MXPA97002714A - Production of peptides in plants as fusions of protein with coating vi - Google Patents

Abstract

The present invention relates to foreign peptide sequences fused to viral structural proteins of recombinant plants and a method for their production. The fusion proteins are economically synthesized in plants at high levels of biologically -contained tobamoviruses. The fusion proteins of the invention have many uses. These uses include the use as antigens to induce the production of antibodies having the desired binding properties, v. g., protective antibodies, or to be used as vaccine antigens for the induction of protective immunity, including immobility against parasitic infections.

Description

"PRODUCTION OF PEPTIDES IN PLANTS AS FUSIONS OF PROTEINS WITH VIRAL COATING" FIELD OF THE INVENTION The present invention relates to the field of genetically engineered peptide production in plants, more specifically, the invention relates to the use of tobamovirus vectors to express the fusion proteins.

REFERENCE TO RELATED REQUESTS The present application is a continuation in part of the application Number 08 / 176,414, filed on December 29, 1993, which is a continuation in part of the application Serial Number 07 / 997,733, filed on December 30, 1992.

BACKGROUND OF THE INVENTION Peptides are a diverse class of molecules that have a variety of important chemical and biological properties. Some examples include: hormones, cytokines, in unregulators, peptide-based enzyme inhibitors, vaccine antigens, adhesions, receptor binding domains, enzyme inhibitors and the like. The cost of chemical synthesis limits the potential applications of synthetic peptides for many useful purposes, such as drug synthesis or large-scale therapeutic vaccine. There is a need for an economical and rapid synthesis of milligram and larger quantities of polypeptides that occur naturally. Towards that sight, any of the animal and bacterial viruses have been successfully used as peptide carriers. The safe and economical plant culture provides an improved alternative host for the cost-effective production of these peptides. During the last decade, considerable progress has been made in expressing foreign genes in proteins, foreign proteins are now routinely produced in many plant species for plant modification or for production of proteins for use after extraction. . Animal proteins have been produced effectively in plants (reviewed in Krebbers et al., 1992). Vectors for genetic manipulation of plants have been derived from various naturally occurring plant viruses including TMV (tobacco mosaic virus). TMV is the typical member of the tobamovir s group. TMV has straight tubular virions of approximately 100 x 18 nanometers with a hollow channel of diameter of 4 nanometers, consisting of approximately 2000 units of individual capsid protein wound helically around a single RNA molecule. The virion particles are 95 percent protein and 5 percent RNA by weight. The TMV genome is composed of a single-stranded RNA of 6395 nucleotides containing five large ORFs. The expression of each gene is regulated independently. The RNA virion serves as the RNA messenger (mRNA) for the 5 'genes, encoding the 126 kDa replicase subunit and overlapping the 183 kDa replicase subunit that is produced by reading through an approximately amber stop codon. 5 percent of the time. The expression of the internal genes is controlled by different promoters in the less-sense RNA that directs the synthesis of the 3 'co-terminal subgenomic mRNAs that occur during duplication (Figure 1). A detailed description of the expression and life cycle of the tobamovirus gene, among other sites, can be found in the article by Dawson and Lehto, Advances in Virus Research 38: 307-342 (1991). It is of interest to provide new and improved vectors for the manipulation of plants. For production of specific proteins, the transient expression of foreign genes in plants using virus-based vectors has several advantages. The products of plant viruses are among the proteins produced most highly in plants. Frequently, a viral gene product is the predominant protein produced in the cells of the plant during the duplication of viruses. Many viruses are able to move quickly from an initial site of infection to almost every cell in the plant. Due to these reasons, plant viruses have been developed in efficient transient expression vectors for foreign genes in plants. The viruses of multicellular plants are relatively small, probably due to the limitation of size in the trajectories that allow the viruses to move towards the adjacent cells in the systematic infection of all the plants. Most plant viruses have single-stranded RNA genomes of less than 10 kb. Genetically altered plant viruses provide an efficient means of transfecting plants with genes that are encoded for peptide carrier fusions.

COMPENDIUM OF THE INVENTION The present invention provides recombinant plant viruses that express fusion proteins that are formed by fusions between a protein with a flat viral coat and a protein of interest. By infecting the cells of the plant with the recombinant plant viruses of the invention, relatively large amounts of protein of interest can be produced in the form of a fusion protein. The fusion protein encoded by the recombinant plant virus can have any of a variety of forms. The protein of interest can be fused to the amino terminus of the protein with viral coating or the protein of interest can be fused to the carboxyl terminus of the protein with viral coating. In other embodiments of the invention, the protein of interest can be internally fused to the coated protein. The viral coated fusion protein may have one or more properties of the protein of interest. The fusion protein with recombinant coating can be used as an antigen for the development of the antibody or to induce a protective immune response. Another aspect of the invention is to provide polynucleotides that encode the genomes of the recombinant plant viruses present. Another aspect of the invention is to provide the coated fusion proteins encoded by the recombinant plant viruses present. Still another embodiment of the invention is to provide plant cells that have been infected by the recombinant plant viruses of the invention.

BRIEF DESCRIPTION OF THE FIGURES Figure 1. Expression of the tobamovirus gene.

The expression of the tobamovirus gene is presented in diagram form.

Figure 2. Plasmid map of TMV Transcription Vector pSNC004.

The infectious RNA genome of the TMV strain Ul is synthesized by in vitro T7 RNA polymerase of pSNC004 linearized with Kpnl.

Figure 3. Diagram of Plasmid Constructions Each step in the construction of the plasmid DNAs encoding the various viral epitope fusion vectors discussed in the examples are presented in diagram form.

Figure 4. Ligament of Monoclonal Antibody (NVS3) to TMV291 The reactivity of NVS3 to the epitome of malaria present in TMV291 is measured in a normal ELISA.

Figure 5. Ligand of Monoclonal Antibody (NYS1) to TMV261.

The reactivity of NYS1 to the malaria epitope present in TMV261 is measured in a normal ELISA.

DESCRIPTION OF THE SPECIFIC MODALITIES Definitions and abbreviations TMV: Tobacco mosaic Tobamovirus TMVCP: Protein with tobacco mosaic tobamovirus coating. Viral particles: High molecular weight aggregates of viral structural proteins with or with genomic nucleic acids. Virion: An infectious viral particle.

