TW200408708A - Internal ribosome entry sites for recombinant protein expression - Google Patents

Abstract

The invention describes compositions and methods for recombinant protein expression in a wide range of cell types, including mammalian, insect, and bacterial cells. The compositions comprise a viral IRES sequence selected from enterovirus 71 (EV71), hepatitis C virus (HCV), or encephalomyocarditis virus (EMCV), or a variant or fragment thereof, or alternatively, a homolog of a viral IRES selected from EV71, HCV, or EMCV, or a variant or fragment thereof. Methods of using the compositions are also described.

Description

200408708 发明, Description of the invention: [Technical field to which the invention belongs] Glycosome binding site. In particular, the present invention relates to the 5, untranslated regions of the viral gene (regions [5'UTRs]), which can serve as internal ribosome entry sites (IRES). The invention relates to the encephalomyocarditis virus (EMCV). Hepatitis C virus

A method for expressing IRES in a recombinant protein system; a composition comprising various RES; and a method for screening antiviral compounds using the IRES of the present invention. [Prior art] The eukaryotic mRN A has a unique structural feature at its 5 'end, called the 5' cap (5 cap), and is a 7-methylguanosine residue via 5, _triscarnic acid Linked to the 5 'terminal residue of the mRNA. Cap_dependent translation first started with the cap-binding protein complex e 丨 b, _4 F bound to the 5 'cap, which can accelerate the binding of the nuclear bran ternary unit 43 s to the 5 · cap region On or in the vicinity of the 5 'cap region, this ribosome complex will scan the mRNA from the 5 cap, until it encounters the start codon AUG, and from then on the mRNA translation (see 1 <: 〇2 & amp 1 <, ^, (1989) £ 1144: 283-292; Kozak, M (1 989) J. Cell. Biol. 1 08: 229-241). A cap-independent translation mechanism has been proposed to explain some highly efficient viral mRN A translations, although a 5 'untranslated region of its mRN A has been predicted to interfere with ribose 200408708 body in mRNA. Scanned height dimension structure. Picornavirus (Picornavirus > mRNA is the first mRNA confirmed to show a cap-independent mechanism (see jacksOn, RJ, (1 988) with "3 3 4 292_ 293)" Small RNA The viral mRNA has structural features including a 5 untranslated region lacking a 5, cap, a special structure, and multiple upstream aug start codons. The 5 untranslated region of this special structure was found to be used as an internal ribosome , Σ-position (IR ES) ', or ribosome take-off and landing field (rib 〇s 〇me 1 anding pad), which can be combined with the ribosome ternary unit 43 $ without the 5' cap structure to start translation The 5 'untranslated region containing IRES usually has three characteristics: a long untranslated region, a stable primary structure, and a potential upstream start codon AUG, where the stable secondary structure is considered to be Deciding on the most important factor in function. A small number of vertebrates have long, highly structured 5, untranslated regions containing multiple start codons AUG. Among these mRNAs, the Drosophila Antp gene has a segment 1 73 5 bases in length 5 The translation region and 15 upstream AUG start codons, and the Ubx gene has a 968 base 5 'untranslated region and 2 upstream AUG start codons. So far, it has been in cells between different species. mRNA finds more iRES, these different

Species include humans (see Macajak, DG · and P. Sarnow, (1991) Nature 3 53:65 3-656; Sarnow, P? (1 989) PNAS 86: 5795-5799; Vagner, S. et al., ( 1 995) Mol. Cell. Biol. 15: 35-44) and yeast (see Zhou, W. et al., (2001;) PNAS 98: 1 53 1-1 536; Paz, I. et al. (1,999) J. Biol. Chem. 274: 2 174b 21 745). IRES was first identified in the virus's mRNA in 200408708, such as poliovirus (see Pe 11 etier, J. and N. Sonenberg · (1988) Nature 334: 320-325), encephalomyocarditis virus ( encephalomyocarditis virus [EMCV]) (see Jang, SK, and E. Wimmer, (1 990) Genes Dev. 4: 1560-1572), and human rhinovirus (HRV)) (see Borman, A. et al "(1993) J. Gen. Virol. 74: 1 775-1788). The Antp and Ubx alleles of Drosophila are also translated by their IRES in their long untranslated regions (see Ye X. et al (1997) Mol. Cell. Biol. 17: 1714-1721; Ho, S.-K. et al., (1992) Genes Dev. 6: 1643-1653. [Summary of the Invention] The present invention provides The internal ribosome binding site (i R ε s) obtained from the 5 'untranslated region of the enterovirus 71 (EV71 I) gene. Enterovirus belongs to the family Picoraviridae and enterovirus 71 is an intestine A member of the genus Virus (see Fields, bn, et ai., Eds., (3rd ed. 1 996) Fundamental Virology, Lippin co 11-Rave n. Philadelphia, PA, p. 477-522), enterovirus 7} type 1 {1 £ 8 activity was compared to IREs of encephalomyocarditis virus and hepatitis c virus. All of these viruses IRES can guide cap-independent translation of mRNA from different cell types, including human, worm, and bacterial cells. Therefore, viral iRKS is useful for nucleic acid vectors because it can guide two The expression of one or more irrelevant proteins. According to the traditional method, to express a recombinant protein in a cell, this protein 5 200408708 gene is placed under the control of a promoter, which provides the RN A polymerase binding required for mRN A synthesis. Position. When two or more recombinant proteins are to be expressed in a cell, each protein gene can be placed under the control of its own promoter in a single nucleic acid vector; in addition, each protein can also be controlled by a separate nucleic acid vector. which performed. Regardless of which method is used, each protein will produce an individual mRNA transcript, and translation of different mRNA transcripts will often lead to non-cooperative performance between different proteins. Putting multiple proteins under the control of the same promoter, the first gene closest to the 5 'cap will be most efficiently translated, presumably due to the cap-dependent mechanism, and the downstream gene, which The level of translation may be extremely low or not translated at all. However, when an IRES is embedded in a nucleic acid vector and is located between the gene closest to the 5 'cap and its downstream genes, there may be two or more proteins that can be efficiently translated in a single transcript. Polycistronic vectors are nucleic acid vectors in which more than one protein is represented by a single vector. In a polycistronic vector, a nucleotide sequence containing at least two cistrons or genes is placed under the control of a single promoter to synthesize mRN A and is between the dicistronics Embed an IRES; produce a single mRNA transcript that includes the first cistron, IRES, and another downstream cistron sequence, rather than individual mRNA transcripts as traditional methods do. During translation, the first cistron is translated by the ribosome scanning mechanism because it is closest to the 5 'cap, while the second cistron and other downstream cistrons are internal ribose The body is translated into IRES. The results show that the mRNA for multiple cistron expression can maintain a constant ratio; the main advantage of this technology is that a single mRNA can simultaneously show two or more proteins, which can avoid the use of proteins that cause non-cooperative expression between proteins. Individual expression constructs plastids and multiple promoters. The virus IRES disclosed in the present invention can guide the aforementioned cap-independent translation and can be applied to a wide range of cell types including mammalian, insect, and bacterial cells. This is quite advantageous, because the baculovirus expression system is widely used to mass-produce recombinant proteins, and many biologically active proteins have been mass-produced using the baculovirus expression system (for a comprehensive review, see the following: Miller, LK ,, ( (1988) Annu. Rev. Microbiol. 42: 177-199; Lucko w VA and MD Summers, (19 8 8)

Bio / T echnology 6: 47-5 5; Luckow V. A., (1 990) In:

