KR20170056902A

KR20170056902A - Pharmaceurical composition for preventing or treating hepatitis C virus infection or disease due to HCV infection

Info

Publication number: KR20170056902A
Application number: KR1020150160303A
Authority: KR
Inventors: 유주연; 김나리
Original assignee: 포항공과대학교 산학협력단
Priority date: 2015-11-16
Filing date: 2015-11-16
Publication date: 2017-05-24
Also published as: KR101783030B1

Abstract

The present invention provides a polypeptide including a transmembrane domain and a proline-rich domain of SCOTIN protein, a polynucleotide coding the polypeptide; a recombinant vector including the polynucleotide, or a pharmaceutical composition for preventing or treating hepatitis C virus (HCV) infection or disease associated with the HCV infection which contains the polypeptide, the polynucleotide, the recombinant vector, or a mixture of the same, or an anti-virus composition against HVC, a pharmacological preparation containing the composition as an active ingredient, and a method for treating or preventing HCV infection by using the composition.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a pharmaceutical composition for preventing or treating hepatitis C virus infection or infection,

The present invention relates to a polypeptide comprising a transmembrane domain and a proline rich domain of SCOTIN protein, a polynucleotide encoding said polypeptide, a recombinant vector comprising said polynucleotide, or a polynucleotide encoding said polypeptide, polynucleotide, A pharmaceutical composition for preventing or treating a hepatitis C virus (HCV) infection or a disease associated with infection, or an antiviral composition for hepatitis C virus (HCV) And a method of treating or preventing hepatitis C virus (HCV) infection using the composition.

Hepatitis C virus (HCV) is a pathogen believed to have infected more than 180,000 people worldwide and is known to cause diseases such as liver fibrosis, cirrhosis, and liver cancer when not effectively treated Hepatology 57: 1333-1342.) (Pawlotsky JM (2004) Pathophysiology (2004). Epidemiology of hepatitis C virus infection: new estimates of age-specific antibody to HCV seroprevalence. of hepatitis C virus infection and related liver disease. Trends Microbiol 12: 96-102.). HCV is an RNA genome-borne virus that consists of four structural proteins and six nonstructural proteins. In HCV-infected cells, HCV proteins are located inside and outside the endoplasmic reticulum (ER) membrane, inducing structural deformation of the membrane of the endoplasmic reticulum, forming a membranous web (MW) with lipid granules,

In HCV-infected cells, vesicular stress is induced by RNA replication and HCV protein synthesis, resulting in unfolded protein response (UPR). In addition, HCV-infected host cells are autophagic in one of the UPRs. In general, autophagy plays a role in eliminating pathogens such as Sindbis virus and Salmonella enterica , whereas HCV The autophagy is advantageously used for proliferation.

The autophagosome induced by HCV infection is necessary for effective proliferation, translation and release of HCV. The autophagosome produced by autophagy itself binds to the HCV proliferative complex and is used as a place for HCV proliferation It is. In addition, HCV-induced autophagy inhibits the expression of interferon cytokines, which are very important biomarkers in host cell antiviral responses.

Development of enzyme inhibitors of nonstructural protein 3 (NS3), nonstructural protein 4 (NS4) or RNA polymerase, nonstructural protein 5B (NS5B), a protein protease in HCV proteins And inhibitors of the proteolytic enzyme NS3 / NS4A, telaprevir and boceprevir, are currently used in combination with interferon and / or ribavirin for HCV genotype 1 infection from the US FDA and EMA Was approved. The NS5A protein of HCV has no enzyme activity but has become a target for the development of HCV therapeutics because it plays an essential role in RNA replication and virus assembly in the life cycle of HCV. NS5A inhibitors, including daclatasvir (BMS790052), which is in phase III clinical trials, are characterized by the ability to inhibit various types of HCV and to induce effective HCV inhibition in small amounts. (Pawlotsky JM (2013) NS5A inhibitors in the treatment of hepatitis C. J Hepatol 59: 375-382.)

The present inventors confirmed that the SCOTIN protein promotes the degradation of the HCV NS5A protein and inhibits HCV proliferation. In particular, the TMPRD portion of the SCOTIN protein binds to the NS5A protein and plays an important role in migration and degradation into the autophagous body By confirming, the expression of SCOTIN protein, specifically the expression of TMPRD domain, was confirmed to be effective for treating HCV infection.

Accordingly, one example of the present invention relates to a polypeptide comprising a transmembrane domain (TM) of SCOTIN protein and a proline-rich domain (PRD).

Another example relates to a polynucleotide encoding a polypeptide comprising a transmembrane domain (TM) of the SCOTIN protein and a proline-rich domain (PRD).

Another example relates to a recombinant vector comprising a polynucleotide encoding said polypeptide.

Another example relates to a cell transformed with the recombinant vector.

Another example relates to a method for producing a polypeptide comprising the step of expressing in a cell a polynucleotide encoding the polypeptide or a recombinant vector comprising the polynucleotide.

Another example is a polypeptide comprising a SCOTIN protein, a transmembrane domain (TM) of a SCOTIN protein and a proline-rich domain (PRD), a polynucleotide encoding the SCOTIN protein or polypeptide, A pharmaceutical for the prevention or treatment of a hepatitis C virus (HCV) infection or a disease associated with infection, comprising a recombinant vector comprising a polynucleotide, a recombinant cell comprising the recombinant vector, or a mixture thereof as an active ingredient &Lt; / RTI >

Another example relates to a pharmaceutical preparation containing the composition as an active ingredient.

Another example is a polypeptide comprising a SCOTIN protein, a transmembrane domain (TM) of a SCOTIN protein and a proline-rich domain (PRD), a polynucleotide encoding the SCOTIN protein or polypeptide, A recombinant vector comprising a polynucleotide, a recombinant cell comprising the recombinant vector, or a mixture thereof as an active ingredient. The present invention also relates to an antiviral composition for hepatitis C virus (HCV).

Another example relates to a method of treating or preventing a hepatitis C virus (HCV) infection comprising administering a pharmaceutically effective amount of the composition.

Another example relates to a method of inhibiting the replication of hepatitis C virus comprising contacting said composition with hepatitis C virus (HCV).

Yet another example is a polypeptide comprising a SCOTIN protein, a transmembrane doamin (TM) of a SCOTIN protein and a proline-rich domain (PRD), a coding polynucleotide thereof, (HCV) infections or diseases associated with infection.

SCOTIN protein is induced by P53 transcription factor activated by DNA damage signal and located in the endoplasmic reticulum and participates in P53 mediated cell death process. The SCOTIN protein has an ER-signal peptide (SS), a cysteine-rich domain (CRD), a transmembrane domain (TM), and a proline-rich domain Domain (proline-rich domain; PRD). These SCOTIN proteins require PRD to be located in the endoplasmic reticulum, and both CRD and PRD are required to promote P53-dependent cell death. (Bourdon JC, Renzing J, Robertson PL, Fernandes KN, Lane DP (2002), Scotin, a novel p53-inducible proapoptotic protein located in the ER and the nuclear membrane J Cell Biol 158: 235-246.

The present inventors have found that the SCOTIN protein inhibits the proliferation of HCV by promoting the degradation of the NS5A protein of HCV. In particular, it is important that the TMPRD portion of the SCOTIN protein binds to the NS5A protein, Respectively. It was confirmed that the expression of SCOTIN protein can be used as a therapeutic agent for HCV infection, and that the increase in expression of the TMPRD domain among SCOTIN proteins can be used as a treatment for HCV infection.

Hereinafter, the present invention will be described in more detail

One example of the present invention relates to polypeptides comprising transmembrane doamin (TM) and proline-rich domain (PRD) of SCOTIN protein. The polypeptide may be a fragment of the SCOTIN protein, or a variant thereof.

The SCOTIN protein is a shisa family member 5 (SHISA5) protein. The SCOTIN protein contains ER-signal peptide (SS), cysteine-rich domain (CRD), transmembrane domain (TM), and proline-rich domain .

The SCOTIN protein may be derived from mammals such as primates such as humans and rodents such as mice, and may be a natural protein or a synthetic or recombinantly produced one. The amino acid sequence of each domain constituting the SCOTIN protein (for example, NCBI Accession No. NP_001271261.1 (SEQ ID NO: 9)) derived from a mouse and the SCOTIN protein (for example, NCBI Accession No. NP_057563 Are shown in Table 1 (m: mouse, h: human).

denomination The sequence (5'-3 ') SEQ ID NO: SS_m MAAPAPSLWTLLLLLLLLPPPP One SS_h MTAPVPAPRILLPLLLLLLLPPP 2 TM_m GFGATVAIGVTIFVVFIATIII 3 TM_h GFGATLAVGLTIFVLSVVTIII 4 PRD_m &Lt; 5 PRD_h CFTCSCCCLYKTCRRPRPVVTTTTSTTVVHAPYPQPPSVPPSYPGPSYQGYHTMPPQPGMPAAPYPMQYPPPYPAQPMGPPAYHETLAGGAAAPYPASQPPYNPAYMDAPKAAL 6 CRD_m GAHGELCRPFGEDNSIPVFCPDFCCGSCSNQYCCSDVLRKIQWNEEMCPEPESSRFSTPAEETPEHLGSALKFRSSFDSDPMS 7 CRD_h GARGEVCMASRGLSLFPESCPDFCCGTCDDQYCCSDVLKKFVWSEERCAVPEASVPASVEPVEQLGSALRFRPGYNDPMS 8

The transmembrane domain included in the polypeptide may include the amino acid sequence of SEQ ID NO: 3 or 4, but is not limited thereto, and may be selected from peptides having a membrane permeability function and a length of 10 to 50 amino acids.

The proline-enriched domain contained in the polypeptide may include, but is not limited to, an amino acid sequence of SEQ ID NO: 5 or 6, and may include 50 to 150 amino acids having a proline content of 10% or more, 15% Length peptide. &Lt; / RTI >

In one example, the SCOTIN protein fragment comprises 132 to 260 consecutive, 132 < RTI ID = 0.0 > (SEQ ID NOS: 3 < / RTI > From 1 to 240, 152 to 260, 152 to 240, 153 to 260, 153 to 240, 173 to 260, 173 to 240, such as 132 to 256, 132 to 236, (Mouse-derived SCOTIN protein), or 136-260, 136-240, 156-260, 152-236, 153-256, 153-236, 173-256, or 173-236 160 to 240, 160 to 260, 160 to 240, 180 to 260, or 180 to 240 (human-derived SCOTIN protein).

In the SCOTIN protein fragment, the transmembrane domain may be located at the N-terminal side, and the proline-rich domain may be located at the C-terminal side. For example, the proline-rich domain may be located at the C-terminus of the membrane-penetrating domain and the transmembrane domain may be located at the N-terminus of the proline-rich domain (for example, the structure of TM-PRD).

In one example, the SCOTIN protein fragment may comprise the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12.

The mutant of the SCOTIN protein fragment means a fragment in which the amino acid sequence portion of the SCOTIN protein fragment except for the transmembrane domain and the proline rich domain of the SCOTIN protein is replaced with a wild-type SCOTIN protein. In one example, the variant of the SCOTIN protein fragment comprises the transmembrane domain and the proline rich domain of the SCOTIN protein described above, wherein the entire amino acid sequence differs from the SCOTIN protein or a fragment of the SCOTIN protein derived therefrom and has a total amino acid length 132 to 260, 132 to 240, 152 to 260, 152 to 240, 153 to 260, 153 to 240, 173 to 260, or 173 to 240 polypeptides. In addition, the polypeptide may further comprise 1 to 20, 5 to 20, 10 to 20, or 15 to 20 amino acids at the N-terminus and / or the C-terminus (eg, C-terminus) .

The variant of the SCOTIN protein fragment includes a membrane-bound domain of SCOTIN protein at the N-terminal side and a proline-rich domain at the C-terminal side, and an amino acid sequence portion excluding the membrane-bound domain and the proline- Terminal end, the C-terminal side of the proline rich domain, between the transmembrane domain and the proline rich domain (i.e., between the C-terminal and the proline rich domain N-terminal of the transmembrane domain) have.

The polypeptide may have an ER locus at the N-terminus (for example, at the N-terminus of the transmembrane domain, or at the N-terminus of the amino acid sequence at the N-terminus of the transmembrane domain) signal peptide < RTI ID = 0.0 > (SS). < / RTI >

The endoplasmic reticulum locus signal peptide may be composed of the amino acid sequence of SEQ ID NO: 1 or 2, but is not limited thereto.

As such, when the polypeptide includes the ER signal peptide, the polypeptide may include the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14.

