal, or a research animal. The mammal may be a human. 
     In an embodiment, the step of administering the composition may include a route selected from the group consisting of: intravenous, intramuscular, intraperitoneal, intradermal, mucosal, cutaneous, and subcutaneous. The step of administering may be achieved through intranasal administration. The intranasal administration may include inhalation or nasal drops. A therapeutic composition may be administered to a recipient by any routes. A therapeutic composition may be introduced by injection, inhalation, oral, or intranasal route of administration. A therapeutic composition may be introduced by a parenteral or mucosal route of administration. Routes may include administering a composition orally, intrapulmonaryly, transdermally, rectally, intravaginally, intraperitoneally, intracisternally, and or ectopically. A mucosal route may include administering a therapeutic composition to any mucosal surface of the body of the recipient. Mucosal administration differs from “systemic” or “parenteral” administration. Systemic administration may include administering compositions to a non-mucosal surface, e.g., intraperitoneal, intramuscular, sub-, or transcutaneous, intra- or transdermal, or intravenous administration. 
     In an embodiment, the step of administering may be achieved by using a formulation with an appropriate pharmaceutically acceptable carrier in a desired dosage. A therapeutic composition may be administered in liquid dosage forms. Liquid dosage forms may be prepared for oral, nasal, inhalation, or transdermal administration. Liquid dosage forms may include, but not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, and suspensions. Liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters or sorbitan, and mixtures of thereof. Besides inert diluents, the compositions may also include ingredients stimulating protein translocation via mucosal tissues and/or absorption/permeability enhancers including but not limited to bile salts, surfactants, fusidic acid derivates, phosphatidylcholines, cyclodextrines, alcohols, low molecular weight polyethylene glycol etc. Liquid dosage forms may be available in forms optimal for use with inhalator devices. 
     Dosage forms for topical or transdermal administration of a therapeutical composition may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalant, or patches. Powders and sprays may content therapeutic proteins admixed with excipients such as talc, silicic acid, aluminum hydroxide, calcium silicates, polyamide powder, or mixture of these substances. Sprays may additionally contain customary propellants, for example, chlorofluorohydrocarbons. The therapeutic proteins may be admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be appropriate. 
     In an embodiment, administering the compositions may include a direct needle-free infusion of the raw, concentrated or partially purified extract of plants containing recombinant protein into human or non-human animal bloodstream. The step of administering may be achieved with the help of existing conventional devices and techniques enhancing absorption/permeability of biological surfaces. These devices or techniques may include but not limited to transdermal patches adapted to the purposes of this invention, or pulmonary delivery through an immunoglobulin transport pathway using conventional inhalators, intranasal spraying, or feeding the subject plant extract. 
     In an embodiment, administering of a plant-derived therapeutic composition may be a preventive treatment of subjects to promote emergency post-infection prophylaxis of a contact with the infectious agent. Administering of a plant-derived composition may be a therapeutic measure for neutralization anthrax toxin produced by bacterial pathogen  Bacillus anthracis  and minimizing complications associated with accumulation of deadly toxin in patients infected with the pathogen bacteria. Administering the plant-derived composition may be used for treatment of a variety of, symptoms and consequences of various forms of anthrax disease arising from infection with pathogenic bacteria  Bacillus anthracis . Plant-derived therapeutic compositions may be useful to treat patients being in contact with anthrax toxin, pathogen, infected animal or human, belonging to a group of risk of biological weapon attack. 
     In an embodiment, the method may further include measuring cell viability in the presence of different concentrations of the anthrax toxin, wherein cell viability is a percentage of surviving cells protected by the antitoxin in comparison to the complete lysis in the control. 
     In an embodiment, the method may further include measuring survival of animals after challenging with lethal concentration of the anthrax toxin or  B. anthracis  spores, followed by administration of protective amounts of the recombinant antitoxin. The survival may be a percentage of live animals protected by the antitoxin in comparison to unprotected objects in the control. 
     A skilled person will recognize that many suitable variations of the methods may be substituted for or used in addition to those described above and in the claims It should be understood that the implementation of other variations and modifications of the embodiments of the invention and its various aspects will be apparent to one skilled in the art, and that the invention is not limited by the specific embodiments described herein and in the claims. The present application mentions various patents, scientific articles, and other publications, each of which is hereby incorporated in its entirety by reference. 
     Further embodiments herein may be formed by supplementing an embodiment with one or more element from any one or more other embodiment herein, and/or substituting one or more element from one embodiment with one or more element from one or more other embodiment herein. Further embodiments herein may be described by reference to any one of the appended claims following claim  1  and reading the chosen claim to depend from any one or more preceding claim. 
     EXAMPLES 
     The following non-limiting examples are provided to illustrate particular embodiments. The embodiments throughout may be supplemented with one or more detail from one or more example below, and/or one or more element from an embodiment may be substituted with one or more detail from one or more example below. 
     Example 1 
     Recombinant Antitoxin Proteins Optimized for Expression in Plants 
     A soluble extracellular domain (aa 35-220) of the human CMG2 protein, sCMG2 (component A1), capable of binding PA protein and neutralization of the anthrax toxin (Scobie et al., 2005) or a fusion of the component A1 with the human IgG1 Fc-fragment (component B1), resulting in a component A1-B1 recombinant antitoxin, were chosen to first to test in a transient plant expression system. Expression cassettes were designed with or without ER targeting and retention signals and two commercial affinity tags (c-myc and 6×His). All cassettes were sub-cloned into a plant transformation vector suitable for transient expression together with commercially available helper plasmids. 
     Further optimization of the human CMG2 extracellular domain (amino acids 35-220) included the removal of a native signal peptide, the transmembrane domain and the cytoplasmic tail and resulted in Component A1 (PgA1). Fusion of a human CMG2 soluble extracellular domain (aa 35-220) with a human IgG1 Fc-fragment (Component B1, PgB1) resulted in Component A1-B1 (PgA1-B1) constructs. All sequences were optimized in a stepwise fashion as described in Table 1 using ELISA and Western blots for experimental quantitative confirmation. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Stepwise optimization of therapeutic proteins for expression in 
               
               
                 plants 
               
            
           
           
               
               
            
               
                 Variable parameter 
                 Outcome 
               
               
                   
               
               
                 1. Sequence analysis and 
                 Analysis of codon usage, mRNA 
               
               
                 genus/species-specific 
                 thermostability, cryptic intron splice 
               
               
                 optimization to improve 
                 sites, polyA signals, 
               
               
                 overall yields of 
                 evaluation and correction of sequences 
               
               
                 recombinant protein 
               
               
                 2. Optimal days post-infiltration 
                 Expression at different time points post 
               
               
                 (transient transformation only) 
                 infiltration 
               
               
                 3.ER localization signal choice 
                 Expression with and without targeting 
               
               
                   
                 signal sequences 
               
               
                 4.ER retention signal presence 
                 Expression with and without C- 
               
               
                   
                 terminal HDEL (SEQ ID NO: 64)/ 
               
               
                   
                 KDEL (SEQ ID NO: 65) peptide 
               
               
                 5. Affinity tags selection 
                 Expression and recovery with 
               
               
                   
                 commercially available tags (c-Myc and 
               
               
                   
                 6xHis) 
               
               
                 6.  N. benthamiana  glycosylation 
                 Expression and functionality in a model 
               
               
                 in knock-out mutants (transient 
                 wild type plant and glycosylation 
               
               
                 transformation only) 
                 knockout mutants that confer 
               
               
                   
                 mammalian type glycans 
               
               
                   
               
            
           
         
       
     
     Using the strategies outlined in Table 1 the nucleic acid sequences encoding the sCMG2-based recombinant subunit antitoxin and the recombinant Fc-fusion antitoxin were optimized for a better plant-specific production. 
     Each of the expression cassettes encoding the recombinant polypeptides also contained specific restriction/ligation sites required for direct subcloning into the plasmid carrier. The 5′ terminal region positioned at the NcoI site CCATGG was introduced in frame with the Kawasaki motif or Kozak-like sequence immediately before the initiation translation ATG codon 5′-gacaccATGG (SEQ ID NO: 39). The respective BglII and SacI sites were identified before and after the stop codon at the 3′ terminal region AGATCTccaa taa GAGCTC-3′ (SEQ ID NO: 40) in both constructs. Additionally, the NotI site as follows: 5′-tcttGCGGCCGCagga-3′ (SEQ ID NO: 41), was identified between Components A1 and B1 in the PgA1-B1 construct. These cloning sites were eliminated from the rest of the sequence body during the process of optimization. 
     PgA1 and PgA1-B1 expression cassettes were synthesized. These cassettes contained the synthetic anthrax toxin receptor sCMG2 or synthetic sCMG2 fused with the Fc fragment inserted into expression cassettes. The expression cassettes also contained C-terminus-specific tags, such as c-Myc and/or 6×His tags, and N-terminus plant-specific intracellular targeting signals. Two targeting signal sequences included the plant BAA gene encoding the amino acid sequence MANKHLSLSLFLVLLGLSASLASG (SEQ ID NO: 42) and the plant APBP1 gene, encoding the amino acid sequence MIVLSVGSASSSPIVVVFSVALLLFYFSETSLG (SEQ ID NO: 43). A flexible linker was introduced into the PgA1-B1 construct between the synthetic sCMG2 and the Fc fragment GGGSGNS (SEQ ID NO: 44). A short peptide was also introduced in front of the flexible linker to facilitate cleavage by a mammal serum protease, such as Thrombin LVPRGS (SEQ ID NO: 45), or Factor Xa protease IEGR (SEQ ID NO: 46). 
     The synthetic expression cassettes were introduced into plant transformation vectors harboring compatible cloning sites for NcoI (harbors ATG, first codon and without targeting signal peptide) and SacI. At the C-terminus of the expression cassettes, affinity tags such as c-Myc and/or 6×His were linked to endoplasmic reticulum (ER) retention signal HDEL (SEQ ID NO: 64) and inserted into the expression cassette immediately prior the stop codon. An Invitrogene/Geneart optimization with Gene with GeneOptimizer® sequence processing included the following parameters: 
     (i) Identification of the optimal sequence elements, such as restriction/ligation sites. 
     (ii) Elimination of cryptic splice sites and RNA destabilizing sequence elements for increased RNA stability. 
     (iii) Addition of RNA stabilizing sequence elements. 
     (iv) Codon optimization and G/C content adaptation for plant expression system. 
     (v) Intron removal. 
     (vi) Avoidance of templates compromising RNA secondary structures. 
     Additionally, regions with very high (&gt;80%) or very low (&lt;30%) GC content were avoided. For expression in plants, an average GC content of 58% was desirable. During the optimization process the following cis-acting sequence motifs were avoided: internal TATA-boxes, chi-sites and ribosomal entry sites. At-rich or GC-rich sequence stretches in codons, other RNA instability motifs, repeat sequences and RNAs secondary structures, cryptic splice donor and acceptor sites in eukaryotes. In some cases, negative cis-acting motifs were not removed. 
     The optimization considered successful if negative cis-acting sites which may negatively influence expression were eliminated, and GC content was adjusted to prolong mRNA half-life. 
     The expression cassettes included nucleic acid sequences encoding the soluble form of extracellular domain (aa 35-220) of human CMG2 protein (sCMG2, component A1) capable of binding PA protein and neutralization of the anthrax toxin; a fusion of the component A1 with human IgG1 Fc-fragment (component B1), resulting in a recombinant antitoxin, a component A1-B1. Expression cassettes were designed and tested in commercial plasmids for stable transformation/expression 
     PgA1 expression cassettes. PgA1 included a soluble extracellular domain of human CMG2 protein, sCMG2 (component A1), capable of binding PA protein and neutralization of the anthrax toxin that has no native signal peptide (amino acids 35-220), transmembrane domain and cytoplasmic tail. These elements were optimized for expression in plants and named sCMG2. PgA1 was designed for transient and stable plant transformation/expression. A 581 bp nucleic acid sequence of PgA1-1(SEQ ID NO: 1) encoding the PgA protein (190 aa) designed for cloning in the MagnICON expression vectors is as follows: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 1) 
               
            
           
           
               
            
               
                 CCATGGAACAACCTTCTTGTCGTCGAGCTTTTGATCTTTATTTTGTTCTT 
               
               
                   
               
               
                 GATAAATCTGGTTCTGTTGCTAATAATTGGATTGAAATTTATAATTTTGT 
               
               
                   
               
               
                 TCAACAACTTGCTGAAAGATTTGTTTCTCCTGAAATGAGACTTTCTTTTA 
               
               
                   
               
               
                 TTGTTTTTTCTTCTCAAGCTACTATTATTCTTCCTCTTACTGGTGATAGA 
               
               
                   
               
               
                 GGAAAAATTTCTAAAGGACTTGAGGATTTGAAAAGAGTTTCTCCTGTTGG 
               
               
                   
               
               
                 TGAAACTTATATTCATGAGGGACTTAAACTTGCTAATGAACAAATTCAAA 
               
               
                   
               
               
                 AAGCTGGTGGTCTTAAAACTTCTTCTATTATTATTGCTCTTACTGATGGA 
               
               
                   
               
               
                 AAACTTGATGGTCTTGTTCCTTCTTATGCTGAAAAAGAAGCTAAAATTTC 
               
               
                   
               
               
                 AAGATCACTTGGTGCTTCTGTTTATTGTGTTGGTGTTCTTGATTTTGAAC 
               
               
                   
               
               
                 AAGCTCAACTTGAAAGAATTGCTGATTCTAAAGAACAAGTTTTTCCTGTT 
               
               
                   
               
               
                 AAGGGTGGATTTCAAGCTCTTAAAGGAATTATTAATTCTATTCTTGCTCA 
               
               
                   
