Abstract:
The present invention relates to polynucleotides comprising a DNA sequence encoding an HCV protein and fragments thereof that contain codons optimized for expression in a vertebrate host. Uses of the polynucleotides include eliciting an immune response specifically recognizing HCV.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     The present application is a 371 of PCT/US97/09884, filed Jun. 6, 1997, which claims benefit of U.S. provisional application 60/020,494 filed Jun. 11, 1996, now abandoned, and U.S. provisional application 60/033,534 filed Dec. 20, 1996, now abandoned. 
    
    
     STATEMENT REGARDING FEDERALLY-SPONSORED R&amp;D 
     Not applicable. 
     REFERENCE TO MICROFICHE APPENDIX 
     Not applicable. 
     FIELD OF THE INVENTION 
     Not applicable. 
     BACKGROUND OF THE INVENTION 
     This invention relates to novel nucleic acid pharmaceutical products, specifically nucleic acid vaccine products. The nucleic acid vaccine products, when introduced directly into muscle cells, induce the production of immune responses which specifically recognize Hepatitis C virus (HCV). 
     Hepatitis C Virus 
     Non-A, Non-B hepatitis (NANBH) is a transmissible disease (or family of diseases) that is believed to be virally induced, and is distinguishable from other forms of virus-associated liver disease, such as those caused by hepatitis A virus (HAV), hepatitis B virus (HBV), delta hepatitis virus (HDV), cytomegalovirus (CMV) or Epstein-Barr virus (EBV). Epidemiologic evidence suggests that there may be three types of NANBH: the water-borne epidemic type; the blood or needle associated type; and the sporadically occurring (community acquired) type. However, the number of causative agents is unknown. Recently, a new viral species, hepatitis C virus (HCV) has been identified as the primary (if not only) cause of blood-associated NANBH (BB-NANBH). Hepatitis C appears to be the major form of transfusion-associated hepatitis in a number of countries, including the United States and Japan. There is also evidence implicating HCV in induction of hepatocellular carcinoma. Thus, a need exists for an effective method for preventing or treating HCV infection: currently, there is none. 
     The HCV may be distantly related to the flaviviridae. The Flavivirus family contains a large number of viruses which are small, enveloped pathogens of man. The morphology and composition of Flavivirus particles are known, and are discussed in M. A. Brinton, in “The Viruses: The Togaviridae And Flaviviridae” (Series eds. Fraenkel-Conrat and Wagner, vol. eds. Schlesinger and Schlesinger, Plenum Press, 1986), pp. 327-374. Generally, with respect to morphology, Flaviviruses contain a central nucleocapsid surrounded by a lipid bilayer. Virions are spherical and have a diameter of about 40-50 nm. Their cores are about 25-30 nm in diameter. Along the outer surface of the virion envelope are projections measuring about 5-10 nm in length with terminal knobs about 2 nm in diameter. Typical examples of the family include Yellow Fever virus, West Nile virus, and Dengue Fever virus. They possess positive-stranded RNA genomes (about 11,000 nucleotides) that are slightly larger than that of HCV and encode a polyprotein precursor of about 3500 amino acids. Individual viral proteins are cleaved from this precursor polypeptide. 
     The genome of HCV appears to be single-stranded RNA containing about 10,000 nucleotides. The genome is positive-stranded, and possesses a continuous translational open reading frame (ORF) that encodes a polyprotein of about 3,000 amino acids. In the ORF, the structural proteins appear to be encoded in approximately the first quarter of the N-terminal region, with the majority of the polyprotein attributed to non-structural proteins. When compared with all known viral sequences, small but significant co-linear homologies are observed with the nonstructural proteins of the Flavivirus family, and with the pestiviruses (which are now also considered to be part of the Flavivirus family). 
     Intramuscular inoculation of polynucleotide constructs, i.e., DNA plasmids encoding proteins have been shown to result in the in situ generation of the protein in muscle cells. By using cDNA plasmids encoding viral proteins, both antibody and CTL responses were generated, providing homologous and heterologous protection against subsequent challenge with either the homologous or cross-strain protection, respectively. Each of these types of immune responses offers a potential advantage over existing vaccination strategies. The use of PNVs (polynucleotide vaccines) to generate antibodies may result in an increased duration of the antibody responses as well as the provision of an antigen that can have both the exact sequence of the clinically circulating strain of virus as well as the proper post-translational modifications and conformation of the native protein (vs. a recombinant protein). The generation of CTL responses by this means offers the benefits of cross-strain protection without the use of a live potentially pathogenic vector or attenuated virus. 
     Therefore, this invention contemplates methods for introducing nucleic acids into living tissue to induce expression of proteins. The invention provides a method for introducing viral proteins into the antigen processing pathway to generate virus-specific immune responses including, but not limited to, CTLs. Thus, the need for specific therapeutic agents capable of eliciting desired prophylactic immune responses against viral pathogens is met for HCV virus by this invention. Of particular importance in this therapeutic approach is the ability to induce T-cell immune responses which can prevent infections even of virus strains which are heterologous to the strain from which the antigen gene was obtained. Therefore, this invention provides DNA constructs encoding viral proteins of the hepatitis C virus core, envelope (E1), nonstructural (NS5) genes or any other HCV genes which encode products which generate specific immune responses including but not limited to CTLs. 
     DNA Vaccines 
     Benvenisty, N., and Reshef, L. [PNAS 83, 9551-9555, (1986)] showed that CaCl 2 -precipitated DNA introduced into mice intraperitoneal ly (i.p.), intravenously (i.v.) or intramuscularly (i.m.) could be expressed. The i.m. injection of DNA expression vectors without CaCl 2  treatment in mice resulted in the uptake of DNA by the muscle cells and expression of the protein encoded by the DNA. The plasmids were maintained episomally and did not replicate. Subsequently, persistent expression has been observed after i.m. injection in skeletal muscle of rats, fish and primates, and cardiac muscle of rats. The technique of using nucleic acids as therapeutic agents was reported in WO90/11092 (Oct. 4, 1990), in which polynucleotides were used to vaccinate vertebrates. 
     It is not necessary for the success of the method that immunization be intramuscular. The introduction of gold microprojectiles coated with DNA encoding bovine growth hormone (BGH) into the skin of mice resulted in production of anti-BGH antibodies in the mice. A jet injector has been used to transfect skin, muscle, fat, and mammary tissues of living animals. Various methods for introducing nucleic acids have been reviewed. Intravenous injection of a DNA:cationic liposome complex in mice was shown by Zhu et al., [Science 261:209-211 (Jul. 9, 1993) to result in systemic expression of a cloned transgene. Ulmer et al., [Science 259:1745-1749, (1993)] reported on the heterologous protection against influenza virus infection by intramuscular injection of DNA encoding influenza virus proteins. 
     The need for specific therapeutic and prophylactic agents capable of eliciting desired immune responses against pathogens and tumor antigens is met by the instant invention. Of particular importance in this therapeutic approach is the ability to induce T-cell immune responses which can prevent infections or disease caused even by virus strains which are heterologous to the strain from which the antigen gene was obtained. This is of particular concern when dealing with HIV as this virus has been recognized to mutate rapidly and many virulent isolates have been identified [see, for example, LaRosa et al., Science 249:932-935 (1990), identifying 245 separate HIV isolates]. In response to this recognized diversity, researchers have attempted to generate CTLs based on peptide immunization. Thus, Takahashi et al., [Science 255:333-336 (1992)] reported on the induction of broadly cross-reactive cytotoxic T cells recognizing an HIV envelope (gp160) determinant. However, those workers recognized the difficulty in achieving a truly cross-reactive CTL response and suggested that there is a dichotomy between the priming or restimulation of T cells, which is very stringent, and the elicitation of effector function, including cytotoxicity, from already stimulated CTLs. 
     Wang et al. reported on elicitation of immune responses in nice against HIV by intramuscular inoculation with a cloned, genomic (unspliced) HIV gene. However, the level of immune responses achieved in these studies was very low. In addition, the Wang et al., DNA construct utilized an essentially genomic piece of HIV encoding contiguous Tat/REV-gp160-Tat/REV coding sequences. As is described in detail below, this is a suboptimal system for obtaining high-level expression of the gp160. It also is potentially dangerous because expression of Tat contributes to the progression of Karposi&#39;s Sarcoma. 
     WO 93/17706 describes a method for vaccinating an animal against a virus, wherein carrier particles were coated with a gene construct and the coated particles are accelerated into cells of an animal. 
     The instant invention contemplates any of the known methods for introducing polynucleotides into living tissue to induce expression of proteins. However, this invention provides a novel immunogen for introducing proteins into the antigen processing pathway to efficiently generate specific CTLs and antibodies. 
     Codon Usage and Codon Context 
     The codon pairings of organisms are highly nonrandom, and differ from organism to organism. This information is used to construct and express altered or synthetic genes having desired levels of translational efficiency, to determine which regions in a genome are protein coding regions, to introduce translational pause sites into heterologous genes, and to ascertain relationship or ancestral origin of nucleotide sequences 
     The expression of foreign heterologous genes in transformed organisms is now commonplace. A large number of mammalian genes, including, for example, murine and human genes, have been successfully inserted into single celled organisms. Standard techniques in this regard include introduction of the foreign gene to be expressed into a vector such as a plasmid or a phage and utilizing that vector to insert the gene into an organism. The native promoters for such genes are commonly replaced with strong promoters compatible with the host into which the gene is inserted. Protein sequencing machinery permits elucidation of the amino acid sequences of even minute quantities of native protein. From these amino acid sequences, DNA sequences coding for those proteins can be inferred. DNA synthesis is also a rapidly developing art, and synthetic genes corresponding to those inferred DNA sequences can be readily constructed. 
     Despite the burgeoning knowledge of expression systems and recombinant DNA, significant obstacles remain when one attempts to express a foreign or synthetic gene in an organism. Many native, active proteins, for example, are glycosylated in a manner different from that which occurs when they are expressed in a foreign host. For this reason, eukaryotic hosts such as yeast may be preferred to bacterial hosts for expressing many mammalian genes. The glycosylation problem is the subject of continuing research. 
     Another problem is more poorly understood. Often translation of a synthetic gene, even when coupled with a strong promoter, proceeds much less efficiently than would be expected. The same is frequently true of exogenous genes foreign to the expression organism. Even when the gene is transcribed in a sufficiently efficient manner that recoverable quantities of the translation product are produced, the protein is often inactive or otherwise different in properties from the native protein. 
     It is recognized that the latter problem is commonly due to differences in protein folding in various organisms. The solution to this problem has been elusive, and the mechanisms controlling protein folding are poorly understood. 
