WO2016051808A1 - 遺伝子組換えカイコにより生産したビオチン化酸化ldl受容体・終末糖化産物受容体 - Google Patents
遺伝子組換えカイコにより生産したビオチン化酸化ldl受容体・終末糖化産物受容体 Download PDFInfo
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- WO2016051808A1 WO2016051808A1 PCT/JP2015/005017 JP2015005017W WO2016051808A1 WO 2016051808 A1 WO2016051808 A1 WO 2016051808A1 JP 2015005017 W JP2015005017 W JP 2015005017W WO 2016051808 A1 WO2016051808 A1 WO 2016051808A1
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- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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Definitions
- the present invention relates to the detection of oxidized LDL-like molecules (modified LDL) associated with arteriosclerosis and the evaluation of the effect of inhibiting the generation of oxidized LDL-like molecules by food intake.
- the present invention also relates to a novel terminal glycation end product receptor (sRAGE; soluble Receptor for Advanced Glycation End products). More specifically, the present invention relates to the detection of advanced glycation end products (AGEs) involved in the development of diabetic vascular disorders, and the evaluation of the effects of inhibiting AGE generation and accumulation by food intake.
- sRAGE novel terminal glycation end product receptor
- Non-Patent Document 1 Denatured LDL such as oxidized LDL is likely to play an important role in the development of atherosclerosis by being detected locally in human and hyperlipidemic model animals. It is strongly suggested.
- Non-patent Document 2 Since it has been reported that there is a correlation between the concentration of denatured LDL such as oxidized LDL in the blood and the onset of arteriosclerosis (Non-patent Document 2), the construction of a system for measuring denatured LDL such as oxidized LDL is a society. Great significance.
- denatured LDL such as oxidized LDL plays an important role in the early stage of arteriosclerosis. Therefore, the amount of denatured LDL such as oxidized LDL in the living body can be an index of lifestyle habit improving effects such as eating habits.
- modified LDL such as oxidized LDL is a general term for molecules generated by oxidative modification of LDL, and is an assembly having a non-uniform structure (FIG. 1). For this reason, various monoclonal antibodies have been developed, but all of them only detect some molecules, and it is difficult to accurately measure the amount of denatured LDL such as in vivo oxidized LDL. There is a need for the development of technologies that can be detected.
- Non-patent Document 3 detection systems using monoclonal antibodies against characteristic molecules (malondialdehyde: MDA, phosphatidylcholine: oxidized PC, etc.) generated when LDL is oxidatively modified have been developed (Non-patent Document 3). ⁇ 5).
- Apolipoprotein B Apolipoprotein B
- ApoB Apolipoprotein B
- monoclonal antibodies using chicken as a host have been produced and put into practical use (Non-patent Document 6). Detection techniques combining this antibody and oxidized LDL receptor have been developed.
- Patent Documents 1 to 5 and Non-Patent Documents 7 to 20 disclose single-chain antibodies and LDL, but none of them describe the production of single-chain antibodies that enable detection of oxidized LDL-like molecules. .
- the present inventors have produced single-chain antibodies against denatured LDL, but have not been able to produce them at the level of analysis and evaluation. Many other technologies related to LDL have been disclosed (Patent Documents 6 to 15), but their sensitivity is insufficient.
- the inventors of the present invention expressed a recognition region (CTLD14) of a receptor (LOX-1) that recognizes a wide variety of LDL molecular species subjected to oxidative modification in large quantities as inclusion bodies in E. coli, and then refolding.
- CTL14 a recognition region of a receptor
- LOX-1 receptor 1
- diabetic vascular disorders nephropathy, retinopathy, neurosis, etc.
- diabetes which is a representative of lifestyle-related diseases
- AGEs advanced glycation products
- RAGE receptor for AGEs
- RAGE receptor for AGEs
- RAGE receptor for AGEs
- RAGE receptor for AGEs
- RAGE recognizes AGEs of various structures, and AGEs are recognized by RAGE, leading to onset. Therefore, AGEs recognized by RAGE are considered to be AGEs that are meaningful to the living body (FIG. 42).
- the present inventors produced a silkworm that co-expressed a target protein (for example, CTLD14, sRAGE) and biotin ligase (BirA), and produced a biotinylated protein with high efficiency by oral administration of biotin.
- Biotinylated CTLD14 obtained from such silkworms had excellent pH stability characteristics.
- a lateral flow assay incorporating an anti-LDL chicken antibody, a single chain antibody, a non-biotinylated CTLD14, and streptavidin is used to oxidize and modify various LDL structures. was detected within 20 minutes.
- E. coli not subjected to glycosylation.
- coli is fragmented in about 1.5 months and loses its activity.
- the sRAGE obtained from the above silkworm has a sugar chain added and stably maintains the activity even when stored for nearly one year. It was revealed that It has been found that AGEs having various structures can be detected by maintaining the directionality through biotin of the obtained silkworm-type biotinylated sRAGE and performing the process.
- the present inventors have made it possible to detect a wide range of oxidized LDL-like molecules, while antibodies against specific molecules can only detect specific molecules.
- an anti-ApoB antibody it is difficult to distinguish from native LDL, and when modification has occurred in ApoB, it was difficult to detect, but in the present invention, it is possible to distinguish from native LDL. Even if there is a modification, it was possible to detect it.
- the present inventors have produced a recombinant silkworm that produces a ligand recognition region (sRAGE) of a terminal glycation end product (AGEs) receptor (RAGE) in the middle silk gland, and efficiently purified it.
- a method was established to produce an improved sRAGE.
- the obtained improved sRAGE has been subjected to glycosylation and is a very stable molecule, and it has been found that a small amount of AGEs can be concentrated and AGEs with various structures can be detected. Completed.
- the improved sRAGE (FIG. 34) produced a transgenic silkworm that produces sRAGE in the middle silk gland, and established a method for efficiently purifying sRAGE in large quantities efficiently from the silk gland extract (FIG. 36). ).
- the present invention is not capable of detecting all molecular species by antibody or instrumental analysis because of the variety of oxidized LDL (generic name for structures caused by oxidative modification) structures that are risk factors for arteriosclerosis.
- oxidized LDL generic name for structures caused by oxidative modification
- LOX-1 lectin like oxidized LDL receptor-1; an oxidized LDL receptor on the vascular endothelium that does not recognize LDL but recognizes a wide range of oxidized LDL
- LOX-1 recognizes oxidized LDL
- vascular endothelial dysfunction that occurs at the earliest stage of arteriosclerosis is induced, so that the oxidized LDL recognized by LOX-1 is a truly important molecule as a risk factor for arteriosclerosis.
- Development of an oxidized LDL detection system utilizing the recognition ability of LOX-1 was advanced (FIG. 1).
- the present invention provides a technique capable of detecting a wide range of molecular species so that the amount of oxidized LDL in vivo can be accurately measured. This is because antibodies against specific molecules can only detect specific molecules and cannot detect a wide range of oxidized LDL-like molecules, but CTLD14 developed to solve this is prepared by refolding.
- CTLD14 developed to solve this is prepared by refolding.
- the simple method there is a problem of stability such as aggregation in several months.
- the total number of days required for all processes is 10 days or more, and the method is complicated. .
- the method of secreting and producing LOX-1 in animal cells requires a special facility such as aseptic culture of cells, has a disadvantage that the culture cost is high, and the productivity is also inferior to that of E. coli.
- anti-ApoB antibody It is difficult to distinguish from native LDL, and there is a problem in measurement accuracy.
- ApoB when modified, there are problems such as difficulty in detection, and the present invention provides a technique for solving these problems.
- the present invention relates to an improved CTLD14 (silk-type); a method for producing improved CTLD14 produced by producing a recombinant silkworm that expresses CTLD14 in the middle silk gland, and expressing and purifying CTLD14 as a soluble protein; anti-LDL chicken Polyclonal and combinations thereof are provided.
- the present invention provides the following.
- ⁇ Single-chain antibody-related sequence, antibody characteristics
- (1) A single chain antibody or fragment thereof comprising the amino acid sequence set forth in any of SEQ ID NOs: 76 to 80 or a variant thereof.
- (2) A detection agent for detecting LDL and modified LDL (also referred to as LDL-modified product) comprising the single-chain antibody or fragment thereof according to item 1, wherein the LDL and modified LDL are acetylated LDL And a detection agent comprising at least one molecule selected from the group consisting of partially oxidized LDL.
- (3) The detection agent according to Item 2, wherein the LDL and modified LDL include acetylated LDL and partially oxidized LDL.
- LDL and the modified LDL include LDL, fully oxidized LDL, aldehyde-modified LDL (eg, malondialdehyde-modified LDL), acetylated LDL, and partially oxidized LDL.
- A2 The method according to Item A1, wherein the refolding is performed at an inclusion body amount of 2 mg to 10 mg (for example, 8.6 mg) per protein.
- A3 The method according to item A1 or A2, wherein the agarose column is a Ni-NTA slurry.
- A4 The method according to any one of Items A1 to A3, wherein the elution solution is a solution containing imidazole.
- A5 The method according to any one of Items A1 to A4, wherein the elution solution is phosphate buffered saline (PBS) containing imidazole.
- PBS phosphate buffered saline
- B1 A system for detecting or quantifying oxidatively modified LDL using the single-chain antibody or fragment thereof according to item 1 and the ligand recognition region of oxidized LDL receptor (LOX-1).
- B2 The system according to item B1, wherein the system uses an antigen-antibody reaction.
- B3 The system according to item B1 or B2, wherein the system is for performing an ELISA.
- B4 The system according to any one of items B1 to B3, wherein the system is for performing a sandwich ELISA.
- B5 The system according to any one of items B1 to B4, wherein the system is for performing a lateral flow assay.
- the system includes a membrane for developing a sample by capillary action, and the membrane is a ligand recognition region of the oxidized LDL receptor (LOX-1) labeled with the sample and metal colloid, silica particles or latex particles.
- LOX-1 oxidized LDL receptor
- a detection unit comprising the single-chain antibody or fragment thereof according to 1, and a control unit comprising a binding molecule for a ligand recognition region of the oxidized LDL receptor (LOX-1) or a binding molecule for a single-chain antibody
- LOX-1 ligand recognition region of oxidized LDL receptor
- the system includes a membrane for developing a sample by capillary action, and the membrane is CTLD14 labeled with a sample and a metal colloid, silica particles or latex particles, or a metal colloid, silica particles or latex particles.
- Conjugate part containing the labeled single-chain antibody or fragment thereof according to item 1; CTLD14 or detection part containing the single-chain antibody or fragment thereof according to item 1; and binding molecule to the CTLD14 The system according to item B5 or B6, including a control unit.
- the CTLD14 is biotinylated, His-tagged, Myc-tagged, Flag-tagged, E-tagged, or Strep-tagged, and in each case, the binding molecule is streptavidin, anti-His antibody
- System using chicken antioxidant LDL antibody> C1
- C1 To detect or quantify oxidatively modified LDL using an anti-denatured LDL antibody, a modified product or a fragment thereof, and a ligand recognition region of oxidized LDL receptor (LOX-1) in a lateral flow assay format System.
- C2 Diseases associated with denatured LDL (eg, dyslipidemia, arteries) using an anti-denatured LDL antibody, a modified product or a fragment thereof, and a ligand recognition region of oxidized LDL receptor (LOX-1) in a lateral flow assay format System for screening for sclerosis, diabetes, ischemic heart disease, cerebrovascular disorder).
- LOX-1 oxidized LDL receptor
- C3 The system according to item C2, wherein the disease associated with denatured LDL is selected from the group consisting of dyslipidemia, arteriosclerosis, diabetes, ischemic heart disease and cerebrovascular disorder.
- C4 Diseases associated with denatured LDL caused by food intake (for example, lipids) using an anti-denatured LDL antibody, a modified product or a fragment thereof and a ligand recognition region of oxidized LDL receptor (LOX-1) in a lateral flow assay format System for the evaluation of preventive effects of abnormalities, arteriosclerosis, diabetes, ischemic heart disease, cerebrovascular disorders).
- LOX-1 oxidized LDL receptor
- C5 The system according to item C4, wherein the disease associated with the modified LDL is selected from the group consisting of dyslipidemia, arteriosclerosis, diabetes, ischemic heart disease and cerebrovascular disorder. ⁇ D.
- CTLD14 (Silkworm type)> (D1) C-type lectin-like domain (CTLD) 14 having a silkworm-type sugar chain and comprising the amino acid sequence shown in SEQ ID NO: 86 or a variant thereof.
- CTLD 14 The CTLD 14 according to Item D1, wherein the silkworm sugar chain includes one or more kinds of sugar chains of a trimannosyl core, a complex sugar chain, an oligomannose sugar chain, or a hybrid sugar chain.
- D3 The silkworm-type sugar chain is added to the structure of trimannosyl core (from asparagine residue to GlcNAc- ⁇ 1,4-GlcNAc- ⁇ 1,4-Man (- ⁇ 1,6-Man) - ⁇ 1,3-Man).
- CTLD14 comprising a sugar chain to which 0 to 4 molecules of GlcNAc2 molecule and Man2 molecule are bound per 2 molecules.
- the silkworm-type sugar chain has the following combination 1) GlcNAc- ⁇ 1,4-GlcNAc- ⁇ 1,4-Man (- ⁇ 1,3-Man) - ⁇ 1,6-Man (- ⁇ 1 , 3-Man) - ⁇ 1,6-Man and GlcNAc- ⁇ 1,4-GlcNAc- ⁇ 1,4-Man (- ⁇ 1,6-Man- ⁇ 1,2-GlcNAc) - ⁇ 1,3-Man- ⁇ 1,2 -In combination with GlcNAc, 2) GlcNAc- ⁇ 1,4-GlcNAc- ⁇ 1,4-Man (- ⁇ 1,3-Man) - ⁇ 1,6-Man (- ⁇ 1,3-Man) - ⁇ 1,6-Man- ⁇ 1,2-Man and , GlcNAc- ⁇ 1,4-GlcNAc- ⁇ 1,4-Man (-- ⁇ 1,4-Glc
- (D6) The CTLD14 according to any one of items D1 to D5, wherein the CTLD14 is biotinylated.
- (D7) A composition for detecting denatured LDL, comprising CTLD14 according to any one of items D1 to D6.
- (D8) The composition according to item D7, wherein the composition is for distinguishing between non-modified LDL and modified LDL and detecting modified LDL.
- (D9) The composition according to Item D8, wherein the non-modified LDL and the modified LDL include LDL and oxidized LDL.
- (D10) The composition according to item D8 or D9, wherein the non-modified LDL and the modified LDL are LDL and oxidized LDL of a plurality of mammals.
- E2 The method according to item E1, wherein the expression is carried out in the posterior silk gland, middle silk gland or whole body of the organism that imparts the same sugar chain as the silkworm or silkworm.
- E3 The method according to item E1 or E2, wherein the expression is performed in the middle silk gland of the organism that imparts the same sugar chain as the silkworm or silkworm.
- E4 The method according to any one of items E1 to E3, wherein the CTLD14 is expressed in a biotinylated state or a biotinable state.
- E5 The method according to any one of items E1 to E4, wherein the organism includes a nucleic acid sequence encoding biotin ligase, and the step C) includes orally administering biotin to a silkworm.
- E6 The method according to any one of items E1 to E5, wherein the CTLD14 is expressed in a biotinylated state.
- E7 The method according to any one of items E1 to E6, wherein the organism comprises a tag sequence that undergoes biotinylation.
- E8 The tag sequence that undergoes biotinylation is BioEase. tag and Avi.
- the method of item E7 which is any of the tags.
- E9 The method according to any one of items E1 to E8, wherein the step A) is achieved by microinjecting an expression vector containing a nucleic acid molecule encoding the CTLD14.
- E10 The method according to any one of items E1 to E9, wherein the nucleic acid molecule comprises the nucleic acid sequence shown in SEQ ID NO: 85 or a variant thereof.
- E10-1) The method according to any one of items E1 to E10, wherein the CTLD 14 has the characteristics described in any one or more of items D1 to D6.
- E11 A silkworm in which a nucleic acid molecule encoding CTLD14 is incorporated so that it can be expressed, or an organism imparting a sugar chain similar to that of a silkworm
- E12 The nucleic acid molecule described above, comprising the nucleic acid sequence shown in SEQ ID NO: 85 or a variant thereof, and the same sugar chain as that of silkworm or silkworm according to item E11.
- E13 The organism according to item E11 or E12, wherein the organism comprises a sequence encoding biotin ligase.
- E14 The organism according to item E13, wherein the biotin ligase is BirA (SEQ ID NO: 116).
- composition according to item F1 or F2, further comprising CTLD14 The composition according to any one of items F1 to F3, wherein the CTLD14 is a CTLD14 containing a silkworm-type sugar chain.
- CTLD14 containing the silkworm-type sugar chain is the CTLD14 according to any one of items D1 to D6.
- F6 The composition according to any one of items F1 to F5, wherein the composition is for detecting LDL and oxidized LDL.
- F7 The composition according to item F6, wherein the LDL and oxidized LDL are LDL and oxidized LDL of plural kinds of mammals.
- (F8) A composition according to item F7, wherein the mammal includes a mouse and a human.
- F9 A kit for detecting denatured LDL, comprising anti-LDL chicken antibody and CTLD14.
- (F12) The kit according to any one of items F9 to F11, wherein the CTLD14 is the CTLD14 according to item D1.
- sRAGE Reconstructed terminal glycation end product receptor
- G1 Reconstructed terminal glycation end product receptor (sRAGE) having a silkworm type sugar chain and comprising the amino acid sequence shown in SEQ ID NO: 97 or a variant thereof.
- G2 The sRAGE according to Item G1, wherein the silkworm sugar chain includes one or more kinds of sugar chains of a trimannosyl core, a complex sugar chain, an oligomannose sugar chain, or a hybrid sugar chain.
- the silkworm-type sugar chain has a trimannosyl core structure (from asparagine residue to GlcNAc- ⁇ 1,4-GlcNAc- ⁇ 1,4-Man (- ⁇ 1,6-Man) - ⁇ 1,3-Man) SRAGE according to item G1 or G2, further comprising a sugar chain to which 0 to 4 molecules of GlcNAc 0 to 2 molecules and Man0 to 4 molecules are bound per molecule.
- the silkworm-type sugar chain has a trimannosyl core (from asparagine residue to GlcNAc- ⁇ 1,4-GlcNAc- ⁇ 1,4-Man (- ⁇ 1,6-Man) - ⁇ 1,3-Man) SRAGE according to item G1 to G2 or G3-1, further comprising a sugar chain to which 0 to 8 molecules out of GlcNAc 0 to 4 molecules and Man0 to 8 molecules are bound per molecule.
- G4-1 Items G1 to G2 or G3-1 in which the composition ratio of the silkworm type sugar chain is oligomannose type: 87 to 97%, complex type: 2 to 8%, hybrid type: 1 to 5% SRAGE according to any one of to G3-2.
- G4-2 Items G1 to G2 and G3-1 in which the composition ratio of the silkworm sugar chain is oligomannose type: 90 to 94%, complex type: 3 to 6%, hybrid type: 2 to 4% SRAGE according to any one of .about.G3-2 or G4-1.
- G4-3) Items G1 to G2, G3-1 in which the composition ratio of the silkworm type sugar chain is oligomannose type: 92.5%, complex type: 4.6%, hybrid type: 2.5% SRAGE according to any one of .about.G3-2 or G4-1 to G4-2.
- the oligomannose type includes (Man) 5 (GlcNAc) 2 , (Man) 7 (GlcNAc) 2 , (Man) 6 (GlcNAc) 2 and (Man) 3 (GlcNAc) 2
- the sRAGE according to any one of G2, G3-1 to G3-2, and G4-1 to G4-3.
- composition ratio in the whole oligomannose-type sugar chain is (Man) 5 (GlcNAc) 2 : 45 to 56%, (Man) 7 (GlcNAc) 2 : 23 to 33%, (Man) 6 (GlcNAc) 2 : 7-15%, and (Man) 3 (GlcNAc) 2 : 1-5% of items G2, G3-1 to G3-2, G4-1 to G4-3 or G5-1
- the sRAGE according to any one of the above.
- composition ratio in the whole oligomannose type sugar chain is (Man) 5 (GlcNAc) 2 : 48 to 54%, (Man) 7 (GlcNAc) 2 : 25 to 31%, (Man) 6 (GlcNAc) 2 : 9 to 13%, and (Man) 3 (GlcNAc) 2 : 2 to 4%, items G2, G3-1 to G3-2, G4-1 to G4-3, or G5-1 to SRAGE of any one of G5-2.
- the composition ratio in the whole oligomannose type sugar chain is (Man) 5 (GlcNAc) 2 : 51%, (Man) 7 (GlcNAc) 2 : 27.5%, (Man) 6 (GlcNAc ) 2 : 10.8%, and (Man) 3 (GlcNAc) 2 : 2.8%, items G2, G3-1 to G3-2, G4-1 to G4-3 or G5-1 to G5- 4. sRAGE of any one of 3. (G6-1) Any one of items G2, G3-1 to G3-2, G4-1 to G4-3, or G5-1 to G5-4, wherein the composite type includes (Man) 3 (GlcNAc) 3 SRAGE according to item.
- G6-2 Items G2, G3-1 to G3-2, G4-1 to G4-3, in which the composition ratio of the complex type sugar chain as a whole is (Man) 3 (GlcNAc) 3 : 2 to 8% SRAGE according to any one of G5-1 to G5-4 or G6-1.
- G6-3 Items G2, G3-1 to G3-2, G4-1 to G4-3 in which the composition ratio of the complex type sugar chain in the whole is (Man) 3 (GlcNAc) 3 : 3 to 6% SRAGE according to any one of G5-1 to G5-4 and G6-1 to G6-2.
- the silkworm type sugar chain binds to asparagine at position 3 and / or asparagine at position 59 of SEQ ID NO: 97, items G1 to G2, G3-1 to G3-2, G4-1 to G4-3, The sRAGE according to any one of G5-1 to G5-4, G6-1 to G6-4, and G7.
- the sRAGE is biotinylated, items G1 to G2, G3-1 to G3-2, G4-1 to G4-3, G5-1 to G5-4, G6-1 to G6- The sRAGE according to any one of 4 or G7 to G8.
- G12 Item G1 to G2, G3-1 to G3-2, G4-1 to G4-3, G5-1 to G5-4, G6-1 to G6-4, or G7 to G9
- a method for detecting AGEs comprising the step of contacting sRAGE with a sample and detecting AGEs.
- G13 A method for producing sRAGE, A) A step of incorporating a nucleic acid molecule encoding sRAGE in an expressible manner into a silkworm or an organism that imparts a sugar chain similar to that of a silkworm; B) placing the silkworm or an organism that imparts a sugar chain similar to that of silkworm under conditions where the gene is expressed, and expressing the sRAGE; and C) obtaining the sRAGE.
- the sRAGE includes items G1 to G2, G3-1 to G3-2, G4-1 to G4-3, G5-1 to G5-4, G6-1 to G6-4, or G7 to G9. 23.
- G23 A silkworm in which a nucleic acid molecule encoding sRAGE is incorporated so that it can be expressed, or an organism imparting a sugar chain similar to that of a silkworm.
- the nucleic acid molecule according to Item G23 which comprises the nucleic acid molecule shown in SEQ ID NO: 96 or a variant thereof, and imparts the same sugar chain as that of a silkworm.
- the organism according to item G23 or G24 wherein the organism contains a sequence encoding biotin ligase.
- H1 A silkworm that incorporates a nucleic acid molecule encoding a target and biotin ligase so as to allow co-expression, or an organism that imparts a sugar chain similar to that of a silkworm.
- H2 The organism according to item H1, wherein the biotin ligase is BirA.
- H3 The organism according to item H1 or H2, wherein the target is a C-type lectin-like domain (CTLD14), a terminal glycation product receptor (sRAGE) or a variant thereof.
- H4 The organism of item H3, wherein the CTLD14 is encoded by SEQ ID NO: 85 and the sRAGE is encoded by SEQ ID NO: 96.
- H5 The organism according to any one of items H1 to H4, wherein the organism includes a tag sequence that undergoes biotinylation.
- H6 The tag sequence that undergoes biotinylation is BioEase. tag and Avi.
- H5 The organism according to item H5, which is any of tags.
- H7 A method for producing a biotinylated protein, A) a step of incorporating a biotin ligase and a nucleic acid molecule encoding a protein into a silkworm or an organism having a sugar chain similar to that of a silkworm so as to allow co-expression; B) placing the silkworm or an organism that imparts a sugar chain similar to that of silkworm under conditions under which the nucleic acid molecule is expressed, and expressing the biotin ligase and the protein; and C) administering biotin to the organism, Obtaining a biotinylated protein.
- H8 The method according to Item H7, wherein the expression is performed in the middle silk gland of the silkworm.
- (H9) The method according to item H7 or H8, wherein the protein is expressed in a biotinylated state.
- (H10) The method according to any one of Items H7 to H9, wherein the step A) is achieved by microinjecting an expression vector containing a nucleic acid molecule encoding the protein.
- the protein comprises a C-type lectin-like domain (CTLD14), a terminal glycation product receptor (sRAGE), a single-chain antibody comprising the amino acid sequence of any of SEQ ID NOs: 76 to 80 or a variant thereof, or a The method according to any one of items H7 to H10, which is a fragment or a variant thereof.
- H12 The method according to any one of items H7 to H11, wherein the organism includes a tag sequence that undergoes biotinylation.
- the tag sequence that undergoes biotinylation is BioEase. tag and Avi.
- a silkworm co-expressing biotin ligase (BirA) and CTLD14 or sRAGE was constructed.
- the expressed protein has an excellent effect of being biotinylated with high efficiency.
- the obtained protein has a silkworm-type sugar chain added and is excellent in stability (for example, stability to pH and long-term stability which is stable for almost one year).
- AGEs with various structures can be detected by immobilizing the silkworm-type biotinylated sRAGE of the present invention while maintaining the orientation via biotin.
- a human single chain antibody library is constructed, and a single chain antibody capable of recognizing oxidized LDL or the like is obtained from a single chain antibody library comprising ⁇ + ⁇ chains by biopanning, and its gene sequence is revealed. It was made. In the present invention, 5 clones were obtained.
- the amino acid sequences of the five single chain antibodies selected in the present invention are all sequences not reported in other groups, but are homologous to the light chain Fab fragment of the antioxidant LDL antibody in its VL region (57-86% )was there.
- the VL region of ⁇ + ⁇ 19 showed 86% homology with the amino acid sequence (Accession No: AAO49738) of the light chain Fab region of the oxidized LDL antibody registered by the group of Jang et al.
- the light chain Fab fragment (Accession No: AAX57559) of the rabbit antibody and the VL region of ⁇ + ⁇ 12 showed 83% homology to malondialdehyde LDL registered by the same group.
- the VL region of ⁇ + ⁇ 5 is also homologous to a recently reported antibody against PCSCK9 (Schiele, 2014).
- PCSK9 is known as a molecule that induces degradation of LDLR (Maxwell, 2004).
- the anti-PCSK9 antibody by Schiele et al. Has a function of lowering the blood level of LDL as a result by binding to the C-terminal region of PCSK9 and inhibiting PCSK9 interaction with LDLR (Schiele, F. , Park, J., Redemann, N., Luippold, G., and Nar, H. (2014) An antibody against the C-terminal domain of PCSK9 lowers LDL cholesterol levels in vivo. J. Mol. -852; and Maxwell, K.N., Breslow, J.L. (2004) Adenoviral-mediated expression of Pcsk9 in miice results in a low-density lipoprotein receptor knockout phenotype. Proc. Natl. Acad. Sci. U. S.A.101, 7100-7105.)
- clones Nos. 5 and 19 SEQ ID NOs: 76 and 78
- clone No5 can be expressed in large quantities.
- a stable supply system of the obtained single chain antibody ( ⁇ + ⁇ 5) was further established. Since establishment of such a stable supply system for single-chain antibodies has not been possible in the past, the present invention also has a remarkable effect in this respect.
- the present invention when the binding characteristics were evaluated, it was found that a wide range of modified LDL recognition ability such as partially oxidized LDL and acetylated LDL was confirmed in addition to fully oxidized LDL and LDL. In the present invention, the ability to recognize oxidized LDL in a low concentration region is excellent. In particular, quantitative evaluation of LDL that has undergone oxidative modification using a sandwich ELISA combined with oxidized LDL receptor (LOX-1) is possible. It was. Furthermore, the present invention can detect oxidized LDL-like molecules derived from a wide range of biological species such as humans and mice.
- LOX-1 oxidized LDL receptor
- the antibody of the present invention is a single-chain antibody with a clear sequence, it is possible to develop an efficient production system using various expression systems, modification such as glycosylation, and functional modification by introducing mutations. It is.
- the present invention relates to a single-chain antibody that recognizes oxidized LDL, a stable supply system of single-chain antibody, a method for detecting denatured LDL using a single-chain antibody, and an oxidized LDL receptor (LOX-1) Provided is a method for the specific detection of combined oxidized LDL-like molecules.
- a large amount of purified CTLD14 can be obtained within a few days by a simple technique by utilizing transgenic silkworms (FIG. 23).
- FOG. 23 transgenic silkworms
- the improved CTLD14 to which the silkworm-type sugar chain of the present invention is added does not recognize native LDL, but widely recognizes modified LDL and has a wider detection dynamic range than CTLD14 prepared from conventional E. coli. Became clear (FIGS. 27 and 28).
- the anti-LDL chicken polyclonal antibody obtained in the present invention was found to be capable of recognizing unmodified LDL, modified LDL in various modified states, and also capable of detecting a type of modified LDL that is not recognized by the anti-ApoB chicken monoclonal antibody. (FIG. 26).
- the combination of anti-LDL chicken polyclonal antibody and CTLD14 enabled detection of denatured LDL, which was difficult to detect by the conventional method (FIG. 28).
- the present invention has a remarkable feature in that the present situation that the AGEs to be subjected to instrumental analysis is less than 10% of the entire AGEs can be overcome.
- a wide range of AGEs can be detected by the conventional detection method using reconstructed RAGE (sRAGE), but sRAGE has a problem in practical use such as fragmentation in about one and a half months and loss of recognition ability ( (See FIG. 38) is also remarkable in that this can be overcome.
- the improved s RAGE of the present invention has undergone glycosylation, is a very stable molecule, and maintains recognition activity over a long period of time (confirmed up to about 1 year at the time of application) when stored at 4 ° C. (FIGS. 38, 40 and 53).
- the improved sRAGE of the present invention enabled the concentration of a small amount of AGEs and the detection of AGEs having various structures (FIG. 40).
- FIG. 1 shows a schematic diagram of LDL and oxidized LDL.
- oxidized LDL modified LDL
- FIG. 1 shows a schematic diagram of LDL and oxidized LDL.
- oxidized LDL modified LDL
- FIG. 2 shows a schematic diagram of a single chain antibody and an exemplary outline of the production procedure.
- a single chain gene antibody library is developed as shown in the examples (for example, pCANTAB5E), displayed on phage, single chain antibodies are selected by biopanning, and live consisting of ⁇ + ⁇ Promising clones were obtained from the rally and the gene sequence was determined. Promising clones were not obtained from the library consisting of ⁇ + ⁇ .
- Single-chain antibodies can be selected in a short period of time without immunizing animals after library construction, and can be employed for glycosylation using various expression systems, modified by mutagenesis, etc. There are also advantages such as being easy.
- FIG. 3A shows evaluation of the prepared phage-displayed single chain antibody by ELISA in a library composed of ⁇ + ⁇ .
- FIG. 3B shows evaluation of the prepared soluble single-chain antibody in the library consisting of ⁇ + ⁇ by ELISA. From the left, each clone No. (5, 12, 14, 15, 19, 31, 40, 79, 96). The rightmost side shows PBS.
- FIG. 4A shows evaluation of the prepared phage-displayed single chain antibody by ELISA in a library consisting of ⁇ + ⁇ . From the left, each clone No. (1, 2, 3, 4, 11, 13, 15, 93, 94). The rightmost side shows PBS.
- FIG. 4B shows evaluation of the prepared soluble single chain antibody by ELISA in a library consisting of ⁇ + ⁇ . From the left, each clone No. (1, 2, 3, 4, 11, 13, 15, 93, 94). The rightmost side shows PBS.
- FIG. 5 shows the amino acid sequences of ⁇ + ⁇ 5, ⁇ + ⁇ 12, ⁇ + ⁇ 19, ⁇ + ⁇ 40, and ⁇ + ⁇ 96 from the top (the amino acid sequence is in single letter code).
- FWR1 indicates framework region 1
- FWR2 indicates framework region 2
- FWR3 indicates framework region 3
- FWR4 indicates framework region 4
- CDR1 indicates complementarity determining region 1
- CDR2 indicates complementarity Determining region 2 is shown
- CDR3 indicates complementarity determining region 3.
- FIG. 6 shows the results of SDS-PAGE of the culture supernatant and cells when cultured using TG1, HB2151 and BLR.
- the leftmost is a molecular weight marker, and the left four bands show TG1 (two left of them are not induced (left shows cells and the right shows supernatant), and two right are induced (left shows cells) , The right shows the supernatant), and the four bands on the left show HB2151 (the left two of them are not induced (the left shows the cells, the right shows the supernatant), and the two on the right Induced (the left shows cells and the right shows supernatant), and the right four bands show BLR (there are two left without induction (of which the left shows the cells and the right shows the supernatant) ), Two on the right are induced (of which the left shows the cells and the right shows the supernatant).