Recombinant DNA Technology and Applications. McGraw-Hill, New York, pp. 97- 1 52; Oeilly, D.R., et al., (1992) Baculovirus Nucleic Acid Vectors: A Laboratory Manual. W.H. Freedman, New York). In the baculovirus expression system, the polyhedrin gene of a baculovirus is usually replaced with a gene encoding a protein of interest. The polyhedrin gene will be highly expressed in infected insects, but it is not It is necessary for the virus to reproduce, so it is an ideal location for the gene of interest. This baculovirus gene will be placed in a separate vector and under the control of a strong polyhedrin promoter or other baculovirus promoter. This vector will be co-transfected with the baculovirus genome into the baculovirus In the host cell, homologous recombination occurs between the vector and the baculovirus genome to produce recombinant baculoviruses that carry the protein of interest. These recombinant baculoviruses will be used to infect host cells to mass produce the protein we want . 7 200408708 Despite the attractiveness of the baculovirus expression system, the IRES of other viruses have not been shown to activate in baculovirus host cells. Therefore, although the IRES molecule of encephalomyocarditis virus has been shown to be highly effective in mammalian systems, published literature has reported that it has failed to promote effective internal translation in baculovirus host cells, presumably because of insect cells There are no cell translation initiation factors required to initiate internal translation in mammalian cells (see Finke 1 stei η Y., Eta 1., (1 999) J. Biotech. 75: 3 3-44 ). Contrary to the above publication, the inventors found that the IRES molecule of encephalomyocarditis virus can function in baculovirus host cells, and the inventors also found that other IR ES that can function in baculovirus host cells can also Acts on other types of cells including mammalian cells and bacterial cells. Therefore, the present invention provides a set that can express recombinant proteins in bacterial, insect and or mammalian cells. The set contains at least one A nucleic acid vector comprising at least one IRES sequence capable of acting in a bacterial cell; at least one nucleic acid vector comprising at least one IRES sequence capable of acting in an insect cell; and at least one nucleic acid vector comprising at least one capable of acting in a mammal The IRES sequence acting in the cell. The invention also provides homology sequences, variant sequences and fragments of IRES of encephalomyocarditis virus, hepatitis C virus and enterovirus type 71, and homology of IRES of encephalomyocarditis virus, hepatitis C virus and enterovirus type 71 Variant sequences and fragments of homologous sequences. The present invention further provides a polycistronic nucleic acid vector, which contains the virus IRES disclosed in the present invention or the homologous sequences, variant sequences and fragments of the virus IRES described above, and can produce multiple recombinations from a single 200408708 mRNA transcript. protein. These polycistronic nucleic acid vectors may be contained in biological vectors capable of expressing multiple genes in host cells. These nucleic acid vectors and biological vectors can be used in gene therapy of patients, and / or recombinant proteins produced thereby. Can be used as a useful therapeutic agent. The present invention also provides a baculovirus vector and a recombinant baculovirus capable of expressing at least two genes in a baculovirus host cell, comprising the virus IRES with IRES activity disclosed in the present invention, or the homologous sequence of the aforementioned virus IRES , Variant sequences and fragments. The ability to express two or more genes from a single baculovirus vector and a recombinant baculovirus simplifies the process of isolating plaques that show genes of interest; moreover, the simultaneous expression of genes of interest and reporter genes will allow Assess the extent of recombinant protein production at the same time as detect / isolate cells producing recombinant protein. The present invention further provides a method for screening antiviral compounds that can disrupt cap-independent translation of viral IRES. This method involves transfecting a nucleic acid vector into a cell, which vector will guide the cap-independent translation of the recombinant protein, and then contacting the test compound with the transfected cell and comparing the transfected cell without the test compound to detect recombination. Changes in protein content. [Embodiment] The present invention provides a nucleotide sequence or c D N A isolated from one of the internal ribosome binding sites (IR E S) of an enterovirus type 71 (EV71) 5 'untranslated region. Enterovirus 71 type 5, untranslated region (5'UTR) has approximately 700 nucleotides. Taking the 5 'untranslated region of enterovirus 71 type (virus strain TW / 2086/98) as an example, the sequence is named SEQ ID: 1'. See FIG. The nucleotide gamma acid sequence or cDNa of the isolation 200408708 of the present invention can be isolated by any conventional technique, for example: it can be replicated by using appropriate probes; by polymerase chain reaction (PCR); or by chemistry Synthesized. As shown below, the 5 'untranslated region of the enterovirus type 71 gene can exhibit IRES activity. IRES of other viruses are well known in this technical field, for example, IRES of encephalomyocarditis virus (EMCV) has been disclosed in jarig, sK, and E. Wimmer, (1990) Genes Dev. 4: 1560-1572; hepatitis C virus (Hepatitis C virus (HCV)) has an IRES of about 3 32 or 34i nucleotides, depending on the specific virus strain (see Tsukiyama-Kohara K., et al., (1 992) J. Virol. 6 6: 1476-148 3; Buratti E., et al.? (1 997) FEES Lett. 41 1: 275-280). As used herein, "IRES activity" refers to the ribosome's translation function using the IRES sequence "Cap-dependent translation effect" refers to the ribosome unit necessary for initiating the translation effect, which will be combined with the 5, or cap region near mRN A, the translation mechanism of the cap region. The "ribosomal scanning" mechanism continues, and the ribosome complex scans the mRNA from the 5 'cap until it encounters the start codon AUG. "--Independent translation" refers to the ribosome necessary to initiate translation The unit will be combined at a certain position on the mRN A and does not require 5, cap Translation mechanism of domains. "IRES" as used herein is a nucleotide sequence that can provide ribosome binding sites for cap-independent translation. The present invention also relates to including encephalomyocarditis virus and hepatitis C virus. And intestinal diseases, type 1 IR e S homology sequence (h 0 m ο 1 0 g), variant sequence (variant) or fragment. "Homologous" as used herein refers to different organisms The internal structure or 10 200408708 showed basic similarities in action. Encephalomyocarditis virus, c-hepatitis virus and enterovirus type 7 IRES homologous sequences may individually have encephalomyocarditis virus, hepatitis C virus and enterovirus 7 Type 1 IRES has similar primary or secondary structures and / or IRES activity. Secondary structures can be predicted by computer programs, such as the Zuker's RNA folding program (see Zuker, M., (1 989) Methods Enzymol 08: 262-2 8 8). The present invention also includes variant sequences of homologous sequences of IRES of encephalomyocarditis virus, hepatitis C virus, and enterovirus type 71 or fragments of homologous sequences. Brain used herein Myocarditis The "variant sequence" of IRES of viruses, hepatitis C virus, and enterovirus type 7 1 refers to a naturally occurring or chemically synthesized nucleotide, and the individual sequences are roughly the same as those of encephalomyocarditis virus, hepatitis C virus, and intestine. Virus 71 has the same IRES, but because of deletion, substitution or insertion of one or more nucleotides, there is a nucleotide sequence different from that of encephalomyocarditis virus, hepatitis C virus and enterovirus 71 type IRES. . Variant sequences of IR ES of encephalomyocarditis virus, hepatitis C virus, and enterovirus 71 type 1 still retain IRES activity, or have stronger IRES activity than encephalomyocarditis virus, hepatitis C virus, and enterovirus 71 type IRES . As used herein, a "fragment" of an IRES of encephalomyocarditis virus, hepatitis C virus, and enterovirus type 7 1 refers to a portion of an IRES nucleotide sequence that contains fewer nucleotide sequences than the entire IRES sequence, and IRES activity that is substantially equal to or greater than the entire IRES nucleotide sequence is maintained. The sequence "similarity" and / or "identity" refers to the degree of correlation between two polynucleotides or peptides. Generally speaking, "identity" means that two or more nucleotide sequences or two or more 200408708 paragraphs or multiple amino acid sequences exactly match, in which the same nucleotide or amino acid is compared. "Similarity" also means that two or more nucleotide sequences or two or more amino acid sequences exactly match, in which the same nucleotides or amino acids are compared, or they have similarities. Chemical and / or physical nucleotides or amino acids. The percentage ratio of identity or similarity can be determined by a program such as' using GAP computer program version 6 to compare information between sequences (see Devereux et al. A ^ c /. Dc / Heart 7? &Amp; v · 12: 387 , 1 984, available at the University of Wisconsin Genetics Computer Group [UWGCG]); the GAP computer program uses the positioning method of Needleman and Wunsch (see J. 5/0 /. 