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COMMON HIGH-SPEED PHOTO-CROSS-LINKING LINKER FOR MOLECULAR INTERACTION ANALYSIS AND IN VITRO SELECTION, AND IN VITRO SELECTION METHOD USING LINKER

外国特許コード F160008912
整理番号 (S2015-0929-N0)
掲載日 2016年12月6日
出願国 世界知的所有権機関(WIPO)
国際出願番号 2016JP060611
国際公開番号 WO 2016159211
国際出願日 平成28年3月31日(2016.3.31)
国際公開日 平成28年10月6日(2016.10.6)
優先権データ
  • 特願2015-072810 (2015.3.31) JP
発明の名称 (英語) COMMON HIGH-SPEED PHOTO-CROSS-LINKING LINKER FOR MOLECULAR INTERACTION ANALYSIS AND IN VITRO SELECTION, AND IN VITRO SELECTION METHOD USING LINKER
発明の概要(英語) The purpose of the present invention is to provide a linker that can be used for both candidate clone screening and assessment of the binding of the resulting candidate clones, without using enzymes, and to provide an in vitro selection method using the linker. The present invention provides a common high-speed photo-cross-linking linker for molecular interaction analysis and in vitro selection comprising a main chain and a side chain. The main chain has: a solid-phase binding site located at the 5' terminus for forming a bond with a solid-phase; solid-phase cleavage site for separating the solid-phase by separating the entire solid-phase binding site; a side chain linking site for linking a side chain; a high-speed photo-cross-linking site for linking the main chain to mRNA having a sequence complementary to the main chain by photo-cross-linking; and a reverse transcription initiation region located adjacent to the side chain linking site at the 3' terminus of the main chain. The side chain has: a fluorescent label; a protein binding site located at the free terminus; and a link forming site that links to the side chain linking site on the main chain.
従来技術、競合技術の概要(英語) BACKGROUND ART
Up to now, the development of antibody pharmaceuticals by each pharmaceutical company have been performed.Antibody, placed for a certain period of antigen molecules is administered to a plurality of times, then, the blood must be purified from a blood, in the in vivo production of the amount of time and effort.In addition, even the smallest molecular weight IgG 150 KDa as large molecular weight, difficult to synthesize a completely in vitro.
On the other hand, the antibody or the antigen recognition site of the peptide aptamers, small molecular weight can be artificially synthesized.
In the case of use as a pharmaceutical GMP level is found in organic synthesis, peptide aptamers as described above in the case of the synthesis of these levels are also possible.In addition, the molecular weight is small can be easy and the chemically-modified, immobilized on the chip or the like can be freely and, further, high stability so compared to the antibody, were fixed to the chip or the like, can be stored at room temperature such advantages as the.
Such peptide aptamers, after creating mRNA, techniques associated with the genotype - phenotype (hereinafter, simply referred to as' association techniques' may also be referred to.) Can be synthesized using and selection.Then, in association with such techniques include, outside the display methods cDNA, phage display, ribosome display method, mRNA display methods and the like are present.Of these, automated, high throughput in consideration of the request, to synthesize cDNA display method is most appropriately on the basis of the peptide aptamers.
Conventional, cDNA for use in display methods, as shown in Fig. 1A - 1D proposed linker, have been used. 1A In the FIG., solid phase binding sites and, with the main chain of the linker and the mRNA T4 RNA ligase for T4 RNA ligase so as to couple the connection portion, the base region and a primer for reverse transcription of the main chain, a peptide binding site and fluorescent labels, the main chain is connected to the side - chain linker is shown (see Patent Document 1, or less, '1 conventional example' referred to.).
In addition, Fig. 1B is, the double-stranded part of the main chain is composed of a conventional example 1 except that the linker has substantially the same structure, composed of the double-stranded portion, to separate the linker from the solid phase linker incorporated restriction sites is shown (Patent Document 1 reference, hereinafter, 'the conventional example 2' that).In addition, Fig. 1C is, in addition to the above conventional example 1, with a linker to a solid phase 1 for separating the first and second cleavage sites for the first 2 linker is shown (see Patent Document 1, hereinafter, 'the conventional example 3' referred to.).Is Fig. 1D, the main chain of 2 linker linked psoralen has been shown (see Patent Document 2, hereinafter, 'the conventional example 4' referred to.).