- FIG. 7 shows the results of Western blotting using anti-E antibody as the primary antibody. Each band shows from the left a molecular weight marker, no induction of ⁇ + ⁇ 5, and induction of ⁇ + ⁇ 5. A band of ⁇ + ⁇ 5 (G3 signal + E tag attached) is in the vicinity of 30.8 kDa.
- FIG. 8 shows the results of SDS-PAGE performed to evaluate purification using anti-E antibody-immobilized sepharose. Each band shows from the left a molecular weight marker, no induction of ⁇ + ⁇ 5, and induction of ⁇ + ⁇ 5. A band of ⁇ + ⁇ 5 (G3 signal + E tag attached) is in the vicinity of 30.8 kDa.
- FIG. 8 shows the results of SDS-PAGE performed to evaluate purification using anti-E antibody-immobilized sepharose. Each band shows from the left a molecular weight marker, no induction of ⁇ + ⁇ 5, and induction of ⁇ + ⁇ 5. A band of ⁇ +
- FIG. 9A shows the results of SDS-PAGE of the insoluble fraction and the soluble fraction when cultured using pET-22b (+) as an expression vector.
- FIG. 9B shows the results of Western blotting using the induced and non-induced anti-His antibody bodies as primary antibodies when cultured using pET-22b (+) as an expression vector.
- FIG. 10 shows the results of SDS-PAGE of ⁇ + ⁇ 5. Among the data on the right side, the upper panel shows ⁇ + ⁇ 5, and from the left, the marker, before induction, after induction, the whole cell lysate fraction, soluble fraction, and insoluble fraction are shown.
- FIG. 11 shows the results of evaluating the antigen recognition activity of a single chain antibody ( ⁇ + ⁇ 5) by direct ELISA.
- antigens LDL (upper left panel), acetylated LDL (upper right panel), partially oxidized LDL (lower left panel), and fully oxidized LDL (lower right panel) were used.
- the vertical axis indicates B / B0 (relative ratio) of absorbance (A450) at 450 nm.
- the horizontal axis indicates the amount of each sample ( ⁇ g / well).
- FIG. 12 shows detection of LDL and oxidized LDL by direct ELISA using a single chain antibody ( ⁇ + ⁇ 5).
- the vertical axis shows the absorbance (A450) at 450 nm.
- the horizontal axis indicates the amount of each sample ( ⁇ g / well).
- FIG. 13 shows the results of evaluating the antigen recognition activity of a single chain antibody ( ⁇ + ⁇ 5) by sandwich ELISA. LDL and oxidized LDL were used as antigens.
- the vertical axis shows the absorbance (A450) at 450 nm.
- the horizontal axis indicates the amount of each sample ( ⁇ g / well).
- FIG. 14 shows an outline of a detection system based on the principle of the lateral flow assay.
- FIG. 14 shows an outline of a detection system based on the principle of the lateral flow assay.
- FIG. 15 shows an outline of a detection system based on the principle of the lateral flow assay.
- a sample pad, a conjugate pad, a pad including a test line, and an absorption pad are used, and a sample is added to each of the sample pad and the conjugate pad, and a conjugate formation reaction is performed.
- the sample added at the sample pad moves by capillary action and moves to the conjugate pad.
- a metal colloid-bound CTLD14 or a single-chain antibody of the present invention is included to produce a conjugate.
- a test line and a control line are included, where the test line includes CTLD14 or single chain antibody and binding occurs when the target is in the sample. Made and detected.
- FIG. 16 shows a schematic example of a detection system based on the principle of the lateral flow assay.
- CTLD14 is labeled with a metal colloid (or latex particle)
- a molecule in which a single-chain antibody is combined with the tag of CTLD14 in the test line in the case of biotinylated CTLD14, streptavidin, His-Tag In this case, an anti-His antibody or the like
- a secondary antibody is applied.
- FIG. 17 shows E. coli cells that were expressed in E. coli and then refolded by a precision method after purification.
- E. coli CTLD14 was treated with SDS-PAGE sample buffer (+) containing ⁇ -mercaptoethanol (2ME) or SDS-PAGE sample buffer (-) not containing 2ME, and then subjected to SDS-PAGE and Coomassie staining. Results are shown.
- CTLD14 When processed in the absence of 2ME, CTLD14 is detected at the molecular weight position of the dimer, whereas when processed in the presence of 2ME, it is detected at the molecular weight position of the monomer. This was refolded by a precision method.
- E. coli CTLD14 correctly forms a dimer which is a quaternary structure of a basic unit on a cell membrane. The yield of CTLD14 that correctly formed dimers was 12 mg per liter culture.
- FIG. 18 shows a schematic diagram of production by transgenic silkworms.
- FIG. 19 shows amino acid information, sugar chain information, and a schematic diagram of dimerization on the right of improved CTLD14 (silk-type sugar chain-added biotinylated CTLD14).
- FIG. 20 shows an example of the structure of a silkworm-type sugar chain (N-linked sugar chain) of CTLD14 or sRAGE (a typical structure of an N-linked sugar chain found in a glycoprotein expressed in the silkworm middle silk gland ( Iizuka et al., FEBSJ., 277, 5806-5820)).
- the combination of sugar chains estimated by CTLD14 is a combination of 2 (one of oligomannose types) and 4 complex types, 3 (one of oligomannose types), and a hybrid type (5 or 6). Although there is, it is not limited to these.
- squares indicate N-acetylglucosamine (GlcNAc), and circles indicate mannose (Man).
- FIG. 21 shows the expression of CTLD14 transgenic silkworms for each strain.
- 1 ⁇ indicates a band of Biotin_CTLD14 (with BioEase tag), and 2 ⁇ indicates CTLD14 with a myc tag.
- A shows Coomassie blue (CBB) staining
- B shows the result of Wester blot (anti-His tag antibody).
- CBB Coomassie blue
- B shows the result of Wester blot (anti-His tag antibody).
- From left negative control, 1 to 4 lines of Biotin-CTLD14, and 1 to 3 lines of Myc-CTLD14 are shown, respectively.
- the left end shows molecular weight (kDa).
- FIG. 22 shows a diagram confirming whether Biotin-CTLD14 is biotinylated in the silkworm body.
- FIG. 23 shows the purification process of CTLD14 produced by transgenic silkworms.
- Lane 1 Ni-agarose elution fraction
- 2 TALON non-adsorbed fraction
- 3 5 mM, elution fraction during imidazole washing
- 4 TALON adsorbed fraction
- 5 50 mM imidazole elution fraction
- 6 100 mM
- 7 The purified silkworm CTLD14 obtained by collecting 200 mM, 8, 9: 500 mM
- 10 1 M imidazole elution fraction
- 11 imidazole non-eluting fraction
- 12 200 mM-500 mM imidazole elution fraction and then dialyzing it against PBS ( ⁇ ) is shown.
- FIG. 24 shows the results of confirming glycosylation of silkworm-derived CTLD14.
- the purified silkworm CTLD (14, 20 ⁇ g) was treated with PNGase F and subjected to 15% SDS-PAGE.
- (+) Means a PNGase F-treated sample
- ( ⁇ ) means a sample that has undergone the same reaction in the absence of PNGase F.
- Left The result of CBBR staining, the amount of sample added is 6.7 ⁇ g / lane.
- a molecular weight shift was confirmed by sugar chain removal by PNGaseF treatment.
- Middle shows results of glycoprotein staining before and after PNGaseF treatment with Glycoprotein Staining (Pierce 24562). The sugar chain was removed by PNGaseF treatment and was no longer detected as a glycoprotein.
- FIG. 25 shows that the protein band at the dimer position is shifted to the monomer position by treatment with 2-mercaptoethanol ( ⁇ -Me). The left is E. E.
- FIG. 26 shows the characteristics of the anti-LDL chicken polyclonal antibody.
- LDL As detection targets, LDL, acetylated (Ac) LDL, malondialdehyde (MDA) LDL, and fully oxidized (fuOx) LDL were used.
- MDA-LDL Commercially available LDL (Biomedical Technologies, BT-903) and AcLDL (Biomedical Technologies, BT-906) were used.
- MDA-LDL was prepared by reacting commercially available LDL with malonated aldehyde.
- Oxidized LDL was prepared by reacting commercially available LDL with copper oxide. What was made to react at 37 degreeC for 4 hours was made into partial oxidation, and what was made to react at 37 degreeC for 20 hours was made into complete oxidation.
- a commercially available anti-ApoB chicken monoclonal antibody (Pharma Foods, HUC20) was used.
- HRP-labeled anti-chicken IgY (Promega, G1351) was used as the secondary antibody.
- a commercially available anti-ApoB mouse monoclonal antibody (Acris BM2149) was used.
- HPR-labeled anti-mouse IgG (Millipore, AP192P) was used.
- LDL detection with anti-LDL chicken polyclonal antibody From the left, fully oxidized LDL (fuOxLDL), MDA-LDL, AcLDL, unmodified LDL, and PBS are shown, and the numbers indicate the concentration of the detection target. It shows that fully oxidized LDL (fuOxLDL), MDA-LDL, AcLDL, and unmodified LDL can all be recognized.
- Middle shows detection of anti-ApoB chicken monoclonal antibody LDL. From the left, fully oxidized LDL (fuOxLDL), MDA-LDL, AcLDL, unmodified LDL, and PBS are shown, and the numbers indicate the concentration of the detection target.
- FIG. 27 shows the results of a combination of Escherichia coli CTLD14 and the anti-LDL chicken antibody of the present application.
- White squares indicate unmodified LDL
- black squares indicate AcLDL
- black triangles indicate MDA LDL
- white circles indicate mo (partially oxidized LDL)
- black circles indicate fu (fully oxidized LDL).
- Left Detection of LDL and modified LDL by sandwich method using CTLD14 derived from E. coli and anti-LDL chicken polychrome.
- FIG. 28 shows the results of a combination of silkworm CTLD14 and the anti-LDL chicken antibody of the present invention.
- White squares indicate unmodified LDL, black squares indicate AcLDL, black triangles indicate MDA-LDL, white circles indicate mo (partially oxidized LDL), and black circles indicate fu (fully oxidized LDL).
- Left Detection of LDL and modified LDL by sandwich method using transgenic silkworm CTLD14 and anti-LDL chicken polychrome. All modified LDLs can be detected. (MoLDL: recognition of partially oxidized LDL is weak). Compared with the combination with E. coli CTLD14, the concentration range for detecting modified LDL without reacting with LDL is extremely wide.
- FIG. 29 shows the results of LC-ESIMS of the tryptic digest of silkworm-type CTLD14 after treatment with silkworm-type CTLD14 without treatment with PNGaseF.
- peptide fragments (1, 2, 3) containing NX-S / T that are expected to be glycosylated and peptide fragments (4, 5, 6) with close chain length, depending on the presence or absence of PNGaseF treatment When the spectra were compared, peptides 1 and 2 containing NCS and NST were not observed without PNaseF treatment, and a signal was observed after PNGaseF treatment, indicating that sugar chains were added. Indicated. On the other hand, peptide 3 showed a signal at the same position regardless of the presence or absence of PNGaseF treatment, indicating that no sugar chain was added.
- FIG. 30 shows the results of LC-ESIMS of a tryptic digest of a sample without CTNG14 treated with PNGaseF.
- FIG. 31 is an enlarged view of the mass spectrum near the elution position 13.1 minutes in FIG. 30 in order to observe the sugar chain structure expected to be added in more detail.
- FIG. 32 is a table summarizing the difference in mass between ions and peptides observed in the mass spectrum near 13.1 minutes shown in FIG. 31 and examined in detail the sugar chain structure expected to be added. It is a result.
- FIG. 33 is a schematic diagram of production by silkworms used in the present invention.
- FIG. 34 shows the amino acid sequence of RAGE expressed in transgenic silkworms.
- FIG. 35 shows the electrophoresis results of the middle silk gland extract of the transgenic silkworm.
- Samples were obtained by removing the middle silk gland from the transgenic silkworm strain 4 (1-4) and the negative control (only the middle silk gland GALA4, no UAS: not expressing sRAGE), followed by the middle silk gland 1 1 ml of PBS containing 1% Triton X-100 was added to the book and mixed by inverting at 4 ° C. for 2 hours. On the left is the result of protein staining with Coomassie Brilliant Blue after electrophoresis on a 4-12% gradient SDS-polyacrylamide gel. The amount of sample added to each lane is 5 ⁇ l / lane on the left side and 10 ⁇ l / lane on the right side.
- M molecular weight marker (benchmark: Invitrogen)
- 1-4 indicates the line number of the produced transgenic silkworm
- Nega negative control.
- the right shows the result of SD Western blotting.
- the sample was developed by SDS-PAGE with a gradient of 4 to 12% and then transferred to a PVDF membrane.
- anti-His rabbit antibody (diluted 1000 times) + anti-His mouse antibody (3000) was then reacted with a secondary antibody, HRP-labeled anti-rabbit & mouse antibody (200,000-fold dilution), and finally reacted with ECL Prime to detect the luminescence and confirm the expression of sRAGE .
- 36 shows the purification process of RAGE.
- sRAGE was eluted by binding a lysate from which the sericin had been removed from the middle silk gland extract by freeze-thawing to Ni-Agarose equilibrated with PBS, and gradually increasing the imidazole concentration in PBS. The results of subjecting each stage sample to SDS-PAGE and staining with Coomassie are shown.
- Lane 1 Lysate, 2: Ni-Agarose non-adsorbed fraction, 3: Ni-Agarose adsorbed fraction, 4: 20 mM imidazole elution fraction, 5: 100 mM imidazole elution fraction, 6: 250 mM imidazole elution fraction, 7 : 500 mM imidazole elution fraction, 8: 1 M imidazole elution fraction, 9: non-elution fraction.
- the eluted fraction was collected with 250 mM-1M imidazole, dialyzed against PBS, and subjected to purification with TALON when necessary.
- Ni-Agarose 250 mM-1 M elution fraction was bound to TALON to increase the purification, and imidazole concentration was increased stepwise to elute sRAGE.
- sRAGE adds a BioEase tag that is a tag for biotinylation. In order to confirm this biotinylation, the binding to Streptavidin / Mutein matrix was confirmed. The result of subjecting the case sample to SDS-PAGE and staining with Coomassie is shown.
- Lane 1 After purification of Ni-Agarose (confirmed to have a sufficient degree of purification even at this stage), 2: mutein-binding fraction, 3: mutein non-adsorbed fraction, 4: TALON adsorbed fraction, 5: TALON Non-adsorbed fraction, 6: 50 mM imidazole elution fraction, 7: 100 mM imidazole elution fraction, 8: 250 mM imidazole elution fraction, 9: 500 mM imidazole elution fraction, 10: 1 M imidazole elution fraction, 11: TALON non-elution Fraction.
- FIG. 37 shows a diagram confirming biotinylation of silkworm-type sRAGE. The left figure shows the result of detecting biotinylation.
- FIG. 38 shows the stability of silkworm sRAGE.
- the left is E.
- the right side shows the silkworm type of the present invention.
- FIG. 39 shows confirmation of glycosylation of silkworm type RAGE. 20 ⁇ g of silkworm-type sRAGE after purification was treated with PNGaseF and subjected to 12% SDS-PAGE.
- (+) Means a PNGase F-treated sample
- ( ⁇ ) means a sample that has undergone the same reaction in the absence of PNGase F.
- Left The result of CBBR staining, the amount of sample added is 6.7 ⁇ g / lane. A molecular weight shift was confirmed by sugar chain removal by PNGaseF treatment.
- Middle shows results of glycoprotein staining before and after PNGaseF treatment with Glycoprotein Staining (Pierce 24562). The sugar chain was removed by PNGaseF treatment and was no longer detected as a glycoprotein.
- Right Confirmed that there is no change in reactivity with anti-RAGE antibody even after PNGaseF treatment.
- FIG. 40 shows detection results of various AGEs by a sandwich ELISA-like method using silkworm-type sRAGE and E. coli-type sRAGE.
- FIG. 41 shows mass spectrometry results of silkworm sRAGE.
- the sRAGE prepared using the silkworm contained in the gel piece was acetamidated, digested with trypsin, and measured by MALDI-TOFMS and LC-ESIMS. Mass spectra were compared for those with and without PNGaseF treatment.
- FIG. 42 shows a schematic diagram of RAGE and AGEs. As shown, AGEs have various molecular forms, and have limitations in detection by antibodies and detection by chemical analysis. In order to detect AGEs having such various forms, multi-recognition ability such as RAGE is important.
- FIG. 43 shows an outline of production of a silkworm-type biotinylated protein. Three constructs were introduced into one individual as indicated. For BirA, four lines were tried.
- FIG. 44 shows the results of Coomassie staining after SDS-PAGE of CTLD14 and sRAGE. “-” Indicates a strain that does not express BirA. Sample No. 72 is a negative control into which only Ser1-GAL4 is introduced. FIG. 45 shows the results of a Western blot for confirming biotinylation. The white band is due to the signal being too strong. “-” Indicates a strain that does not express BirA. Sample No. 72 is a negative control into which only Ser1-GAL4 is introduced. FIG.
- FIG. 46 shows the outline of the experiment for confirming the biotinylation efficiency and the results thereof.
- L represents Lysate before processing
- F represents a flow through fraction after processing
- R represents a Resin binding fraction.
- Sample No. 72 is a negative control into which only Ser1-GAL4 is introduced.
- FIG. 47 shows an overview of the yield and biotinylation efficiency of CTLD14 and sRAGE subjected to TALON and Mutein column purification.
- FIG. A table summarizing the stability of E. coli CTLD14 and silkworm CTLD14 is shown.
- E. The E. coli type has limited applications because the buffer that can be used is limited. However, the silkworm type is stable at pH 7 to 9.5 in the absence of salt, and is stable in Tris buffer.
- FIG. 49 shows the results of detection of oxidized LDL in human plasma diluted 1/1000.
- sample 1 no OxLDL added
- sample 2 (1 ⁇ g OxLDL / ml added
- sample 3 (3 ⁇ g OxLDL / ml added)
- sample 4 (6 ⁇ g OxLDL / ml added)
- sample 5 (12 ⁇ g OxLDL added) / Ml addition
- the right graph shows the relative absorbance when Sample 1 (without OxLDL / ml addition) is 1.
- FIG. 50 shows an outline of an immunochromatographic detection system using a simple half strip using a silkworm-type biotinylated CTLD14.
- FIG. 51 shows the results of immunochromatography using a simple half strip. Lane 1: 0 ⁇ g / well, Lane 2: 0.03 ⁇ g / well, Lane 3: 0.3 ⁇ g / well, Lane 4: 3 ⁇ g / well.
- FIG. 52 shows the results of expressing the single-chain antibody ⁇ + ⁇ 5 in the middle silk gland of the transgenic silkworm. This is a result of preparing a middle silk gland extract and subjecting it to SDS-PAGE for each of 4 clones of a line added with a myc tag at the N-terminal and a line added at the C-terminal. The left figure shows the results of Coomassie staining, and the right figure shows the results of detection with anti-His antibody.
- FIG. 52 shows the results of expressing the single-chain antibody ⁇ + ⁇ 5 in the middle silk gland of the transgenic silkworm. This is a result of preparing a middle silk gland extract and subjecting it to SDS-PAGE for each of 4 clones
- FIG. 54 shows the sugar chain structure of silkworm sRAGE.
- N1, N2, N3 and N4-1 are oligomannose types
- N4-2 is a complex type
- N4-3 is a hybrid type sugar chain.
- FIG. 55 shows the composition ratio, quantitative value, and sugar composition of each sugar chain identified in Example 22.
- FIG. 56 shows the results of measuring the interaction of biotinylated sRAGE or biotinylated sRAGE with AGEs by surface plasmon resonance.
- the figure on the left shows the result of immobilizing silkworm-type sRAGE without biotinylation to sensor chip CM5 by amine coupling and analyzing the interaction with fructose-treated BSA (F-AGE), which is a typical AGE, by single cycle kinetics. is there.
- the right figure shows biotinylated sRAGE with one molecule of biotin added to the C-terminal side, immobilized on the sensor chip SA via the biotin, and the interaction with F-AGE, which is a typical AGE, is shown in single cycle kinetics. This is the result of analysis.
- the upper right is E.E.
- the lower right figure shows the analysis result of the interaction between silkworm biotinylated sRAGE and AGEs.
- PBS (-) was used as the buffer, and the flow rate was 30 ⁇ l / min.
- 0.25, 0.5, 1, 2, 4 ⁇ M F-AGE was added, the addition time was 60 seconds, the dissociation time was 120 seconds, and the interaction was measured.
- the time interval for adding F-AGE at each concentration was 60 seconds.
- the vertical axis represents Resonance Unit (RU), and the horizontal axis represents time (seconds).
- antibody broadly refers to polyclonal antibodies, monoclonal antibodies, multispecific antibodies, chimeric antibodies, and anti-idiotype antibodies, and functional fragments thereof (eg, F (ab ′) 2 , and Fab fragments), as well as other recombinantly produced conjugates or functional equivalents (eg, chimeric antibodies, humanized antibodies, multifunctional antibodies, bispecific or oligospecific antibodies, single chain antibodies) Single chain antibody (scFV), diabody, sc (Fv) 2 (single chain (Fv) 2 ), scFv-Fc).
- antibodies may be covalently linked or recombinantly fused to enzymes such as alkaline phosphatase, horseradish peroxidase, alpha galactosidase, and the like.
- such antibodies may be covalently linked or recombinantly fused to enzymes such as alkaline phosphatase, horseradish peroxidase, alpha galactosidase, and the like.
- enzymes such as alkaline phosphatase, horseradish peroxidase, alpha galactosidase, and the like.
- anti-denatured LDL antibody refers to any antibody against denatured LDL.
- an antibody refers to a full-length antibody (eg, polyclonal antibody, monoclonal antibody, etc.), and the others may be referred to as a variant or a fragment.
- the antibody used in the present invention may be bound to its target (LDL or oxidized LDL-like molecule), and its origin, type, shape, etc. are not limited. Specifically, it can be produced based on known antibodies such as non-human animal antibodies (eg, mouse antibodies, rat antibodies, camel antibodies), human antibodies, chimeric antibodies, and humanized antibodies. In the present invention, a single chain antibody is used.
- the binding of the antibody to the target is preferably discriminative or specific binding.
- the modified antibody may be a combination of an antibody and various molecules such as polyethylene glycol. Antibody variants can be obtained by chemically modifying antibodies using known techniques.
- single chain antibody is also referred to as “scFv (single chain Fv)”, in which the variable region regions (V H and V L ) of the heavy chain and light chain of the antibody are linked with an appropriate linker peptide. It corresponds to that.
- a single chain antibody protein can be expressed by constructing such a construct at the gene level and introducing it into E. coli using a protein expression vector.
- fragment refers to a polypeptide or polynucleotide having a sequence length of 1 to n ⁇ 1 with respect to a full-length polypeptide or polynucleotide (length is n).
- the length of the fragment can be appropriately changed according to the purpose.
- the lower limit of the length is 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, and more amino acids, and lengths expressed in integers not specifically listed here (eg, 11 etc.) are also suitable as lower limits. possible.
- examples include 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 and more nucleotides.
- Non-integer lengths may also be appropriate as a lower limit.
- the lengths of polypeptides and polynucleotides can be represented by the number of amino acids or nucleic acids, respectively, as described above, but the above numbers are not absolute, and the upper limit is not limited as long as they have the same function.
- the above-mentioned number as the lower limit is intended to include the upper and lower numbers (or, for example, up and down 10%) of the number.
- “about” may be added before the number. However, it should be understood herein that the presence or absence of “about” does not affect the interpretation of the value.
- the length of a fragment useful herein can be determined by whether or not at least one of the functions of a full-length protein that serves as a reference for the fragment is retained.
- homology of a gene refers to the degree of identity of two or more gene sequences to each other. Therefore, the higher the homology between two genes, the higher the sequence identity or similarity. Whether two genes have homology can be examined by direct sequence comparison or, in the case of nucleic acids, hybridization methods under stringent conditions.
- the DNA sequence between the gene sequences is typically at least 50% identical, preferably at least 70% identical, more preferably at least 80%, 90% , 95%, 96%, 97%, 98% or 99% are identical, the genes are homologous.
- the comparison of similarity, identity, and homology between amino acid sequences and base sequences is calculated using default parameters using BLAST, which is a sequence analysis tool.
- the identity search can be performed using, for example, NCBI BLAST 2.2.9 (issued 2004.12).
- the identity value usually refers to a value when the BLAST is used and aligned under default conditions. However, if a higher value is obtained by changing the parameter, the highest value is set as the identity value. When identity is evaluated in a plurality of areas, the highest value among them is set as the identity value.
- the “corresponding” amino acid has or is expected to have the same action as a predetermined amino acid in a protein or polypeptide as a reference for comparison in a certain protein molecule or polypeptide molecule.
- An amino acid For example, in LOX-1, the amino acids at positions 232 and 249.
- the “corresponding” gene refers to a gene having, or expected to have, the same action as that of a predetermined gene in a species as a reference for comparison in a certain species.
- a gene corresponding to a gene eg, LOX-1
- genes corresponding to human genes can be found in other animals (mouse, rat, pig, rabbit, guinea pig, cow, sheep, etc.). Such corresponding genes can be identified using techniques well known in the art.
- the corresponding gene in an animal is the sequence of that animal (eg, mouse, rat, pig, rabbit, guinea pig, cow, sheep, etc.) using the sequence of the gene serving as the reference for the corresponding gene as a query sequence. It can be found by searching the database.
- the “variant” refers to a substance in which a part of the original substance such as a polypeptide or polynucleotide is changed. Such variants include substitutional variants, addition variants, deletion variants, truncated variants, allelic variants, and the like. Alleles are genetic variants that belong to the same locus and are distinguished from each other. Therefore, an “allelic variant” refers to a variant having an allelic relationship with a certain gene. “Species homologue or homolog” means homology (preferably at least 60% homology, more preferably at least 80%, at a certain amino acid level or nucleotide level within a certain species. 85% or higher, 90% or higher, 95% or higher homology).
- ortholog is also called an orthologous gene, which is a gene derived from speciation from a common ancestor with two genes. For example, taking the hemoglobin gene family with multiple gene structures as an example, the human and mouse alpha hemoglobin genes are orthologs, but the human alpha and beta hemoglobin genes are paralogs (genes generated by gene duplication). . Since orthologs are useful for the estimation of molecular phylogenetic trees, orthologs may also be useful in the present invention.
- the “conservative (modified) variant” is applied to both the amino acid sequence and the nucleic acid sequence.
- Conservatively modified variants with respect to a particular nucleic acid sequence refer to nucleic acids that encode the same or essentially the same amino acid sequence, and are essentially identical if the nucleic acid does not encode an amino acid sequence. An array.
- Such base sequence modification methods include restriction enzyme digestion, DNA polymerase, Klenow fragment, DNA ligase treatment and other ligation treatments, site-specific base substitution methods using synthetic oligonucleotides (specification) Site-directed mutagenesis; MarkZollerandMichael Smith, Methods in Enzymology, 100, 468-500 (1983)), but can also be modified by methods usually used in the field of molecular biology. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine.
- each codon in a nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan), produces a functionally identical molecule. It is understood that it can be modified.
- each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence.
- such modifications can be made to avoid substitution of cysteine, an amino acid that greatly affects the conformation of the polypeptide.
- Certain amino acids can be substituted with other amino acids in the protein structure, such as, for example, the binding site of a ligand molecule, without an apparent reduction or loss of interaction binding capacity. It is the protein's ability to interact and the nature that defines the biological function of a protein. Thus, specific amino acid substitutions can be made in the amino acid sequence or at the level of its DNA coding sequence, resulting in proteins that still retain their original properties after substitution. Thus, various modifications can be made in the peptide disclosed herein or in the corresponding DNA encoding this peptide without any apparent loss of biological utility.
- Such a nucleic acid can be obtained by a well-known PCR method, and can also be chemically synthesized. These methods may be combined with, for example, a site-specific displacement induction method or a hybridization method.
- hydrophobicity index of amino acids can be taken into account.
- the importance of the hydrophobic amino acid index in conferring interactive biological functions in proteins is generally recognized in the art (Kyte. J and Doolittle, RFJ. Mol. Biol. 157 ( 1): 105-132, 1982).
- the hydrophobic nature of amino acids contributes to the secondary structure of the protein produced and then defines the interaction of the protein with other molecules (eg, enzymes, substrates, receptors, DNA, antibodies, antigens, etc.).
- Each amino acid is assigned a hydrophobicity index based on their hydrophobicity and charge properties.
- one amino acid can be replaced by another amino acid having a similar hydrophobicity index and still result in a protein having a similar biological function (eg, a protein equivalent in ligand binding capacity). It is well known.
- the hydrophobicity index is preferably within ⁇ 2, more preferably within ⁇ 1, and even more preferably within ⁇ 0.5. It is understood in the art that such amino acid substitutions based on hydrophobicity are efficient. As described in US Pat. No. 4,554,101, the following hydrophilicity indices have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.
- an amino acid can be substituted with another that has a similar hydrophilicity index and can still provide a biological equivalent.
- the hydrophilicity index is preferably within ⁇ 2, more preferably within ⁇ 1, and even more preferably within ⁇ 0.5.
- “conservative substitution” refers to substitution in which the hydrophilicity index and / or hydrophobicity index of the amino acid to be replaced with the original amino acid is similar as described above.
- conservative substitutions are well known to those skilled in the art and include, for example, substitutions within the following groups: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine; However, it is not limited to these.
- amino acid additions, deletions, or modifications can also be made to produce functionally equivalent polypeptides.
- Amino acid substitution refers to substitution of one or more of the original peptide with, for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3 amino acids.
- Addition of amino acid means adding one or more, for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3 amino acids to the original peptide chain.
- Deletion of amino acids refers to deletion of one or more, for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3 amino acids from the original peptide.
- Amino acid modifications include, but are not limited to, amidation, carboxylation, sulfation, halogenation, alkylation, phosphorylation, hydroxylation, acylation (eg, acetylation), and the like.
- the amino acid to be substituted or added may be a natural amino acid, a non-natural amino acid, or an amino acid analog. Natural amino acids are preferred.
- substitution, addition and / or deletion of a polypeptide or polynucleotide refers to an amino acid or a substitute thereof, or a nucleotide or a substitute thereof, respectively, relative to the original polypeptide or polynucleotide.
- Replacing, adding, or removing Such substitution, addition and / or deletion techniques are well known in the art, and examples of such techniques include site-directed mutagenesis techniques. Can these changes in the reference nucleic acid molecule or polypeptide occur at the 5 'or 3' end of this nucleic acid molecule as long as the desired function (eg, LOX-1 recognition ability, etc.) is retained?
- Or can occur at the amino-terminal or carboxy-terminal site of the amino acid sequence representing this polypeptide, or can occur anywhere between those terminal sites, interspersed individually between residues in the reference sequence.
- such a number can be one or several, and preferably within 20%, within 15%, within 10%, within 5%, or less than 150, less than 100, less than 100, It can be 50 or less, 25 or less, and the like.
- tag sequence refers to a substance for sorting molecules by a specific recognition mechanism such as a receptor-ligand, more specifically, to bind a specific substance.
- a substance that plays the role of a binding partner for example, biotin-avidin, biotin-streptavidin or the like.
- a specific substance to which a tag sequence is bound can be selected by contacting the substrate to which the binding partner of the tag sequence is bound.
- tag sequences are well known in the art. Representative tag sequences include, but are not limited to, myc tag, His tag, HA, Avi tag and the like.
- the “detection agent” broadly means any factor capable of detecting a target substance or state (for example, denatured LDL, LOX-1, AGEs, cells, tissues, pathological conditions, etc.).
- protein protein
- polypeptide oligopeptide
- peptide refers to a polymer of amino acids having an arbitrary length.
- This polymer may be linear, branched, or cyclic.
- the amino acid may be natural or non-natural and may be a modified amino acid.
- the term can also encompass one assembled into a complex of multiple polypeptide chains.
- the term also encompasses natural or artificially modified amino acid polymers. Such modifications include, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification (eg, conjugation with a labeling component).
- This definition also includes, for example, polypeptides containing one or more analogs of amino acids (eg, including unnatural amino acids, etc.), peptide-like compounds (eg, peptoids) and other modifications known in the art. Is done.
- amino acid may be natural or non-natural as long as the object of the present invention is satisfied.
- amino acid derivative or “amino acid analog” refers to an amino acid that is different from a naturally occurring amino acid but has the same function as the original amino acid. Such amino acid derivatives and amino acid analogs are well known in the art. As used herein, it is understood that amino acid derivatives and amino acid analogs can be used alternatively as long as they can provide the same biological function as an amino acid.
- natural amino acid means an L-isomer of a natural amino acid.
- Natural amino acids are glycine, alanine, valine, leucine, isoleucine, serine, methionine, threonine, phenylalanine, tyrosine, tryptophan, cysteine, proline, histidine, aspartic acid, asparagine, glutamic acid, glutamine, ⁇ -carboxyglutamic acid, arginine, ornithine , And lysine. Unless otherwise indicated, all amino acids referred to herein are L-forms, but forms using D-form amino acids are also within the scope of the present invention.
- “unnatural amino acid” means an amino acid that is not normally found naturally in proteins.
- unnatural amino acids include norleucine, para-nitrophenylalanine, homophenylalanine, para-fluorophenylalanine, 3-amino-2-benzylpropionic acid, homo-arginine D-form or L-form and D-phenylalanine.