     The problems related to translational efficiency are believed to be related to codon context effects. The protein coding regions of genes in all organisms are subject to a wide variety of functional constraints, some of which depend on the requirement for encoding a properly functioning protein, as well as appropriate translational start and stop signals. However, several features of protein coding regions have been discerned which are not readily understood in terms of these constraints. Two important classes of such features are those involving codon usage and codon context. 
     It is known that codon utilization is highly biased and varies considerably between different organisms. Codon usage patterns have been shown to be related to the relative abundance of tRNA isoacceptors. Genes encoding proteins of high versus low abundance show differences in their codon preferences. The possibility that biases in codon usage alter peptide elongation rates has been widely discussed. While differences in codon use are associated with differences in translation rates, direct effects of codon choice on translation have been difficult to demonstrate. Other proposed constraints on codon usage patterns include maximizing the fidelity of translation and optimizing the kinetic efficiency of protein synthesis. 
     Apart from the non-random use of codons, considerable evidence has accumulated that codon/anticodon recognition is influenced by sequences outside the codon itself, a phenomenon termed “codon context.” There exists a strong influence of nearby nucleotides on the efficiency of suppression of nonsense codons as well as missense codons. Clearly, the abundance of suppressor activity in natural bacterial populations, as well as the use of “termination” codons to encode selenocysteine and phosphoserine require that termination be context-dependent. Similar context effects have been shown to influence the fidelity of translation, as well as the efficiency of translation initiation. 
     Statistical analyses of protein coding regions of  E. coli  have demonstrate another manifestation of “codon context.” The presence of a particular codon at one position strongly influences the frequency of occurrence of certain nucleotides in neighboring codons, and these context constraints differ markedly for genes expressed at high versus low levels. Although the context effect has been recognized, the predictive value of the statistical rules relating to preferred nucleotides adjacent to codons is relatively low. This has limited the utility of such nucleotide preference data for selecting codons to effect desired levels of translational efficiency. 
     The advent of automated nucleotide sequencing equipment has made available large quantities of sequence data for a wide variety of organisms. Understanding those data presents substantial difficulties. For example, it is important to identify the coding regions of the genome in order to relate the genetic sequence data to protein sequences. In addition, the ancestry of the genome of certain organisms is of substantial interest. It is known that genomes of some organisms are of mixed ancestry. Some sequences that are viral in origin are now stably incorporated into the genome of eukaryotic organisms. The viral sequences themselves may have originated in another substantially unrelated species. An understanding of the ancestry of a gene can be important in drawing proper analogies between related genes and their translation products in other organisms. 
     There is a need for a better understanding of codon context effects on translation, and for a method for determining the appropriate codons for any desired translational effect. There is also a need for a method for identifying coding regions of the genome from nucleotide sequence data. There is also a need for a method for controlling protein folding and for insuring that a foreign gene will fold appropriately when expressed in a host. Genes altered or constructed in accordance with desired translational efficiencies would be of significant worth. 
     Another aspect of the practice of recombinant DNA techniques for the expression by microorganisms of proteins of industrial and pharmaceutical interest is the phenomenon of “codon preference”. While it was earlier noted that the existing machinery for gene expression is genetically transformed host cells will “operate” to construct a given desired product, levels of expression attained in a microorganism can be subject to wide variation, depending in part on specific alternative forms of the amino acid-specifying genetic code present in an inserted exogenous gene. A “triplet” codon of four possible nucleotide bases can exist in 64 variant forms. That these forms provide the message for only 20 different amino acids (as well as transcription initiation and termination) means that some amino acids can be coded for by more than one codon. Indeed, some amino acids have as many as six “redundant”, alternative codons while some others have a single, required codon. For reasons not completely understood, alternative codons are not at all uniformly present in the endogenous DNA of differing types of cells and there appears to exist a variable natural hierarchy or “preference” for certain codons in certain types of cells. 
     As one example, the amino acid leucine is specified by any of six DNA codons including CTA, CTC, CTG, CTT, TTA, and TTG (which correspond, respectively, to the mRNA codons, CUA, CUC, CUG, CUU, UUA and UUG). Exhaustive analysis of genome codon frequencies for microorganisms has revealed endogenous DNA of  E. coli  most commonly contains the CTG leucine-specifying codon, while the DNA of yeasts and slime molds most commonly includes a TTA leucine-specifying codon. In view of this hierarchy, it is generally held that the likelihood of obtaining high levels of expression of a leucine-rich polypeptide by an  E. coli  host will depend to some extent on the frequency of codon use. For example, a gene rich in TTA codons will in all probability be poorly expressed in  E. coli,  whereas a CTG ricb gene will probably highly express the polypeptide. Similarly, when yeast cells are the projected transformation host cells for expression of a leucine-rich polypeptide, a preferred codon for use in an inserted DNA would be TTA. 
     The implications of codon preference phenomena on recombinant DNA techniques are manifest, and the phenomenon may serve to explain many prior failures to achieve high expression levels of exogenous genes in successfully transformed host organisms—a less “preferred” codon may be repeatedly present in the inserted gene and the host cell machinery for expression may not operate as efficiently. This phenomenon suggests that synthetic genes which have been designed to include a projected host cell&#39;s preferred codons provide a preferred form of foreign genetic material for practice of recombinant DNA techniques. 
     Protein Trafficking 
     The diversity of function that typifies eukaryotic cells depends upon the structural differentiation of their membrane boundaries. To generate and maintain these structures, proteins must be transported from their site of synthesis in the endoplasmic reticulum to predetermined destinations throughout the cell. This requires that the trafficking proteins display sorting signals that are recognized by the molecular machinery responsible for route selection located at the access points to the main trafficking pathways. Sorting decisions for most proteins need to be made only once as they traverse their biosynthetic pathways since their final destination, the cellular location at which they perform their function, becomes their permanent residence. 
     Maintenance of intracellular integrity depends in part on the selective sorting and accurate transport of proteins to their correct destinations. Over the past few years the dissection of the molecular machinery for targeting and localization of proteins has been studied vigorously. Defined sequence motifs have been identified on proteins which can act as ‘address labels’. A number of sorting signals have been found associated with the cytoplasmic domains of membrane proteins. 
     SUMMARY OF THE INVENTION 
     This invention relates to novel formulations of nucleic acid pharmaceutical products, specifically nucleic acid vaccine products. The nucleic acid products, when introduced directly into muscle cells, induce the production of immune responses which specifically recognize Hepatitis C virus (HCV). 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIGS. 1A and 1B show the nucleotide sequence (SEQ. ID. NO. 1) of the V1Ra vector. 
     FIG. 2 is a diagram of the V1Ra vector. 
     FIG. 3 is a diagram of the Vtpa vector. 
     FIG. 4 is the VUb vector. 
     FIG. 5 shows an optimized nucleotide sequence (SEQ. ID. NO. 2) of the HCV core antigen and the encoded amino acid sequence (SEQ. ID. NO. 3). 
     FIG. 6 shows V1Ra.HCV1CorePAb, Vtpa.HCV1CorePAb and VUb.HCV1CorePAb. A sequence coding for an epitope derived from influenza virus nucleoprotein residues 366-374 and an antibody epitope sequence derived from SV40 T antigen resides 684-698 is illustrated by SEQ. ID. NO. 4 (starting GAA) and its complementary sequence SEQ. ID. NO. 16 (starting CTT). 
     FIG. 7 shows the Hepatitis C Virus Core Antigen nucleotide sequence (SEQ. ID. NO. 5) and amino acid sequence (SEQ. ID. NO. 3). 
     FIG. 8 shows codon utilization in human protein-coding sequences (from Lathe et al.). 
     FIG. 9 shows an optimized nucleotide sequence (SEQ. ID. NO. 6) and the encoded amino acid sequence (SEQ. ID. NO. 7) of the HCV E1 protein. 
     FIGS. 10A and 10B show an optimized nucleotide sequence (SEQ. ID. NO. 8) and the encoded amino acid sequence (SEQ. ID. NO. 9) of the HCV E2 protein. 
     FIGS. 11A,  11 B and  11 C show an optimized nucleotide sequence (SEQ. ID. NO. 10) and the encoded amino acid sequence (SEQ. ID. NO. 11) of the HCV E1+E2 proteins. 
     FIGS. 12A,  12 B and  12 C show an optimized nucleotide sequence (SEQ. ID. NO. 12) and the encoded amino acid sequence (SEQ. ID. NO. 13) of the HCV NS5a protein. 
     FIGS. 13A,  13 B,  13 C and  13 D show an optimized nucleotide sequence (SEQ. ID. NO. 14) and the encoded amino acid sequence (SEQ. ID. NO. 15) of the HCV NS5b protein. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     This invention relates to novel fonnulations of nucleic acid pharmaceutical products, specifically nucleic acid vaccine products. The nucleic acid vaccine products, when introduced directly into muscle cells, induce the production of immune responses which specifically recognize Hepatitis C virus (HCV). 
     Non-A, Non-B hepatitis (NANBH) is a transmissible disease (or family of diseases) that is believed to be virally induced, and is distinguishable from other forms of virus-associated liver disease, such as those caused by hepatitis A virus (HAV), hepatitis B virus (HBV), delta hepatitis virus (HDV), cytomegalovirus (CMV) or Epstein-Barr virus (EBV). Epidemiologic evidence suggests that there may be three types of NANBH: the water-bome epidemic type; the blood or needle associated type; and the sporadically occurring (community acquired) type. However, the number of causative agents is unknown. Recently, a new viral species, hepatitis C virus (HCV) has been identified as the primary (if not only) cause of blood-associated NANBH (BB-NANBH). Hepatitis C appears to be the major form of transfusion-associated hepatitis in a number of countries, including the United States and Japan. There is also evidence implicating HCV in induction of hepatocellular carcinoma. Thus, a need exists for an effective method for preventing or treating HCV infection: currently, there is none. 
     The HCV may be distantly related to the flaviviridae. The Flavivirus family contains a large number of viruses which are small, enveloped pathogens of man. The morphology and composition of Flavivirus particles are known, and are discussed in M. A. Brinton, in “The Viruses: The Togaviridae And Flaviviridae” (Series eds. Fraenkel-Conrat and Wagner, vol. eds. Schlesinger and Schlesinger, Plenum Press, 1996), pp. 327-374. Generally, with respect to morphology, Flaviviruses contain a central nucleocapsid surrounded by a lipid bilayer. Virions are spherical and have a diameter of about 40-50 nm. Their cores are about 25-30 nm in diameter. Along the outer surface of the virion envelope are projections measuring about 5-10 nm in length with terminal knobs about 2 nm in diameter. Typical examples of the family include Yellow Fever virus, West Nile virus, and Dengue Fever virus. They possess positive-stranded RNA genomes (about 11,000 nucleotides) that are slightly larger than that of HCV and encode a polyprotein precursor of about 3500 amino acids. Individual viral proteins are cleaved from this precursor polypeptide. 