The amino acid sequences of the available SCOTIN proteins and fragments of the SCOTIN protein provided on the basis thereof are shown in Table 2 below.

denomination The sequence (5'-3 ') SEQ ID NO: SCOTIN MAAPAPSLWTLLLLLLLLPPPPGAHGELCRPFGEDNSIPVFCPDFCCGSCSNQYCCSDVLRKIQWNEEMCPEPESSRFSTPAEETPEHLGSALKFRSSFDSDPMSGFGATVAIGVTIFVVFIATIIICFTCSCCCLYKMCCPQRPVVTNTTTTTVVHAPYPQPQPQPVAPSYPGPTYQGYHPMPPQPGMPAAPYPTQYPPPYLAQPTGPPPYHESLAGASQPPYNPTYMDSLKTIP 9 hSCOTIN MTAPVPAPRILLPLLLLLLLTPPPGARGEVCMASRGLSLFPESCPDFCCGTCDDQYCCSDVLKKFVWSEERCAVPEASVPASVEPVEQLGSALRFRPGYNDPMSGFGATLAVGLTIFVLSVVTIIICFTCSCCCLYKTCRRPRPVVTTTTSTTVVHAPYPQPPSVPPSYPGPSYQGYHTMPPQPGMPAAPYPMQYPPPYPAQPMGPPAYHETLAGGAAAPYPASQPPYNPAYMDAPKAAL 10 SCOTIN
(TMPRD) GFGATVAIGVTIFVVFIATIIICFTCSCCCLYKMCCPQRPVVTNTTTTTVVHAPYPQPQPQPVAPSYPGPTYQGYHPMPPQPGMPAAPYPTQYPPPYLAQPTGPPPYHESLAGASQPPYNPTYMDSLKTIP 11 hSCOTIN (TMPRD) GFGATLAVGLTIFVLSVVTIIICFTCSCCCLYKTCRRPRPVVTTTTSTTVVHAPYPQPPSVPPSYPGPSYQGYHTMPPQPGMPAAPYPMQYPPPYPAQPMGPPAYHETLAGGAAAPYPASQPPYNPAYMDAPKAAL 12 SCOTIN
(SSTMPRD) MAAPAPSLWTLLLLLLLPPPPGFGATVAIGVTIFVVFIATIIICFTCSCCCLYKMCCPQRPVVTNTTTTTVVHAPYPQPQPQPVAPSYPGPTYQGYHPMPPQPGMPAAPYPTQYPPPYLAQPTGPPPYHESLAGASQPPYNPTYMDSLKTIP 13 hSCOTIN (SSTMPRD) MTAPVPAPRILLPLLLLLLLTPPPGFGATLAVGLTIFVLSVVTIIICFTCSCCCLYKTCRRPRPVVTTTTSTTVVHAPYPQPPSVPPSYPGPSYQGYHTMPPQPGMPAAPYPMQYPPPYPAQPMGPPAYHETLAGGAAAPYPASQPPYNPAYMDAPKAAL 14 SCOTIN
(SSCRD) MAAPAPSLWTLLLLLLLPPPPGAHGELCRPFGEDNSIPVFCPDFCCGSCSNQYCCSDVLRKIQWNEEMCPEPESSRFSTPAEETPEHLGSALKFRSSFDSDPMS 15 SCOTIN
(SSTMCRD) MAAPAPSLWTLLLLLLLPPPPGAHGELCRPFGEDNSIPVFCPDFCCGSCSNQYCCSDVLRKIQWNEEMCPEPESSRFSTPAEETPEHLGSALKFRSSFDSDPMSGFGATVAIGVTIFVVFIATIIICF 16 SCOTIN
(SSPRD) MAAPAPSLWTLLLLLLLLPPPPCFTCSCCCLYKMCCPQRPVVTNTTTTTVVHAPYPQPQPQPVAPSYPGPTYQGYHPMPPQPGMPAAPYPTQYPPPYLAQPTGPPPYHESLAGASQPPYNPTYMDSLKTIP 17

The term "protein" as used herein refers to a molecule comprising a polymer of amino acids linked together by a peptide bond (s). The protein may comprise two or more amino acids.

The term "TMPRD" or "TMPRD domain" refers to a protein in which the transmembrane domain (TM) of the SCOTIN protein and the proline-rich domain (PRD) are fused.

The term " TMCRD "or" TMCRD domain "means a protein in which a transmembrane domain (TM) of SCOTIN protein and a cysteine-rich domain (CRD) are fused.

Another example of the present invention relates to a polynucleotide encoding a polypeptide comprising a transmembrane domain (TM) and a proline-rich domain (PRD) of the SCOTIN protein as described above.

The nucleotide sequences encoding each domain constituting the SCOTIN protein derived from mouse and the SCOTIN protein derived from human are shown in Table 3 (m: mouse (NM_001284332.1), h: human (NM_016479.4)).

denomination The sequence (5'-3 ') SEQ ID NO: SS_m ATGGCTGCGCCGGCGCCCTCTCTGTGGACCCTATTGCTGCTGCTGTTGCTGCTGCCGCCGCCTCCG 18 SS_h ATGACTGCGCCGGTCCCCGCGCCGCGGATCCTGTTGCCGTTGCTGTTGCTGCTGCTGCTAACGCCGCCTCCG 19 TM_m GGGTTCGGAGCGACCGTCGCCATTGGCGTGACCATCTTTGTGGTGTTTATTGCCACTATCATCATC 20 TM_h GGGTTCGGAGCGACCTTGGCCGTTGGCCTGACCATCTTTGTGCTGTCTGTCGTCACTATCATCATC 21 PRD_m TGCTTCACCTGCTCCTGCTGCTGTCTGTATAAGATGTGCTGCCCCCAACGCCCTGTCGTGACCAACACCACAACTACTACCGTGGTTCATGCCCCTTACCCTCAGCCTCAACCTCAACCTGTGGCCCCCAGCTATCCTGGACCAACATACCAGGGCTACCATCCCATGCCCCCCCAGCCAGGAATGCCAGCAGCACCCTACCCAACGCAGTACCCACCACCCTACCTGGCCCAGCCCACAGGGCCGCCACCCTACCATGAGTCCTTGGCTGGAGCCAGCCAGCCTCCATACAACCCGACCTACATGGATTCCCTAAAGACAATTCCC 22 PRD_h TGCTTCACCTGCTCCTGCTGCTGCCTTTACAAGACGTGCCGCCGACCACGTCCGGTTGTCACCACCACCACATCCACCACTGTGGTGCATGCCCCTTATCCTCAGCCTCCAAGTGTGCCGCCCAGCTACCCTGGACCAAGCTACCAGGGCTACCACACCATGCCGCCTCAGCCAGGGATGCCAGCAGCACCCTACCCAATGCAGTACCCACCACCTTACCCAGCCCAGCCCATGGGCCCACCGGCCTACCACGAGACCCTGGCTGGAGGAGCAGCCGCGCCCTACCCCGCCAGCCAGCCTCCTTACAACCCGGCCTACATGGATGCCCCGAAGGCGGCCCTC 23 CRD_m GGTGCCCATGGTGAGCTGTGCAGGCCCTTTGGTGAAGACAATTCGATCCCAGTGTTCTGTCCTGATTTCTGTTGTGGTTCCTGTTCCAACCAATACTGCTGCTCGGACGTGCTGAGGAAAATCCAGTGGAATGAGGAAATGTGTCCTGAGCCAGAGTCCAGCAGATTTTCCACCCCCGCGGAGGAGACACCCGAACATCTGGGTTCAGCGCTGAAATTTCGATCCAGTTTTGACAGTGACCCTATGTCA 24 CRD_h GGTGCACGTGGTGAGGTGTGTATGGCTTCCCGTGGACTCAGCCTCTTCCCCGAGTCCTGTCCAGATTTCTGCTGTGGTACCTGTGATGACCAATACTGCTGCTCTGACGTGCTGAAGAAATTTGTGTGGAGCGAGGAAAGGTGTGCTGTGCCTGAGGCCAGCGTGCCTGCCAGTGTAGAGCCGGTGGAGCAGCTGGGCTCGGCGCTGAGGTTTCGCCCTGGCTACAACGACCCCATGTCA 25

In one example, when the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 3 or 4, the polynucleotide sequence encoding the transmembrane domain may comprise the nucleotide sequence of SEQ ID NO: 20 or 21, But is not limited thereto.

When the proline-rich domain is composed of the amino acid sequence of SEQ ID NO: 5 or 6, the polynucleotide sequence encoding the proline-rich domain may include the nucleotide sequence of SEQ ID NO: 22 or 23, but is not limited thereto .

When the polypeptide further comprises an ER-signal peptide (SS) at the N-terminal side, the ER signal peptide may be composed of the amino acid sequence of SEQ ID NO: 1 or 2, The polynucleotide sequence encoding the signal peptide may comprise, but is not limited to, the nucleotide sequence of SEQ ID NO: 18 or 19.

The polynucleotide encoding the polypeptide comprising the membrane-penetrating domain and the proline-rich domain of the SCOTIN protein may comprise the nucleotide sequence of SEQ ID NOS: 28 to 31, but is not limited thereto.

denomination The sequence (5'-3 ') order
number SCOTIN ATGGCTGCGCCGGCGCCCTCTCTGTGGACCCTATTGCTGCTGCTGTTGCTGCTGCCGCCGCCTCCGGGTGCCCATGGTGAGCTGTGCAGGCCCTTTGGTGAAGACAATTCGATCCCAGTGTTCTGTCCTGATTTCTGTTGTGGTTCCTGTTCCAACCAATACTGCTGCTCGGACGTGCTGAGGAAAATCCAGTGGAATGAGGAAATGTGTCCTGAGCCAGAGTCCAGCAGATTTTCCACCCCCGCGGAGGAGACACCCGAACATCTGGGTTCAGCGCTGAAATTTCGATCCAGTTTTGACAGTGACCCTATGTCAGGGTTCGGAGCGACCGTCGCCATTGGCGTGACCATCTTTGTGGTGTTTATTGCCACTATCATCATCTGCTTCACCTGCTCCTGCTGCTGTCTGTATAAGATGTGCTGCCCCCAACGCCCTGTCGTGACCAACACCACAACTACTACCGTGGTTCATGCCCCTTACCCTCAGCCTCAACCTCAACCTGTGGCCCCCAGCTATCCTGGACCAACATACCAGGGCTACCATCCCATGCCCCCCCAGCCAGGAATGCCAGCAGCACCCTACCCAACGCAGTACCCACCACCCTACCTGGCCCAGCCCACAGGGCCGCCACCCTACCATGAGTCCTTGGCTGGAGCCAGCCAGCCTCCATACAACCCGACCTACATGGATTCCCTAAAGACAATTCCC 26 hSCOTIN ATGACTGCGCCGGTCCCCGCGCCGCGGATCCTGTTGCCGTTGCTGTTGCTGCTGCTGCTAACGCCGCCTCCGGGTGCACGTGGTGAGGTGTGTATGGCTTCCCGTGGACTCAGCCTCTTCCCCGAGTCCTGTCCAGATTTCTGCTGTGGTACCTGTGATGACCAATACTGCTGCTCTGACGTGCTGAAGAAATTTGTGTGGAGCGAGGAAAGGTGTGCTGTGCCTGAGGCCAGCGTGCCTGCCAGTGTAGAGCCGGTGGAGCAGCTGGGCTCGGCGCTGAGGTTTCGCCCTGGCTACAACGACCCCATGTCAGGGTTCGGAGCGACCTTGGCCGTTGGCCTGACCATCTTTGTGCTGTCTGTCGTCACTATCATCATCTGCTTCACCTGCTCCTGCTGCTGCCTTTACAAGACGTGCCGCCGACCACGTCCGGTTGTCACCACCACCACATCCACCACTGTGGTGCATGCCCCTTATCCTCAGCCTCCAAGTGTGCCGCCCAGCTACCCTGGACCAAGCTACCAGGGCTACCACACCATGCCGCCTCAGCCAGGGATGCCAGCAGCACCCTACCCAATGCAGTACCCACCACCTTACCCAGCCCAGCCCATGGGCCCACCGGCCTACCACGAGACCCTGGCTGGAGGAGCAGCCGCGCCCTACCCCGCCAGCCAGCCTCCTTACAACCCGGCCTACATGGATGCCCCGAAGGCGGCCCTC 27 SCOTIN
(TMPRD) GGGTTCGGAGCGACCGTCGCCATTGGCGTGACCATCTTTGTGGTGTTTATTGCCACTATCATCATCTGCTTCACCTGCTCCTGCTGCTGTCTGTATAAGATGTGCTGCCCCCAACGCCCTGTCGTGACCAACACCACAACTACTACCGTGGTTCATGCCCCTTACCCTCAGCCTCAACCTCAACCTGTGGCCCCCAGCTATCCTGGACCAACATACCAGGGCTACCATCCCATGCCCCCCCAGCCAGGAATGCCAGCAGCACCCTACCCAACGCAGTACCCACCACCCTACCTGGCCCAGCCCACAGGGCCGCCACCCTACCATGAGTCCTTGGCTGGAGCCAGCCAGCCTCCATACAACCCGACCTACATGGATTCCCTAAAGACAATTCCC 28 hSCOTIN (TMPRD) GGGTTCGGAGCGACCTTGGCCGTTGGCCTGACCATCTTTGTGCTGTCTGTCGTCACTATCATCATCTGCTTCACCTGCTCCTGCTGCTGCCTTTACAAGACGTGCCGCCGACCACGTCCGGTTGTCACCACCACCACATCCACCACTGTGGTGCATGCCCCTTATCCTCAGCCTCCAAGTGTGCCGCCCAGCTACCCTGGACCAAGCTACCAGGGCTACCACACCATGCCGCCTCAGCCAGGGATGCCAGCAGCACCCTACCCAATGCAGTACCCACCACCTTACCCAGCCCAGCCCATGGGCCCACCGGCCTACCACGAGACCCTGGCTGGAGGAGCAGCCGCGCCCTACCCCGCCAGCCAGCCTCCTTACAACCCGGCCTACATGGATGCCCCGAAGGCGGCCCTC 29 SCOTIN
(SSTMPRD) ATGGCTGCGCCGGCGCCCTCTCTGTGGACCCTATTGCTGCTGCTGTTGCTGCTGCCGCCGCCTCCGGGGTTCGGAGCGACCGTCGCCATTGGCGTGACCATCTTTGTGGTGTTTATTGCCACTATCATCATCTGCTTCACCTGCTCCTGCTGCTGTCTGTATAAGATGTGCTGCCCCCAACGCCCTGTCGTGACCAACACCACAACTACTACCGTGGTTCATGCCCCTTACCCTCAGCCTCAACCTCAACCTGTGGCCCCCAGCTATCCTGGACCAACATACCAGGGCTACCATCCCATGCCCCCCCAGCCAGGAATGCCAGCAGCACCCTACCCAACGCAGTACCCACCACCCTACCTGGCCCAGCCCACAGGGCCGCCACCCTACCATGAGTCCTTGGCTGGAGCCAGCCAGCCTCCATACAACCCGACCTACATGGATTCCCTAAAGACAATTCCC 30 hSCOTIN (SSTMPRD) ATGACTGCGCCGGTCCCCGCGCCGCGGATCCTGTTGCCGTTGCTGTTGCTGCTGCTGCTAACGCCGCCTCCGGGGTTCGGAGCGACCTTGGCCGTTGGCCTGACCATCTTTGTGCTGTCTGTCGTCACTATCATCATCTGCTTCACCTGCTCCTGCTGCTGCCTTTACAAGACGTGCCGCCGACCACGTCCGGTTGTCACCACCACCACATCCACCACTGTGGTGCATGCCCCTTATCCTCAGCCTCCAAGTGTGCCGCCCAGCTACCCTGGACCAAGCTACCAGGGCTACCACACCATGCCGCCTCAGCCAGGGATGCCAGCAGCACCCTACCCAATGCAGTACCCACCACCTTACCCAGCCCAGCCCATGGGCCCACCGGCCTACCACGAGACCCTGGCTGGAGGAGCAGCCGCGCCCTACCCCGCCAGCCAGCCTCCTTACAACCCGGCCTACATGGATGCCCCGAAGGCGGCCCTC 31 SCOTIN
(SSCRD) ATGGCTGCGCCGGCGCCCTCTCTGTGGACCCTATTGCTGCTGCTGTTGCTGCTGCCGCCGCCTCCGGGTGCCCATGGTGAGCTGTGCAGGCCCTTTGGTGAAGACAATTCGATCCCAGTGTTCTGTCCTGATTTCTGTTGTGGTTCCTGTTCCAACCAATACTGCTGCTCGGACGTGCTGAGGAAAATCCAGTGGAATGAGGAAATGTGTCCTGAGCCAGAGTCCAGCAGATTTTCCACCCCCGCGGAGGAGACACCCGAACATCTGGGTTCAGCGCTGAAATTTCGATCCAGTTTTGACAGTGACCCTATGTCA 32 SCOTIN
(SSTMCRD) ATGGCTGCGCCGGCGCCCTCTCTGTGGACCCTATTGCTGCTGCTGTTGCTGCTGCCGCCGCCTCCGGGTGCCCATGGTGAGCTGTGCAGGCCCTTTGGTGAAGACAATTCGATCCCAGTGTTCTGTCCTGATTTCTGTTGTGGTTCCTGTTCCAACCAATACTGCTGCTCGGACGTGCTGAGGAAAATCCAGTGGAATGAGGAAATGTGTCCTGAGCCAGAGTCCAGCAGATTTTCCACCCCCGCGGAGGAGACACCCGAACATCTGGGTTCAGCGCTGAAATTTCGATCCAGTTTTGACAGTGACCCTATGTCAGGGTTCGGAGCGACCGTCGCCATTGGCGTGACCATCTTTGTGGTGTTTATTGCCACTATCATCATCTGCTTC 33 SCOTIN
(SSPRD) ATGGCTGCGCCGGCGCCCTCTCTGTGGACCCTATTGCTGCTGCTGTTGCTGCTGCCGCCGCCTCCGTGCTTCACCTGCTCCTGCTGCTGTCTGTATAAGATGTGCTGCCCCCAACGCCCTGTCGTGACCAACACCACAACTACTACCGTGGTTCATGCCCCTTACCCTCAGCCTCAACCTCAACCTGTGGCCCCCAGCTATCCTGGACCAACATACCAGGGCTACCATCCCATGCCCCCCCAGCCAGGAATGCCAGCAGCACCCTACCCAACGCAGTACCCACCACCCTACCTGGCCCAGCCCACAGGGCCGCCACCCTACCATGAGTCCTTGGCTGGAGCCAGCCAGCCTCCATACAACCCGACCTACATGGATTCCCTAAAGACAATTCCC 34