- amino acid analog refers to a molecule that is not an amino acid but is similar to the physical properties and / or function of an amino acid. Examples of amino acid analogs include ethionine, canavanine, 2-methylglutamine and the like.
- Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
- Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides can also be referred to by the generally recognized one letter code.
- polynucleotide As used herein, “polynucleotide”, “oligonucleotide”, and “nucleic acid” are used interchangeably herein and refer to a nucleotide polymer of any length. The term also includes “oligonucleotide derivatives” or “polynucleotide derivatives”. “Oligonucleotide derivatives” or “polynucleotide derivatives” refer to oligonucleotides or polynucleotides that include derivatives of nucleotides or that have unusual linkages between nucleotides, and are used interchangeably.
- oligonucleotide examples include, for example, 2′-O-methyl-ribonucleotide, an oligonucleotide derivative in which a phosphodiester bond in an oligonucleotide is converted to a phosphorothioate bond, and a phosphodiester bond in an oligonucleotide.
- oligonucleotide derivatives in which ribose and phosphodiester bond in oligonucleotide are converted to peptide nucleic acid bond uracil in oligonucleotide is C— Oligonucleotide derivatives substituted with 5-propynyluracil, oligonucleotide derivatives wherein uracil in the oligonucleotide is substituted with C-5 thiazole uracil, cytosine in the oligonucleotide is C-5 propynylcytosine Substituted oligonucleotide derivatives, oligonucleotide derivatives in which cytosine in the oligonucleotide is substituted with phenoxazine-modified cytosine, oligonucleotide derivatives in which the ribose in DNA is substituted with 2'-
- a particular nucleic acid sequence may also be conservatively modified (eg, degenerate codon substitutes) and complementary sequences, as well as those explicitly indicated. Is contemplated. Specifically, a degenerate codon substitute creates a sequence in which the third position of one or more selected (or all) codons is replaced with a mixed base and / or deoxyinosine residue. (Batzer et al., Nucleic Acid Res. 19: 5081 (1991); Ohtsuka et al., J. Biol. Chem. 260: 2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8: 91-). 98 (1994)).
- nucleotide may be natural or non-natural.
- Nucleotide derivative or “nucleotide analog” refers to a substance that is different from a naturally occurring nucleotide but has a function similar to that of the original nucleotide.
- nucleotide derivatives and nucleotide analogs are well known in the art. Examples of such nucleotide derivatives and nucleotide analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2′-O-methyl ribonucleotides, peptide-type nucleic acids (PNA). Not.
- complex molecule refers to a molecule formed by linking a plurality of types of molecules such as polypeptides, polynucleotides, lipids, sugars, and small molecules.
- complex molecules include, but are not limited to, glycolipids and glycopeptides.
- a polypeptide having an amino acid exemplified by SEQ ID NO: 97 or the like, or a variant or fragment thereof, as long as it has biological activity involved in diagnosis, each variant or fragment is referred to as Encoding nucleic acid molecules can also be used.
- a complex molecule containing such a nucleic acid molecule can also be used.
- nucleic acid is also used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.
- Particular nucleic acid sequences also include “splice variants”.
- a particular protein encoded by a nucleic acid implicitly encompasses any protein encoded by a splice variant of that nucleic acid.
- a “splice variant” is the product of alternative splicing of a gene. After transcription, the initial nucleic acid transcript can be spliced such that different (another) nucleic acid splice products encode different polypeptides.
- the production mechanism of splice variants varies, but includes exon alternative splicing.
- Other polypeptides derived from the same nucleic acid by read-through transcription are also encompassed by this definition. Any product of a splicing reaction (including recombinant forms of splice products) is included in this definition.
- gene refers to a factor that defines a genetic trait. Usually arranged in a certain order on the chromosome. A gene that defines the primary structure of a protein is called a structural gene, and a gene that affects its expression is called a regulatory gene. As used herein, “gene” may refer to “polynucleotide”, “oligonucleotide” and “nucleic acid” and / or “protein” “polypeptide”, “oligopeptide” and “peptide”.
- homology of a gene refers to the degree of identity of two or more gene sequences to each other. Therefore, the higher the homology between two genes, the higher the sequence identity or similarity. Whether two genes have homology can be examined by direct sequence comparison or, in the case of nucleic acids, hybridization methods under stringent conditions.
- the DNA sequence between the gene sequences is typically at least 50% identical, preferably at least 70% identical, more preferably at least 80%, 90% , 95%, 96%, 97%, 98% or 99% are identical, the genes are homologous.
- Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides can also be referred to by the generally recognized one letter code.
- the comparison of similarity, identity, and homology between amino acid sequences and base sequences is calculated using default parameters using BLAST, which is a sequence analysis tool.
- the identity search can be performed using, for example, NCBI BLAST 2.2.9 (issued 2004.12).
- the identity value usually refers to a value when the BLAST is used and aligned under default conditions. However, if a higher value is obtained by changing the parameter, the highest value is set as the identity value. When identity is evaluated in a plurality of areas, the highest value among them is set as the identity value.
- polynucleotide that hybridizes under stringent conditions refers to well-known conditions commonly used in the art.
- a polynucleotide can be obtained by using a colony hybridization method, a plaque hybridization method, a Southern blot hybridization method or the like using a polynucleotide selected from among the polynucleotides of the present invention as a probe.
- hybridization was performed at 65 ° C. in the presence of 0.7 to 1.0 M NaCl using a filter on which colony or plaque-derived DNA was immobilized, and then a 0.1 to 2-fold concentration was obtained.
- a polynucleotide that can be identified by washing the filter under conditions of 65 ° C using an SSC (saline-sodium citrate) solution (composition of 1-fold concentration of SSC solution is 150 mM sodium chloride, 15 mM sodium citrate).
- SSC saline-sodium citrate
- concentration of 1-fold concentration of SSC solution is 150 mM sodium chloride, 15 mM sodium citrate.
- Hybridization was performed in Molecular Cloning 2nd ed. , Current Protocols in Molecular Biology, Supplements 1-38, DNA Cloning 1: Core Techniques, APRacical Approach, Second Edition, Oxford, etc.
- the sequence containing only the A sequence or only the T sequence is preferably excluded from the sequences that hybridize under stringent conditions.
- polypeptide used in the present invention is encoded by a nucleic acid molecule that hybridizes under stringent conditions to a nucleic acid molecule encoding a polypeptide specifically described in the present invention. Also encompassed are polypeptides.
- hybridizable polynucleotide refers to a polynucleotide that can hybridize to another polynucleotide under the above hybridization conditions.
- the polynucleotide capable of hybridizing is a polynucleotide having at least 60% homology with the base sequence of DNA encoding a polypeptide having the amino acid sequence represented by SEQ ID NO: 97, preferably 80% The polynucleotide which has the above homology, More preferably, the polynucleotide which has 95% or more of homology can be mentioned.
- Tm (° C.) 81.5 + 16.6 (log [Na + ]) + 0.41 (% G + C) ⁇ 600 / N ⁇ 0.72 (% formamide)
- N is the length of the duplex formed
- [Na + ] is the molar concentration of sodium ions in the hybridization or wash solution
- % G + C is the (guanine + Cytosine) base percentage.
- the melting temperature decreases by about 1 ° C. for each 1% mismatch.
- refolding refers to the wrapping of cycloamylose while unraveling an abnormal structure of a polypeptide that has lost its original function due to abnormal folding and preventing reaggregation with a surfactant. This refers to refolding to the original correct structure of the polypeptide by utilizing its ability to contact or by dilution dialysis under the control of redox potential.
- purification column refers to a solid or semi-solid support that has affinity or can bind to molecules such as proteins (for example, antibodies), such as metal chelate agarose. Column etc. can be mentioned. Each molecule can be separated by the difference in affinity or binding force between molecules. Depending on the properties of the molecule to be purified, a person skilled in the art will select an appropriate support. For example, a Ni-NTA agarose column can efficiently purify proteins to which a His tag or the like has been added.
- Ni-NTA slurry refers to a resin in which nickel ions are chelated to nitrilotriacetic acid (NTA) immobilized on agarose.
- phosphate buffered saline is an aqueous solution having a pH of 7 to 8 containing NaCl, KCl, Na 2 HPO 4 , and KH 2 PO 4.
- concentration and pH of each component are as follows.
- PBS (+) means containing calcium ions and magnesium ions
- PBS ( ⁇ ) means calcium ions.
- PBS means “PBS ( ⁇ )” unless explicitly stated otherwise.
- typically Dulbecco's PBS (-) can be used.
- the composition of Dulbecco's PBS ( ⁇ ) is NaCl 8 g, KCl 0.2 g, Na 2 HPO 4 1.15 g, KH 2 PO 4 0.2 g / L, (pH 7.4).
- cellulose ester membrane tube refers to a dialysis tube produced by extruding a polymer mixture of cellulose acetate.
- receptor refers to an organism comprising one or more binding domains that are reversibly and specifically complexed with one or more ligands. Where the complexation has a biological structure. Receptors can be completely outside the cell (extracellular receptors), inside the cell membrane (but directing the receptor portion to the extracellular environment and cytosol), or completely inside the cell (intracellular Of the receptor). They can also function independently of cells. Receptors in the cell membrane allow cells to communicate (eg, signal transduction) with space outside their boundaries and to function in the transport of molecules and ions to the inside and outside of the cell. As used herein, a receptor may be a full length receptor or a fragment of a receptor.
- Oxidized LDL receptor refers to any receptor that receives oxidized LDL. Oxidized LDL receptors include, but are not limited to, for example, LOX-1 as described herein, as well as scavenger receptors such as SR-A, SR-B, CD36, and the like. Although sometimes referred to in the art as “denatured LDL receptor”, it is identical to “oxidized LDL receptor”.
- modified LDL is also referred to as “LDL modified product” or “modified LDL” (which are used interchangeably), and LDL is active oxygen, oxidative enzyme, Fe in the body. Any LDL modification product having various molecular modifications generated by contact with 3+ or the like, or by cell-dependent chemical changes by vascular endothelial cells, macrophages and the like.
- OxLDL oxidized LDL
- MDA-LDL malondialdehyde-modified LDL
- CRA crotonaldehyde
- HNE 4-hydroxynonenal
- HEL hexanoyl
- small particle LDL LDL having a diameter of 255 nm or less
- saccharified LDL acetylated LDL (AcLDL)
- AcLDL acetylated LDL
- oxidized LDL shows abnormal values, arteriosclerosis, ischemic heart disease (myocardial infarction, angina, etc.), cerebrovascular disorder (cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage, transient ischemic attack, etc.), Diseases such as aortic aneurysm, renal infarction, and hyperlipidemia are expected, but not limited thereto (see “Today's clinical test 2007-2008” publisher, Nankodo, Inc.).
- MDA-LDL normal range: 10 to 80 L
- oxidized phosphatidylcholine normal range: 8.4 U / mL to 17.6 U / mL
- LDL low density lipoprotein
- LOX-1 lectin-like Oxidized LDL receptor
- LOX-1 feeding such as human LOX-1 (also referred to as hLOX-1), bovine LOX-1 (also referred to as b-LOX1), porcine LOX-1, mouse LOX-1, and rabbit LOX-1 Examples include, but are not limited to, animal LOX-1.
- bLOX-1 is a glycoprotein having a molecular weight of about 50 kDa consisting of 273 amino acid residues belonging to the C-type lectin family, the N-terminal is in the cytoplasm, and the C-terminal is a cell membrane once-through type II It is a type membrane protein.
- hLOX-1 is also a glycoprotein with a molecular weight of about 50 kDa consisting of 273 amino acid residues belonging to the C-type lectin family, having a N-terminal in the cytoplasm, and a C-terminal single-penetrating cell membrane. It is a type II membrane protein.
- LOX-1 is structurally composed of the following four domains: an N-terminal cytoplasmic domain, a hydrophobic transmembrane domain, a neck domain, and a C-type lectin-like domain.
- Bovine LOX-1 (bLOX-1) is a glycoprotein having a molecular weight of about 50 kDa consisting of 273 amino acid residues belonging to the C-type lectin family, the N-terminus is in the cytoplasm, and the C-terminus is outside the cell membrane.
- Human LOX-1 (hLOX-1) is also a type II membrane protein of about 30 kDa consisting of 273 amino acid residues belonging to the C-type lectin family (molecular weight of about 40 kDa due to addition of sugar chains at positions 139 and 183).
- This receptor is structurally composed of the following four domains: an N-terminal cytoplasmic domain, a hydrophobic transmembrane domain, a neck domain, and a C-type lectin-like domain (also referred to herein as CTLD). .
- This CTLD is highly conserved among species, particularly at the position of 6 cysteine residues, and is a functional domain for recognizing LOX-1 ligands. Six cysteine residues in this CTLD are involved in the intramolecular disulfide bond of hLOX-1. In addition to this conserved CTLD, neck domains in hLOX-1 and other known species have high sequence identity. Also, according to Xie et al.
- CTLD is a minimal domain sufficient to bind denatured LDL, even if CTLD is not glycosylated. It has been shown that LDL can be recognized and bound.
- LOX-1 is expressed not only in vascular endothelial cells but also in macrophages and activated vascular smooth muscle cells and has undergone various structurally unrelated macromolecules (modified LDL, bacteria, senescent erythrocytes, apoptosis) Cells and platelets), and plays an important role in various biological phenomena such as biological defense mechanisms and inflammatory mechanisms, and its expression is hyperlipidemia, diabetes under various conditions Pathologies such as hyperglycemia, hypertension, hypertensive nephrosclerosis, arteriosclerosis, ischemia-reperfusion injury, vascular balloon injury; and oxidized LDL, angiotensin II, endothelin, TNF- ⁇ , late glycation reaction product ( AGEs), TGF- ⁇ , 8-
- C-type lectin-like domain is also referred to as “CTLD”, and has homology with sugar chain recognition sites of members belonging to the C-type lectin family.
- CTLD is very well conserved among these members, among species, and the position of the 6 cysteine residues is completely conserved.
- CTLD is a functional domain for recognizing the ligand of LOX-1, and six cysteine residues in it are involved in three intramolecular disulfide bonds of hLOX-1. Therefore, the mutant LOX-1-surfactant complex preferably retains cysteines at positions 144, 155, 172, 243, 256, and 264 in the amino acid sequence of SEQ ID NO: 82.
- cysteine at position 140 in hLOX-1 is involved in an intermolecular disulfide bond and forms a dimer of hLOX-1.
- cysteine at position 140 is not essential for recognition of denatured LDL, it is not always necessary to retain the cysteine at position 140 when expressed, and it may be mutated.
- hLOX-1 sugar chains are added at N at position 183 and N at position 139.
- the molecular weight of hLOX-1 with a sugar chain added is 50 kDa.
- hLOX-1 is usually glycosylated, it can recognize and bind to denatured LDL, just like normal glycosylated hLOX-1, even if it is not glycosylated.
- the CTLD is a minimal domain necessary and sufficient to bind denatured LDL.
- the 4 C-terminal residues (LRaq) of LOX-1 are essential for ligand recognition and uptake, and the 7 C-terminal residues (KANLRAQ) are essential for hLOX-1 folding and transport.
- hLOX-1 W150, R208, R229, R231, R248, etc. are essential amino acids for ligand recognition and uptake.
- LOX-1 has also been reported to be cleaved from the cell membrane, released as a soluble form, and also present in the blood of healthy individuals.
- CTLD-like polypeptide refers to “CTLD”, “PR (Protease-Resistant (protease resistance))-CTLD”, “CTLD14” (CTLD + C-terminal side of the neck domain).
- a polypeptide having 14 amino acids “PR-CTLD14”, “CTLD + neck” (a polypeptide having CTLD + neck domain), or all polypeptides encompassed by “PR-CTLD + neck” or variants thereof .
- CTLD molecule is understood to include CTLD-like polypeptides as well as any complexes thereof. Therefore, it is understood that CTLD molecules include LOX-1 full length, LOX-1 extracellular region full length (S61 to Q273), CTLD14 (129-143), CTLD (143-273), and the like.
- CTLD14 and PR-CTLD14 are (1) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 84; (2) the amino acid sequence shown in SEQ ID NO: 84 above.
- CTLD-like polypeptide including CTLD14 may contain an unnatural amino acid, an amino acid analog, an amino acid derivative, or the like as long as the activity of natural LOX-1 is maintained.
- terminal glycation product or “late glycation reaction product” (both in English, Advanced Glycation End Products) are also referred to as AGEs, and are the source of damage to the quality of life of diabetic patients. It is involved in the development and development of diabetic vascular disorders known as vascular complications. Eye, nerve, and kidney disorders due to vascular complications are called diabetic retinopathy, neuropathy, and nephropathy (a total of three major complications), which are characteristic pathologies for diabetic patients. Reducing sugars represented by glucose react non-enzymatically with amino groups of proteins and amino acids to form glycation products such as Schiff bases or Amadori rearrangement compounds.
- the reaction so far is reversible and is called the first reaction. Thereafter, a terminal glycation product is formed through complicated and irreversible reactions such as condensation, cleavage, and cross-linking. Such a series of reactions is called glycation.
- AGEs is also a generic name for structures generated through such a process. Examples of the AGEs structure present in the living body include, but are not limited to, carboxymethyllysine (CML), carboxyethyllysine (CEL), pentosidine, pyralin, imidazoline, methylglyoxal, and crosslin.
- a product obtained by glycation of albumin, immunoglobulin, ovalbumin or the like present in plasma is also AGE, and is widely used in experimental systems as AGE.
- BSA bovine serum albumin
- R-AGE BSA glycated with ribose
- F-AGE BSA treated with fructose
- G- AGE BSA saccharified with glucose
- Hemoglobin A1c used as an indicator of blood glucose control is an Amadori transfer compound, but is included in AGEs. Arbitrary proteins can also be converted to AGEs.
- CML albumin and CEL albumin included in AGEs are both AGEs in which albumin has undergone glycation.
- Such an AGE generation reaction can occur in the living body either in the circulating blood, in the extracellular matrix, or in the cell.
- AGEs present in the blood vessels of diabetic patients include: those that are fluorescent and have a cross-linked structure (such as pentosidine and croslin) and those that are neither fluorescent nor cross-linked (such as carboxymethyllysine, pyralin, methylglyoxal (MG) -imidazolone) ).
- reference substances include pyralin (normal range: less than 23 pmol / mL in plasma), pentosidine (normal range: 0.00915 to 0.0431 ⁇ g / mL in plasma (when measured by ELISA)) (See “Today's clinical test 2007-2008” issuance office, Nankodo Co., Ltd.).
- complex means any construct that includes two or more parts.
- the other part may be a polypeptide or other substance (eg, sugar, lipid, nucleic acid, other hydrocarbon, etc.).
- two or more parts constituting the complex may be bonded by a covalent bond, or bonded by other bonds (for example, hydrogen bond, ionic bond, hydrophobic interaction, van der Waals force, etc.). May be.
- bonds for example, hydrogen bond, ionic bond, hydrophobic interaction, van der Waals force, etc.
- the “complex” includes a molecule formed by linking a plurality of molecules such as a polypeptide, a polynucleotide, a lipid, a sugar, and a small molecule.
- ligand is a binding partner for a specific receptor or family of receptors.
- the ligand can be an endogenous ligand for the receptor, or alternatively, a synthetic ligand for the receptor, such as a drug, drug candidate, or pharmacological means.
- AGE molecules refers to any molecule contained in the AGE.
- Lys-AGE glutaraldehyde-modified lysine-modified AGE
- glucose-modified AGE G-AGE
- R-AGE ribose-modified AGE
- F-AGE fructose-modified AGE
- the “molecule exhibiting AGE-like activity” refers to a molecule having at least one of the above-mentioned AGE activity (referred to as “AGEs-like activity” in the present specification).
- AGEs-like activity examples include, but are not limited to, binding activity (ligand activity) to RAGE.
- AGE receptor Receptor for AGE
- RAGE receptor for AGE
- (1) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 8; (2) SEQ ID NO: 102 above A polypeptide comprising an amino acid sequence comprising one or several amino acid substitutions, additions and / or deletions in the amino acid sequence represented by (2), and exhibiting the activity of natural RAGE; A polypeptide comprising an amino acid sequence having at least 90% sequence identity with the sequence and exhibiting the activity of natural RAGE; (4) an amino acid having at least 80% sequence homology with the amino acid sequence shown in SEQ ID NO: 102 above A polypeptide comprising the sequence and exhibiting the activity of native RAGE; (5) SEQ ID NO: 10 (6) a nucleic acid molecule that hybridizes under stringent conditions with a nucleic acid sequence complementary to the nucleic acid sequence shown in SEQ ID NO: 101.
- Nucleic acid sequence shown in serial SEQ ID NO: 101 and comprises an amino acid sequence encoded by a nucleic acid molecule having at least 80% sequence homology, and polypeptides displaying the activity of natural RAGE, is one of.
- RAGE is also a type I membrane protein with a molecular weight of about 35 kDa, which was identified from bovine lung in 1992 and belongs to the immunoglobulin superfamily that binds to AGE (complete RAGE with sugar chain modification has a molecular weight of 55 kDa).
- the extracellular domain of RAGEs has a structure in which one V-type immunoglobulin domain, followed by two immunoglobulin fold structures of two C-type immunoglobulin domains (C1 region and C2 region) are combined. Yes.
- RAGE also contains a single-pass membrane domain and a 43 amino acid cytoplasmic domain.
- RAGE interacts with various classes of ligands (AGEs, S100 / calgranulin, amphoterin and amyloid- ⁇ peptide).
- the V domain is an essential site for ligand binding, and the cytoplasmic domain is essential for RAGE-mediated intracellular signaling.
- the mutant RAGE-surfactant complex of the present invention has positions 38, 99, 144, 208, 259 and 301 in the amino acid sequence of SEQ ID NO: 97. It is preferred to retain a cysteine residue corresponding to the position.
- RAGE is expressed only at low levels in normal tissues and the vascular system. However, this receptor is upregulated where the ligand accumulates. RAGE expression is increased in endothelial cells, smooth muscle cells, pericytes, renal mesangial cells and infiltrating mononuclear phagocytes in the diabetic vasculature. RAGE expression is also increasing in pathological sites such as arteriosclerotic lesions where AGEs are accumulated.
- AGEs-RAGE interactions alter cellular properties important in vascular homeostasis. For example, after RAGE binds to AGEs, vascular endothelial cells increase the expression of VCAM-1, tissue factor, and IL-6, and their permeability to macromolecules. In mononuclear phagocytes, RAGE activates the expression of cytokines and growth factors and induces cell migration in response to soluble AGEs, whereas tactility occurs with fixed ligands.
- RAGE ligand recognition region or “sRAGE (soluble Receptor Advanced Advanced Products)” is used interchangeably and refers to a region that is recognized by the RAGE ligand.
- the sRAGE that is, the RAGE ligand recognition region refers to all or part of the extracellular region of RAGE.
- sRAGE is typically composed of positions 22 to 332 of SEQ ID NO: 97 or 102, but is not limited thereto.
- RAGE-like polypeptide refers to “RAGE8”, “mRAGE8”, “RAGE1”, “mRAGE1”, “RAGE2”, “mRAGE2”, “RAGE3”, “mRAGE3”.
- RAGE molecule is understood to include RAGE-like polypeptides as well as any complexes thereof. Therefore, it is understood that RAGE molecules include RAGE-like polypeptides, such as RAGE (full length), RAGE extracellular region (positions 22-332 of SEQ ID NO: 102), RAGE143, RAGE223, RAGE226, and the like. .
- a molecule containing a “RAGE ligand recognition region” is a molecule other than full-length RAGE among “RAGE molecules” (including “RAGE-like polypeptide”), for example, “RAGE8”, “mRAGE8”, “RAGE1”.
- the RAGE-like polypeptide may contain an unnatural amino acid, an amino acid analog, an amino acid derivative, or the like as long as the activity of natural RAGE is retained.
- RAGE-like polypeptide since it is important to form an intramolecular disulfide bond, it corresponds to positions 38, 99, 144, 208, 259 and 301 in the amino acid sequence of SEQ ID NO: 102. Cysteine is preferably retained. .
- a “ligand” is a binding partner for a specific receptor or family of receptors.
- the ligand can be an endogenous ligand for the receptor, or alternatively, a synthetic ligand for the receptor, such as a drug, drug candidate, or pharmacological means.
- antigen-antibody reaction is used in the broadest sense used in the art, and particularly refers to a reaction based on specific binding between an antigen and an antibody.
- Reagents and methods are also provided for detecting and quantifying antigen (eg, LDL) in a sample by using an immunoblot (Western blot) format as a detection system.
- binding means a physical or chemical interaction between two proteins or compounds or related proteins or compounds, or a combination thereof. Bonds include ionic bonds, non-ionic bonds, hydrogen bonds, van der Waals bonds, hydrophobic interactions, and the like.
- a physical interaction (binding) can be direct or indirect, where indirect is through or due to the effect of another protein or compound. Direct binding refers to an interaction that does not occur through or due to the effects of another protein or compound and does not involve other substantial chemical intermediates.
- a first substance or factor “specifically interacts” with a second substance or factor means that the first substance or factor has a second Interacting with a higher affinity than a substance or factor other than a substance or factor (especially another substance or factor present in a sample containing a second substance or factor).
- Specific interactions for a substance or factor include, for example, when a nucleic acid and protein are involved, such as hybridization in a nucleic acid, antigen-antibody reaction in a protein, ligand-receptor reaction, enzyme-substrate reaction, and the like. Examples include, but are not limited to, protein-lipid interactions, nucleic acid-lipid interactions, and the like, such as reactions with transcription factor binding sites.
- the first substance or factor “specifically interacts” with the second substance or factor means that the first substance or factor has the second substance Or having at least a part of complementarity to the factor.
- the first substance or factor “specifically interacts” with the second substance or factor includes, for example, an antigen-antibody reaction interaction, receptor- Examples include, but are not limited to, interaction by a ligand reaction and enzyme-substrate interaction.
- the first substance or factor “specifically interacts” with the second substance or factor means that the transcription factor and the transcription factor Interaction between the binding region of the nucleic acid molecule to be included.
- a “factor that specifically interacts” with a biological agent such as a polynucleotide or a polypeptide means an affinity for the biological agent such as the polynucleotide or the polypeptide,
- the affinity for other unrelated (especially less than 30% identity) polynucleotides or polypeptides is typically equivalent or higher, preferably significantly (eg, statistically significant) ) Includes the expensive.
- Such affinity can be measured, for example, by hybridization assays, binding assays, and the like.
- contacting means bringing a compound into physical proximity, either directly or indirectly, to a polypeptide or polynucleotide of the present invention.
- the polypeptide or polynucleotide can be present in many buffers, salts, solutions, and the like.
- Contact includes placing the compound in, for example, a beaker, microtiter plate, cell culture flask or microarray (eg, gene chip) containing a polypeptide encoding a nucleic acid molecule or fragment thereof.
- solid phase refers to a material used interchangeably herein with “substrate” and “substrate” from which the device of the present invention is constructed.
- the material of the substrate can be any solid, either covalently or noncovalently, that has the property of binding to the biomolecules used in the present invention or that can be derivatized to have such properties. Materials.
- Suitable substrates include beads, gold particles, plates (eg, microtiter plates), test tubes, chips, magnetic particles, membranes, fibers, glass slides, metal films, filters, tubes, balls, diamond-like carbon coated stainless steel. However, it is not limited to these.
- any material capable of forming a solid surface can be used, for example, glass, silica, silicone, ceramic, silicon dioxide, plastic, metal (also alloys) Natural) and synthetic polymers (including, but not limited to) polystyrene, cellulose, amylose, chitosan, dextran, and nylon.
- the substrate may be formed from a plurality of layers of different materials.
- inorganic insulating materials such as glass, quartz glass, alumina, sapphire, forsterite, silicon carbide, silicon oxide, and silicon nitride can be used.
- Polyethylene ethylene, polypropylene, polyisobutylene, polyethylene terephthalate, unsaturated polyester, fluorine-containing resin, polyvinyl chloride, polyvinylidene chloride, polyvinyl acetate, polyvinyl alcohol, polyvinyl acetal, acrylic resin, polyacrylonitrile, polystyrene, acetal resin
- Organic materials such as polycarbonate, polyamide, phenol resin, urea resin, epoxy resin, melamine resin, styrene / acrylonitrile copolymer, acrylonitrile butadiene styrene copolymer, silicone resin, polyphenylene oxide, and polysulfone can be used.
- a membrane used for blotting such as a nylon membrane, a nitrocellulose membrane, and a PVDF membrane can also be used.
- a material having hardness such as glass.
- a preferable material for the substrate varies depending on various parameters such as a measuring instrument, and those skilled in the art can appropriately select an appropriate material from the various materials described above.
- “Sandwich ELISA” refers to a specific form of ELISA, in which a molecule capable of specifically binding to an antibody or antigen (eg, LOX-1) is bound to a solid material, subjected to a sample containing the antigen, and then non- To remove the bound antigen, the solid material surface is washed, and then a labeled antibody (eg, a labeled antibody to which an enzyme is linked) is bound to the bound antigen (if present) and the antibody-antigen- A format that forms an antibody sandwich.
- an antibody or antigen eg, LOX-1
- a labeled antibody eg, a labeled antibody to which an enzyme is linked
- enzymes that can be linked to antibodies are alkaline phosphatase, horseradish peroxidase, luciferase, urease and ⁇ -galactosidase.
- the enzyme-linked antibody reacts with the substrate to generate a color reaction product that can be measured.
- unlabeled antibodies can also be used. In this case, detection can be performed by binding a labeled secondary antibody to an unlabeled antibody.
- “Lateral flow assay” is a type of immunoassay, and there are non-competitive and competitive types.
- the sample moves across the support, for example by capillary action, and encounters the mobile labeled antibody, which binds to the analyte to form a conjugate, which then crosses the support.
- an immobilized second antibody which binds to the analyte and detects the labeled antibody, thereby detecting the immobilized analyte.
- the labeled form of the analyte migrates across the support and competes with the unlabeled analyte for binding to the immobilized antibody.
- the “LOX-1 ligand recognition region” refers to a region that recognizes a ligand of C type Lectin-like LDL receptor (LOX-1), which is an oxidized LDL receptor.
- the LOX ligand recognition region is assumed to include a C-type Lectin-like region (CTLD) that is a minimum region essential for recognition, and a C-type Lectin-like region (CTLD) that is a minimum region essential for recognition.
- C-type Lectin-like region C-type Lectin-like region (CTLD) that is a minimum region essential for recognition.
- C-type Lectin-like region C-type Lectin-like region
- CTLD14 N-terminal biotinylated CTLD14
- biotinylated CTLD14 may be preferable.
- silkworm means a silkworm having a normal meaning, and is a kind of insect belonging to the order Lepidoptera (Lepidoptera) and Bombycidae.
- the official Japanese name is silkworm (scientific name: Bombix mori), and silkworm is the name of this larva, but generally also refers to this species in general.
- Mulberry is used as a diet, and silk is produced to make a cocoon.
- Silkworms are also called rabbits and are not wild insects. The ancestors of silkworms are considered to be Bombyxmandarina living in East Asia. Silkworms and mulberry are considered to be different species academically, but these hybrids have fertility. In this specification, silkworms include mulberry.
- an organism that imparts a sugar chain similar to that of silkworms refers to an organism having the ability to add a sugar chain similar to that of silkworms, such as a gene encoding an enzyme that adds a sugar chain similar to that of silkworms And can include transgenic organisms.
- silk gland is a pair of left and right organs existing in the body of a mature cocoon, and a large amount of proteins (amino acids) taken from mulberry leaves are converted into two types of silk proteins (fibroin, sericin). Means an organ that changes into The silk glands are paired on the left and right, and secrete liquid silk, which is the raw material for the silk thread.
- the silk gland is divided into three parts: a posterior silk gland, a middle silk gland, and an anterior silk gland.
- synthesis is possible using any silk gland, but in consideration of handling after synthesis, the rear silk gland and the middle silk gland are usually used, and preferably the middle silk gland is used. It is not limited. It can also be expressed throughout the body and recovered from the whole body.
- the posterior silk gland is an elongate part at the end, and when stretched, it becomes about 20 cm.
- fibroin protein which will later become the center of the eyebrows.
- the middle silk gland is a thick part bent in an S-shape in the center, and when it is extended, it becomes about 6 cm. Concentrates and stores fibroin protein sent from the posterior silk gland and arranges it into a shape that is easy to make into fibers. It also secretes another silk protein, sericin. When spitting out mayu, it acts as an adhesive that binds fibroin proteins.
- the front silk gland is a thin tube connected to the spout with a length of about 4 cm, and becomes thinner as it goes further. Liquid fibroin protein molecules are stretched and aligned in a certain direction, and gather together to further remove moisture. At the tip of the tube, it merges with the other pair of tubes, and is spouted from the spout to form one mayu yarn.
- ⁇ ⁇ Silkworms stop eating mulberry at the end of the age of 5 (ripe).
- the body of the mature cocoon is filled with a pair of organs (silk glands) filled with a liquid (liquid silk) that is the raw material of mayu thread.
- the silk gland is connected to the spout at the mouth of the silkworm through a thin spout.
- the liquid silk is stretched and hardened by passing through a thin splint tube to form a mayu yarn.
- the silkworm gland is pulled out from the silk gland one after another by a series of movements in which the larvae spouted from the sphincter are attached to a nearby object, the head and chest are moved in the shape of figure 8, and pulled. It is.