The Invention The present invention provides novel recombinant plant viruses that encode the expression of fusion proteins consisting of a fusion between a protein with viral coat of the plant and a protein of interest. The recombinant plant viruses of the invention provide systematic expression of the fusion protein, systematically infecting the cells in a plant. Therefore, by using the recombinant plant viruses of the invention, large amounts of a protein of interest can be produced. The fusion proteins of the invention comprise two portions: (i) a protein with viral coating of the plant and (ii) a protein of interest. The portion of the protein with viral coating of the plant can be derived from the same protein with viral coat of the plant that serves as a coating protein for the virus from which the genome of the expression vector is derived primarily, ie the protein with coating is native with respect to the recombinant viral genome. Alternatively, the portion of the protein coated with the fusion protein can be heterologous, that is, non-native, with respect to the recombinant viral genome. In a preferred embodiment of the invention, the 17.5 KDa coated protein of tobacco mosaic virus is used together with a vector derived from tobacco mosaic virus. The protein portion of interest of the fusion protein for expression may consist of a peptide from virtually any amino acid sequence, as long as the protein of interest does not significantly interfere with (1) the ability to bind to a receptor molecule, including antibodies and the receptor of the T cell, (2) the ability to bind to the active site of an enzyme (3) the ability to induce an immune response (4) hormonal activity, (5) the immunoregulatory activity, and (6) the activity of metal chelation. The portion of the protein of interest of the present fusion proteins may possess additional chemical, biological properties that have not been listed. Portions of the protein of interest of the fusion proteins present having the desired properties can be obtained by using all or part of the sequence of the amino acid residue of a protein known to have properties as desired. For example, the amino acid sequence of the surface antigen of hepatitis B can be used as a portion of protein of interest from a fusion protein of the invention to produce a fusion protein having similar antigenic properties, surface antigen of hepatitis B. The detailed structural and functional information about many proteins of interest is well known, this information can be used by the person skilled in the art in order to provide coated fusion proteins having the desired properties of the protein of interest. The portion of the protein of interest of the fusion proteins present may vary in size from an amino acid residue to several hundred amino acid residues, preferably the sequence of the portion of interest of the fusion protein present is of a smaller size of 100 amino acid residues, more preferably the sequence of the portion of interest is less in length than 50 amino acid residues. It will be appreciated by those skilled in the art that, in some embodiments of the invention, the portion of the protein of interest may need to be longer than 100 amino acid residues in order to maintain the desired properties. Preferably, the size of the protein portion of interest of the fusion proteins of the invention is minimized, when possible (but retains the desired biological / chemical properties). Even though the portion of the protein of interest of the fusion proteins of the invention can be derived from any of the variety of proteins, proteins are particularly preferred for use as antigens. For example, the fusion protein, or a portion thereof, can be injected into a mammal, together with appropriate adjuvants in order to produce an immune response directed against the portion of the protein of interest in the fusion protein. The immune response against the portion of the protein of interest of the fusion protein has numerous applications such as including applications, protection against infection, and the generation of antibodies useful in immunoassays. The location (or locations) in the fusion protein of the invention when the protein portion with viral coating binds to the protein of interest is referred to herein as fusion binding. A given fusion protein can have one or two fusion bonds. The fusion junction may be located at the carboxyl terminus of the protein portion coated with the fusion protein (attached to the amino terminus of the portion of the protein of interest). Fusion binding can be placed at the amino terminus of the portion of the protein coated with the fusion protein (attached to the carboxyl terminus of the protein of interest). In other embodiments of the invention, the fusion protein can have two fusion bonds. In those fusion proteins having two fusion junctions, the protein of interest is internally positioned with respect to the carboxyl and amino terminal amino acid residues of the portion of the protein coated with the fusion protein, i.e. internal fusion protein. The internal fusion proteins may comprise a whole sequence of protein amino acid residues with plant virus coating (or a portion thereof) that is "interrupted" by a protein of interest, ie, the amino terminal segment. or the coated protein portion binds to a fusion junction with the amino acid residue of the amino terminal of the protein of interest and the carboxyl terminal segment of the coated protein binds to a fusion junction with the residue of amino terminal acid of the protein of interest. When the coated fusion protein for expression is an internal fusion protein, the fusion junctions may be placed at a variety of sites within a coated protein. Suitable sites for fusion junctions can be determined either through routine systematic variation of the fusion junction locations in order to obtain an internal fusion protein with the desired properties. Suitable sites for fusion binding can also be determined by analysis of the three-dimensional structure of the coated protein in order to determine the sites for "insertion" of the protein of interest, which do not significantly interfere with structural and biological functions of the portion of the protein with the fusion protein coating. Detailed three-dimensional structures of the virally coated proteins of the pilanta and their orientation in the virus have been determined and made publicly available to a person skilled in the art. For example, a resolution model of the protein coated with the Mosaic Virus c.the Cucumber Green Beetle (a coated protein bearing strong structural similarities to other proteins with tobamovirus coating) and the virus can be found in the article by Wang and Stubbs J. Mol. Biol. 239: 371 384 (1994). Detailed structural information on the virus and protein coated with the Tobacco Mosaic Virus can be found among other sites in the articles by Namba et al., J. Mol. Biol. 208: 307 325 (1989) and Pattanayek and Stubbs J. Mol. Biol. 228: 516-528 (1992). The knowledge of the three-dimensional structure of a virus particle of the plant and the process of assembling the virus particle allows a person skilled in the art to design different protein fusions with coating of the invention, including insertions, and partial substitutions. For example, if the protein of interest is of a hydrophilic nature, it may be appropriate to fuse the peptide with the TMVCP region that is known to be oriented as a surface circuit region. Also, helical alpha segments that maintain contacts with the subunit could be substituted for appropriate regions of the TMVCP helices or nucleic acid binding domains expressed in the genome-oriented TMVCP region. The polynucleotide sequences encoding the fusion proteins present can provide a stop codon "leaky" in the fusion junction. The stop codon may be present as a codon immediately adjacent to the fusion junction or may be placed proximal (e.g., within 9 bases) to the fusion junction. A "leaky" stop codon can be included in the polynucleotides encoding the coated fusion proteins present in order to obtain a desired ratio of fusion protein. with a protein with wild-type coating. A "leaky" stop codon does not always result in a translation termination and is periodically translated. The frequency of initiation or termination at a given start / stop codon depends on the context. The ribosome scans from the 5 'end of a messenger RNA for the first ATG codon. If it is a non-optimal sequence context, the ribosome will pass at a certain fraction of the time to the next available start codon and will start the translation downstream of the first one. Similarly, the first termination codon found during the transfer will not work 100 percent of the time if it is in a specific sequence context. Consequently, many of the naturally occurring proteins are known to exist as a population that has heterogeneous extensions of the N and / or C terminus. Thus, including a "leaky" stop codon in a binding coding region by fusion in a recombinant viral vector encoding a coated fusion protein, the vector can be used to produce both a fusion protein and a second smaller protein, e.g., the virally coated protein. A "leaky" stop codon can be used at or near fusion junctions of fusion proteins wherein the portion of the protein of interest binds to the carboxyl terminus of the coated protein region, whereby a Only recombinant viral vector can produce both the coated fusion proteins and the coated proteins. In addition, the "leaky" start codon can be used at or near the fusion junctions of the fusion proteins wherein the portion of the protein of interest binds to the amino terminus of the protein region coated by which a similar result is achieved. In the case of TMVCP, the extensions at terminals N and C are on the surface of the viral particles and can be expected to project away from the helical axis. An example of a "leaky" stop sequence occurs at the board of the 126/183 kDa TMV reading frames and was described more than 15 years ago (Pelham, H.R.B., 1978). Skuzeski et al., (1991) defined the 3 'context requirements necessary for this region to confer termination leakage in a heterologous protein marker gene (beta-glucuronidase) such as CAR-YYA O cytidine, A = adenine, Y = pyrimidine) . In another embodiment of the invention, the fusion junctions in the coated fusion proteins present are designed so that they comprise an amino acid sequence that is a substrate for the protease. By providing a fusion protein with coating having this fusion binding, the protein of interest can conveniently be derived from the fusion of the protein with coating using an appropriate proteolytic enzyme. The proteolytic enzyme can be contacted with the fusion protein either in vitro or in vivo. The expression of the coated fusion proteins present can be driven by any of a variety of functional promoters in the genome of the viral vector of the recombinant plant. In a preferred embodiment of the invention, the fusion proteins present are expressed from viral subgenomic protomors of the plant using vectors as described in U.S. Patent Number 5,316,931. Recombinant DNA technologies have allowed the life cycle of numerous plant RNA viruses to be artificially amplified through the DNA phase that facilitates manipulation of the viral genome. These techniques can be applied by a person skilled in the art in order to produce and use the recombinant plant viruses of the invention. All cDNA of the TMV genome was cloned and functionally linked to a bacterial promoter in an E. coli plasmid (Dawson et al., 1986). Transfers of infectious recombinant plant viral RNA can also be produced using other well-known techniques, for example, with RNA polymerases commercially available from T7, T3 or SP6. Accurate duplicates of the RNA virion can be produced in vitro with RNA polymerase and a m7GpppG dinucleotide cap. This not only allows the manipulation of the viral genome for inverse genetics, but also allows the manipulation of the virus towards a vector to express the foreign genes. A method to produce virus RNA vectors from the plant based on the manipulation of RNA fragments with RNA ligase has been shown to be impractical and not widely used (Pelcher, L.E., 1982). Detailed information on how to produce and use recombinant RNA plant viruses can be found, among other sites, in U.S. Patent No. 5,316,931 (Donson et al.), Which is incorporated herein by reference. The invention provides polynucleotides that encode recombinant RNA plant vectors for the expression of the fusion proteins present. The invention also provides polynucleotides comprising a portion or portions of the vectors present. The vectors described in US Pat. No. 5,316,931 are particularly preferred for expressing the fusion proteins of the invention. In addition to providing the described viral coat fusion proteins, the invention also provides virus particles comprising the fusion proteins present. The coating of the virus particles of the invention may consist entirely of coated fusion protein. In another embodiment of the virus particles of the invention, the coating of virus particles can consist of a mixture of fusion proteins with coating and protein with no fusion coating, wherein the ratio of the two proteins can be varied. Since the proteins coated with tobamoviorus can self-assemble into virus particles, the virus particles of the invention can be assembled either in vivo or in vitrc. The virus particles can also be conveniently disassembled using well-known techniques in order to simplify the purification of the present fusion proteins or portions thereof. The invention also provides recombinant plant cells comprising the present coated fusion proteins and / or virus particles comprising the coated fusion proteins present. These plant cells can be produced either by infecting the cells of the plant (either in culture or in whole plants) with infectious virus particles of the invention or with polynucleotides encoding the genomes of the infectious virus particle of the virus. invention. The recombinant plant cells of the invention have many uses. These uses include serving as a source for the fusion-coated proteins of the invention. The portion of the protein of interest of the fusion proteins present may comprise many different amino acid residue sequences, and correspondingly many different possible biological / chemical properties, however, in a preferred embodiment of the invention, the protein portion of The interest of the fusion protein is useful as a vaccine antigen. The surface of the TMV particles and other tobamoviruses contain continuous epitopes of high antigenicity and segment mobility thereby making the TMV particles especially useful for producing a desired immune response. These properties make the virus particles of the invention especially useful as carriers in the presentation of foreign epitopes to mammalian immune systems. Although the recombinant RNA viruses of the invention can be used to produce numerous coated fusion proteins for use as vaccine antigens or vaccine antigen precursors, it is of specific interest to provide vaccines against malaria. Human malaria is caused by the protozoan species Plasmodiun falciparum, P. vivax, P. vale and P. malariae and is transmitted in the form of a sporozoite by Anopheles mosquitoes. The control of this disease will possibly require safe and stable vaccines. Several peptide epitopes expressed during the various stages of the parasite's life cycle are thought to contribute to the induction of protective immunity in particularly resistant individuals living in endemic areas and persons experimentally immunized with irradiated sporozoites.