One example of the present invention relates to a recombinant vector comprising a polynucleotide encoding said polypeptide.

The recombination vector may have a cleavage map as shown in Fig.

The recombinant vector may be a viral or non-viral vector.

The viral vector may be, but is not limited to, adenovirus, adeno-associated virus, helper-dependent adenovirus, retroviral vector, and the like.

The term "vector" means means for expressing a gene of interest in a host cell. The vector includes elements for expression of a target gene and may include a replication origin, a promoter, an operator, a transcription termination terminator, and the like into the genome of the host cell. Ribosome binding sites (RBS) for translation into selectable markers and / or proteins to confirm successful introduction into the host cell and / or appropriate enzyme sites for introduction of the IRES (e.g., IRES (Internal Ribosome Entry Site), and the like. The vector may be engineered in a conventional genetic engineering manner to have the fusion polynucleotide (fusion promoter) described above as a promoter. The vector may further comprise a transcription control sequence other than the promoter (e.g., an enhancer, etc.).

The recombinant vector may be constructed by a variety of methods known in the art.

The transfer (introduction) of the recombinant vector into a cell can be carried out using a carrier method well known in the art. For example, microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, gene bombardment, and the like can be used as the delivery method. For example, liposome-mediated transfection (Lipofector reagent). < / RTI >

Another example of the present invention provides a recombinant cell comprising the polynucleotide or a recombinant vector. The recombinant cell may be a recombinant cell obtained by transforming a host cell with the recombinant vector.

The host cell may be selected from the group consisting of viruses, bacteria, plant cells, insect cells, mammalian cells, for example, mammalian cells other than human, but is not limited to, Lt; / RTI > cells.

The above-mentioned SCOTIN protein, SCOTIN protein fragment or its mutant has an effect of binding to the NS5A protein necessary for HCV proliferation and transferring the NS5A protein to an autotrophic somatic cell. Thus, the above-mentioned SCOTIN protein, SCOTIN protein fragment or variant thereof has an antiviral effect on HCV and / or a preventive or therapeutic effect on HCV infection or diseases related thereto.

Another example of the present invention is a polypeptide comprising a SCOTIN protein, a transmembrane domain (TM) of a SCOTIN protein and a proline-rich domain (PRD), a polynucleotide encoding the SCOTIN protein or polypeptide , A recombinant vector comprising the polynucleotide, a recombinant cell comprising the recombinant vector, or a mixture thereof, as an active ingredient. The present invention also provides an antiviral composition for hepatitis C virus (HCV).

Another example of the present invention is a polypeptide comprising a SCOTIN protein, a transmembrane domain (TM) of a SCOTIN protein and a proline-rich domain (PRD), a polynucleotide encoding the SCOTIN protein or polypeptide , A prophylactic or therapeutic agent for a hepatitis C virus (HCV) infection or infection associated with an effective component, a recombinant vector comprising the polynucleotide, a recombinant cell comprising the recombinant vector, or a mixture thereof &Lt; / RTI > The disease associated with the HCV infection may be hepatitis C, cirrhosis, cirrhosis, or liver cancer.

For example, the SCOTIN protein may be a protein consisting of SEQ ID NO: 9 and / or SEQ ID NO: 10. The nucleotide sequence encoding the SCOTIN protein may be a nucleotide sequence of SEQ ID NO: 26 or 27, but is not limited thereto.

The transmembrane domain included in the polypeptide may be composed of the amino acid sequence of SEQ ID NO: 3 or 4, but is not limited thereto. The polynucleotide sequence encoding the transmembrane domain may include, but is not limited to, the nucleotide sequence of SEQ ID NO: 20 or 21.

The proline-enriched domain contained in the polypeptide may be composed of the amino acid sequence of SEQ ID NO: 5 or 6, but is not limited thereto. The polynucleotide sequence encoding the proline-rich domain may comprise the nucleotide sequence of SEQ ID NO: 22 or 23, but is not limited thereto.

The polypeptide comprises a sequence selected from the group consisting of 132 to 260 consecutive, 132 to 240, 152 consecutive amino acids in the SCOTIN protein comprising a proline enrichment domain consisting of the amino acid sequence of SEQ ID NO: 5 or 6 and the transmembrane domain consisting of the amino acid sequence of SEQ ID NO: From 1 to 260, 152 to 240, 153 to 260, 153 to 240, 173 to 260, 173 to 240, such as 132 to 256, 132 to 236, 152 to 256, (Mouse-derived SCOTIN protein), or 136 to 260, 136 to 240, 156 to 260, 156 to 240, 153 to 256, 153 to 236, 173 to 256, or 173 to 236 160 to 260, 160 to 240, 180 to 260, or 180 to 240 (human-derived SCOTIN protein). The polypeptide may comprise the amino acid sequence of SEQ ID NOS: 11 to 14, but is not limited thereto.

The polypeptide contained in the composition may further include an ER-signal peptide (SS) at the N-terminus.

The envelope position signal peptide contained in the protein contained in the composition may be composed of the amino acid sequence of SEQ ID NO: 1 or 2, but is not limited thereto. The polynucleotide sequence encoding the ER locus signal peptide may include, but is not limited to, the nucleotide sequence of SEQ ID NO: 18 or 19.

The recombinant vector contained in the composition may have a cleavage map as shown in Fig.

The recombinant vector may be a viral or non-viral vector.

In the above composition, the active ingredient may be contained in a pharmaceutically effective amount capable of exhibiting a desired preventive and / or therapeutic effect.

When the protein contained in the composition is over-expressed using a subgenomic HCV replicon cell line that specifically reflects the HCV proliferation process, HCV proliferation is inhibited, and HCV proliferation is activated when the expression of the protein is decreased.

In this regard, the amount of NS5A protein, which plays an essential role in HCV proliferation, is decreased by the overexpression of the protein without HCV RNA or other HCV protein, and is increased by the expression of the HCV. This shows that the NS5A protein itself is regulated by the protein.

The term "over-expressing" means expressing greater than the amount expressed in normal cells. In addition, the term " small expression "or" low expression "means expression in an amount less than that expressed in normal cells.

When the autophagy-dependent pathway involved in intracellular proteolysis is inhibited, the effect of decreasing the NS5A protein by the protein is lost, and when the proteasome-dependent pathway is inhibited, the effect of decreasing the NS5A protein by the protein is maintained. This means that the reduction effect of the protein on the NS5A protein is mediated by autophagy.

In the present invention, the binding of SCOTIN protein to the NS5A protein was confirmed by the GST-pulldown assay, and intracellular SCOTIN protein can be confirmed not only in the endoplasmic reticulum but also in the autophagocyte.

In order to clarify the domains required for the movement of SS, CRD, TM, and PRD domains constituting the SCOTIN protein to autophagy bodies, a deletion mutant protein (deletion mutant protein) Was essential for movement to autophagic bodies.

In addition, it was confirmed that when the SCOTIN protein was overexpressed, the NS5A protein enhanced the migration to the autophagic body and the disinfectant. Therefore, it was confirmed that only TMPRD polypeptide including SS among the SS, CRD, TM, and PRD domains constituting the SCOTIN protein showed an NS5A protein reduction effect similar to that of the SCOTIN protein. This indicates that only a polypeptide including TMPRD is a full-length SCOTIN Because they show a similar level of protein binding to the NS5A protein. Therefore, it was confirmed that only the polypeptide including TMPRD showed HCV proliferation inhibitory effect similar to that of SCOTIN protein. Suggesting that the SCOTIN protein may function as an HCV proliferation inhibitor through the TMPRD domain.

In the present invention, it was confirmed that domain 1, domain 2, and domain 3 of the NS5A protein are required for binding to the SCOTIN protein and for controlling the degradation by the SCOTIN protein. This means that SCOTIN protein binds through domain 2 and promotes degradation.

The "NS5A protein" was amplified from the portion of the hepatitis C virus strain J33 genomic RNA encoding the NS5A protein. The accession number of the RNA was D14484.1, and the accession number of the protein was BAA03375.1. Table 5 below shows the total amino acid sequence of the NS5A protein and the amino acid sequence of each domain constituting the NS5A protein.

denomination The sequence (5'-3 ') SEQ ID NO: NS5A SGSWLRDVWDWICTVLTDFKTWLQSKLLPRLPGVPFFSCQRGYKGVWRGEGIMQTTCPCGAQIAGHVKNGSMRIVGPRTCSNTWHGTFPINAYTTGPCSPSPAPNYSRALWRVAAEEYVEVTRVGDFHYVTGVTTDNVKCPCQVPAPEFFTELDGVRLHRYAPACKPLLRDEVSFQVGLNQYLVGSQLPCEPEPDVAVLTSMLTDPSHITAETAKRRLARGSPPSLASSSASQLSAPSLKATCTIHHDSPDADLIEANLLWRQEMGGNITRVESENKVVILDSFEPIRAEEDEREVSVPAEILRRSRKFPAAMPIWARPDYNPPLLESWKDPDYVPPVVHGCPLPPTKAAPIPPPRRKRTIVLTESTVSSALAELATKTFGGSGSSAADSGTATAPPDQTSDDGDKESDVESYSSMPPLEGEPGDPDLSDGSWSTVSEEASEDVVCC 35 Domain 1 SGSWLRDVWDWICTVLTDFKTWLQSKLLPRLPGVPFFSCQRGYKGVWRGEGIMQTTCPCGAQIAGHVKNGSMRIVGPRTCSNTWHGTFPINAYTTGPCSPSPAPNYSRALWRVAAEEYVEVTRVGDFHYVTGVTTDNVKCPCQVPAPEFFTELDGVRLHRYAPACKPLLRDEVSFQVGLNQYLVGSQLPCEPEPDVAVLTSMLTDPSHITAET 36 Domain 2 AKRRLARGSPPSLASSSASQLSAPSLKATCTIHHDSPDADLIEANLLWRQEMGGNITRVESENKVVILDSFEPIRAEEREEREVSVPAEILRRSRKFPAAMPIWARPDYNPPLLESWKDPDYVPPVVHGC 37 Domain 3 &Lt; RTI ID = 0.0 > 38

Another example of the present invention provides a pharmaceutical preparation containing the composition as an active ingredient.

The pharmaceutical preparations according to the present invention may be formulated into oral preparations such as powders, granules, tablets, capsules, ointments, suspensions, emulsions, syrups and aerosols or percutaneous preparations such as suppositories and sterilized injection solutions Parenteral formulations, and the like.

The pharmaceutical preparations of the present invention may be pharmaceutically acceptable and additionally contain adjuvants such as physiologically acceptable carriers, excipients and diluents. Examples of carriers, excipients and diluents that can be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium Silicates, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, diluents or excipients such as fillers, extenders, binders, humectants, disintegrants, surfactants and the like which are usually used can be used

When the pharmaceutical preparation according to the present invention is used for parenteral administration, for example, a topical administration such as a liquid preparation, a gel preparation, a cleaning composition, a tablet for insertion, a suppository form, cream, ointment, dressing solution, spray, It may be a sterile aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a freeze-dried preparation, a suppository, a cream, an ointment, a jelly, a foam, a cleanser or an insert, May be a skin external agent such as a liquid preparation, a gel preparation, a cleaning composition, and a tablet for insertion. The formulation can be prepared, for example, by adding a solubilizer, an emulsifying agent, a buffering agent for pH control, etc. to the sterilized water. Examples of the non-aqueous solvent or suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like.