48: 443, 1 970), and then through Smith and Modified by Waterman (see / Uv. AppL Math 2: 482, 1 98 1). Other programs for calculating the consistency and similarity between two sequences are known techniques. For the purpose of the invention, the homologous sequence, variant sequence or fragment of IRES of enterovirus type 71, hepatitis C virus, and brain to myositis virus is different from that of enterovirus type 71, hepatitis C virus, and encephalomyocarditis virus, respectively. The ire S alignment must show at least 20% nucleotide identity, at least 30% nucleotide identity, or at least 40 ° / 〇 nucleotide identity; although the present invention undoubtedly includes Res of enterovirus type 71, hepatitis C virus, and encephalomyocarditis virus showed at least 50 °. , 60%, 70%, 80%, and 90% of the nucleotide sequence identity. No, the Θ source sequence, variant sequence or fragment of the IR E s of enterovirus 71, hepatitis C virus, and encephalomyocarditis virus are respectively the same as those of enterovirus 71, hepatitis C relief, and encephalomyocarditis virus. , Showing the nucleotide sequence similarity range_ from at least 50%, 60%, 70%, 80% to at least 90% of the nucleotide sequence 柞 倬 12 200408708. Similarly, enterovirus type 71, hepatitis c virus, and encephalomyocarditis virus I: ES variant sequence or homologous sequence of κ and enterovirus 71, hepatitis C virus, and encephalomyocarditis virus IREs, respectively Compared with the homologous sequence, the nucleotide identity is at least 20%, and the identity is increased by 0% successively, until the nucleotide identity is at least 90%; Compared with the homologous sequences of hepatitis C virus and encephalomyocarditis virus iRes, the nucleotide similarity is at least 50%, and the similarity is increased by 10% successively, up to at least 90. /. Nucleotide similarity so far. Naturally occurring homologous sequences, variant sequences and fragments are included in the present invention. Homologous myocarditis virus, hepatitis C virus and enterovirus 71 type 1 r £ s homologous sequences, variant sequences or fragments can be obtained from mutant encephalomyocarditis virus, hepatitis C virus and enterovirus 71 IRES nuclear gamma sequences . The following techniques are routine work in this field: mutations are introduced into specific positions by synthesizing nucleotides containing mutant genes, which are flanked by restriction enzyme cuts in order to be able to join the gene fragment to the original sequence. After conjugation, the resulting reconstructed sequence contains the nucleotides of the desired insertion, substitution, or deletion; see Molecular Cloning-Laboratory Manuals, Volumes 1 to 3 by Sambrook et al. (Molecular Cloning · A Laboratory Manual (Second Edition, 1989)), Cold Spring Harbor Press, Cold Spring Harbor, New York. In addition, oligonucleotide-directed site-specific mutagenesis (oligonucleotide-directed site-specific mutagenesis) programs can be used to provide a modified nucleotide sequence that can be changed to Predetermined sequence. The methods mentioned above as examples of manufacturing changes are conventional techniques (see Walder RY et al., (1 986) Gew 13 200408708 42: 1 3 3-1 3 9; Bauer CE, et al., (1 985) Gene 37:73 -81; Craik CS, (Jan · 1 985) BioTechniques, 12-19; Smith et al., (1981) Genetic Engineering: Principles and Methods, Plenum Press; Kunkel TA, (19 8 5) Proc Nail. A cad. Sci. USA 82: 488-492; Kunkel TA, et al., (1 987) Methods in / 7 mo /. 154: 367-382; US Patent Nos. 4,518,584 and 4,737,462 ; All of the above are incorporated for reference). Other known methods can also be used. IRES activity can be determined by its ability to independently translate mRNA, which is not related to the mRNA and the vesicular region. Several reports support the hypothesis that IRES activity is different depending on the cell type (see 0.11 to 8., 61 & 1., (2000); \ ^ / · Cell. Biol. 20: 275 5-2759; Stoneley M., et al., (1998) Oncogene 16: 423-428; Pozner A.? Et al., (2000) Mol. Cell · 5h / · 20: 2297-23 07), these reports propose IRES active lines It is different due to the reaction with specific protein factors in different cells. The IRES of the encephalomyocarditis virus, hepatitis C virus and enterovirus type 71 of the present invention or a homologous sequence, variant sequence or fragment thereof can guide the independent translation of caps of different cell types including mammalian cells, bacteria and insect cells. The IRES of the encephalomyocarditis virus, hepatitis C virus, and enterovirus type 71 of the present invention or a homologous sequence, variant sequence or fragment thereof also has IRES activity in other eukaryotic cells such as yeasts and plant cells. The invention further includes DNA constructs, such as plastids and recombinant expression vectors, comprising IRES of encephalomyocarditis virus, hepatitis C virus and enterovirus 71, or homologous sequences, variant sequences or fragments thereof. In a recombinant expression vector, 14 200408708 IRES of encephalomyocarditis virus, hepatitis C virus and enterovirus type 71, or a homologous sequence, variant sequence or fragment thereof, can guide the expression of at least one recombinant protein. The construction and expression of traditional recombinant nucleic acid vectors are conventional techniques. For details, please refer to Molecular Cloning: A Laboratory Manual (Second Edition, 1 9) by Sambro Ok et al. 8 9)), Cold Spring Harbor Press, Cold Spring Harbor, New York. Such nucleic acid vectors can be contained in biological vectors such as viruses and bacteria, preferably in non-pathogenic or less toxic microorganisms, including attenuated viruses, bacteria, parasites and virus-like particles . In the context of the present invention, the IRES of encephalomyocarditis virus, hepatitis C virus, and enterovirus type 71, or the nucleotide sequence of a homologous sequence, variant sequence or fragment thereof, is placed in a nucleic acid vector in a gene of interest or cis The upstream of the aposton is used to guide the independent translation of the performance product. Variant sequences of homologous sequences of IRES of encephalomyocarditis virus, hepatitis C virus and enterovirus 71 or fragments of homologous sequences can also be used. This nucleic acid vector can be in the form of a monocistronic (monocistronic, under the control of a promoter for mRNA production, showing a single gene of interest) or a multicistronic form (multicistronic, for the same promoter for mRN A generation) Under control, showing at least two genes of interest). Such a nucleic acid vector may include several "IRES-cistronic" molecules arranged in tandem, and at least one of the IRES positions contains the IRES of the encephalomyocarditis virus, hepatitis C virus, and enterovirus type 71 or a homologous sequence thereof. , A variant sequence or fragment, or a nucleotide sequence of a variant sequence or a fragment of a homologous sequence of a homologous sequence of IR ES of encephalomyocarditis virus, hepatitis C virus and enterovirus type 71. 15 200408708 The nucleic acid vector of the present invention comprises a nucleotide depleted acid sequence linked to and acting on a promoter, which comprises an encephalomyocarditis virus, hepatitis C virus and enterovirus 71 type IRES linked to at least one cistron Or a homologous sequence, a variant sequence or a fragment thereof, or a nucleotide sequence of a variant sequence or a fragment of a homologous sequence of an IRES homologous sequence of encephalomyocarditis virus, hepatitis c virus, and enterovirus type 71 type I. The promoter is necessary for the synthesis of mRN A by the DNA sequence, and usually eukaryotic mRN A has a 5 'cap. As used herein, "cistron" refers to the polynucleic acid sequence or gene of the protein, peptide or peptide of interest. "Operably linked" means that the elements are in a spatial relationship that allows them to function in a predetermined manner. For example, a cistron and a promoter are “connected to and function with” a promoter, meaning that the junction between the two is such that translation of the cistron must be achieved in a promoter-compatible environment. Similarly, a nucleotide sequence of IR ES is connected to and interacts with a cistron, which means that the junction between the two must be performed in an IRES-compatible environment in order to achieve cistronic translation. This nucleic acid vector also contains one or more additional "IRES-cistronic" molecules arranged in tandem. A cistron can be encoded as a receptor, an ion channel, a protein subunit, an enzyme, an antibody, a protein ligand, a protein that imparts antibiotic resistance to a cell, a growth factor, a hormone, or any other protein of interest. Peptide or peptide gene. In one embodiment of the present invention, at least one of the cistrons in the nucleic acid vector of the present invention contains a therapeutic gene that can prevent or delay the establishment and / or development of congenital or acquired diseases, such as cystic fibrous cysts (cystic fibrosis) Hemophilia A or 16 200408708 B, Duchenne or Becker type myopathy, cancer, AIDS and other bacterial or infectious diseases caused by pathogenic microorganisms . Examples of these therapeutic agents include, but are not limited to the following: cytokine; interleukin; interferon; factors or cofactors involved in coagulation, such as factor VIII Factor nine, factor IX, von Willebrand factor, antithrombin III, protein C, thrombin, and blood-sucking 4 to anticoagulant enzymes (hirudin ); Enzyme inhibitors, such as viral protease inhibitors; ion channel activators or inhibitors; proteins that prevent the onset or evolution of cancer, such as the expression products of tumor suppressor genes (p 5 3 genes, Rb genes, etc.), poisons , Antibodies, or immunotoxins (immu notoxin); or proteins capable of inhibiting viral infection or reproduction, for example, the epitope of a problem virus or a variant protein that has been altered to compete with the original viral protein. In another embodiment of the present invention, at least one cistron in the nucleic acid vector of the present invention includes a reporter gene, such as a gene, which encodes a galactose r enzyme (β-galactosidase), firefly luciferin (firefl luciferase) 'green fluorescent protein, recj fluorescent protein fromm Discosoma sp. C DsRed], or secreted alkaline phosphatase ( SEAP]). Other known reporter genes can also be used. Reporter genes can accelerate the detection of cells expressing functional proteins from nucleic acid vectors. Detection of reporter proteins can be achieved by providing an enzyme 17 200408708 response Substrate, this reaction will produce products that can be easily observed with the naked eye, cold light, fluorescence or microscope. The products of other reported genes, such as green fluorescent protein, can be directly used in a suitable fluorescent or luminescent environment. Observed by a microscope. The promoters used in the present invention include viral promoters and cellular promoters, and are all known techniques. Viral promoters include Cytomegalovirus (CMV) promoter, baculovirus polyhedrin promoter, adenovirus type 2 major late promoter, and SV40 virus promoter. Examples of cell promoters include Drosophila actin 5C distal promoter and mouse cadmium binding substance promoter (metallothionein 1 promoter). Other promoters that can be used in the nucleic acid vector of the present invention can be used by those skilled in the art Also included in the nucleic acid vector is a polyadenylation signal downstream of the last cistron of interest. The polyadenylation signal includes early or late polyadenylation signals, which are taken from the SV40 virus , Adenovirus type 5 E 1 B strain, and human growth hormone gene. This nucleic acid vector may also include enhancer sequences, such as enhancers of SV 40 virus and cytomegalovirus. To identify the nucleic acid vectors that have been obtained Cells, usually the selectable marker (selectable marker) and the gene of interest Was introduced into cells. Selectable markers include genes confer drug resistance genes such as anti-cell Appi resistant (ampicillin), spectin (to hygromycin and) and Yue ammonia σ day old ticket (methotrexate) a. For the organization of selectable markers, see 18 200408708