When the fusion proteins of the invention, portions thereof, or viral particles comprising the fusion proteins are used in vivo, the proteins are typically administered in a composition comprising a pharmaceutical carrier. A pharmaceutical carrier can be any suitable non-toxic substance suitable for the delivery of the desired compounds to the body. Sterile water, alcohol, fats, waxes and inert solids can be included in the carrier. The pharmaceutically acceptable adjuvants (stabilizing agents, dispersing agents) can also be incorporated into the pharmaceutical composition. In addition, when the fusion proteins present or a portion thereof are used for the generation of an immune response, protective or otherwise, the formulation for administration may comprise one or more immunological adjuvants in order to stimulate a desired immune response. When the fusion proteins of the invention, or portions thereof, are used in vivo, they can be administered to a human or animal patient, in a variety of ways. The pharmaceutical compositions can be administered orally or parenterally, i.e., subcutaneously, intramuscularly or intravenously. Thus, this invention provides compositions for parenteral administration comprising a solution of the fusion protein (or a derivative thereof) or a "cocktail" thereof which is dissolved in an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, e.g., water, stabilized water, 0.4 percent saline, 0.3 percent glycerin, and the like. These solutions are sterile and are generally free of particulate matter. These compositions can be sterilized by conventional well-known sterilization techniques. The compositions may contain pharmaceutically acceptable excipients as required to approximate physiological conditions such as stabilizing and pH adjusting agents, toxicity adjusting agents and the like, eg, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of the fusion protein (or a portion thereof) in these formulations can vary widely depending on the specific amino acid sequence of the proteins present and the desired biological activity, v.gr, from less than about 0.5 percent, usually at least about 1 percent up to as much as 15 percent to 20 percent by weight and will be selected principally based on fluid volumes, viscosities, etc. in accordance with the specific management mode selected. Current methods for preparing parenterally administrable compositions and the necessary adjustments for administration to patients will be known or apparent to those skilled in the art and are described in greater detail, for example, in Remington's Pharmaceutical Science, current edition, Mack Publishing Company, of Easton, Pa, which is incorporated herein by reference. Having described the invention in the foregoing, it will be better understood by reference to the following examples. The examples are offered by way of illustration and are not intended to be construed as limiting the scope of the invention,.

EXAMPLES Biological Deposits The following present examples are based on a full-length insertion of wild type TMV (strain Ul) cloned in the vector pUC18 with a sequence of the T7 promoter at the 5 'end and a Kpnl site at the end 3 '(pSNCC04, Figure 2) or a similar plasmid pTMV304. Using the polymerase chain reaction (PCR) technique and the primers D29 (SEQ ID NO: 1) and D1094 (SEQ ID NO: 2) an amplification product of 277 Xmal / HindIII was inserted with the 6140 bp fragment of Xmal / Kpnl of pTMV304 between the Kpnl and HindIII sites of the common cloning vector pUC18 to create pSNC004. Plasmid pTMV304 can be obtained from American Type Culture Collection, of Rockville, Maryland (ATCC deposit Number 45138). The genome of the wild-type TMV strain can be synthesized from pTMV304 using the SP6 polymerase, or from pSNC004 using the T7 polymerase. The wild-type TMV strain can also be obtained from American Type Culture Collection, of Rockville, Maryland (ATCC Deposit Number PV135). Plasmid pBGC152, Kumagai, M. and others (1993), is a derivative of pTMV304 and is only used as a cloning intermediate in the examples that will be described below. The construction of each plasmid vector that is described in the examples presented below is diagrammed in Figure 3.

Example 1.

Propagation and purification of the TMV strain Ul The TMVCP fusion vectors described in the following examples are based on Ul or the wild-type TMV strain and are therefore compared to the parental virus as a control. Nicotiana tabacum cv Xanthi (which will be referred to below as tobacco) was grown for 4 to 6 weeks after germination, and two of the expanded leaves of 4 to 8 centimeters were inoculated with a solution of 50 micrograms per milliliter of TMV Ul emptying 100 microlitres on the leaves sprayed with carborundum and slightly fronting the surface with a gloved hand. Six were grown. tobacco plants for 27 days after inoculation accumulating 177 grams of fresh weight of leaf biomass harvested not including the two lower inoculated leaves. A purified TMV Ul Sample, ID Number KTMV204.B4 (745 milligrams) was recovered at a yield of 4.2 milligrams of virion per gram of fresh weight by two cycles of differential centrifugation and precipitation with PEG according to the method of Gooding and others. (1967). Tobacco plants infected with TMV Ul accumulated more than 230 micromoles of coated protein per kilogram of leaf tissue.

Example 2 Production of a B-cell epitope of genetically-melted malaria in the surface loop region of the TMVCP The NVS3 monoclonal antibody was produced by immunizing a mouse with sporozoites irradiated with P. vivax. The NVS3 mAb was passively transferred to monkeys provided with protective immunity to sporozoiro infection with this human parasite. Using the synthetic peptide top scan technique, the exact amino acid sequence present on the surface of the P. vivax sporozoite and recognized by NVS3 was defined as AGDR (Seq ID No. Pl). The AGDR epitope is contained within a circumsporozoite (CS) protein repeat unit (Charoenvit et al., 1991a), the major immunodominant protein that coats the sporozoite. The construction of a modified tobamovirus genetically engineered to bring this epitope from the B cell of molten malaria to the surface of the virus particles, is noted herein. Construction of plasmid pBGC291. The 2.1 kb EcoRI-PstI fragment of pTMV204 described in the article by. Dawson, et al. (1986) was cloned into pBstSK- (Stratagene Cloning Systems) to form pBGCll. A 0.27 kb fragment of pBGCll was PCR amplified using the 5 'primer, TB2ClaI5' (SEQ ID NO: 3) and the 3 'primer, CP.ME2 + (SEQ ID NO: 4). The amplified product of 0.27 kb was used as the 5 'primer and C / OAvrlI (SEQ ID NO: 5) was the 3' primer for PCR amplification. The amplified product was cloned into the SmaI site of pBstKS + (Stratagene Cloning Systems) to form pBGC243. To eliminate the BstXI and SacII sites of the polylinker, pBGC234 was formed by digesting pBstKS + (Stratagene Cloning Systems) with BstXI followed by treatment with T4 DNA polymerase and self-ligating. The 1.3 kb fragment of HindIII-Kpnl was cloned into pBGC234 to form pBGC235. pBGC304 is also named pTMV304 (ATCC deposit Number 45138). The 0.3 kb Pacl-Accl fragment from pBGC243 was cloned into pBGC235 to form pBGC244. The 0.02 kb polylinker fragment of pBGC243 (Smal-EcoRV) was removed to form pBGC280. A 0.02 kb synthetic PstI fragment encoding the AGDR P. vivax repeat was formed by annealing AGDR3p (SEQ ID NO: 6) with AGDR3m (SEQ ID NO: 7) and the resulting double-stranded type fragment was cloned into pBGC280 to form pBGC282. The 1.0 kb Ncol-Kpnl fragment from pBGC282 was cloned into pSNC004 to form pBGC291. The protein sequence with TMV291 virus coating produced by transcription of plasmid pBGC291 in vitro was mentioned in (SEQ ID NO: 16). The epitope (AGDR) 3 was calculated as being about 6.2 weight percent of the virion.

Propagation and purification of the epitope expression vector. Infectious transcripts were synthesized from pBGC291 linearized with Kpnl using T7 RNA polymerase and cap (7mGpppG) according to the manufacturer (New England Biolabs). An increased amount of the recombinant virus was obtained by passage and purification of Sample ID Number TMV291.1B1 as described in Example 1. Twenty tobacco plants were grown for 29 days after inoculation by accumulating 1060 grams of fresh biomass weight of harvested leaf that does not include the two lower inoculated leaves. Purified Sample ID TMV291.1B2 was recovered (474 milligrams) at a yield of 0.4 milligram of virion per gram of fresh weight. Therefore, 25 micrograms of the 12-mer peptide per gram of fresh weight extracted was obtained. Tobacco plants infected with TMV291 accumulated more than 21 micromoles of the peptide per kilogram of leaf tissue. Analysis of the product. The conformation of the AGDR epitope contained in the TMV291 virus is specifically recognized by the monoclonal antibody NVS3 in ELISA assays (Figure 4). By Western drying analysis, NVS3 reacted only with the TMV291 cp > at 18.6 kD and did not react with the cp or wild-type fusion present in TMV261. The genomic sequence of the epitope coding region was confirmed by directly sequencing the viral RNA extracted from Sample ID Number TMV291.1B2.