More specifically, the pharmaceutical preparation may comprise a carrier for additionally formulating the composition. The carrier may be a binder, a lubricant, a suspending agent, a solubilizer, a buffer, a preservative, a lubricant, an isotonic agent, an excipient, a stabilizer, a dispersant, a suspending agent,

In embodiments where the pharmaceutical preparations of the present invention are applied to humans, the pharmaceutical preparations of the present invention may be administered alone, but are generally administered in a pharmacological manner selected in consideration of the mode of administration and standard phamaceutical practice May be administered in admixture with a carrier. For example, the pharmaceutical preparations of the present invention may be in the form of tablets containing starch or lactose, or in capsules containing the active ingredient alone or as an excipient, or as an elixir or a suspending agent containing a chemical that tastes or colors 0.0 > oral, < / RTI > intraoral, or sublingually.

The dose of the pharmaceutical preparation of the present invention may be varied depending on the age, body weight, sex, dosage form, health condition and disease severity of the patient, and may be determined once or several times per day It may be administered in divided doses. For example, a daily dosage of 0.001 to 10000 mg / kg, 0.01 to 10000 mg / kg, 0.1 to 10000 mg / kg, 0.5 to 10000 mg / kg, based on the content of the active ingredient (i.e., SCOTIN protein or its coding polynucleotide or mixture thereof) Kg, 0.1 to 1000 mg / kg, 0.001 to 1000 mg / kg, 0.01 to 1000 mg / kg, 0.1 to 1000 mg / kg, Kg, 0.5 to 500 mg / kg, 0.001 to 300 mg / kg, 0.01 to 300 mg / kg, 0.1 to 300 mg / kg, or 0.5 to 300 mg / kg. The above-mentioned dosage is an average case, and the dose may be high or low depending on individual differences.

If the daily dose of the pharmaceutical preparation of the present invention is less than the above dose, no significant effect can be obtained, and if it exceeds the above range, it is not only economical but also causes an undesirable side effect due to deviation from the commercial dose range It is preferable to set the above range.

Another embodiment of the invention provides a method of treating or preventing a hepatitis C virus (HCV) infection comprising administering a pharmaceutically effective amount of the composition.

The composition used in the above method for treating or preventing hepatitis C virus (HCV) infection is the same as described above.

Another embodiment of the present invention provides a method of inhibiting replication of hepatitis C virus comprising contacting said composition with hepatitis C virus (HCV).

The method of inhibiting the replication of hepatitis C virus may be performed in vivo or in vitro.

The composition used in the method for inhibiting the replication of hepatitis C virus is the same as described above.

The above-mentioned treatment or prevention method and the method of inhibiting the replication of hepatitis C virus may be performed in vitro or may be performed on an animal other than human.

Another example of the present invention is a polypeptide comprising a SCOTIN protein, a transmembrane doamin (TM) of a SCOTIN protein and a proline-rich domain (PRD), a coding polynucleotide thereof or a mixture thereof Gt; Hepatitis C < / RTI > virus (HCV) infection.

The present invention relates to a polypeptide comprising a transmembrane domain and a proline rich domain of SCOTIN protein, a polynucleotide encoding said polypeptide, a recombinant vector comprising said polynucleotide, or a polynucleotide encoding said polypeptide, polynucleotide, A pharmaceutical composition for preventing or treating a hepatitis C virus (HCV) infection or a disease associated with infection, or an antiviral composition for hepatitis C virus (HCV) And a method for treating or preventing HCV infection using the composition, wherein the protein comprising the transmembrane domain of the SCOTIN protein and the proline-rich domain is selected from the group consisting of NS5A (hepatitis C virus) Promoting the degradation of proteins, Bar to suppress the expression, the use of self-NS5A protein degradation controlled study with phagocytosis, or may be usefully employed in preventive and / or therapeutic treatment against HCV infection.

FIG. 1 is a graph showing the results of confirming HCV proliferation inhibitory effect through overexpression of SCOTIN protein according to an embodiment of the present invention.
FIG. 2 is a graph showing the results of confirming that HCV proliferation is increased when SCOTIN protein is inhibited through SCOTIN siRNA according to an embodiment of the present invention.
3 is a photograph showing the result of confirming the amount of NS5A protein when overexpressing SCOTIN protein according to an embodiment of the present invention.
FIG. 4 is a photograph showing the result of confirming the amount of NS5A protein when SCOTIN protein was expressed in small amount according to an embodiment of the present invention.
FIG. 5 is a photograph showing changes in the amount of NS5A protein caused by overexpression of SCOTIN protein according to an embodiment of the present invention, which is observed by 3-MA, BFA, and MG-132 treatment.
FIG. 6 is a photograph showing the binding between the NS5A protein and the SCOTIN protein according to an embodiment of the present invention, using a GST-pulldown assay.
FIG. 7 is a photograph showing the intracellular location of the NS5A protein and the SCOTIN protein on the GFP-LC3 spot according to an embodiment of the present invention by immunofluorescence staining.
FIG. 8 is a graph showing the results of analysis of the intracellular location of the NS5A protein on the GFP-LC3 spot by overexpression of the SCOTIN protein according to an embodiment of the present invention by 80 and 63 immunofluorescence staining, respectively to be.
9 is a photograph showing the intracellular location of the NS5A protein and the SCOTIN protein in the GFP-LAMP1 position according to an embodiment of the present invention by immunofluorescence staining.
10 is a schematic diagram of fragments of SCOTIN protein and SCOTIN protein according to an embodiment of the present invention.
FIG. 11 is a photograph showing a fragment of SCOTIN protein and GFP-LC3 spots according to an embodiment of the present invention and a result of confirming the intracellular location of SCOTIN protein by immunofluorescence staining.
FIG. 12 is a photograph showing the result of Western blot analysis of the SCOTIN protein fragment and the SCOTIN protein effect on the amount of NS5A protein according to an embodiment of the present invention.
FIG. 13 is a photograph showing the binding between the NS5A protein and SCOTIN protein fragments or SCOTIN protein according to an embodiment of the present invention in a GST-pulldown assay.
FIG. 14 is a photograph showing the results of confirming HCV RNA level by RT-qPCR by fragment expression of SCOTIN protein according to an embodiment of the present invention.
FIG. 15 is a result of Western blot analysis of HCV NS5A protein level by fragment expression of SCOTIN protein according to an embodiment of the present invention,
16 is a schematic diagram of an NS5A deletion protein according to an embodiment of the present invention.
FIG. 17 is a photograph showing the results of Western blot analysis of changes in the amount of NS5A deletion protein and NS5A protein by the haplotype of SCOTIN protein according to an embodiment of the present invention.
FIG. 18 is a photograph showing the binding between SCOTIN protein and GST-NS5A deletion protein or GST-NS5A protein according to an embodiment of the present invention in a GST-pulldown assay.
FIG. 19 is a photograph showing the effect of mouse overexpression of SCOTIN protein on human-derived SCOTIN protein (hSCOTIN protein) overexpression and 3-MA treatment according to one embodiment of the present invention on HCV proliferation.
20 shows a cleavage map of a SCOTIN-V5 vector according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Experimental Example 1. Plasmid preparation

Nucleotide sequences of SEQ ID NOs: 26 to 34 encoding SCOTIN protein or SCOTIN protein fragment were amplified by PCR and cloned into pDEST-51 or pcDNA3.1 vector to construct a plasmid expressing SCOTIN protein or recombinant SCOTIN protein. The amino acid sequences of the SCOTIN protein or SCOTIN protein fragment are shown in Table 6 below.

denomination The sequence (5'-3 ') Amino acid sequence number Coding
Nucleotide
SEQ ID NO: SCOTIN MAAPAPSLWTLLLLLLLLPPPPGAHGELCRPFGEDNSIPVFCPDFCCGSCSNQYCCSDVLRKIQWNEEMCPEPESSRFSTPAEETPEHLGSALKFRSSFDSDPMSGFGATVAIGVTIFVVFIATIIICFTCSCCCLYKMCCPQRPVVTNTTTTTVVHAPYPQPQPQPVAPSYPGPTYQGYHPMPPQPGMPAAPYPTQYPPPYLAQPTGPPPYHESLAGASQPPYNPTYMDSLKTIP 9 26 hSCOTIN MTAPVPAPRILLPLLLLLLLTPPPGARGEVCMASRGLSLFPESCPDFCCGTCDDQYCCSDVLKKFVWSEERCAVPEASVPASVEPVEQLGSALRFRPGYNDPMSGFGATLAVGLTIFVLSVVTIIICFTCSCCCLYKTCRRPRPVVTTTTSTTVVHAPYPQPPSVPPSYPGPSYQGYHTMPPQPGMPAAPYPMQYPPPYPAQPMGPPAYHETLAGGAAAPYPASQPPYNPAYMDAPKAAL 10 27 SCOTIN
(TMPRD) GFGATVAIGVTIFVVFIATIIICFTCSCCCLYKMCCPQRPVVTNTTTTTVVHAPYPQPQPQPVAPSYPGPTYQGYHPMPPQPGMPAAPYVVTNTTTTTVVHAPYPQPQPQPVAPSYPGPYNPTYMDSLKTIP 11 28 hSCOTIN (TMPRD) GFGATLAVGLTIFVLSVVTIIICFTCSCCCLYKTCRRPRPVVTTTTSTTVVHAPYPQPPSVPPSYPGPSYQGYHTMPPQPGMPAAPYPMQYPPPYPAQPMGPPAYHETLAGGAAAPYPASQPPYNPAYMDAPKAAL 12 29 SCOTIN
(SSTMPRD) MAAPAPSLWTLLLLLLLPPPPGFGATVAIGVTIFVVFIATIIICFTCSCCCLYKMCCPQRPVVTNTTTTTVVHAPYPQPQPQPVAPSYPGPTYQGYHPMPPQPGMPAAPYPTQYPPPYLAQPTGPPPYHESLAGASQPPYNPTYMDSLKTIP 13 30 hSCOTIN (SSTMPRD) MTAPVPAPRILLPLLLLLLLTPPPGFGATLAVGLTIFVLSVVTIIICFTCSCCCLYKTCRRPRPVVTTTTSTTVVHAPYPQPPSVPPSYPGPSYQGYHTMPPQPGMPAAPYPMQYPPPYPAQPMGPPAYHETLAGGAAAPYPASQPPYNPAYMDAPKAAL 14 31 SCOTIN
(SSCRD) MAAPAPSLWTLLLLLLLPPPPGAHGELCRPFGEDNSIPVFCPDFCCGSCSNQYCCSDVLRKIQWNEEMCPEPESSRFSTPAEETPEHLGSALKFRSSFDSDPMS 15 32 SCOTIN
(SSTMCRD) MAAPAPSLWTLLLLLLLPPPPGAHGELCRPFGEDNSIPVFCPDFCCGSCSNQYCCSDVLRKIQWNEEMCPEPESSRFSTPAEETPEHLGSALKFRSSFDSDPMSGFGATVAIGVTIFVVFIATIIICF 16 33 SCOTIN
(SSPRD) MAAPAPSLWTLLLLLLLLPPPPCFTCSCCCLYKMCCPQRPVVTNTTTTTVVHAPYPQPQPQPVAPSYPGPTYQGYHPMPPQPGMPAAPYPTQYPPPYLAQPTGPPPYHESLAGASQPPYNPTYMDSLKTIP 17 34

Experimental Example 2. Real time Reverse transcription The polymerization reaction (real-time RT- PCR )

The total RNA of the Huh-neo-5-15 cell line (Bartenschlager, R., University of Heidelberg, Germany) was extracted using RNAiso plus (Takara, Japan) and the extracted RNA was purified using the Improm-II reverse transcription system , USA).

Specifically, for the real-time reverse transcription polymerase chain reaction, 1 ug of the extracted RNA and 5 pmol of each primer specifically recognizing each target RNA shown in Table 4, SYBR premix Ex-Taq 2.5 μl (Takara, Japan), 50 × Rox 0.2 ul (Takara, Japan), and 1 ul of cDNA were reacted in a One-step ^TM Real-time PCR system (Applied Biosystem).

The PRC reaction was carried out for 15 cycles at 95 ° C for 15 minutes, followed by 40 cycles of 1 cycle consisting of 95 ° C for 15 seconds, 57 ° C for 15 seconds, and 72 ° C for 15 seconds, followed by a melting curve stage 95 ° C for 15 seconds, 72 ° C for 1 minute, and 95 ° C for 15 seconds. The sequences of the primers that specifically recognize each target RNA used in the experiment are shown in Table 7 below.

Primer The sequence (5'-3 ') SEQ ID NO: NS4_F ACAACAGGCAGCGTGGTCATT 39 NS4_R TTCCACATGTGCTTTGCCCA 40 SCOTIN_F TGCTGTGGTACCTGTGATGACCAA 41 SCOTIN_R AGGAGCAGGTGAAGCAGATGATGA 42 RPL32_F AACCCAGAGGCATTGACAAC 43 RPL32_R GTTGCACATCAGCAGCACTT 44

Experimental Example 3. Western Blat And immunofluorescence staining

Western blot was prepared by dissolving Huh7 cells (professor Jang Ki-Seung, POSTECH) with lysis buffer (25 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate) The seafood was separated by SDS-PAGE and then analyzed by GFP (Santacruz), SCOTIN (Santacruz), ACTIN (Santacruz), Lamin B2 (Santacruz), c-MYC (Santacruz), V5 (Invitrogen), GST Virogen) and Flag (Sigma) were used to confirm the amount of each protein.

In addition, immunofluorescence staining is performed by incubating Huh-7 cell line (Professor Chang Seung-Ki, POSTECH) on cover-glass and fixing cells with 4% paraformaldehyde. The cells were then treated with 0.2% TritonX-100 to increase the permeability of the cells and the anti-Rabbit IgG-Alexa Fluor 568 (supplied by FLAC (Santacruz), MYC (Santacruz), SCOTIN (Lane, DP of Dundee, UK) Invitrogen antibody, and anti-mouse IgG-Alexa Fluor 405 (Invitrogen) antibody. Hoechst 33258 (Sigma) was used for nuclear staining. Fluorescence signals were confirmed by fluorescence microscopy. Through this, the intracellular location of the target protein could be confirmed.

Experimental example 4. GST-pulldown assay

HEK293 cells were lysed with lysis buffer (25 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate) to obtain cell lysates. 1 mg of the cell lysate was made up to 1 ml with dissolution buffer and then rotated with glutathion-sepharose beads (Amersham) for 20 minutes at 4 for 12 hours. The beads were washed 3 times with 1 ml of dissolution buffer, and then treated with laemmli sample buffer (Biorad) at 95 ° C for 10 min. The amount of protein was confirmed by Western blotting. For the input, 20 ug of cell lysate from each sample was used.