Thamma, Mammalian Cell Technology · Publishers, Stoneham, Mass. How to choose selective targets. The selectable markers can be placed on another plastid and introduced into the cell between the nucleic acid carriers, or they can be located on the same nucleic acid carrier in the same nucleic acid vector, the selective markers and the interest are controlled by different promoters or IRES, or Under the same Kai control. If you want to secrete the gene product of interest into the cell segment's secretion signal sequence, place it near the upstream of the nucleic acid interesting gene with the correct reading structure. There are many secretory signal sequences such as human serum albumin, daughter, factor signal sequences, and signals from immunoglobulin chains, but only some of them are indicated. In addition, the secretion signal sequence can be synthesized according to rules, such as the rules established by von Heinje (\ Biochem. (1 983) 13: 1 7-2 1; J. Mol. Biol. (1 9 8 5) Nuc. Acids Res. (1 986) 14: 4683-4690). The present invention also includes a method of expressing at least a cistron by cap-independent action, which comprises introducing a nucleic acid vector into a body including a segment connected to a promoter and interacting with the nucleotide including one connected to at least one cistron And its acting virus, hepatitis C virus and enterovirus type 71 IRES, or its homologous sequence or fragment, or a variant sequence or homologous sequence of the homologous sequence of encephalomyocarditis virus, hepatitis C disease type 71 IRES Source sequence B U11 erw 〇rth: Recorded as a skilled artisan at the same time in vivo. If the gene can be moved separately or outside the IRES, a vector can be used for internal learning. For example, the human growth factor sequence 'established here.' See £ 7 / Plant J. 184: 99-105; a cell of interest Here, this sequence contains the sequence of the encephalomyocarditis etiology, the mutated and enterovirus fragments, and the nuclear 19 200408708 gallic acid sequence. This nucleic acid vector may further contain one or more additional "IRES-cistronic" molecules arranged in tandem, and at least two cistrons are expressed by cap-independent action. Nucleic acid vectors can be introduced into cultured host cells. For example, by calcium phosphate-mediated transfection, see Wigler et al. 5 (1 97 8) Cell 14: 725; Corsaro and Pearson (1981) Somatic Cell Genetics 7: 603; Graham and Van der Eb. (1 97 3) FzWog ;; 52: 456). Other techniques for introducing nucleic acid vectors into host cells, such as the electric perforation method (e 1 ectr ο ρ 〇 〇rati ο η, see N euma η neta 1., (1982) J 1:84 1 -845) can also be used use. Transfected cells will be allowed to grow for a period of time to express the gene of interest. Drugs can be applied to select cells that exhibit a selectable marker for continued growth. Host cells containing a nucleic acid vector of the invention will be grown in a suitable growth medium. As used herein, "appropriate growth medium" refers to a medium containing nutrients necessary for cell growth. Nutrients required for cell growth include carbon sources, nitrogen sources, essential amino acids, vitamins, minerals, and growth factors. The growth medium includes drugs that have been screened from nucleic acid vectors for selective labeling of cells. Doing colonization into the host genome When you choose the interest… — ~~,-一 | 一, Qing ncj ’, you can establish a stable cell line. Chan Sen Tong's stable cell line was established after a drug screening process over a period of three days to three weeks. As described above, the present invention provides tp cc-producing 1 1RES sequences that are active in a wide range of cell types including mucoid cells, insects and / or mammalian cells. Therefore, the present invention 20 200408708 relates to a set for expressing recombinant proteins in bacterial, insect and / or mammalian cells, the set comprising: at least one nucleic acid vector comprising at least one IRES sequence; at least one nucleic acid vector comprising at least one IRES sequence capable of acting in insect cells; and at least one nucleic acid vector comprising at least one IRES sequence capable of acting in mammalian cells. In an embodiment of the present invention, the kit includes: at least one nucleic acid vector containing at least one IRES sequence of enterovirus type 71; at least one nucleic acid vector containing at least one IRES sequence of hepatitis C virus; and at least one A nucleic acid vector comprising at least one IRES sequence of an encephalomyocarditis virus. In another embodiment, the kit comprises a single nucleic acid vector comprising at least one IRES sequence capable of acting on bacteria, insects and / or mammalian cells. In yet another embodiment of the present invention, the kit includes two nucleic acid vectors, and the aforementioned two nucleic acid vectors each include at least one IRES sequence capable of acting on bacteria, insects and / or mammalian cells. As mentioned above, the nucleic acid vectors of the present invention can be contained in biological vectors, such as viruses and bacteria, preferably in non-pathogenic or attenuated microorganisms, including attenuated viruses, bacteria, parasites and the like Virus particles. Examples include poxviruses such as vaccinia virus; adenoviruses; baculoviruses; herpesviruses; adeno-associated viruses; and retroviruses. Such carriers are described in detail in the literature. In one embodiment of the present invention, the nucleic acid vector of the present invention may be included in a recombinant baculovirus capable of infecting a baculovirus host cell and expressing a gene of interest 21 200408708