- silk-type sugar chain refers to a sugar chain structure unique to glycoproteins produced in silkworms, and is typically a trimannosyl core (self), an oligomannose sugar chain, and a complex sugar chain. Or a hybrid type thereof (see FIGS. 19 and 20).
- silkworm-type glycoproteins are produced using the middle silk gland, and unless otherwise specified, the “silk-type sugar chain” refers to the specific sugar chain type produced in this middle silk gland.
- trimannosyl core As such a silkworm type sugar chain, for example, two N-acetylglucosamine (GlcNAc) bonded to asparagine (Asn) and then three molecules of mannose (Man) are combined (referred to as trimannosyl core) It has a structure branched from the core (shown by the formula (1)), and various sugar chains are further bonded.
- sugar chains that bind to asparagine referred to as N-glycoside-linked sugar chains
- sugar chains that bind to serine, threonine, etc. referred to as 0-glycoside-linked sugar chains.
- the above-mentioned N-glycoside-linked sugar chain has various structures [Biochemical Experimental Method 23-Glycoprotein Sugar Chain Research Method (Academic Publishing Center) Atsuko Takahashi (1989)]. It is preferable to have a basic common core structure shown below. However, this point is the same when the above-mentioned glycoprotein is not an antibody.
- the silkworm type is as follows (Iizuka et al. FEBS Journal 276 (2009) 5806-5820). That is, in the production method described above, the silkworm glycoprotein described above has a sugar chain structure represented by the following chemical formula (1) (also referred to as trimannosyl core).
- sugar chain structure is a feature common to silkworm-type sugar chains, and as shown in the examples, it has excellent stability, and modified LDL or AGEs. This is because it is estimated that this sugar chain plays a certain role.
- sugar chain (1) is also indicated as N4-1 or sugar chain 000.1.
- the end of the sugar chain that binds to asparagine is called the reducing end, and the opposite side is called the non-reducing end.
- Examples of the bond of fucose to N-acetylglucosamine at the reducing end include ⁇ 1,3 bond and ⁇ 1,6 bond.
- the N-glycoside-linked sugar chain has a high mannose type (oligomannose type) in which only mannose binds to the non-reducing end of the core structure, and galactose-N-acetylglucosamine (hereinafter referred to as Gal-GlcNAc) on the non-reducing end side of the core structure.
- Gal-GlcNAc galactose-N-acetylglucosamine
- a complex type having a structure such as sialic acid and bisecting N-acetylglucosamine on the non-reducing terminal side of Gal-GlcNAc in parallel also referred to as complex type. Both are synonymous.
- the above-mentioned glycoprotein has a sugar chain structure represented by the following chemical formula (2), (3) or (4)
- sugar chain (3) is also indicated as sugar chain 100.1.
- the sugar chain (4) is also indicated as N4-2 or sugar chain 100.2.
- glycoprotein (2) is typically recognized.
- a glycoprotein having an N-glycoside-linked sugar chain containing a sugar chain structure represented by the chemical formula (3) or (4) may be included.
- the proportion of glycoprotein having an N-glycoside-linked sugar chain containing a sugar chain structure represented by chemical formula (2), (3) or (4) is 10 mol% or more, 20 mol% or more, or 30 mol% or more.
- the N-glycoside-linked sugar chain of the glycoprotein is imparted by silkworm-type sugar chains as compared to the N-glycoside-linked sugar chain of glycoproteins produced by general cells that do not produce silkworm type. Because it is strengthened.
- this structure is “GlcNAc- ⁇ 1,4-GlcNAc- ⁇ 1,4-Man (- ⁇ 1,3-Man- ⁇ 1,2-Man) - ⁇ 1,6-Man (- ⁇ 1,3-Man ) - ⁇ 1,6-Man ”.
- the sugar chain (6) is also indicated as N2 or sugar chain M6.1. In the present invention, this (6) is typically recognized in addition to (2).
- the silkworm-type sugar chain contained in CTLD14 of the present invention includes the following combinations 1) GlcNAc- ⁇ 1,4-GlcNAc- ⁇ 1,4-Man (- ⁇ 1,3-Man) - ⁇ 1,6-Man as viewed from the asparagine residue.
- sugar chain (8) is N1 or sugar chain M7.2. Are also displayed.
- the sugar chain structure analysis of sRAGE revealed that about 90% or more are oligomannose type sugar chains and contain several% of complex type and hybrid type sugar chains. Furthermore, it has the outstanding effect that stability improves by sugar chain addition.
- FIG. 20 and FIG. 54 can be referred to.
- the silkworm sugar chain of the RAGE of the present invention has a trimannosyl core (from asparagine residue to GlcNAc- ⁇ 1,4-GlcNAc- ⁇ 1,4-Man (- ⁇ 1,6-Man) - ⁇ 1, In addition to the structure of 3-Man), it includes sugar chains to which 0 to 4 molecules of GlcNAc 0 to 2 molecules and Man0 to 4 molecules are bound per molecule.
- the silkworm sugar chain of the RAGE of the present invention has a trimannosyl core (from an asparagine residue to GlcNAc- ⁇ 1,4-GlcNAc- ⁇ 1,4-Man (- ⁇ 1,6-Man) - ⁇ 1, In addition to the structure of 3-Man), it includes sugar chains to which 0 to 8 molecules out of GlcNAc 0 to 4 molecules and Man 0 to 8 molecules are bound per molecule.
- sugar chain may be added to only one of the two sites, or may be added to both.
- sugar chains are added to both of the sites capable of undergoing glycosylation at two locations.
- the added sugar chains may be the same sugar chain or different.
- sugar chain to be added include sugar chains having the structures (1) to (8) ([Chemical Formula 1] to [Chemical Formula 8]).
- silkworm-type sugar chains are classified into oligomannose type, complex type and hybrid type.
- sugar chain structure of sRAGE 87% to 97% is an oligomannose type, 2% to 8% is a complex type, and 1% to 5% is a hybrid type.
- 90% to 94% are oligomannose types, 3% to 6% are composite types, and 2% to 4% are hybrid types.
- 92.5% is oligomannose, 4.6% is complex, and 2.5% is hybrid.
- the sugar composition of silkworm-type sRAGE is (Man) 5 (GlcNAc) 2 (N3 in FIG. 54) is 46% to 56%, and (Man) 7 (GlcNAc) 2 (in FIG. 54).
- N1 is 23% to 33%
- (Man) 6 (GlcNAc) 2 (N2 in FIG. 54) is 7% to 15%
- 1) is 1% to 5%
- (Man) 3 (GlcNAc) 3 (N4-2 in FIG. 54) is 2% to 8%
- (Man) 4 (GlcNAc) 3 (in FIG. 54) N4-3) is 1% to 5%.
- the sugar composition is (Man) 5 (GlcNAc) 2 is between 48% and 54%, (Man) 7 (GlcNAc) 2 is between 25% and 31%, and (Man) 6 (GlcNAc) 2 is 9% to 13%, (Man) 3 (GlcNAc) 2 is 2% to 4%, (Man) 3 (GlcNAc) 3 is 3% to 6%, (Man) 4 (GlcNAc) 3 is 2% to 4%.
- the sugar composition is (Man) 5 (GlcNAc) 2 is 51.4%, (Man) 7 (GlcNAc) 2 is 27.5% and (Man) 6 (GlcNAc) 2 Is 10.8%, (Man) 3 (GlcNAc) 2 is 2.8%, (Man) 3 (GlcNAc) 3 is 4.6%, (Man) 4 (GlcNAc) 3 is 2.5%.
- sugars constituting the sugar chain bound to glycoprotein there are not so many kinds of sugars constituting the sugar chain bound to glycoprotein, and common ones are glucose, galactose, mannose, fucose, N-acetylglucosamine, N-acetylgalactosamine, N-acetylneuraminic acid, There are about 7-8 types such as xylose.
- the structural style is also limited to some extent, and slight differences in the structure are identified and precisely recognized to control various life phenomena.
- fucose means normal fucose, which is 6-deoxy-galactose which is a kind of deoxy sugar, the chemical formula is C 6 H 12 O 5 , molecular weight 164.16, melting point 163 It is classified into hexose and monosaccharide at °C and specific rotation -76 °.
- the L form is in the form of L-fucoside and exists widely in animals and plants. In mammals and plants, it is found on cell surface N-linked sugar chains.
- N-acetylglucosamine and strawberry mean normal N-acetylglucosamine (N-acetyl-D-glucosamine, GlcNAc, NAG), a monosaccharide derived from glucose, It is an important substance for the chemical mechanism. Chemically this material is an amide between glucosamine and acetic acid. N-acetylglucosamine is a component of glycosaminoglycan (mucopolysaccharide) such as glycoprotein and hyaluronic acid in mammals.
- N-acetylglucosamine forms the skeleton of an N-linked glycoprotein in which oligosaccharide chains centering on mannose bind to asparagine (chitobiose structure), and is the main constituent sugar of sugar chains having a more complex structure.
- galactose means normal galactose, and its molecular formula and molecular weight are C 6 H 12 O 6 , 180, which is the same as glucose.
- the configuration is 2nd (second from the top in the Fischer projection formula), 5th -OH is in the same direction 3rd and 4th is in the opposite direction, D-galactose has the same orientation as 5th D-glyceraldehyde Yes. It is the 4-epimer of glucose. In nature, D-galactose is almost all.
- mannose means normal mannose, which is a kind of monosaccharide classified as aldohexose, and has the same molecular formula and molecular weight as C 6 H 12 O 6 , 180 as glucose. is there.
- the configuration is 2nd (second from the top in the Fischer projection formula), 3rd -OH is the same direction 4th and 5th is the opposite direction, D-mannose has the same orientation as 5th D-glyceraldehyde Yes. It is the 2-epimer of glucose. In nature, D-mannose is almost all.
- the sugar chain is typically composed of neutral sugars such as galactose, mannose, and fucose, amino sugars such as N-acetylglucosamine, and acidic sugars such as sialic acid.
- neutral sugars or amino sugars are released by acid hydrolysis of sugar chains with trifluoroacetic acid or the like, and the composition ratio can be analyzed.
- a specific method includes a method using a sugar composition analyzer (BioLC) manufactured by Dionex. BioLC is an HPAEC-PAD (high performance anion-exchange chromatographic-pulsed amplometric detection) method [J. Liq. Chromatogr. , 6, 1577 (1983)].
- the composition ratio can also be analyzed by fluorescence labeling with 2-aminopyridine. Specifically, a known method [Agric. Biol. Chem. , 55 (1), 283-284 (1991)], the sample can be fluorescently labeled with 2-aminopyridylation and analyzed by HPLC to calculate the composition ratio.
- sugar chain structure analysis The structure analysis of sugar chains is performed by a two-dimensional sugar chain mapping method [Anal. Biochem. , 171, 73 (1988), Biochemical Experimental Method 23-Glycoprotein Glycan Research Method (Academic Publishing Center) edited by Etsuko Takahashi (1989)].
- the two-dimensional sugar chain mapping method for example, the retention time or elution position of reversed-phase chromatography sugar chains is plotted on the X axis, and the retention time or elution position of sugar chains by normal phase chromatography is plotted on the Y axis. This is a method for estimating the sugar chain structure by comparing with those of known sugar chains.
- the glycoprotein is hydrazine-degraded to release the sugar chain from the glycoprotein, and the fluorescent labeling of the sugar chain with 2-aminopyridine (hereinafter abbreviated as PA) [J. Biochem. , 95, 197 (1984)], and then the sugar chain is separated from excess PA reagent by gel filtration, and reverse phase chromatography is performed. Next, normal phase chromatography is performed on each peak of the separated sugar chain. Based on these results, the results were plotted on a two-dimensional sugar chain map, and sugar chain standard (TaKaRa), literature [Anal. Biochem. , 171, 73 (1988)], the sugar chain structure can be estimated. Further, mass analysis such as MALDI-TOF-MS of each sugar chain can be performed to confirm the structure estimated by the two-dimensional sugar chain mapping method.
- PA 2-aminopyridine
- glycoproteins are expressed in silk gland cells of insects classified as Lepidoptera such as silkworms.
- the above-mentioned insect is preferably a silkworm.
- silkworms breeding methods have been established in the sericulture industry, and furthermore, the ability to extrude cocoons from silk glands is excellent, so that the above-described glycoprotein can be efficiently obtained in large quantities.
- Silkworms typically make 0.3-0.5 g of silkworm per head, but most of this is silk protein such as fibroin synthesized in the posterior silk gland and sericin synthesized in the middle silk gland. As described above, silkworms are organisms having excellent protein synthesis ability, and if this ability is used, recombinant proteins for analysis and evaluation can be produced in large quantities at low cost. .
- the above silk gland cells are preferably middle silk gland cells. Because sericin is synthesized in the middle silk gland, if the above-mentioned glycoprotein is expressed in sericin, the recombinant protein is localized in the sericin layer that exists around fibroin and is relatively soluble in water. Therefore, the recombinant protein can be efficiently expressed in the middle silk gland, and the recombinant protein contained in the cocoon can be easily extracted without modifying its three-dimensional structure.
- the glycoprotein production method according to the present embodiment is the silk gland of the above-mentioned insect (eg, silkworm) individual, wherein the step of expressing the glycoprotein is genetically modified to express the glycoprotein of the present invention. It is preferable to include a step of producing a koji containing the glycoprotein of the present invention.
- the generation of a cocoon containing the above-described glycoprotein in the silk gland of an insect individual typically causes the above-described glycoprotein to be expressed in the silk gland cells of the insect.
- the process of extracting the target glycoprotein includes the process of discharging the cocoon containing the glycoprotein from the silk gland, and the process of extracting the glycoprotein from the discharged cocoon. preferable. Because fibroin or sericin is synthesized in the silk gland, if the above-mentioned glycoprotein is expressed in fibroin or sericin, it is efficient in the silk gland to localize the recombinant protein in the fibroin layer or sericin layer. This is because the recombinant protein can be expressed well, and the recombinant protein contained in the straw can be extracted.
- the step of expressing the glycoprotein includes an insect in which the gene encoding the glycoprotein is provided so as to be expressed downstream of the sericin promoter in the insect genome. It is preferable to include a step of using an individual and a step of generating a cocoon containing the above-mentioned glycoprotein in the sericin part as the cocoon.
- the step of extracting the glycoprotein described above includes the step of immersing the cocoon in the extract and extracting the glycoprotein from the sericin portion of the cocoon into the extract. It is preferable to contain. This is because the silk thread constituting the cocoon has a structure in which a sericin layer is present around the fibroin layer present in the center.
- Silk glands that synthesize silk proteins are functionally and morphologically distinguished into posterior silk glands, middle silk glands, and anterior silk glands, but the sericin that composes the sericin layer is synthesized in the middle silk glands. Fibroin constituting the fibroin layer is synthesized in the posterior silk gland. If the tissue that expresses the glycoprotein is the middle silk gland, the glycoprotein is secreted into the sericin layer of the silk. On the other hand, when the tissue expressing the glycoprotein is the posterior silk gland, the glycoprotein is secreted into the fibroin layer of the silk.
- a promoter that causes gene expression in the middle silk gland cells may be assembled upstream.
- promoters that cause gene expression in middle silk gland cells include the promoter of sericin 1 gene or sericin 2 gene.
- the middle silk gland or posterior silk gland is a commonly used synthetic pathway and can be used in the present invention as well. Alternatively, it may be expressed throughout the body.
- when expressed throughout the body there are methods of grinding the whole body and squeezing body fluids.
- transgenic silkworms that secrete glycoproteins into the sericin layer can be produced by a single gene transfer operation.
- transgenic silkworms that secrete glycoproteins into the sericin layer can also be produced by multiple gene transfer operations.
- transgenic silkworms that secrete glycoproteins into the fibroin layer are not intended to exclude the method of producing transgenic silkworms that secrete glycoproteins into the fibroin layer.
- a fibroin layer using a vector incorporating a promoter that causes gene expression in the posterior silk gland cells for example, upstream of each structural gene
- transgenic silkworms that secrete antibodies may be prepared.
- promoters that cause gene expression in posterior silk gland cells include promoters of genes such as fibroin L chain, fibroin H chain, or fibrohexamarin.
- the above-described expression vector set can be used to generate a plurality of genes when producing a transgenic silkworm that secretes a glycoprotein into the fibroin layer. You may produce a transgenic silkworm by introduction
- These expression vectors have a function for introducing a gene into the silkworm chromosome.
- a gene can be introduced into the silkworm chromosome.
- a plasmid vector having a pair of inverted repeats present at the end of a DNA-type transposon, and a gene sequence inserted into a chromosome in a region sandwiched between a pair of inverted repeats An expression vector incorporating a glycoprotein gene and a promoter.
- insect-derived DNA-type transposons piggyBac, mariner (Insect Mol. Biol. 9, 145-155, 2000), and Minos (Insect Mol. Biol.
- helper plasmid is a recombinant plasmid vector lacking one or both of the inverted repeats of piggyBac and incorporating substantially only the transposase gene region of piggyBac.
- a promoter for expressing transposase may be an endogenous transposase promoter as it is, or a silkworm / actin promoter, a Drosophila HSP70 promoter, or the like may be used.
- a marker gene can be simultaneously incorporated into a vector into which a polynucleotide to be inserted has been incorporated.
- the promoter and the nucleic acid sequence can be combined so that expression of the nucleic acid sequence of the structural gene existing downstream of the promoter is induced by binding of the transcriptional control factor to the promoter.
- the combination of the transcription control factor and the target sequence include GAL4 and UAS, TetR and TRE, and the like.
- the glycoprotein produced by the method of the present invention is not limited in any way, as long as it is produced by the method of the present invention.
- SEQ ID NO: 89 biotinylation site
- tag sequence for example, BioEase tag, His tag, etc.
- the glycoprotein produced by the glycoprotein production method of the present invention includes both glycoproteins having a signal sequence and glycoproteins having no signal sequence.
- silkworm eggs having a promoter of a nucleic acid sequence encoding a protein specifically expressed in the silk gland and a nucleic acid sequence encoding a glycoprotein that is directly controlled by the promoter include, for example, silk gland specific Silkworm eggs having a nucleic acid sequence to which a nucleic acid sequence encoding a target glycoprotein is operably linked downstream of a promoter of a nucleic acid sequence encoding a protein to be expressed.
- silkworm eggs a nucleic acid sequence in which a nucleic acid sequence encoding a target glycoprotein is operably linked downstream of a promoter of a nucleic acid sequence encoding a protein specifically expressed in the silk gland is introduced into a silkworm egg. Can be manufactured.
- a silkworm egg having a promoter of a nucleic acid sequence encoding a protein specifically expressed in the silk gland and a nucleic acid sequence encoding a recombinant glycoprotein that is indirectly controlled by the promoter for example, (I) a nucleic acid sequence in which a nucleic acid sequence encoding a transcription factor is functionally linked downstream of a promoter of a nucleic acid sequence encoding a protein that is expressed specifically in the silk gland, and (ii) a target promoter of the transcription control factor
- the nucleic acid sequence encoding the recombinant antibody whose expression is directly or indirectly controlled by the promoter of the nucleic acid sequence encoding the protein specifically expressed in the silk gland promotes the secretion of the antibody and is recovered.
- a signal sequence is preferable. Specific embodiments of the signal sequence are described elsewhere herein, and any known one can be used.
- transgenic silkworms are selected from the next-generation (F1 generation) silkworms. Selection of transgenic silkworms is performed using, for example, PCR or Southern blotting.
- a marker gene when incorporated, it is possible to select using the phenotype. For example, when a fluorescent protein gene such as GFP is used as a marker gene, it can be performed by irradiating an F1 generation silkworm egg or larva with excitation light and detecting the fluorescence emitted by the fluorescent protein.
- a transgenic silkworm can be produced by the method as described above.
- the glycoprotein When collecting the target glycoprotein from the silkworm of the transgenic silkworm, the glycoprotein is collected from the sericin layer if it is localized in the sericin layer, and from the fibroin layer if it is contained in the fibroin layer.
- the recovery of glycoprotein from the sericin layer can be carried out particularly easily. Since sericin constituting the sericin layer is hydrophilic, the recombinant protein localized in this layer can be extracted without using a solution that denatures the protein.
- the extract for extracting the target glycoprotein from the sericin layer is not particularly limited as long as the glycoprotein can be extracted. For example, it may be a neutral salt solution, or a solution containing a surfactant or other reagents for efficiently performing extraction.
- a method of immersing fragmented cocoons in the extract and stirring them can be used.
- a process for finely pulverizing the soot may be performed, or a mechanical process such as an ultrasonic process may be used in combination with the extraction.
- the silk gland cells of the silkworm described above may be transformed so as to appropriately express a transferase (for example, ⁇ -galactose transferase, N-acetylglucosamine transferase, etc.) so as to be further glycosylated. .
- a reporter gene may be incorporated in order to confirm expression.
- a reporter gene is not particularly limited as long as its expression can be detected.
- a CAT gene a lacZ gene, a luciferase gene, a ⁇ -glucuronidase gene (GUS) commonly used by those skilled in the art.
- GUS ⁇ -glucuronidase gene
- the expression level of the reporter gene can be measured by methods known to those skilled in the art depending on the type of the reporter gene. For example, when the reporter gene is a CAT gene, the expression level of the reporter gene can be measured by detecting acetylation of chloramphenicol by the gene product.
- the reporter gene is a lacZ gene
- the fluorescent compound by the catalytic action of the gene expression product
- ⁇ -clonidase gene GUS
- Glucuron (ICN) luminescence or 5-bromo-4-chloro-3-indolyl catalyzed by the gene expression product is detected.
- X-Gluc - ⁇ -glucuronide
- the expression level of the reporter gene can be measured by detecting the fluorescence of the GFP protein.
- the method for producing a recombinant glycoprotein of the present invention includes a step of recovering a target glycoprotein synthesized in the silkworm.
- the synthesized glycoprotein of interest is secreted to the middle silk gland or posterior silk gland in an active state without being insolubilized. Therefore, the target glycoprotein can be recovered from the middle silk gland or posterior silk gland.
- a silkworm that has reached the spitting stage is dissected, and the middle silk gland or the rear silk gland is extracted into 20 mM Tris-HCl pH 7.4.
- the target glycoprotein in the silk gland can be recovered by scratching the silk gland with tweezers or a scalpel.
- the glycoprotein of interest of the present invention can also be recovered from, for example, silkworm spun by a transgenic silkworm.
- a recovery method a method well known to those skilled in the art, for example, a method in which sputum is dissolved in 60% LiSCN and dialyzed with 20 mM Tris, 5 M urea (Inoue, S., Tsuda, H., Tanaka, H., Magoshi, Y and Mizuno (2001) Serialology 4,157-163.) Can be used.
- a method using a surfactant or a method of dissolving in an aqueous solution can be used.
- the silkworm in the present invention is not particularly limited, but for mass production of a target glycoprotein, a nucleic acid sequence region (coding region, promoter region, non-coding region) encoding a protein constituting a silk thread such as fibroin protein. It is preferable to use silkworms in which the production of the protein constituting the silk thread is suppressed by mutation (including the translation region).
- Such a silkworm is a mutant silkworm in which the production of the protein constituting the silk thread is suppressed by mutation of the nucleic acid sequence region encoding the protein constituting the silk thread, preferably the production of the protein constituting the silk thread by the mutation Nd-s D is more preferable, but Nd-s D is preferred, but it depends on whether the cause of the suppression of the production of the protein that constitutes the silk thread is artificial or a mutation that occurs in nature. Regardless of whether or not to do so, any silkworm in which the production of the protein constituting the silk thread is suppressed may be used.
- a silkworm known to those skilled in the art as a sericin silkworm.
- sericin silkworm mass production of the target glycoprotein in the middle silk gland becomes possible, and purification of the protein synthesized from the nucleic acid sequence encoding the recombinant antibody introduced into the chromosome becomes easy.
- sericin silkworm it is preferable to use sericin silkworm in terms of production amount.
- silkworms in the present invention silkworms having the property of producing non-dormant eggs
- silkworms having the property of producing dormant eggs for example, the practical varieties Gunma, 200, Shunchu, Kangetsu, Nishikiaki, Kanwa, etc.
- a dormant egg refers to an egg in which embryonic development stops temporarily after spawning
- a non-diapause egg refers to an egg in which postnatal embryonic development does not stop and larvae hatch.
- a method for producing a non-dormant egg for example, in Gunma, a method of causing a non-dormant egg to lay on an adult produced from the dormant egg by culturing the dormant egg at 15 ° C. to 21 ° C., preferably a dormant egg Is cultivated at 16 ° C. to 20 ° C. to allow the adults produced from the dormant eggs to lay non-dormant eggs, and more preferably, the dormant eggs are cultured at 18 ° C. so that the adults produced from the dormant eggs are non-destructive.
- a method for giving birth to dormant eggs most preferably a method for cultivating dormant eggs at 18 ° C. so that the larvae produced from the dormant eggs are fully bred and the grown adults are made to lay non-dormant eggs. Can do.
- a method in which a dormant egg is cultivated at 15 ° C. to 21 ° C. to allow an adult produced from the dormant egg to produce a non-dormant egg preferably the dormant egg is cultured at 16 ° C. to 20 ° C.
- non-dormant eggs are laid down on the adults generated from the dormant eggs, more preferably, non-dormant eggs are laid on the adults generated from the dormant eggs by culturing dormant eggs at 18 ° C.
- the larvae generated from the diapause eggs are bred in full light by culturing the dormant eggs at 25 ° C.
- a method of causing the grown adult to lay non-dormant eggs are caused by the grown adult to lay non-dormant eggs.
- Egg culturing can be performed, for example, by placing it in an incubator at 18 ° C. to 25 ° C. or in a room at a constant temperature, and raising larvae can be performed using an artificial feed in a 20 ° C. to 29 ° C. breeding room .
- the above-mentioned dormant egg culture of the present invention can be carried out by those skilled in the art according to a general silkworm egg culture method.
- the culture is performed according to the method described in “Ministry of Education (1978) Soybean Manufacture. Pp193, Jikkyo Publishing, Tokyo”.
- breeding of silkworm larvae in the present invention can be performed by those skilled in the art by a well-known method.
- breeding is performed according to the method described in “Ministry of Education (1978) Soybean Manufacture. Pp193, Jikkyo Publishing, Tokyo”.
- whether or not a spawned egg is a non-dormant egg can be determined by the color of the egg.
- dormant eggs are colored dark brown and non-dormant eggs are yellowish white. Therefore, in the present invention, an egg laid is determined to be a non-dormant egg by not being dark brown, more preferably yellowish white.
- the method for introducing a nucleic acid molecule into a silkworm egg in the present invention is not limited to this method.
- Nucleic acid molecules are introduced from the holes.
- the tube for injecting nucleic acid molecules can be inserted into the egg through the hole so that the insertion angle is substantially perpendicular to the side of the egg on the ventral side.
- the eggshell can be pierced by a method using a needle.
- the material, strength, etc. of the needle are not particularly limited.
- the needle in the present invention usually refers to a rod-like needle having a sharp tip, but is not limited to this shape, and the overall shape is not particularly limited as long as it can make a hole in the eggshell.
- a pyramidal material with a sharp tip or a triangular pyramid shaped material with a sharp tip is also included in the “needle” of the present invention.
- a tungsten needle can be preferably used.
- the thickness (diameter) of the needle of the present invention may be any thickness that can open a hole through which a capillary, which will be described later, can pass, and is usually 2 to 20 ⁇ m, preferably 5 to 10 ⁇ m.
- a method of chemically making a hole in an eggshell for example, a method of making a hole using a chemical (such as hypochlorous acid) can be mentioned.
- the position for opening the hole is not particularly limited as long as the insertion angle with respect to the side surface on the ventral side of the egg when a tube for injecting a nucleic acid molecule is inserted from the hole, but is not particularly limited. Is the ventral side or the opposite side, more preferably the ventral side, and even more preferably the middle part of the egg on the ventral side from the rear end.
- substantially vertical means 70 ° to 120 °, preferably 80 ° to 100 °.
- the “position to become a germ cell in the future” is usually a position close to the egg surface on the ventral side of the egg (usually a position of 0.01 mm to 0.05 mm from the egg table), Preferably, it is a position near the egg surface in the center of the ventral side of the egg and a position slightly behind the rear pole.
- the material, strength, inner diameter, etc. of the tube for injecting nucleic acid molecules of the present invention are not particularly limited, but when a hole is made physically or chemically before inserting the tube for injecting nucleic acid molecules. Is preferably of a thickness (outer diameter) that can pass through the vacated hole.
- Examples of the tube for injecting nucleic acid molecules of the present invention include a glass capillary.
- a hole is physically or chemically formed in the silkworm egg, and the tube for inserting the nucleic acid molecule is inserted with respect to the ventral side surface of the egg.
- the step of inserting into the egg through the hole so as to be almost vertical and injecting the nucleic acid molecule is performed using a manipulator in which the needle and the tube for injecting the nucleic acid molecule are integrated.
- the present invention is typically implemented using an apparatus that has the manipulator as one of its components.
- Such devices include a dissecting microscope, an illumination device, a movable stage, a coarse manipulator fixed to the microscope with metal fittings, a micromanipulator attached to this manipulator, and air pressure for injecting nucleic acid molecules. It consists of an injector that adjusts. The pressure used for the injector is supplied from a nitrogen cylinder, and the pressure switch can be turned on by a foot switch. Injection is performed on an egg fixed on a substrate such as a glass slide, and the position of the egg is determined by a movable stage. Further, the glass capillary of the micromanipulator is operated by an operation unit connected by four tubes.
- the actual procedure is to determine the position of the tungsten needle relative to the egg with a coarse manipulator, and move the egg horizontally with the stage lever to make a hole. Subsequently, the lever of the operation part of the micromanipulator is operated to guide the tip of the glass capillary to the position of the hole, and the capillary is again inserted into the egg by the lever of the stage. In this case, it is necessary to insert a glass capillary perpendicular to the ventral side surface of the egg. Turn on the foot switch, inject the nucleic acid molecule, and operate the lever to pull the capillary from the egg.
- the hole is covered with an instant adhesive and protected with an incubator at a constant temperature and constant humidity.
- the apparatus used in the present invention preferably includes the apparatus described in Japanese Patent No. 1654050 or an apparatus obtained by improving the apparatus.
- a silkworm egg used for introducing a nucleic acid molecule may be fixed to a substrate.
- a substrate of the present invention for example, a slide glass, a plastic plate or the like can be used, but is not particularly limited thereto.
- substrate there is no restriction
- substrate there is no restriction
- substrate there is no restriction
- substrate there is no restriction
- substrate there is no restriction
- the directionality for fixing the silkworm eggs to the substrate is preferably such that the orientation of the dorsal belly is constant.
- the silkworm egg is fixed to the substrate of the present invention by, for example, laying eggs on a commercially available mount (rose seed mount) pre-applied with aqueous glue, adding water to the mount to peel off the egg, and then getting wet
- the eggs are aligned on a substrate and air-dried.
- the eggs are preferably fixed on the slide glass with the direction of the eggs aligned.
- the egg can be fixed to the base by using a double-sided tape or an adhesive.
- Whether or not a nucleic acid molecule has been introduced into a silkworm egg is, for example, measured by extracting the injected nucleic acid molecule again from the egg (Nagaraju, J., Kanda, T., Yukuhiro, K., Chavancy, G., Tamura, T. and Couble, P. (1996) Attempt of transgenesis of the silkworm (Bombyx mori L) by egg-injection of foreign DNA. Appl. Entomol. Zool., 31, 589-598) and injected nucleic acid molecules To see expression in eggs (Tamura, T., Kanda, T., Takiya, S., Okano, K. and Maekawa, H. (1990). Transient expression of chimeric CAT genes injected into early embryos of the domesticated silkworm, Bombyx mori. Jpn. J. Genet., 65, 401-410).
- a “purified” biological factor refers to a product from which at least a part of the factor that naturally accompanies the biological factor has been removed.
- the purity of the biological agent in a purified biological agent is higher (ie, enriched) than the state in which the biological agent is normally present.
- purified is preferably at least 75% by weight, more preferably at least 85% by weight, even more preferably at least 95% by weight, and most preferably at least 98% by weight, Means the presence of the same type of biological agent.
- a “corresponding” amino acid or nucleic acid has or has the same action as a predetermined amino acid or nucleotide in a reference polypeptide or polynucleotide in a polypeptide molecule or polynucleotide molecule.
- a reference polypeptide or polynucleotide in a polypeptide molecule or polynucleotide molecule in particular, in the case of an enzyme molecule, it means an amino acid that is present at the same position in the active site and contributes similarly to the catalytic activity.
- an antisense molecule can be a similar part in an ortholog corresponding to a particular part of the antisense molecule.
- Corresponding amino acids are identified as, for example, cysteinylation, glutathioneation, SS bond formation, oxidation (eg, oxidation of methionine side chain), formylation, acetylation, phosphorylation, glycosylation, myristylation, etc.
- the corresponding amino acid can be an amino acid responsible for dimerization.
- Such “corresponding” amino acids or nucleic acids may be regions or domains spanning a range. Thus, in such cases, it is referred to herein as a “corresponding” region or domain.
- a “corresponding” gene eg, a polypeptide molecule or a polynucleotide molecule
- a “corresponding” gene has or is expected to have, in a species, the same effect as a given gene in a species to which comparison is made.
- Gene for example, a polypeptide molecule or a polynucleotide molecule
- a gene corresponding to a gene can be an ortholog of that gene.