Example 1. Measurement of inhibitory effect of HCV proliferation by SCOTIN protein

Huh-neo-5-15 replicon cells were stably transfected with Huh-7 cells to ensure stable expression of the HCV-proliferating complex in Lohmann V (1999) Replication of Subgenomic Hepatitis C Virus RNAs in a Hepatoma Cell Line. Science 285: 110-113. , Which was obtained from Bartenschlager, R. (University of Heidelberg, Germany).

Specifically, 1 ug of (+) and 3 ug of SCOTIN-V5 vector were introduced into Huh-neo-5-15 replicon cells by liposome infusion, cultured in 10% FBS DMEM media for 48 hours, and RNAiso plus (Takara, Japan). The SCOTIN-V5 vector was a vector containing the SCOTIN protein coding nucleotide (SEQ ID NO: 26) among the vectors prepared in Experimental Example 1 (see FIG. 20), and the negative SCOTIN-V5 vector was a control to be.

The level of HCV RNA in the replicon cells was determined by measuring the level of NS4 mRNA and the relative expression of NS4 mRNA was measured using the primer set of SEQ ID NOS: 39 and 40 described in Table 7 using RT-qPCR . The results obtained above are shown in Fig.

As can be seen in FIG. 1, when the SCOTIN protein was overexpressed (++), it was confirmed that the HCV RNA level (NS4 mRNA level) was markedly reduced as compared with the control (-) cell not transformed with the SCOTIN-V5 vector I could.

Next, HCV RNA levels (measured at the level of NS4 mRNA) and SCOTIN mRNA levels were measured after transfection of control siRNA and SCOTIN siRNA into Huh-neo-5-15 replicon cells with the levels of SEQ ID NOs: 39 and 40 QPCR using the primer set and the primer set of SEQ ID NOS: 41 and 42 in the manner described in Experimental Example 2.

For siCON, control siRNA (++) was 100 pmol and (+) was 50 pmol. For siSCOTIN (++), 33 pmol of SCOTIN siRNA # 1, 33 pmol of SCOTIN siRNA # 2, and 33 pmol of SCOTIN siRNA # 3, 16.7 pmol of SCOTIN siRNA # 1, 16.7 pmol of SCOTIN siRNA # 2, and 16.7 pmol of SCOTIN siRNA # 3 were mixed and used.

The primers and conditions used for the RT-qPCR were as described in Experimental Example 2 and Table 7 above. The control siRNA and SCOTIN siRNA used at this time are summarized in Table 8 below. The results obtained are shown in Fig.

siRNA The sequence (5'-3 ') SEQ ID NO: SCOTIN siRNA # 1 GUACCUGUGAUGACCAAUATT 45 SCOTIN siRNA # 2 GAGCUGUCUUAGCUCAAAUTT 46 SCOTIN siRNA # 3 GGUCUGUACACUUGUUUAUTT 47 control siRNA UUCUCCGAACGUGUCACGUUU 48

As can be seen in FIG. 2, SCOTIN siRNA (siSCOTIN) treatment showed a decrease in SCOTIN RNA level (right) compared with control siRNA (siCON) treatment, while HCV RNA level (NS4 mRNA level) (Left). That is, when the expression of SCOTIN protein is inhibited, the level of HCV RNA is increased. As a result, HCV proliferation is induced by inhibiting the expression of SCOTIN protein.

Example 2. Measurement of reduction effect of NS5A protein by SCOTIN protein

VEGF (-); empty plasmid (pDEST) 4 ug / (+); empty plasmid 3 ug + SCOTIN-V5 (-); V5 1ug / (++) empty plasmid 2ug + SCOTIN-V5 2ug / (+++) SCOTIN-V5 4ug) and the cellular FLAG-NS5A protein level was confirmed by Western blotting (see Experimental Example 3). The obtained results are shown in Fig.

As can be seen in FIG. 3, it was confirmed that the level of NS5A protein was significantly decreased as the expression of SCOTIN protein increased (IB: FLAG). The level of GST protein introduced with the intracellular proteins Lamin B2 and FLAG-NS5A did not change in the SCOTIN protein (IB: Lamin B2), indicating that the NS5A protein was specifically affected by overexpression of the SCOTIN protein do.

Then, FLAG-labeled NS5A was transfected into control siRNA, each of the three kinds of SCOTIN siRNAs listed in Table 8, or all three of them, and Huh-7 cells, and the level of intracellular FLAG-NS5A protein was determined by western blot ) (See Experimental Example 3), and the results are shown in FIG.

As shown in FIG. 4, it was confirmed that the level of FLAG-NS5A protein was increased by SCOTIN protein expression by SCOTIN siRNA action. The level of LacZ-V5 protein introduced with the intracellular proteins ACTIN and FLAG-NS5A was not altered by the small expression of SCOTIN protein, suggesting that the expression of SCOTIN protein was specific to FALG-NS5A protein .

Example 3. Control effect of NS5A protein by SCOTIN protein

Protein degradation in cells can be divided into proteasome-dependent pathways and autopoiesis-dependent pathways. Therefore, Western blotting was performed to determine whether treatment of NS5A protein by overexpression of SCOTIN protein when treated with MG132 or autophagic inhibitors 3-methyladeine (3-MA) and bafilomycin A1 (BFA) inhibiting proteasome- And the results are shown in Fig. DMSO was a control for MG132, BFA and DW was used as a control for 3-MA (see Experimental Example 3).

As can be seen from FIG. 5, NS5A protein reduction effect was observed by overexpression of SCOTIN protein even after treatment with MG132. However, it was confirmed that after 3-MA or BFA treatment, the effect of decreasing NS5A protein by SCOTIN protein overexpression disappeared. These results indicate that the SCOTIN protein controls the NS5A protein through an autophagy dependent pathway.

Then, to examine whether SCOTIN protein directly regulates the NS5A protein, GST-NS5A protein and SCOTIN-MYC protein were introduced into HEK293 cells, and GST pulldown assay was performed to confirm binding of NS5A protein and SCOTIN protein, The results are shown in Fig. 6 (see Experimental Example 4).

As shown in FIG. 6, the GST-NS5A protein binds to the SCOTIN-MYC protein and is pulldowned with glutathione sepharose beads (+ / +), which means that the SCOTIN protein binds to the NS5A protein.

The protein degradation by autophagy is carried out through the process of protein migration into autophagic bodies and autophagy bodies binding to lysosomes. The NS5A protein and SCOTIN protein are transported to autophagic bodies, together with the GFP-LC3 vector, -7 cells were transfected and confirmed by immunofluorescence staining (see Experiment 4), and the results are shown in FIGS. 7 and 8

As can be seen in FIG. 7, the SCOTIN protein is located in an autopagophoric entity appearing in the form of a GFP-LC3 spot, while the NS5A protein was found at a different location (center) within the GFP-LC3 spot and within the cell. In addition, when the NS5A protein was overexpressed with the SCOTIN protein, it was confirmed that the NS5A protein was located in the GFP-LC3 spot together with the SCOTIN protein (below).

In addition, as shown in FIG. 8, NS5A protein of the 40% cell was located at the GFP-LC3 spot (+/-) in the cell group overexpressing the NS5A protein only, but 73% cell Of the NS5A protein was located in the GFP-LC3 spot (+ / +). This means that the SCOTIN protein increases the migration of the NS5A protein to the autolysed body.

Then, NS5A expression vector and SCOTIN expression vector were transformed into Huh-7 cells together with GFP-LAMP1 vector to determine the intracellular location of NS5A protein and SCOTIN protein in the lysosomes by immunofluorescence staining And the results are shown in Fig. 9 (see Experimental Example 5).

As shown in FIG. 9, when the SCOTIN protein was expressed together with the NS5A protein (below), the NS5A protein was located at the same position as the lysosomal marker protein GFP-LAMP1 (middle), unlike the NS5A protein expression I could confirm. These results indicate that the SCOTIN protein can promote the degradation in lysosomes by increasing the migration of the NS5A protein to the lysosomes binding to autophagic bodies.

Example 3. NS5A Protein control and HCV Necessary for proliferation control SCOTIN Identification of domains

The SCOTIN protein consists of SS, CRD, TM, and PRD domains. In order to examine the SCOTIN domain required for NS5A protein control, SCOTIN protein fragments having the respective domains were prepared as shown in FIG. Next, each SCOTIN protein fragment expression vector or SCOTIN protein expression vector was transformed into Huh7 cells together with the GFP-LC3 expression vector, followed by immunofluorescence staining (see Experimental Example 3), and the results are shown in FIG. 11 .

11, SCOTIN protein fragments and SCOTIN-MYC proteins containing TMPRD were observed at the same positions as the GFP-LC3 spots (fourth and fifth lines), but other SCOTIN protein fragments (CRD, TMCRD, PRD ) Was observed at a different location from the GFP-LC3 spot (lines 1 to 3). This means that the TMPRD domain is required for the transfer of the SCOTIN protein to autophagic bodies.

Next, a SCOTIN protein fragment expression vector or a SCOTIN protein expression vector was transformed into Huh7 cells together with a FLAG-NS5A expression vector, followed by Western blotting (see Experimental Example 3), and the results are shown in FIG.

As shown in FIG. 12, the amount of FLAG-NS5A protein decreased when SCOTIN protein and SCOTIN-MYC protein containing TMPRD were expressed, while the amount of FLAG-NS5A protein . This means that the TMPRD domain is the minimal domain required for NS5A protein control.

Next, the GST-NS5A expression vector was transformed into HEK293 cells together with a SCOTIN protein fragment expression vector or a SCOTIN-MYC expression vector, followed by GST-pulldown assay (see Experimental Example 4) Respectively.

As can be seen in FIG. 13, SCOTIN protein and SCOTIN-MYC protein containing TMPRD appeared in the pulldown part of GST-NS5A, but not in the pulldown part of GST-NS5A in other SCOTIN protein fragments. This result also implies that the TMPRD domain is a domain involved in binding to the NS5A protein.

Subsequently, the SCOTIN protein fragment expression vector or SCOTIN-MYC expression vector was transformed into Huh-neo-5-15 replicon cells, and the intracellular HCV RNA level was confirmed by RT-qPCR and the level of intracellular NS5A protein And confirmed by Western blotting (see Experimental Example 3), and the results are shown in Figs. 14 and 15, respectively.

As can be seen in FIGS. 14 and 15, SCOTIN protein fragments and SCOTIN-MYC proteins containing TMPRD reduced NS5A protein and HCV RNA levels, but the other recombinant SCOTIN proteins showed no significant change compared to the control group I could confirm. This means that the SCOTIN protein inhibitory effect on HCV proliferation is mediated by the TMPRD domain.

Example 4. Identification of NS5A domain important for control by SCOTIN protein

The HCV NS5A protein is divided into three domains (He Y, Staschke KA, Tan SL (2006) HCV NS5A: A Multifunctional Regulator of Domain 1 (D1), Domain 2 In order to determine which domain the control of NS5A protein by SCOTIN protein is mediated through, GST tagging (Genome and Molecular Biology, Norfolk, UK) ) NS5A deletion protein was prepared. The prepared protein is shown in Fig.

NS5A deletion protein expression vector or NS5A (Full) expression vector was transformed into Huh7 cell together with the SCOTIN-V5 expression vector, followed by Western blotting (see Experimental Example 3), and the result is shown in FIG.

As shown in FIG. 17, the amount of protein decreased by SCOTIN protein expression in the NS5A (D1 + D2) deletion protein and the NS5A (full) protein, while the GST protein and the NS5A (D1) It is possible to confirm that it does not change. This means that domain 2 is required for NS5A protein reduction by the SCOTIN protein.

Next, in order to identify the domain of the NS5A protein important for binding to the SCOTIN protein, a GST-pulldown assay was performed by transfecting SCOTIN-V5 protein and HEK293 cells with GST-tagging NS5A deletion protein or GST-tagging NS5A protein (See Experimental Example 4), and the results are shown in Fig.

As shown in FIG. 18, it was confirmed that only the GST tagging NS5A (D1 + D2) deletion protein and the GST tagging NS5A (Full) protein bind to the SCOTIN protein and the GST tagging NS5A (D1) protein does not bind to the SCOTIN protein. This shows that domain 2 of the NS5A protein is required for binding to the SCOTIN protein.

Example 5. SCOTIN By protein HCV multiplication Inhibitory action Self-predation Dependency

After introducing the SCOTIN-V5 vector or hSCOTIN-V5 (SCOTIN's human homolog) vector into the Huh-neo-5-15 replicon cells by liposome infusion, the autophagy inhibitor 3-MA was treated and the level of HCV RNA in replicon cells RT-qPCR. The results are shown in Fig.

As can be seen from FIG. 19, it was confirmed that the overexpression of human SCOTIN protein (hSCOTIN protein) significantly reduced the level of HCV RNA as the overexpression of mouse SCOTIN protein. In addition, it was confirmed that when the autogranular inhibitor 3-MA was treated, the effect disappears. This demonstrates that autophagy is important in inhibiting HCV proliferation of SCOTIN protein.