     The genome of HCV appears to be single-stranded RNA containing about 10,000 nucleotides. The genome is positive-stranded, and possesses a continuous translational open reading frame (ORF) that encodes a polyprotein of about 3,000 amino acids. In the ORF, the structural proteins appear to be encoded in approximately the first quarter of the N-terminal region, with the majority of the polyprotein attributed to non-structural proteins. When compared with all known viral sequences, small but significant co-linear homologies are observed with the nonstructural proteins of the Flavivirus family, and with the pestiviruses (which are now also considered to be part of the Flavivirus family). 
     Intramuscular inoculation of polynucleotide constructs, i.e., DNA plasmids encoding proteins halve been shown to result in the generation of the encoded protein in situ in muscle cells. By using cDNA plasmids encoding viral proteins, both antibody and CTL responses were generated, providing homologous and heterologous protection against subsequent challenge with either the homologous or cross-strain protection, respectively. Each of these types of immune responses offers a potential advantage over existing vaccination strategies. The use of PNVs (polynucleotide vaccines) to generate antibodies may result in an increased duration of the antibody responses as well as the provision of an antigen that can have both the exact sequence of the clinically circulating strain of virus as well as the proper post-translational modifications and conformation of the native protein (vs. a recombinant protein). The generation of CTL responses by this means offers the benefits of cross-strain protection without the use of a live potentially pathogenic vector or attenuated virus. 
     The standard techniques of molecular biology for preparing and purifying DNA constructs enable the preparation of the DNA therapeutics of this invention. While standard techniques of molecular biology are therefore sufficient for the production of the products of this invention, the specific constructs disclosed herein provide novel therapeutics which surprisingly produce cross-strain protection, a result heretofore unattainable with standard inactivated whole virus or subunit protein vaccines. 
     The amount of expressible DNA to be introduced to a vaccine recipient will depend on the strength of the transcriptional and translational promoters used in the DNA construct, and on the immunogenicity of the expressed gene product. In general, an immunologically or prophylactically effective dose of about 1 μg to 1 mg, and preferably about 10 μg to 300 μg is administered directly into muscle tissue. Subcutaneous injection, intradermal introduction, impression through the skin, and other modes of administration such as intraperitoneal, intravenous, or inhalation delivery are also contemplated. It is also contemplated that booster vaccinations are to be provided. 
     The DNA may be naked, that is, unassociated with any proteins, adjuvants or other agents which impact on the recipients immune system. In this case, it is desirable for the DNA to be in a physiologically acceptable solution, such as, but not limited to, sterile saline or sterile buffered saline. Alternatively, the DNA may be associated with surfactants, liposomes, such as lecithin liposomes or other liposomes known in the art, as a DNA-liposome mixture, (see for example WO93/24640) or the DNA may be associated with an adjuvant known in the art to boost immune responses, such as a protein or other carrier. Agents which assist in the cellular uptake of DNA, such as, but not limited to, calcium ions, detergents, viral proteins and other transfection facilitating agents may also be used to advantage. These agents are generally referred to as transfection facilitating agents and as pharmaceutically acceptable carriers. As used herein, the term gene refers to a segment of nucleic acid which encodes a discrete polypeptide. The term pharmaceutical, and vaccine are used interchangeably to indicate compositions useful for inducing immune responses. The terms construct, and plasmid are used interchangeably. The term vector is used to indicate a DNA into which genes may be cloned for use according to the method of this invention. 
     The following examples are provided to further define the invention, without limiting the invention to the specifics of the examples. 
     EXAMPLE 1 
     V1J Expression Vectors 
     V1J is derived from vectors V1 and pUC18, a commercially available plasmid. V1 was digested with SspI and EcoRI restriction enzymes producing two fragments of DNA. The smaller of these fragments, containing the CMVintA promoter and Bovine Growth Hormone (BGH) transcription termination elements which control the expression of heterologous genes, was purified from an agarose electrophoresis gel. The ends of this DNA fragment were then “blunted” using the T4 DNA polymerase enzyme in order to facilitate its ligation to another “blunt-ended” DNA fragment. 
     pUC18 was chosen to provide the “backbone” of the expression vector. It is known to produce high yields of plasmid, is well-characterized by sequence and function, and is of minimum size. We removed the entire lac operon from this vector, which was unnecessary for our purposes and may be detrimental to plasmid yields and heterologous gene expression, by partial digestion with the HaeII restriction enzyme. The remaining plasmid was purified from an agarose electrophoresis gel, blunt-ended with the T4 DNA polymerase, treated with calf intestinal alkaline phosphatase, and ligated to the CMVintA/BGH element described above. Plasmids exhibiting either of two possible orientations of the promoter elements within the pUC backbone were obtained. One of these plasmids gave much higher yields of DNA in  E. coli  and was designated V1J. This vector&#39;s structure was verified by sequence analysis of the junction regions and was subsequently demonstrated to give comparable or higher expression of heterologous genes compared with V1. The ampicillin resistance marker was replaced with the neomycin resistance marker to yield vector V1Jneo. 
     An Sfi I site was added to V1Jneo to facilitate integration studies. A commercially available 13 base pair Sfi I linker (New England BioLabs) was added at the Kpn I site within the BGH sequence of the vector. V1Jneo was linearized with Kpn I, gel purified, blunted by T4 DNA polymerase, and ligated to the blunt Sfi I linker. Clonal isolates were chosen by restriction mapping and verified by sequencing through the linker. The new vector was designated V1Jns. Expression of heterologous genes in V1Jns (with Sfi I) was comparable to expression of the same genes in V1Jneo (with Kpn I). 
     Vector V1Ra (Sequence is shown in FIG. 1; map is shown in FIG. 2) was derived from vector V1R, a derivative of the V1Jns vector. Multiple cloning sites (BglII, KpnI, EcoRV, EcoRI, SalI, and NotI) were introduced into V1R to create the V1Ra vector to improve the convenience of subcloning. V1Ra vector derivatives containing the tpa leader sequence and ubiquitin sequence were generated (Vtpa (FIG. 3) and Vub (FIG.  4 ), respectively). Expression of viral antigen from Vtpa vector will target the antigen protein into the exocytic pathway, thus producing a secretable form of the antigen proteins. These secreted proteins are likely to be captured by professional antigen presenting cells, such as macrophages and dendritic cells, and processed and presented by class II molecules to activate CD4+ Th cells. They also are more likely to efficiently simulate antibody responses. Expression of viral antigen through VUb vector will produce a ubiquitin and antigen fusion protein. The uncleavable ubiquitin segment (glycine to alanine change at the cleavage site, Butt et al., JBC 263:16364, 1988) will target the viral antigen to ubiquitin-associated proteasomes for rapid degradation. The resulting peptide fragments will be transported into the ER for antigen presentation by class I molecules. This modification is attempted to enhance the class I molecule-restricted CTL responses against the viral antigen (Townsend et al, JEM 168:1211, 1988). 
     EXAMPLE 2 
     Design and Construction of the Synthetic Genes 
     A. Design of Synthetic Gene Sepments for HCV Gene Expression 
     Gene segments were converted to sequences having identical translated sequences (except where noted) but with alternative codon usage as defined by R. Lathe in a research article from  J. Molec. Biol. Vol.  183, pp. 1-12 (1985) entitled “Synthetic Oligonucleotide Probes Deduced from Amino Acid Sequence Data: Theoretical and Practical Considerations”. The methodology described below was based on our hypothesis that the known inability to express a gene efficiently in mammalian cells is a consequence of the overall transcript composition. Thus, using alternative codons encoding the same protein sequence may remove the constraints on HCV gene expression. Inspection of the codon usage within HCV genome revealed that a high percentage of codons were among those infrequently used by highly expressed human genes. The specific codon replacement method employed may be described as follows employing data from Lathe et al.: 
     1. Identify placement of codons for proper open reading frame. 
     2. Compare wild type codon for observed frequency of use by human genes (refer to Table 3 in Lathe et al.). 
     3. If codon is not the most commonly employed, replace it with an optimal codon for high expression based on data in Table 5. 
     4. Inspect the third nucleotide of the new codon and the first nucleotide of the adjacent codon immediately 3′- of the first. If a 5′-CG-3′ pairing has been created by the new codon selection, replace it with the choice indicated in Table 5. 
     5. Repeat this procedure until the entire gene segment has been replaced. 
     6. Inspect new gene sequence for undesired sequences generated by these codon replacements (e.g., “ATTTA” sequences, inadvertent creation of intron splice recognition sites, unwanted restriction enzyme sites, etc.) and substitute codons that eliminate these sequences. 
     7. Assemble synthetic gene segments and test for improved expression. 
     B. HCV Core Antigen Sequence 
     The consensus core sequence of HCV was adopted from a generalized core sequence reported by Bukh et al. (PNAS, 91:8239, 1994). This core sequence contains all the identified CTL epitopes in both human and mouse. The gene is composed of 573 nucleotides and encodes 191 amino acids. The predicted molecular weight is about 23 kDa. 
     The codon replacement was conducted to eliminate codons which may hinder the expression of the HCV core protein in transfected mammalian cells in order to maximize the translational efficiency of DNA vaccine. Twenty three point two percent (23.2%) of nucleotide sequence (133 out of 573 nucleotides) were altered, resulting in changes of 61.3% of the codons (117 out 191 codons) in the core antigen sequence. The optimized nucleotide sequence of HCV core is shown in FIG.  5 . 
     C. Construction of the Synthetic Core Gene 
     The optimized HCV core gene (FIG. 5) was constructed as a synthetic gene annealed from multiple synthetic oligonucleotides. To facilitate the identification and evaluation of the synthetic gene expression in cell culture and its immunogenicity in mice, a CTL epitope derived from influenza virus nucleoprotein residues 366-374 and an antibody epitope sequence derived from SV40 T antigen residues 684-699 were tagged to the carboxyl terminal of the core sequence (FIG.  6 ). For clinical use it may be desired to express the core sequence without the nucleoprotein 366-374 and SV40 T 684-698 sequences. For this reason, the sequence of the two epitopes is flanked by two EcoRI sites which will be used to excise this fragment of sequence at a later time. Thus an embodiment of the invention for clinical use could consist of the V1Ra.HCV1CorePAb, Vtpa.HCV1CorePAb, or VUb.HCV1CorePAb plasmids that had been cut with EcoRI, annealed, and ligated to yield plasmids V1Ra.HCV1Core, Vtpa.HCV1Core, and VUb.HCV1Core. 
     The synthetic gene was built as three separate segments in three vectors, nucleotides 1 to 80 in V1Ra, nucleotides 80 to 347 (BstX1 site) in pUC18, and nucleotides 347 to 573 plus the two epitope sequence in pUC18. All the segments were verified by DNA sequencing, and joined together in V1Ra vector. 
     D. HCV Gene Expression Constructs 
     In each case, the junction sequences from the 5′ promoter region (CMVintA) into the cloned gene is shown. The position at which the junction occurs is demarcated by a “/”, which does not represent any discontinuity in the sequence. 
     The nomenclature for these constructs follows the convention: “Vector name-HCV strain-gene”. 
     