- mouse, rat CTLD14 or LOX-1 or RAGE can find the corresponding CTLD14 or LOX-1 or RAGE (sRAGE or soluble form of sRAGE), respectively, in humans.
- corresponding genes can be identified using techniques well known in the art.
- the corresponding gene in an animal eg, a mouse
- fragment refers to a polypeptide or polynucleotide having a sequence length of 1 to n ⁇ 1 with respect to a full-length polypeptide or polynucleotide (length is n).
- the length of the fragment can be appropriately changed according to the purpose.
- the lower limit of the length is 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50 and more amino acids, and lengths expressed in integers not specifically listed here (eg 11 etc.) are also suitable as lower limits obtain.
- examples include 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 and more nucleotides.
- Non-integer lengths may also be appropriate as a lower limit.
- a fragment falls within the scope of the present invention as long as the full-length fragment functions as a marker, as long as the fragment itself also functions as a marker.
- heterologous refers to nucleotide or amino acid sequences that are different or non-corresponding sequences, or sequences from different species.
- the human nucleotide or amino acid sequence is heterologous to the mouse nucleotide or amino acid sequence, and the human LOX-1 or RAGE nucleic acid or amino acid sequence is relative to the human albumin nucleotide or amino acid sequence.
- fragment refers to a polypeptide or polynucleotide having a sequence length of 1 to n ⁇ 1 with respect to a full-length polypeptide or polynucleotide (length is n).
- the length of the fragment can be appropriately changed according to the purpose.
- the lower limit of the length is 3, 4, 5, 6, 7, 8, 9, 10, Examples include 15, 20, 25, 30, 40, 50 and more amino acids, and lengths expressed in integers not specifically listed here (eg, 11 etc.) are also suitable as lower limits.
- examples include 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 and more nucleotides.
- Non-integer lengths may also be appropriate as a lower limit.
- the lengths of polypeptides and polynucleotides can be represented by the number of amino acids or nucleic acids, respectively, as described above, but the above numbers are not absolute, and the upper limit is not limited as long as they have the same function.
- the above-mentioned number as the lower limit is intended to include the upper and lower numbers (or, for example, up and down 10%) of the number.
- “about” may be added before the number. However, it should be understood herein that the presence or absence of “about” does not affect the interpretation of the value.
- the length of a fragment useful herein can be determined by whether or not at least one of the functions of a full-length protein that serves as a reference for the fragment is retained.
- biological function refers to a specific function that a gene, nucleic acid molecule or polypeptide may have in vivo when referring to a gene or a nucleic acid molecule or polypeptide related thereto.
- the antibody include, but are not limited to, generation of specific antibodies, enzyme activity, and imparting resistance.
- the present invention include, but are not limited to, a function that LOX-1 recognizes oxidized LDL and the like, and a function that RAGE recognizes a marker such as hemopexin.
- a biological function can be exerted by “biological activity”.
- biological activity refers to activity that a certain factor (eg, polynucleotide, protein, etc.) may have in vivo, and exhibits various functions (eg, transcription promoting activity). For example, an activity in which another molecule is activated or inactivated by interaction with one molecule. When two factors interact, their biological activity depends on the binding between the two molecules and the resulting biological change, eg, when one molecule is precipitated with an antibody, the other When molecules also coprecipitate, the two molecules are considered bound. Therefore, seeing such coprecipitation is one method of judgment.
- a factor is an enzyme
- the biological activity includes the enzyme activity.
- an agent is a ligand
- the ligand includes binding to the corresponding receptor. Such biological activity can be measured by techniques well known in the art.
- “activity” indicates or reveals binding (either direct or indirect); affects the response (ie, has a measurable effect in response to some exposure or stimulus); Refers to various measurable indicators, such as the affinity of a compound that directly binds to a polypeptide or polynucleotide of the invention, or the amount of protein upstream or downstream after some stimulation or event or other Similar measures of function are mentioned.
- marker refers to a certain condition (disease associated with degenerative LDL or diabetes related to AGEs, diabetic nephropathy, diabetic retinopathy, diabetic complications such as diabetic neuropathy, etc.
- a substance that serves as an indicator to track whether or not there is a risk of Such markers can include genes, gene products, metabolites, enzymes, and the like.
- dyslipidemia arteriosclerosis, diabetes, ischemic heart disease (myocardial infarction, angina, etc.), cerebrovascular disorder (cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage, transient ischemic attack, etc.), aorta Diseases related to degenerative LDL such as aneurysm, renal infarction, lipid abnormalities such as hyperlipidemia, diabetes, hyperglycemia, hypertension, hypertensive nephrosclerosis, arteriosclerosis, ischemia reperfusion injury, vascular balloon injury Diagnosis, preliminary detection, prediction or pre-diagnosis of a disease state such as using a drug, agent, factor or means specific to a marker associated with the condition, or a composition, kit or system containing the same, etc.
- ischemic heart disease myocardial infarction, angina, etc.
- cerebrovascular disorder Cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage
- diagnosis, preliminary detection, prediction or pre-diagnosis for a certain condition for example, diseases such as diabetes, diabetic nephropathy, diabetic retinopathy, diabetic complications such as diabetic neuropathy
- diagnosis, preliminary detection, prediction or pre-diagnosis for a certain condition for example, diseases such as diabetes, diabetic nephropathy, diabetic retinopathy, diabetic complications such as diabetic neuropathy
- a drug, agent, factor or means specific to a marker associated with the condition or a composition, kit or system containing the same.
- gene product refers to a protein or mRNA encoded by a gene.
- gene products not directly related to sugar metabolism ie, proteins not related to sugar metabolism such as insulin
- proteins not related to sugar metabolism such as insulin
- the “subject” refers to an organism (for example, human) that is a target of diagnosis or detection of the present invention.
- sample refers to any substance obtained from a subject or the like, and includes, for example, body fluids (blood, saliva, urine, tears, etc.). Blood, urine, and tears are preferably used, and the specificity varies depending on the marker, but those skilled in the art can appropriately select a preferable sample based on the description of the present specification.
- drug drug
- drug may also be a substance or other element (eg energy such as light, radioactivity, heat, electricity).
- Such substances include, for example, proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (eg, DNA such as cDNA, genomic DNA, RNA such as mRNA), poly Saccharides, oligosaccharides, lipids, small organic molecules (for example, hormones, ligands, signaling substances, small organic molecules, molecules synthesized by combinatorial chemistry, small molecules that can be used as pharmaceuticals (for example, small molecule ligands, etc.)) , These complex molecules are included, but not limited thereto.
- a polynucleotide having a certain sequence homology to the sequence of the polynucleotide (for example, 70% or more sequence identity) and complementarity examples include, but are not limited to, a polypeptide such as a transcription factor that binds to the promoter region.
- Factors specific for a polypeptide typically include an antibody specifically directed against the polypeptide or a derivative or analog thereof (eg, a single chain antibody), and the polypeptide is a receptor.
- specific ligands or receptors in the case of ligands, and substrates thereof when the polypeptide is an enzyme include, but are not limited to.
- interaction refers to two substances. Force (for example, intermolecular force (van der Waals force), hydrogen bond, hydrophobic interaction between one substance and the other substance. Etc.). Usually, two interacting substances are in an associated or bound state.
- a “factor that specifically interacts” with a biological agent such as a polynucleotide or a polypeptide means an affinity for the biological agent such as the polynucleotide or the polypeptide,
- the affinity for other unrelated (especially less than 30% identity) polynucleotides or polypeptides is typically equivalent or higher, preferably significantly (eg, statistically significant) ) Includes the expensive.
- Such affinity can be measured, for example, by hybridization assays, binding assays, and the like.
- a first substance or factor “specifically interacts” with a second substance or factor means that the first substance or factor has a second Interacting with a higher affinity than a substance or factor other than a substance or factor (especially another substance or factor present in a sample containing a second substance or factor).
- Specific interactions for a substance or factor include, for example, a transcription factor and its protein when both nucleic acid and protein are involved, such as ligand-receptor reaction, hybridization in nucleic acid, antigen-antibody reaction in protein, enzyme-substrate reaction, etc. Examples include, but are not limited to, protein-lipid interactions, nucleic acid-lipid interactions, and the like, such as reactions with transcription factor binding sites.
- the first substance or factor “specifically interacts” with the second substance or factor means that the first substance or factor has the second substance Or having at least a part of complementarity to the factor.
- the first substance or factor “specifically interacts” with the second substance or factor includes, for example, an antigen-antibody reaction interaction, receptor- Examples include, but are not limited to, interaction by a ligand reaction and enzyme-substrate interaction.
- the first substance or factor “specifically interacts” with the second substance or factor means that the transcription factor and the transcription factor Interaction between the binding region of the nucleic acid molecule to be included.
- binding means a physical or chemical interaction between two proteins or compounds or related proteins or compounds, or a combination thereof. Bonds include ionic bonds, non-ionic bonds, hydrogen bonds, van der Waals bonds, hydrophobic interactions, and the like.
- a physical interaction (binding) can be direct or indirect, where indirect is through or due to the effect of another protein or compound. Direct binding refers to an interaction that does not occur through or due to the effects of another protein or compound and does not involve other substantial chemical intermediates.
- contacting refers to physically bringing a compound, either directly or indirectly, against a polypeptide or polynucleotide that can function as a marker, ligand, etc. of the present invention. Means close.
- the polypeptide or polynucleotide can be present in many buffers, salts, solutions, and the like.
- Contact includes placing the compound in, for example, a beaker, microtiter plate, cell culture flask or microarray (eg, gene chip) containing a polypeptide encoding a nucleic acid molecule or fragment thereof.
- the “label” refers to a presence (for example, a substance, energy, electromagnetic wave, etc.) for distinguishing a target molecule or substance from others.
- a labeling method include RI (radioisotope) method, fluorescence method, biotin method, chemiluminescence method and the like.
- the labeling is performed with fluorescent substances having different fluorescence emission maximum wavelengths. The difference in the maximum fluorescence emission wavelength is preferably 10 nm or more.
- DiI is a fat-soluble fluorescent dye that is inserted into the lipid bilayer of LDL or oxidized LDL and can introduce a fluorescent label without impairing the molecular characteristics.
- Alexa Fluor is desirable as the fluorescent material. Alexa Fluor is a water-soluble fluorescent dye obtained by modifying coumarin, rhodamine, fluorescein, cyanine, etc., and is a series corresponding to a wide range of fluorescent wavelengths. It is stable, bright and has low pH sensitivity.
- Examples of combinations of fluorescent dyes having a fluorescence maximum wavelength of 10 nm or more include a combination of Alexa 555 and Alexa 633, a combination of Alexa 488 and Alexa 555, and the like.
- Any nucleic acid can be used as long as it can bind to its base moiety.
- a cyanine dye eg, CyDye TM series Cy3, Cy5, etc.
- rhodamine 6G reagent N-acetoxy-N2— Acetylaminofluorene (AAF), AAIF (iodine derivative of AAF) or the like is preferably used.
- Examples of the fluorescent substance having a difference in fluorescence emission maximum wavelength of 10 nm or more include a combination of Cy5 and rhodamine 6G reagent, a combination of Cy3 and fluorescein, a combination of rhodamine 6G reagent and fluorescein, and the like.
- the target object can be modified so that it can be detected by the detection means used. Such modifications are known in the art, and those skilled in the art can appropriately carry out such methods depending on the label and the target object.
- diagnosis refers to identifying various parameters related to a disease, disorder, or condition in a subject and determining the current state or future of such a disease, disorder, or condition.
- conditions within the body can be examined, and such information can be used to formulate a disease, disorder, condition, treatment to be administered or prevention in a subject.
- various parameters such as methods can be selected.
- diagnosis in a narrow sense refers to diagnosis of the current state, but includes “predictive diagnosis”, “pre-diagnosis”, and the like in a broad sense. Early diagnosis is sometimes referred to as “early diagnosis”.
- pre-diagnosis uses CTLD14, a molecule capable of recognizing modified LDL, etc.
- Diseases related to degenerative LDL such as heart disease (myocardial infarction, angina pectoris), cerebrovascular disorder (cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage, transient ischemic attack, etc.), aortic aneurysm, renal infarction, etc.
- heart disease myocardial infarction, angina pectoris
- cerebrovascular disorder Cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage, transient ischemic attack, etc.
- aortic aneurysm aortic aneurysm
- renal infarction etc.
- pre-onset stages such as hyperlipidemia, lipid abnormalities, diabetes, hyperglycemia, hypertension, hypertensive nephrosclerosis, arteriosclerosis, ischemia reperfusion injury, vascular balloon injury,
- lipid abnormalities arteriosclerosis, diabetes, ischemic heart disease (myocardial infarction, angina etc.), cerebrovascular disorder (cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage, transient brain Ischemic attack), aortic aneurysm, renal infarction, etc.
- ischemic heart disease myocardial infarction, angina etc.
- cerebrovascular disorder Cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage, transient brain Ischemic attack
- aortic aneurysm renal infarction, etc.
- Diagnosis of pathological conditions such as diseases related to sex LDL, lipid abnormalities such as hyperlipidemia, diabetes, hyperglycemia, hypertension, hypertensive nephrosclerosis, arteriosclerosis, ischemia reperfusion injury, vascular balloon injury
- lipid abnormalities such as hyperlipidemia, diabetes, hyperglycemia, hypertension, hypertensive nephrosclerosis, arteriosclerosis, ischemia reperfusion injury, vascular balloon injury
- diabetes diabetic nephropathy, diabetic retinopathy
- diabetes When referring to diabetic complications such as diabetic neurosis, this refers to detecting the pre-onset stage of diabetic complications such as diabetes, diabetic nephropathy, diabetic retinopathy, diabetic neuropathy, etc.
- the state of the body can be examined in advance, and using such information, the disease, disorder, condition in the subject Various parameters can be selected, such as the formulation or method for treatment or prevention to be administered.
- “predictive diagnosis” or “preliminary diagnosis” includes diagnosis at a stage that cannot be diagnosed by other conventional methods, and is therefore used in part with the concept of “early diagnosis”.
- the diagnostic method of the present invention is industrially useful because, in principle, the diagnostic method of the present invention can be used from the body and can be performed away from the hands of medical personnel such as doctors.
- diagnosis, prior diagnosis or diagnosis may be referred to as “support”.
- detection agent broadly refers to any factor that can detect a target substance (eg, denatured LDL, AGEs, etc.).
- diagnosis agent broadly refers to any factor that can diagnose a target condition (for example, a disease).
- treatment refers to preventing a disease or disorder from deteriorating, preferably maintaining the status quo, more preferably reducing, when a certain disease or disorder becomes such a condition. Preferably, it means elimination.
- prevention means that a certain disease or disorder is prevented from becoming such a state before becoming such a state.
- lipid abnormality arteriosclerosis, diabetes, ischemic heart disease (myocardial infarction, angina pectoris, etc.), cerebrovascular disorder (cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage, transient Cerebral ischemic attack), aortic aneurysm, renal infarction, degenerative LDL-related diseases, lipid abnormalities such as hyperlipidemia, diabetes, hyperglycemia, hypertension, hypertensive nephrosclerosis, arteriosclerosis, false It is possible to prevent pathological conditions such as after blood reperfusion injury and vascular balloon injury, or to take preventive measures.
- diabetic complications such as diabetic nephropathy, diabetic retinopathy, diabetic neurode
- the present invention provides a single chain antibody or fragment thereof or a variant thereof comprising the amino acid sequence set forth in any of SEQ ID NOs: 76 to 80 or a variant thereof.
- the single chain antibody comprises the amino acid sequence shown in either SEQ ID NO: 76 or SEQ ID NO: 78 or a variant thereof. More preferably, the single chain antibody comprises the amino acid sequence set forth in SEQ ID NO: 76 or a variant thereof.
- the single chain antibody of the present invention or a fragment thereof can specifically bind to LDL and / or a modified product thereof.
- the amino acid sequence variants described in any of SEQ ID NOs: 76 to 80 include variants containing amino acid mutations, insertions, and deletions.
- the variant comprises at least 60%, at least 70%, at least 70% of the amino acid sequence of any of SEQ ID NOs: 1-5 over at least 50, at least 100, at least 150, at least 200, at least 250 adjacent amino acid sequences. It has 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% amino acid homology or identity.
- the present invention relates to a single-chain antibody or fragment thereof or variant thereof comprising the amino acid sequence set forth in any of SEQ ID NOS: 76 to 80 or a variant thereof, and LDL and / or a modified product thereof (modified A detection agent for detecting LDL) is provided.
- This detection agent may further comprise a molecule capable of recognizing oxidatively modified LDL in addition to the single-chain antibody or a fragment thereof.
- Molecules capable of recognizing oxidatively modified LDL include, but are not limited to, CTLD14, anti-LDL chicken antibodies, and combinations thereof.
- the detection agent of the present invention can detect LDL and / or a modified product thereof (modified LDL) with high sensitivity.
- the detection agent of the present invention contains LDL and / or a modified product thereof (modified LDL) at least 100 ng / 100 ⁇ l well, at least 90 ng / 100 ⁇ l well, at least 80 ng / 100 ⁇ l well, at least 70 ng / 100 ⁇ l well, at least 60 ng.
- / 100 ⁇ l well or less at least 50 ng / 100 ⁇ l well or less, at least 40 ng / 100 ⁇ l well or less, at least 30 ng / 100 ⁇ l well or less, at least 29 ng / 100 ⁇ l well or less, at least 25 ng / 100 ⁇ l well or less, at least 20 ng / 100 ⁇ l well or less, at least 15 ng / Sensation of 100 ⁇ l well or less, at least 10 ng / 100 ⁇ l well or less, or most preferably at least 5 ng / 100 ⁇ l well or less Can be detected in degrees.
- the present invention provides a method for detecting or quantifying LDL and / or a modified product thereof (denatured LDL), wherein (A) a sample to be detected or quantified is any of SEQ ID NOs: 76 to 80 A step of contacting the single-chain antibody or fragment or variant thereof comprising the amino acid sequence or variant thereof according to (1), and (B) the amino acid sequence or variant thereof according to any one of SEQ ID NOs: 76 to 80 Detection of the binding or the level of the binding to the target of the presence of LDL and / or a modified product thereof (denatured LDL) in the sample Alternatively, a method is provided comprising a step of indicating a presence level.
- any technique known in the art can be used as a technique for bringing the sample of the present invention into contact with the above-mentioned single-chain antibody, a fragment thereof or a variant thereof. However, it is not limited to these methods.
- any technique known in the art can be used as a technique for detecting the binding of the above single-chain antibody or a fragment thereof or a variant thereof to LDL and / or a modified product thereof (modified LDL).
- any technique for detecting the label can be utilized.
- the presence / absence or level of the binding indicates the presence or level of LDL and / or a modified product thereof (modified LDL) in the sample.
- the quantification of the binding of the single-chain antibody or fragment or variant thereof to LDL and / or a modified product thereof can be realized by any method for quantifying in the detection. it can. Such quantification may be relative (level) or displayed as an absolute amount. For example, as such a technique, for a known amount of LDL and / or a modified product thereof (denatured LDL), the intensity of the label to be detected is plotted, a calibration curve is created, and extrapolated from the detection data of the sample Although a technique is mentioned, it is not limited to it.
- any molecule can be used as long as a molecule to be immobilized, for example, a CTLD molecule or denatured LDL can be immobilized.
- a mechanism such as an enzyme linked immunosorbent assay (ELISA)
- ELISA enzyme linked immunosorbent assay
- a microtiter plate is generally used as the solid phase (substrate).
- the material of the substrate can be any solid, either covalently or noncovalently, that has the property of binding to the biomolecules used in the present invention or that can be derivatized to have such properties. Materials.
- Examples of the material include polyethylene, ethylene, polypropylene, polyisobutylene, polyethylene terephthalate, unsaturated polyester, fluorine-containing resin, polyvinyl chloride, polyvinylidene chloride, polyvinyl acetate, polyvinyl alcohol, polyvinyl acetal, acrylic resin, polyacrylonitrile.
- Use organic materials such as polystyrene, acetal resin, polycarbonate, polyamide, phenol resin, urea resin, epoxy resin, melamine resin, styrene / acrylonitrile copolymer, acrylonitrile butadiene styrene copolymer, silicone resin, polyphenylene oxide, polysulfone, etc. Can do.
- the fixing method to the solid phase can be carried out by any known method in this field. For example, it is preferable to use a bond by a silanol group (SiOH) on a solid surface, a hydrophobic bond by a matrix of a material and an ionic bond by a modified functional group, or a bond through an aromatic ring contained in the matrix. it can.
- the solid phase may be dried or used in a wet state. It becomes easy to preserve
- solid phase refers to a material that is used interchangeably with “substrate” and “substrate” herein and from which the device of the present invention is constructed.
- the solid phase when detecting using the principle of surface plasmon resonance, is preferably a glass substrate base material having a metal thin film containing gold, silver or aluminum on one side.
- a mechanism such as an enzyme-linked immunosorbent assay (ELISA) is used, a microtiter plate is generally used as the solid phase (substrate).
- a frequency conversion element for example, a crystal oscillator or a surface acoustic wave element
- the receptor is directly coupled.
- One side of the quartz plate is coated with silicone, and the other side is provided with a gold electrode as a solid phase.
- the material of the substrate can be any solid, either covalently or noncovalently, that has the property of binding to the biomolecules used in the present invention or that can be derivatized to have such properties. Materials.
- Suitable substrates include beads, gold particles, semiconductor nanoparticles (eg, CdTe nanoparticles, CdSe nanoparticles, GaN nanoparticles, ZnS nanoparticles, InP nanoparticles, etc.), silica nanoparticles, polystyrene nanoparticles, acrylic nanoparticles. Particles, latex nanoparticles, carbon nanoparticles, plates (eg, microtiter plates), test tubes, chips, magnetic particles, membranes, fibers, glass slides, metal thin films, filters, tubes, balls, diamond-like carbon coated stainless steel, etc. For example, but not limited to.
- semiconductor nanoparticles eg, CdTe nanoparticles, CdSe nanoparticles, GaN nanoparticles, ZnS nanoparticles, InP nanoparticles, etc.
- silica nanoparticles eg., silica nanoparticles, polystyrene nanoparticles, acrylic nanoparticles.
- any material capable of forming a solid surface can be used, for example, glass, silica, silicone, ceramic, silicon dioxide, plastic, metal (also alloys) Natural) and synthetic polymers (including, but not limited to) polystyrene, cellulose, amylose, chitosan, dextran, and nylon.
- the substrate may be formed from a plurality of layers of different materials.
- inorganic insulating materials such as glass, quartz glass, alumina, sapphire, forsterite, silicon carbide, silicon oxide, and silicon nitride can be used.
- Polyethylene ethylene, polypropylene, polyisobutylene, polyethylene terephthalate, unsaturated polyester, fluorine-containing resin, polyvinyl chloride, polyvinylidene chloride, polyvinyl acetate, polyvinyl alcohol, polyvinyl acetal, acrylic resin, polyacrylonitrile, polystyrene, acetal resin
- Organic materials such as polycarbonate, polyamide, phenol resin, urea resin, epoxy resin, melamine resin, styrene / acrylonitrile copolymer, acrylonitrile butadiene styrene copolymer, silicone resin, polyphenylene oxide, and polysulfone can be used.
- a membrane used for blotting such as a nylon membrane, a nitrocellulose membrane, and a PVDF membrane can also be used.
- a material having hardness such as glass.
- a preferable material for the substrate varies depending on various parameters such as a measuring instrument, and those skilled in the art can appropriately select an appropriate material from the various materials described above.
- chip refers to a microminiature integrated circuit that has various functions and becomes a part of the system.
- solid phase on which the receptor is immobilized is referred to as a receptor chip and / or a receptor microchip.
- detection or “quantification” of polynucleotide or polypeptide expression can be accomplished using suitable methods including, for example, mRNA measurement and immunological measurement methods.
- molecular biological measurement methods include Northern blotting, dot blotting, and PCR.
- immunological measurement method include an ELISA method using a microtiter plate, an RIA method, a fluorescent antibody method, a Western blot method, and an immunohistochemical staining method.
- the quantitative method include an ELISA method and an RIA method. It can also be performed by a gene analysis method using an array (eg, DNA array, protein array).
- the DNA array is widely outlined in (edited by Shujunsha, separate volume of cell engineering "DNA microarray and latest PCR method”).
- gene expression analysis methods include, but are not limited to, RT-PCR, RACE method, SSCP method, immunoprecipitation method, two-hybrid system, and in vitro translation.
- Such further analysis methods are described in, for example, Genome Analysis Experimental Method / Yusuke Nakamura Lab Manual, Editing / Yusuke Nakamura Yodosha (2002), etc., all of which are incorporated herein by reference. Is done.
- the term “probe” refers to a substance that serves as a search means used in biological experiments such as screening in vitro and / or in vivo.
- a nucleic acid molecule containing a specific base sequence or a specific nucleic acid molecule examples include, but are not limited to, peptides containing amino acid sequences, specific antibodies or fragments thereof.
- the probe is used as a marker detection means.
- Nucleic acid sequences used as probes are nucleic acid sequences that are at least 70% homologous, more preferably at least 80% homologous, more preferably at least 90% homologous, at least 95% homologous to the sequences described above. Is included.
- search refers to finding another nucleobase sequence having a specific function and / or property using a nucleobase sequence electronically or biologically or by other methods.
- Electronic search includes BLAST (Altschul et al., J. Mol. Biol. 215: 403-410 (1990)), FASTA (Pearson & Lipman, Proc. Natl. Acad. Sci., USA 85: 2444-). 2448 (1988)), Smith and Waterman method (Smith and Waterman, J. Mol. Biol. 147: 195-197 (1981)), and Needleman and Wunsch method (Needleman and Wunsch, J. Mol. Biol. 48:44). -453 (1970)), but is not limited thereto.
- Bio searches include stringent hybridization, macroarrays with genomic DNA affixed to nylon membranes, microarrays affixed to glass plates (microarray assays), PCR and in situ hybridization, etc. It is not limited to. In the present specification, it is intended that the gene used in the present invention should include a corresponding gene identified by such an electronic search or biological search.
- the “primer” refers to a substance necessary for the start of the reaction of the polymer compound to be synthesized in the polymer synthase reaction.
- a nucleic acid molecule for example, DNA or RNA
- the primer can be used as a marker detection means.
- Nucleic acid sequences used as probes are nucleic acid sequences that are at least 70% homologous, more preferably at least 80% homologous, more preferably at least 90% homologous, at least 95% homologous to the sequences described above. Is included.
- a sequence suitable as a primer may vary depending on the nature of the sequence intended for synthesis (amplification), but those skilled in the art can appropriately design a primer according to the intended sequence. Such primer design is well known in the art, and may be performed manually or using a computer program (eg, LASERGENE, PrimerSelect, DNAStar).
- the “kit” refers to a unit that is usually divided into two or more sections and a portion to be provided (eg, antibody, label, etc.) is provided.
- This kit form is preferred when it is intended to provide a composition that should not be provided in a mixed form but is preferably used in a mixed state immediately prior to use.
- Such a kit preferably comprises a provided part (eg, instructions or instructions describing how the reagent should be processed.
- the kit is a reagent kit. When used as a kit, the kit usually includes instructions describing how to use the antibody.
- the “instruction sheet” describes the method for using the present invention for a doctor or other user.
- This instruction manual includes a word indicating that the detection method of the present invention, how to use a diagnostic agent, or administration of a medicine or the like is given.
- the instruction sheet may include a word for instructing administration (for example, by injection) to skeletal muscle as an administration site.
- This instruction is prepared in accordance with the format prescribed by the national supervisory authority (for example, the Ministry of Health, Labor and Welfare in Japan and the Food and Drug Administration (FDA) in the United States, etc.) It is clearly stated that it has received.
- the instruction sheet is a so-called package insert and is usually provided as a paper medium, but is not limited thereto, and is in the form of, for example, an electronic medium (for example, a home page or e-mail provided on the Internet). But it can be provided.
- in vivo refers to the inside of a living body. In a particular context, “in vivo” refers to the location where a target substance is to be placed.
- in vitro refers to a state in which a part of a living body has been removed or released “outside a living body” (for example, in a test tube) for various research purposes. A term that contrasts with in vivo.
- ex vivo refers to a series of operations ex vivo when a certain treatment is performed outside the body but is intended to be returned to the body afterwards.
- the prescription procedure as a medicine such as the in vitro diagnostic agent of the present invention is known in the art, and is described in, for example, the Japanese Pharmacopoeia, the US Pharmacopoeia, and the pharmacopoeia of other countries. Thus, one skilled in the art can determine the amount to be used without undue experimentation as described herein.
- the modified LDL used in the present invention is oxidized LDL (OxLDL), malondialdehyded LDL (MDA-LDL), acrolein modified LDL, nonenal modified LDL, crotonaldehyde (CRA) modified LDL, 4- It may be hydroxynonenal (HNE) modified LDL, hexanoyl (HEL) modified LDL, small particle LDL, saccharified LDL, acetylated LDL or a variant thereof.
- OxLDL oxidized LDL
- MDA-LDL malondialdehyded LDL
- CRA crotonaldehyde
- HNE hydroxynonenal
- HEL hexanoyl
- small particle LDL small particle LDL
- saccharified LDL acetylated LDL or a variant thereof.
- a method for producing the single chain antibody or a fragment thereof is provided.
- the Escherichia coli used in the present invention is preferably selected from the group consisting of DH5 ⁇ strain, BL21 (DE3) strain, HB2151 strain, BLR strain, and TG1 strain. Most preferably, the E. coli is the BL21 (DE3) strain.
- the expression vector include, but are not limited to, a T7 promoter type E. coli protein expression vector (pET-22b (+) and the like). Most preferably, the expression vector is pET-22b (+).
- the inclusion body can be taken out using a technique known in the art. For example, it can be removed by centrifugation after sonication, French press, or homogenization. Inclusion bodies are found mainly in the pellets. Inclusion bodies can be solubilized by mixing reducing agents such as DTT and denaturing agents such as guanidine, guanidine derivatives, urea, or urea derivatives in the inclusion body suspension to extract single chain antibodies or fragments thereof. it can.
- the refolding condition is represented by a value obtained by converting the amount of inclusion bodies to be subjected to one purification (50 ml refolding) into a protein concentration measured by the BCA method, and this is expressed as “per protein”.
- “2 to 10 mg per protein (for example, 8.6 mg)” means that the value obtained by converting the amount of inclusion bodies to be used for one purification (50 ml refolding) to the protein concentration measured by the BCA method is 2 to 10 mg. (For example, 8.6 mg).
- “2 to 10 mg per protein (for example, 8.6 mg)” is important for obtaining a good yield.
- refolding of a single-chain antibody or a fragment thereof that can be used uses a cycloamylose (CA) method (Machida et al., FEBS Lett. 486 (2000): 131-135).
- CA cycloamylose
- refolding is performed by the CA method, it is performed in a solution containing an ionic surfactant containing cycloamylose and DL-cystine.
- the lower limit of the degree of polymerization of cycloamylose is 17 or more, preferably 25 or more, more preferably 40 or more, and the upper limit of the degree of polymerization is 150 or less, preferably 100 or less, more preferably 40 or less.
- ionic surfactants that may be used include cetyltrimethylammonium bromide, sodium dodecyl sulfate, sodium deoxycholate, 3-[(3-colamidopropyl) dimethylammonio] -1 -Propanesulfonic acid, hexadecyltrimethylammonium bromide (hereinafter sometimes abbreviated as CTAB), myristylsulfobetaine (hereinafter also abbreviated as SB3-14), and the like. Not. SB3-14 is preferred, and CTAB is more preferred.
- Dialysis is performed once or a plurality of times (for example, 2 to 4 times) using a cellulose ester membrane tube (Spectra / Por® Biotech CE, molecular weight cut-off 8000, 10 mm width).
- the dialysis solution is, for example, phosphate buffered saline (PBS), and as the PBS, typically Dulbecco's PBS (-) can be used (Composition of Dulbecco's PBS (-)) Is NaCl 8 g, KCl 0.2 g, Na 2 HPO 4 1.15 g, KH 2 PO 4 0.2 g / L, (pH 7.4)).
- a volume of at least 20 times, at least 50 times, at least 100 times, at least 200 times, at least 300 times, or preferably at least 400 times the sample volume is used.
- PBS PBS
- ⁇ PBS
- This PBS (-) does not contain calcium ions and magnesium ions.
- the present invention provides a system or kit for detecting or quantifying LDL or a modified product thereof (modified LDL) using the single-chain antibody of the present invention or a fragment thereof.
- the single-chain antibody or fragment thereof or a variant thereof that can be used in this aspect, detection or quantification techniques, and means, kits, systems, etc. used therefor include the above-mentioned section (Single-chain antibody or fragment thereof), ( Any form described in the sections (Detecting agents comprising single chain antibodies or fragments thereof), (Stable supply systems for single chain antibodies or fragments thereof) and other sections can be used.
- the kit or system can be a kit or system for implementing a direct ELISA.
- the present invention provides a molecule capable of recognizing a single-chain antibody of the present invention or a fragment thereof and an LDL that has undergone oxidative modification such as a ligand recognition region of oxidized LDL receptor (LOX-1). And a system for detecting or quantifying denatured LDL, such as LDL that has undergone oxidative modification.
- LDL oxidized LDL receptor
- the single-chain antibody or fragment thereof that can be used in this aspect include the above-mentioned section (Single-chain antibody or fragment thereof), (Detection agent containing single-chain antibody or fragment thereof), (Single-chain antibody). Any form described in the section on stable supply of antibodies or fragments thereof and in other sections can be used.