               
               
                 ATCTTGTACTGAAGATCTCCAATAAGAGCTC 
               
            
           
         
       
     
     A 575 bp nucleic acid sequence of PgA1-2 (SEQ ID NO: 2) encoding the PgA1 protein (190 aa) designed for stable expression is as follows: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 2) 
               
            
           
           
               
            
               
                 CCATGGAACAACCTTCTTGTCGTCGAGCTTTTGATCTTTATTTTGTTCTT 
               
               
                   
               
               
                 GATAAATCTGGTTCTGTTGCTAATAATTGGATTGAAATTTATAATTTTGT 
               
               
                   
               
               
                 TCAACAACTTGCTGAAAGATTTGTTTCTCCTGAAATGAGACTTTCTTTTA 
               
               
                   
               
               
                 TTGTTTTTTCTTCTCAAGCTACTATTATTCTTCCTCTTACTGGTGATAGA 
               
               
                   
               
               
                 GGAAAAATTTCTAAAGGACTTGAGGATTTGAAAAGAGTTTCTCCTGTTGG 
               
               
                   
               
               
                 TGAAACTTATATTCATGAGGGACTTAAACTTGCTAATGAACAAATTCAAA 
               
               
                   
               
               
                 AAGCTGGTGGTCTTAAAACTTCTTCTATTATTATTGCTCTTACTGATGGA 
               
               
                   
               
               
                 AAACTTGATGGTCTTGTTCCTTCTTATGCTGAAAAAGAAGCTAAAATTTC 
               
               
                   
               
               
                 AAGATCACTTGGTGCTTCTGTTTATTGTGTTGGTGTTCTTGATTTTGAAC 
               
               
                   
               
               
                 AAGCTCAACTTGAAAGAATTGCTGATTCTAAAGAACAAGTTTTTCCTGTT 
               
               
                   
               
               
                 AAGGGTGGATTTCAAGCTCTTAAAGGAATTATTAATTCTATTCTTGCTCA 
               
               
                   
               
               
                 ATCTTGTACTGAAGATCTCCAATAA 
               
            
           
         
       
     
     PgA1-B1 expression cassettes. A soluble extracellular domain of human CMG2 protein, component A1 as described above, fused with human IgG1 Fc-fragment (component B1), resulting in a component A1-B1 recombinant antitoxin optimized for expression in plants. PgA1-B1 expression cassettes were designed for transient and stable plant transformation. 
     A 1286 bp nucleic acid sequence of PgA1-3:B1-3 (SEQ ID NO: 16) encoding the PgA1-B1 protein (425 aa) designed for transient expression vectors is as follows: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 16) 
               
            
           
           
               
            
               
                 CCATGGAACAACCTTCTTGTCGTCGAGCTTTTGATCTTTATTTTGTTCTT 
               
               
                   
               
               
                 GATAAATCTGGTTCTGTTGCTAATAATTGGATTGAAATTTATAATTTTGT 
               
               
                   
               
               
                 TCAACAACTTGCTGAAAGATTTGTTTCTCCTGAAATGAGACTTTCTTTTA 
               
               
                   
               
               
                 TTGTTTTTTCTTCTCAAGCTACTATTATTCTTCCTCTTACTGGTGATAGA 
               
               
                   
               
               
                 GGAAAAATTTCTAAAGGACTTGAGGATTTGAAAAGAGTTTCTCCTGTTGG 
               
               
                   
               
               
                 TGAAACTTATATTCATGAGGGACTTAAACTTGCTAATGAACAAATTCAAA 
               
               
                   
               
               
                 AAGCTGGTGGTCTTAAAACTTCTTCTATTATTATTGCTCTTACTGATGGA 
               
               
                   
               
               
                 AAACTTGATGGTCTTGTTCCTTCTTATGCTGAAAAAGAAGCTAAAATTTC 
               
               
                   
               
               
                 AAGATCACTTGGTGCTTCTGTTTATTGTGTTGGTGTTCTTGATTTTGAAC 
               
               
                   
               
               
                 AAGCTCAACTTGAAAGAATTGCTGATTCTAAAGAACAAGTTTTTCCTGTT 
               
               
                   
               
               
                 AAGGGTGGATTTCAAGCTCTTAAAGGAATTATTAATTCTATTCTTGCTCA 
               
               
                   
               
               
                 ATCTTGTACTGCGGCCGCAGGAGGTGGATCTGGAAATTCTGACAAGACCC 
               
               
                   
               
               
                 ACACCTGCCCTCCTTGCCCTGCTCCTGAGCTCCTCGGTGGTCCTTCTGTC 
               
               
                   
               
               
                 TTCCTCTTCCCTCCTAAGCCTAAGGACACCCTCATGATCTCTCGTACCCC 
               
               
                   
               
               
                 TGAGGTCACCTGCGTCGTCGTCGACGTCTCTCACGAGGACCCTGAGGTCA 
               
               
                   
               
               
                 AGTTCAACTGGTACGTCGACGGTGTCGAGGTCCACAACGCTAAGACCAAG 
               
               
                   
               
               
                 CCTCGTGAGGAGCAGTACAACTCTACCTACCGTGTCGTCTCTGTCCTCAC 
               
               
                   
               
               
                 CGTCCTCCACCAGGACTGGCTCAACGGTAAGGAGTACAAGTGCAAGGTCT 
               
               
                   
               
               
                 CTAACAAGGCTCTCCCTGCTCCTATCGAGAAGACCATCTCTAAGGCTAAG 
               
               
                   
               
               
                 GGTCAGCCTCGTGAGCCTCAGGTCTACACCCTCCCTCCTTCTCGTGAGGA 
               
               
                   
               
               
                 GATGACCAAGAACCAGGTCTCTCTCACCTGCCTCGTCAAGGGTTTCTACC 
               
               
                   
               
               
                 CTTCTGACATCGCTGTCGAGTGGGAGTCTAACGGTCAGCCTGAGAACAAC 
               
               
                   
               
               
                 TACAAGACCACCCCTCCTGTCCTCGACTCTGACGGTTCTTTCTTCCTCTA 
               
               
                   
               
               
                 CTCTAAGCTCACCGTCGACAAGTCTCGTTGGCAGCAGGGTAACGTCTTCT 
               
               
                   
               
               
                 CTTGCTCTGTCATGCACGAGGCTCTCCACAACCACTACACCCAGAAGTCT 
               
               
                   
               
               
                 CTCTCTCTCTCTCCTGGTAAGGACCTCTAAGAGCTC 
               
            
           
         
       
     
     A 1283 bp nucleic acid sequence of PgA1-4:B1-4 (SEQ ID NO: 17) encoding the PgA1-B1 protein (442 aa) designed for stable transformation is as follows: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 17) 
               
            
           
           
               
            
               
                 CCATGGAACAACCTTCTTGTCGTCGAGCTTTTGATCTTTATTTTGTTCTT 
               
               
                   
               
               
                 GATAAATCTGGTTCTGTTGCTAATAATTGGATTGAAATTTATAATTTTGT 
               
               
                   
               
               
                 TCAACAACTTGCTGAAAGATTTGTTTCTCCTGAAATGAGACTTTCTTTTA 
               
               
                   
               
               
                 TTGTTTTTTCTTCTCAAGCTACTATTATTCTTCCTCTTACTGGTGATAGA 
               
               
                   
               
               
                 GGAAAAATTTCTAAAGGACTTGAGGATTTGAAAAGAGTTTCTCCTGTTGG 
               
               
                   
               
               
                 TGAAACTTATATTCATGAGGGACTTAAACTTGCTAATGAACAAATTCAAA 
               
               
                   
               
               
                 AAGCTGGTGGTCTTAAAACTTCTTCTATTATTATTGCTCTTACTGATGGA 
               
               
                   
               
               
                 AAACTTGATGGTCTTGTTCCTTCTTATGCTGAAAAAGAAGCTAAAATTTC 
               
               
                   
               
               
                 AAGATCACTTGGTGCTTCTGTTTATTGTGTTGGTGTTCTTGATTTTGAAC 
               
               
                   
               
               
                 AAGCTCAACTTGAAAGAATTGCTGATTCTAAAGAACAAGTTTTTCCTGTT 
               
               
                   
               
               
                 AAGGGTGGATTTCAAGCTCTTAAAGGAATTATTAATTCTATTCTTGCTCA 
               
               
                   
               
               
                 ATCTTGTACTGCGGCCGCAGGAGGTGGATCTGGAAATTCTGACAAGACCC 
               
               
                   
               
               
                 ACACCTGCCCTCCTTGCCCTGCTCCTGAGCTCCTCGGTGGTCCTTCTGTC 
               
               
                   
               
               
                 TTCCTCTTCCCTCCTAAGCCTAAGGACACCCTCATGATCTCTCGTACCCC 
               
               
                   
               
               
                 TGAGGTCACCTGCGTCGTCGTCGACGTCTCTCACGAGGACCCTGAGGTCA 
               
               
                   
               
               
                 AGTTCAACTGGTACGTCGACGGTGTCGAGGTCCACAACGCTAAGACCAAG 
               
               
                   
               
               
                 CCTCGTGAGGAGCAGTACAACTCTACCTACCGTGTCGTCTCTGTCCTCAC 
               
               
                   
               
               
                 CGTCCTCCACCAGGACTGGCTCAACGGTAAGGAGTACAAGTGCAAGGTCT 
               
               
                   
               
               
                 CTAACAAGGCTCTCCCTGCTCCTATCGAGAAGACCATCTCTAAGGCTAAG 
               
               
                   
               
               
                 GGTCAGCCTCGTGAGCCTCAGGTCTACACCCTCCCTCCTTCTCGTGAGGA 
               
               
                   
               
               
                 GATGACCAAGAACCAGGTCTCTCTCACCTGCCTCGTCAAGGGTTTCTACC 
               
               
                   
               
               
                 CTTCTGACATCGCTGTCGAGTGGGAGTCTAACGGTCAGCCTGAGAACAAC 
               
               
                   
               
               
                 TACAAGACCACCCCTCCTGTCCTCGACTCTGACGGTTCTTTCTTCCTCTA 
               
               
                   
               
               
                 CTCTAAGCTCACCGTCGACAAGTCTCGTTGGCAGCAGGGTAACGTCTTCT 
               
               
                   
               
               
                 CTTGCTCTGTCATGCACGAGGCTCTCCACAACCACTACACCCAGAAGTCT 
               
               
                   
               
               
                 CTCTCTCTCTCTCCTGGTAAGGACCTCGATCTCCAAAAGCTTATTAGCGA 
               
               
                   
               
               
                 GGAGGATCTTCATCACCATCACCATCACTAAGAGCTC 
               
            
           
         
       
     
     PgA1-B2 expression cassettes. A soluble extracellular domain of human CMG2 protein, component A1 as described above, fused with human IgA1 Fc-fragment (component B2), resulting in a component A1-B2 recombinant antitoxin optimized for expression in plants. PgA1-B2 expression cassettes were designed for transient and stable plant transformation/expression. 
     A 1377 bp nucleic acid sequence of PgA1-3:B2-3(SEQ ID NO: 18) encoding the PgA1-B2 Nt  protein (458 aa) designed for transient expression vectors is as follows: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 18) 
               
            
           
           
               
            
               
                 CCATGGAACAACCTTCTTGTCGTCGAGCTTTTGATCTTTATTTTGTTCTT 
               
               
                   
               
               
                 GATAAATCTGGTTCTGTTGCTAATAATTGGATTGAAATTTATAATTTTGT 
               
               
                   
               
               
                 TCAACAACTTGCTGAAAGATTTGTTTCTCCTGAAATGAGACTTTCTTTTA 
               
               
                   
               
               
                 TTGTTTTTTCTTCTCAAGCTACTATTATTCTTCCTCTTACTGGTGATAGA 
               
               
                   
               
               
                 GGAAAAATTTCTAAAGGACTTGAGGATTTGAAAAGAGTTTCTCCTGTTGG 
               
               
                   
               
               
                 TGAAACTTATATTCATGAGGGACTTAAACTTGCTAATGAACAAATTCAAA 
               
               
                   
               
               
                 AAGCTGGTGGTCTTAAAACTTCTTCTATTATTATTGCTCTTACTGATGGA 
               
               
                   
               
               
                 AAACTTGATGGTCTTGTTCCTTCTTATGCTGAAAAAGAAGCTAAAATTTC 
               
               
                   
               
               
                 AAGATCACTTGGTGCTTCTGTTTATTGTGTTGGTGTTCTTGATTTTGAAC 
               
               
                   
               
               
                 AAGCTCAACTTGAAAGAATTGCTGATTCTAAAGAACAAGTTTTTCCTGTT 
               
               
                   
               
               
                 AAGGGTGGATTTCAAGCTCTTAAAGGAATTATTAATTCTATTCTTGCTCA 
               
               
                   
               
               
                 ATCTTGTACTGCGGCCGCAGGAGGTGGATCTGGAAATTCTGACGTCACCG 
               
               
                   
               
               
                 TCCCTTGCCCTGTCCCTTCTACCCCTCCTACCCCTTCTCCTTCTACCCCT 
               
               
                   
               
               
                 CCTACCCCTTCTCCTTCTTGCTGCCACCCTCGTCTCTCTCTCCACCGTCC 
               
               
                   
               
               
                 TGCTCTCGAGGACCTCCTCCTCGGTTCTGAGGCTAACCTCACCTGCACCC 
               
               
                   
               
               
                 TCACCGGTCTCCGTGACGCTTCTGGTGTCACCTTCACCTGGACCCCTTCT 
               
               
                   
               
               
                 TCTGGTAAGTCTGCTGTCCAGGGTCCTCCTGAGCGTGACCTCTGCGGTTG 
               
               
                   