       
         
               
             
           
               
                              V1Ra.HICV1.CorePAb 
               
               
                 ---IntA--AGA TCT ACC ATG AGC (SEQ. ID. NO.  
               
               
                 17)--HCV.Core.--GCC GAA TTC GCT TCC (SEQ. ID. NO. 
               
               
                 18)--PAb Sequence--TAA ACC CGG GAA TTC TAA A GTC 
               
               
                 GAC (SEQ. ID. NO. 19)--BGH--- 
               
               
                   
               
               
                              Vtpa.HCV1.CorePAb 
               
               
                 ---IntA--ATC ACC ATG GAT (SEQ. ID. NO. 20)--tpa 
               
               
                 leader--GAG ATC-TTC ATG AGC (SEQ. ID. NO. 21)-- 
               
               
                 HCV.Core.--GCC GAA TTC GCT TCC--(SEQ. ID. NO. 
               
               
                 18) PAb Sequence--TAA ACC CGG GAA TTC TAA A GTC 
               
               
                 GAC (SEQ. ID. NO. 19)--BGH--- 
               
               
                   
               
               
                              VUb.HCV1.CorePAb. 
               
               
                 ---IntA--AGA TCC ACC ATG CAG (SEQ. ID. NO. 22) 
               
               
                 --Ubiquitin--GGT GCA GAT CTG ATG AGC (SEQ. ID. NO. 
               
               
                 23)--HCV.Core.--GCC GAA TTC GCT TCC--(SEQ. ID. NO. 
               
               
                 18) PAb Sequence--TAA ACC CGG GAA TTC TAA A GTC 
               
               
                 GAC--BGH-- 
               
               
                   
               
               
                              V1Ra.HCV1.Core 
               
               
                 ---IntA--AGA TCT ACC ATG AGC (SEQ. ID. NO. 17)-- 
               
               
                 HCV.Core.--GCC TAA A GTC GAC (SEQ. ID. NO. 24)-- 
               
               
                 BGH--- 
               
               
                   
               
               
                              Vtpa.HCV1.Core 
               
               
                 ---IntA--ATC ACC ATG GAT (SEQ. ID. NO. 20)--tpa 
               
               
                 leader--GAG ATC-TTC ATG AGC (SEQ. ID. NO. 21)-- 
               
               
                 HCV.Core.--GCC TAA A GTC GAC (SEQ. ID. NO. 24)-- 
               
               
                 BGH--- 
               
               
                   
               
               
                 VUb.HCV1.Core 
               
               
                 ---IntA--AGA TCC ACC ATG CAG (SEQ. ID. NO. 22)-- 
               
               
                 Ubiquitin--GGT GCA GAT CTG ATG AGC (SEQ. ID. NO. 
               
               
                 23)--HCV.Core.--GCC TAA A GTC GAC (SEQ. ID. NO. 
               
               
                 24)--BGH-- 
               
             
          
         
       
     
     E. Other Synthetic HCV Genes 
     Using similar codon optimization techniques, synthetic genes encoding the HCV E1 (FIG.  9 ), HCV E2 (FIG.  10 ), HCV E1+E2 (FIG.  11 ), HCV NS5a (FIG. 12) and HCV NS5b (FIG. 13) proteins were created. 
     
       
         
           
             25 
           
           
             1 
             3610 
             DNA 
             Artificial Sequence 
             
               Modified Vector Sequence 
             
           
            1
gatattggct attggccatt gcatacgttg tatccatatc ataatatgta catttatatt    60
ggctcatgtc caacattacc gccatgttga cattgattat tgactagtta ttaatagtaa   120
tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg   180
gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg   240
tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta   300
cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt   360
gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac   420
tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt   480
tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac   540
cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt   600
cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat   660
ataagcagag ctcgtttagt gaaccgtcag atcgcctgga gacgccatcc acgctgtttt   720
gacctccata gaagacaccg ggaccgatcc agcctccgcg gccgggaacg gtgcattgga   780
acgcggattc cccgtgccaa gagtgacgta agtaccgcct atagagtcta taggcccacc   840
cccttggctt cttatgcatg ctatactgtt tttggcttgg ggtctataca cccccgcttc   900
ctcatgttat aggtgatggt atagcttagc ctataggtgt gggttattga ccattattga   960
ccactcccct attggtgacg atactttcca ttactaatcc ataacatggc tctttgccac  1020
aactctcttt attggctata tgccaataca ctgtccttca gagactgaca cggactctgt  1080
atttttacag gatggggtct catttattat ttacaaattc acatatacaa caccaccgtc  1140
cccagtgccc gcagttttta ttaaacataa cgtgggatct ccacgcgaat ctcgggtacg  1200
tgttccggac atgggctctt ctccggtagc ggcggagctt ctacatccga gccctgctcc  1260
catgcctcca gcgactcatg gtcgctcggc agctccttgc tcctaacagt ggaggccaga  1320
cttaggcaca gcacgatgcc caccaccacc agtgtgccgc acaaggccgt ggcggtaggg  1380
tatgtgtctg aaaatgagct cggggagcgg gcttgcaccg ctgacgcatt tggaagactt  1440
aaggcagcgg cagaagaaga tgcaggcagc tgagttgttg tgttctgata agagtcagag  1500
gtaactcccg ttgcggtgct gttaacggtg gagggcagtg tagtctgagc agtactcgtt  1560
gctgccgcgc gcgccaccag acataatagc tgacagacta acagactgtt cctttccatg  1620
ggtcttttct gcagtcaccg tccttagatc taggtaccag atatcagaat tcagtcgaca  1680
gcggccgcga tctgctgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt  1740
gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat  1800
tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcagcacag  1860
caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggg  1920
tacggccgca gcggccttaa ttaaggccgc agcggccgta cccaggtgct gaagaattga  1980
cccggttcct cgacccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc  2040
cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta  2100
taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg  2160
ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcaatgc  2220
tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac  2280
gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac  2340
ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg  2400
aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga  2460
aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt  2520
agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag  2580
cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacgtgatcc  2640
cgtaatgctc tgccagtgtt acaaccaatt aaccaattct gattagaaaa actcatcgag  2700
catcaaatga aactgcaatt tattcatatc aggattatca ataccatatt tttgaaaaag  2760
ccgtttctgt aatgaaggag aaaactcacc gaggcagttc cataggatgg caagatcctg  2820
gtatcggtct gcgattccga ctcgtccaac atcaatacaa cctattaatt tcccctcgtc  2880
aaaaataagg ttatcaagtg agaaatcacc atgagtgacg actgaatccg gtgagaatgg  2940
caaaagctta tgcatttctt tccagacttg ttcaacaggc cagccattac gctcgtcatc  3000
aaaatcactc gcatcaacca aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa  3060
tacgcgatcg ctgttaaaag gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa  3120
cactgccagc gcatcaacaa tattttcacc tgaatcagga tattcttcta atacctggaa  3180
tgctgttttc ccggggatcg cagtggtgag taaccatgca tcatcaggag tacggataaa  3240
atgcttgatg gtcggaagag gcataaattc cgtcagccag tttagtctga ccatctcatc  3300
tgtaacatca ttggcaacgc tacctttgcc atgtttcaga aacaactctg gcgcatcggg  3360
cttcccatac aatcgataga ttgtcgcacc tgattgcccg acattatcgc gagcccattt  3420
atacccatat aaatcagcat ccatgttgga atttaatcgc ggcctcgagc aagacgtttc  3480
ccgttgaata tggctcataa caccccttgt attactgttt atgtaagcag acagttttat  3540
tgttcatgat gatatatttt tatcttgtgc aatgtaacat cagagatttt gagacacaac  3600
gtggctttcc                                                         3610
 
           
             2 
             573 
             DNA 
             Artificial Sequence 
             
               Optimized sequence encoding HCV core antigen 
             
           
            2
atgagcacca accccaagcc ccagaggaag accaagagga acaccaacag gaggccccag    60
gatgtgaagt tccctggggg aggccagatt gtgggagggg tctacctgct gcccaggagg   120
ggccccaggc tgggggtgag ggctaccagg aagacctctg agaggtccca gcccaggggc   180
aggaggcagc ccatccccaa ggccaggagg cctgagggcc gctcctgggc ccagcctggc   240
tacccctggc ccctgtatgg caatgaaggc tttggctggg ctggctggct gctgtccccc   300
aggggctcca ggccctcctg gggccccaca gaccccagga ggaggtccag gaacctgggc   360
aaggtgattg acaccctgac ctgtggcttt gctgacctga tgggctacat ccccctggtg   420
ggggctcctg tgggaggggt ggctagggct ctggctcatg gggtgagggt gctggaggat   480
ggggtgaact atgctactgg caacctgcct ggctgctcct tctccatctt cctgctggcc   540
ctgctctcct gcctgacagt gcctgcttct gcc                                573
 