- the present invention provides a kit or system for detection or quantification of denatured LDL, such as LDL that has undergone oxidative modification, in which (A) is immobilized.
- a solid phase having a molecule capable of recognizing LDL subjected to oxidative modification such as a ligand recognition region of LOX-1 (eg, CTLD14) and a labeled single-chain antibody of the present invention or a fragment thereof or a variant thereof,
- recognizing LDL that has undergone oxidative modification such as a solid phase having a fixed single-chain antibody of the present invention or a fragment or variant thereof, and a labeled LOX-1 ligand recognition region (eg, CTLD14).
- the presence or absence or level of the label indicates the presence or level of LDL that has undergone oxidative modification.
- the modified LDL, CTLD molecule, solid phase, label, contact, binding detection technique or means, etc. that can be used in this kit of the present invention, such as calculation of the level of binding, etc. (detection including single chain antibody or fragment thereof) It is understood that any of the embodiments described in Agents and detection methods) can be utilized. As these formats, an indirect ELISA (sandwich ELISA) format is typically used, but is not limited thereto.
- Molecules capable of recognizing oxidatively modified LDL that can be used are typically oxidized LDL receptor (LOX-1), CTLD14, and anti-LDL chicken antibodies, single chain antibodies of the invention (SEQ ID NO: 76, 77, 78, 79 or 80) or a modified form thereof.
- LOX-1 oxidized LDL receptor
- CTLD14 CTLD14
- anti-LDL chicken antibodies single chain antibodies of the invention (SEQ ID NO: 76, 77, 78, 79 or 80) or a modified form thereof.
- an N-terminal cytoplasmic domain typically among the four domains of LOX-1: an N-terminal cytoplasmic domain, a hydrophobic transmembrane domain, a neck domain, and a C-type lectin-like domain (also referred to herein as CTLD), It may be a molecule containing CTLD, and may be CTLD14, LOX-1 full length, LOX-1 extracellular region full length or a variant thereof, preferably CTLD14 is used. Although it is because stability is long with respect to long-term storage, it is not limited to this.
- the system is for performing an antigen-antibody reaction, eg, for performing an ELISA, a sandwich ELISA, or a lateral flow assay.
- a membrane for developing a sample by capillary action recognizes a ligand of an oxidized LDL receptor (LOX-1) labeled with a sample and a metal colloid, silica particles or latex particles.
- LOX-1 oxidized LDL receptor
- a conjugate part comprising the single-chain antibody or fragment thereof labeled with a metal colloid, silica particle or latex particle; a ligand recognition region of oxidized LDL receptor (LOX-1), or one of the above
- a lateral flow assay comprising a detection part comprising a single chain antibody or a fragment thereof; and a control part comprising a binding molecule for the ligand recognition region of the oxidized LDL receptor (LOX-1) or a binding molecule for a single chain antibody System.
- a membrane for developing a sample by capillary action is provided, and the membrane is CTLD14 labeled with a sample and a metal colloid, silica particles or latex particles, or a metal colloid, silica particles or latex particles.
- CTLD14 labeled with a sample and a metal colloid, silica particles or latex particles, or a metal colloid, silica particles or latex particles.
- a system for performing a lateral flow assay comprising a conjugate part comprising the single-chain antibody or fragment thereof labeled with a control part comprising a binding molecule for CTLD14.
- metal colloid examples include, but are not limited to, gold colloid, platinum colloid, palladium colloid, and silver colloid.
- latex particles include polystyrene-based polymers, carboxy-modified, fluorescent latex particles, and magnetic latex particles.
- silica-based particles include silica particles and fluorescent silica particles, but are not limited thereto.
- CTLTD14 is biotinylated, His-tagged, Myc-tagged, Flag-tagged, E-tagged, or Strep-tagged, and in each case streptavidin, anti-His antibody, anti-Myc antibody, anti-Flag
- An antibody, an anti-E tag antibody, Strep-Tactin or the like is used as a binding molecule, but is not limited thereto.
- the present invention also recognizes anti-denaturing LDL antibodies, variants thereof or fragments thereof and LDL that has undergone oxidative modification such as a ligand recognition region of LOX-1 (eg, CTLD14).
- a system is provided for detecting or quantifying denatured LDL, such as LDL that has undergone oxidative modification, using the molecules that can be produced in a lateral flow assay format.
- Such a system of the lateral flow assay format is advantageous because it is not provided for the detection of conventional denatured LDL and can detect cases that could not be detected by a conventional assay system.
- the advantages of the present invention are that (1) the reaction time is typically within 30 minutes, and (2) special equipment is used. For example, it can be detected visually without using it, and (3) it is not necessary to add a reagent for detection, and the reaction can be completed only by adding a sample to be measured.
- the present invention also provides an anti-denatured LDL antibody, a variant thereof, or a fragment thereof and a molecule capable of recognizing LDL that has undergone oxidative modification such as a ligand recognition region of LOX-1 (eg, CTLD14).
- a system for the assessment of diseases associated with degenerative LDL such as lipid abnormalities, arteriosclerosis, diabetes, ischemic heart disease, cerebrovascular disorders, etc. for use in a flow assay format.
- Oxidatively modified LDL is expressed under various conditions under abnormal conditions such as hyperlipidemia, diabetes, hyperglycemia, hypertension, hypertensive nephrosclerosis, arteriosclerosis, ischemia reperfusion injury, vascular balloon By pathologies such as post-injury; and by stimuli such as oxidized LDL, angiotensin II, endothelin, TNF- ⁇ , late glycation products (AGEs), TGF- ⁇ , 8-iso-prostaglandin F 2 ⁇ , shear stress Therefore, the information can be used for the evaluation according to the present invention (Folia Pharmacol. Jpn. 127, 103-107 (2006), JP 2009-082076).
- modified LDL present in a living body oxidized LDL, malondialdehyde-modified LDL (MDA-LDL), acrolein-modified LDL, nonenal-modified LDL, small particle LDL (LDL having a diameter of 255 nm or less), saccharified LDL, and the like can be mentioned. However, it is not limited to these.
- oxidized LDL shows abnormal values, arteriosclerosis, ischemic heart disease (myocardial infarction, angina, etc.), cerebrovascular disorder (cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage, transient ischemic attack, etc.), Diseases such as aortic aneurysm, renal infarction, and hyperlipidemia are expected, but not limited to these (see “Today's Clinical Examination 2007-2008” publisher: Nanedo Co., Ltd.).
- MDA-LDL normal range: 10 to 80 U / L
- oxidized phosphatidylcholine normal range: 8.4 U / mL to 17.6 U / mL
- a subject for example, a healthy subject not suffering from the disease and a subject suspected of suffering from the disease ( Patient) denatured LDL is detected in both samples, and the amount of complex of the polypeptide of the invention and denatured LDL is compared between these samples to determine whether degenerative LDL in a subject suspected of the disease.
- a relative value for the control subject is calculated.
- MDA-LDL normal range: 10 to 80 U / L
- oxidized phosphatidylcholine normal range: 8.4 U / mL to 17.6 U / mL
- the diseases caused by degenerated LDL are typically arteriosclerosis, coronary artery disease, arteritis, coronary spasm angina based on arteriosclerosis, dilated cardiomyopathy, ischemic heart disease (myocardial infarction, angina) ), Cerebrovascular disorders (cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage, transient cerebral ischemic attack, etc.), aortic aneurysm, renal infarction, hyperlipidemia and the like, but are not limited thereto.
- the present invention also provides a lateral flow of an anti-denatured LDL antibody, a variant or fragment thereof, and a molecule capable of recognizing LDL that has undergone oxidative modification such as a ligand recognition region of LOX-1 (eg, CTLD14).
- a system for evaluating the preventive effect of diseases associated with degenerative LDL such as lipid abnormalities caused by food intake, arteriosclerosis, diabetes, ischemic heart disease, cerebrovascular disorder, and the like used in an assay format.
- the anti-denatured LDL antibody refers to any antibody against denatured LDL, and is a single chain antibody or fragment thereof comprising the amino acid sequence set forth in any of SEQ ID NOs: 76 to 80 described herein or a variant thereof.
- anti-LDL chicken antibodies can be mentioned, but are not limited thereto.
- the chicken anti-LDL antibody that can be used here is, for example, antigen (LDL) 0.3 mg (Adjuband FCA) 6 times every 2 weeks, after inoculation into chicken skin, after 5th check of titer increase And it can extract
- the antibody activity can be measured by immobilizing 1 ⁇ g / ml of antigen (LDL) at 100 ⁇ l / well and according to a normal ELISA (HRP-labeled anti-chicken IgY is used as the labeled secondary antibody). ).
- an immunoassay when used for the antigen detection method, it is preferable to use a carrier on which an antibody is immobilized.
- an immunoassay system using the antibody immobilized on the carrier as the primary antibody can be easily constructed.
- two types of antibodies specific for an antigen and having different epitopes are prepared, one is immobilized on a carrier as a primary antibody, and the other is enzyme-labeled as a secondary antibody to construct a sandwich EIA system.
- immunoassay systems based on binding inhibition methods and competitive methods can be constructed.
- a substrate is used as a carrier, immunoassay using an antibody chip is possible. According to the antibody chip, the concentration of a plurality of markers can be measured simultaneously, and rapid measurement is possible.
- CTLD14 (Silkworm type) and production method thereof>
- the present invention provides a C-type lectin-like domain (CTLD) 14 having a silkworm type sugar chain and comprising the amino acid sequence shown in SEQ ID NO: 86 or a variant thereof.
- the silkworm sugar chain includes one or more kinds of sugar chains of a trimannosyl core, a complex sugar chain, an oligomannose sugar chain, or a hybrid sugar chain.
- the silkworm-type sugar chain has a trimannosyl core (from asparagine residue to GlcNAc- ⁇ 1,4-GlcNAc- ⁇ 1,4-Man (- ⁇ 1,6-Man) - ⁇ 1,3-Man).
- each molecule contains a sugar chain to which 0 to 4 molecules of GlcNAc2 molecule and Man2 molecule are bound.
- the silkworm-type sugar chain has the following combination 1) GlcNAc- ⁇ 1,4-GlcNAc- ⁇ 1,4-Man (- ⁇ 1,3-Man) - ⁇ 1,6-Man as viewed from the asparagine residue.
- the silkworm sugar chain binds to asparagine at position 111 and / or asparagine at position 155 of SEQ ID NO: 86.
- the CTLD 14 of the present invention is biotinylated.
- biotinylation By performing biotinylation, measurement results can be obtained with high sensitivity in bioassays, and high-density integration in a certain direction on beads and membranes via streptavidin becomes possible.
- the CTLD14 of the present invention can be used as a composition for detecting denatured LDL. Therefore, the present invention provides a composition for detecting denatured LDL including CTLD14 containing the silkworm-type sugar chain of the present invention.
- the composition for detecting denatured LDL of the present invention is for distinguishing non-denatured LDL from denatured LDL and detecting denatured LDL.
- the improved CTLD14 containing the silkworm-type sugar chain of the present invention widely recognizes modified LDL, but has high specificity for non-modified LDL. This makes it possible to determine with high sensitivity the degeneration LDL used for diagnosis of diabetes, arteriosclerosis and the like. Compared to the prior art, some of the universality, sensitivity, and measurement accuracy are outstanding, and it is particularly excellent in that molecules having a structure that causes onset in a living body can be widely detected. In addition, there are advantages that manufacturing is simple and mass production is relatively possible.
- the present invention relates to a method for producing C-type lectin-like domain (CTLD) 14, which is a step of incorporating a nucleic acid molecule encoding CTLD14 into an organism that imparts a sugar chain similar to silkworm or silkworm, in an expressible manner. ; B) A method comprising the steps of placing the silkworm or an organism that imparts a sugar chain similar to that of a silkworm under conditions where the gene is expressed to express the CTLD14; and C) obtaining the CTLD14. .
- C-type lectin-like domain C-type lectin-like domain
- any organism may be used as long as it can impart a silkworm-type sugar chain, and any organism that imparts a sugar chain similar to that of silkworm is used. It is understood that can be done.
- the expression is performed in the middle silk gland of the organism that imparts the same sugar chain as the silkworm or silkworm.
- the CTLD14 is expressed in a biotinylated state or in a biotinable state.
- biotinylation can be achieved by incorporating a sequence to which biotin can bind. For example, it can be achieved by adding lysine (K) by recombination.
- the step A) is achieved by microinjecting an expression vector containing a nucleic acid molecule encoding the CTLD14.
- the expression vector can be introduced by any other method described in the present specification or other known methods other than microinjection.
- the nucleic acid molecule comprises the nucleic acid sequence shown in SEQ ID NO: 85 or a variant thereof.
- the present invention provides a reconstructed terminal glycation end product receptor (sRAGE) having the silkworm type sugar chain and comprising the amino acid sequence shown in SEQ ID NO: 97 or a variant thereof.
- sRAGE reconstructed terminal glycation end product receptor
- the silkworm type sugar chain in the present invention includes a trimannosyl core, a complex type sugar chain, an oligomannose type sugar chain or a sugar chain of a hybrid type sugar chain.
- the silkworm-type sugar chain has a trimannosyl core (from asparagine residue to GlcNAc- ⁇ 1,4-GlcNAc- ⁇ 1,4-Man (- ⁇ 1,6-Man) - ⁇ 1,3-Man).
- each molecule contains a sugar chain to which 0 to 4 molecules out of GlcNAc 0 to 2 molecules and Man0 to 4 molecules are bound.
- the silkworm sugar chain of the RAGE of the present invention has a trimannosyl core (from an asparagine residue to GlcNAc- ⁇ 1,4-GlcNAc- ⁇ 1,4-Man (- ⁇ 1,6-Man) - ⁇ 1, In addition to the structure of 3-Man), it includes sugar chains to which 0 to 8 molecules out of GlcNAc 0 to 4 molecules and Man 0 to 8 molecules are bound per molecule.
- the sRAGE silkworm sugar chain includes, but is not limited to, sugar chains having the structures (1) to (8) ([Chemical Formula 1] to [Chemical Formula 8]).
- the sugar chain composition ratio of silkworm-type sRAGE accounts for 90% or more of oligomannose type and less than 5% of complex type and hybrid type.
- the sugar composition of the silkworm-type sRAGE is (Man) 5 (GlcNAc) 2 (N3 in FIG. 54) is 46% to 56%, and (Man) 7 (GlcNAc) 2 (N1 in FIG. 54) is 23%.
- (Man) 6 (GlcNAc) 2 (N2 in FIG. 54) is 7% to 15%, and
- (Man) 3 (GlcNAc) 2 (N4-1 in FIG. 54) is 1%.
- the sugar composition is (Man) 5 (GlcNAc) 2 is between 48% and 54%, (Man) 7 (GlcNAc) 2 is between 25% and 31%, and (Man) 6 ( GlcNAc) 2 is 9% to 13%, (Man) 3 (GlcNAc) 2 is 2% to 4%, (Man) 3 (GlcNAc) 3 is 3% to 6%, Man) 4 (GlcNAc) 3 is between 2% and 4%.
- the sugar composition is (Man) 5 (GlcNAc) 2 is 51.4%, (Man) 7 (GlcNAc) 2 is 27.5%, and (Man) 6 (GlcNAc). ) 2 is 10.8%, (Man) 3 (GlcNAc) 2 is 2.8%, (Man) 3 (GlcNAc) 3 is 4.6%, and (Man) 4 ( GlcNAc) 3 is 2.5%.
- SRAGE that has been subjected to addition of the sugar chain or a sugar chain obtained by adding a sugar to the sugar chain is excellent in stability and has the ability to recognize AGEs having various structures.
- the silkworm sugar chain binds to asparagine at position 3 and / or asparagine at position 59 of SEQ ID NO: 97.
- the sRAGE of the present invention is biotinylated.
- biotinylation By performing biotinylation, measurement results can be obtained with high sensitivity in bioassays, and high-density integration in a certain direction on beads and membranes via streptavidin becomes possible.
- the sRAGE of the present invention can be used as a composition for detecting end-stage glycation products (AGEs). Therefore, this invention provides the composition for detecting AGEs containing sRAGE containing the silkworm type sugar chain of this invention.
- the AGEs to be analyzed can be raised from about 10% of the conventional level to almost all levels, and more accurate diagnosis It has a remarkable effect in that it can be performed.
- a wide range of AGEs can be detected by the conventional detection method based on reconstructed RAGE (sRAGE), but sRAGE has a problem in practical use such as fragmentation in about one and a half months and loss of recognition ability. This is also remarkable in that this can be overcome by using the sRAGE of the present invention.
- the improved sRAGE of the present invention has undergone glycosylation, is a very stable molecule, and has maintained recognition activity for about 1 year over a long period of time when stored at 4 ° C. Further, by the improved sRAGE of the present invention, it was possible to concentrate a small amount of AGEs and detect AGEs having various structures.
- the present invention also provides a method for producing sRAGE, wherein A) a nucleic acid molecule encoding sRAGE is incorporated so as to allow expression into a silkworm or an organism imparting a sugar chain similar to silkworm; A method comprising: placing a silkworm or an organism that imparts a sugar chain similar to a silkworm under conditions under which the gene is expressed, and expressing the sRAGE; and C) obtaining the sRAGE.
- any organism may be used as long as it can impart a silkworm-type sugar chain, and any organism that imparts a sugar chain similar to that of silkworm is used. It is understood that can be done.
- the expression is performed in the middle silk gland of the organism that imparts the same sugar chain as the silkworm or silkworm.
- the step A) is achieved by microinjecting an expression vector containing a nucleic acid molecule encoding the sRAGE.
- the expression vector can be introduced by any other method described in the present specification or other known methods other than microinjection.
- the nucleic acid molecule comprises the nucleic acid sequence shown in SEQ ID NO: 96 or a variant thereof.
- a promoter of a nucleic acid sequence encoding a protein that is specifically expressed in the middle silk gland, and any protein whose expression is directly or indirectly controlled by the promoter Transgenic silkworm having a nucleic acid sequence to be encoded or an organism imparting a sugar chain similar to that of silkworm, wherein said arbitrary protein is expressed in silk gland or secreted to transgenic silkworm or silkworm that is secreted into silkworm silkworm It may be achieved by a step of producing an organism to be imparted; and (b) a step of recovering the arbitrary protein from an organism that imparts a sugar chain similar to the produced transgenic silkworm.
- a promoter of a nucleic acid sequence encoding a protein that is specifically expressed in the middle silk gland, and Silkworm eggs having a nucleic acid sequence encoding any protein whose expression is directly or indirectly controlled by the promoter are produced.
- selecting organisms that impart a sugar chain similar to transgenic silkworms or silkworms expressing any protein Can be realized.
- transgenic silkworms or organisms to which sugar chains similar to silkworms are added can be selected using, for example, a selection marker.
- a selection marker in the present invention a marker generally used by those skilled in the art, for example, a fluorescent protein such as CFP, GFP, YFP, and DsRed can be used.
- CFP, GFP, YFP, and DsRed can be used as a selection marker.
- transgenic silkworms can be detected simply by observing with a stereoscopic fluorescence microscope. Further, since the fluorescent colors are different, a plurality of markers can be used simultaneously.
- a method for recovering any protein from the produced transgenic silkworm or an organism that imparts a sugar chain similar to that of a silkworm includes, for example, the provision of a sugar chain similar to that of a transgenic silkworm or silkworm.
- a method of recovering an arbitrary protein from the spider spun by the living organism includes, for example, the provision of a sugar chain similar to that of a transgenic silkworm or silkworm.
- a method of recovering an arbitrary protein from the spider spun by the living organism As a recovery method, a method well known to those skilled in the art, for example, a method of recovering by dissolving cocoon in 60% LiSCN and dialyzing with 20 mM Tris, 5M urea (Inoue, S., Tsuda, H., Tanaka, H., Magoshi, Y and Mizuno (2001) Serialology 4,157-163.) Can be used. Further, as other protein recovery methods, for example, a method using a surfactant or a method of dissolving in an a
- a promoter of a nucleic acid sequence encoding a protein that is specifically expressed in the middle silk gland and a sugar chain similar to that of a silkworm egg or silkworm having a nucleic acid sequence encoding an arbitrary protein whose expression is indirectly controlled by the promoter.
- Examples of the egg of the organism to be given include, for example, (i) a nucleic acid sequence in which a nucleic acid sequence encoding a transcriptional regulatory factor is functionally linked downstream of a promoter of a nucleic acid sequence encoding a protein that is specifically expressed in the middle silk gland And (ii) a silkworm egg having a nucleic acid sequence to which a nucleic acid sequence encoding an arbitrary protein is operably linked downstream of a target promoter of the transcription control factor or an egg of an organism that imparts a sugar chain similar to a silkworm Can be mentioned.
- a silkworm egg or silkworm having a promoter of a nucleic acid sequence encoding a protein expressed specifically in the middle silk gland and an arbitrary protein whose expression is directly controlled by the promoter As an egg of a living organism, for example, a silkworm egg having a nucleic acid sequence to which a nucleic acid sequence encoding an arbitrary protein is operably linked downstream of a promoter of a nucleic acid sequence encoding a protein expressed specifically in the middle silk gland or Examples include eggs of organisms that impart sugar chains similar to silkworms.
- nucleic acid sequence in which a nucleic acid sequence encoding an arbitrary protein is operably linked downstream of a promoter of a nucleic acid sequence encoding a protein expressed specifically in the middle silk gland is introduced into the egg.
- operably linked refers to being linked so that expression of a target protein is realized, and typically, a transcriptional regulatory factor binds to a promoter, This means that the promoter and the nucleic acid sequence are linked so that expression of the nucleic acid sequence existing downstream of the protein is induced.
- transcriptional regulatory factors and target sequences include GAL4 and UAS, TetR and TRE, and the like.
- the nucleic acid sequence used in the present invention can be prepared by methods such as hybridization technology, polymerase chain reaction (PCR) technology, site-directed mutationagesis method, DNA synthesis and the like. Whether or not the prepared nucleic acid sequence has promoter activity can be examined by those skilled in the art by a well-known reporter assay using a reporter gene. Reporter genes that can be used are described elsewhere in this specification and any known one can be used.
- the glycoprotein of the present invention is preferably a protein that does not cause irreversible denaturation in silk thread.
- examples of such proteins include proteins that do not have a secretion signal from silk gland cells to the silk gland lumen.
- DNA is introduced into silkworm eggs or eggs of living organisms to which sugar chains similar to those of silkworms are introduced, for example, by transposon injection into early-stage eggs as a vector (Tamura, T., Thibert, C., Royer, C ., Kanda, T., Abraham, E., Kamba, M., Komoto, N., Thomas, J.-L., Mauchamp, B., Chavancy, G., Shirk, P., Fraser, M., Prud Subscribe, J.-C. and Couble, P., 2000, Nature Biotechnology 18, 81-84).
- a vector Tamura, T., Thibert, C., Royer, C ., Kanda, T., Abraham, E., Kamba, M., Komoto, N., Thomas, J.-L., Mauchamp, B., Chavancy, G., Shirk, P., Fraser, M., Prud Subscribe, J.-C. and Couble, P., 2000, Nature Biotechnology 18, 81
- a vector (helper vector) having a nucleic acid sequence encoding a transposon transferase can be introduced into a silkworm egg or an egg of an organism to which a sugar chain similar to that of a silkworm is imparted together with a vector into which is inserted.
- Helper vectors include pHA3PIG (Tamura, T., Thibert, C., Royer, C., Kanda, T., Abraham, E., Kamba, M., Komoto, N., Thomas, J.-L., Mauchamp, B., Chavancy, G., Shirk, P., Fraser, M., Prud Subscribe, J.-C. and Couble, P., 2000, Nature Biotechnology 18, 81-84). It is not limited.
- piggyBac is preferable, but is not limited thereto, and mariner, minos, etc. can also be used (Shimizu, K., Kamba, M., Sonobe, H ., Kanda, T., Klinakis, A. G., Savakis, C. and Tamura, T. (2000) Insect Mol. Biol., 9, 277-281; Wang W, Swevers L, Iatrou K. (2000) Insect Mol Biol 9 (2): 145-55).
- a transgenic silkworm or an organism that imparts a sugar chain similar to that of a silkworm by using a baculovirus vector (Yamao, M., N. Katayama, H. Nakazawa, M. Yamakawa, Y. Hayashi et al., 1999, Genes Dev 13: 511-516).
- the silkworm or the organism that imparts the same sugar chain as that of the silkworm in the present invention is not particularly limited, but for mass production of the target glycoprotein, a protein constituting a silk thread such as fibroin protein is used. It is preferable to use silkworms in which the production of the protein constituting the silk thread is suppressed by the mutation of the coding gene region (including the coding region, promoter region, and untranslated region) or organisms to which sugar chains similar to silkworms are added. .
- Examples of such silkworms or organisms that give the same sugar chain as silkworms include silkworms or silkworms of mutant strains in which the production of the protein constituting the silk thread is suppressed by mutation of the gene region encoding the protein constituting the silk thread.
- Organisms that impart a similar sugar chain preferably silkworms of the naked silkworm strain in which the production of proteins that constitute silk is suppressed by the mutation, or organisms that impart a sugar chain similar to that of silkworms, more preferably Nd-s D
- Any silkworm or organism that imparts the same sugar chain as that of the silkworm can be used.
- Such a silkworm or an organism imparting a sugar chain similar to that of a silkworm is a silkworm well known to those skilled in the art as a sericin silkworm.
- sericin silkworm the protein synthesized from any gene introduced into the chromosome can be further purified.
- the silkworm or the organism imparting the same sugar chain as the silkworm in the present invention is not only a silkworm having the property of producing non-dormant eggs but also a silkworm having the property of producing dormant eggs (for example, practical varieties).
- Arumuma, 200, Shunchu, Kangetsu, Nishikiaki, Kanwa, etc. can also be used.
- a dormant egg refers to an egg in which embryonic development stops temporarily after spawning
- a non-diapause egg refers to an egg in which postnatal embryonic development does not stop and larvae hatch.
- non-dormant eggs are laid and DNA is introduced into the non-dormant eggs.
- a method for producing a non-dormant egg for example, in Gunma, a method of causing a non-dormant egg to lay on an adult produced from the dormant egg by culturing the dormant egg at 15 ° C. to 21 ° C., preferably a dormant egg Is cultivated at 16 ° C. to 20 ° C. to allow the adults produced from the dormant eggs to lay non-dormant eggs, and more preferably, the dormant eggs are cultured at 18 ° C.
- a method for giving birth to dormant eggs most preferably a method for cultivating dormant eggs at 18 ° C. so that the larvae produced from the dormant eggs are fully bred and the grown adults are made to lay non-dormant eggs.
- a method in which a dormant egg is cultivated at 15 ° C. to 21 ° C. to allow an adult produced from the dormant egg to produce a non-dormant egg preferably the dormant egg is cultured at 16 ° C. to 20 ° C.
- non-dormant eggs are laid down on the adults generated from the dormant eggs, more preferably, non-dormant eggs are laid down on the adults generated from the dormant eggs by culturing dormant eggs at 18 ° C.
- the larvae generated from the diapause eggs are bred in full light by culturing the dormant eggs at 25 ° C.
- a method of causing the grown adult to lay non-dormant eggs Egg culturing can be carried out, for example, by placing it in an incubator at 18 ° C. to 25 ° C. or in a constant temperature room, and larvae can be raised using artificial feed in a breeding room at 20 ° C. to 29 ° C. .
- the day length condition means a daily light / dark cycle during egg culture or larval rearing. Such conditions include a light condition and a dark condition.
- the all-light condition means a 24-hour light condition without darkness.
- the day length condition can be changed according to the variety.
- Those skilled in the art can culture the dormant egg of the present invention according to a general silkworm egg culture method. For example, the culture is performed according to the method described in “Ministry of Education (1978) Soybean Manufacture. Pp193, Jikkyo Publishing, Tokyo”.
- breeding of silkworm larvae in the present invention can be performed by those skilled in the art by a well-known method. For example, breeding is performed according to the method described in “Ministry of Education (1978) Soybean Manufacture. Pp193, Jikkyo Publishing, Tokyo”.
- whether or not a spawned egg is a non-dormant egg can be determined by the color of the egg.
- dormant eggs are colored dark brown and non-dormant eggs are yellowish white. Therefore, in the present invention, an egg laid is determined to be a non-dormant egg by not being dark brown, more preferably yellowish white.
- the present invention provides a silkworm incorporating a nucleic acid molecule encoding CTLD14 or sRAGE so that it can be expressed, or an organism imparting a sugar chain similar to that of a silkworm.
- the nucleic acid molecule used in the present invention includes the nucleic acid sequence shown in SEQ ID NO: 85 or SEQ ID NO: 96 or a variant thereof.
- the silkworm of the present invention or an organism imparting a sugar chain similar to the silkworm is a promoter of a nucleic acid sequence encoding CTLD14 or sRAGE that is specifically expressed in the middle silk gland, and directly or indirectly by the promoter.
- a transgenic silkworm or an organism that imparts a sugar chain similar to that of a silkworm is provided.
- CTLD14 or sRAGE can be produced in large quantities by using the transgenic silkworm of the present invention or an organism that imparts a sugar chain similar to that of the silkworm.
- the transgenic silkworm of the present invention or an organism that imparts a sugar chain similar to that of the silkworm is preferably operable downstream of the promoter of the nucleic acid sequence encoding CTLD14 or sRAGE that is specifically expressed in the middle silk gland.
- transgenic silkworm or silkworm having a nucleic acid sequence encoding CTLD14 or sRAGE operably linked downstream of a target promoter of the transcriptional regulator, A nucleic acid sequence to which a nucleic acid sequence encoding CTLD14 or sRAGE is operably linked downstream of a promoter of an organism imparting the same sugar chain or a nucleic acid sequence encoding CTLD14 or sRAGE specifically expressed in the middle silk gland Transgenic silkworm also having A organisms that confer a similar carbohydrate and silkworm.
- the present invention provides a silk gland or cocoon of an organism (a silk gland or cocoon producing organism) that imparts a sugar chain similar to the transgenic silkworm or silkworm of the present invention.
- a silk gland or wrinkle is useful as a silk gland or wrinkle containing a large amount of CTLD14 or sRAGE.
- the present invention also provides nucleic acid sequences or nucleic acid molecules (eg, DNA) for use in the methods of the present invention.
- a nucleic acid sequence or nucleic acid molecule include (a) a nucleic acid sequence encoding a transcriptional regulator operably linked downstream of the promoter of the nucleic acid sequence encoding sericin, and (b) a target of the transcriptional regulatory factor.
- Nucleic acid sequences and the like may be mentioned, and a kit comprising these combinations may be provided.
- the present invention also provides a vector in which the nucleic acid sequences (a) to (c) are inserted between the inverted terminal repeats of the transposon.
- a kit comprising the vector and a vector (helper vector) having a nucleic acid sequence encoding a transposon transferase is provided.
- the present invention provides a method, kit and composition for detecting, predicting, preliminary detection or diagnosis of AGE-related diseases, disorders, etc. by detecting terminal glycation products (AGEs).
- AGEs terminal glycation products
- the marker of the present invention it is understood that the section ⁇ Improved sRAGE (Silkworm type) and production method thereof> above can be referred to, and any embodiment described in this section can be adopted.
- diabetes include diabetes, diabetic nephropathy, diabetic retinopathy, diabetic complications such as diabetic neuropathy, neurodegenerative diseases such as Alzheimer's disease, rheumatoid arthritis, posterior longitudinal ligament ossification, osteoporosis, non-alcohol Examples include, but are not limited to, steatohepatitis, periodontitis, muscle atrophy, age-related macular degeneration, skin diseases, skin aging, arteriosclerosis, and the like.
- diabetic nephropathy is a microvascular disorder, but it is known that diabetes leads not only to microvascular disorders but also to the development of macrovascular disorders such as arteriosclerosis. .
- the sample may be acquired by any means. Usually, when a person in charge other than the doctor is engaged in the measurement, it may have been acquired by the doctor in some form.
- the step of determining whether or not there is a possibility of diabetes from the measurement result can be carried out by determining whether or not it is abnormal compared to the normal value as compared with the normal value.
- it is possible to diagnose these diseases and the like by conducting a comparison experiment between a diabetes model and a control, based on an illustrative example.
- the reagent for diagnosing AGE-related diseases or disorders of the present invention may contain other reagents.
- a carrier such as a bead, a blocking solution, a buffer solution such as PBS, and a color development It may contain a substrate or the like.
- the healthy value used in the disease diagnosis method of the present invention is set, for example, by collecting concentration data of AGEs in body fluids in healthy persons who have been definitely diagnosed as not developing diabetes and based on the concentration value. Can do.
- a healthy value can be set based on the concentration value in the healthy person. It is also possible to set a plurality of healthy values in stages and quantitatively determine the presence or absence of diabetes or the risk of future onset.
- urine or blood is preferably used as the body fluid.
- urine collected from the subject serum or plasma (body fluid component) prepared from blood is preferably used as the measurement sample.
- Serum or plasma can be prepared from blood by a known method such as centrifugation.
- the RAGE ligand recognition region is a RAGE extracellular region (positions 22-332 of SEQ ID NO: 102), RAGE8, mRAGE8, RAGE1, mRAGE1, RAGE2, mRAGE2, RAGE3, mRAGE3, RAGE4, mRAGE4, RAGE7, mRAGE7, RAGE143, mRAGE143, RAGE223, mRAGE223, RAGE226 and mRAGE226, a molecule comprising at least one of these, or a variant thereof or a complex thereof.