               
               
                 CTACTCTGTCTCTTCTGTCCTCCCTGGTTGCGCTGAGCCTTGGAACCACG 
               
               
                   
               
               
                 GTAAGACCTTCACCTGCACCGCTGCTTACCCTGAGTCTAAGACCCCTCTC 
               
               
                   
               
               
                 ACCGCTACCCTCTCTAAGTCTGGTAACACCTTCCGTCCTGAGGTCCACCT 
               
               
                   
               
               
                 CCTCCCTCCTCCTTCTGAGGAGCTCGCTCTCAACGAGCTCGTCACCCTCA 
               
               
                   
               
               
                 CCTGCCTCGCTCGTGGTTTCTCTCCTAAGGACGTCCTCGTCCGTTGGCTC 
               
               
                   
               
               
                 CAGGGTTCTCAGGAGCTCCCTCGTGAGAAGTACCTCACCTGGGCTTCTCG 
               
               
                   
               
               
                 TCAGGAGCCTTCTCAGGGTACCACCACCTTCGCTGTCACCTCTATCCTCC 
               
               
                   
               
               
                 GTGTCGCTGCTGAGGACTGGAAGAAGGGTGACACCTTCTCTTGCATGGTC 
               
               
                   
               
               
                 GGTCACGAGGCTCTCCCTCTCGCTTTCACCCAGAAGACCATCGACCGTCT 
               
               
                   
               
               
                 CGCTGGTAAGCCTACCCACGTCAACGTCTCTGTCGTCATGGCTGAGGTCG 
               
               
                   
               
               
                 ACGGTACCTGCTACCCAATAAGAGCTC 
               
            
           
         
       
     
     A 1273 bp nucleic acid sequence of PgA1-4:B2-4(SEQ ID NO: 19) encoding the PgA1-B2 protein (454 aa) designed for stable transformation vectors is as follows: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 19) 
               
            
           
           
               
            
               
                 CCATGGAACAACCTTCTTGTCGTCGAGCTTTTGATCTTTATTTTGTTCTT 
               
               
                   
               
               
                 GATAAATCTGGTTCTGTTGCTAATAATTGGATTGAAATTTATAATTTTGT 
               
               
                   
               
               
                 TCAACAACTTGCTGAAAGATTTGTTTCTCCTGAAATGAGACTTTCTTTTA 
               
               
                   
               
               
                 TTGTTTTTTCTTCTCAAGCTACTATTATTCTTCCTCTTACTGGTGATAGA 
               
               
                   
               
               
                 GGAAAAATTTCTAAAGGACTTGAGGATTTGAAAAGAGTTTCTCCTGTTGG 
               
               
                   
               
               
                 TGAAACTTATATTCATGAGGGACTTAAACTTGCTAATGAACAAATTCAAA 
               
               
                   
               
               
                 AAGCTGGTGGTCTTAAAACTTCTTCTATTATTATTGCTCTTACTGATGGA 
               
               
                   
               
               
                 AAACTTGATGGTCTTGTTCCTTCTTATGCTGAAAAAGAAGCTAAAATTTC 
               
               
                   
               
               
                 AAGATCACTTGGTGCTTCTGTTTATTGTGTTGGTGTTCTTGATTTTGAAC 
               
               
                   
               
               
                 AAGCTCAACTTGAAAGAATTGCTGATTCTAAAGAACAAGTTTTTCCTGTT 
               
               
                   
               
               
                 AAGGGTGGATTTCAAGCTCTTAAAGGAATTATTAATTCTATTCTTGCTCA 
               
               
                   
               
               
                 ATCTTGTACTGCGGCCGCAGGAGGTGGATCTGGAAATTCTGACGTCACCG 
               
               
                   
               
               
                 TCCCTTGCCCTGTCCCTTCTACCCCTCCTACCCCTTCTCCTTCTACCCCT 
               
               
                   
               
               
                 CCTACCCCTTCTCCTTCTTGCTGCCACCCTCGTCTCTCTCTCCACCGTCC 
               
               
                   
               
               
                 TGCTCTCGAGGACCTCCTCCTCGGTTCTGAGGCTAACCTCACCTGCACCC 
               
               
                   
               
               
                 TCACCGGTCTCCGTGACGCTTCTGGTGTCACCTTCACCTGGACCCCTTCT 
               
               
                   
               
               
                 TCTGGTAAGTCTGCTGTCCAGGGTCCTCCTGAGCGTGACCTCTGCGGTTG 
               
               
                   
               
               
                 CTACTCTGTCTCTTCTGTCCTCCCTGGTTGCGCTGAGCCTTGGAACCACG 
               
               
                   
               
               
                 GTAAGACCTTCACCTGCACCGCTGCTTACCCTGAGTCTAAGACCCCTCTC 
               
               
                   
               
               
                 ACCGCTACCCTCTCTAAGTCTGGTAACACCTTCCGTCCTGAGGTCCACCT 
               
               
                   
               
               
                 CCTCCCTCCTCCTTCTGAGGAGCTCGCTCTCAACGAGCTCGTCACCCTCA 
               
               
                   
               
               
                 CCTGCCTCGCTCGTGGTTTCTCTCCTAAGGACGTCCTCGTCCGTTGGCTC 
               
               
                   
               
               
                 CAGGGTTCTCAGGAGCTCCCTCGTGAGAAGTACCTCACCTGGGCTTCTCG 
               
               
                   
               
               
                 TCAGGAGCCTTCTCAGGGTACCACCACCTTCGCTGTCACCTCTATCCTCC 
               
               
                   
               
               
                 GTGTCGCTGCTGAGGACTGGAAGAAGGGTGACACCTTCTCTTGCATGGTC 
               
               
                   
               
               
                 GGTCACGAGGCTCTCCCTCTCGCTTTCACCCAGAAGACCATCGACCGTCT 
               
               
                   
               
               
                 CGCTGGTAAGCCTACCCACGTCAACGTCTCTGTCGTCATGGCTGAGGTCG 
               
               
                   
               
               
                 ACGGTACCTGCTACTAAAGATCT 
               
            
           
         
       
     
     PgA1-B3 expression cassettes. A soluble extracellular domain of human CMG2 protein, component A1 as above, fused with human IgM Fc-fragment (component B3), resulting in a component A1-B3 recombinant antitoxin optimized for expression in plants. PgA1-B3 expression cassettes were designed for transient and stable plant transformation and expression. 
     A 1662 bp nucleic acid sequence of PgA1-3:B3-3 (SEQ ID NO: 20) encoding the PgA1-B3 protein (553 aa) for transient expression vectors is as follows: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 20) 
               
            
           
           
               
            
               
                 CCATGGAACAACCTTCTTGTCGTCGAGCTTTTGATCTTTATTTTGTTCTT 
               
               
                   
               
               
                 GATAAATCTGGTTCTGTTGCTAATAATTGGATTGAAATTTATAATTTTGT 
               
               
                   
               
               
                 TCAACAACTTGCTGAAAGATTTGTTTCTCCTGAAATGAGACTTTCTTTTA 
               
               
                   
               
               
                 TTGTTTTTTCTTCTCAAGCTACTATTATTCTTCCTCTTACTGGTGATAGA 
               
               
                   
               
               
                 GGAAAAATTTCTAAAGGACTTGAGGATTTGAAAAGAGTTTCTCCTGTTGG 
               
               
                   
               
               
                 TGAAACTTATATTCATGAGGGACTTAAACTTGCTAATGAACAAATTCAAA 
               
               
                   
               
               
                 AAGCTGGTGGTCTTAAAACTTCTTCTATTATTATTGCTCTTACTGATGGA 
               
               
                   
               
               
                 AAACTTGATGGTCTTGTTCCTTCTTATGCTGAAAAAGAAGCTAAAATTTC 
               
               
                   
               
               
                 AAGATCACTTGGTGCTTCTGTTTATTGTGTTGGTGTTCTTGATTTTGAAC 
               
               
                   
               
               
                 AAGCTCAACTTGAAAGAATTGCTGATTCTAAAGAACAAGTTTTTCCTGTT 
               
               
                   
               
               
                 AAGGGTGGATTTCAAGCTCTTAAAGGAATTATTAATTCTATTCTTGCTCA 
               
               
                   
               
               
                 ATCTTGTACTGCGGCCGCAGGAGGTGGATCTGGAAATTCTGTCCCTCTCC 
               
               
                   
               
               
                 CTGTCATCGCTGAGCTCCCTCCTAAGGTCTCTGTCTTCGTCCCTCCTCGT 
               
               
                   
               
               
                 GACGGTTTCTTCGGTAACCCTCGTAAGTCTAAGCTCATCTGCCAGGCTAC 
               
               
                   
               
               
                 CGGTTTCTCTCCTCGTCAGATCCAGGTCTCTTGGCTCCGTGAGGGTAAGC 
               
               
                   
               
               
                 AGGTCGGTTCTGGTGTCACCACCGACCAGGTCCAGGCTGAGGCTAAGGAG 
               
               
                   
               
               
                 TCTGGTCCTACCACCTACAAGGTCACCTCTACCCTCACCATCAAGGAGTC 
               
               
                   
               
               
                 TGACTGGCTCTCTCAGTCTATGTTCACCTGCCGTGTCGACCACCGTGGTC 
               
               
                   
               
               
                 TCACCTTCCAGCAGAACGCTTCTTCTATGTGCGTCCCTGACCAGGACACC 
               
               
                   
               
               
                 GCTATCCGTGTCTTCGCTATCCCTCCTTCTTTCGCTTCTATCTTCCTCAC 
               
               
                   
               
               
                 CAAGTCTACCAAGCTCACCTGCCTCGTCACCGACCTCACCACCTACGACT 
               
               
                   
               
               
                 CTGTCACCATCTCTTGGACCCGTCAGAACGGTGAGGCTGTCAAGACCCAC 
               
               
                   
               
               
                 ACCAACATCTCTGAGTCTCACCCTAACGCTACCTTCTCTGCTGTCGGTGA 
               
               
                   
               
               
                 GGCTTCTATCTGCGAGGACGACTGGAACTCTGGTGAGCGTTTCACCTGCA 
               
               
                   
               
               
                 CCGTCACCCACACCGACCTCCCTTCTCCTCTCAAGCAGACCATCTCTCGT 
               
               
                   
               
               
                 CCTAAGGGTGTCGCTCTCCACCGTCCTGACGTCTACCTCCTCCCTCCTGC 
               
               
                   
               
               
                 TCGTGAGCAGCTCAACCTCCGTGAGTCTGCTACCATCACCTGCCTCGTCA 
               
               
                   
               
               
                 CCGGTTTCTCTCCTGCTGACGTCTTCGTCCAGTGGATGCAGCGTGGTCAG 
               
               
                   
               
               
                 CCTCTCTCTCCTGAGAAGTACGTCACCTCTGCTCCTATGCCTGAGCCTCA 
               
               
                   
               
               
                 GGCTCCTGGTCGTTACTTCGCTCACTCTATCCTCACCGTCTCTGAGGAGG 
               
               
                   
               
               
                 AGTGGAACACCGGTGAGACCTACACCTGCGTCGTCGCTCACGAGGCTCTC 
               
               
                   
               
               
                 CCTAACCGTGTCACCGAGCGTACCGTCGACAAGTCTACCGGTAAGCCTAC 
               
               
                   
               
               
                 CCTCTACAACGTCTCTCTCGTCATGTCTGACACCGCTGGTACCTGCTACC 
               
               
                   
               
               
                 CAATAAGAGCTC 
               
            
           
         
       
     
     A 1658 bp nucleic acid sequence of PgA-4:B3-4 (SEQ ID NO: 21) encoding the PgA1-B3 protein (549 aa) for stable transformation vectors is as follows: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 21) 
               
            
           
           
               
            
               
                 CCATGGAACAACCTTCTTGTCGTCGAGCTTTTGATCTTTATTTTGTTCTT 
               
               
                   
               
               
                 GATAAATCTGGTTCTGTTGCTAATAATTGGATTGAAATTTATAATTTTGT 
               
               
                   
               
               
                 TCAACAACTTGCTGAAAGATTTGTTTCTCCTGAAATGAGACTTTCTTTTA 
               
               
                   
               
               
                 TTGTTTTTTCTTCTCAAGCTACTATTATTCTTCCTCTTACTGGTGATAGA 
               
               
                   
               
               
                 GGAAAAATTTCTAAAGGACTTGAGGATTTGAAAAGAGTTTCTCCTGTTGG 
               
               
                   
               
               
                 TGAAACTTATATTCATGAGGGACTTAAACTTGCTAATGAACAAATTCAAA 
               
               
                   
               
               
                 AAGCTGGTGGTCTTAAAACTTCTTCTATTATTATTGCTCTTACTGATGGA 
               
               
                   
               
               
                 AAACTTGATGGTCTTGTTCCTTCTTATGCTGAAAAAGAAGCTAAAATTTC 
               
               
                   
               
               
                 AAGATCACTTGGTGCTTCTGTTTATTGTGTTGGTGTTCTTGATTTTGAAC 
               
               
                   
               
               
                 AAGCTCAACTTGAAAGAATTGCTGATTCTAAAGAACAAGTTTTTCCTGTT 
               
               
                   
               
               
                 AAGGGTGGATTTCAAGCTCTTAAAGGAATTATTAATTCTATTCTTGCTCA 
               
               
                   
               
               
                 ATCTTGTACTGCGGCCGCAGGAGGTGGATCTGGAAATTCTGTCCCTCTCC 
               
               
                   
               
               
                 CTGTCATCGCTGAGCTCCCTCCTAAGGTCTCTGTCTTCGTCCCTCCTCGT 
               
               
                   
               
               