Example 3 Production of a B-cell epitope of genetically-melted malaria with the C terminal of TMVCP Significant progress has been made in designing effective subunit vaccines using rodent models of malaria disease caused by non-human pathogens such as P. yoelii or P. berghei The antibody NYSl monoclonal recognizes the repeat epitope QGPGAP (SEQ ID NO: 18), present in the CS protein of P. yoelii, and provides a very high level of immunity for the challenge of sporozoite when passively transferred to mice (Charoenvit, Y. and others 1991b). The construction of a genetically modified tobamovirus to carry this B-cell epitope of molten malaria on the surface of the virus particles is disclosed herein. Construction of plasmid pBGC261. A 0.5 kb fragment of pBGCII, which was amplified by PCR using the 5 'primer Tb2ClaI5' number (SEQ ID NO: 3) and the 3 * Numere C / OAvrlI primer (SEQ ID NO: 5). The amplified product was cloned into the SmaI site of pBstKS + (Estrategenous Cloning Systems) to form pBGC218. PBGC219 was formed by cloning the 0.15 kb AccI-Nsil fragment of pBGC218 in pBGC235. A 0.05 kb synthetic Avrll fragment was formed by annealing PYCS. lp (SEQ ID NO: 8) with PYCS. lm (SEQ ID NO: 9) and the resulting double-stranded type fragment, which encodes the "leaky" stop signal and the B cell malaria epitope, P. yoelii, was cloned into the Avrll site of pBGC219 to form pBFC221. The 1.0 kb NCoI-Kpnl fragment from pBGC221 was cloned into pBGC152 to form pBGC261. TMV261 virus, produced by transcription of plasmid pBGC261 in vitro, contains a "leaky" stop signal at the C-terminus of the coated protein gene and is therefore predicted to synthesize proteins with recombinant and wild-type coatings to a ratio of 20: 1. The fusion of recombinant TMVCP synthesized by TMV261 is listed in (SEQ ID NO: 19) with the stop codon decoded as amino acid Y (amino acid residue 160). The wild-type sequence synthesized by the same virus is listed in (SEQ ID NO: 21). The epitope (QGPGAP) 2 is calculated as being present at 0.3 percent of the virion's weight.

Propagation and purification of the epitope expression vector. We synthesized the infectious transcripts of pBGC261 linearized with Kpnl-using SP6 RNA polymerase and cap (7mGpppG) according to the manufacturer (Gibco / BRL Life Technologies). An increased amount of the recombinant virus was obtained by passage and purification of Sample ID Number TMV261.Blb as described in Example 1. Six tobacco plants were grown for 27 days after inoculation by accumulating 205 grams of fresh weight of the leaf biomass harvested not including the two lower inoculated leaves. Purified sample ID Number T V161.1B2 was recovered (252 milligrams) at a yield of 1.2 milligrams of virion per gram of fresh weight. Thus, 4 micrograms of the fresh extracted 12-mer peptide were obtained. Tobacco plants infected with TMV262 accumulated more than 3.9 micromoles of the peptide per kilogram of leaf tissue. Analysis of the product. The content of the QGPGAP epitope in the TMV261 virus was determined by ELISA with the monoclonal antibody NYSl (Figure 5). From the titration curve, 50 micrograms per milliliter of TMV261 gave the same reading of O.D. (1.0) as 0.2 microgram per milliliter per (QGPGAP) 2: The measured value of approximately 0.4 percent of the virion weight as an epitope matches the calculated value of 0.3 percent. By western drying analysis, NYSl reacted only with the cp fusion of TMV261 at 19 kD and did not react with the fusion of cp or wild-type cp present in TMV291. The genomic sequence of the epitope coding region was confirmed by directly sequencing the viral RNA extracted from sample ID Number TMV261.1B2.

Example 4 Production of a CTL epitope of malaria genetically fused to the C terminal of the TMVCP. The immunity of malaria induced in mice by irradiated sporozoites of P. yoelii also depends on the CD8 + T lymphocytes. Clone B is a cytotoxic T lymphocyte cell (CTL) clone shown to recognize an epitope present in both CS proteins, P. yoelii and P. Berghei. Clone B recognizes the following amino acid sequence; SYVPSAEQILEFVKQISSQ (SEQ ID MO: 23) and when adoptively transferred to mice protects against infection of both sporozoi species of malaria (Weiss et al., 1992). The construction of a genetically modified tobamovirus designed to carry this CTL epitope of molten malaria on the surface of the virus particles is disclosed herein. Construction of plasmid pBGC289. A 0.5 kb fragment of pBGCII was amplified by PCR using the 5 'primer, TB2ClaI5 * (SEQ ID N: 3) and the 3' primer, C / -5AvrII (SEQ ID NO: 10). The amplified product was cloned into the SmaI site of pBstKS + (Stratagene Cloning Systems) to form pBGC214. PBGC215 was formed by cloning the 0.15 kb AccI-Nsil fragment of pBGC214 in pBGC235. The 0.9 kb Ncol-Kpnl fragment from pBGC215 was cloned into pBGC152 to form PBGC216. A synthetic 0.07 kb fragment was formed by annealing PYCS.2p (SEQ ID NO: 11) with PYCS .2m (SEQ ID NO: 12) and the resulting double-stranded type fragment, which encodes the CTL malaria epitope, P yoelii, was cloned into the Avrll site of pBGC215 blunt-ended by treatment with the mung bean nuclease and creating a unique Astil site to form PBGC262. A synthetic AatlI fragment of 0.03 kb was formed by annealing TLS.1EXP (SEQ ID NO: 13) with TLS .1EXM (SEQ ID NO: 14) and the resulting double-stranded type fragment and coding for the "leaky" stop sequence and a fill sequence used to facilitate cloning was cloned into pBGC262 which was digested with AatlI to form pBGC263. PBGC262 was digested with AatlI and ligated into itself by removing the 0.02 kb filler fragment to form pEGC264. The 1.0 kb Ncol-Kpnl fragment from pBGC264 was cloned into pSNC004 to form pBGC289. The TMV289 virus produced by plasmid transcription, pBGC289 in vitro, contains a "leaky" stop signal resulting in the removal of four amino acids from the C-terminus of the protein gene with TMV wild-type coatings and therefore it is predicted that synthesizes a protein with truncated coating and a protein coated with a CTL epitope fused at the C-terminal at a ratio of 20: 1. The recombinant TMVCP / CTL epitope fusion presented in TMV289 is listed in SEQ ID NO: 25 with the stop codon decoded as the amino acid and Y acid (amino acid residue 156). The wild-type sequence minus four C-terminal amino acids is listed in SEQ ID NO: 26. The amino acid sequence of the TMV216 virus-coated protein produced by transcription of plasmid pBGC216 in vitro is also truncated by four amino acids. The epitope SYVPSAEQILEFVKQISSQ (SEQ ID NO: 23) is calculated as being present at approximately 0.5 percent of the virion weight using the same assumptions confirmed by quantitative ELISA analysis of the properties read of TMV261 in Example 3.

Propagation and purification of the epitope expression vector. The infectious transcripts of pBGC289 linearized with Kpnl were synthesized using T7 RNA polymerase and cap (7mGpppG) according to the manufacturer (New England Biolabs). An increased amount of the recombinant virus was obtained by passing the Sample ID No. TMV289.11Bla as described in Example 1. Fifteen tobacco plants were grown for 33 days after the inoculation, accumulating 595 grams of fresh weight of the biomass of harvested leaf not including the two lower inoculated leaves. The Purified Sample ID. TMV289.1132 number was recovered (383 milligrams) at a yield of 0.6 milligram of virion per gram of fresh weight. Thus, 3 micrograms of the 19-mer peptide were obtained per gram of fresh weight extracted. Tobacco plants infected with TMV289 accumulated more than 1.4 micromoles of the peptide per kilogram of leaf tissue. Product Analysis Partial confirmation of the sequence of the epitope coding region of TMV289 was obtained by restriction digestion analysis of cDNA amplified by PCR, using viral RNA isolated from Sample ID. TMV289.11B2 number. The presence of proteins in TMV289 with a predicted mobility of the cp fusion at 20 kD and truncated cp at 17.1 kD, was confirmed by denaturing polyacrylamide gel electrophoresis.

LITERATURE CITED P. G. Ahlquist and R. C. French, 1986. Vector of RNA transformation. European Patent Application Number 194,809.

G. Bruening, 1978. Group of comovirus, C .M. I. /A.A.B. Description of plant viruses, Number 199. m. Culross and Son Ltd., Coupar Angus, of Perthshire, Scotland.

P. J. G. Butler, M. A. May, 1987. Molecular architecture and assembly of tobacco mosaic virus particles. The molecular biology of positive chain RNA viruses. (D. J. Rowlands, M. A. Mayo, and B. W. J. Mahy, editors), Academic Press, London, pages 237-257.

Y. Charoenvit, W.E. Collins, T.R., Jones, P. Millet, L.

Yuan, R.L. Beaudoin, J.R. Broderson and S.L. Hoffman, 1991a.

Inability of the malaria vaccine to induce antibodies to a protective epitope within its sequence.

Science 251: 668-671.