poison. The baculovirus expression system has been disclosed in U.S. Patent Nos. 4,745,05 1, 4,879,236, and 5,147,788, and see Miller, LK, (1 988) Annu. Rev. Microbiol. 42: 1 77- 1 99; Luckow , VA, (1 990) In: Recombinant DNA Technology and Applications. McGraw-Hill, New York, pp. 97- 1 52; and O'Reilly, DR, et al., (19 92) Baculo virus Nucleic acid vectors: A

Laboratory Manual. W.H. Freedman, New York, all of which are listed for reference. Generally, the production of a recombinant baculovirus capable of infecting a baculovirus host cell and expressing a gene of interest involves transfection of the recombinant vector and the baculovirus genome DNA into the baculovirus host cell. Recombinant baculovirus vectors are usually derived from baculovirus genomic DNA fragments containing a polyhedrin promoter and a polyhedrin gene. In the recombinant baculovirus vector, the gene of interest is placed under the control of a polyhedrin promoter or other baculovirus promoter 'to replace some or all of the sequence of the polyhedrin gene. The recombinant baculovirus vector of the present invention comprises a nucleotide sequence connected to and acting on a polyhedrin promoter or other baculovirus promoters, and comprises a encephalomyocarditis connected to and acting on at least one cistron. Variations of IRES of viruses, hepatitis C virus and enterovirus type 71, or homologous sequences, variant sequences or fragments thereof, or variations of homologous sequences of IRES of encephalomyocarditis virus, hepatitis C virus, and enterovirus type 71 A nucleotide sequence of a sequence or a fragment of a homologous sequence. The recombinant baculovirus vector of the present invention further comprises one or more additional "IRES-cistronic" molecules. When the recombinant vector and the baculovirus genome DNA enter the host cell, the recombination vector and the baculovirus group 22 200408708 due to the homologous recombination phenomenon of the group DNA, that is, the gene of interest will be incorporated into the rod In the rhabdovirus genome, recombinant baculoviruses capable of expressing genes of interest will be released into extracellular media. However, neither transfection nor homologous recombination can achieve 100% efficiency. The result will be a group of mixed cells that can produce recombinant baculovirus and a group that does not produce recombinant baculovirus. Recombinant baculoviruses capable of expressing genes of interest in baculovirus host cells will be subsequently selected by appropriate screening methods or gene selection techniques. One method for selecting recombinant baculoviruses is the use of a plaque assay. The plaque assay was designed to produce distinct plaques on monolayer host cells under conditions in which each plaque was produced by a single virus-infected cell. Phytolytic plaques are produced by infecting baculovirus host cells with a diluted cell culture medium obtained from a transfected recombinant vector and a baculovirus genome DNA. To see lytic plaques produced by infected cells, the infected cells can be coated with Place on agar substrate or use a microscope. Viral plaques can be isolated and used to evaluate recombinant baculoviruses that can express genes of interest. In this technical field, there are many screening methods that can be used to confirm that lysobacteria isolated from co-transfection contain recombinant baculovirus. The preferred method is to detect the synthesis of target proteins, such as Western blotting, immunization, etc. Enzyme labeling assay (ELISA) or biochemical test method for expressed proteins. Southern blot analysis (S0 u t h e r n b 1 0 t a n a 1 y s s s) and polymerase chain reaction (PCR) also confirmed that the target gene exists in the recombinant baculovirus genome. The present invention also relates to a nucleic acid vaccine, which contains a 23 200408708 nucleic acid vector or biological vector of the present invention, which can guide the expression of a demand gene in a patient, and the demand gene is mostly a gene lacking or losing function in the patient. . The nucleic acid vaccine of the present invention can be inhaled into the lungs by intravenous injection or intramuscular injection, or by aerosolization; and the nucleic acid vaccine of the present invention can be applied in combination with the previously-disclosed electroporation technique of the living body to improve the transfection of the vaccine in vivo rate. This in vivo electroporation technique has been disclosed in the following literature, including Neuma η neta 1., “Electroporation” (1 982) 1: 8 4 1-8 4 5 and Widera et al., “Increased DN A vaccine delivery and immiinogenicity by electroporation z · / 7 v / v < 9 ”c / / mm < 9 / (2000) 164: 4635-4640 〇 In short, this live electroporation technique is based on the location of each nucleic acid vaccine injection (tumor or rolling). Meat) to perform an electroporation step to help deliver the nucleic acid vaccine. Those skilled in the art should understand that the parameters of this live electroporation vaccine delivery procedure may vary depending on the animal species, vaccine dose delivered, host disease cell volume, etc .; and can be obtained without undue experimentation. Best or most useful operating conditions. The present invention also relates to a method for treating a patient, or a method that is beneficial to the patient, by administering a sufficient number of nucleic acid vectors or biological vectors' to induce an essential gene in the patient. Performance. The administered nucleic acid vector or biological vector can provide the desired gene expression ' which is a gene lacking or losing function in a patient. The nucleic acid plant or biological carrier can be administered directly into a patient, for example, by intravenous or intramuscular injection, or by inhalation into the lungs by aerosolization. In addition, a set of in vitro gene therapy implementation steps can be used, which includes removing cells or tissues from a patient, introducing the nucleic acid vector or biological vector into the removed cells or tissues 24 200408708, and then transplanting the cells or tissues back Within the disease (see Knoell DL., Et al., (19 9 8) Am. J. Health Syst. Pharm. 55: 899-904; Raymon HK, et al., (1997) Exp. Neurol. 144: 82 -91; Culver KW, et al., (1 990) Hum. Gene Ther. 1: 3 99-4 1 0; Kasid A., et al., (1990) Proc. Natl. Acad. Sci. USA 87: 473 -477). The nucleic acid vector or biological vector can be introduced into the removed cells or tissues by transfection or infection, for example, by the method described above. A patient is defined here as any human or non-human animal that requires a specific protein, peptide or peptide, or any experimental material that is beneficial to its treatment, including human and non-human animals. The non-human animals being treated here include all domestic or wild animals. It goes without saying that anyone skilled in the art will recognize that the choice of protein, peptide, or peptide will depend on the disease or condition being treated in a particular system. The present invention relates to a method for screening antiviral compounds capable of interfering with cap-independent translation of the virus I RE S. Virus I R K S can support the infection, replication, and reproduction of the virus when it infects host cells. It generates important viral proteins through cap-independent translation. Therefore, the method of the present invention utilizes a polycistronic nucleic acid vector, which contains a nucleotide sequence connected to and interacts with a promoter, and includes a encephalomyocarditis virus, C, which is connected to and interacts with at least one cistron. Hepatitis virus and enterovirus 71 type IRES or homologous sequences, variant sequences or fragments thereof, or encephalomyocarditis virus, hepatitis C virus and enterovirus type 1 IR ES homologous sequences of variant sequences or homology A nucleotide sequence of a fragment of a sequence. This nucleic acid vector may further contain one or more additional "IRES-cis 25 200408708 second daughter" molecules arranged in tandem for at least two cistron expressions. This method includes transfection of a cistron nucleic acid vector into a cell, which vector will guide at least one IRES or its homologous sequence, variant sequence or Fragment 'or the mutated sequence of the homologous sequence of iRES of encephalomyocarditis virus, hepatitis C virus and enterovirus type 7 丨, or the cap-independent translation of the recombinant protein of the fragment of the homologous sequence; the test compound is then exposed to this Stained cells; and compared with transfected cells without test compounds to detect: reduced production of recombinant protein. The test compound can be any chemical protein, peptide, peptide, or nucleic acid (DNA or RNA) ◦ The test compound can be naturally occurring or synthesized by conventional methods. In this R example, the method of the present invention will be used to screen antiviral compounds against enterovirus 71, hepatitis C virus and encephalomyocarditis. This meme will be illustrated by the following examples, which should not be limited in any way. Example 1 Encephalomyocarditis virus with IRES activity in insect cells has been reported. IRES of encephalomyocarditis virus has been reported to be very efficient in mammalian systems' but not active in insect cells (see Finkelstein γ. Et al. (1999) J. Biotech. 75: 3 3-44), the inventors have unexpectedly found that the IRES of encephalomyocarditis virus indeed works in insect cells. A recombinant baculovirus expression system was used to test the IRES activity of cerebrocardiac myositis virus in insect cells. The baculovirus vector was constructed using the vector pBlueBac4.5 (Invitrogen), and the green fluorescent protein 26 200408708 (enhanced green fluorescent protein [EGFP]) was inserted into the multiple cloning site containing pBlueBac4.5 and placed on the rod. Under the control of the polyhedrin promoter (P p H) of the parvovirus, the resulting control group vector was named pBac-EGFP (see Figures 2A and 2B). Another vector, named pBac-IR-EGFP, contains an encephalomyocarditis virus IRES sequence (see Jang, S.K., and E. Wimmer, (1 990) Genes