                 GACGGTTTCTTCGGTAACCCTCGTAAGTCTAAGCTCATCTGCCAGGCTAC 
               
               
                   
               
               
                 CGGTTTCTCTCCTCGTCAGATCCAGGTCTCTTGGCTCCGTGAGGGTAAGC 
               
               
                   
               
               
                 AGGTCGGTTCTGGTGTCACCACCGACCAGGTCCAGGCTGAGGCTAAGGAG 
               
               
                   
               
               
                 TCTGGTCCTACCACCTACAAGGTCACCTCTACCCTCACCATCAAGGAGTC 
               
               
                   
               
               
                 TGACTGGCTCTCTCAGTCTATGTTCACCTGCCGTGTCGACCACCGTGGTC 
               
               
                   
               
               
                 TCACCTTCCAGCAGAACGCTTCTTCTATGTGCGTCCCTGACCAGGACACC 
               
               
                   
               
               
                 GCTATCCGTGTCTTCGCTATCCCTCCTTCTTTCGCTTCTATCTTCCTCAC 
               
               
                   
               
               
                 CAAGTCTACCAAGCTCACCTGCCTCGTCACCGACCTCACCACCTACGACT 
               
               
                   
               
               
                 CTGTCACCATCTCTTGGACCCGTCAGAACGGTGAGGCTGTCAAGACCCAC 
               
               
                   
               
               
                 ACCAACATCTCTGAGTCTCACCCTAACGCTACCTTCTCTGCTGTCGGTGA 
               
               
                   
               
               
                 GGCTTCTATCTGCGAGGACGACTGGAACTCTGGTGAGCGTTTCACCTGCA 
               
               
                   
               
               
                 CCGTCACCCACACCGACCTCCCTTCTCCTCTCAAGCAGACCATCTCTCGT 
               
               
                   
               
               
                 CCTAAGGGTGTCGCTCTCCACCGTCCTGACGTCTACCTCCTCCCTCCTGC 
               
               
                   
               
               
                 TCGTGAGCAGCTCAACCTCCGTGAGTCTGCTACCATCACCTGCCTCGTCA 
               
               
                   
               
               
                 CCGGTTTCTCTCCTGCTGACGTCTTCGTCCAGTGGATGCAGCGTGGTCAG 
               
               
                   
               
               
                 CCTCTCTCTCCTGAGAAGTACGTCACCTCTGCTCCTATGCCTGAGCCTCA 
               
               
                   
               
               
                 GGCTCCTGGTCGTTACTTCGCTCACTCTATCCTCACCGTCTCTGAGGAGG 
               
               
                   
               
               
                 AGTGGAACACCGGTGAGACCTACACCTGCGTCGTCGCTCACGAGGCTCTC 
               
               
                   
               
               
                 CCTAACCGTGTCACCGAGCGTACCGTCGACAAGTCTACCGGTAAGCCTAC 
               
               
                   
               
               
                 CCTCTACAACGTCTCTCTCGTCATGTCTGACACCGCTGGTACCTGCTACT 
               
               
                   
               
               
                 AAAGATCT 
               
            
           
         
       
     
     PgB4-A1 expression cassettes. A coat protein VP2 of the human polyoma virus (JC virus) capable of self-assembly into VLPs (component B4) was fused with the soluble extracellular domain of human CMG2 protein, (component A1), resulting in a component B4-A1 recombinant antitoxin. PgB4-A1 expression cassettes were optimized for transient and stable plant transformation and expression. 
     A 1064 bp nucleic acid sequence of PgB4-2: A1-3 (SEQ ID NO: 22) encoding the PgB4-A1 protein (354 aa) for transient or stable plant transformation/expression vectors is as follows: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 22) 
               
            
           
           
               
            
               
                 CCATGGGTGCTGCTCTCGCTCTCCTCGGTGACCTCGTCGCTACCGTCTCT 
               
               
                   
               
               
                 GAGGCTGCTGCTGCTACCGGTTTCTCTGTCGCTGAGATCGCTGCTGGTGA 
               
               
                   
               
               
                 GGCTGCTGCTACCATCGAGGTCGAGATCGCTTCTCTCGCTACCGTCGAGG 
               
               
                   
               
               
                 GTATCACCTCTACCTCTGAGGCTATCGCTGCTATCGGTCTCACCCCTGAG 
               
               
                   
               
               
                 ACCTACGCTGTCATCACCGGTGCTCCTGGTGCTGTCGCTGGTTTCGCTGC 
               
               
                   
               
               
                 TCTCGTCCAGACCGTCACCGGTGGTTCTGCTATCGCTCAGCTCGGTTACC 
               
               
                   
               
               
                 GTTTCTTCGCTGACTGGGACCACAAGGTCTCTACCGTCGGTCTCTTCCAG 
               
               
                   
               
               
                 CAGCCTGCTATGGCTCTCCAGCTCTTCAACCCTGAGGACTACTACGACAT 
               
               
                   
               
               
                 CCTCTTCCCTGGTGTCAACGCTTTCGTCAACAACATCCACTACCTCGACC 
               
               
                   
               
               
                 CTCGTCACTGGGGTCCTTCTCTCTTCTCTACCATCTCTCAGGCTTTCTGG 
               
               
                   
               
               
                 AACCTCGTCCGTGACGACCTCCCTGCTCTCACCTCTCAGGAGATCCAGCG 
               
               
                   
               
               
                 TCGTACCCAGAAGCTCTTCGTCGAGTCTCTCGCTCGTTTCCTCGAGGAGA 
               
               
                   
               
               
                 CCACCTGGGCTATCGTCAACTCTCCTGCTAACCTCTACAACTACATCTCT 
               
               
                   
               
               
                 GACTACTACTCTCGTCTCTCTCCTGTCCGTCCTTCTATGGTCCGTCAGGT 
               
               
                   
               
               
                 CGCTCAGCGTGAGGGTACCTACATCTCTTTCGGTCACTCTTACACCCAGT 
               
               
                   
               
               
                 CTATCGACGACGCTGACTCTATCCAGGAGGTCACCCAGCGTCTCGACCTC 
               
               
                   
               
               
                 AAGACCCCTAACGTCCAGTCTGGTGAGTTCATCGAGCGTTCTATCGCTCC 
               
               
                   
               
               
                 TGGTGGTGCTAACCAGCGTTCTGCTCCTCAGTGGATGCTCCCTCTCCTCC 
               
               
                   
               
               
                 TCGGTCTCTACGGTACCGTCACCCCTGCTCTCGAGGCTTACGAGGACGGT 
               
               
                   
               
               
                 CCTAACAAGAAGAAGCGTCGTAAGGAGGGTCCTCGTGCTTCTTCTAAGAC 
               
               
                   
               
               
                 CTCTTACAAGCGTCGTTCTCGTTCTTCTCGTTCTGGAGGTGGATCTGGAA 
               
               
                   
               
               
                 ATTCTGCGGCCGCA 
               
            
           
         
       
     
     PgB5-A1 expression cassettes.  Arabidopsis thaliana  oleosin (OLE) protein is capable of targeting itself, as well as, another covalently fused protein to plant cell oil bodies, thus providing accumulation of the target protein in plant lipid fraction making it easily extractable (“component B5”). OLE protein was used for cloning as a fusion with CMG2 (“component A1”), resulting in “component B5-A1” recombinant antitoxin, and named PgB5-A1. PgB5-A1 constructs were optimized for transient and stable plant transformation/expression. 
     A 530 bp nucleic acid sequence PgB5-2: A1-3 (SEQ ID NO: 22) encoding the PgB5 protein (176 aa) for cloning as a fusion with the ATR-encoding DNA fragment in transient or stable plant transformation/expression vectors is as follows: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 22) 
               
            
           
           
               
            
               
                 CCATGGCCGACACGGCCAGGGGCACGCACCACGACATCATCGGCAGGGAC 
               
               
                   
               
               
                 CAGTACCCGATGATGGGCAGGGACAGGGACCAGTACCAGATGTCCGGCAG 
               
               
                   
               
               
                 GGGCTCCGACTACTCCAAGTCCAGGCAGATCGCCAAGGCCGCCACGGCCG 
               
               
                   
               
               
                 TGACGGCCGGCGGCTCCCTCCTCGTGCTCTCCTCCCTCACGCTCGTGGGC 
               
               
                   
               
               
                 ACGGTGATCGCCCTCACGGTGGCCACGCCGCTCCTCGTGATCTTCTCCCC 
               
               
                   
               
               
                 GATCCTCGTGCCGGCCCTCATCACGGTGGCCCTCCTCATCACGGGCTTCC 
               
               
                   
               
               
                 TCTCCTCCGGCGGCTTCGGCATCGCCGCCATCACGGTGTTCTCCTGGATC 
               
               
                   
               
               
                 TACAAGTACGCCACGGGCGAGCACCCGCAGGGCTCCGACAAGCTCGACTC 
               
               
                   
               
               
                 CGCCAGGATGAAGCTCGGCTCCAAGGCCCAGGACCTCAAGGACAGGGCCC 
               
               
                   
               
               
                 AGTACTACGGCCAGCAGCACACGGGCGGCGAGCACGACAGGGACAGGACG 
               
               
                   
               
               
                 AGGGGCGGCCAGCACACGACGGCGGCCGCA 
               
            
           
         
       
     
     PgA2 expression cassettes. PA-binding domain of LF (LFn, amino acids 28-263) of the  B. anthracis  three-component anthrax toxin (component A2), capable of binding the PA region was optimized for expression in plants. PgA2 designs for transient and stable plant transformation/expression are shown as follows. 
     A 722 bp nucleic acid sequence of PgA2-1(SEQ ID NO: 3) encoding the PgA2 protein (237 aa) for cloning in transient expression vector is as follows: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 3) 
               
            
           
           
               
            
               
                 CCATGGGTGACGTCGGTATGCACGTCAAGGAGAAGGAGAAGAACAAGGAC 
               
               
                   
               
               
                 GAGAACAAGCGTAAGGACGAGGAGCGTAACAAGACCCAGGAGGAGCACCT 
               
               
                   
               
               
                 CAAGGAGATCATGAAGCACATCGTCAAGATCGAGGTCAAGGGTGAGGAGG 
               
               
                   
               
               
                 CTGTCAAGAAGGAGGCTGCTGAGAAGCTCCTCGAGAAGGTCCCTTCTGAC 
               
               
                   
               
               
                 GTCCTCGAGATGTACAAGGCTATCGGTGGTAAGATCTACATCGTCGACGG 
               
               
                   
               
               
                 TGACATCACCAAGCACATCTCTCTCGAGGCTCTCTCTGAGGACAAGAAGA 
               
               
                   
               
               
                 AGATCAAGGACATCTACGGTAAGGACGCTCTCCTCCACGAGCACTACGTC 
               
               
                   
               
               
                 TACGCTAAGGAGGGTTACGAGCCTGTCCTCGTCATCCAGTCTTCTGAGGA 
               
               
                   
               
               
                 CTACGTCGAGAACACCGAGAAGGCTCTCAACGTCTACTACGAGATCGGTA 
               
               
                   
               
               
                 AGATCCTCTCTCGTGACATCCTCTCTAAGATCAACCAGCCTTACCAGAAG 
               
               
                   
               
               
                 TTCCTCGACGTCCTCAACACCATCAAGAACGCTTCTGACTCTGACGGTCA 
               
               
                   
               
               
                 GGACCTCCTCTTCACCAACCAGCTCAAGGAGCACCCTACCGACTTCTCTG 
               
               
                   
               
               
                 TCGAGTTCCTCGAGCAGAACTCTAACGAGGTCCAGGAGGTCTTCGCTAAG 
               
               
                   
               
               
                 GCTTTCGCTTACTACATCGAGCCTCAGCACCGTGACGTCCTCCAGCTCTA 
               
               
                   
               
               
                 CGCTCCTGAGGCTTAAGAGCTC 
               
            
           
         
       
     
     A 728 bp nucleic acid sequence of PgA2-2(SEQ ID NO: 4) encoding the PgA2 protein (241 aa) for stable plant transformation vectors is as follows: 
     
       
         
           
               
            
               
                 SEQ ID NO: 4) 
               
            
           
           
               
            
               
                 CCATGGGTGACGTCGGTATGCACGTCAAGGAGAAGGAGAAGAACAAGGAC 
               
               
                   
               
               
                 GAGAACAAGCGTAAGGACGAGGAGCGTAACAAGACCCAGGAGGAGCACCT 
               
               
                   
               
               
                 CAAGGAGATCATGAAGCACATCGTCAAGATCGAGGTCAAGGGTGAGGAGG 
               
               
                   
               
               
                 CTGTCAAGAAGGAGGCTGCTGAGAAGCTCCTCGAGAAGGTCCCTTCTGAC 
               
               
                   
               
               
                 GTCCTCGAGATGTACAAGGCTATCGGTGGTAAGATCTACATCGTCGACGG 
               
               
                   
               
               
                 TGACATCACCAAGCACATCTCTCTCGAGGCTCTCTCTGAGGACAAGAAGA 
               
               
                   
               
               
                 AGATCAAGGACATCTACGGTAAGGACGCTCTCCTCCACGAGCACTACGTC 
               
               
                   
               
               
                 TACGCTAAGGAGGGTTACGAGCCTGTCCTCGTCATCCAGTCTTCTGAGGA 
               
               
                   
               
               
                 CTACGTCGAGAACACCGAGAAGGCTCTCAACGTCTACTACGAGATCGGTA 
               
               
                   
               
               
                 AGATCCTCTCTCGTGACATCCTCTCTAAGATCAACCAGCCTTACCAGAAG 
               
               
                   
               
               