<110> POSTECH ACADEMY-INDUSTRY FOUNDATION <120> Pharmaceuric composition for preventing or treating hepatitis C virus infection or disease due to HCV infection <130> DPP20146990KR <160> 48 <170> Kopatentin 2.0 <210> 1 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> SS_m <400> 1 Met Ala Ala Pro Ala Pro Ser Leu Trp Thr Leu Leu Leu Leu Leu Leu 1 5 10 15 Leu Leu Pro Pro Pro 20 <210> 2 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> SS_h <400> 2 Met Thr Ala Pro Val Pro Ala Pro Arg Ile Leu Leu Pro Leu Leu Leu 1 5 10 15 Leu Leu Leu Leu Thr Pro Pro 20 <210> 3 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> TM_m <400> 3 Gly Phe Gly Ala Thr Val Ala Ile Gly Val Thr Ile Phe Val Val Phe 1 5 10 15 Ile Ala Thr Ile Ile Ile 20 <210> 4 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> TM_h <400> 4 Gly Phe Gly Ala Thr Leu Ala Val Gly Leu Thr Ile Phe Val Leu Ser 1 5 10 15 Val Val Thr Ile Ile Ile 20 <210> 5 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> PRD_m <400> 5 Cys Phe Thr Cys Ser Cys Cys Cys Leu Tyr Lys Met Cys Cys Pro Gln 1 5 10 15 Arg Pro Val Val Thr Asn Thr Thr Thr Thr Val Val His Ala Pro 20 25 30 Tyr Pro Gln Pro Gln Pro Gln Pro Val Ala Pro Ser Tyr Pro Gly Pro 35 40 45 Thr Tyr Gln Gly Tyr His Pro Met Pro Pro Gln Pro Gly Met Pro Ala 50 55 60 Ala Pro Tyr Pro Thr Gln Tyr Pro Pro Pro Tyr Leu Ala Gln Pro Thr 65 70 75 80 Gly Pro Pro Pro Tyr His Glu Ser Leu Ala Gly Ala Ser Gln Pro Pro 85 90 95 Tyr Asn Pro Thr Tyr Met Asp Ser Leu Lys Thr Ile Pro 100 105 <210> 6 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> PRD_h <400> 6 Cys Phe Thr Cys Ser Cys Cys Cys Leu Tyr Lys Thr Cys Arg Arg Pro 1 5 10 15 Arg Pro Val Val Thr Thr Thr Thr Ser Thr Val Val His Ala Pro 20 25 30 Tyr Pro Gln Pro Pro Ser Val Pro Pro Ser Tyr Pro Gly Pro Ser Tyr 35 40 45 Gln Gly Tyr His Thr Met Pro Pro Gln Pro Gly Met Pro Ala Ala Pro 50 55 60 Tyr Pro Met Gln Tyr Pro Pro Pro Tyr Pro Ala Gln Pro Met Gly Pro 65 70 75 80 Pro Ala Tyr His Glu Thr Leu Ala Gly Aly Ala Ala Pro Tyr Pro 85 90 95 Ala Ser Gln Pro Pro Tyr Asn Pro Ala Tyr Met Asp Ala Pro Lys Ala 100 105 110 Ala Leu <210> 7 <211> 83 <212> PRT <213> Artificial Sequence <220> <223> CRD_m <400> 7 Gly Ala His Gly Glu Leu Cys Arg Pro Phe Gly Glu Asp Asn Ser Ile 1 5 10 15 Pro Val Phe Cys Pro Asp Phe Cys Cys Gly Ser Cys Ser Asn Gln Tyr 20 25 30 Cys Cys Ser Asp Val Leu Arg Lys Ile Gln Trp Asn Glu Glu Met Cys 35 40 45 Pro Glu Pro Glu Ser Ser Arg Phe Ser Thr Pro Ala Glu Glu Thr Pro 50 55 60 Glu His Leu Gly Ser Ala Leu Lys Phe Arg Ser Ser Phe Asp Ser Asp 65 70 75 80 Pro Met Ser <210> 8 <211> 80 <212> PRT <213> Artificial Sequence <220> <223> CRD_h <400> 8 Gly Ala Arg Gly Glu Val Cys Met Ala Ser Arg Gly Leu Ser Leu Phe 1 5 10 15 Pro Glu Ser Cys Pro Asp Phe Cys Cys Gly Thr Cys Asp Asp Gln Tyr 20 25 30 Cys Cys Ser Asp Val Leu Lys Lys Phe Val Trp Ser Glu Glu Arg Cys 35 40 45 Ala Val Pro Glu Ala Ser Val Pro Ala Ser Val Glu Pro Val Glu Gln 50 55 60 Leu Gly Ser Ala Leu Arg Phe Arg Pro Gly Tyr Asn Asp Pro Met Ser 65 70 75 80 <210> 9 <211> 236 <212> PRT <213> Artificial Sequence <220> <223> SCOTIN <400> 9 Met Ala Ala Pro Ala Pro Ser Leu Trp Thr Leu Leu Leu Leu Leu Leu 1 5 10 15 Leu Leu Pro Pro Pro Pro Gly Ala His Gly Glu Leu Cys Arg Pro Phe 20 25 30 Gly Glu Asp Asn Ser Ile Pro Val Phe Cys Pro Asp Phe Cys Cys Gly 35 40 45 Ser Cys Ser Asn Gln Tyr Cys Cys Ser Asp Val Leu Arg Lys Ile Gln 50 55 60 Trp Asn Glu Glu Met Cys Pro Glu Pro Glu Ser Ser Arg Phe Ser Thr 65 70 75 80 Pro Ala Glu Glu Thr Pro Glu His Leu Gly Ser Ala Leu Lys Phe Arg 85 90 95 Ser Ser Phe Asp Ser Asp Pro Met Ser Gly Phe Gly Ala Thr Val Ala 100 105 110 Ile Gly Val Thr Ile Phe Val Val Phe Ile Ala Thr Ile Ile Ile Cys 115 120 125 Phe Thr Cys Ser Cys Cys Cys Leu Tyr Lys Met Cys Cys Pro Gln Arg 130 135 140 Pro Val Val Thr Asn Thr Thr Thr Thr Val Val His Ala Pro Tyr 145 150 155 160 Pro Gln Pro Gln Pro Gln Pro Val Ala Pro Ser Tyr Pro Gly Pro Thr 165 170 175 Tyr Gln Gly Tyr His Pro Met Pro Pro Gln Pro Gly Met Pro Ala Ala 180 185 190 Pro Tyr Pro Thr Gln Tyr Pro Pro Pro Tyr Leu Ala Gln Pro Thr Gly 195 200 205 Pro Pro Pro Tyr His Glu Ser Leu Ala Gly Ala Ser Gln Pro Pro Tyr 210 215 220 Asn Pro Thr Tyr Met Asp Ser Leu Lys Thr Ile Pro 225 230 235 <210> 10 <211> 240 <212> PRT <213> Artificial Sequence <220> <223> hSCOTIN <400> 10 Met Thr Ala Pro Val Pro Ala Pro Arg Ile Leu Leu Pro Leu Leu Leu 1 5 10 15 Leu Leu Leu Leu Thr Pro Pro Pro Gly Ala Arg Gly Glu Val Cys Met 20 25 30 Ala Ser Arg Gly Leu Ser Leu Phe Pro Glu Ser Cys Pro Asp Phe Cys 35 40 45 Cys Gly Thr Cys Asp Asp Gln Tyr Cys Cys Ser Asp Val Leu Lys Lys 50 55 60 Phe Val Trp Ser Glu Glu Arg Cys Ala Val Pro Glu Ala Ser Val Pro 65 70 75 80 Ala Ser Val Glu Pro Val Glu Gln Leu Gly Ser Ala Leu Arg Phe Arg 85 90 95 Pro Gly Tyr Asn Asp Pro Met Ser Gly Phe Gly Ala Thr Leu Ala Val 100 105 110 Gly Leu Thr Ile Phe Val Leu Ser Val Val Thr Ile Ile Ile Cys Phe 115 120 125 Thr Cys Ser Cys Cys Cys Leu Tyr Lys Thr Cys Arg Arg Pro Arg Pro 130 135 140 Val Val Thr Thr Thr Thr Ser Thr Thr Val Val His Ala Pro Tyr Pro 145 150 155 160 Gln Pro Pro Ser Val Pro Pro Ser Tyr Pro Gly Pro Ser Tyr Gln Gly 165 170 175 Tyr His Thr Met Pro Pro Gln Pro Gly Met Pro Ala Ala Pro Tyr Pro 180 185 190 Met Gln Tyr Pro Pro Pro Tyr Pro Ala Gln Pro Met Gly Pro Pro Ala 195 200 205 Tyr His Glu Thr Leu Ala Gly Gly Ala Ala Ala Pro Tyr Pro Ala Ser 210 215 220 Gln Pro Pro Tyr Asn Pro Ala Tyr Met Asp Ala Pro Lys Ala Ala Leu 225 230 235 240 <210> 11 <211> 131 <212> PRT <213> Artificial Sequence <220> <223> SCOTIN (TMPRD) <400> 11 Gly Phe Gly Ala Thr Val Ala Ile Gly Val Thr Ile Phe Val Val Phe 1 5 10 15 Ile Ala Thr Ile Ile Ile Cys Phe Thr Cys Ser Cys Cys Cys Leu Tyr 20 25 30 Lys Met Cys Cys Pro Gln Arg Pro Val Val Thr Asn Thr Thr Thr Thr 35 40 45 Thr Val Val His Ala Pro Tyr Pro Gln Pro Gln Pro Gln Pro Val Ala 50 55 60 Pro Ser Tyr Pro Gly Pro Thr Tyr Gln Gly Tyr His Pro Met Pro Pro 65 70 75 80 Gln Pro Gly Met Pro Ala Ala Pro Tyr Pro Thr Gln Tyr Pro Pro Pro 85 90 95 Tyr Leu Ala Gln Pro Thr Gly Pro Pro Pro Tyr His Glu Ser Leu Ala 100 105 110 Gly Ala Ser Gln Pro Pro Tyr Asn Pro Thr Tyr Met Asp Ser Leu Lys 115 120 125 Thr Ile Pro 130 <210> 12 <211> 136 <212> PRT <213> Artificial Sequence <220> <223> hSCOTIN (TMPRD) <400> 12 Gly Phe Gly Ala Thr Leu Ala Val Gly Leu Thr Ile Phe Val Leu Ser 1 5 10 15 Val Val Thr Ile Ile Ile Cys Phe Thr Cys Ser Cys Cys Cys Leu Tyr 20 25 30 Lys Thr Cys Arg Arg Pro Pro Val Val Thr Thr Thr Thr Ser Thr 35 40 45 Thr Val Val His Ala Pro Tyr Pro Gln Pro Pro Ser Val Pro Ser Ser 50 55 60 Tyr Pro Gly Pro Ser Tyr Gln Gly Tyr His Thr Met Pro Pro Gln Pro 65 70 75 80 Gly Met Pro Ala Ala Pro Tyr Pro Met Gln Tyr Pro Pro Pro Tyr Pro 85 90 95 Ala Gln Pro Met Gly Pro Pro Ala Tyr His Glu Thr Leu Ala Gly Gly 100 105 110 Ala Ala Ala Pro Tyr Pro Ala Ser Gln Pro Pro Tyr Asn Pro Ala Tyr 115 120 125 Met Asp Ala Pro Lys Ala Ala Leu 130 135 <210> 13 <211> 153 <212> PRT <213> Artificial Sequence <220> <223> SCOTIN (SSTMPRD) <400> 13 Met Ala Ala Pro Ala Pro Ser Leu Trp Thr Leu Leu Leu Leu Leu Leu 1 5 10 15 Leu Leu Pro Pro Pro Gly Phe Gly Ala Thr Val Ala Ile Gly Val 20 25 30 Thr Ile Phe Val Val Phe Ile Ala Thr Ile Ile Ile Cys Phe Thr Cys 35 40 45 Ser Cys Cys Cys Leu Tyr Lys Met Cys Cys Pro Gln Arg Pro Val Val 50 55 60 Thr Asn Thr Thr Thr Thr Thr Val Val His Ala Pro Tyr Pro Gln Pro 65 70 75 80 Gln Pro Gln Pro Val Ala Pro Ser Tyr Pro Gly Pro Thr Tyr Gln Gly 85 90 95 Tyr His Pro Met Pro Pro Gln Pro Gly Met Pro Ala Ala Pro Tyr Pro 100 105 110 Thr Gln Tyr Pro Pro Tyr Leu Ala Gln Pro Thr Gly Pro Pro Pro 115 120 125 Tyr His Glu Ser Leu Ala Gly Ala Ser Gln Pro Pro Tyr Asn Pro Thr 130 135 140 Tyr Met Asp Ser Leu Lys Thr Ile Pro 145 150 <210> 14 <211> 160 <212> PRT <213> Artificial Sequence <220> <223> hSCOTIN (SSTMPRD) <400> 14 Met Thr Ala Pro Val Pro Ala Pro Arg Ile Leu Leu Pro Leu Leu Leu 1 5 10 15 Leu Leu Leu Leu Thr Pro Pro Gly Phe Gly Ala Thr Leu Ala Val 20 25 30 Gly Leu Thr Ile Phe Val Leu Ser Val Val Thr Ile Ile Ile Cys Phe 35 40 45 Thr Cys Ser Cys Cys Cys Leu Tyr Lys Thr Cys Arg Arg Pro Arg Pro 50 55 60 Val Val Thr Thr Thr Thr Ser Thr Thr Val Val His Ala Pro Tyr Pro 65 70 75 80 Gln Pro Pro Ser Val Pro Pro Ser Tyr Pro Gly Pro Ser Tyr Gln Gly 85 90 95 Tyr His Thr Met Pro Pro Gln Pro Gly Met Pro Ala Ala Pro Tyr Pro 100 105 110 Met Gln Tyr Pro Pro Pro Tyr Pro Ala Gln Pro Met Gly Pro Pro Ala 115 120 125 Tyr His Glu Thr Leu Ala Gly Gly Ala Ala Ala Pro Tyr Pro Ala Ser 130 135 140 Gln Pro Pro Tyr Asn Pro Ala Tyr Met Asp Ala Pro Lys Ala Ala Leu 145 150 155 160 <210> 15 <211> 105 <212> PRT <213> Artificial Sequence <220> <223> SCOTIN (SSCRD) <400> 15 Met Ala Ala Pro Ala Pro Ser Leu Trp Thr Leu Leu Leu Leu Leu Leu 1 5 10 15 Leu Leu Pro Pro Pro Pro Gly Ala His Gly Glu Leu Cys Arg Pro Phe 20 25 30 Gly Glu Asp Asn Ser Ile Pro Val Phe Cys Pro Asp Phe Cys Cys Gly 35 40 45 Ser Cys Ser Asn Gln Tyr Cys Cys Ser Asp Val Leu Arg Lys Ile Gln 50 55 60 Trp Asn Glu Glu Met Cys Pro Glu Pro Glu Ser Ser Arg Phe Ser Thr 65 70 75 80 Pro Ala Glu Glu Thr Pro Glu His Leu Gly Ser Ala Leu Lys Phe Arg 85 90 95 Ser Ser Phe Asp Ser Asp Pro Met Ser 100 105 <210> 16 <211> 129 <212> PRT <213> Artificial Sequence <220> <223> SCOTIN (SSTMCRD) <400> 16 Met Ala Ala Pro Ala Pro Ser Leu Trp Thr Leu Leu Leu Leu Leu Leu 1 5 10 15 Leu Leu Pro Pro Pro Pro Gly Ala His Gly Glu Leu Cys Arg Pro Phe 20 25 30 Gly Glu Asp Asn Ser Ile Pro Val Phe Cys Pro Asp Phe Cys Cys Gly 35 40 45 Ser Cys Ser Asn Gln Tyr Cys Cys Ser Asp Val Leu Arg Lys Ile Gln 50 55 60 Trp Asn Glu Glu Met Cys Pro Glu Pro Glu Ser Ser Arg Phe Ser Thr 65 70 75 80 Pro Ala Glu Glu Thr Pro Glu His Leu Gly Ser Ala Leu Lys Phe Arg 85 90 95 Ser Ser Phe Asp Ser Asp Pro Met Ser Gly Phe Gly Ala Thr Val Ala 100 105 110 Ile Gly Val Thr Ile Phe Val Val Phe Ile Ala Thr Ile Ile Ile Cys 115 120 125 Phe <210> 17 <211> 131 <212> PRT <213> Artificial Sequence <220> <223> SCOTIN (SSPRD) <400> 17 Met Ala Ala Pro Ala Pro Ser Leu Trp Thr Leu Leu Leu Leu Leu Leu 1 5 10 15 Leu Leu Pro Pro Pro Cys Phe Thr Cys Ser Cys Cys Cys Leu Tyr 20 25 30 Lys Met Cys Cys Pro Gln Arg Pro Val Val Thr Asn Thr Thr Thr Thr 35 40 45 Thr Val Val His Ala Pro Tyr Pro Gln Pro Gln Pro Gln Pro Val Ala 50 55 60 Pro Ser Tyr Pro Gly Pro Thr Tyr Gln Gly Tyr His Pro Met Pro Pro 65 70 75 80 Gln Pro Gly Met Pro Ala Ala Pro Tyr Pro Thr Gln Tyr Pro Pro Pro 85 90 95 Tyr Leu Ala Gln Pro Thr Gly Pro Pro Pro Tyr His Glu Ser Leu Ala 100 105 110 Gly Ala Ser Gln Pro Pro Tyr Asn Pro Thr Tyr Met Asp Ser Leu Lys 115 120 125 Thr Ile Pro 130 <210> 18 <211> 66 <212> DNA <213> Artificial Sequence <220> <223> SS_m <400> 18 atggctgcgc cggcgccctc tctgtggacc ctattgctgc tgctgttgct gctgccgccg 60 cctccg 66 <210> 19 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> SS_h <400> 19 atgactgcgc cggtccccgc gccgcggatc ctgttgccgt tgctgttgct gctgctgcta 60 acgccgcctc cg 72 <210> 20 <211> 66 <212> DNA <213> Artificial Sequence <220> <223> TM_m <400> 20 gggttcggag cgaccgtcgc cattggcgtg accatctttg tggtgtttat tgccactatc 60 atcatc 66 <210> 21 <211> 66 <212> DNA <213> Artificial Sequence <220> <223> TM_h <400> 21 gggttcggag cgaccttggc cgttggcctg accatctttg tgctgtctgt cgtcactatc 60 atcatc 66 <210> 22 <211> 327 <212> DNA <213> Artificial Sequence <220> <223> PRD_m <400> 22 tgcttcacct gctcctgctg ctgtctgtat aagatgtgct gcccccaacg ccctgtcgtg 60 accaacacca caactactac cgtggttcat gccccttacc ctcagcctca acctcaacct 120 gtggccccca gctatcctgg accaacatac cagggctacc atcccatgcc cccccagcca 180 ggaatgccag cagcacccta cccaacgcag tacccaccac cctacctggc ccagcccaca 240 gggccgccac cctaccatga gtccttggct ggagccagcc agcctccata caacccgacc 300 tacatggatt ccctaaagac aattccc 327 <210> 23 <211> 342 <212> DNA <213> Artificial Sequence <220> <223> PRD_h <400> 23 tgcttcacct gctcctgctg ctgcctttac aagacgtgcc gccgaccacg tccggttgtc 60 accaccacca catccaccac tgtggtgcat gccccttatc ctcagcctcc aagtgtgccg 120 cccagctacc ctggaccaag ctaccagggc taccacacca tgccgcctca gccagggatg 180 ccagcagcac cctacccaat gcagtaccca ccaccttacc cagcccagcc catgggccca 240 ccggcctacc acgagaccct ggctggagga gcagccgcgc cctaccccgc cagccagcct 300 ccttacaacc cggcctacat ggatgccccg aaggcggccc tc 342 <210> 24 <211> 249 <212> DNA <213> Artificial Sequence <220> <223> CRD_m <400> 