           
             3 
             191 
             PRT 
             Hepatitis C Virus 
           
            3
Met Ser Thr Asn Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn
 1               5                  10                  15
Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly
            20                  25                  30
Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Arg Ala
        35                  40                  45
Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro
    50                  55                  60
Ile Pro Lys Ala Arg Arg Pro Glu Gly Arg Ser Trp Ala Gln Pro Gly
65                  70                  75                  80
Tyr Pro Trp Pro Leu Tyr Gly Asn Glu Gly Phe Gly Trp Ala Gly Trp
                85                  90                  95
Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro Thr Asp Pro
            100                 105                 110
Arg Arg Arg Ser Arg Asn Leu Gly Lys Val Ile Asp Thr Leu Thr Cys
        115                 120                 125
Gly Phe Ala Asp Leu Met Gly Tyr Ile Pro Leu Val Gly Ala Pro Val
    130                 135                 140
Gly Gly Val Ala Arg Ala Leu Ala His Gly Val Arg Val Leu Glu Asp
145                 150                 155                 160
Gly Val Asn Tyr Ala Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile
                165                 170                 175
Phe Leu Leu Ala Leu Leu Ser Cys Leu Thr Val Pro Ala Ser Ala
            180                 185                 190
 
           
             4 
             103 
             DNA 
             Artificial Sequence 
             
               Modified Vector Sequence 
             
           
            4
gaattcgctt ccaatgagaa catggagacc atgaaccagc cctaccacat ctgccgcggc    60
ttcacctgct tcaagaagta aacccgggaa ttctaaagtc gac                     103
 
           
             5 
             573 
             DNA 
             Hepatitis C Virus 
           
            5
atgagcacga atcctaaacc tcaaagaaaa accaaacgta acaccaaccg ccgcccacag    60
gacgtcaagt tcccgggcgg tggtcagatc gttggtggag tttacttgtt gccgcgcagg   120
ggccccaggt tgggtgtgcg cgcgactagg aagacttccg agcggtcgca acctcgtgga   180
aggcgacagc ctatccccaa ggctcgccgg cccgagggca ggtcctgggc tcagcccggg   240
tacccttggc ccctctatgg caatgagggc ttcgggtggg caggatggct cctgtccccc   300
cgcggctctc ggcctagttg gggccccact gacccccggc gtaggtcgcg caatttgggt   360
aaggtcatcg ataccctcac gtgcggcttc gccgacctca tggggtacat cccgctcgtc   420
ggcgcccccg tagggggcgt cgccagggcc ctggcgcatg gcgtcagggt tctggaggac   480
ggggtgaact atgcaacagg gaatttgccc ggttgctctt tctctatctt cctcctggct   540
ctgctgtcct gcctgaccgt cccagcttct gct                                573
 
           
             6 
             582 
             DNA 
             Artificial Sequence 
             
               Optimized sequence encoding HCV E1 protein 
             
           
            6
atgtatgagg tgaggaatgt ctctggcgtc taccatgtga ccaatgactg ctccaactcc    60
tgcattgtct atgaggctgc tgacatgatc atgcacaccc ctggctgtgt gccatgtgtg   120
agggagggca actcctccag gtgctgggtg gccctgaccc ccaccctggc tgccaggaac   180
tcctccatcc ccaccaccac catcaggagg catgtggacc tgctggtggg cgctgctgcc   240
ctgtgctctg ccatgtatgt gggcgacctg tgtggctctg tcttcctggt gtcccagctg   300
ttcaccttct cccccaggag gtatgagact gtgcaggact gcaactgctc cctgtaccct   360
ggccatgtct ctggccacag gatggcctgg gacatgatga tgaactggtc ccccaccact   420
gccctggtgg tctcccagct gctgaggatc ccccaggctg tggtggacat ggtggtgggc   480
gcccactggg gcgtgctggc tggcctggcc tactactcca tggtgggcaa ctgggccaag   540
gtgctgattg tgatgctgct gtttgctggc gtggatggct aa                      582
 
           
             7 
             193 
             PRT 
             Hepatitis C Virus 
           
            7
Met Tyr Glu Val Arg Asn Val Ser Gly Val Tyr His Val Thr Asn Asp
 1               5                  10                  15
Cys Ser Asn Ser Cys Ile Val Tyr Glu Ala Ala Asp Met Ile Met His
            20                  25                  30
Thr Pro Gly Cys Val Pro Cys Val Arg Glu Gly Asn Ser Ser Arg Cys
        35                  40                  45
Trp Val Ala Leu Thr Pro Thr Leu Ala Ala Arg Asn Ser Ser Ile Pro
    50                  55                  60
Thr Thr Thr Ile Arg Arg His Val Asp Leu Leu Val Gly Ala Ala Ala
65                  70                  75                  80
Leu Cys Ser Ala Met Tyr Val Gly Asp Leu Cys Gly Ser Val Phe Leu
                85                  90                  95
Val Ser Gln Leu Phe Thr Phe Ser Pro Arg Arg Tyr Glu Thr Val Gln
            100                 105                 110
Asp Cys Asn Cys Ser Leu Tyr Pro Gly His Val Ser Gly His Arg Met
        115                 120                 125
Ala Trp Asp Met Met Met Asn Trp Ser Pro Thr Thr Ala Leu Val Val
    130                 135                 140
Ser Gln Leu Leu Arg Ile Pro Gln Ala Val Val Asp Met Val Val Gly
145                 150                 155                 160
Ala His Trp Gly Val Leu Ala Gly Leu Ala Tyr Tyr Ser Met Val Gly
                165                 170                 175
Asn Trp Ala Lys Val Leu Ile Val Met Leu Leu Phe Ala Gly Val Asp
            180                 185                 190
Gly
 
           
             8 
             1044 
             DNA 
             Artificial Sequence 
             
               Optimized sequence encoding HCV E2 protein 
             
           
            8
atgaccacct atgtctctgt gggccatgcc tcccagacca ccaggagggt ggcctccttc    60
ttctcccctg gctctgccca gaagatccag ctggtgaaca ccaatggctc ctggcacatc   120
aacaggactg ccctgaattg caacgagtcc atcaacactg gcttctttgc tgccctgttc   180
tatgtgaaga agttcaactc ctctggctgc tctgagagga tggcctcctg caggcccatt   240
gacaggtttg cccagggctg gggccccatc acccatgctg agtccaggtc ctctgaccag   300
aggccatact gctggcacta tgccccccag ccatgtggca ttgtgcctgc cctgcaggtc   360
tgtggccctg tctactgctt caccccatcc cctgtggtgg tgggcaccac tgacaggttt   420
ggcgtgccca cctacaactg gggcgacaat gagactgatg tgctgctgct gaacaacacc   480
aggccccccc agggcaactg gtttggctgc acctggatga actccactgg cttcaccaag   540
acctgtggcg gccccccatg caacattggc ggcgctggca acaacaccct gacctgcccc   600
actgactgct tcaggaagca tcctgaggcc acctacacca agtgtggctc tggcccatgg   660
ctgaccccca ggtgcatggt ggactaccca tacaggctgt ggcactaccc atgcaccttc   720
aacttcacca tcttcaagat caggatgtat gtgggcggcg tggagcacag gctgaatgct   780
gcctgcaact ggaccagggg cgagaggtgc aacattgagg acagggacag gtctgagctg   840
tcccccctgc tgctgtccac cactgagtgg cagatcctgc catgctcctt caccaccctg   900
cctgccctgt ccactggcct gatccatctg catcagaaca ttgtggatgt gcagtacctg   960
tacggcgtgg gctccgctgt ggtctccatt gtgatcaagt gggagtatgt gctgctgctg  1020
ttcctgctgc tggctgatgc ctaa                                         1044
 
           
             9 
             347 
             PRT 
             Hepatitis C Virus 
           
            9
Met Thr Thr Tyr Val Ser Val Gly His Ala Ser Gln Thr Thr Arg Arg
 1               5                  10                  15
Val Ala Ser Phe Phe Ser Pro Gly Ser Ala Gln Lys Ile Gln Leu Val
            20                  25                  30
Asn Thr Asn Gly Ser Trp His Ile Asn Arg Thr Ala Leu Asn Cys Asn
        35                  40                  45
Glu Ser Ile Asn Thr Gly Phe Phe Ala Ala Leu Phe Tyr Val Lys Lys
    50                  55                  60
Phe Asn Ser Ser Gly Cys Ser Glu Arg Met Ala Ser Cys Arg Pro Ile
65                  70                  75                  80
Asp Arg Phe Ala Gln Gly Trp Gly Pro Ile Thr His Ala Glu Ser Arg
                85                  90                  95
Ser Ser Asp Gln Arg Pro Tyr Cys Trp His Tyr Ala Pro Gln Pro Cys
            100                 105                 110
Gly Ile Val Pro Ala Leu Gln Val Cys Gly Pro Val Tyr Cys Phe Thr
        115                 120                 125
Pro Ser Pro Val Val Val Gly Thr Thr Asp Arg Phe Gly Val Pro Thr
    130                 135                 140
Tyr Asn Trp Gly Asp Asn Glu Thr Asp Val Leu Leu Leu Asn Asn Thr
145                 150                 155                 160
Arg Pro Pro Gln Gly Asn Trp Phe Gly Cys Thr Trp Met Asn Ser Thr
                165                 170                 175
Gly Phe Thr Lys Thr Cys Gly Gly Pro Pro Cys Asn Ile Gly Gly Ala
            180                 185                 190
Gly Asn Asn Thr Leu Thr Cys Pro Thr Asp Cys Phe Arg Lys His Pro
        195                 200                 205
Glu Ala Thr Tyr Thr Lys Cys Gly Ser Gly Pro Trp Leu Thr Pro Arg
    210                 215                 220
Cys Met Val Asp Tyr Pro Tyr Arg Leu Trp His Tyr Pro Cys Thr Phe
225                 230                 235                 240
Asn Phe Thr Ile Phe Lys Ile Arg Met Tyr Val Gly Gly Val Glu His
                245                 250                 255
Arg Leu Asn Ala Ala Cys Asn Trp Thr Arg Gly Glu Arg Cys Asn Ile
            260                 265                 270
Glu Asp Arg Asp Arg Ser Glu Leu Ser Pro Leu Leu Leu Ser Thr Thr
        275                 280                 285
Glu Trp Gln Ile Leu Pro Cys Ser Phe Thr Thr Leu Pro Ala Leu Ser
    290                 295                 300
Thr Gly Leu Ile His Leu His Gln Asn Ile Val Asp Val Gln Tyr Leu
305                 310                 315                 320
Tyr Gly Val Gly Ser Ala Val Val Ser Ile Val Ile Lys Trp Glu Tyr
                325                 330                 335
Val Leu Leu Leu Phe Leu Leu Leu Ala Asp Ala
            340                 345
 