- a sample such as serum, plasma, or urine is mixed with sRAGE-immobilized beads, and then the beads are collected and reacted with a plurality of fluorescently labeled antibodies (change the fluorescence wavelength of the fluorescent label for each antibody).
- a fluorescence measurement can be performed later, and a plurality of markers can be detected rapidly at the same time.
- a more specific protocol is as follows.
- sRAGE is immobilized on beads such as agarose, silica, and magnetism. Immobilization can be carried out simply and firmly by immobilizing the avidin on the beads via a biotin tag of sRAGE.
- Reaction with sRAGE beads Add sRAGE beads to the sample, and mix by inverting at low temperature for 30 minutes to bind the sRAGE ligand in the sample. Collect sRAGE beads by centrifugation (use magnet for magnetic beads) and wash with TBS.
- the antibody against the molecule to be detected labeled with a fluorescent dye to the reaction product in 3.2.
- Two or more types of molecules can be detected simultaneously by a combination of fluorescent dyes having different absorption wavelengths (for example, Alexa 488, 555, 633, or a combination of Cy3 and Cy5). After reacting for 1 hour, the beads are collected and washed.
- the fluorescence of the collected beads is measured with a fluorescence detector. By selecting the excitation wavelength and absorption length, multiple molecules can be detected simultaneously.
- a membrane for developing a sample by capillary action is provided, and the membrane recognizes a ligand of RAGE labeled with a sample and a metal colloid, silica particles or latex particles.
- An immunochromatography system including, but not limited to, a conjugate part containing a region or a fragment thereof; a detection part containing a ligand recognition region of RAGE or a fragment thereof can be envisaged.
- Lab-on-a Chip can be detected at a stage before it is expressed at the protein level.
- a PANASONIC sensing chip or the like can be used (for example, PANASONIC Technical Journal 57, 21-26 (2011) can be cited as a reference).
- the expression at the gene level in blood cells can be carried out with reference to the literature on type 2 diabetes diagnosis (for example, Patent Publication 2010-261920) using the gene expression level in leukocytes by DNA microarray as an index.
- type 2 diabetes diagnosis for example, Patent Publication 2010-261920
- the target gene can be detected from blood in one step by utilizing a sensing chip.
- Gene detection can also be performed. Basically, it can be carried out by performing a specialized design so that the SNP sensor part detects the corresponding gene in the PANASONIC sensor chip procedure.
- the sample is tear fluid.
- tears are mailed and a simple determination is made and then a fundus examination is performed.
- the presence or absence of AGE in a sample or blood, urine, etc.
- diagnosis of diseases or injuries such as diabetic complications such as diabetes or diabetic nephropathy, diabetic retinopathy, diabetic neuropathy, and the like is performed.
- the concentration of at least one of the markers in a subject's sample is compared with a healthy value to determine whether or not a disease or disorder has developed or in the future.
- the risk of onset can be determined.
- Predictive diagnosis '' and ⁇ pre-diagnosis '' of a disease or disorder and ⁇ determining the risk of future onset '' of a disease or disorder are used interchangeably and in the future when no disease or disorder has developed Determining whether or not there is a possibility (risk) of the disease or disorder or the degree of the possibility (risk).
- the present invention can construct a system in order to implement the method of the present invention.
- system means any system for performing detection, predictive diagnosis, pre-diagnosis, diagnosis, etc., and generally consists of one or a plurality of components, and when there are a plurality of components, These elements are systems that satisfy the three conditions of acting and related to each other and exhibiting harmonious behavior and function as a whole.
- the system can be in any form, such as a device, composition, diagnostic agent. Accordingly, the system can be used, for example, from a large-scale system including a measuring device to a system including chromatography, a kit using an immune reaction, a composition including an antibody (that is, a diagnostic agent that is an in-vitro drug including a monoclonal antibody of a marker) ) And the like.
- the present invention includes sRAGE for detecting AGEs in a sample from a subject, wherein the subject is associated with a disease associated with AGEs (eg, diabetes, diabetic nephropathy, diabetic retinopathy, A system or composition for pre-diagnosis or diagnosis of diabetic complications such as diabetic neurosis) is provided.
- a disease associated with AGEs eg, diabetes, diabetic nephropathy, diabetic retinopathy,
- these compositions or systems can use any factor or means as long as the AGEs can be identified.
- any equivalent factors or means known in the art can be used, not just the factors or means specifically described herein.
- the factor used in the present invention is selected from the group consisting of nucleic acid molecules, polypeptides, lipids, sugar chains, small organic molecules and complex molecules thereof, preferably the factor is a protein or complex.
- a molecule eg, glycoprotein, lipid protein, etc.
- the factor is an antibody (eg, a polyclonal antibody or a monoclonal antibody).
- Such factors are preferably labeled or labelable. This is because it is easy to diagnose.
- the means used are mass spectrometer, nuclear magnetic resonance analyzer, X-ray analyzer, SPR, chromatography (eg, HPLC, thin layer chromatography, gas chromatography), immunology (E.g., Western blotting, ELISA, RIA), biochemical means (e.g., pI electrophoresis, Southern blotting, two-dimensional electrophoresis), electrophoresis instrument, chemical analysis instrument, fluorescence two-dimensional differential electrophoresis ( 2DE-DIGE), isotope labeling method (ICAT), tandem affinity purification method (TAP method), physical means, laser microdissection, and combinations thereof.
- mass spectrometer nuclear magnetic resonance analyzer
- X-ray analyzer eg., X-ray analyzer
- SPR chromatography
- immunology E.g., Western blotting, ELISA, RIA
- biochemical means e.g., p
- the system of the present invention further includes a standard for AGEs.
- a standard for AGEs is preferably used to confirm whether the detection means of AGEs is functioning normally.
- the present invention may further comprise means for purifying the sample of interest.
- purification means include chromatography. Since purification can increase the accuracy of the diagnosis, it can be used in preferred embodiments, but this is not essential.
- the subject includes a mammal, and in one embodiment, the subject includes a rodent.
- rodents for example, rats, mice, etc.
- model animals particularly model animals for diabetes or diabetic nephropathy (for example, streptozocin (Stz) mice, etc.) have been prepared.
- the subject comprises a human.
- the factor or means used in the present invention has the ability to quantify the AGE of the present invention.
- Such quantification may be a means or factor that can draw a calibration curve properly when a standard curve is drawn.
- Preferable examples include antibodies, mass spectrometry, and chromatographic analysis. Therefore, in one embodiment, the system of the present invention further comprises a quantification means for quantifying AGEs.
- the quantification unit includes a determination unit that compares the standard curve and the measurement result to determine whether the AGEs are within a normal value range.
- determination means can be realized using a computer.
- a method for measuring the concentration of AGEs a method generally used for protein quantification can be used as it is as long as the method can specifically measure the concentration of AGEs.
- various immunoassays, mass spectrometry (MS), chromatography, electrophoresis and the like can be used.
- One preferred embodiment in the detection or diagnosis of the present invention is to capture AGEs on a carrier and measure the concentration of the captured AGEs. That is, a substance having affinity for AGEs is immobilized on a carrier, and AGEs are captured on the carrier through the substance having affinity. According to the present embodiment, the influence of contaminants contained in the sample can be reduced, and the concentration of AGEs can be measured with higher sensitivity and higher accuracy.
- the detection method include known techniques such as EIA (enzyme immunoassay), RIA (radioimmunoassay), ELISA (enzyme linked immunosorbent assay) and the like.
- an immunoassay when used as a method for measuring AGEs, it is preferable to use a carrier on which an antibody is immobilized.
- an immunoassay system using the antibody immobilized on the carrier as the primary antibody can be easily constructed.
- two types of antibodies specific to AGEs or sRAGE and having different epitopes are prepared, one is immobilized on a carrier as a primary antibody, and the other is enzyme-labeled as a secondary antibody to construct a sandwich EIA system. it can.
- immunoassay systems based on binding inhibition methods and competitive methods can be constructed.
- a substrate when used as a carrier, immunoassay using an antibody chip is possible.
- the present invention can provide a reagent in which sRAGE is immobilized on a substrate.
- Compositions for detecting AGEs can also be provided using such reagents.
- AGE can be captured on a carrier by ionic bonds or hydrophobic interactions in addition to antibodies. Ion binding and hydrophobic interaction are not as specific as bioaffinity such as antigen and antibody, and substances other than AGEs are also captured, but according to mass spectrometry, it is quantified by a mass spectrometer spectrum that reflects molecular weight. No problem.
- a protein chip using a substrate as a carrier surface-enhanced laser desorption / ionization-time-of-flight mass spectrometry (hereinafter referred to as "SELDI-TOF").
- the concentration of AGEs can be measured more accurately.
- substrates that can be used, cation exchange substrates, anion exchange substrates, normal phase substrates, reverse phase substrates, metal ion substrates, antibody substrates, etc. can be used, but cation exchange substrates, particularly weak cation exchanges.
- a substrate and a metal ion substrate are preferably used.
- the present invention provides a composition for detecting denatured LDL, comprising an anti-LDL chicken antibody.
- a composition for detecting denatured LDL comprising an anti-LDL chicken antibody.
- the ability to discriminate between oxidized LDL (modified LDL) and native LDL is significantly improved as compared with antibodies produced in other animal species (for example, mice and the like).
- the anti-LDL chicken antibody of the present invention is a polyclonal antibody. It was shown that by using a polyclonal antibody as an anti-LDL chicken antibody, all of fully oxidized LDL (fuOxLDL), MDA-LDL, AcLDL, and native LDL can be recognized to the same extent.
- composition of the present invention can further comprise CTLD14.
- the present invention provides a composition for detecting denatured LDL, comprising an anti-LDL chicken antibody and CTLD14. Since all such denatured LDL can be detected comprehensively and stably, true risk factors such as the onset of arteriosclerosis can be detected.
- the CTLD14 used in the composition of the present invention may be a CTLD14 containing a silkworm type sugar chain.
- CTLD14 containing such a silkworm type sugar chain as long as it contains a silkworm type sugar chain, what kind of thing may be used and a heterogeneous thing may be used,
- the silkworm type sugar of this invention CTLD14 having a chain and containing the amino acid sequence shown in SEQ ID NO: 86 or a variant thereof can also be used.
- the composition of the present invention is for detecting LDL and oxidized LDL.
- the structure that can be detected is limited, or even when it is widely recognized from LDL to modified LDL, it is nonspecific and has low reactivity. There was a problem and it was not practical. There was nothing that could be used in common from experimental animals to humans. Therefore, the antibody of the present invention can broadly recognize mammalian LDL and oxidized LDL from mice to humans, and therefore can broadly recognize from LDL to modified LDL, and compared with non-denatured LDL. Because of its high specificity and high reactivity, it can be used for analysis and evaluation. This has not been known so far and is provided for the first time in the present invention.
- the composition for detecting denatured LDL of the present invention is for distinguishing non-denatured LDL from denatured LDL and detecting denatured LDL.
- the unmodified LDL and the modified LDL are LDL and oxidized LDL, respectively.
- the LDL and oxidized LDL that can be distinguished by using the present invention are multiple mammalian LDL and oxidized LDL.
- the antibody of the present invention has a broad range of mammalian LDL and oxidized LDL from mice to humans.
- LDL and oxidized LDL of multiple types of mammals can be distinguished. Accordingly, the plural types of mammals include mice and humans.
- the present invention provides a kit for detecting denatured LDL, comprising an anti-LDL chicken antibody and CTLD14.
- the anti-LDL chicken antibody is a polyclonal antibody.
- the CTLD 14 is the CTLD 14 provided in the present invention, and may contain a silkworm type sugar chain.
- the anti-LDL chicken antibody contained in the kit of the present invention is a polyclonal antibody
- CTLD14 is CTLD14 provided in the present invention, and contains a silkworm type sugar chain.
- the present invention is for detecting LDL and oxidized LDL.
- the LDL and oxidized LDL are multiple mammalian LDL and oxidized LDL, preferably the multiple mammals include mice and humans.
- the multiple mammals include mice and humans.
- CTLD14 having a silkworm-type sugar chain of the present invention and an anti-LDL chicken polyclonal antibody has an effect of remarkably high sensitivity, stability, and wide detection of denatured LDL. Is done.
- a silkworm or a living organism imparting a sugar chain similar to that of a silkworm incorporating a nucleic acid molecule encoding a target protein and a biotin ligase so as to be co-expressed is provided.
- the target protein is biotinylated by co-expressing biotin ligase.
- the biotin ligase is BirA (SEQ ID NO: 106).
- tag sequences that undergo biotinylation include BioEase. tag, Avi. tag, any sequence that can undergo biotinylation, includes but is not limited to. These silkworms can efficiently produce biotinylated CTLD14 and biotinylated sRAGE by oral administration of biotin.
- the obtained biotinylated CTLD14 is excellent in pH stability, modified with metal colloids, and subjected to lateral flow assay incorporating anti-LDL chicken antibody, single chain antibody, streptavidin, and oxidatively modified LDL with various structures. Detection is possible within 20 minutes.
- oligomannose type sugar chains mainly composed of (Man) 5 (GlcNAc) 2 account for 90% or more, and several% complex type and hybrid type sugar chains It was made clear that Furthermore, stability is improved by addition of a sugar chain (stable for almost one year), and AGEs with various structures can be detected by immobilization while maintaining directionality via biotin.
- biotinylated protein by silkworm co-expressing biotin ligase
- A) a step of incorporating a biotin ligase and a nucleic acid molecule encoding a protein into a silkworm or an organism to which a sugar chain similar to that of silkworm is imparted so as to allow co-expression.
- B) placing the silkworm or an organism that imparts a sugar chain similar to that of silkworm under conditions under which the nucleic acid molecule is expressed, and expressing the biotin ligase and the protein; and C) administering biotin to the organism.
- a target protein for example, CTLD14, sRAGE, etc.
- a target protein for example, CTLD14, sRAGE, etc.
- the obtained biotinylated CTLD14 is excellent in pH stability, modified with metal colloids, and subjected to lateral flow assay incorporating anti-LDL chicken antibody, single chain antibody, streptavidin, and oxidatively modified LDL with various structures. Detection is possible within 20 minutes.
- oligomannose type sugar chains mainly composed of (Man) 5 (GlcNAc) 2 account for 90% or more, and several% complex type and hybrid type sugar chains It was made clear that Furthermore, stability is improved by addition of a sugar chain (stable for almost one year), and AGEs with various structures can be detected by immobilization while maintaining directionality via biotin.
- biotin to silkworms is preferably oral administration of a diet supplemented with biotin, but can also be administered by a method such as injection.
- the step A) is achieved by microinjecting an expression vector containing a biotin ligase and a nucleic acid molecule encoding a protein.
- the expression vector can be introduced by any other method described in the present specification or other known methods other than microinjection.
- the present invention provides highly efficient biotinylated CTLD14. Due to the highly efficient biotinylation, it is possible to detect LDL of various structures subjected to oxidative modification within 20 minutes in a short time by a lateral flow assay incorporating streptavidin.
- the biotinylation efficiency of CTLD14 of the present invention is about 10% or more, about 20% or more, about 30% or more, about 40% or more, or about 50% or more. In a preferred embodiment, the biotinylation efficiency of CTLD14 of the present invention is from about 30% to about 40%.
- the obtained biotinylated CTLD14 is excellent in pH stability, modified with metal colloids, and subjected to lateral flow assay incorporating anti-LDL chicken antibody, single chain antibody, streptavidin, and oxidatively modified LDL with various structures. Detection is possible within 20 minutes.
- the CTLD14 of the present invention has undergone silkworm glycosylation.
- the sugar chain structure of CTLD14 of the present invention includes the sugar chain structures of (1) to (6) ([Chemical Formula 1] to [Chemical Formula 6]), as well as the above (1) to (6) ([Chemical Formula 1] to A sugar chain in which a further sugar is added to the sugar chain of [Chemical Formula 6]) is also included.
- the CTLD14 of the invention has the amino acid sequence of SEQ ID NO: 86 with two sites that can undergo glycosylation. The sugar chains added to these sugar chain addition sites may be the same or different. When the sugar chains are different, the added sugar chain can be any combination of the sugar chains of the above (1) to (6) ([Chemical Formula 1] to [Chemical Formula 6]).
- the present invention provides highly efficient biotinylated sRAGE. Due to high-efficiency biotinylation, it can be immobilized on a substrate or the like that maintains its orientation via biotin, and AGEs with various structures can be detected.
- the biotinylation efficiency of the sRAGE of the present invention is about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, or about 60% or more. In a preferred embodiment, the biotinylation efficiency of the sRAGE of the present invention is about 60% or greater.
- oligomannose type sugar chains mainly composed of (Man) 5 (GlcNAc) 2 occupy 90% or more, including several% complex type and hybrid type sugar chains It revealed that. Furthermore, stability is improved by addition of a sugar chain (stable for almost one year), and AGEs with various structures can be detected by immobilization while maintaining directionality via biotin.
- the sRAGE of the present invention is subjected to silkworm type glycosylation.
- the sugar chain structure of CTLD14 of the present invention includes the sugar chain structures of (1) to (8) ([Chemical Formula 1] to [Chemical Formula 8]) and the above (1) to (8) ([Chemical Formula 1] to A sugar chain in which a further sugar is added to the sugar chain of [Chemical Formula 8]) is also included.
- the sRAGE of the invention has the amino acid sequence of SEQ ID NO: 100 with two sites that can undergo glycosylation. The sugar chains added to these sugar chain addition sites may be the same or different. When the sugar chains are different, the added sugar chain may be any combination of the sugar chains of the above (1) to (8) ([Chemical Formula 1] to [Chemical Formula 8]).
- the present invention provides a method for producing a single-chain antibody or a fragment thereof, wherein (A) a silkworm or an organism that imparts a sugar chain similar to that of a silkworm is encoded with the single-chain antibody or a fragment thereof. A step of incorporating the nucleic acid molecule to be expressed; (B) placing the silkworm or a living organism that imparts a sugar chain similar to that of the silkworm under conditions under which the nucleic acid molecule is expressed, and expressing the single-chain antibody or a fragment thereof. And (C) obtaining the single chain antibody or fragment thereof.
- the expressed protein formed inclusion bodies and required a refolding process, but in the production by the silkworm of the present invention, it is expressed as a soluble protein. Does not require a refolding process.
- the protein produced according to the present invention can be subjected to silkworm sugar chain addition.
- sugar chain to be added include the above trimannosyl core, complex type, oligomannose type and hybrid type, and preferably the structures of (1) to (8) ([Chemical Formula 1] to [Chemical Formula 8]). It is a sugar chain having any of the following. *
- the single chain antibody or a fragment thereof includes the amino acid sequence set forth in any of SEQ ID NOs: 76 to 80 or a variant thereof.
- Example 1 Preparation of single-chain antibody gene library
- the method of Marks et al. And Hawkins et al. was modified to generate single chain antibody gene libraries (Marks et al., J. Mol. Biol. 222: 581-597 (1991); Hawkins et al., Blood 83: 3279-3288 ( 1994)).
- the sequencing reaction was carried out using a Bigdye® terminator v1.1 cycle sequencing kit (Applied Biosystems) according to the protocol.
- the primer of Table 1 was used according to the kind of plasmid DNA.
- forward direction of a primer is referred to as forward (forward, FORWARD, or FOR), and the reverse direction is referred to as reverse (reverse, REVERSE, or REV) or back (BACK).
- reverse (REVERSE, REVERSE, or REV) and BACK (BACK) have the same meaning with respect to a primer in this specification.
- First-strand cDNA was synthesized from human splen poly A + RNA (BD Biosciences) using First-strand cDNA synthesis Kit (GE healthcare).
- the primers shown in Table 2 were used according to the type of antibody gene ( ⁇ chain, ⁇ chain, ⁇ chain, ⁇ chain).
- the kappa chain was amplified by the PCR method using Human V ⁇ reverse primer (Table 4) and Human J ⁇ forward primer (Table 4) as mixed primers.
- the ⁇ chain was amplified by a PCR method using Human V ⁇ reverse primer (Table 5) and Human J ⁇ forward primer (Table 5) as mixed primers.
- PCR was 94 ° C. for 3 minutes, then 94 ° C. for 1 minute, 60 ° C. for 1 minute, 72 ° C. for 1 minute for 25 cycles, then 72 ° C. for 10 minutes.
- the reaction was carried out at 50 ° C. for 2 hours with the reaction solution for SfiI and cleaved with SfiI. After returning to room temperature, the addition solution for NotI was added, reacted at 37 ° C. for 1 hour, and cleaved with NotI.
- the collected product was electrophoresed on an agarose gel, purified and desalted with Wizard (registered trademark) SV Gel and PCR Clean-Up System (Promega).
- the purified product was ligated to phagemid DNA pCANTAB5E.
- the obtained phagemid DNA was subjected to ethanol precipitation as a single chain antibody gene library, dissolved in an appropriate amount of sterilized water, and used for electroporation.
- Example 2 Screening of single-chain antibody binding to LDL
- the prepared LDL was completely oxidized according to a standard method (Steinbrecher, 1984) to prepare oxidized LDL as an antigen. Specifically, CuSO 4 was added to 1 mg / ml LDL (dialyzed with PBS) to a final concentration of 5 ⁇ M, reacted at 37 ° C. for 20 hours under aseptic conditions, and then EDTA was added to a final concentration of 1 mM. added. Next, after dialyzing with PBS and sterilizing by filtration, NaN 3 was added to 0.02% and stored at 4 ° C.
- Clone 5,12,19,40 was A 450 values more clones 19 and 79 is a negative control, and 96 as oxidized LDL recognition single-stranded, and a further evaluation (Fig. 3B).
- Fig. 3B a further evaluation
- PBS PBS
- a 450 1 was used as a reference value, 52 clones showing values higher than that were selected, and soluble single-chain antibodies were prepared to evaluate the binding ability. The indicated clone was not obtained (FIG. 4B).
- the clone names of the five single-chain antibodies obtained from the library consisting of ⁇ + ⁇ were ⁇ + ⁇ 5, ⁇ + ⁇ 12, ⁇ + ⁇ 19, ⁇ + ⁇ 40, and ⁇ + ⁇ 96, and the amino acid sequences are shown in FIG.
- a single colony was inoculated into 2 ml of LB medium and pre-cultured for 8 hours.
- the culture broth was inoculated with 1% in 25 ml LB medium and cultured with shaking for 14 hours, and then phagemid DNA was prepared using QIAGEN midi (QIAGEN).
- the electrophoresis tank is Mupid (registered trademark) -EXu (ADVANCE)
- the buffer is TBE, which has a stronger buffer action than TAE
- the electrophoresis temperature is 4 ° C
- the voltage is 50 V
- the electrophoresis is performed for 4 hours or more with ethidium bromide. After staining, the fragment containing the target DNA was excised.
- the detection was performed using ECL plus Western Blotting Detection Reagents (GE healthcare).
- Anti-E antibody or anti-His antibody was used as the primary antibody, and HRP-labeled anti-mouse IgG (Fc specific) was used as the secondary antibody.
- Example 4 Purification of single chain antibody using anti-E antibody-immobilized Sepharose
- the anti-E antibody was immobilized on Sepharose with reference to the protocol of NHS-activated sepharose 4 Fast Flow (GE healthcare).
- NHS-activated sepharose 4 Fast Flow GE healthcare
- the anti-E antibody solution was ultrafiltered (15,000 rpm, 24 minutes, 4 ° C.) with microcon® YM-30 (MILLIPORE), and 240 ⁇ l of 0.2M NaHCO 3 , 0.5M NaCl (pH 8.3).
- the buffer was exchanged by adding The purified anti-E antibody was mixed with washed NHS-activated sepharose, attached to a rotator and stirred at room temperature for 3 hours. Next, it was centrifuged (3,000 rpm, 5 minutes, 4 ° C.), and allowed to stand in ice for 5 minutes to remove the supernatant.
- Tris-HCl pH 8.5
- TBS Tris-HCl
- the periplasm fraction was prepared by the following method.
- the cells recovered from the 25 ml culture solution were resuspended in 0.5 ml ice-cold 1 ⁇ TES, 0.75 ml ice-cold 1/5 ⁇ TES was added, and vortexed.
- the supernatant was recovered as a periplasmic fraction by standing for 30 minutes in ice and centrifuging (15,000 rpm, 10 minutes).
- IPTG concentration and culture time were examined in the same manner at 0.02 mM, 9 hours, 0.05 mM, 9 hours, and 0.2 mM, 3 hours, respectively.
- E-tag is added to the C-terminus of the expressed single chain antibody. Since affinity purification using anti-E antibody is specific and has high binding power, it was considered that the target protein could be concentrated from the lysed cells. Therefore, first, the production of the target protein in BLR was confirmed by Western blot using anti-E antibody as the primary antibody. As a result, a signal was detected at the expected molecular weight, indicating that the target protein was present without being degraded (FIG. 7). Next, purification was attempted using anti-E antibody-immobilized sepharose, but it was determined that the band could not be confirmed by Coomassie staining, and an evaluable amount of protein could not be recovered (FIG. 8).
- Example 5 Expression of a single chain antibody using pET-22b (+)
- Construction of pET-22b (+) ⁇ + ⁇ In order to obtain a sufficient amount of single chain antibody protein, the pET system was used in the host-vector system. For ⁇ + ⁇ 5, using the g + k reverse primer (Table 8) and the g + kaFOR primer (Table 8), SalI and NdeI were added to the 5 ′ side and the XhoI restriction enzyme site was added to the 3 ′ side by PCR. The reaction conditions were 94 ° C. for 3 minutes, then 94 ° C. for 30 seconds, 55 ° C., 30 seconds, 72 ° C. and 30 seconds for 25 cycles.
- pET-22b (+) was similarly digested and ligated to construct pET-22b (+)- ⁇ + ⁇ 5 and pET-22b (+)- ⁇ + ⁇ 19.
- Escherichia coli DH5 ⁇ was transformed with the obtained plasmid DNA, applied to 2 ⁇ YT (Amp) agar medium, and cultured at 37 ° C. for 16 hours. Whether or not the target gene was contained was confirmed by colony PCR, and plasmid DNA was prepared from the positive colony. Furthermore, the DNA base sequence of the inserted fragment was confirmed.
- the expression host BL21 (DE3) was transformed with pET-22b (+)- ⁇ + ⁇ 5 and pET-22b (+)- ⁇ + ⁇ 19.
- BL21 (DE3) retaining pET-22b (+)- ⁇ + ⁇ 5 was inoculated into 2 ml LB (Amp) from the agar medium and pre-cultured at 37 ° C. for 12 hours. 1 ml of the culture solution is inoculated into 100 ml LB (Amp), cultured at 37 ° C. so that A 600 becomes about 0.6, IPTG is added so that the final concentration becomes 1 mM, and further at 37 ° C. for 4 hours. Cultured.
- the cells were collected (8,000 rpm, 5 minutes) and washed 3 times with 10 ml PBS. Three times the wet weight of PBS was added and sonicated. Centrifugation (15,000 rpm, 15 minutes) was performed, and the collected supernatant was subjected to SDS-PAGE with the soluble fraction and the precipitate as the insoluble fraction. The same operation was performed for ⁇ + ⁇ 19.
- Centrifugation was performed using a swing rotor (3,000 rpm, 5 minutes, 4 ° C.), and the imidazole elution fraction was collected to obtain a purified single-chain antibody.
- aggregation after elution resulted in a significant yield loss.
- Dialysis speed and efficiency conditions were optimized to prevent reaggregation after elution.
- a 1/25 volume of 50% nickel agarose slurry in the refolding solution was added and gently stirred at 4 ° C. for 30 minutes with a rotator. Thereafter, the mixture was centrifuged at 1,000 ⁇ g for 5 minutes to recover nickel agarose.
- Nickel agarose was washed 2-3 times with an equivalent amount of PBS, 500 ⁇ l of PBS containing 2M imidazole was added and eluted, and further eluted several times with PBS containing 1M imidazole.
- the eluted fraction was dialyzed with PBS 400 times the sample volume using a cellulose ester membrane tube to obtain a purified single chain antibody.
- the purity and concentration of the obtained single chain antibody were evaluated by SDS-PAGE (FIG. 10). Yield was 3-5 mg per liter of culture.
- the single-chain antibody ⁇ + ⁇ 5 recognizes all of LDL, acetylated LDL, partially oxidized LDL, and fully oxidized LDL used as antigens, but it becomes clear that the specificity to each antigen is different.
- ⁇ + ⁇ 5 is considered to have a feature that recognizes oxidized LDL more strongly than LDL at a low concentration.
- the detection limit of each antigen was 5 ng / 100 ⁇ l well for LDL, 10 ng / 100 ⁇ l well for acetylated LDL, 10 ng / 100 ⁇ l well for partially oxidized LDL, and 20 ng / 100 ⁇ l well for fully oxidized LDL. (FIG. 11). The detection limit was calculated as the detection limit at the lowest concentration at which detection was possible among measurement samples with different dilution stages.
- the plate was washed 3 times with PBS (200 ⁇ l / well), added with blocking buffer (0.25% BSA / PBS) 250 ⁇ l / well, and allowed to stand at room temperature for 2 hours.
- the plate was washed 3 times with PBS (200 ⁇ l / well), and 0.05-10 ⁇ g / ml of antigen solution (LDL or oxidized LDL) was added at 100 ⁇ l / well and reacted at room temperature for 2 hours.
- the plate was washed 5 times with PBS (200 ⁇ l / well), 100 ⁇ l / well of a single-chain antibody of 2.5 ⁇ g / ml / PBS was added, and allowed to react at room temperature for 1 hour.
- the plate was washed with PBS (200 ⁇ l / well) in the same manner as in the direct method.
- the TMB reaction was performed, and the absorbance at 450 nm was measured after stopping the reaction (FIG. 13).
- Example 7 Detection system for malondialdehyde-modified LDL
- Example 6 According to Example 6, the same experiment is performed using malondialdehyde-modified LDL as a target instead of oxidized LDL or the like.
- MDA-LDL malondialdehyde modified LDL
- Example 8 Detection system based on the principle of lateral flow assay
- a detection system using the principle of the lateral flow assay is constructed (FIGS. 14 to 16).
- CTLD14 or single chain antibody labeled with metal colloid was used as a detection reagent. Lateral flow membrane in which CTLD14 or single-chain antibody is applied to the detection part, and streptavidin (when CTLD14 is biotinylated) and anti-His antibody (when CTLD14 contains a His tag) are applied to the control part Is used. Samples such as serum, plasma or whole blood are added to the sample pad and the denatured LDL activity in the sample is assessed by test run color development or test line / control line color development ratio.
- Example 9 Oxidatively modified LDL detection and quantification system using chicken anti-LDL antibody and LOX-1 in lateral flow format
- this example develops an oxidatively modified LDL detection and quantification system using anti-LDL chicken antibody and LOX-1 in a lateral flow format.
- the anti-LDL chicken antibody was produced by requesting Japan Lamb Corporation. Details are as follows. The anti-LDL chicken antibody was administered 6 times every 2 weeks of antigen (LDL) 0.3 mg (Adjuvant FCA), and after the intradermal injection, the increase in titer was checked after the 5th and the 6th booster Later, it was collected from a whole blood sample. The antibody activity was measured by immobilizing 1 ⁇ g / ml of antigen (LDL) at 100 ⁇ l / well and following a normal ELISA. Here, HRP-labeled anti-chicken IgY (for example, available from Promega etc.) was used as the labeled secondary antibody.
- CTLD14 or single chain antibody labeled with metal colloid was used as a detection reagent. Lateral flow membranes were used in which CTLD14 or single-chain antibody was applied to the detection part, and anti-LDL chicken antibody was applied to the control part. Samples such as serum, plasma or whole blood were added to the sample pad and denatured LDL activity in the samples was assessed by test run color development, or test line / control line color development ratio.
- Example 10 System for screening for lipid abnormalities, arteriosclerosis, diabetes using anti-LDL chicken antibody and LOX-1 in a lateral flow assay format
- screening for diseases associated with degenerative LDL such as dyslipidemia, arteriosclerosis, diabetes, ischemic heart disease, cerebrovascular disorder using anti-LDL chicken antibody and LOX-1 in a lateral flow assay format
- diseases associated with degenerative LDL such as dyslipidemia, arteriosclerosis, diabetes, ischemic heart disease, cerebrovascular disorder using anti-LDL chicken antibody and LOX-1 in a lateral flow assay format
- the presence of denatured LDL can be detected visually within 30 minutes, and based on this, screening for diseases related to degenerative LDL such as lipid abnormalities, arteriosclerosis, diabetes, ischemic heart disease, cerebrovascular disorder, etc. It shows that it can be done in a short time.
- Example 11 Evaluation system of preventive effect by food intake using anti-LDL chicken antibody and LOX-1 in a lateral flow assay format
- anti-LDL chicken antibody and LOX-1 are used in a lateral flow assay format to prevent diseases associated with degenerative LDL such as lipid abnormalities caused by food intake, arteriosclerosis, diabetes, ischemic heart disease, and cerebrovascular disorder.
- diseases associated with degenerative LDL such as lipid abnormalities caused by food intake, arteriosclerosis, diabetes, ischemic heart disease, and cerebrovascular disorder.
- the presence of denatured LDL can be detected visually within 30 minutes, and based on this, diseases related to denatured LDL such as lipid abnormalities caused by food intake, arteriosclerosis, diabetes, ischemic heart disease, cerebrovascular disorder, etc. It is shown that the preventive effect of can be evaluated in a short time.