In addition to the above-described linker, is integrated into the main chain of the linker cnvK, light mRNA and cross-linking with the main chain of the linker that has been proposed (see Patent Document 3, hereinafter, 'the conventional example 5' referred to.).
However, the current Japanese leading cause of death in cancer (tumor), in the mixture is measured to its death, has been increasing year by.Whether or not suffering from a tumor, and the main treatment in order to observe the course after, and to exist in the tumor in the body, in blood or urine, or above a certain level of a particular substance (tumor marker) is detected as.Then, the type of tumor markers and their blood or urine by measuring the level, of whether or not the degree of progression of the tumor can be known.
Here, the tumor marker is, in most of the carbohydrate antigen.And normal cells by, the chain length is on the cell surface changes by glycosyltransferases, sugar chain specific for cancer cells, such sugar chain tumor markers for identifying cancer (hereinafter, 'sugar-chain tumor marker' is referred.) And (see non-patent document 5) are.Here, an antigenic determinant of a tumor marker sugar chain (hereinafter, referred to as' epitope '.) Is, schematically Fig. 2 (A) has a structure such as that depicted in general, from its structure, as shown in the following Table 1, 1-type sugar chain, 2-type sugar chain, sugar chain mother nucleus, is divided into core protein.
TABLE 1
Here, the 1 and 2 is-type sugar chain-type sugar chain, as shown in the above Table 1 the film is known, any of these, present on the cell surface sugar chain elongated by glycosyltransferase, are also longer than the case of normal cells.In addition, of the virus core protein and protein that is present around the nucleic acid.
(B) 2 to FIG., 1 of the backbone structure of a sugar chain structure of the type CA19-9(Carbohydrate Antigen 19-9) antigen, such as a carbohydrate antigen of example 1 schematically.Is CA19-9, 5 monosaccharide having the structure of 874 molecule of molecular weight, each monosaccharide is connected to the source by a glycosidic bond.In the figure, is NeuNac N- acetylneuraminic acid, gal is galactose, fucose Fuc, GlcNAc is N- acetyl glucosamine -D- respectively.Then, as shown in Fig. 2 (B) CA19-9 GlcNAc is included, other than the disk CA19-9 also included in the carbohydrate antigen is known (see non-patent document 5, hereinafter, 'the conventional example 6' referred to.).
In addition, the antibody, generally the light and heavy - chain and therefore, the molecular weight of the immunoglobulin (IgG), known as large as 170KDa.On the other hand, does not have a light chain of the camelidae antibody, and a heavy chain only, as small as 12KDa molecular weight, high plasticity and a three-dimensional structure, superior in thermal stability is known.Or less, such a region (domain) may also be referred to a molecule having a 'VHH'.
  • 出願人(英語)
  • ※2012年7月以前掲載分については米国以外のすべての指定国
  • SAITAMA UNIVERSITY
  • 発明者(英語)
  • NEMOTO NAOTO
  • MOCHIZUKI YUKI
  • KUMACHI SHIGEFUMI
国際特許分類(IPC)
指定国 National States: AE AG AL AM AO AT AU AZ BA BB BG BH BN BR BW BY BZ CA CH CL CN CO CR CU CZ DE DK DM DO DZ EC EE EG ES FI GB GD GE GH GM GT HN HR HU ID IL IN IR IS JP KE KG KN KP KR KZ LA LC LK LR LS LU LY MA MD ME MG MK MN MW MX MY MZ NA NG NI NO NZ OM PA PE PG PH PL PT QA RO RS RU RW SA SC SD SE SG SK SL SM ST SV SY TH TJ TM TN TR TT TZ UA UG US UZ VC VN ZA ZM ZW
ARIPO: BW GH GM KE LR LS MW MZ NA RW SD SL SZ TZ UG ZM ZW
EAPO: AM AZ BY KG KZ RU TJ TM
EPO: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
OAPI: BF BJ CF CG CI CM GA GN GQ GW KM ML MR NE SN ST TD TG
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