                 TTCCTCGACGTCCTCAACACCATCAAGAACGCTTCTGACTCTGACGGTCA 
               
               
                   
               
               
                 GGACCTCCTCTTCACCAACCAGCTCAAGGAGCACCCTACCGACTTCTCTG 
               
               
                   
               
               
                 TCGAGTTCCTCGAGCAGAACTCTAACGAGGTCCAGGAGGTCTTCGCTAAG 
               
               
                   
               
               
                 GCTTTCGCTTACTACATCGAGCCTCAGCACCGTGACGTCCTCCAGCTCTA 
               
               
                   
               
               
                 CGCTCCTGAGGCTGGAGATCTCCAATAA 
               
            
           
         
       
     
     PgA2-B1 expression cassettes. PA-binding domain of LF (LF, amino acids 28-263) of the  B. anthracis  three-component anthrax toxin (component A2), capable of binding the PA region was optimized for expression in plants as fusion with human IgG1 Fc-fragment (component B1), resulting in a component A2-B1 recombinant antitoxin. PgA2-B1 designs for transient and stable plant transformation/expression are shown as follows. 
     A 721 bp nucleic acid sequence encoding PgA2-4: B1-3 (SEQ ID NO: 37) the PgA2 protein (239 aa) for cloning as a fusion with the Fc-encoding DNA fragment in the transient (as in SEQ ID NO: 16) or stable transformation/expression vectors (as in SEQ ID NO: 17): 
     
       
         
           
               
            
               
                 (SEQ ID NO: 37) 
               
            
           
           
               
            
               
                 CCATGGGTGACGTCGGTATGCACGTCAAGGAGAAGGAGAAGAACAAGGAC 
               
               
                   
               
               
                 GAGAACAAGCGTAAGGACGAGGAGCGTAACAAGACCCAGGAGGAGCACCT 
               
               
                   
               
               
                 CAAGGAGATCATGAAGCACATCGTCAAGATCGAGGTCAAGGGTGAGGAGG 
               
               
                   
               
               
                 CTGTCAAGAAGGAGGCTGCTGAGAAGCTCCTCGAGAAGGTCCCTTCTGAC 
               
               
                   
               
               
                 GTCCTCGAGATGTACAAGGCTATCGGTGGTAAGATCTACATCGTCGACGG 
               
               
                   
               
               
                 TGACATCACCAAGCACATCTCTCTCGAGGCTCTCTCTGAGGACAAGAAGA 
               
               
                   
               
               
                 AGATCAAGGACATCTACGGTAAGGACGCTCTCCTCCACGAGCACTACGTC 
               
               
                   
               
               
                 TACGCTAAGGAGGGTTACGAGCCTGTCCTCGTCATCCAGTCTTCTGAGGA 
               
               
                   
               
               
                 CTACGTCGAGAACACCGAGAAGGCTCTCAACGTCTACTACGAGATCGGTA 
               
               
                   
               
               
                 AGATCCTCTCTCGTGACATCCTCTCTAAGATCAACCAGCCTTACCAGAAG 
               
               
                   
               
               
                 TTCCTCGACGTCCTCAACACCATCAAGAACGCTTCTGACTCTGACGGTCA 
               
               
                   
               
               
                 GGACCTCCTCTTCACCAACCAGCTCAAGGAGCACCCTACCGACTTCTCTG 
               
               
                   
               
               
                 TCGAGTTCCTCGAGCAGAACTCTAACGAGGTCCAGGAGGTCTTCGCTAAG 
               
               
                   
               
               
                 GCTTTCGCTTACTACATCGAGCCTCAGCACCGTGACGTCCTCCAGCTCTA 
               
               
                   
               
               
                 CGCTCCTGAGGCTGCGGCCGC 
               
            
           
         
       
     
     Helper element (component C)/PgC1 expression cassettes. Human immunoglobulin J polypeptide, linker protein for immunoglobulin Alpha and Mu polypeptides (IgJ, amino acids 34-159) (component C1), that has no native signal peptide (amino acids 1-33), was optimized for expression in plants. IgJ is capable of helping IgA and/or IgM subunits to self-assemble into quaternary structure. PgC1 cassettes were designed for transient and stable plant transformation/expression. 
     A 395 bp nucleic acid sequence of PgC1-2 (SEQ ID NO: 32) encoding the PgC1 protein (128 aa) for transient expression vectors is as follows: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 32) 
               
            
           
           
               
            
               
                 CCATGGGAAAGTGCAAGTGCGCTCGTATCACCTCTCGTATCATCCGTTCT 
               
               
                   
               
               
                 TCTGAGGACCCTAACGAGGACATCGTCGAGCGTAACATCCGTATCATCGT 
               
               
                   
               
               
                 CCCTCTCAACAACCGTGAGAACATCTCTGACCCTACCTCTCCTCTCCGTA 
               
               
                   
               
               
                 CCCGTTTCGTCTACCACCTCTCTGACCTCTGCAAGAAGTGCGACCCTACC 
               
               
                   
               
               
                 GAGGTCGAGCTCGACAACCAGATCGTCACCGCTACCCAGTCTAACATCTG 
               
               
                   
               
               
                 CGACGAGGACTCTGCTACCGAGACCTGCTACACCTACGACCGTAACAAGT 
               
               
                   
               
               
                 GCTACACCGCTGTCGTCCCTCTCGTCTACGGTGGTGAGACCAAGATGGTC 
               
               
                   
               
               
                 GAGACCGCTCTCACCCCTGACGCTTGCTACCCTGACTAAGAGCTC 
               
            
           
         
       
     
     A 401 bp nucleic acid sequence of PgC1-3 (SEQ ID NO: 33) encoding the PgC1 Nt  protein (132 aa) for stable transformation vectors is as follows: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 33) 
               
            
           
           
               
            
               
                 CCATGGGAAAGTGCAAGTGCGCTCGTATCACCTCTCGTATCATCCGTTCT 
               
               
                   
               
               
                 TCTGAGGACCCTAACGAGGACATCGTCGAGCGTAACATCCGTATCATCGT 
               
               
                   
               
               
                 CCCTCTCAACAACCGTGAGAACATCTCTGACCCTACCTCTCCTCTCCGTA 
               
               
                   
               
               
                 CCCGTTTCGTCTACCACCTCTCTGACCTCTGCAAGAAGTGCGACCCTACC 
               
               
                   
               
               
                 GAGGTCGAGCTCGACAACCAGATCGTCACCGCTACCCAGTCTAACATCTG 
               
               
                   
               
               
                 CGACGAGGACTCTGCTACCGAGACCTGCTACACCTACGACCGTAACAAGT 
               
               
                   
               
               
                 GCTACACCGCTGTCGTCCCTCTCGTCTACGGTGGTGAGACCAAGATGGTC 
               
               
                   
               
               
                 GAGACCGCTCTCACCCCTGACGCTTGCTACCCTGACGCAGATCTCCAATA 
               
               
                   
               
               
                 A 
               
            
           
         
       
     
     VLP helper element/PgC2 expression cassettes. A coat protein VP1 of human polyoma virus capable of self-assembling into VLPs and necessary for the inclusion of JC virus VP2 protein into the same VLP assembly (“component C2”), resulting in a component C2, was optimized for expression in plants. PgC2 design for Magnifection and stable plant transformation is shown as follows. 
     A 1133 bp nucleic acid PgC2-2 (SEQ ID NO: 34) encoding the PgC2 protein (374 aa) for cloning in MagnICON expression vector or in pIV1.2/pIV1.3 Impact Vectors (Plant Research International, Wageningen, The Netherlands): 
     
       
         
           
               
            
               
                 (SEQ ID NO: 34) 
               
            
           
           
               
            
               
                 CCATGGCCCCAACAAAGAGAAAAGGAGAAAGGAAGGACCCAGTGCAAGTT 
               
               
                   
               
               
                 CCAAAACTTCTCATAAGAGGAGGAGTAGAAGTTCTTGAAGTTAAAACTGG 
               
               
                   
               
               
                 AGTTGACTCAATTACAGAGGTAGAATGCTTCTTAACTCCAGAAATGGGTG 
               
               
                   
               
               
                 ACCCAGATGAGCATCTTAGGGGTTTTAGTAAGTCAATATCTATATCAGAT 
               
               
                   
               
               
                 ACATTTGAAAGTGACTCCCCAAATAGGGACATGCTTCCTTGTTACAGTGT 
               
               
                   
               
               
                 GGCCAGGATTCCACTACCTAATCTAAATGAGGATCTAACTTGTGGAAATA 
               
               
                   
               
               
                 TACTCATGTGGGAGGCTGTGACATTAAAGACTGAGGTTATAGGAGTGACA 
               
               
                   
               
               
                 AGTTTGATGAATGTGCATTCTAATGGTCAAGCAACTCATGACAATGGTGC 
               
               
                   
               
               
                 AGGTAAGCCAGTGCAGGGTACAAGTTTTCATTTCTTTTCTGTTGGAGGTG 
               
               
                   
               
               
                 AGGCTTTAGAATTACAGGGAGTGCTTTTTAATTACAGAACAAAGTACCCA 
               
               
                   
               
               
                 GATGGAACAATTTTTCCAAAGAATGCCACAGTGCAATCTCAAGTCATGAA 
               
               
                   
               
               
                 CACAGAGCATAAGGCGTACCTAGATAAGAACAAAGCATATCCTGTTGAAT 
               
               
                   
               
               
                 GTTGGGTTCCTGATCCAACTAGAAATGAAAACACAAGATATTTTGGTACA 
               
               
                   
               
               
                 CTAACAGGAGGAGAAAATGTTCCTCCAGTTCTTCATATAACAAACACTGC 
               
               
                   
               
               
                 CACAACAGTGTTGCTTGATGAATTTGGTGTTGGACCACTTTGTAAAGGTG 
               
               
                   
               
               
                 ACAACTTATACTTGTCAGCTGTTGATGTCTGTGGTATGTTTACAAACAGG 
               
               
                   
               
               
                 TCTGGTTCCCAGCAGTGGAGAGGACTCTCCAGATATTTTAAGGTGCAGCT 
               
               
                   
               
               
                 AAGGAAGAGGAGGGTTAAGAACCCATACCCAATTTCTTTCCTTCTTACTG 
               
               
                   
               
               
                 ATTTGATTAACAGAAGGACTCCTAGAGTTGATGGACAGCCTATGTATGGT 
               
               
                   
               
               
                 ATGGATGCTCAAGTAGAGGAGGTTAGAGTTTTTGAGGGAACAGAGGAGCT 
               
               
                   
               
               
                 TCCAGGAGACCCAGACATGATGAGATACGTTGACAAATATGGACAGTTGC 
               
               
                   
               
               
                 AGACAAAGATGCTGGCGGCCGCAGATCTCCAAAAGCTTATTAGCGAGGAG 
               
               
                   
               
               
                 GATCTTCATCACCATCACCATCACTAAGAGCTC 
               
            
           
         
       
     
     PgB1, PgB2, and PgB2 expression cassettes. Human IgG1, IgA1, and IgM Fc-fragment (components B1, B2, and B3) polypeptides were used as carrier proteins. B1, B2 and B3 constructs were optimized for transient and stable plant transformation/expression. 
     A 687 bp nucleic acid sequence of PgB1-1(SEQ ID NO: 6) encoding the PgB1 polypeptide (229 aa) for cloning as a fusion with A1 or A2 Component in transient or stable plant transformation/expression vectors is as follows: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 6) 
               
            
           
           
               
            
               
                 GATAAAACTCATACTTGTCCTCCTTGTCCTGCTCCTGAACTTCTTGGTGG 
               
               
                   
               
               
                 TCCTTCTGTTTTTCTTTTTCCTCCTAAACCTAAAGATACTCTTATGATTT 
               
               
                   
               
               
                 CTCGTACTCCTGAAGTTACTTGTGTTGTTGTTGATGTTTCTCATGAAGAT 
               
               
                   
               
               
                 CCTGAAGTTAAATTTAATTGGTATGTTGATGGTGTTGAAGTTCATAATGC 
               
               
                   
               
               
                 TAAAACTAAACCTCGTGAAGAACAATATAATTCTACTTATCGTGTTGTTT 
               
               
                   
               
               
                 CTGTTCTTACTGTTCTTCATCAAGATTGGCTTAATGGTAAAGAATATAAA 
               
               
                   
               
               
                 TGTAAAGTTTCTAATAAAGCTCTTCCTGCTCCTATTGAAAAAACTATTTC 
               
               
                   
               
               
                 TAAAGCTAAAGGTCAACCTCGTGAACCTCAAGTTTATACTCTTCCTCCTT 
               
               
                   
               
               
                 CTCGTGAAGAAATGACTAAAAATCAAGTTTCTCTTACTTGTCTTGTTAAA 
               
               
                   
               
               
                 GGTTTTTATCCTTCTGATATTGCTGTTGAATGGGAATCTAATGGTCAACC 
               
               
                   
               
               
                 TGAAAATAATTATAAAACTACTCCTCCTGTTCTTGATTCTGATGGTTCTT 
               
               
                   
               
               
                 TTTTTCTTTATTCTAAACTTACTGTTGATAAATCTCGTTGGCAACAAGGT 
               
               
                   
               
               
                 AATGTTTTTTCTTGTTCTGTTATGCATGAAGCTCTTCATAATCATTATAC 
               
               
                   
               
               
                 TCAAAAATCTCTTTCTCTTTCTCCTGGTAAAGATCTT 
               
            
           
         
       
     
     A 774 bp nucleic acid sequence of PgB2-1 (SEQ ID NO: 7) encoding the PgB2 polypeptide (258 aa) for cloning as a fusion with A1 or A2 Component in transient or stable plant transformation/expression vectors is as follows: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 7) 
               
            
           