24 ggtgcccatg gtgagctgtg caggcccttt ggtgaagaca attcgatccc agtgttctgt 60 cctgatttct gttgtggttc ctgttccaac caatactgct gctcggacgt gctgaggaaa 120 atccagtgga atgaggaaat gtgtcctgag ccagagtcca gcagattttc cacccccgcg 180 gggagacac ccgaacatct gggttcagcg ctgaaatttc gatccagttt tgacagtgg 240 cctatgtca 249 <210> 25 <211> 240 <212> DNA <213> Artificial Sequence <220> <223> CRD_h <400> 25 ggtgcacgtg gtgaggtgtg tatggcttcc cgtggactca gcctcttccc cgagtcctgt 60 ccagatttct gctgtggtac ctgtgatgac caatactgct gctctgacgt gctgaagaaa 120 tttgtgtgga gcgaggaaag gtgtgctgtg cctgaggcca gcgtgcctgc cagtgtagag 180 ccggtggagc agctgggctc ggcgctgagg tttcgccctg gctacaacga ccccatgtca 240 240 <210> 26 <211> 708 <212> DNA <213> Artificial Sequence <220> <223> SCOTIN <400> 26 atggctgcgc cggcgccctc tctgtggacc ctattgctgc tgctgttgct gctgccgccg 60 cctccgggtg cccatggtga gctgtgcagg ccctttggtg aagacaattc gatcccagtg 120 ttctgtcctg atttctgttg tggttcctgt tccaaccaat actgctgctc ggacgtgctg 180 aggaaaatcc agtggaatga ggaaatgtgt cctgagccag agtccagcag attttccacc 240 cccgcggagg agacacccga acatctgggt tcagcgctga aatttcgatc cagttttgac 300 agtgacccta tgtcagggtt cggagcgacc gtcgccattg gcgtgaccat ctttgtggtg 360 tttattgcca ctatcatcat ctgcttcacc tgctcctgct gctgtctgta taagatgtgc 420 tgcccccaac gccctgtcgt gaccaacacc acaactacta ccgtggttca tgccccttac 480 cctcagcctc aacctcaacc tgtggccccc agctatcctg gaccaacata ccagggctac 540 catcccatgc ccccccagcc aggaatgcca gcagcaccct acccaacgca gtacccacca 600 ccctacctgg cccagcccac agggccgcca ccctaccatg agtccttggc tggagccagc 660 cagcctccat acaacccgac ctacatggat tccctaaaga caattccc 708 <210> 27 <211> 720 <212> DNA <213> Artificial Sequence <220> <223> hSCOTIN <400> 27 atgactgcgc cggtccccgc gccgcggatc ctgttgccgt tgctgttgct gctgctgcta 60 acgccgcctc cgggtgcacg tggtgaggtg tgtatggctt cccgtggact cagcctcttc 120 cccgagtcct gtccagattt ctgctgtggt acctgtgatg accaatactg ctgctctgac 180 gtgctgaaga aatttgtgtg gagcgaggaa aggtgtgctg tgcctgaggc cagcgtgcct 240 gccagtgtag agccggtgga gcagctgggc tcggcgctga ggtttcgccc tggctacaac 300 gaccccatgt cagggttcgg agcgaccttg gccgttggcc tgaccatctt tgtgctgtct 360 gtcgtcacta tcatcatctg cttcacctgc tcctgctgct gcctttacaa gacgtgccgc 420 cgaccacgtc cggttgtcac caccaccaca tccaccactg tggtgcatgc cccttatcct 480 cagcctccaa gtgtgccgcc cagctaccct ggaccaagct accagggcta ccacaccatg 540 ccgcctcagc cagggatgcc agcagcaccc tacccaatgc agtacccacc accttaccca 600 gcccagccca tgggcccacc ggcctaccac gagaccctgg ctggaggagc agccgcgccc 660 taccccgcca gccagcctcc ttacaacccg gcctacatgg atgccccgaa ggcggccctc 720 720 <210> 28 <211> 393 <212> DNA <213> Artificial Sequence <220> <223> SCOTIN (TMPRD) <400> 28 gggttcggag cgaccgtcgc cattggcgtg accatctttg tggtgtttat tgccactatc 60 atcatctgct tcacctgctc ctgctgctgt ctgtataaga tgtgctgccc ccaacgccct 120 gtcgtgacca acaccacaac tactaccgtg gttcatgccc cttaccctca gcctcaacct 180 caacctgtgg cccccagcta tcctggacca acataccagg gctaccatcc catgcccccc 240 cagccaggaa tgccagcagc accctaccca acgcagtacc caccacccta cctggcccag 300 cccacagggc cgccacccta ccatgagtcc ttggctggag ccagccagcc tccatacaac 360 ccgacctaca tggattccct aaagacaatt ccc 393 <210> 29 <211> 408 <212> DNA <213> Artificial Sequence <220> <223> hSCOTIN (TMPRD) <400> 29 gggttcggag cgaccttggc cgttggcctg accatctttg tgctgtctgt cgtcactatc 60 atcatctgct tcacctgctc ctgctgctgc ctttacaaga cgtgccgccg accacgtccg 120 gttgcacca ccaccacatc caccactgtg gtgcatgccc cttatcctca gcctccaagt 180 gtgccgccca gctaccctgg accaagctac cagggctacc acaccatgcc gcctcagcca 240 gggatgccag cagcacccta cccaatgcag tacccaccac cttacccagc ccagcccatg 300 ggcccaccgg cctaccacga gaccctggct ggaggagcag ccgcgcccta ccccgccagc 360 cagcctcctt acaacccggc ctacatggat gccccgaagg cggccctc 408 <210> 30 <211> 459 <212> DNA <213> Artificial Sequence <220> <223> SCOTIN (SSTMPRD) <400> 30 atggctgcgc cggcgccctc tctgtggacc ctattgctgc tgctgttgct gctgccgccg 60 cctccggggt tcggagcgac cgtcgccatt ggcgtgacca tctttgtggt gtttattgcc 120 actatcatca tctgcttcac ctgctcctgc tgctgtctgt ataagatgtg ctgcccccaa 180 cgccctgtcg tgaccaacac cacaactact accgtggttc atgcccctta ccctcagcct 240 caacctcaac ctgtggcccc cagctatcct ggaccaacat accagggcta ccatcccatg 300 cccccccagc caggaatgcc agcagcaccc tacccaacgc agtacccacc accctacctg 360 gcccagccca cagggccgcc accctaccat gagtccttgg ctggagccag ccagcctcca 420 tacaacccga cctacatgga ttccctaaag acaattccc 459 <210> 31 <211> 480 <212> DNA <213> Artificial Sequence <220> <223> hSCOTIN (SSTMPRD) <400> 31 atgactgcgc cggtccccgc gccgcggatc ctgttgccgt tgctgttgct gctgctgcta 60 acgccgcctc cggggttcgg agcgaccttg gccgttggcc tgaccatctt tgtgctgtct 120 gtcgtcacta tcatcatctg cttcacctgc tcctgctgct gcctttacaa gacgtgccgc 180 cgaccacgtc cggttgtcac caccaccaca tccaccactg tggtgcatgc cccttatcct 240 cagcctccaa gtgtgccgcc cagctaccct ggaccaagct accagggcta ccacaccatg 300 ccgcctcagc cagggatgcc agcagcaccc tacccaatgc agtacccacc accttaccca 360 gcccagccca tgggcccacc ggcctaccac gagaccctgg ctggaggagc agccgcgccc 420 taccccgcca gccagcctcc ttacaacccg gcctacatgg atgccccgaa ggcggccctc 480 480 <210> 32 <211> 315 <212> DNA <213> Artificial Sequence <220> <223> SCOTIN (SSCRD) <400> 32 atggctgcgc cggcgccctc tctgtggacc ctattgctgc tgctgttgct gctgccgccg 60 cctccgggtg cccatggtga gctgtgcagg ccctttggtg aagacaattc gatcccagtg 120 ttctgtcctg atttctgttg tggttcctgt tccaaccaat actgctgctc ggacgtgctg 180 aggaaaatcc agtggaatga ggaaatgtgt cctgagccag agtccagcag attttccacc 240 cccgcggagg agacacccga acatctgggt tcagcgctga aatttcgatc cagttttgac 300 agtgacccta tgtca 315 <210> 33 <211> 387 <212> DNA <213> Artificial Sequence <220> <223> SCOTIN (SSTMCRD) <400> 33 atggctgcgc cggcgccctc tctgtggacc ctattgctgc tgctgttgct gctgccgccg 60 cctccgggtg cccatggtga gctgtgcagg ccctttggtg aagacaattc gatcccagtg 120 ttctgtcctg atttctgttg tggttcctgt tccaaccaat actgctgctc ggacgtgctg 180 aggaaaatcc agtggaatga ggaaatgtgt cctgagccag agtccagcag attttccacc 240 cccgcggagg agacacccga acatctgggt tcagcgctga aatttcgatc cagttttgac 300 agtgacccta tgtcagggtt cggagcgacc gtcgccattg gcgtgaccat ctttgtggtg 360 tttattgcca ctatcatcat ctgcttc 387 <210> 34 <211> 393 <212> DNA <213> Artificial Sequence <220> <223> SCOTIN (SSPRD) <400> 34 atggctgcgc cggcgccctc tctgtggacc ctattgctgc tgctgttgct gctgccgccg 60 cctccgtgct tcacctgctc ctgctgctgt ctgtataaga tgtgctgccc ccaacgccct 120 gtcgtgacca acaccacaac tactaccgtg gttcatgccc cttaccctca gcctcaacct 180 caacctgtgg cccccagcta tcctggacca acataccagg gctaccatcc catgcccccc 240 cagccaggaa tgccagcagc accctaccca acgcagtacc caccacccta cctggcccag 300 cccacagggc cgccacccta ccatgagtcc ttggctggag ccagccagcc tccatacaac 360 ccgacctaca tggattccct aaagacaatt ccc 393 <210> 35 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> NS5A <400> 35 Ser Gly Ser Trp Leu Arg Asp Val Trp Asp Trp Ile Cys Thr Val Leu 1 5 10 15 Thr Asp Phe Lys Thr Trp Leu Gln Ser Lys Leu Leu Pro Arg Leu Pro 20 25 30 Gly Val Pro Phe Phe Ser Cys Gln Arg Gly Tyr Lys Gly Val Trp Arg 35 40 45 Gly Glu Ile Met Gln Thr Thr Cys Pro Cys Gly Ala Gln Ile Ala 50 55 60 Gly His Val Lys Asn Gly Ser Met Arg Ile Val Gly Pro Arg Thr Cys 65 70 75 80 Ser Asn Thr Trp His Gly Thr Phe Pro Ile Asn Ala Tyr Thr Thr Gly 85 90 95 Pro Cys Ser Pro Ser Pro Ala Pro Asn Tyr Ser Arg Ala Leu Trp Arg 100 105 110 Val Ala Ala Glu Glu Tyr Val Glu Val Thr Arg Val Gly Asp Phe His 115 120 125 Tyr Val Thr Gly Val Thr Thr Asp Asn Val Lys Cys Pro Cys Gln Val 130 135 140 Pro Ala Pro Glu Phe Phe Thr Glu Leu Asp Gly Val Arg Leu His Arg 145 150 155 160 Tyr Ala Pro Ala Cys Lys Pro Leu Leu Arg Asp Glu Val Ser Phe Gln 165 170 175 Val Gly Leu Asn Gln Tyr Leu Val Gly Ser Gln Leu Pro Cys Glu Pro 180 185 190 Glu Pro Asp Val Ala Val Leu Thr Ser Met Leu Thr Asp Pro Ser His 195 200 205 Ile Thr Ala Glu Thr Ala Lys Arg Arg Leu Ala Arg Gly Ser Pro Pro 210 215 220 Ser Leu Ala Ser Ser Ala Ser Gln Leu Ser Ala Pro Ser Leu Lys 225 230 235 240 Ala Thr Cys Thr Ile His His Asp Ser Pro Asp Ala Asp Leu Ile Glu 245 250 255 Ala Asn Leu Leu Trp Arg Gln Glu Met Gly Gly Asn Ile Thr Arg Val 260 265 270 Glu Ser Glu Asn Lys Val Val Ile Leu Asp Ser Phe Glu Pro Ile Arg 275 280 285 Ala Glu Glu Asp Glu Arg Glu Val Ser Val Pro Ala Glu Ile Leu Arg 290 295 300 Arg Ser Lys Phe Pro Ala Ala Met Pro Ile Trp Ala Arg Pro Asp 305 310 315 320 Tyr Asn Pro Pro Leu Leu Glu Ser Trp Lys Asp Pro Asp Tyr Val Pro 325 330 335 Pro Val Val His Gly Cys Pro Leu Pro Pro Thr Lys Ala Ala Pro Ile 340 345 350 Pro Pro Arg Arg Lys Arg Thr Ile Val Leu Thr Glu Ser Thr Val 355 360 365 Ser Ser Ala Leu Ala Glu Leu Ala Thr Lys Thr Phe Gly Gly Ser Gly 370 375 380 Ser Ser Ala Asp Ser Gly Thr Ala Thr Ala Pro Pro Asp Gln Thr 385 390 395 400 Ser Asp Asp Gly Asp Lys Glu Ser Asp Val Glu Ser Tyr Ser Ser Met 405 410 415 Pro Pro Leu Glu Gly Glu Pro Gly Asp Pro Asp Leu Ser Asp Gly Ser 420 425 430 Trp Ser Thr Val Ser Glu Glu Ala Ser Glu Asp Val Val Cys Cys 435 440 445 <210> 36 <211> 213 <212> PRT <213> Artificial Sequence <220> <223> NS5A Domain 1 <400> 36 Ser Gly Ser Trp Leu Arg Asp Val Trp Asp Trp Ile Cys Thr Val Leu 1 5 10 15 Thr Asp Phe Lys Thr Trp Leu Gln Ser Lys Leu Leu Pro Arg Leu Pro 20 25 30 Gly Val Pro Phe Phe Ser Cys Gln Arg Gly Tyr Lys Gly Val Trp Arg 35 40 45 Gly Glu Ile Met Gln Thr Thr Cys Pro Cys Gly Ala Gln Ile Ala 50 55 60 Gly His Val Lys Asn Gly Ser Met Arg Ile Val Gly Pro Arg Thr Cys 65 70 75 80 Ser Asn Thr Trp His Gly Thr Phe Pro Ile Asn Ala Tyr Thr Thr Gly 85 90 95 Pro Cys Ser Pro Ser Pro Ala Pro Asn Tyr Ser Arg Ala Leu Trp Arg 100 105 110 Val Ala Ala Glu Glu Tyr Val Glu Val Thr Arg Val Gly Asp Phe His 115 120 125 Tyr Val Thr Gly Val Thr Thr Asp Asn Val Lys Cys Pro Cys Gln Val 130 135 140 Pro Ala Pro Glu Phe Phe Thr Glu Leu Asp Gly Val Arg Leu His Arg 145 150 155 160 Tyr Ala Pro Ala Cys Lys Pro Leu Leu Arg Asp Glu Val Ser Phe Gln 165 170 175 Val Gly Leu Asn Gln Tyr Leu Val Gly Ser Gln Leu Pro Cys Glu Pro 180 185 190 Glu Pro Asp Val Ala Val Leu Thr Ser Met Leu Thr Asp Pro Ser His 195 200 205 Ile Thr Ala Glu Thr 210 <210> 37 <211> 129 <212> PRT <213> Artificial Sequence <220> <223> NS5A Domain 2 <400> 37 Ala Lys Arg Arg Leu Ala Arg Gly Ser Pro Pro Ser Leu Ala Ser Ser 1 5 10 15 Ser Ala Ser Gln Leu Ser Ala Pro Ser Leu Lys Ala Thr Cys Thr Ile 20 25 30 His His Asp Ser Pro Asp Ala Asp Leu Ile Glu Ala Asn Leu Leu Trp 35 40 45 Arg Gln Glu Met Gly Gly Asn Ile Thr Arg Val Glu Ser Glu Asn Lys 50 55 60 Val Val Ile Leu Asp Ser Phe Glu Pro Ile Arg Ala Glu Glu Asp Glu 65 70 75 80 Arg Glu Val Ser Val Pro Ala Glu Ile Leu Arg Arg Ser Ser Lys Phe 85 90 95 Pro Ala Ala Met Pro Ile Trp Ala Arg Pro Asp Tyr Asn Pro Pro Leu 100 105 110 Leu Glu Ser Trp Lys Asp Pro Asp Tyr Val Pro Pro Val Val His Gly 115 120 125 Cys <210> 38 <211> 105 <212> PRT <213> Artificial Sequence <220> <223> NS5A Domain 3 <400> 38 Pro Leu Pro Pro Thr Lys Ala Ala Pro Ile Pro Pro Pro Arg Arg Lys 1 5 10 15 Arg Thr Ile Val Leu Thr Glu Ser Ser Val Ser Ser Ala Leu Ala Glu 20 25 30 Leu Ala Thr Lys Thr Phe Gly Gly Ser Gly Ser Ser Ala Ala Asp Ser 35 40 45 Gly Thr Ala Thr Ala Pro Pro Asp Gln Thr Ser Asp Asp Gly Asp Lys 50 55 60 Glu Ser Asp Val Glu Ser Ser Ser Ser Met Pro Pro Leu Glu Gly Glu 65 70 75 80 Pro Gly Asp Pro Asp Leu Ser Asp Gly Ser Trp Ser Thr Val Ser Glu 85 90 95 Glu Ala Ser Glu Asp Val Val Cys Cys 100 105 <210> 39 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> NS4_F <400> 39 acaacaggca gcgtggtcat t 21 <210> 40 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NS4_R <400> 40 ttccacatgt gctttgccca 20 <210> 41 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> SCOTIN_F <400> 41 tgctgtggta cctgtgatga ccaa 24 <210> 42 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> SCOTIN_R <400> 42 aggagcaggt gaagcagatg atga 24 <210> 43 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> RPL32_F <400> 43 aacccagagg cattgacaac 20 <210> 44 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> RPL32_R <400> 44 gttgcacatc agcagcactt 20 <210> 45 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> SCOTIN siRNA # 1 <400> 45 guaccuguga ugaccaauat t 21 <210> 46 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> SCOTIN siRNA # 2 <400> 46 gagcugucuu agcucaaaut t 21 <210> 47 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> SCOTIN siRNA # 3 <400> 47 ggucuguaca cuuguuuaut t 21 <210> 48 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> control siRNA <400> 48 uucccgaac gugucacguu u 21