           
             10 
             1620 
             DNA 
             Artificial Sequence 
             
               Optimized sequence encoding HCV E1 +
      E2 proteins 
             
           
            10
atgtatgagg tgaggaatgt ctctggcgtc taccatgtga ccaatgactg ctccaactcc    60
tgcattgtct atgaggctgc tgacatgatc atgcacaccc ctggctgtgt gccatgtgtg   120
agggagggca actcctccag gtgctgggtg gccctgaccc ccaccctggc tgccaggaac   180
tcctccatcc ccaccaccac catcaggagg catgtggacc tgctggtggg cgctgctgcc   240
ctgtgctctg ccatgtatgt gggcgacctg tgtggctctg tcttcctggt gtcccagctg   300
ttcaccttct cccccaggag gtatgagact gtgcaggact gcaactgctc cctgtaccct   360
ggccatgtct ctggccacag gatggcctgg gacatgatga tgaactggtc ccccaccact   420
gccctggtgg tctcccagct gctgaggatc ccccaggctg tggtggacat ggtggtgggc   480
gcccactggg gcgtgctggc tggcctggcc tactactcca tggtgggcaa ctgggccaag   540
gtgctgattg tgatgctgct gtttgctggc gtggatggca ccacctatgt ctctgtgggc   600
catgcctccc agaccaccag gagggtggcc tccttcttct cccctggctc tgcccagaag   660
atccagctgg tgaacaccaa tggctcctgg cacatcaaca ggactgccct gaattgcaac   720
gagtccatca acactggctt ctttgctgcc ctgttctatg tgaagaagtt caactcctct   780
ggctgctctg agaggatggc ctcctgcagg cccattgaca ggtttgccca gggctggggc   840
cccatcaccc atgctgagtc caggtcctct gaccagaggc catactgctg gcactatgcc   900
ccccagccat gtggcattgt gcctgccctg caggtctgtg gccctgtcta ctgcttcacc   960
ccatcccctg tggtggtggg caccactgac aggtttggcg tgcccaccta caactggggc  1020
gacaatgaga ctgatgtgct gctgctgaac aacaccaggc ccccccaggg caactggttt  1080
ggctgcacct ggatgaactc cactggcttc accaagacct gtggcggccc cccatgcaac  1140
attggcggcg ctggcaacaa caccctgacc tgccccactg actgcttcag gaagcatcct  1200
gaggccacct acaccaagtg tggctctggc ccatggctga cccccaggtg catggtggac  1260
tacccataca ggctgtggca ctacccatgc accttcaact tcaccatctt caagatcagg  1320
atgtatgtgg gcggcgtgga gcacaggctg aatgctgcct gcaactggac caggggcgag  1380
aggtgcaaca ttgaggacag ggacaggtct gagctgtccc ccctgctgct gtccaccact  1440
gagtggcaga tcctgccatg ctccttcacc accctgcctg ccctgtccac tggcctgatc  1500
catctgcatc agaacattgt ggatgtgcag tacctgtacg gcgtgggctc cgctgtggtc  1560
tccattgtga tcaagtggga gtatgtgctg ctgctgttcc tgctgctggc tgatgcctaa  1620
 
           
             11 
             539 
             PRT 
             Hepatitis C Virus 
           
            11
Met Tyr Glu Val Arg Asn Val Ser Gly Val Tyr His Val Thr Asn Asp
 1               5                  10                  15
Cys Ser Asn Ser Cys Ile Val Tyr Glu Ala Ala Asp Met Ile Met His
            20                  25                  30
Thr Pro Gly Cys Val Pro Cys Val Arg Glu Gly Asn Ser Ser Arg Cys
        35                  40                  45
Trp Val Ala Leu Thr Pro Thr Leu Ala Ala Arg Asn Ser Ser Ile Pro
    50                  55                  60
Thr Thr Thr Ile Arg Arg His Val Asp Leu Leu Val Gly Ala Ala Ala
65                  70                  75                  80
Leu Cys Ser Ala Met Tyr Val Gly Asp Leu Cys Gly Ser Val Phe Leu
                85                  90                  95
Val Ser Gln Leu Phe Thr Phe Ser Pro Arg Arg Tyr Glu Thr Val Gln
            100                 105                 110
Asp Cys Asn Cys Ser Leu Tyr Pro Gly His Val Ser Gly His Arg Met
        115                 120                 125
Ala Trp Asp Met Met Met Asn Trp Ser Pro Thr Thr Ala Leu Val Val
    130                 135                 140
Ser Gln Leu Leu Arg Ile Pro Gln Ala Val Val Asp Met Val Val Gly
145                 150                 155                 160
Ala His Trp Gly Val Leu Ala Gly Leu Ala Tyr Tyr Ser Met Val Gly
                165                 170                 175
Asn Trp Ala Lys Val Leu Ile Val Met Leu Leu Phe Ala Gly Val Asp
            180                 185                 190
Gly Thr Thr Tyr Val Ser Val Gly His Ala Ser Gln Thr Thr Arg Arg
        195                 200                 205
Val Ala Ser Phe Phe Ser Pro Gly Ser Ala Gln Lys Ile Gln Leu Val
    210                 215                 220
Asn Thr Asn Gly Ser Trp His Ile Asn Arg Thr Ala Leu Asn Cys Asn
225                 230                 235                 240
Glu Ser Ile Asn Thr Gly Phe Phe Ala Ala Leu Phe Tyr Val Lys Lys
                245                 250                 255
Phe Asn Ser Ser Gly Cys Ser Glu Arg Met Ala Ser Cys Arg Pro Ile
            260                 265                 270
Asp Arg Phe Ala Gln Gly Trp Gly Pro Ile Thr His Ala Glu Ser Arg
        275                 280                 285
Ser Ser Asp Gln Arg Pro Tyr Cys Trp His Tyr Ala Pro Gln Pro Cys
    290                 295                 300
Gly Ile Val Pro Ala Leu Gln Val Cys Gly Pro Val Tyr Cys Phe Thr
305                 310                 315                 320
Pro Ser Pro Val Val Val Gly Thr Thr Asp Arg Phe Gly Val Pro Thr
                325                 330                 335
Tyr Asn Trp Gly Asp Asn Glu Thr Asp Val Leu Leu Leu Asn Asn Thr
            340                 345                 350
Arg Pro Pro Gln Gly Asn Trp Phe Gly Cys Thr Trp Met Asn Ser Thr
        355                 360                 365
Gly Phe Thr Lys Thr Cys Gly Gly Pro Pro Cys Asn Ile Gly Gly Ala
    370                 375                 380
Gly Asn Asn Thr Leu Thr Cys Pro Thr Asp Cys Phe Arg Lys His Pro
385                 390                 395                 400
Glu Ala Thr Tyr Thr Lys Cys Gly Ser Gly Pro Trp Leu Thr Pro Arg
                405                 410                 415
Cys Met Val Asp Tyr Pro Tyr Arg Leu Trp His Tyr Pro Cys Thr Phe
            420                 425                 430
Asn Phe Thr Ile Phe Lys Ile Arg Met Tyr Val Gly Gly Val Glu His
        435                 440                 445
Arg Leu Asn Ala Ala Cys Asn Trp Thr Arg Gly Glu Arg Cys Asn Ile
    450                 455                 460
Glu Asp Arg Asp Arg Ser Glu Leu Ser Pro Leu Leu Leu Ser Thr Thr
465                 470                 475                 480
Glu Trp Gln Ile Leu Pro Cys Ser Phe Thr Thr Leu Pro Ala Leu Ser
                485                 490                 495
Thr Gly Leu Ile His Leu His Gln Asn Ile Val Asp Val Gln Tyr Leu
            500                 505                 510
Tyr Gly Val Gly Ser Ala Val Val Ser Ile Val Ile Lys Trp Glu Tyr
        515                 520                 525
Val Leu Leu Leu Phe Leu Leu Leu Ala Asp Ala
    530                 535
 