           
               
            
               
                 GATGTTACTGTTCCTTGTCCTGTTCCTTCTACTCCTCCTACTCCTTCTCC 
               
               
                   
               
               
                 TTCTACTCCTCCTACTCCTTCTCCTTCTTGTTGTCATCCTCGTCTTTCTC 
               
               
                   
               
               
                 TTCATCGTCCTGCTCTTGAAGATCTTCTTCTTGGTTCTGAAGCTAATCTT 
               
               
                   
               
               
                 ACTTGTACTCTTACTGGTCTTCGTGATGCTTCTGGTGTTACTTTTACTTG 
               
               
                   
               
               
                 GACTCCTTCTTCTGGTAAATCTGCTGTTCAAGGTCCTCCTGAACGTGATC 
               
               
                   
               
               
                 TTTGTGGTTGTTATTCTGTTTCTTCTGTTCTTCCTGGTTGTGCTGAACCT 
               
               
                   
               
               
                 TGGAATCATGGTAAAACTTTTACTTGTACTGCTGCTTATCCTGAATCTAA 
               
               
                   
               
               
                 AACTCCTCTTACTGCTACTCTTTCTAAATCTGGTAATACTTTTCGTCCTG 
               
               
                   
               
               
                 AAGTTCATCTTCTTCCTCCTCCTTCTGAAGAACTTGCTCTTAATGAACTT 
               
               
                   
               
               
                 GTTACTCTTACTTGTCTTGCTCGTGGTTTTTCTCCTAAAGATGTTCTTGT 
               
               
                   
               
               
                 TCGTTGGCTTCAAGGTTCTCAAGAACTTCCTCGTGAAAAATATCTTACTT 
               
               
                   
               
               
                 GGGCTTCTCGTCAAGAACCTTCTCAAGGTACTACTACTTTTGCTGTTACT 
               
               
                   
               
               
                 TCTATTCTTCGTGTTGCTGCTGAAGATTGGAAAAAAGGTGATACTTTTTC 
               
               
                   
               
               
                 TTGTATGGTTGGTCATGAAGCTCTTCCTCTTGCTTTTACTCAAAAAACTA 
               
               
                   
               
               
                 TTGATCGTCTTGCTGGTAAACCTACTCATGTTAATGTTTCTGTTGTTATG 
               
               
                   
               
               
                 GCTGAAGTTGATGGTACTTGTTAT 
               
            
           
         
       
     
     A 1059 bp nucleic acid sequence of PgB3-1 (SEQ ID NO: 8) encoding the PgB3 polypeptide (353 aa) for cloning as a fusion with A1 or A2 Component in transient or stable plant transformation/expression vectors is as follows: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 8) 
               
            
           
           
               
            
               
                 GTTCCTCTTCCTGTTATTGCTGAACTTCCTCCTAAAGTTTCTGTTTTTGT 
               
               
                   
               
               
                 TCCTCCTCGTGATGGTTTTTTTGGTAATCCTCGTAAATCTAAACTTATTT 
               
               
                   
               
               
                 GTCAAGCTACTGGTTTTTCTCCTCGTCAAATTCAAGTTTCTTGGCTTCGT 
               
               
                   
               
               
                 GAAGGTAAACAAGTTGGTTCTGGTGTTACTACTGATCAAGTTCAAGCTGA 
               
               
                   
               
               
                 AGCTAAAGAATCTGGTCCTACTACTTATAAAGTTACTTCTACTCTTACTA 
               
               
                   
               
               
                 TTAAAGAATCTGATTGGCTTTCTCAATCTATGTTTACTTGTCGTGTTGAT 
               
               
                   
               
               
                 CATCGTGGTCTTACTTTTCAACAAAATGCTTCTTCTATGTGTGTTCCTGA 
               
               
                   
               
               
                 TCAAGATACTGCTATTCGTGTTTTTGCTATTCCTCCTTCTTTTGCTTCTA 
               
               
                   
               
               
                 TTTTTCTTACTAAATCTACTAAACTTACTTGTCTTGTTACTGATCTTACT 
               
               
                   
               
               
                 ACTTATGATTCTGTTACTATTTCTTGGACTCGTCAAAATGGTGAAGCTGT 
               
               
                   
               
               
                 TAAAACTCATACTAATATTTCTGGATGATTGGAATTCTGGTGAACGTTTT 
               
               
                   
               
               
                 ACTTGTACTGTTACTCATACTGATCTTCCTTCTCCTCTTAAACAAACTAT 
               
               
                   
               
               
                 TTCTCGTCCTAAAGGTGTTGCTCTTCATCGTCCTGATGTTTATCTTCTTC 
               
               
                   
               
               
                 CTCCTGCTCGTGAACAACTTAATCTTCGTGAATCTGCTACTATTACTTGT 
               
               
                   
               
               
                 CTTGTTACTGGTTTTTCTCCTGCTGATGTTTTTGTTCAATGGATGCAACG 
               
               
                   
               
               
                 TGGTCAACCTCTTTCTCCTGAAAAATATGTTACTTCTGCTCCTATGCCTG 
               
               
                   
               
               
                 AACCTCAAGCTCCTGGTCGTTATTTTGCTCATTCTATTCTTACTGTTTCT 
               
               
                   
               
               
                 GAAGAAGAATGGAATACTGGTGAAACTTATACTTGTGTTGTTGCTCATGA 
               
               
                   
               
               
                 AGCTCTTCCTAATCGTGTTACTGAACGTACTGTTGATAAATCTACTGGTA 
               
               
                   
               
               
                 AACCTACTCTTTATAATGTTTCTCTTGTTATGTCTGATACTGCTGGTACT 
               
               
                   
               
               
                 TGTTAT 
               
            
           
         
       
     
     Table 2 lists and describes all sequences included herein. 
     
       
         
           
               
               
               
             
               
                   
               
               
                   
                   
                 SEQ 
               
               
                 SEQ NAME 
                 DESCRIPTION 
                 ID NO 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 PgA1-1 construct 
                 Extracellular domain 
                 1 
               
               
                   
                 of human CMG2 for 
               
               
                   
                 transient expression 
               
               
                   
                 (DNA) 
               
               
                 PgA1-2 construct 
                 Extracellular domain 
                 2 
               
               
                   
                 of human CMG2 for 
               
               
                   
                 stable transformation 
               
               
                   
                 (DNA) 
               
               
                 PgA2-1 construct 
                 PA-binding domain of 
                 3 
               
               
                   
                 Lethal factor LF 
               
               
                   
                 for transient 
               
               
                   
                 expression (DNA) 
               
               
                 PgA2-2 construct 
                 PA-binding domain of 
                 4 
               
               
                   
                 Lethal factor LF 
               
               
                   
                 for stable 
               
               
                   
                 transformation(DNA) 
               
               
                 A2-3-PA-LF construct 
                 Native PA-binding 
                 5 
               
               
                   
                 domain LF of  B. anthracis   
               
               
                   
                 lethal 
               
               
                   
                 factor LF (DNA) 
               
               
                   
                 Accession No: 
               
               
                   
                 M29081.1 
               
               
                 PgB1-1 construct 
                 Human Fc-IgG1 
                 6 
               
               
                   
                 fragment optimized 
               
               
                   
                 for medicinal plant 
               
               
                   
                 (DNA) 
               
               
                 PgB2-1 construct 
                 Human Fc-IgA1 
                 7 
               
               
                   
                 fragment optimized 
               
               
                   
                 for medicinal plant 
               
               
                   
                 (DNA) 
               
               
                 PgB3-1 construct 
                 Human Fc-IgM 
                 8 
               
               
                   
                 fragment optimized 
               
               
                   
                 for medicinal plant 
               
               
                   
                 (DNA) 
               
               
                 PgB1-2 construct 
                 Native Fc-IgG 
                 9 
               
               
                   
                 fragment (DNA) 
               
               
                 PgB2-2 construct 
                 Native Fc-IgA 
                 10 
               
               
                   
                 fragment (DNA) 
               
               
                 PgB3-2 construct 
                 Native Fc-IgM 
                 11 
               
               
                   
                 fragment (DNA) 
               
               
                 PgB4-1 construct 
                 Native VP2 coat 
                 12 
               
               
                   
                 protein of JC virus 
               
               
                   
                 (DNA) 
               
               
                 PgB5 construct 
                 Native  A. thaliana   
                 13 
               
               
                   
                 oleosin (DNA) 
               
               
                 PgC1-1 construct 
                 Native IgJ gene 
                 14 
               
               
                 C2-1 construct 
                 Native VP1 coat 
                 15 
               
               
                   
                 protein of JC virus 
               
               
                   
                 (DNA) 
               
               
                 PgA1-3:B1-3 construct 
                 sCMG2-IgG1(Fc) for 
                 16 
               
               
                   
                 transient expression 
               
               
                   
                 (DNA) 
               
               
                 PgA1-4:B1-4 construct 
                 CMG2-IgG1(Fc) for 
                 17 
               
               
                   
                 stable transformation 
               
               
                   
                 (DNA) 
               
               
                 PgA1-3:B2-4 construct 
                 sCMG2-IgA1 (Fc) 
                 18 
               
               
                   
                 for transient 
               
               
                   
                 expression (DNA) 
               
               
                 PgA1-4:B2-4 construct 
                 sCMG2-IgA1 (Fc) 
                 19 
               
               
                   
                 for stable 
               
               
                   
                 transformation 
               
               
                   
                 (DNA) 
               
               
                 PgA1-3:B3-3 construct 
                 CMG2-IgM1 (Fc) 
                 20 
               
               
                   
                 for transient 
               
               
                   
                 expression (DNA) 
               
               
                 PgA1-4:B3-4 construct 
                 sCMG2-IgM1 (Fc) 
                 21 
               
               
                   
                 for stable 
               
               
                   
                 transformation 
               
               
                   
                 (DNA) 
               
               
                 PgA1-3:B4-2 construct 
                 VP2 polyoma 
                 22 
               
               
                   
                 (JCvirus)-sCMG2 
               
               
                   
                 (DNA) 
               
               
                 PgA1-3:B5-2 
                   A. thaliana  oleosin 
                 23 
               
               
                   
                 (OLE)-sCMG2 (DNA) 
               
               
                 A1-CMG2 protein 
                 Native example of 
                 24 
               
               
                   
                 human CMG2 protein 
               
               
                 A2-protein-PA-binding LF 
                 Native example of PA 
                 25 
               
               
                 protein 
                 binding domain of  B. anthracis   
               
               
                   
                 lethal 
               
               
                   
                 factor LF protein 
               
               
                 B1-protein 
                 Native Fc-IgG protein 
                 26 
               
               
                   
                 Accession No. 
               
               
                   
                 AY172957 
               
               
                 B2-protein 
                 Native Fc-IgA protein 27 
               
               
                   
                 Accession No. S71043 
               
               
                 B3-protein 
                 Native Fc-IgM 
                 28 
               
               
                   
                 protein Accession No. 
               
               
                   
                 X67301 S50847 
               
               
                 B4-protein 
                 Native VP2 coat 
                 29 
               
               
                   
                 protein of JC virus 
               
               
                   
                 Accession No. 
               
               
                   
                 NC_001699 
               
               
                 B5-protein 
                 Native  A. thaliana   
                 30 
               
               
                   
                 oleosin 
               
               
                   
                 Accession No. X62353 
               
               
                   
                 S38026 
               
               
                 PgA1-1:B1-1 protein 
                 Amino acid sequence 
                 31 
               
               
                   
                 PgA1B1 
               
               
                 C1-2 construct 
                 Human IgJ 
                 32 
               
               
                   
                 polypeptide (linker 
               
               
                   
                 for IGgs Alpha and 
               
               
                   
                 Mu) for transient 
               
               
                   
                 expression 
               
               
                 C1-3 construct 
                 Human IgJ 
                 33 
               
               
                   
                 polypeptide (linker 
               
               
                   
                 for Igs Alpha and Mu) 
               
               
                   
                 for stable 
               
               
                   
                 transformation 
               
               
                   
                 (DNA) 
               
               
                 C2-2 construct 
                 A coat protein of 
                 34 
               
               
                   
                 human polyoma virus 
               
               
                 C1-protein 
                 IgJ protein 
                 35 
               
               
                   
                 Accession No. 
               
               
                   
                 NM_144646 
               
               
                 C2-protein 
                 VP1 coat protein of 
                 36 
               
               
                   
                 JC virus 
               
               
                   
                 Accession No. 
               