Y. Charoenvit, S. Mellouk, C. Cole, R. Bechara, M.F. Leef, M. Sedegah, L. Yuan, F.A. Robey, R.L. Beaudoin and S.L. Hoffman 1991b. Monoclonal antibodies but not policlona-.es protect against Plasmodium yoelii sporozoites. J. Immunol. 146: 1020-1025.

W. 0. Dawson, D. L. Beck, D. A. Knorr and G. L. Grantham, 1986. The cloning of whole genome cDNA from tobacco mosaic virus and production of infectious transcripts. Proc. Nati Acad. Sci. USA 83: 1832-1836.

W. 0. E'awson, P. Bubrick and G. L. Grantham. 1988. Modifications of the protein gene with tobacco mosaic virus coating that affect duplication, movement and sympathomatology. Phytopathology 78: 783-789. . 0. Dawson, D. J. Lewandowski, M. E. Hilf, P. Bubrick, A.J. Raffo, J. J. Shaw, G. L. Grantham, and P. R. Desjardins, 1989. The tobacco mosaic hybrid-virus expresses and loses an added gene. Virology 172: 285-292.

J. Donson, C. M. Kearney, M. E. Hilf, and W. 0. Dawson 1991. Systematic expression of the bacterial gene by means of a vector based on tobacco mosaic virus. Proc. Nati Acad. Sci. USA 88: 7204-7208.

J. Donson, W. O. Dawson, G. L. Gratham, T. H. Turpén, A. M. Turpén, S. J. Garger and L. K. Grill, 1992. Recombinant viral vectors having heterologous subgenomic promoters for systematic expression of foreign genes. US Patent Application Serial Number 923,692.

R. French, M. Janda, and P. Ahlquist 1986. Bacterial gene inserted in a modified RNA virus. Efficient expression in the cells of the monocotyledonous plant. Science 231: 1294-1297.

A. J. Gibbs, 1977. Tobamovirus group, C.M. I. /A.A.B. Descriptions of plant viruses Number 184. Wm. Culross and Son Ltd., Coupar Angus, Perthshire, Scotland.

P. Goelet, G. P. Lomonossoff, P.J.G. Butler, Aka, - M.E., and J. Karn, 1982. Nucleotide sequence of tobacco mosaic virus RNA. Proc. Nati Acad. Sci. USA 79: 5818-5822.

G.V. Gooding, Jr., and T.T. Hebert 1967. A simple technique for the purification of tobacco mosaic virus in large quantities. Phytopathology 57: 1285. that is, the division number is graduated up to four as seen in Figure 14B, in order to produce four sub-polygons in accordance with the curved reference surface, and store the coordinate values of the apexes of the sub-regions. polygons in the target data RAM 97.

However, when the value of Z is low and the polygon is placed close to the depth direction, that is, where the polygon is large in the default presentation section, the sub-CPU 95 due to the polygon in 16, that is, the number of divisions of 16 co or is seen in Figure 14C, in order to produce 16 sub-polygons in accordance with the curved reference surface and store the coordinate values of the apices of the 16 sub-polygons in RAM 97 of destination data.

For example, when the polygon is divided into 16 polygons, as shown in Figure 14C, the 16 sub-polygons have 25 apices PO to P24, as shown in Figure 15A. Therefore, the sub-CPU 95 forms coordinate values and color data values (Xi, Yi, Zi, RGBi) (i = 0, ..., 24) of the apices in such a manner as shown in FIG. Figure 15B and store them in destination data RAM 97.

It should be noted that the present example of division makes use of quadrangular polygons (su-polygons).

In this way, the shape of an object is varied according to the size (presentation size) when presented in the default presentation section and when the presentation size is small, an object that has a small apex number is presented as a predetermined object and the number of operations necessary to present it is reduced. On the other hand, when the presentation size is large, an object having a comparatively large number of apex is presented as a predetermined object so that a user can feel a difference in the shape between the original object and the presented object.

Further, by transmitting the data of a polygon of a comparatively large size to the programmable packet engine 48 and dividing the polygon in accordance with the display area by the programmable packet engine 48, the amount of data to be supplied to the CPU 44 Main to the programmable packet motor 48 through the main bus 41 can be reduced in order to reduce the load on the main bus 41.

Even though the amount of data (amount of output data) sent from the programmable packet engine 48 to the graphic processing unit 49 increases in accordance with a division number of a polygon as shown in Figure 16, the amount of data (amount of input data) supplied from the main CPU 44 to the programmable packet engine 48 through the main bus 41 is set independently of the polygon's division number (in the packet of Figure 13, six words per polygon ). Consequently, the load to the main bus 41 can be set. Furthermore, by this means, the data is compressed as indicated by the data compression ratio of Figure 16 and the amount of data to be handled (amount of data communicated along the main bus 41 and so on). it is reduced of course.

It should be noted that, since the graphics processing unit 49 performs the processing of the individual polygons while retaining the data of four apices of each one of them, when the graphics processing unit 49 uses the data of the apices supplied above by four or more again, the programmable packet engine 48 supplies the apex data one more time. Correspondingly, the amount of output data (number of words) from the programmable packet engine 48 is larger than the number of apices of the polygons obtained by division. For example, a case where the data of the polygon of Figure 15 divided into 16 sub-polygons having in total 25 apices PO to P24 that are to be supplied to the graphics processing unit 49 is examined of course. First, the programmable packet engine 48 supplies the data from the apex PO to P3 to the graphic processing unit 49 and the graphic processing unit 49 performs the processing of the polygon having the apex PO to P3. Then, the programmable packet engine 48 supplies the data of the apices P4 and P5, and the processing unit 49 performs the processing of the polygon having the apices Pl, P3, P4 and P5. Similarly, the programmable packet engine 48 supplies the data of the apices P6 to P9 in order, and the graphics processing unit 49 successively carries out the processing of the polygon having the apices P4 to P7 and the polygon having the apices P6 to P9. Then, the programmable packet engine 48 supplies data from the apices P2, P3, PÍO and Pll, to the graphics processing unit 49, and the graphics processing unit 49 carries out the processing of the polygon having the apices P2, P3, PÍO y Pll. In addition, the programmable packet engine 48 supplies the apex data P5 and P12, and the graphic processing unit 49 carries out the processing of the polymer having the apexes P3, P5, Pll and P12. Similarly, the programmable packet engine 48 supplies the data of the apices P7, P9, P13 and P14, and the graphic processing unit 49 successively carries out the processing of the polymer having the apices P5, P7, P12 and P13 and the polygon that has the apices P7, P9, P13 and P14. Then, the programmable packet engine 48 supplies data from the PÍO to P24 apices to the graphics processing unit 49 and the graphics processing unit 49 successively performs the processing of the individual polygons in a similar manner. Since the programmable packet engine 48 supplies the apex data of the individual polygons to the graphic processing unit 49 in a manner as described above, the data of the 15 apices including the apices P2, P3, P5, P7 and P9, and the apexes PID to P19 are supplied twice to the graphic processing unit 49. Correspondingly, since the data giving a total of 40 appendages (= 25 + 15) are supplied to the graphics processing unit 49, the amount of data output from the programmable packet engine 48 when the polygon is divided into two. . 16 is 40 words, as shown in Figure 16. The data supplied in this way are called web mesh. Although in the embodiment described above the parameters of the curved surface representative of the curved reference surface are supplied to the programmable packet engine 48, a parameter representative of the position of a light source can be supplied together with the coordinate values. from the apices of the polygon to the programmable packet engine 48 so that, after the polygon is divided according to the value of Z, the brightness values of the polygons (sub-polygons) obtained by division can be calculated from the representative parameter of the position of the light source. For example, the data that include a luminous source parameter representative of the position of a light source together with the coordinate values (Xi, Yi, Zi, (i = 0, ..., 3) of the apex PO to P3 of this polygon, as shown in Figure 17A, a normal vector (Nx, Ny, Nz) and an identifier (Code in Figure 17A) that designates a program to carry out the division processing of a polygon, are recorded previously on the CD-ROM 40, and the data is read and stored in the main memory 45. Then, after the conversion of the coordinates into the main memory is carried out, and the data is read and stored. Then, after the conversion to the coordinates is carried out, the polygon by the graphics transfer engine 71, that data is supplied as a packet to the programmable packet engine 48 as shown in FIG. Figure 17B by the pack engine 72. Then the programmable packet engine 48 performs after the division of the polygon to the calculation of the brightness values of the individual sub-polygons from the parameter of the light source. It should be noted that when a point light source is used, the coordinates (Lx, Ly, Lz) of the light source of point and color information (Lr, Lg, Lb) of the light source are supplied as parameters of the source luminous to the programmable pack 48 motor.