           
             12 
             1350 
             DNA 
             Artificial Sequence 
             
               Optimized sequence encoding HCV NS5a protein 
             
           
            12
atgtctggct cctggctgag ggatgtctgg gactggatct gcactgtgct gactgacttc    60
aagacctggc tgcattccaa gctgctgccc aggctgcctg gcgacccatt cttctcctgc   120
cagaggggct acaggggcgt ctggaggggc gatggcgtga tgcagaccac ctgcccatgt   180
ggcgcccaga tcactggcca tgtgaagaat ggctccatga ggattgtggg ccccaagacc   240
tgctccaaca cctggcatgg caccttcccc atcaatgcct acaccactgg cccatgcacc   300
ccatcccctg cccccaacta ctccagggcc ctgtggaggg tggctgctga ggagtatgtg   360
gaggtgacca gggtgggcga cttccactat gtgactggca tgaccactga caatgtgaag   420
tgcccatgcc aggtgcctgc ccctgagttc ttcactgagg tggatggcgt gaggctgcac   480
aggtatgccc ctgcctgcaa gcccctgctg agggatgagg tgaccttcca ggtgggcctg   540
aaccagttcc ctgtgggctc ccagctgcca tgtgagcctg agcctgatgt gactgtgctg   600
acctccatgc tgactgagcc atcccacatc actgctgaga ctgccaagag gaggctggcc   660
aggggctccc ctccatccct ggcctcctcc tctgcctccc agctgtctgc tccatccctg   720
aaggccacct gcaccaccag gcatgactcc cctgatgctg acctgattga ggccaacctg   780
ctgtggaggc aggagatggg cggcaacatc accagggtgg agtctgagaa caaggtggtg   840
atcctggact cctttgagcc cctgagggct gaggaggatg agagggaggt ctctgtggct   900
gctgagatcc tgaggaagtc caggaagttc ccccctgccc tgcccatctg ggcgaggcca   960
tcctacaacc cacccctgct ggagtcctgg aaggaccctg actatgtgcc ccctgtggtg  1020
catggctgcc ccctgccccc caccatggcc ccacccatcc ccccacccag gaggaagagg  1080
actgtggtgc tgactgagtc cactgtctcc tctgccctgg ctgagctggc caccaagacc  1140
ttcggctcct ctggctcctc tgctgtggac tctggcactg ccacggcccc ccctgaccag  1200
ccatctgatg atggcgacag gggctctgat gatgagtcct actcctccat gccccccctg  1260
gagggcgagc ctggcgaccc tgacctgtct gatggctcct ggtccactgt ctctgaggag  1320
gcctctgagg atgtggcctg ctgctcctaa                                   1350
 
           
             13 
             449 
             PRT 
             Hepatitis C Virus 
           
            13
Met Ser Gly Ser Trp Leu Arg Asp Val Trp Asp Trp Ile Cys Thr Val
 1               5                  10                  15
Leu Thr Asp Phe Lys Thr Trp Leu His Ser Lys Leu Leu Pro Arg Leu
            20                  25                  30
Pro Gly Asp Pro Phe Phe Ser Cys Gln Arg Gly Tyr Arg Gly Val Trp
        35                  40                  45
Arg Gly Asp Gly Val Met Gln Thr Thr Cys Pro Cys Gly Ala Gln Ile
    50                  55                  60
Thr Gly His Val Lys Asn Gly Ser Met Arg Ile Val Gly Pro Lys Thr
65                  70                  75                  80
Cys Ser Asn Thr Trp His Gly Thr Phe Pro Ile Asn Ala Tyr Thr Thr
                85                  90                  95
Gly Pro Cys Thr Pro Ser Pro Ala Pro Asn Tyr Ser Arg Ala Leu Trp
            100                 105                 110
Arg Val Ala Ala Glu Glu Tyr Val Glu Val Thr Arg Val Gly Asp Phe
        115                 120                 125
His Tyr Val Thr Gly Met Thr Thr Asp Asn Val Lys Cys Pro Cys Gln
    130                 135                 140
Val Pro Ala Pro Glu Phe Phe Thr Glu Val Asp Gly Val Arg Leu His
145                 150                 155                 160
Arg Tyr Ala Pro Ala Cys Lys Pro Leu Leu Arg Asp Glu Val Thr Phe
                165                 170                 175
Gln Val Gly Leu Asn Gln Phe Pro Val Gly Ser Gln Leu Pro Cys Glu
            180                 185                 190
Pro Glu Pro Asp Val Thr Val Leu Thr Ser Met Leu Thr Glu Pro Ser
        195                 200                 205
His Ile Thr Ala Glu Thr Ala Lys Arg Arg Leu Ala Arg Gly Ser Pro
    210                 215                 220
Pro Ser Leu Ala Ser Ser Ser Ala Ser Gln Leu Ser Ala Pro Ser Leu
225                 230                 235                 240
Lys Ala Thr Cys Thr Thr Arg His Asp Ser Pro Asp Ala Asp Leu Ile
                245                 250                 255
Glu Ala Asn Leu Leu Trp Arg Gln Glu Met Gly Gly Asn Ile Thr Arg
            260                 265                 270
Val Glu Ser Glu Asn Lys Val Val Ile Leu Asp Ser Phe Glu Pro Leu
        275                 280                 285
Arg Ala Glu Glu Asp Glu Arg Glu Val Ser Val Ala Ala Glu Ile Leu
    290                 295                 300
Arg Lys Ser Arg Lys Phe Pro Pro Ala Leu Pro Ile Trp Ala Arg Pro
305                 310                 315                 320
Ser Tyr Asn Pro Pro Leu Leu Glu Ser Trp Lys Asp Pro Asp Tyr Val
                325                 330                 335
Pro Pro Val Val His Gly Cys Pro Leu Pro Pro Thr Met Ala Pro Pro
            340                 345                 350
Ile Pro Pro Pro Arg Arg Lys Arg Thr Val Val Leu Thr Glu Ser Thr
        355                 360                 365
Val Ser Ser Ala Leu Ala Glu Leu Ala Thr Lys Thr Phe Gly Ser Ser
    370                 375                 380
Gly Ser Ser Ala Val Asp Ser Gly Thr Ala Thr Ala Pro Pro Asp Gln
385                 390                 395                 400
Pro Ser Asp Asp Gly Asp Arg Gly Ser Asp Asp Glu Ser Tyr Ser Ser
                405                 410                 415
Met Pro Pro Leu Glu Gly Glu Pro Gly Asp Pro Asp Leu Ser Asp Gly
            420                 425                 430
Ser Trp Ser Thr Val Ser Glu Glu Ala Ser Glu Asp Val Ala Cys Cys
        435                 440                 445
Ser
 
           
             14 
             1773 
             DNA 
             Artificial Sequence 
             
               Optimized sequence encoding HCV NS5b protein 
             
           
            14
atgtcctaca cctggactgg cgccctgatc accccatgtg ctgctgagga gtccaagctg    60
cccatcaacc ccctgtccaa ctccctgctg aggcatcaca acatggtcta tgccaccacc   120
tccaggtctg ctggcctgag gcagaagaag gtgacctttg acaggctgca tgtgcctgat   180
gaccactaca gggatgtgct gaaggagatg aaggccaagg cctccactgt gaaggcgaag   240
ctgctgtctg tggaggaggc ctgcaagctg acccctcccc actctgccag gtccaagttt   300
ggctatggcg ccaaggatgt gaggaacctg tcctccaagg ctgtgaacca catccactct   360
gtctggaagg acctgctgga ggacactgag acccccattg acaccaccat catggccaag   420
aatgaggtct tctgtgtgca gcctgagaag ggcggcagga agcctgccag gctgattgtc   480
ttccctgagc tgggcgtgag ggtgtgtgag aagatggccc tgtatgatgt ggtctccacc   540
ctgccccagg ctgtgatggg ctcctcctat ggcttccagt actcccctgg ccagagggtg   600
gagttcctgg tgaatgcctg gaagtccaag aagaacccca tgggctttgc ctactgcacc   660
aggtgctttg actccactgt gactgagtct gacatcaggg tggaggagtc catctaccag   720
tgctgtgacc tggctcctga ggccaggcag gtgatcaggt ccctgactga gaggctgtac   780
attggcggcc ccctgaccaa ctccaagggc cagaactgtg gctacaggag gtgcagggcc   840
tctggcgtgc tgaccactaa ctgtggcaac accctgacct gctacctgaa ggcctctgct   900
gcttgcaggg ctgccaagct gcatgactgc accatgctgg tctgtggcga tgacctggtg   960
gtgatctgtg agtctgctgg cacccaggag gatgctgcct ccctgagggt cttcactgag  1020
gccatgacca ggtactctgc cccccctggc gaccctcccc agcctgagta tgacctggag  1080
ctgatcacct cctgctcctc caatgtctct gtggcccatg atgcctctgg caagagggtc  1140
tactacctga ccagggaccc caccaccccc ctggccaggg ctgcctggga gactgccagg  1200
cacacccctg tgaactcctg gctgggcaac atcatcatgt atgcccccac cctgtgggcc  1260
aggatgatcc tgatgaccca cttcttctcc atcctgctgg cccaggagca gctggagaag  1320
gccctgggct gccagattta tggcgccacc tacttcattg agcccctgga cctgccccag  1380
atcatccaga ggctgcatgg cctgtctgcc ttctccctgc actcctactc ccctggcgag  1440
atcaacaggg tggcctcctg cctgaggaag ctgggcgtgc cccccctgag ggtgtggagg  1500
cacagggcca ggtctgtgag ggccaagctg ctgtcccagg gcggcagggc tgccacctgt  1560
ggcaagtacc tgttcaactg ggctgtgagg accaagctga agctgacccc catccctgct  1620
gcctcccagc tggacctgtc tggctggttt gtggctggct actctggcgg cgacatctac  1680
cactccctgt ccagggccag gcccaggtgg ttcatgtggt gcctgctgct gctgtctgtg  1740
ggcgtgggca tctacctgct gcccaacagg tga                               1773
 