               
                   
                 NC_001699 
               
               
                 PgA2-4:B1-3 construct 
                 PA-binding domain of 
                 37 
               
               
                   
                 lethal factor-Fc-IgG1 
               
               
                   
                 (DNA) 
               
               
                 PgA1-5:B1-5 construct 
                 sCMG2-hIgG1(Fc) 
                 38 
               
               
                   
                 gene optimized for 
               
               
                   
                 expression in plants 
               
               
                   
                 (DNA) 
               
               
                 Kozak-like sequence 
                 Regulatory element 
                 39 
               
               
                   
                 (DNA) 
               
               
                 BGLII and SACI sites 
                 Restriction sites 
                 40 
               
               
                 NOTI site 
                 Restriction site 
                 41 
               
               
                 BAA targeting peptide 
                 Targeting peptide 
                 42 
               
               
                 ABP1 targeting peptide 
                 Targeting peptide 
                 43 
               
               
                 Flexible linker 
                 Linker 
                 44 
               
               
                 Trombin cleavage peptide 
                 Cleavage peptide 
                 45 
               
               
                 Factor Faa protease 
                 Cleavage peptide 
                 46 
               
               
                 cleavage peptide 
               
               
                 PgA1-3 construct 
                 Extracellular domain 
                 47 
               
               
                   
                 of human CMG2 
               
               
                   
                 (DNA) 
               
               
                 PgB1-3 construct 
                 Human Fc-IgG1 
                 48 
               
               
                   
                 fragment (DNA) 
               
               
                 PgA1-4 construct 
                 Extracellular domain 
                 49 
               
               
                   
                 of human CMG2 
               
               
                   
                 (DNA) 
               
               
                 PgB1-4 construct 
                 Human Fc-IgG1 
                 50 
               
               
                   
                 fragment (DNA) 
               
               
                 PgB2-3 construct 
                 Human Fc-IgA1 
                 51 
               
               
                   
                 fragment (DNA) 
               
               
                 PgB2-4 construct 
                 Human Fc-IgA1 
                 52 
               
               
                   
                 fragment (DNA) 
               
               
                 PgB3-3 construct 
                 Fc-IgM fragment 
                 53 
               
               
                   
                 (DNA) 
               
               
                 PgB3-4 construct 
                 Fc-IgM fragment 
                 54 
               
               
                   
                 (DNA) 
               
               
                 PgB4-2 construct 
                 VP2 coat protein of 
                 55 
               
               
                   
                 JC virus (DNA 
               
               
                 PgB5-2 construct 
                   A. thaliana  oleosin 
                 56 
               
               
                   
                 (DNA) 
               
               
                 PgA2-4 construct 
                 Human Fc-IgA1 
                 57 
               
               
                   
                 fragment (DNA) 
               
               
                 PgA1-5 construct 
                 Extracellular domain 
                 58 
               
               
                   
                 of human CMG2 
               
               
                   
                 (DNA) 
               
               
                 PgB1-5 construct 
                 Human Fc-IgG1 
                 59 
               
               
                   
                 fragment DNA 
               
               
                 PgA1-5 protein 
                 Extracellular domain 
                 60 
               
               
                   
                 of human CMG2 
               
               
                   
                 protein 
               
               
                 PgB1-5 protein 
                 Human Fc-IgG1 
                 61 
               
               
                   
                 fragment protein 
               
               
                 NPTII forward primer 
                 PCR primer 
                 62 
               
               
                 NPTII reverse primer 
                 PCR primer 
                 63 
               
               
                   
               
            
           
         
       
     