Dev · 4: 1 560-1 572) immediately before the green fluorescent protein coding sequence (see Figures 3A and 3B). A bicistronic vector carrying DsRed red fluorescent protein (DsRed) and green fluorescent protein obtained from Lentinula edodes was constructed. In the vector pBacDS-IRE-EGFP, the baculovirus polyhedrin promoter Promote mRNA synthesis containing the nucleotide sequences of the DsRed red fluorescent protein and the green fluorescent protein gene, and the RES of encephalomyocarditis virus is embedded between the DsRed red fluorescent protein gene and the green fluorescent protein gene (see Figure 4) . It can be predicted that the DsRed red fluorescent protein gene will be expressed through a cap-dependent mechanism, and the green glory protein gene will be expressed through an IRES-driven cap of the encephalomyocarditis virus. Recombinant baculoviruses were generated using the Invhrgei ^ MaxBac 2.0 Recombinant Virus Expression System. The host cells of baculovirus, sf9 cells, were carried by papac-EGFP, pBac-IR-EGF] ^ pBacDs-IR-EGFP Two days after the recombinant virus infection, the green fluorescent protein (maximum excitation wavelength 488 nm; maximum emission wavelength 507 nm) and / or Ds red fluorescent protein (maximum excitation wavelength 5 5 8 _) were analyzed by fluorescence microscopy. (Maximum emission wavelength) As shown in Figure 2C, a cell line infected with a recombinant baculovirus carrying the vector pBac-EGFP expresses green fluorescent protein via a cap-dependent mechanism; Section 3C_Exhibit 27 200408708 does not infect the carrier vector pBac-lR-EGFP Recombinant baculovirus cells are slightly less efficient at expressing green fluorescent protein. It is speculated that the IRES of encephalomyocarditis virus is close to the polyhedrin promoter and interferes with the cap-dependent translation of green fluorescent protein; infection carries Recombinant baculovirus cells of the bicistronic vector pBacDs-IR-EGFP show both DsRed red fluorescent protein (see left of Figure 5) and green fluorescent protein (see right of Figure 5) in the same cell ). Therefore, contrary to previous publications, RES of encephalomyocarditis virus can guide the IRES-dependent translation of recombinant proteins in worm cells. Example 2 Eniremyositis virus, hepatitis C virus, and enterovirus type 71 ires are active in a wide range of cell types. Encephalomyocarditis virus, hepatitis C virus, and enterovirus type 71 IRES are analyzed in different cell types. Its activities include insect cells (S f 9 cells), mammalian cells (COS-7 cells and Huh7 cells), and fine cells (BL21 cells). The vector pTriEX-4 (Novagen) was used to generate a bicistronic vector expressing recombinant proteins in these three types of cells. The vector pTriEX-4 contains a cytomegalovirus immediate early promoter, which is used in Active in mammalian cells; pi 0 promoter of baculovirus AcMNPV, which is active in insect cells; and T7 promoter of phage, which is active in bacterial cells. As shown in Figure 6, the β-galactosidase (β_galactosidase) gene and the secreted alkaline phosphatase (SEAP) gene are placed in three promoters of the vector pTriEX-4 28 200408708 One under control to synthesize mRNA. Enterovirus type 7i IRES (see Figure 1), Hepatitis C virus IRES (see Ding 3111 ^ 江 〇 ^-