The programmable package engine calculates, from the parameters of the light source supplied to it, the coordinates (pO, qO) of a point of intersection between a perpedicular from the light source and a two-dimensional plane (p, q) that includes the polygon shown in Figure 18 a and the height h of the light source from the two-dimensional plane (p, q) and also calculates, from the coordinates values (p, q) of the apices of the individual sub-polygons, the L values of brightness (which increase in inverse proportion to the square of the distance from the light source) at the apices of the sub-polygons according to the following expression: L = h2 / (h2 + (p - p0) 2 + (q - qO ) 2) For example, when a polygon is divided into eight sub-polygons, the programmable packet engine 48 calculates the brightness value for each of the apices of the sub-polygons. Then, the graphics processing unit 49 calculates the brightness values of the sub-polygons from the brightness values of the apices of the sub-polygons and effects the presentation of the polygon as shown in Figure 18B. Similarly, when a polygon is divided into 32 sub-polygons, the programmable packet engine 48 calculates the brightness values for the individual apices of the sub-polygons. Then, the graphic processing unit 49 calculates the brightness values of the sub-polygons from the brightness values of the apices of the sub-polygons, performs the presentation of the polygon as shown in Figure 18C. By calculating the brightness values for the individual polygons of a predetermined polygon in this manner, the brightness value of the surface of an object can be finely varied. Then, the brightness values calculated in this way for the individual apices (25 apices in this (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO:: AAAGTCTCTG TCTCCTGCAG GGAACCTAAC AGTTAC 36 (2) INFORMATION FOR SEQ ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: unknown (D) TOPOLOGY: unknown ( ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 5: ATTATGCATC TTGACTACCT AGGTTGCAGG ACCAGA 36 (2) INFORMATION FOR SEQ ID NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: unknown (D) TOPOLOGY: unknown ( ii) TYPE OF MOLECULE DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6: GGCGATCGGG CTGGTGACCG TGCA 24 (2) INF?: ^ MATION FOR SEQ ID NO: 7: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 7: CGGTCACCAG CCCGATCGCC TGCA 24 (2) INFORMATION FOR SEQ ID NO: 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 45 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: unknown (D) TOPOLOGY: unknown ( ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 8: CTAGCAATTA. CAAGGTCCAG GTGCACCTCA AGGTCCTGGA GCTCC 45 (2) INFORMATION FOR SEQ ID NO: 9: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 45 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: unknown (D) TOPOLOGY: unknown ( ii) TYPE OF MOLECULE: DNA (genomic) (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 66 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: unknown (D) TOPOLOGY: unknown (ii) ) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 12: TATAGTTACT GACTCGAGAT TTGCTTAACG AATTCCAAGA TCTGCTCTGC AGATGGAACA 60 TATGAC 66 (2) INFORMATION FOR SEQ ID NO: 13: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: unknown (D) TOPOLOGY: unknown ( ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 13: CGACCTAGGT GATGACGTCA TAGCAATTAA CGT 33 (2) INFORMATION FOR SEQ ID NO: 14: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: unknown (D) TOPOLOGY: unknown ( ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 14: TAATTGCTAT GACGTCATCA CCTAGGTCGA CGT 33 (2) INFOPMATION FOR SEQ ID NO: 15: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 4 amino acids (B) TYPE: aminoino acids (C) TYPE OF CHAIN: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 15: Ala Gly Asp Arg 1 (2) INFOPMATION FOR SEQ ID NO: 16: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 510 pairs of base (B) TYPE: nucleic acid (C) TYPE OF CHAIN: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: DNA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM: Fusion pBGC291 (ix) PARTICULARITY : (A) NAME / KEY: CDS (B) LOCATION: 1..510 (xi) DESCRIPTION OF SEQUENCE: ID NO: 16: ATG TCT TAC AGT ATC ACT ACT CCA TCT CAG TTC GTG TTC TTG TCA TCA 48 Met Ser Tyr Ser lie Thr Thr Pro Ser Gln Phe Val Phe Leu Ser Ser 1 5 10 15 GCG TGG GCC GAC CCA ATA GAG TTA ATT AAT TTA TGT ACT AAT GCC TTA 96 Wing Trp Wing Asp Pro lie Glu Leu lie Asn Leu Cys Thr Asn Wing Leu 20 25 30 GGA AAT CAG TTT CA AC AC CA GCT CGA ACT GTC GTT CAA AGA CAA 144 Gly Asn Gl.n Phe Gln Thr Gln Gln Wing Arg Thr Val Val Gln Arg Gln i S 40 45 TTC AGT GAG GTG TGG AAA CCT TCA CCA CAA GTA ACT GTT AGG TTC CCT 192 Phe Ser GLu Val Trp Lys Pro Ser Pro Gln Val Thr Val Arg Phe Pro 50 55 60 GCA GGC GkT CGG GCT GGT GAC CGT GCA GAC GAC AGA GAC TTT AAG GTG 240 Wing Gly Asp Arg Wing Gly Asp Arg Wing Gly Asp Arg Asp Phe Lys Val 65 70 75 80 TAC AGG TAC AAT GCG GTA TTA GAC CCG CTA GTC GÁ ACE GCT CTG TTA GGT 288 Tyr Arg Tyr Asn Wing Val Leu Asp Pro Leu Val Thr Wing Leu Leu Gly 85 90 95 GCA TTC GAC ACT AGA AAT AGA ATA GAA GTT GAA AAT CAG GCG AAC 336 Wing Phe Asp Thr Arg Asn Arg lie lie Glu Val Glu Asn Gln Wing Asn 100 105 110 CCC ACG ACT GCC GAA ACG TTA GAT GCT ACT CGT AGA GTA GAC GAC GCA 384 Pro Thr r: hr Wing Glu Thr Leu Asp Wing Thr Arg Arg Val Asp Asp Wing 115 120 125? CG GTG GCC ATA AGG AGC GCG ATA AAT AAT TTA ATA GTA GA A TTG ATC 432 Thr Val Ala lie Arg Ser Ala lie Asn Aen Leu lie Val Glu Leu lie 130 135 140 AGA GGA &CC GGA TCT TAT AAT CGG AGC TCT TTC GAG AGC TCT TCT GGT 480 Arg Gly Thr Gly Ser Tyr Asn Arg Being Ser Phe Glu Being Ser Gly 145 150 155 160 TTG GTT TGG ACC TCT GGT CCT GCA ACT TGA 510 Leu Val Trp Thr Be Gly Pro Wing Thr - (2) INFORMATION FOR SEQ ID NO: 17: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 169 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (vi) SOURCE ORIGINAL: (A) ORGANISM: pBGC291 Fusion (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 17 Met Ser Tyr Ser He Thr Thr Pro Ser Gln Phe Val Phe Leu Ser Ser 1 5 10 15 Ala Trr Ala Asp Pro He Glu Leu He Asn Leu Cys Thr Asn Ala Leu 20 25 30 Gly Asr. Gln Phe Gln Thr Gln Gln Wing Arg Thr Val Val Gln Arg Gln 35 40 45 Phe Ser Glu Val Trp Lys Pro Ser Pro Gln Val Thr Val Arg Phe Pro 5C 55 60 Wing Gly Asp Arg Wing Gly Asp Arg Wing Gly Asp Arg Asp Phe Lys Val 70 3 Tyr Arg Tyr Asn Wing Val Leu Asp Pro Leu Val Thr Wing Leu Leu Gly 85 90 Wing Phe Asp Thr Arg Asn Arg He lie Glu Val Glu Asn Gln Wing Asn 100 105 Pro Thr Thr Wing Glu Thr Leu Asp Wing Thr Arg Arg Val Asp Asp Wing 120 x "" Thr Val Wing He Arg Wing Wing As Asn Leu lie Val Glu Leu He 130 135? Rg Gly Thr Gly Ser Tyr Asn Arg Ser