Claims

A polypeptide consisting of 132 to 260 or 136 to 260 amino acids comprising the transmembrane domain (TM) of the SCOTIN protein and the proline-rich domain (PRD).

3. The polypeptide of claim 1, wherein the polypeptide comprises an ER-signal peptide (SS) at the N-terminus.

3. The polypeptide of claim 1, wherein the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 3 or 4, and wherein the proline enriched domain comprises the amino acid sequence of SEQ ID NO: 5 or 6.

3. The polypeptide of claim 2, wherein the ER locus signal peptide comprises the amino acid sequence of SEQ ID NO: 1 or 2.

The polypeptide according to claim 1, wherein the polypeptide comprises the sequence of any one of the amino acid sequences of SEQ ID NOS: 11 to 14.

A polynucleotide encoding a polypeptide consisting of 132 to 260 or 136 to 260 amino acids comprising a transmembrane domain (TM) of the SCOTIN protein and a proline-rich domain (PRD).

7. The polynucleotide of claim 6, wherein the polypeptide comprises an ER-signal peptide (SS) at the N-terminus.

7. The polynucleotide according to claim 6, wherein the polynucleotide sequence encoding the transmembrane domain comprises the nucleotide sequence of SEQ ID NO: 20 or 21, and the polynucleotide sequence encoding the proline rich domain comprises the nucleotide sequence of SEQ ID NO: 22 or 23 Polynucleotide. &Lt; / RTI >

8. The polynucleotide of claim 7, wherein the polynucleotide sequence encoding the ER locus signal peptide comprises the nucleotide sequence of SEQ ID NO: 18 or 19.

8. The polynucleotide of claim 7, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NOS: 28 to 31.

8. A recombinant vector comprising the polynucleotide of claim 7.

12. The recombinant vector according to claim 11, wherein the recombinant vector has a cleavage map as disclosed in Fig.

12. The recombinant vector of claim 11, wherein the recombinant vector is a viral or non-viral vector.

14. The recombinant vector of claim 13, wherein said viral vector is selected from the group consisting of adenovirus, adeno-associated virus, helper-dependent adenovirus, and retroviral vector.

A cell transformed with the recombinant vector of claim 11.

16. The cell of claim 15, wherein the cell is a cell of a mammal.

SCOTIN protein, a polypeptide consisting of 132-260 or 136-260 amino acids comprising the transmembrane domain (TM) of the SCOTIN protein and the proline-rich domain (PRD), the SCOTIN protein or polypeptide , A polynucleotide encoding the polynucleotide, a recombinant vector comprising the polynucleotide, a recombinant cell comprising the recombinant vector, or a mixture thereof, as an active ingredient, in association with hepatitis C virus (HCV) infection or infection A pharmaceutical composition for the prevention or treatment of diseases.

18. The pharmaceutical composition according to claim 17, wherein the polypeptide comprises an ER-signal peptide (SS) at the N-terminus.

18. The pharmaceutical composition of claim 17, wherein said transmembrane domain comprises the amino acid sequence of SEQ ID NO: 3 or 4 and said proline enriched domain comprises the amino acid sequence of SEQ ID NO: 5 or 6.

19. The pharmaceutical composition of claim 18, wherein said ER locus signal peptide comprises the amino acid sequence of SEQ ID NO: 1 or 2.

18. The pharmaceutical composition according to claim 17, wherein the polypeptide comprises the sequence of any one of the amino acid sequences of SEQ ID NOS: 11 to 14.

18. The pharmaceutical composition according to claim 17, wherein the SCOTIN protein comprises the amino acid sequence of SEQ ID NO: 9 or 10.

18. The pharmaceutical composition according to claim 17, wherein the disease associated with hepatitis C virus infection is hepatitis C, cirrhosis, cirrhosis, or liver cancer.

A pharmaceutical preparation containing the composition according to any one of claims 17 to 23 as an active ingredient.

25. The pharmaceutical preparation according to claim 24, wherein the pharmaceutical preparation further comprises at least one selected from the group consisting of a carrier, an excipient and a diluent.

The pharmaceutical composition according to claim 24, wherein the pharmaceutical preparation is in the form of tablets, pills, powders, sachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, soft or hard gelatin capsules, Wherein the pharmaceutical formulation is a formulation selected from the group consisting of sterile disintegrants.

SCOTIN protein, a polypeptide consisting of 132-260 or 136-260 amino acids comprising the transmembrane domain (TM) of the SCOTIN protein and the proline-rich domain (PRD), the SCOTIN protein or polypeptide , A recombinant vector comprising the polynucleotide, a recombinant cell comprising the recombinant vector, or a mixture thereof, as an active ingredient, to a hepatitis C virus (HCV) composition comprising a polynucleotide encoding the polynucleotide, a recombinant vector comprising the polynucleotide, .

28. The antiviral composition for hepatitis C virus according to claim 27, wherein the polypeptide comprises an ER-signal peptide (SS) at the N-terminus.

28. The antiviral composition for hepatitis C virus according to claim 27, wherein said transmembrane domain is composed of the amino acid sequence of SEQ ID NO: 3 or 4 and said proline enrichment domain consists of the amino acid sequence of SEQ ID NO: 5 or 6 .

29. The antiviral composition for hepatitis C virus according to claim 28, wherein said ER locus signal peptide comprises the amino acid sequence of SEQ ID NO: 1 or 2.

28. The antiviral composition for hepatitis C virus according to claim 27, wherein said polypeptide comprises a sequence of any one of the amino acid sequences of SEQ ID NOS: 11-14.

28. The antiviral composition for hepatitis C virus according to claim 27, wherein the SCOTIN protein comprises the amino acid sequence of SEQ ID NO: 9 or 10.

33. A method of inhibiting replication of hepatitis C virus comprising contacting a composition according to any one of claims 27-32 with hepatitis C virus (HCV).