Kohara K., et al., (1 992) J. Virol. 66: 1 476 1 483) or IRES of encephalomyocarditis virus (see Jang, SK, and E. Wimmer *, (1990) Genes Dev. 4: 1 560- 1 572) were embedded between β-galactosidase gene * SEAP genes to promote the expression of IRES-dependent proteins of SEAP genes, and these bicistronic nucleic acid vectors were named vectors pC} S- EV71, pGS-HCV and pGS-EMCV. In order to detect IR ES activity in S f 9 insect cells, recombinant baculoviruses carrying vectors pGS_EV7 1, p G S-H C V or p G S-E M C V were generated according to the vector pr j n system operating manual (Novagen). SF9 cells were first infected with these recombinant baculoviruses, and the cell culture medium was collected 72 hours after infection, and their S E A P activity was analyzed. Recombinant baculovirus as a positive control group is produced by combining baculovirus genomic DNA with a recombinant vector pTriEX-4 carrying the SEap gene but without any leading IRES sequence; unmodified baculovirus as a negative control group AcMNPV is used to infect Sf9 cells. As shown in Figure 7, the RRs of enterovirus 71, hepatitis C virus, and encephalomyocarditis virus were stronger in Sf9 cells than the negative control group, and enteric virus 7 IRES showed The strongest activity. In order to detect IRES activity in mammalian cells, the vectors pGS_EMCV, pGS-HCV or pGS-EV71 were transfected into coS-7 cells (monkey kidney cell line) according to the outline outlined in the vector pTriEX system operating manual (Novagen). ) And Huh7 cells (a human liver cancer cell line). In mammalian cells, the mRNA of a nucleic acid vector is produced from a cytomegalovirus promoter. After 48 hours of transfection 200408708, the SEAP protein activity of the transfected cell culture medium was tested. In two groups of mammalian cell lines, the IRES of enterovirus 71, hepatitis C virus, and encephalomyocarditis virus compared to the negative control group (named pC MV V-gal monocistronic nucleic acid vector, its performance β-galactosidase gene and put it under the control of cytomegalovirus promoter (see Figure 8) all showed activity, of which ireS of enterovirus 71 type 1 was again expressed in two groups of mammalian cell lines Out of the strongest activity. In order to detect IRES activity in bacterial cells, the vectors PGS-EMC V, pGS-HCV or pGS-EV71 were transfected into BL21 cells, respectively, according to the outline outlined in the vector pTriEX system operating manual (Novagen). In bacterial animal cells, the mRNA of the nucleic acid vector is produced from the T7 promoter, which can be induced by IPTG to produce a large amount of mRNA. After induction with 0.4 mM IPTG for 3 hours, cells were collected and tested for their sera protein activity. As shown in Fig. 9, in the bacterial cells, compared with BL21 cells (first row) which were not transfected and BL21 cells (second row) which were transfected without the reporter gene vector pTriEX-4, the brain The IRES of myocarditis virus are highly active without the addition of IPTG and induced by the addition of IPTG (third and fourth lines, respectively). This is the first time that IRES of encephalomyocarditis virus has been shown to be active in bacterial cells. Hepatitis C virus and enterovirus 71 type 1 IRES are also active in bacterial cells (fifth and sixth lines, respectively). f Example 3 Interferon alpha (IFN-a) disrupts IRES cap-independent translation of enterovirus 71 and hepatitis C virus (cap-independent 30 200408708 translation) The bicistronic nucleic acid vector of IRES is used to screen for antiviral compounds that can disrupt the IRES cap-independent translation of the virus. Antiviral compounds are expected to bind to IRES and thus interfere with the performance of the SEAP protein, as shown in Figure 10. Others have shown that the first (cap-dependent) cistron is parallel to the level of steady state of the mRNA and is not affected by proteins encoded on the mRNA (see Hennecke, M., et al.? (200 1) Nucleic Acids Res. 29: 3 3 27-3 3 3 4). Therefore, cap-dependent cistron translation can be used as an internal standard for examining differences in mRNA levels. The bicistronic nucleic acid vectors pGS-EV71 and pGS-HCV described in Example 2 were transfected into Huh7 cells and cultured with different amounts of interferon alpha. 48 hours after transfection, the culture medium of the transfected cells was collected and tested for SEAP protein activity. The cells as the control group were each transfected with the above bicistronic nucleic acid vector, but were cultured without the addition of oc interferon. As shown in Figures 11 and 12, the unit of interferon alpha of 500 units inhibited the IRES activity of enterovirus type 71 and hepatitis C virus, respectively. The description of the present invention can be thoroughly understood through inspiration from the enumerated documents in the description of the present invention, and all the documents listed therein are incorporated by reference in their entirety. The examples in the description of the present invention are provided to illustrate specific implementations of the present invention and should not be used to limit the scope of the present invention. Those skilled in the art can recognize that many other embodiments are also included in the scope of the present invention. The description and the embodiments of the present invention are for illustration only. The true scope and spirit of the present invention will be indicated by the scope of the accompanying patent application. . 31 200408708 [Schematic description] Figure 1: Nucleotide sequence of 5 'untranslated region of enterovirus 71 (EV 7 1), which is derived from enterovirus 71 TW / 2 08 6/98 virus Strain genes. Figure 2: Figures 2A, 2B show the recombinant baculovirus vector pBac-EGFP, which is used to generate recombinant baculovirus. Figure 2C shows the expression of EGFP protein in S f 9 insect cells infected with recombinant baculovirus under a fluorescent microscope. Figure 3: Figures 3A and 3B show the recombinant baculovirus vector pBac-IR-EGFP, where the IRES of encephalomyocarditis virus is immediately before the sequence encoding the EGFP protein. Figure 3C shows the expression of EGFP protein in Sf'9 insect cells infected with recombinant baculovirus under a fluorescent microscope. Figure 4: Recombinant baculovirus vector pBac-DR-IR-EGFP is shown, in which the sequences encoding the DsRed and EGFP proteins are placed under the control of a polyhedrin promoter for mRNA synthesis, and the encephalomyocarditis virus ( EMCV) IR ES is placed between sequences encoding DsRed and EGFP proteins to drive the cap-independent translation of the EGFP gene. Figure 5: Sf9 insect cells infected with the recombinant baculovirus carrying the vector pBac-DR-IR-EGFP were observed under a fluorescent microscope. The left side is a cell expressing Ds albumin and the right side is a cell expressing EGFP fluorescent protein. 32 200408708 Figure 6: Shown is a bicistronic nucleic acid vector, which is used to express β-galactosidase (β-gal) and secretory test scales in mammalian, insect and bacterial cells. Acidic water enzyme (secreted alkaline phosphatase [SEAP]). The IRES sequences of encephalomyocarditis virus (EMCV), (: hepatitis virus (HCV), and enterovirus 71 (EV71) are embedded between the β-gal gene and the SEAP gene to drive SEAP's cap-independent translation effect. It corresponds to The bicistronic vectors were named vectors pGS-EMCV and pGS-HCV pGS-EV71. Figure 7: Sf9 insect cells display encephalomyocarditis virus (EMC V), hepatitis virus (HCV), and enterovirus 71 (EV71) IRES activity. This Sf9 insect cell was infected by vectors pGS-EMCV pGS-HCV and PGS-EV71 recombinant baculovirus. Figure 8: Encephalomyocarditis (EMCV) displayed on COS-7 and Huh 7 cells ), IHC activity of hepatitis C virus (HCV) and enterovirus type (EV71). Figure 9 ... IRES activity was shown on BL21 cells. The cells analyzed were transfected BL 2 1 cells (first block) ; BL21 cells (transplant) that have been transfected into the vector pTriEX-4 that does not contain the leader gene; cells that have been transfected with the vector pGS-EMCV and have not been induced by IPTG (third transport); 0.4 mM IPTG-induced cells (fourth 襕); cells that have been transformed with pGS-HCV and induced by 0.4 mM IPTG ( Activating β, detoxification by basal nucleus and Λ poison of C 7 1 Unreported column 5 33 200408708); cells that have been transfected with PGS-EV71 and induced by 0.4 (sixth rot). Figure 10: Illustrate the process of screening antiviral compounds, which, when combined with polycistronic nucleic acid vectors, disrupt the virus' IRES-type translation. Figure 11 ·· IRES of hepatitis C virus shows the antiviral activity of interferon alpha. Section 12 Figure: IRES in enterovirus 71 shows antiviral activity of interferon alpha. M Μ 1 PTG is independent of the cap used (IFN-α) (IF Ν-α) 34

Claims (1)