Being Phe Glu Being Ser Gly -1Í? 45C 150 Xü: 3 Leu Val Trp Thr Ser Gly Pro Wing Thr 165 (2) INFORMATION FOR SEQ ID NO: 18: (i ) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 6 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 18: Gln Gly Pro Gly Ala Pro 1 5 (2) INFORMATION FOR SEQ ID NO: 19: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 525 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: unknown (D) TOPOLOGY: unknown (ii) ) TYPE OF MOLECULE: DNA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM: pBGC261 stoppage "with leaks"ULARITY: (A) NAME / KEY: CDS (B) LOCATION: 1..525 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 19: - - ATG TCT TAC AGT ATC ACT ACT CCA TCT CAG TTC GTG TC TTG TCA TCA 48 Met Ser Tyr Ser He Thr Thr Pro Ser Gln Phe Val Phe Leu Ser Ser 1 5 10 15 GCG TGG GCC GAC CCA ATA GAG TTA ATT AAT TTA TGT ACT AAT GCC TTA 96 Wing Trp Wing Asp Pro He Glu Leu He Asn Leu Cys Thr Asn Wing Leu 20 25 30 GGA AAT CAG TTT CAÁ ACÁ CAÁ CAÁ GCT CGA ACT GTC GTT CAA AGA CAÁ 144 Gly Asn Gln Phe Gln Thr Gln Gln Ala Arg Thr Val Val Gln Arg Gln 35 40 45 TTC AGT GAG GTG TGG AAA CCT TCA CCA CAA GTA ACT GTT AGG TTC CCT 192 Phe Ser Glu Val Trp Lys Pro Ser Pro Gln Val Thr Val Arg Phe Pro 50 55 60 GAC AGT GAC TTT AAG GTG TAC AGG TAC AAT GCG GTA TTA GAC CCG CTA 240 Asp Ser Asp Phe Lys Val Tyr Arg Tyr Asn Wing Val Leu Asp Pro Leu 65 70 75 80 GTC ACÁ GCA CTG TTA GGT GCA TTC GAC ACT AGA AAT AGA ATA ATA GAA 288 Val Thr Ala Leu Leu Gly Wing Phe Asp Thr Arg Asn Arg He He Glu 85 90 95 GTT GAA AAT CAG GCG AAC CCC ACG ACT GCC GAA ACG TTA GAT GCT ACT 336 Val Glu Asn Gln Wing Asn Pro Thr Thr Wing Glu Thr Leu Asp Wing Thr 100 105 110 CGT AGA GTA GAC GAC GCA ACG GTG GCC ATA AGG AGC GCG ATA AAT AAT 384 Arg Arg Val Asp Aep Wing Thr Val Wing He Arg Wing He Asn Asn 115 120 125 TTA ATA GTA GAA TTG ATC AGA GGA ACC GGA TCT TAT AAT CGG AGC TCT 432 Leu He Val Glu Leu He Arg Gly Thr Gly Ser Tyr Asn Arg Ser Ser 130 135 140 TTC GAG? TC TCT TCT GGT TTG GTT TGG ACC TCT GGT CCT GCA ACC TAG_480_Phe Glu Ser Ser Gly Leu Val Trp Thr Ser Gly Pro Wing Thr Tyr 145 150 155 160 CA TTA CAA GGT CCA GGT GCA CCT CAA GGT CCT GGA GCT CCC TAG_52_Gln Leu Gln Gly Pro Gly Wing Pro Gln Gly Pro Gly Ala Pro 165 170 175 (2) INFORMATION FOR SEQ ID NO: 20: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 174 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (vi) SOURCE ORIGINAL : (A) ORGANISM: pBGC261 stoppage "with leaks" (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 20: Met Ser Tyr Ser He Thr Thr Pro Ser Gln Phe Val Phe Leu Ser Ser 1 5 10 15 Wing Trp Wing Asp Pro He Glu Leu He Asn Leu Cys Thr Asn Wing Leu 20 25 30 Gly Asn Gln Phe Gln Thr Gln Gln Wing Arg Thr Val Val Gln Arg Gln 35 40 45 Phe Ser Glu Val Trp Lys Pro Ser Pro Gln Val Thr Val Arg Phe Pro 5C 55 60 Val Thr Ala Leu Leu Gly Wing Phe Asp Thr Arg Asn Arg He He Glu 85 90 95 Val Glv Asn Gln Wing Asn Pro Thr Thr Wing Glu Thr Leu Asp Wing Thr 100 105 110 Arq Ar He Asn Asn Leu Ha Val Glu Leu He Arg Gly Thr Gly Ser Tyr Asn Arg Ser Ser 130 135 140 Phe Glu S »r Ser Ser Gly Leu Val Trp Thr Ser Gly Pro Wing Thr Tyr 145 ~ 150 '155 160 Gln Leu Gln Gly Pro Gly Wing Pro Gln Gly Pro Gly Ala Pro 165 70 INFORMATION FOR SEQ ID NO: 21: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 480 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: DNA (genomic) (vi) ORGINAL SOURCE: (A) ORGANISM: pBGC261 - No fusion (xi) PARTICULARITY: (A) NAME / KEY: CDS (B) LOCATION: 1.488 (xi) DESCRIPTION OF THE SEQUENCE : SEQ ID NO: 21 ATG TCT TAC AGT ATC ACT ACT CCA TCT CAG TTC GTG TTC TTG TCA TCA Met Ser Tyr Ser He Thr Thr Pro Ser Gln Phe Val Phe Leu Ser Ser 1 5 10 15 GCG TGG GCC GAC CCA ATA GAG TTA ATT ATA TTA TGT ACT AAT GCC TTA Wing Trp Wing Asp Pro He Glu Leu He Asn Leu Cye Thr Asn Wing Leu 20 25 30 GGA AF.T CAG TTT CA ACÁ CAÁ CAÁ GCT CGA ACT GTC GTT CAA AGA CAÁ 1 Gly Aen Gln Phe Gln Thr Gln Gln Ala Arg Thr Val Val Gln Arg Gln 35 40 45 TTC AGT GAG GTG TGG AAA CCT TCA CCA CA GTA ACT GTT AGG TTC CCT 1 Phe Ser Glu Val Trp Lys Pro Ser Pro Gln Val Thr Val Arg Phe Pro 50 55 60 GAC AG! T GAC TTT AAG GTG TAC AGG TAC AAT GCG GTA TTA GAC CCG CTA Asp Ser Asp Phe Lys Val Tyr Arg Tyr Asn Wing Val Leu Asp Pro Leu 65 70 75 80 GTC ACA GCA CTG TTA GGT GCA TTC GAC ACT AGA AAT AGA ATA ATA GAA Val Thr Ala Leu Leu Gly Wing Phe Asp Thr Arg Asn Arg He He Glu 85 90 95 GTT G? A AAT CAG GCG AAC CCC ACG ACT GCC GAA ACG TTA GAT GCT ACT Val Glu Asn Gln Wing Asn Pro Thr Thr Wing Glu Thr Leu Asp Wing Thr 100 105 110 CGT AGA GTA GAC GAC GCA ACG GTG GCC ATA AGG AGC GCG ATA AAT AAT Arg Arg Val Asp Asp Wing Thr Val Wing He Arg Ser Wing He Asn Asn 115 120 125 TTA ITTA GATA TTG ATC AGA GGA ACC GGA TCT TAT AAT CGG AGC TCT Leu He Val Glu Leu He Arg Gly Thr Gly Ser Tyr Asn Arg Ser Ser 130 135 140 TTC GAG AGC TCT TCT GGT TTG GTT TGG ACC TCT GGT CCT GCA ACC TAG Phe Glu Ser Ser Gly Leu Val Trp Thr Ser Gly Pro Thr Wing 145 150 155 160 (2) INFORMATION FOR SEQ ID NO: 22: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH : 159 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (vi) ORIGINAL SOURCE: (A) ORGANISM: pBGC261 - No fusion (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 22: Met Ser Tyr Ser He Thr Thr Pro Ser Gln Phe Val Phe Leu Ser Sering Trp Wing Asp Pro He Glu Leu He Asn Leu Cys Thr Asn Wing Leu 20 25 30 Gly Asn Gln Phe Gln Thr Gln Gln Wing Arg Thr Val Val Gln Arg Gln 35 40 45 Phe £ er Glu Val Trp Lys Pro Ser Pro Gln Val Thr Val Arg Phe Pro 50 55 -60 Asp £ er Asp Phe Lys Val Tyr Arg Tyr Asn Wing Val Leu Asp Pro Leu 65 70 75 80 Val rhr Ala Leu Leu Gly Ala Phe Asp Thr Arg Asn Arg He He Glu 85 90 95 Val Glu Asn Gln Wing Asn Pro Thr Thr Wing Glu Thr Leu Asp Wing Thr 100 105 110 Arg Arg Val Aep Asp Wing Thr Val Wing He Arg Wing He Asn Asn 115 120 125 Leu He Val Glu Leu He Arg Gly Thr Gly Ser Tyr Asn Arg Being 130 135 140 Phe Glu Ser Ser Gly Leu Val Trp Thr Ser Gly Pro Thr Wing 145 150 155 (2) INFORMATION FOR SEQ ID NO: 23: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 19 amino acids (B) TYPE : amino acids (C) TYPE OF CHAIN: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 23: Ser Tyr Val Pro Be Wing Glu Gln lie Leu Glu Phe Val Lys Gln lie 1 5 10 15 Being Ser Gln (2) INFORMATION FOR SEQ ID NO: 24: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 537 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: unknown (D) TOPOLOGY: unknown (ii) ) TYPE OF MOLECULE: DNA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM: pBGC289 unemployment "with leaks" (ix) PARTICULARITY: (A) NAME / KEY: (B) LOCATION: 1..537 (xi) ) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 24: - ATG TCT TAC AGT ATC ACT ACT CCA TCT CAG TTC GTG TTC TTG TCA TCA 48 Met Ser Tyr Ser He Thr Thr Pro Ser Gln Phe Val Phe Leu Ser Ser 1 5 10 15 GCG TGG G ^ C GAC CCA ATA GAG TTA A ^ T AAT TTA TGT ACT AAT GCC TTA 96 Wing Trp Wing Asp Pro He Glu Leu He Asn Leu Cys Thr Asn Wing Leu 20 25 30 GGA AAT CAG TTT FACILITATION CAA FACILITATION CAG GCT CGA ACT GTC GTT CAA AGA CAA 144 Gly 7.sn Gln Phe Gln Thr Gln Gln Wing Arg Thr Val Val Gln Arg Gln 35 40 45 TTC AGT GAG GTG TGG AAA CCT TCA CCA CAA GTA ACT GTT AGG TTC CCT 192 Phe Ser Glu Val Trp Lys Pro Ser Pro Gln Val Thr Val Arg Phe Pro 50 55 60 GAC AGT GAC TTT AAG GTG TAC AGG TAC AAT GCG GTA TTA GAC CCG CTA "240 Asp Ser? Sp Phe Lys Val Tyr Arg Tyr Asn Wing Val Leu Asp Pro Leu 65 70 75 80 GTC AC GCA CTG TTA GGT GCA TTC GAC ACT AGA AAT AGA ATA ATA GAA 288 Val Thr Ala Leu Leu Gly Ala Phe Asp Thr Arg Asn Arg He He Glu 85 90. 