           
             15 
             590 
             PRT 
             Hepatitis C Virus 
           
            15
Met Ser Tyr Thr Trp Thr Gly Ala Leu Ile Thr Pro Cys Ala Ala Glu
 1               5                  10                  15
Glu Ser Lys Leu Pro Ile Asn Pro Leu Ser Asn Ser Leu Leu Arg His
            20                  25                  30
His Asn Met Val Tyr Ala Thr Thr Ser Arg Ser Ala Gly Leu Arg Gln
        35                  40                  45
Lys Lys Val Thr Phe Asp Arg Leu His Val Pro Asp Asp His Tyr Arg
    50                  55                  60
Asp Val Leu Lys Glu Met Lys Ala Lys Ala Ser Thr Val Lys Ala Lys
65                  70                  75                  80
Leu Leu Ser Val Glu Glu Ala Cys Lys Leu Thr Pro Pro His Ser Ala
                85                  90                  95
Arg Ser Lys Phe Gly Tyr Gly Ala Lys Asp Val Arg Asn Leu Ser Ser
            100                 105                 110
Lys Ala Val Asn His Ile His Ser Val Trp Lys Asp Leu Leu Glu Asp
        115                 120                 125
Thr Glu Thr Pro Ile Asp Thr Thr Ile Met Ala Lys Asn Glu Val Phe
    130                 135                 140
Cys Val Gln Pro Glu Lys Gly Gly Arg Lys Pro Ala Arg Leu Ile Val
145                 150                 155                 160
Phe Pro Glu Leu Gly Val Arg Val Cys Glu Lys Met Ala Leu Tyr Asp
                165                 170                 175
Val Val Ser Thr Leu Pro Gln Ala Val Met Gly Ser Ser Tyr Gly Phe
            180                 185                 190
Gln Tyr Ser Pro Gly Gln Arg Val Glu Phe Leu Val Asn Ala Trp Lys
        195                 200                 205
Ser Lys Lys Asn Pro Met Gly Phe Ala Tyr Cys Thr Arg Cys Phe Asp
    210                 215                 220
Ser Thr Val Thr Glu Ser Asp Ile Arg Val Glu Glu Ser Ile Tyr Gln
225                 230                 235                 240
Cys Cys Asp Leu Ala Pro Glu Ala Arg Gln Val Ile Arg Ser Leu Thr
                245                 250                 255
Glu Arg Leu Tyr Ile Gly Gly Pro Leu Thr Asn Ser Lys Gly Gln Asn
            260                 265                 270
Cys Gly Tyr Arg Arg Cys Arg Ala Ser Gly Val Leu Thr Thr Asn Cys
        275                 280                 285
Gly Asn Thr Leu Thr Cys Tyr Leu Lys Ala Ser Ala Ala Cys Arg Ala
    290                 295                 300
Ala Lys Leu His Asp Cys Thr Met Leu Val Cys Gly Asp Asp Leu Val
305                 310                 315                 320
Val Ile Cys Glu Ser Ala Gly Thr Gln Glu Asp Ala Ala Ser Leu Arg
                325                 330                 335
Val Phe Thr Glu Ala Met Thr Arg Tyr Ser Ala Pro Pro Gly Asp Pro
            340                 345                 350
Pro Gln Pro Glu Tyr Asp Leu Glu Leu Ile Thr Ser Cys Ser Ser Asn
        355                 360                 365
Val Ser Val Ala His Asp Ala Ser Gly Lys Arg Val Tyr Tyr Leu Thr
    370                 375                 380
Arg Asp Pro Thr Thr Pro Leu Ala Arg Ala Ala Trp Glu Thr Ala Arg
385                 390                 395                 400
His Thr Pro Val Asn Ser Trp Leu Gly Asn Ile Ile Met Tyr Ala Pro
                405                 410                 415
Thr Leu Trp Ala Arg Met Ile Leu Met Thr His Phe Phe Ser Ile Leu
            420                 425                 430
Leu Ala Gln Glu Gln Leu Glu Lys Ala Leu Gly Cys Gln Ile Tyr Gly
        435                 440                 445
Ala Thr Tyr Phe Ile Glu Pro Leu Asp Leu Pro Gln Ile Ile Gln Arg
    450                 455                 460
Leu His Gly Leu Ser Ala Phe Ser Leu His Ser Tyr Ser Pro Gly Glu
465                 470                 475                 480
Ile Asn Arg Val Ala Ser Cys Leu Arg Lys Leu Gly Val Pro Pro Leu
                485                 490                 495
Arg Val Trp Arg His Arg Ala Arg Ser Val Arg Ala Lys Leu Leu Ser
            500                 505                 510
Gln Gly Gly Arg Ala Ala Thr Cys Gly Lys Tyr Leu Phe Asn Trp Ala
        515                 520                 525
Val Arg Thr Lys Leu Lys Leu Thr Pro Ile Pro Ala Ala Ser Gln Leu
    530                 535                 540
Asp Leu Ser Gly Trp Phe Val Ala Gly Tyr Ser Gly Gly Asp Ile Tyr
545                 550                 555                 560
His Ser Leu Ser Arg Ala Arg Pro Arg Trp Phe Met Trp Cys Leu Leu
                565                 570                 575
Leu Leu Ser Val Gly Val Gly Ile Tyr Leu Leu Pro Asn Arg
            580                 585                 590
 
           
             16 
             103 
             DNA 
             Artificial Sequence 
             
               Modified Vector Sequence 
             
           
            16
cttaagcgaa ggttactctt gtacctctgg tacttggtcg ggatggtgta gacggcgccg    60
aagtggacga agttcttcat ttgggccctt aagatttcag ctg                     103
 
           
             17 
             15 
             DNA 
             Artificial Sequence 
             
               Modified Vector Sequence 
             
           
            17
agatctacca tgagc                                                     15
 
           
             18 
             15 
             DNA 
             Artificial Sequence 
             
               Modified Vector Sequence 
             
           
            18
gccgaattcg cttcc                                                     15
 
           
             19 
             25 
             DNA 
             Artificial Sequence 
             
               Modified Vector Sequence 
             
           
            19
taaacccggg aattctaaag tcgac                                          25
 
           
             20 
             12 
             DNA 
             Artificial Sequence 
             
               Modified Vector Sequence 
             
           
            20
atcaccatgg at                                                        12
 
           
             21 
             15 
             DNA 
             Artificial Sequence 
             
               Modified Vector Sequence 
             
           
            21
gagatcttca tgagc                                                     15
 
           
             22 
             15 
             DNA 
             Artificial Sequence 
             
               Modified Vector Sequence 
             
           
            22
agatccacca tgcag                                                     15
 
           
             23 
             18 
             DNA 
             Artificial Sequence 
             
               Modified Vector Sequence 
             
           
            23
ggtgcagatc tgatgagc                                                  18
 
           
             24 
             13 
             DNA 
             Artificial Sequence 
             
               Modified Vector Sequence 
             
           
            24
gcctaaagtc gac                                                       13
 
           
             25 
             4261 
             DNA 
             Artificial Sequence 
             
               Modified Vector Sequence 
             
           
            25
gatattggct attggccatt gcatacgttg tatccatatc ataatatgta catttatatt    60
ggctcatgtc caacattacc gccatgttga cattgattat tgactagtta ttaatagtaa   120
tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg   180
gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg   240
tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta   300
cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt   360
gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac   420
tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt   480
tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac   540
cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt   600
cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat   660
ataagcagag ctcgtttagt gaaccgtcag atcgcctgga gacgccatcc acgctgtttt   720
gacctccata gaagacaccg ggaccgatcc agcctccgcg gccgggaacg gtgcattgga   780
acgcggattc cccgtgccaa gagtgacgta agtaccgcct atagagtcta taggcccacc   840
cccttggctt cttatgcatg ctatactgtt tttggcttgg ggtctataca cccccgcttc   900
ctcatgttat aggtgatggt atagcttagc ctataggtgt gggttattga ccattattga   960
ccactcccct attggtgacg atactttcca ttactaatcc ataacatggc tctttgccac  1020
aactctcttt attggctata tgccaataca ctgtccttca gagactgaca cggactctgt  1080
atttttacag gatggggtct catttattat ttacaaattc acatatacaa caccaccgtc  1140
cccagtgccc gcagttttta ttaaacataa cgtgggatct ccacgcgaat ctcgggtacg  1200
tgttccggac atgggctctt ctccggtagc ggcggagctt ctacatccga gccctgctcc  1260
catgcctcca gcgactcatg gtcgctcggc agctccttgc tcctaacagt ggaggccaga  1320
cttaggcaca gcacgatgcc caccaccacc agtgtgccgc acaaggccgt ggcggtaggg  1380
tatgtgtctg aaaatgagct cggggagcgg gcttgcaccg ctgacgcatt tggaagactt  1440
aaggcagcgg cagaagaaga tgcaggcagc tgagttgttg tgttctgata agagtcagag  1500
gtaactcccg ttgcggtgct gttaacggtg gagggcagtg tagtctgagc agtactcgtt  1560
gctgccgcgc gcgccaccag acataatagc tgacagacta acagactgtt cctttccatg  1620
ggtcttttct gcagtcaccg tccttagatc taccatgagc accaacccca agccccagag  1680
gaagaccaag aggaacacca acaggaggcc ccaggatgtg aagttccctg ggggaggcca  1740
gattgtggga ggggtctacc tgctgcccag gaggggcccc aggctggggg tgagggctac  1800
caggaagacc tctgagaggt cccagcccag gggcaggagg cagcccatcc ccaaggccag  1860
gaggcctgag ggccgctcct gggcccagcc tggctacccc tggcccctgt atggcaatga  1920
aggctttggc tgggctggct ggctgctgtc ccccaggggc tccaggccct cctggggccc  1980
cacagacccc aggaggaggt ccaggaacct gggcaaggtg attgacaccc tgacctgtgg  2040
ctttgctgac ctgatgggct acatccccct ggtgggggct cctgtgggag gggtggctag  2100
ggctctggct catggggtga gggtgctgga ggatggggtg aactatgcta ctggcaacct  2160
gcctggctgc tccttctcca tcttcctgct ggccctgctc tcctgcctga cagtgcctgc  2220
ttctgccgaa ttcgcttcca atgagaacat ggagaccatg aaccagccct accacatctg  2280
ccgcggcttc acctgcttca agaagtaaac ccgggaattc taaagtcgac agcggccgcg  2340
atctgctgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt  2400
gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca  2460
ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcagcaca gcaaggggga  2520
ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg gtacggccgc  2580
agcggcctta attaaggccg cagcggccgt acccaggtgc tgaagaattg acccggttcc  2640
tcgacccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag  2700
catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac  2760
caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc  2820
ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcaatg ctcacgctgt  2880
aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc  2940
gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga  3000
cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta  3060
ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta  3120
tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga  3180
tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg  3240
cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacgtgatc ccgtaatgct  3300
ctgccagtgt tacaaccaat taaccaattc tgattagaaa aactcatcga gcatcaaatg  3360
aaactgcaat ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg  3420
taatgaagga gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc  3480
tgcgattccg actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag  3540
gttatcaagt gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagctt  3600
atgcatttct ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact  3660
cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc  3720
gctgttaaaa ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag  3780
cgcatcaaca atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt  3840
cccggggatc gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat  3900
ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc  3960
attggcaacg ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata  4020
caatcgatag attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata  4080
taaatcagca tccatgttgg aatttaatcg cggcctcgag caagacgttt cccgttgaat  4140
atggctcata acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga  4200
tgatatattt ttatcttgtg caatgtaaca tcagagattt tgagacacaa cgtggctttc  4260
c                                                                  4261