     Example 2 
     Construction of the Expression Cassette for Production of Recombinant ATR-FC Fusion Proteins in Plants 
     A synthetic gene encoding PgA1B1 (SEQ ID NO: 38) was assembled from synthetic nucleotides and/or PCR products. The fragment was cloned into the pMK-RQ (KanR) plasmid using SfiI and SfiI cloning sites and resulted in a plasmid 12AA3X5C_PgA1B1_pMK-RQ ( FIG. 3A ). The plasmid includes an origin of replication Col E1, the restriction sites NarI, DraII, NcoI, Eco57I, and SfiI in the body of vector pMK-RQ, and the restriction sites NcoI, XhoI, HindIII, and BamHI in the 1297 bp synthetic PgA1B1 gene (SEQ ID NO: 39). The plasmid DNA was purified from transformed bacteria and concentration determined with UV spectroscopy. The final construct was verified by sequencing. The sequence congruence within the used restriction sites was 100%. 
     The sequence encoding PgA1B1 (SEQ ID NO: 38) was then cloned into an expression cassette using NcoI and BglII cloning sites. The expression cassette also included nucleic acid sequence encoding the human IgG1 Fc fragment, P rbcS  promoter and T rbcS  termination signals ( FIG. 3B ). The expression cassette also included the restriction sites XbaI, NcoI, BglII, and SacI for cloning of the expression cassette into the plant vector. The expression cassette includes PgA1B1, a synthetic nucleotide sequence encoding a soluble recombinant protein (component A1-B1) capable of binding an anthrax toxin PA protein. The recombinant protein includes ATR, which is covalently linked to the Fc fragment of an immunoglobulin G (Fc-IgG) using a short flexible polypeptide linker or hinge. The expression cassette also includes c-myc and His, peptide tags for easy purification of the recombinant protein. The cassette includes the KDEL (SEQ ID NO: 65) short signal peptide directing the expressed recombinant protein to a specific compartment of a plant cell. The expression cassette was than cloned into the pBI121 binary vector (based on the pBIN19 vector) using XbaI and SacI cloning sites (Bevan, M, 1984,  Nucl Acids Res: 12, 8711-8721). 
       FIG. 4A  schematically shows a plant expression vector. As shown, the vector includes a backbone derived from a conventional pBI binary vector covalently linked to the T-DNA surrounded by the right border, RB, and the left border, LB. The T-DNA includes the nptll gene for kanamycin selection. The nptII gene is linked to the expression cassette that includes the Rubisco promoter, P rbcS ; the recombinant protein PgA1B1; the purification tag, Tag; the endoplasmic reticulum compartment sorting signal, KDEL (SEQ ID NO: 65); and the translation termination signal of the Rubisco gene, RbcT.  FIG. 4B  is a diagram illustrating ways of using vectors for production of PgA1B1 recombinant proteins in plant. DNA of the expression vectors can be introduced into plant cells either by using  Agrobacterium , or by direct DNA transfer. This figure shows that the plant expression vector can be used for stable or transient transformation of plants. In the transient system, plant expression vectors are not integrated into the genomes of the transformed plants. In this system, the recombinant proteins can be produced after a short period of tissue incubation with the vectors. In the stable transformation system, only plant cells with the T-DNAs of the expression vectors integrated into the plant genomes are selected. Such transgenic plants can transfer the T-DNAs to the progeny which may also produce the recombinant proteins. 
     Example 3 
     Plants Transformation 
     Stable Transformation of Tobacco Plants 
     Stable transformation of tobacco was performed as described in Golovkin et al., 2007, which is incorporated herein by reference in entirety. Tobacco ( Nicotiana tabacum  cv Wisconsin 38) were used for all experiments. Tobacco seeds were surface sterilized and incubated on the solidified with 0.7% agar Murashige-Skoog medium (Physiol. Plant, 1962, 15:473) supplemented with 1% sucrose, pH 5.8 at 24° C., with 16/8 h light/dark photoperiod. 2-3 weeks old seedlings were excised and transferred to flasks containing the MS medium supplemented with 0.7% agar and 3% sucrose. Sterile leaf explants were transformed by using the described above kanamycin-resistant construct encoding recombinant ATR-Fc protein in the  Agrobacterium tumefaciens  strain LB4404. Leaves of 5-6 week-old aseptically grown plants were cut into segments 0.5 to 0.7 cm in size and inoculated for 10 minutes with the  Agrobacterium  cell suspension diluted to OD 600nm  0.5. Inoculated leaf explants were blotted dry and plated onto Petri dishes with solidified MS medium supplemented with 3% sucrose and 0.7% agar. After 2 days of co-cultivation at 24° C. in the dark, plant explants were transferred to the selection/regeneration MS medium supplemented with 3% sucrose, 1 mg/l BAP, 0.1 mg/l NAA, 100 mg/l kanamycin, 300 mg/l timentin, 0.7% agar and incubated at 24° C. and 16/8 h light/dark photoperiod. After 5-6 weeks of selection, the putative transgenic green shoots were formed ( FIGS. 5A-5B ). These shoots were excised and transferred to Magenta boxes containing MS medium supplemented with 100 mg/l kanamycin, 3% sucrose, 0.7% agar and 200 mg/l timentin for rooting,  FIG. 5C , where transgenic shoots were developing on the medium supplemented with kanamycin. Control wild type tobacco plants failed to grow under similar conditions ( FIG. 5A ). Plants that rooted in the presence of 100 mg/l kanamycin were tested for expression of recombinant products. The best transgenic lines were chosen for root induction and then were transferred to soil to mature and set seeds ( FIG. 5D ). Plants were grown in 10-15 cm pot in the greenhouse at 25° C. and 16/8 day/night period. 
     Selected transgenic lines were screened by a PCR reaction for the T-DNA integration based on the detection of the nptII marker encoding resistance to kanamycin ( FIG. 4A ) linked to recombinant PgA1B1 gene. The nptII-specific primers were as follows: 5-′TGAATGAACTGCAGGACGA-3′ (forward; SEQ ID NO: 62) and 5′-AGCCAACGTATGTCCTGAT-3′ (reverse; SEQ ID NO: 63). The PCR reaction condition included 30 cycles of polymerization at the annealing temperature 56° C. using Taq polymerase protocol from Promega. As shown on  FIG. 5E , over 50% tested plants were positive as they produced PCR products of expected molecular size of approximately 500 bp. 
     Transgenic plants were further screened with Western blotting for the presence of the ATR-Fc protein fused with the c-myc tag ( FIG. 3B ). A total crude protein extracts of selected tobacco lines were separated on SDS-PACE and tested by Western blot analysis using the c-myc monoclonal antibody mAb (ATCC, Manassas) and the goat anti-mouse horse radish conjugated antibody (Ab) (Upstate NY) ( FIG. 5F ). As shown on  FIG. 5F , the recombinant product of the expected size of approximately 50 kDa was detected as a single band in all but one tested samples containing a total soluble protein (TSP). The correct molecular weights were verified by pre-stained Universal Kaleidoscope protein marker (BioRad). 
     Transformation of  Echinacea  plants 
     Plant material. Seeds of Echinacea plants were obtained from Horizon Herbs Co. (Williams, Oreg.). Seeds were sterilized with 70% ethanol for 1 min and 25% commercial bleach solution for 10 min. After washing with sterile distilled water, seeds were placed in the germination MS medium containing 10 g/L sucrose and 8 g/L agar. Efficient germination and in vitro culturing was carried out at 24° C. at 16 h-light/8 h-dark photoperiods and 40 μE/m2/S1 light intensity. Cotyledons and young leaves were used for transformation experiments. 
     Transformation.  Agrobacterium tumefaciens  strain LBA4404 carrying the pBI binary vector for PgA1B1 protein expression was used for transformation. Cotyledons and young leaves were cut into 3-5 mm explants and incubated in  Agrobacterium  suspension (OD 600nm  0.5) for 10 min. After blotting dry with sterile filter paper, explants were transferred to solid MS co-cultivation medium supplemented with 100 μM acetosyringone and incubated in the dark for 2 days at 24° C. After co-cultivation, explants were transferred to the first selection/regeneration MS medium supplemented with 30 g/L sucrose, 1 mg/L BAP, 1 mg/L thidiazuron, 0.3 mg/L NAA, 30 mg/L kanamycin, 300 mg/L timentin and 8 g/L agar. After 10 days, explants were transferred to the second selection/regeneration MS medium supplemented with 30 g/L sucrose, 1 mg/L BAP, 1 mg/L thidiazuron, 0.1 mg/L NAA, 50 mg/L kanamycin, 300 mg/L timentin and 8 g/L agar. After 4-5 weeks of culturing on the second selection medium, putatively transformed green shoots were formed ( FIGS. 6A-6B and 6D ). It was noticed that the cotyledon explants produced 2.5 times more putative transgenic shoots than the leaf explants. Healthy green shoots that reached 1-2 cm in height were excised and transferred to the MS medium supplemented with 30 g/L sucrose, 8 g/L agar, 300 mg/L timentin and 50 mg/L kanamycin for rooting. Putative transgenic shoots produced roots after 1-2 weeks of cultivation and showed good growth on the selection medium ( FIG. 6C ). 
     Transgenic plants were further screened with Western blotting for the presence of the PgA1B1 -c-myc fusions using the c-myc monoclonal antibody mAb (ATCC, Manassas) and the goat anti-mouse horse radish conjugated antibody (Ab) (Upstate NY) ( FIG. 6E ). As shown on  FIG. 6E , the recombinant product of the expected size of approximately 50 kDa was detected in many samples. 
     Transformation of  Kalanchoe  Plants. 
     Plant material. Fresh leaves of  Kalanchoe pinnata  were obtained from Tropilab Inc. (St. Petersburg, Fla.) and surface sterilized by immersion in 70% ethanol for 1 min, followed by soaking in 25% of bleach solution for 8 min. After rinsing 3 times in sterile distilled water, and blotting dry with sterile filter paper, leaf segments (1×1 cm) were cultured on MS basal medium supplemented with 30 g/l sucrose, 1 mg/l BAP, 0.1 mg/l NAA and 7 g/l agar. Explants were cultivated at 24° C. at 16 h-light /8 h-dark photoperiods and 40 μE/m2/S1 light intensity. Explants developed shoots after 5-6 weeks in culture. Shoots were excised and transferred to the Magenta boxes containing basal MS medium supplemented with 30 g/L sucrose and 7 g/L agar. Shoots formed roots and produced whole plants within 3-5 weeks. For propagation of plant material stem segments with axillary buds were transferred to the fresh MS medium supplemented with 30 g/L sucrose and 7 g/L agar. 
     Transformation.  Agrobacterium tumefaciens  strain LBA4404 containing the pBI-PgA1B1 construct was used for transformation experiments. Agrobacteria were grown at 28° C. on solid LB plates supplemented with 50 mg/L kanamycin and 20 mg/L rifampicin. A single colony was used to inoculate 20 mL of LB liquid medium with the same antibiotics.  Agrobacterium  culture was incubated 1-2 days at 150 rpm on a shaker. The suspension of  Agrobacterium  was diluted with a liquid MS medium to obtain OD 600  0.5. 
     For transformation experiments leaves of aseptically propagated 2 moths-old plants were cut into 0.6 to 0.7 cm pieces and inoculated with  Agrobacterium  suspensions for 10 min. After blotting dry with sterile filter paper, explants were transferred to the co-cultivation MS medium supplemented with 100 μM acetosyringone and incubated in the dark for 2 days at 24° C. After co-cultivation, explants were transferred to the selection regeneration MS basal medium supplemented with 2 mg/L BAP, 0.1 mg/L NAA, 50 mg/L kanamycin and 300 mg/L timentin. All explants were sub-cultured every 2-3 weeks onto the fresh medium with the same combination of plant hormones and antibiotics. After 5-6 weeks, explants produced shoots on the selection medium ( FIG. 7A ).  FIG. 7A  shows the transformed explant developing shoots (left) and the non-transformed explant bleaching on the selection medium containing 50 mg/L of kanamycin. Putative transgenic green regenerants were transferred to MS medium supplemented with 50 mg/L kanamycin and 300 mg/L timentin for rooting ( FIG. 7B ). In the presence of 50 mg/L kanamycin transgenic kalanchoe shoots showed good growth and development of roots compare to the non-transgenic shoots that did not produce roots and eventually died.  FIG. 7C  shows the transgenic kalanchoe plant rooted on the selection medium. Transgenic kalanchoe plants had showed no morphological abnormalities and resembled non-transgenic plants of the same age. 
       FIG. 8  is a schematic drawing explaining the process of production of plant-derived composition. As shown in this figure, vectors that include the expression constructs encoding recombinant ATR proteins are introduced into either crop or medicinal plants. The recombinant ATR proteins are extracted and may be used in plant-derived therapeutic compositions either alone or in combination with enhancers and adjuvants. 
     Example 4 
     Analysis of Activity of Synthetic Recombinant Proteins 
     Extraction of Soluble Protein from Transgenic Tobacco Plants. 
     Total and soluble plant proteins were extracted from transgenic tobacco and medicinal plants as described by Golovkin et al., 2007. Plant tissue sample were collected, immediately frozen in liquid nitrogen and stored at −80° C. until extraction. Recombinant product was extracted from frozen plant tissues directly using equal amount (V/W) of Laemmli loading buffer for the total/insoluble extract or soluble buffer containing 0.1M Na phosphate pH7.4, 0.3M NaCl, 3% Glycerol, 0.1 mM EDTA, 2 mM β-ME and 0.05% of plant protease inhibitors cocktail (Sigma) for a total soluble protein, concentrated and brought into an equal volume of loading buffer. Protein was extracted from the transgenic tobacco line shown on was used for further analysis ( FIG. 9A ). 
     Extraction of Protein from Transgenic  Echinacea  Plants. 
     Total plant protein was extracted from transgenic  Echinacea  plants using similar protocol as in Golovkin et al., 2007. Plant tissue sample were collected, immediately frozen in liquid nitrogen and store at −80° C. until extraction. Recombinant product was extracted from frozen plant tissues directly with equal amount (V/W) of Laemmli loading buffer and further used for Western blot analysis ( FIG. 6E ). 
     Purification of Soluble Recombinant Proteins 
     About 200 g of frozen leaf material was grounded in 5 volumes of the extraction buffer containing 50-100 mM Na Phosphate, pH7.4, 0.3M NaCl, 0.2% Tween-20, 1.5mM β-Mercaptoethanol, 0.05% Plant Protein Inhibitors Cocktail (Sigma) using Brinkman Polytron Homogenizer at 27,000 rpm. Insoluble parts were pelleted (Beckman) at 16,000 rpm for 20 min at 4° C. Following the flow-filtering through Miracloth (Calbiochem), the PgA1B1 protein was purified in a single-step protocol, using protein A agarose as described earlier (Spitsin et al., 2009; Andrianov et al., 2010).  FIG. 9C  illustrates extraction of the plant PgA1B1 recombinant protein from tobacco leaf tissue and purification of the recombinant protein on the protein-A agarose column. In this figure, “M” is a protein molecular weight marker, “IgG” is a purified conventional IgG antibody with the heavy (“H”) and light (“L”) chains eluted from the protein-A agarose column and used as a standard. “Total” stands for a total plant protein extract before purification. “FT” indicates flow through the fraction from the column. “Eluate” marks the final purified plant-derived PgA1B1 product identified as the “PgA1B1” 48 kDa band monomer of the PgA1B1 protein and the upper “dimer” band. 
     In vitro characterization and quantification of protein expression was performed with ELISA and Western blot analysis ( FIG. 9A ) essentially as described by Golovkin et al., 2007.  FIG. 9A  shows a total protein extracted from the transgenic tobacco plant on PAAG gel (left panel) and immunodetection of PgA1B1 protein expressed in plants using Western blotting (right panel). In this figure, “M” is a protein molecular weight marker, “neg” is an extract of untransformed plant, “Tr” marks total proteins from PgA1B1-expressing transgenic plants, “+” is a positive control from bacteria, “-” is a total protein extract from a wild type plant, “Transgenic” refers to protein extracts from different transgenic tobacco lines. “PgA1B1” indicates the position of the 48 kDa TBL-Fc fusion protein and upper band represents the corresponding “dimer”. 
       FIG. 9B  demonstrates comparison of a total and soluble PgA1B1 protein in transgenic tobacco plants using Western blot analysis. In this figure, “+” is a positive control, “-” is a negative control from wild type plants, “Sol” and “Tot” stands for soluble and total protein fractions, respectively. Analysis as shown in  FIG. 9B  confirmed that almost all fusion proteins are expressed as soluble proteins in plants. Highly pure preparations of plant-derived PgA1B1 protein were extracted from plant tissue minimal concentration of 1.5 mg/ml and yield of at least 3 mg per Kg of raw plant tissue weight. 
     Example 5 
     Affinity Binding of the Anthrax Toxin by Plant-Derived Toxin Binding Ligand 
     As shown earlier by VWA/I domain of the native CMG2 protein may bind PA in a divalent cation-dependent manner (Bradley et al., 2001; Lacy et al., 2004; Scobie et al. 2003). The ability of plant-derived PgAB fusion protein to bind PA was confirmed by two kinds of experiments. 
     Affinity Pulls Down Assay of PA Protein from Solution 
     Affinity of the PA protein to the plant-derived PgA1B1 protein was demonstrated by using protein A agarose beads. Specific binding of 0.5 μg of commercial anthrax PA protein (List Laboratories, Campbell, Calif.) was mixed with 5 μ(=7.25 μg) of purified PgA1B1 protein was done in 100 μl of TBS buffer containing 0.05 Tween-20 (TBSTS), 3% BSA, and 1 mM MgCl 2  by incubating it at 4° C. overnight with gentle shaking. A positive control, 2 μl (aprox. 8-10 μg) of commercial anti-PA goat antiserum (List Laboratories, Campbell, Calif.) was used instead of PgA1B1 protein. Bound protein complexes were rescued from the solution using MagnaBind Protein A Beads (Pierce, Rockford, Ill.). The beads were eluted and analyzed by Western immunoassay using anti-PA mAbs (Biodesign, Saco, Me.). Both anti-PA mAbs and plant-derived PgA1B1 were shown to efficiently bind PA protein. Under experimental conditions, a plant-derived PgA1B1 recombinant protein was more efficient in binding anthrax toxin PA component then the control commercial antibody. 
     Detection of Binding PA Protein to Plant-Derived TBL by ELISA 
       FIG. 10A  illustrates an analysis of the PA binding by the recombinant PgA1B1 protein. Concentration dependency of the plant-derived PgA1B1 binding to the surface-immobilized of PA protein was detected by sandwich ELISA. ELISA was performed in a 96-well plate coated with 0.3 μg/well of the PA protein (List Laboratories, Campbell, Calif.) preparation in 50 μl TBS per well at 4° C. overnight followed by blocking with TBS containing 0.025% Tween (TBST2.5) and 3% BSA. The PA protein preparation was incubated with increasing concentrations of plant PgA1B1 in the presence (dark bars) of or without magnesium ions (Mg 2+ ) (light bars). Bound PgA1B1 was detected using c-myc-specific mAbs. Dilutions of PgA1B1 protein starting at 300 ng/well in 50 μl TBST2.5 with 1 mM MgCl 2  were added and incubated 1 h to set up binding, then washed 2×5′ in TBST2.5. Primary c-myc specific antibody (Invitrogen, Carlsbad, Calif.) were applied at 1/1000-1/2000 dilution for an hour washed vigorously&#39; in TBST2.5. After incubation with secondary AP-anti-mouse conjugate (Sigma, Saint Louis, Mo.) at 1/2000 dilution, plates were developed by using pNPP Substrate (Sigma, Saint Louis, Mo.) as recommended by manufacturer and OD 405  nm determined. Strong affinity of PgA1B1 to PA was demonstrated in the presence of Mg 2+  ions, which is characteristic to a native ATR/sCMG2 protein (Scobie et al. 2003). 
     In vitro Protection of Macrophage Cells Against Anthrax Toxin with Plant ATR. 
     Referring to  FIG. 10B  neutralizing-antibody activity was determined in host monocyte-macrophages cells J774A.1 (American Type Culture Collection, Manassas, VA) essentially as described by Little et al., 1990. Various concentrations of purified plant PgA1B1 (diamonds) or an irrelevant serum from mice immunized with PBS buffer (Negative serum, squares) were premixed with the anthrax lethal toxin before addition to the monocyte-macrophage J774A.1 cells. Cells were incubated with lethal concentrations of the PA-LF toxin in the presence of different concentrations of either the recombinant PgA1B1 protein or sera from mice immunized with PBS buffer (Negative serum). Lactate dehydrogenase activity was measured by a Cytotoxicity Detection Kit (Roche, Indianapolis, Ind.). Cell viability was calculated as a percentage of surviving cells to the complete lysis achieved with 1% Triton X-100. Data points represent the deviation of mean values in triplicate samples. PA and LF components of the anthrax toxin (at 0.1 μg/ml) were combined with plant purified PgA1B1 and negative serum. The antiserum-toxin mixtures were added to J774A.1 at 4×10 4  cells per well. Lactate dehydrogenase activity was measured by the Cytotoxicity Detection kit (Roche, Indianapolis, Ind.). The percentage of cell lysis was calculated as the mean of “A 490  of serum/PA toxin/target cell mix” minus “A490 of target cell control” divided by “A490 of cells treated with 2% Triton X-100” minus “A490 of cells incubated with medium.” Neutralization of anthrax toxin was observed for purified plant-derived PgA1B1 at working concentration of 0.6 μg/ml where 50% cells survived confirming its capability to protect cells against the anthrax toxin. That is in a good agreement with previously described result for native sCMG2 protein (Scobie et al., 2005; Vuyisich et al., 2008; Wycoff et al., 2011; Thomas et al., 2012). No neutralization activity was detected in negative control serum. 
     Example 6 
     Administration of Antitoxin Composition to a Subject 
     Administration of a therapeutic antitoxin composition into bloodstream of animal subjects could be done with the help of commercial needle-free technique developed by Apogee Technologies (Norwood, Mass.), based on using micro needle patches for transdermal administration of protein-based therapeuticals. Up to 1.5 μg/cm 2  of c-myc tagged PgA1B1 plant-derived protein, produced as described in Example 1, was loaded onto the micro-needle patch in a formulation recommended by manufacturer. The patches containing micro-needle array are then manually applied on the skin and released upon a pressure applied on the center of the patch for 1 min to facilitate micro-needle insertion. After 30 min the amount of intramuscular recombinant protein could be estimated histologically using c-myc-specific antibody demonstrating complete dissolution of the formulation in the animal. 
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     Lacy D. B., Wigelsworth D. J., Scobie H. M., Young J. A. and Collier R. J. Crystal structure of the von Willebrand factor A domain of human capillary morphogenesis protein 2: an anthrax toxin receptor.  Proc Natl Acad Sci USA,  101: 6367-6372 (2004). 
     Manayani D. J., Thomas D., Dryden K. A., Reddy V., Silath M. E., Marlett J. M., Rainey G. J. A., Pique M. E., Scobie H. M., Yeager M., Young J. A. T. A viral nanoparticle with dual function as an anthrax antitoxin and vaccine.  PLoS Pathog  3:e142 (2007). 
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     Scobie H. M., et al. A soluble receptor decoy protects rats against anthrax lethal toxin challenge.  The Journal of Infectious Diseases,  192: 1047-51 (2005). 
     Spitsin S., Andrianov V., Pogrebnyak N. et al., Immunological assessment of plant-derived avian flu H5/HA1 variants.  Vaccine,  27:1289-1292 (2009). 
     Thomas D., Naughton J., Cote C., Welkos S., Manchester M., Young J. A. T. Delayed toxicity associated with soluble anthrax toxin receptor decoy-Ig fusion protein treatment.  PLoS ONE,  7:e34611 (2012). 
     Vuyisich M., Gnanakaran S., Lovchik J. A., Lyons C. R., Gupta G. A dual-purpose protein ligand for effective therapy and sensitive diagnosis of anthrax.  Protein J  27:292-302 (2008). 
     Wycoff K. L., Belle A., Deppe D., Schaefer L, Maclean J. M., Haase S., Trilling A. K., Liu S., Leppla S. H., Geren I. N., Pawlik J., Peterson J. W. Recombinant Anthrax Toxin Receptor-Fc Fusion Proteins Produced in Plants Protect Rabbits against Inhalational Anthrax.  Antimicrobial Agents and Chemotherapy  55:132-139 (2011). 
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     The references cited throughout this application are incorporated for all purposes apparent herein and in the references themselves as if each reference was fully set forth. For the sake of presentation, specific ones of these references are cited at particular locations herein. A citation of a reference at a particular location indicates a manner(s) in which the teachings of the reference are incorporated. However, a citation of a reference at a particular location does not limit the manner in which all of the teachings of the cited reference are incorporated for all purposes. 
     It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but is intended to cover all modifications which are within the spirit and scope of the invention as defined by the appended claims; the above description; and/or shown in the attached drawings.