95 GTT GAA Í.AT CAG GCG AAC CCC ACG ACT GCC GAA ACG TTA GAT GCT ACT 336 Val Glu Asn Gln Wing Aen Pro Thr Thr Wing Glu Thr Leu Asp Wing Thr 100 10 5 110 CGT AGA GTA GAC GAC GG ACG GTG GCC ATA AGG AGC GCG ATA AAT AAT 384 Arg Arg Val Aep Asp Wing Thr Val Wing He Arg Wing He Asn Asn 115 120 125 TTA ATA C-TA GAA TTG ATC AGA GGA ACC GGA TCT TAT AAT CGG AGC TCT 432 Leu He Val Glu Leu He Arg Gly Thr Gly Ser Tyr Asn Arg Ser Ser 130 135 140 TTC GAG AGC TCT TCT GGT TTG GTT TGG ACG TCA TAG CAA TTA ACG TCA 480 Phe Glu Being Ser Gly Leu Val Trp Thr Ser Tyr Gln Leu Thr Ser 145 150 155 160 TAT GTT CCA TCT GCA GAG CAG ATC TTG GAA TTC GTT AAG CAA ATC TCG 528 Tyr Val Pro Be Wing Glu Gln He Leu Glu Phe Val Lys Gln He Ser 165 170 175 AT CAG TAG_537_Ser Gln (2) INFORMATION FOR SEQ ID NO: 25: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 178 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TI PO OF MOLECULE: protein (vi) JENTE ORIGINAL: (A) ORGANIZATION: pBGC289 of unemployment "with leaks" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 25: Met Ser Tyr Ser He Thr Thr Pro Ser Gln Phe Val Phe Leu Ser Ser 1 5 10 15 Wing Trp Wing Asp Pro He Glu Leu He Asn Leu Cys Thr Asn Wing Leu 20 25 30 Gly Asn Gln Phe Gln Thr Gln Gln Wing Arg Thr Val Val Gln Arg Gln 35 40 45 Phe Ser Glu Val Trp Lys Pro Ser Pro Gln Val Thr Val Arg Phe Pro 50 55 60 Asp Ser Asp Phe Lys Val Tyr Arg Tyr Asn Wing Val Leu Asp Pro Leu 65 70 75 80 Val Thr Ala Leu Leu Gly Ala Phe Asp Thr Arg Asn Arg He He Glu Val Glu Asn Gln Wing Asn Pro Thr Thr Wing Glu Thr Leu Asp Wing Thr 100 105 110 Arg? Rg Val Asp Asp Wing Thr Val Wing He Arg Wing He Asn Asn 115 120 125 Leu He Val Glu Leu He Arg Gly Thr Gly Ser Tyr Asn Arg Ser Ser 130 135 140 Phe Glu Ser Ser Gly Leu Val Trp Thr Ser Tyr Gln Leu Thr Ser 145 150 155 160 Tyr Val Pro Be Wing Glu Gln He Leu Glu Phe Val Lys Gln He Ser 165 170 175 Ser Oln (2) INFORMATION FOR SEQ ID NO: 26: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 468 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: unknown (D) TOPOLOGY: unknown (ii) ) TYPE OF MOLECULE: DNA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM: pBGC289 - No fusion (ix) PARTICULARITY: (A) NAME / KEY: CDS (B) LOCATION: 1. 468 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 26: ATG C: TAC AGT ATC ACT ACT CCA TCT CAG TTC GTG TTC TTG TCA TCA Met Se :: Tyr Ser He Thr Thr Pro Ser Gln Phe Val Phe Leu Ser Ser 1 5 10 15 GCG TG3 GCC GAC CCA ATA GAG TTA ATT AAT TTA TGT ACT AAT GCC TTA Wing Trp Wing Asp Pro He Glu Leu He Asn Leu Cys Thr Asn Wing Leu 20 25 30 GGA AAT CAG TTT FULL FULL FACE GCT FACT GTC GTT CAA AGA ' CAA Gly Asn Gln Phe Gln Thr Gln Gln Wing Arg Thr Val Val Gln Arg Gln 35 40 45 TTC AGT GAG GTG TGG AAA CCT TCA CCA CAA GTA ACT GTT AGG TTC CCT Phe Ser Glu Val Trp Lys Pro Ser Pro Gln Val Thr Val Arg Phe Pro SO 55 60 GAC AGT GAC TTT AAG GTG TA C AGG TAC AAT GCG GTA TTA GAC CCG CTA Asp Ser Asp Phe Lys Val Tyr Arg Tyr Asn Wing Val Leu Asp Pro Leu 65 70 75 80 GTC ACÁ GCA CTG TTA GGT GCA TTC GAC ACT AGA AAT AGA ATA ATA GAA Val Thr Ala Leu Leu Gly Wing Phe Asp Thr Arg Aen Arg He He Glu 85 90 95 GTT 3AA pp CAG GCG AAC CCC ACG ACT GCC GAA ACG TTA GAT GCT ACT Val Glu Asn Gln Wing Asn Pro Thr Thr Wing Glu Thr Leu Asp Wing Thr 100 105 110 CGT AGA GTA GAC GAC GCA ACG GTG GCC ATA AGG AGC GCG ATA AAT AAT Arg Arg Val Asp Asp Wing Thr Val Wing He Arg Ser Wing He Asn Asn 115 0 125 TTA ATA GTA GAA TTG ATC AGA GGA ACC GGA TCT TAT AAT CGG AGC TCT Leu He Val Glu Leu He Arg Gly Thr Gly Ser Tyr Asn Arg Ser Ser 130 135 140 TTC GAG AGC TCT TCT GGT TTG GTT TGG ACG TCA TAG Phe Glu Being Ser Gly Leu Val Trp Thr Ser 145 150 155 - (2) INFORMATION FOR SEQ ID NO: 27: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 155 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (vi) SOURCE ORIGINAL: (A) ORGANIZATION: pBGC289 - Non-merger (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 27 Met Ser Tyr Ser He Thr Thr Pro Ser Gln Phe Val Phe Leu Ser Ser 1 5 10 15 Wing T: rp Wing Asp Pro He Glu Leu He Asn Leu Cys Thr Asn Wing Leu 20 25 30 Gly Asn Gln Phe Gln Thr Gln Gln Wing Arg Thr Val Val Gln Arg Gln 35 40 45 Phe Ser Glu Val Trp Lys Pro Ser Pro Gln Val Thr Val Arg Phe Pro 50 55 60 Asp Ser Asp Phe Lys Val Tyr Arg Tyr Asn Wing Val Leu Asp Pro Leu 65 70 75 80 Val Thr Ala Leu Leu Gly Wing Phe Asp Thr Arg Asn Arg He He Glu 85 90 95 Val Glu Asn Gln Wing Asn Pro Thr Thr Wing Glu Thr Leu Asp Wing Thr 100 105 110 Arg Arg Val Asp Asp Wing Thr Val Wing He Arg Ser Ala He Asn Asn 115 120 125 Leu He Val Glu Leu He Arg Gly Thr Gly Ser Tyr Asn Arg Ser Ser 130 135 140 Phe Glu Ser Ser Gly Leu Val Trp Thr Ser 145 150 155

Claims (13)

R E I V I N D I C A C I O N E S:

1. A polynucleotide-encoding fusion protein, the fusion protein consists essentially of a protein coated with tobamovirus fused to a fusion binding interest protein.

2. A polynucleotide according to claim 1, wherein the fusion is a fusion of the amino terminal.

3. A polynucleotide according to claim 1, wherein the fusion is a carboxy terminal fusion.

4. A polynucleotide according to claim 1, wherein the fusion is an internal fusion.

5. A polynucleotide according to claim 1, wherein the fusion junction comprises a "leaky" stop codon.

6. A polynucleotide according to claim 1, wherein the fusion junction comprises a "leaky" start codon.

7. A polynucleotide according to claim 1, wherein the protein of interest is an antigen.

8. A polynucleotide according to claim 1, wherein the coated protein is a coated protein of tobacco mosaic virus.

9. A recombinant plant viral genome comprising a polynucleotide according to claim 1.

10. A recombinant plant virus particle comprising a genome according to claim 9.

11. A polypeptide encoded by a polynucleotide in accordance with with claim 1.

12. A recombinant plant virus, wherein the coated protein is encoded by a polynucleotide according to claim 1.

13. A plant cell comprising a polynucleotide according to claim 9.