200408708 Scope of patent application: 1. A nucleic acid vector capable of expressing at least two cistrons, comprising: a. — A nucleotide sequence comprising at least two cistrons connected to the promoter and acting on it And b. At least one nucleotide sequence connected to and interacting with at least one of the at least two cistrons, comprising a member selected from the group consisting of enterovirus type 71 (EV71), hepatitis C virus (HCV), IRES of encephalomyocarditis virus (EMCV), or a variant sequence or fragment thereof; wherein the nucleotide sequence, its variant sequence or fragment, can provide IRES activity. 2. A nucleic acid vector according to item 1 of the scope of patent application, wherein at least one of the at least two cistrons includes a reporter gene. 3. A nucleic acid vector according to item 1 of the patent application, wherein at least one of the at least two cistrons comprises a therapeutic gene. 4. A biological vector capable of expressing at least two cistrons, comprising a nucleic acid vector as described in item 1 of the scope of patent application. 5. A biological vector as described in item 4 of the scope of patent application, wherein the biological vector is selected from the group consisting of poxvirus, adenovirus, herpesvirus, and adeno-associated virus (adeno- associated virus), retrovirus and baculovirus. 35 200408708 6. A nucleic acid vector for the expression of at least two cistrons, comprising: a. A nucleotide sequence comprising at least two cistrons connected to and acting on the promoter; and b, at least one nucleotide sequence connected to and interacting with at least one of the at least two cistrons, which comprises one selected from the group consisting of enterovirus 71 (EV71), hepatitis C virus (HCV), and encephalomyocarditis virus (EMCV) IRES homologous sequence, or a variant sequence or fragment thereof; wherein the homologous sequence, its variant sequence or fragment, can provide IRES activity 0 7. A nucleic acid vector as described in item 6 of the scope of patent application Wherein at least one of the at least two cistrons includes a reporter gene. 8. A nucleic acid vector according to item 6 of the patent application, wherein at least one of the at least two cistrons comprises a therapeutic gene. 9. A biological vector for at least two cistron expressions, comprising a nucleic acid vector as described in item 6 of the scope of patent application. 10. A biological vector according to item 9 of the scope of the patent application, wherein the biological vector is selected from the group consisting of poxvirus, adenovirus, herpesvirus, and adeno-associated virus (adeno- associated virus), retrovirus, and baculovirus 36 200408708. 11. A host cell comprising a nucleic acid vector as described in item 1 of the scope of patent application. 12. A host cell according to item 11 of the application, wherein the host cell is an insect cell. 13. A host cell according to item 11 of the application, wherein the host cell is a mammalian cell. 14. A host cell according to item 11 of the application, wherein the host cell is a bacterial cell. 15. A host cell comprising a nucleic acid vector as described in item 6 of the patent application. 16. A host cell according to item 15 of the application, wherein the host cell is an insect cell. 17. A host cell according to item 15 of the application, wherein the host cell is a mammalian cell. 18. A host cell as described in item 15 of the application, wherein 37 200408708 the host cell is a bacterial cell. 19. A method for expression of at least two cistrons, comprising introducing a nucleic acid vector into a host cell, said nucleic acid vector comprising: a.-A segment that is connected to a promoter and interacts with at least two cistrons Nucleotide sequence; and b. At least one nucleotide sequence connected to and interacting with at least one of the at least two cistrons, which comprises one selected from the group consisting of enterovirus 71 type 1 (EV71), hepatitis C Virus (HCV), encephalomyocarditis virus (EMC V) IRES, or a variant sequence or fragment thereof; wherein the nucleotide sequence, its variant sequence or fragment, can provide IRES activity. 20. A method by which at least two cistrons can be expressed, comprising introducing a nucleic acid vector into a host cell, said nucleic acid vector comprising: a. A segment that is connected to a promoter and interacts with it comprising at least two cistrons Nucleotide sequence; and b. At least one nucleotide sequence connected to and interacting with at least one of the at least two cistrons, which comprises one selected from the group consisting of enterovirus 71 type 1 (EV71), hepatitis C Homology sequence of IRES of virus (HCV) and moon diarrhea myocarditis virus (EMCV), or a variant sequence or fragment thereof; wherein the homology sequence, the variant sequence or fragment thereof, can provide IRES activity 21. One type is available for at least two A cistronic expression baculovirus delivery vector, 38 200408708, comprising: a. A cistronic nucleotide sequence connected to the baculovirus promoter and interacting with it; and b. At least one segment from the above At least one of at least two cistrons has a functioning nucleotide sequence that includes an IRES selected from the group consisting of enteropathy (EV71), hepatitis C virus (HCV), encephalomyocarditis, or a variant sequence or fragment thereof; Where the , Variant sequences or fragments thereof, may be provided based IRES living 22. Application of one of the item 21 patentable scope baculovirus, wherein the at least two cistron at least one packet-based gene. 23. A rod-shaped disease as described in item 21 of the patent application scope, wherein at least one of the at least two cistrons is a gene. Contains at least two nucleotide sequences that are linked to and are not toxic to Type 71 virus (EM CV). Poisonous transmission includes a report Poisonous transmission includes a treatment 24; A recombinant baculovirus comprising a baculovirus genome host cell expressing at least two cistrons, the recombination rod contains ... a. A segment that is connected to the baculovirus promoter and includes a cis-transformer The nucleotide sequence of the daughter; and b. At least one nucleotide sequence which functions with at least one of the at least two cistrons, which comprises one selected from the group consisting of enteroviruses and is Poison 71 39 200408708 (EV71), IRES of hepatitis C virus (HCV), encephalomyocarditis virus (EMCV), or a variant sequence or fragment thereof; wherein the nucleotide sequence, its variant sequence or fragment, provides IRES activity. 25. A method for generating a baculovirus capable of expressing at least two cistrons, comprising: a. Introducing a baculovirus delivery vector and a baculovirus genomic DNA as described in item 21 of the patent application scope Into a baculovirus host cell to cause homologous recombination; and b. Isolate a recombinant baculovirus. 26. A baculovirus host cell expressing at least two cistrons, comprising a baculovirus as described in item 24 of the scope of patent application. 27. A baculovirus delivery vector for the expression of at least two cistrons, comprising: a. A nucleotide sequence comprising at least two cistrons connected to and acting on a baculovirus promoter; And b. At least one nucleotide sequence that is connected to and interacts with at least one of the at least two cistrons, and comprises a nucleotide selected from the group consisting of enterovirus type 71 (EV71), hepatitis C virus (HCV), The homologous sequence of IRES of encephalomyocarditis virus (EMCV), or a variant sequence or fragment thereof; wherein the homologous sequence, the variant sequence or fragment thereof, can provide IRES activity. 40 200408708 28. A baculovirus delivery vector as described in item 27 of the scope of patent application, wherein at least one of the at least two cistrons includes a reporter gene. 29. The baculovirus delivery vector according to item 27 in the scope of the patent application, wherein at least one of the at least two cistrons comprises a therapeutic gene. 30. A recombinant baculovirus comprising a baculovirus genome and capable of expressing at least two cistrons in a host cell, the recombinant baculovirus comprising: a. A segment connected to and acting on a baculovirus promoter A nucleotide sequence comprising at least two cistrons; and b. At least one nucleotide sequence connected to and interacting with at least one of the at least two cistrons, comprising one selected from enterovirus type 71 (EV71), the homologous sequence of IRES of hepatitis C virus (HCV), encephalomyocarditis virus (EMC V), or a variant sequence or fragment thereof; wherein the homologous sequence, the variant sequence or fragment thereof, provides IR ES activity. 31. A method for generating a baculovirus capable of expressing at least two cistrons, comprising: a. Introducing a baculovirus delivery vector and baculovirus genomic DNA to a baculovirus as described in item 27 of the scope of patent application 40 200408708 to cause homologous recombination; and b. Isolation of recombinant baculovirus. 32. A baculovirus host cell expressing at least two cistrons, comprising a baculovirus as described in patent application scope 30. 33. A kit capable of expressing recombinant proteins in bacterial, insect and / or mammalian cells, comprising at least one nucleic acid vector comprising at least one IRES sequence capable of acting in a bacterial cell; at least one nucleic acid vector, the The nucleic acid vector comprises at least one IRES sequence capable of acting in insect cells; and at least one nucleic acid vector comprising at least one IR ES sequence capable of acting in mammalian cells. 34. The set of patent application scope 33, wherein the at least one nucleic acid vector comprises at least one IRES sequence selected from the group consisting of enterovirus 71 (EV71), hepatitis C virus (HCV), and encephalomyocarditis virus (EMCV) . 35. The set according to the scope of patent application 33, wherein the set comprises a single nucleic acid vector comprising at least one IR ES sequence capable of acting on bacteria, insects and / or mammalian cells. 36. The set according to the scope of patent application 33, wherein the set comprises two nucleic acid vectors, each of which contains at least one IR ES sequence capable of acting on bacteria, insects and / or mammalian cells . 42 200408708 37. A method of treating a patient, comprising administering a nucleic acid vector as described in any one of the scope of patent application 1 or 6. 38. A method of treating a patient, comprising administering a biological carrier as described in any one of claims 4 or 9 of the scope of patent application. 39. A method for treating a patient, comprising: a. Removing cells or tissues from the patient; b. Introducing a nucleic acid vector as described in the scope of patent application item 1 or 6 into the cells or tissues removed above ; And c. Implant the cell or tissue back into the patient. 40. A method for treating a patient, comprising: a. Removing cells or tissues from the patient; b. Introducing a biological carrier according to any one of the claims 4 or 9 of the scope of the patent application to the removed cells Or tissue; and c. Implant the cell or tissue back into the patient. 41. A method for screening an antiviral compound capable of disrupting cap-independent translation of a member selected from the group consisting of enterovirus 71 (EV71), hepatitis C virus (HCV), and encephalomyocarditis virus (EMC V) IRES, comprising: a. Transfection of a nucleic acid vector into a cell as described in any one of the scope of patent applications 1 or 6; 43 200408708 b. contacting a test compound with the above-mentioned transfected cells; and c. detecting a decrease in recombinant protein production Amount compared to transfected cells without test compound. 42. A nucleic acid vaccine comprising at least one nucleic acid vector as described in any one of the scope of patent application 1 or 6. 43. A nucleic acid vaccine comprising at least one biological carrier as described in any one of claims 4 or 9 of the scope of patent application. 44. The nucleic acid vaccine according to any one of claims 42 or 43 of the scope of patent application, which is introduced into a patient's cell or tissue by electroporation in vivo. 45. A nucleic acid vector or nucleic acid vaccine delivery system, comprising at least: a. Removing a cell or a tissue from a patient; b. introducing a nucleic acid vector as described in any one of the scope of patent applications 1 or 6 into the cells or tissues removed above; and c. implanting the cells or tissues back into the patient. 46. A nucleic acid vector or nucleic acid vaccine delivery system, comprising at least: a. Removing a cell or a tissue from a patient; b. A biological agent according to any one of the 4 or 9 scope of the patent application Sexual vectors are introduced into the cells or tissues removed as described above; and 44 200408708 C. The cells or tissues are implanted back into the patient. 47. A recombinant protein, which is prepared with a nucleic acid vector according to any one of claims 1 or 6 of the scope of patent application. 48. A recombinant protein prepared from a nucleic acid vector according to any one of claims 4 or 9 of the scope of patent application. 49. A therapeutic agent delivery system, comprising at least: a. Removing a cell or a tissue from a patient; b. Introducing a recombinant protein according to any one of claims 47 or 48 in the scope of patent application Into the cells or tissues removed above; and c. Implant the cells or tissues back into the patient. 45