- CTLD14 was synthesized under the condition that the inclusion bodies were denatured under reducing conditions using a conventional E. coli expression system. The protocol is shown below.
- Precision method A refolding method using a dilution dialysis method was performed.
- the dialysis buffer 1-5 shows a 5 from buffer 1 below, the premix of the components of the buffer 1-5 (Of these, all reagents other than glutathione are mixed.) -All reagents were mixed except glutathione (reduction, oxidation). • pH adjusted to appropriate for each buffer • Stored at 4 ° C.
- sample preparation The protein concentration was adjusted to 1 mg / ml (dilution buffer: buffer C: buffer C: 6M guanidine HCl, 50 mM Tris, pH 8.0) A tube (dialysis tube, Spectra / Por 7, RC, MWCO 8,000) was used. Each tidal buffer (1-5) (see 1 to 5 below) was prepared before use. • Thawed the required volume of glutathione (reduction, oxidation) solution. • Mixed dialysis buffer premix and glutathione solution. • Matched to the appropriate volume and mixed well.
- buffer 2 50 mM Tris-Cl, pH 8.5, 3 M guanidine HCl, 0.4 M L-arginine, 0.4 M NaCl, 10% Glycerol, 0.5 mM oxidized glutathione, 5 mM reduced glutathione
- buffer 3 50 mM Tris-Cl, pH 8.0, 2 M guanidine HCl, 0.4 M L-arginine, 0.4 M NaCl, 10% Glycerol, 0.5 mM oxidized glutathione, 5 mM reduced glutathione
- Example 12 Production by transgenic silkworm
- CTLD14_vector was microinjected into silkworm eggs to obtain CTLD14-expressing silkworms. In addition, they were crossed with a strain that can be expressed in the middle silk gland, and a transgenic silkworm expressing CTLD14 in the middle silk gland (which is easy to extract and high in purity) was produced. A schematic diagram thereof is shown in FIG. The detailed protocol is shown below.
- CTLD14-F 5′-AATCTCCAAGAAACACTGAAG-3 ′ (SEQ ID NO: 87), CTLD14s-R: 5′-TCACTGTGCCTCTTAGGTTTGC-3 ′ (SEQ ID NO: 88)
- a plasmid containing the LOX-1 gene hLOX
- hLOX LOX-1 gene
- CTLD14 sequence possessed by pCR8 was introduced into pCDNA TM 6 BioEase TM -DEST by LR reaction and transformed into E. coli DH5 ⁇ .
- the introduced CTLD14 sequence was confirmed by DNA and sequencing, and a plasmid having CTLD14 to which a biotinylated tag (BioEase TM ) was added was constructed.
- UAS vector was constructed by inserting a fragment encoding a signal peptide of fibroin H chain amplified by PCR and a fragment encoding CTLD14 into the downstream. Using this expression vector, transgenic silkworms were produced.
- the w1-pnd strain of the white eye / white egg / non-dormant strain maintained at the National Institute of Agrobiological Sciences was used as the host strain.
- Strains expressing GAL4 in the middle silk gland of the resulting transgenic silkworm (Ken-ichiro Tatematsu, Isao Kobayashi, Keiro Uchino, Hideki Sezutsu Tetsuya Iizuka, Naoyuki Yonemura, Toshiki Tamura, Transgenic Research, 2010, 473 Of UAS (Upstream Activation Sequence).
- individuals having both the GAL4 construct and the UAS construct were selected using a selection marker.
- the 5th instar larvae were dissected and the middle silk gland was removed. Proteins were extracted by shaking with 1 mL of PBS + 1% Triton X-100 extract per bottle at 4 ° C. for 2 hours.
- the purified CTLD14 is treated with SDS-PAGE sample buffer (+) containing ⁇ -mercaptoethanol (2ME) or SDS-PAGE sample buffer (-) not containing 2ME, and then subjected to SDS-PAGE and Coomassie staining. Dimer formation was confirmed.
- FIGS. The analysis result of what was expressed is shown in FIGS. As shown in FIG. 21, a plurality of lines expressing CTLD14 (biotin tag, myc tag; His tag as a common tag for purification) having different tags was established. The expression of each line was confirmed. As shown in FIG. 21, detection by His tag was performed in left: Coomassie staining, right: Western blot. As shown by “ ⁇ 1” in the figure, clear expression of Biotin_CTLD14 (with BioEase tag) is observed. Further, the molecular weight was also consistent with the estimated molecular weight (29 kDa only for the protein portion, and 29 kDa or more with the addition of sugar chains).
- CTLD14 biotin tag, myc tag; His tag as a common tag for purification
- Biotin-CTLD14 was biotinylated in the silkworm body.
- FIG. 19 shows the amino acid sequence of the improved CTLD14 (skin-type sugar chain-added biotinylated CTLD14) produced in this example and information on sugar chain modification.
- the structure of the N-linked sugar chain of the silkworm middle silk gland expression protein is as follows, and CTLD14 is specifically expressed in the silkworm middle silk gland.
- the glycoprotein sugar chain expressed in the middle silk gland has a complex type sugar chain that has almost no boutmannose type (with fucose) sugar chain or high mannose type sugar chain other than the middle silk gland and has a terminal GlcNAc, Or it is an oligomannose type sugar chain.
- glycopeptide it was determined that the glycosylation site was aspartic acid in two places, NCS and NST, and the total mass number of the two sugar chains was about 2500. Therefore, it is estimated that the same thing is included.
- the structure of the N-linked sugar chain of the silkworm middle silk gland expressed protein is shown in FIG.
- Samples prepared using silkworm-type CTLD14 contained in electrophoresis gel pieces are digested with trypsin after reductive alkylation, and the peptides obtained are subjected to matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectrometer and liquid Chromatograph (LC) —electrospray ionization kingdon trap (ESI) mass spectrometer.
- MALDI-TOF matrix-assisted laser desorption / ionization time-of-flight
- LC liquid Chromatograph
- ⁇ -cyano-4-hydroxycinnamic acid was used as a matrix in 4800 plus TOF / TOF Analyzer manufactured by AB SCIEX, and the sample was irradiated with a nitrogen gas laser at 337 nm for ionization.
- MS / MS spectra were collected.
- LC-ESIMS uses Orbitrap Veros Pro (Thermo Fischer Scientific), and EASY Spray column (PepMap C75, 75 ⁇ m), and Nano LC (EASY nLC), ThermoFisher Scientific
- the concentration of acetonitrile containing 1% formic acid was increased from 0% to 100% in 30 minutes, and a gradient program was maintained for 10 minutes.
- the peptides were ionized and their respective MS / MS spectra were acquired. In both methods, ions were detected in the positive ion mode.
- Mass spectra and MS / MS spectra were compared between those treated with PNGase (PNGase-plus) and those not treated (PNGase-plus).
- CTLD Ions corresponding to the two amino acid sequences 109-139 and 154-180 including the region where N-linked sugar chains are supposed to be added were not observed in either PNGase-plus or PNGase-minus samples. However, ionization was observed only at PNGase-plus at m / z which is 1 larger than the mass of ions corresponding to amino acid sequences 109-139 and 154-180. This is because the N-linked sugar chain is cleaved by the enzyme and asparagine is changed to aspartic acid (http://www.ncbi.nlm.nih.gov/books/NBK56012/), and the sugar chain is bound to asparagine. It can be said that it is doing.
- FIGS. 29 to 32 are summaries of the results of mass spectrometry of the silkworm CTLD14.
- FIG. 29 shows the results of LC-ESIMS of the tryptic digest of silkworm-type CTLD14 after treatment with silkworm-type CTLD14 without treatment with PNGaseF.
- peptide fragments (1, 2, 3) containing NX-S / T that are expected to be glycosylated and peptide fragments (4, 5, 6) with close chain length, depending on the presence or absence of PNGaseF treatment When the spectra were compared, peptides 1 and 2 containing NCS and NST were not observed without PNaseF treatment, and a signal was observed after PNGaseF treatment, indicating that sugar chains were added. Indicated. On the other hand, peptide 3 showed a signal at the same position regardless of the presence or absence of PNGaseF treatment, indicating that no sugar chain was added.
- FIG. 30 shows the results of LC-ESIMS of a tryptic digest of a sample without CTNG14 treated with PNGaseF.
- FIG. 31 is an enlarged view of the mass spectrum near the elution position 13.1 minutes in FIG. 30 in order to observe the sugar chain structure expected to be added in more detail.
- FIG. 32 is a table summarizing the difference in mass between ions and peptides observed in the mass spectrum near 13.1 minutes shown in FIG. 31 and examined in detail the sugar chain structure expected to be added. It is a result.
- 29-32 show that (trimannosyl core 892) ⁇ 2 + (GlcNAc203) ⁇ 2 + (Man162) ⁇ 2 or (trimannosyl core 892) ⁇ 2 + (GlcNAc 203 ) X1 + (Man162) x3 combinations are estimated.
- CTLD14 in which the protein band at the dimer position was shifted to the monomer position by 2-mercaptoethanol ( ⁇ -ME) treatment, also formed a dimer in the silkworm body (FIG. 25).
- Example 13 Examination of antibody for evaluation using silkworm-type CTLD14
- an evaluation system using silkworm CTLD14 was developed.
- an antibody combined with CTLD14 is required.
- LDL LDL
- anti-ApoB a large protein that constitutes LDL
- mouse monoclonal antibodies rabbit polyclonal antibodies, and the like.
- the disadvantage of antibodies obtained by immunizing mammals is that LDL (ApoB) has a high homology among mammals, so that a good antibody cannot be obtained. Therefore, there was no antibody that could be used in common from experimental animals to humans.
- Anti-ApoB chicken monoclonal antibody A commercially available antibody (Pharma Foods, HUC20) was purchased and used.
- Anti-ApoB mouse monoclonal antibody A commercially available antibody (Acris BM2149) was purchased and used.
- LDL detection protocol As detection targets, LDL, acetylated (Ac) LDL, malondialdehyde (MDA) LDL, partially oxidized (moOx) LDL, and fully oxidized (fuOx) LDL were used.
- LDL acetylated LDL
- MDA malondialdehyde
- moOx partially oxidized LDL
- fuOx fully oxidized LDL
- MDA-LDL was prepared by reacting commercially available LDL with malonated aldehyde.
- Oxidized LDL was prepared by reacting commercially available LDL with copper oxide. What was made to react at 37 degreeC for 4 hours was made into partial oxidation, and what was made to react at 37 degreeC for 20 hours was made into complete oxidation.
- a commercially available anti-ApoB chicken monoclonal antibody (Pharma Foods, HUC20) was used.
- An anti-chicken IgY-HRP conjugate (Promega, G1351) was used as the secondary antibody.
- a commercially available anti-ApoB mouse monoclonal antibody (Acris, BM2149) was used.
- HRP-labeled anti-mouse IgG (Millipore, AP192P) was used.
- LDLs to be detected LDLs to be detected (LDL, acetylated LDL, partially oxidized LDL, fully oxidized LDL) were added to polystyrene 96-well microwell plates at a concentration of 0.2, 1, 5 ⁇ g / ml, and 100 ⁇ l / well was added at 4 ° C. Adsorbed overnight. The plate was washed 3 times with PBS (200 ⁇ l / well), added with blocking buffer (0.25% BSA / PBS) 250 ⁇ l / well, and allowed to stand at room temperature for 2 hours.
- PBS 200 ⁇ l / well
- blocking buffer 0.25% BSA / PBS
- anti-ApoB chicken polyclonal antibody diluted 3,000 times with blocking buffer, or anti-ApoB chicken monoclonal antibody diluted 8,000 times with blocking buffer was added 100 ⁇ l / well And allowed to react for 1 hour at room temperature.
- the plate was washed 5 times with PBS (200 ⁇ l / well), 100 ⁇ l / well of HRP-labeled anti-chicken IgY (Promega, G1351) diluted 2,000 times with blocking buffer was added, and allowed to react at room temperature for 1 hour.
- TMB 3,3 ′, 5,5′-tetramethyl-benzidine
- FIG. 26 The results are shown in FIG. On the left, LDL detection with anti-LDL chicken polyclonal antibody is shown. It can be seen that fully oxidized LDL (fuOxLDL), MDA-LDL, AcLDL, and native LDL can all be recognized.
- FIG. 26 the detection of LDL by an anti-ApoB chicken monoclonal antibody is shown. Although the ability to recognize fully oxidized LDL (FoxLDL) is high, it can be seen that the recognition intensity is not constant depending on the type of modification.
- the right side of FIG. 26 shows detection of LDL by anti-ApoB mouse monoclonal antibody. It can be seen that the recognition ability is generally low, and there is no great difference in recognition ability between oxidized LDL and native LDL.
- LDL is highly homologous between mammals, and it is difficult to obtain excellent antibodies when immunizing mice, but chicken polyclonal antibodies obtained by immunizing chickens that are birds can be widely used for detection of mammalian samples It seems that the combination with CTLD14 can be widely used from detection in animal experiments (mouse, rat, rabbit, etc.) to detection in humans.
- Example 14 Assay development using CTLD14 containing silkworm-type sugar chain
- Example 13 Assay development using CTLD14 containing silkworm-type sugar chain
- an oxidized LDL detection technique using silkworm CTLD14 and anti-LDL chicken antibody was performed.
- the combination of E. coli CTLD14 and the anti-LDL chicken antibody of the present invention was used.
- E. coli type CTLD14 was prepared as described in Comparative Example 1.
- Silkworm type CTLD14 was produced as described in Example 12.
- the anti-LDL chicken polyclonal antibody was produced as in Example 9.
- anti-ApoB chicken monoclonal antibody As the anti-ApoB chicken monoclonal antibody, a commercially available product (Pharmaceuticals, HUC20) was used.
- CTLD14 expressed and purified in silkworms and Escherichia coli based on the methods described in Comparative Example 1 and Example 12 as described above was used.
- the plate was washed 3 times with PBS (200 ⁇ l / well), and 0.05-8 ⁇ g / ml LDLs (LDL, acetylated LDL, partially oxidized LDL, fully oxidized LDL) were added at 100 ⁇ l / well for 2 hours at room temperature. Reacted.
- the plate was washed 5 times with PBS (200 ⁇ l / well), and anti-LDL chicken polyclonal antibody diluted 6,000 times with blocking buffer or anti-ApoB chicken monoclonal antibody diluted 8,000 times with blocking buffer was added 100 ⁇ l / well. And allowed to react at room temperature for 1 hour.
- the plate was washed 5 times with PBS (200 ⁇ l / well), 100 ⁇ l / well of HRP-labeled anti-chicken IgY (Promega, G1351) diluted 2,000 times with blocking buffer was added, and allowed to react at room temperature for 1 hour. After the plate was washed 5 times with PBS (200 ⁇ l / well), 50 ⁇ l / well of TMB was added to confirm the color development state, and 50 ⁇ l / well of 1N HCl was added to stop the reaction. Thereafter, the absorbance at 450 nm of each well of the plate was measured.
- FIG. 27 shows the result of combination of E. coli CTLD14 and the anti-LDL chicken antibody of the present application.
- the left side of FIG. 27 shows detection by sandwich method using E. coli CTLD14 and anti-LDL chicken polyclonal antibody. In a low concentration region (0.1 ⁇ g / well, solution concentration of 1 ⁇ g / ml or less), it can be seen that specific detection of oxidatively modified LDL is possible (moLDL: LDL for partially oxidized LDL). No significant difference).
- the right side of FIG. 27 shows detection by sandwich method using E. coli CTLD14 and anti-apoB chicken monoclonal antibody.
- fuOxLDL Detection of fully oxidized LDL is good, but recognition of MDA-LDL, AcLDL, and moOxLDL is weak. It can be seen that detection of MDA-LDL, which is a typical LDL, is also difficult.
- FIG. 28 shows the result of combination of silkworm CTLD14 and the anti-LDL chicken antibody of the present invention.
- FIG. 28 left shows detection by a sandwich method using silkworm-type CTLD14 and anti-LDL chicken polyclonal antibody. All modified LDLs can be detected.
- MoOxLDL recognition of partially oxidized LDL is weak.
- the right side of FIG. 28 shows detection by a sandwich method using transgenic silkworm CTLD14 and anti-apoB chicken monochrome.
- FuOxLDL Detection of fully oxidized LDL is good, but MDA-LDL and AcLDL are difficult to detect.
- glycated LDL is also expected to be detectable.
- Example 15 Further evaluation system development using CTLD14 containing silkworm type sugar chain
- Example 14 an experiment using saccharified LDL as a target is performed.
- glycated LDL can also be detected by the sandwich method using silkworm-type CTLD14 and anti-LDL chicken polyclonal antibody, as in Example 14.
- OrigamiB (Method and material) OrigamiB (DE3) was used as a host bacterial cell.
- NdeI is used on the 5 ′ side
- XhoI BamHI, SmaI, HindIII can also be used
- a biotinylated amino acid sequence recognized by biotin ligase is added to the C-terminal side of the target protein.
- RAGE since the N-terminal side is an extracellular region and an essential region for AGE recognition, a biotinylated amino acid sequence was added to the C-terminal side so that it can be immobilized on the C-terminal side when chipping or the like .
- nucleic acid sequence encoding the biotinylated amino acid sequence “GLNDIFEAQKIEWHE” was linked to the C-terminal side of the amino acid sequence of SEQ ID NO: 97 to prepare a plasmid placed under the control of the T7 promoter. .
- This plasmid and a plasmid that expresses the biotin ligase gene (a plasmid in which the biotin ligase gene is inserted into the pAC vector, which is a p15A-based plasmid that can stably coexist in the same cell because the incompatibility group differs from the ColE1-based pET vector) ) Were used to transform host bacterial cells.
- the medium used for culturing the transformed host cells was a modified LB medium (0.5%) supplemented with 50 ⁇ g / ml ampicillin, 34 ⁇ g / ml chloramphenicol, 15 ⁇ g / ml kanamycin, 12.5 ⁇ g / ml tetracycline. NaCl, 0.5% yeast extract, 1% tryptone).
- a pre-culture solution cultured overnight at 25 ° C. was added to the medium to a final concentration of 5%, and the mixture was stirred and cultured at 25 ° C. It should be noted that the amount of addition to the main culture can be made 5% or more to shorten the culture time until the start of induction.
- the proportion obtained as soluble decreased when the addition amount exceeded 5%.
- the absorbance at A 600 reached 0.5 to 0.7, 1 mM IPTG, 50 ⁇ M d-biotin (type D [(+)-cis-hexahydro-2-oxo-1H-thieno- (3 , 4) -imidazole-4-valeric acid)] (both at the final concentration)
- induction of the target protein and biotinylation are started, and the target protein is dissolved in biotin by stirring for 18 hours.
- the target protein becomes almost insoluble at 100%, but by reducing the culture temperature to 25 ° C., about 80% of the expressed target protein can be expressed as a soluble protein. there were.
- a fraction containing soluble biotinylated RAGE was collected and dialyzed with a lysis buffer, and then bound to a streptavidin mutein matrix (Roche) column using AviTag (C powder) ("GLNDIFEAQKIEWHE" (SEQ ID NO: 98)).
- Purified soluble biotinylated RAGE was obtained by elution with 3 mM d-biotin, and finally 5 mg of purified soluble biotinylated RAGE could be obtained from 1 L culture.
- Example 16 Improvement of stability by addition of sugar chain
- sRAGE vector was microinjected into silkworm eggs to obtain RAGE-expressing silkworms.
- a transgenic silkworm expressing RAGE in the middle silk gland was created by crossing with a strain that can be expressed in the middle silk gland.
- FIG. 33 A schematic diagram is shown in FIG. 33 and the detailed protocol is presented below.
- UAS Upstream Activation Sequence
- Ser-UAS / 3xP3EGFP Upstream Activation Sequence
- a UAS vector was constructed by inserting a fragment encoding a signal peptide of fibroin H chain amplified by PCR and a fragment encoding sRAGE downstream of. Using this expression vector, transgenic silkworms were produced.
- the white-eye, white-egg, and non-dormant w1-pnd strains maintained at the National Institute of Agrobiological Sciences were used as host strains.
- Strains expressing GAL4 in the middle silk gland of the resulting transgenic silkworm (Ken-ichiro Tatematsu, Isao Kobayashi, Keiro Uchino, Hideki Sezutsu, Tetsuya Iizuka, Naoyuki Yonemura, Toshiki Tamura, Transgenic Research, 19, 473 (2010) ) And UAS (Upstream Activation Sequence).
- individuals having both the GAL4 construct and the UAS construct were selected using a selection marker.
- the 5th instar larvae were dissected and the middle silk gland was removed. Proteins were extracted by shaking with 1 mL of PBS + 1% Triton X-100 per bottle at 4 ° C. for 2 hours.
- the analysis result of what was expressed was shown in FIG.
- the middle silk gland was taken out from the 4 transgenic silkworm lines (1-4) and the negative control (only the middle silk gland GALA4, no UAS: sRAGE was not expressed), followed by 1 per middle silk gland.
- 1 ml of PBS containing% Triton X-100 was added and mixed by inverting at 4 ° C. for 2 hours.
- On the left is the result of protein staining with Coomassie Brilliant Blue after electrophoresis on a 4-12% gradient SDS-polyacrylamide gel.
- the amount of sample added to each lane is 5 ⁇ l / lane on the left side and 10 ⁇ l / lane on the right side.
- M molecular weight marker (benchmark: Invitrogen), 1-4 indicates the line number of the transgenic silkworm produced, and Nega: negative control. On the right is the result of Western blotting.
- the sample was developed by SDS-PAGE with a gradient of 4 to 12% and transferred to a PVDF membrane.
- Anti-His rabbit antibody (diluted 1000 times) + anti-His mouse antibody (diluted 3000 times) was used as the primary antibody. )
- HRP-labeled anti-rabbit IgG & anti-mouse IgG antibody (200,000-fold dilution)
- reaction with ECL Prime to detect the luminescence and confirm the expression of sRAGE .
- the molecular weight was also consistent with the estimated molecular weight (44 kDa for the protein portion alone, and a sugar chain was added, resulting in a molecular weight of 44 kDa or more).
- FIG. 36 silkworm-type sRAGE was purified. From 13 ml of lysate, 1.8 mg of purified protein was obtained (about 0.3 mg / head per silkworm). Even if the process of binding and elution to TALON is omitted, a sufficiently usable sRAGE having a purification degree of about 95% can be obtained. In this case, the yield is three times or more.
- FIG. 36 As shown in FIG. 37, biotinylation of silkworm-type sRAGE was confirmed. As a result of confirmation with an anti-RAGE antibody and HRP-labeled streptavidin after Western blotting, it was confirmed that sRAGE derived from both strains was also biotinylated. See also FIG.
- FIG. 1 The arrangement of sRAGE produced in this example is shown in FIG.
- the structure of the N-linked sugar chain of the silkworm middle silk gland expressed protein is as follows, and sRAGE is specifically expressed in the silkworm middle silk gland.
- the glycoprotein sugar chain expressed in the middle silk gland has almost no Schwarzmannose-type (with fucose) or high mannose-type sugar chains found outside the middle silk gland. Oligomannose type sugar chain.
- the glycosylation site was determined to be NIT and NGS aspartic acid. Examples of the structure of the N-linked sugar chain of the silkworm middle silk gland expressed protein are shown in FIGS.
- the sRAGE having silkworm-type sugar chains contained in the electrophoretic gel pieces and those not treated with PNGaseF were subjected to trypsin after reductive alkylation.
- the obtained peptides were measured with a matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectrometer and a liquid chromatograph (LC) -electrospray ionization kingdon trap (ESI) mass spectrometer.
- MALDI-TOF matrix-assisted laser desorption / ionization time-of-flight
- LC liquid chromatograph
- ESI electrospray ionization kingdon trap
- MALDI-TOFMS used the following. Apparatus: AB SCIEX 4800 plus TOF / TOF Analyzer Matrix: ⁇ -Cyano-4-hydroxycinnamic acid Laser: Nitrogen gas laser 337 nm MS / MS: MS / MS spectra were collected in descending order of ion intensity.
- LC-ESIMS used the following.
- MS Thermo Fischer Scientific Orbitrap Veros Pro Equipment
- LC Nano LC (EASY nLC) manufactured by Thermo Fischer Scientific Column: EASY Spray column (PepMapC18, 3 ⁇ m, 75 ⁇ mx15cm, Thermo Fisher Scientific) Eluent: 300 nL / min, A) 0.1% formic acid, B) Acetonitrile with 0.1% formic acid, B concentration increased from
- candidate sugar chains were narrowed down by GALAXY search from the Glucose unit (GU) value and molecular weight determined by the ODS column and the Amide column. Finally, the candidate sugar chain standard and the sample are co-fired, separated by an ODS column, the match between the candidate sugar chain and the sample is confirmed, and the sugar chain as shown in FIG. It has been found that *
- Example 17 Experiment of FIG. 38 Stability of sRAGE containing silkworm-type sugar chain
- Example 17 Experiment of FIG. 38 Stability of sRAGE containing silkworm-type sugar chain
- E. coli type and silkworm type sRAGE 10 ⁇ g (10 ⁇ l) were treated with 5 ⁇ l of SDS-PAGE sample buffer and stored at ⁇ 20 ° C.
- E. coli type sRAGE and silkworm type sRAGE stored for 90 days at 4 ° C. after purification were treated in the same manner, and the sample was electrophoresed with 12% SDS-PAGE, followed by Coomassie staining.
- FIG. The stability of silkworm sRAGE is shown.
- the left is E.
- the right side shows the silkworm type of the present invention. 1 day, 40 days storage and 1 day, 90 days storage are shown, respectively.
- SRAGE containing a silkworm-type sugar chain is a very stable molecule. While the E. coli type fragmented in 40 days, it was maintained at full length for a long time (confirmed up to half a year at the time of filing) at 4 ° C.
- Example 18 Sandwich ELISA-like detection
- the plate was washed 3 times with TBST (200 ⁇ l / well) and 0.01-8 ⁇ g / ml AGEs (R-AGEs, F-AGEs, G-AGEs, Ctl-AGEs) were added at 100 ⁇ l / well at room temperature. The reaction was performed for 2 hours. The plate was washed 5 times with TBST (200 ⁇ l / well), 100 ⁇ l / well of an anti-BSA mouse monoclonal antibody diluted 6,000 times with Protein free blocking buffer was added, and reacted at room temperature for 1 hour.
- the plate was washed 5 times with TBST (200 ⁇ l / well), 100 ⁇ l / well of HRP-labeled anti-mouse IgG antibody diluted 5,000 times with Protein free blocking buffer was added, and reacted at room temperature for 1 hour. After the plate was washed 5 times with TBST (200 ⁇ l / well), 50 ⁇ l / well of TMB was added to confirm the color development state, and 1N HCl 50 ⁇ / well was added to stop the reaction. Thereafter, the absorbance at 450 nm of each well of the plate was measured.
- FIG. 1 shows the E. coli after storage for 1 week after purification. It shows the recognition activity of E. coli sRAGE, and the right shows the recognition activity of silkworm sRAGE after storage for 3 months after purification.
- the right figure shows the AGE detection results by silkworm sRAGE 90 days after purification. The cognitive ability is maintained even after 90 days, and E. coli at 1 week after purification. It was confirmed that the same detection as that of the E. coli type was possible.
- the sRAGE of the present invention can be maintained extremely stably and can be detected with high sensitivity.
- Example 19 Production of silkworm-type CTLD14 and silkworm-type sRAGE by a silkworm co-expressing biotin ligase
- silkworm-type CTLD14 and silkworm-type sRAGE were tried to be produced by silkworms expressing biotin ligase (BirA) specifically for the middle silk gland.
- USA-Biotin tag CTLD14 (or USA-Biotin tag sRAGE) with BioEase tag CTLD14 (or BioEase tag sRAGE) inserted downstream of the target sequence USA
- BioEase tag CTLD14 (or BioEase tag sRAGE) inserted downstream of the target sequence USA
- USA-GAL4 with biotin ligase (BirA) inserted downstream of the target sequence USA
- BirA biotin ligase
- This silkworm expresses Biotin tag CTLD14 (or Biotin tag sRAGE) and BirA specifically in the middle silk gland.
- the 5th instar larvae were fed a diet supplemented with 20 ⁇ g of biotin per gram to supply biotin necessary for biotinylation of CTLD14 or sRAGE.
- the middle silk gland was removed just before making a cocoon, and the protein was extracted with Triton X-100 / PBS ( ⁇ ) (see FIG. 43).
- FIG. 44 shows the results of confirming the expression of CTLD14 and sRAGE by Coomassie staining after SDS-PAGE.
- “-” Is a line that does not express BirA.
- an experimental group of a normal diet and a biotin diet was provided, and in the biotin diet group, a diet to which 20 ⁇ g of biotin was added per gram was given.
- the middle silk gland was removed just before making a cocoon, and an extract treated with Triton X-100 / PBS ( ⁇ ) was prepared and subjected to SDS-PAGE.
- Line 72 is a negative control in which only Ser1-GAL4 is introduced and USB-CTLD14 (or USB-sRAGE) is not introduced.
- sRAGE expression was confirmed at the position of the molecular weight of sRAGE (about 50 kDa) in the line introduced with sRAGE, compared to line 72.
- CTLD14 was not as expressed as sRAGE.
- a large molecular weight signal is also detected at the same time, which appears to be a dimer of CTLD14. Furthermore, a very strong signal was observed with the administration of biotin bait. It was confirmed that biotinylation efficiency was dramatically improved by BirA co-expression and biotin addition.
- the right figure shows the result of Western blot of the obtained purified protein.
- HRP-labeled anti-rabbit IgG antibody HRP-labeled anti-rabbit IgG antibody as the secondary antibody
- CTLD14 it was confirmed that the protein observed at the molecular weight at the dimer position stably detected by SDS-PAGE was a dimer of CTLD14, and it was also shown that silkworm-type CTLD14 exists stably as a dimer. It was done.
- E. E. coli type CTLD14 has properties that make it difficult to modify metal colloids, such as precipitation by desalting treatment and precipitation by pH change.
- silkworm-type CTLD14 remained active without precipitation even after conversion from PBS ( ⁇ ) to phosphate buffer (PB) containing no salt.
- silkworm-type CTLD14 is an E. coli strain.
- the Tris buffer which is likely to cause precipitation, was stable.
- the usable pH range was as wide as 7.4 to 9.2.
- E. E. coli CTLD14 could not obtain stable activity with Tris buffer (7.4 to 9.0). Therefore, the silkworm-type CTLD14 is stable regardless of the presence / absence of salt, the pH range, and the type of buffer (FIG. 48), and it is clear that there is a wide range of selection when used in the detection / evaluation system. became.
- Example 20 Detection of oxidized LDL in human plasma by silkworm CTLD14
- silkworm CTLD14 it was examined whether or not trace amounts of oxidized LDL in human plasma can be detected by silkworm CTLD14.
- Detection was performed by sandwich ELISA using CTLD14 and anti-LDL chicken polyclonal antibody as follows.
- the plate was washed 5 times with PBS (200 ⁇ l / well) and anti-ApoB chicken polyclonal antibody diluted 6,000 times with blocking buffer, or anti-ApoB chicken monoclonal antibody diluted 8,000 times with blocking buffer, 100 Add ⁇ l / well and react at room temperature for 1 hour.
- samples (samples 1 to 5) obtained by adding oxidized LDL at the following concentrations to plasma derived from healthy Asians were used.
- the detection result is shown in FIG. From the significant difference with respect to the sample when diluted 1000-fold and used in the detection system, a low concentration of oxidized LDL with a detection limit of 1 ug / ml to 3 ug / ml in human plasma was determined by sandwich ELISA using silkworm type CTLD14. It became clear that detection was possible.
- the concentration of oxidized LDL is about 0.2% of LDL (1.2 to 2.2 ⁇ g) relative to a large excess of LDL (600 to 1100 ⁇ g / ml), and abnormal lipids, The ratio of oxidized LDL increases due to ischemic heart disease and the like. Since this method was able to detect 1 to 3 ug / ml of oxidized LDL in human samples, it was shown that it was possible to detect and quantify oxidized LDL slightly elevated from healthy subjects.
- Example 21 Oxidized LDL detection system by immunochromatography based on the principle of lateral flow assay using biotinylated CTLD14
- a detection system based on the principle of lateral flow assay using biotinylated CTLD14 was examined (FIG. 50).
- platinum colloids and palladium colloids were also examined.
- the platinum colloid-labeled CTLD14 was the most excellent in terms of the stability and detection sensitivity of the labeled CTLD14.
- the platinum colloid-labeled CTLD14 was stable at 4 ° C. for several months. Since the platinum colloid is dense and black, it was also detected in black by immunochromatography.
- the sample (oxidized LDL / PBS (-)) was added with platinum colloid-labeled biotinylated CTLD14 dispersed by sonication immediately before mixing, and a half-strip was formed to make capillary action. (Photo in the center of FIG. 51). The reaction was completed in 10-20 minutes. The spot on the test line was visible even when the oxidized LDL was 30 ng. This indicates that 30 ng / well oxidized LDL can be detected in a short time within 20 minutes.
- control line indicates that the development was normal and at the same time the spot intensity of the test line increases depending on the sample concentration, whereas the spot intensity of the control line decreases, It is expected that a certain amount of quantitative evaluation will be possible with the colorimetric ratio / color of the control line.
- Example 22 Identification of sugar chain structure
- the sugar chain structure of silkworm sRAGE was identified.
- FIG. 55 shows the composition ratio, quantitative value, and sugar composition of each sugar chain identified.
- N1, N2 and N3 oligomannose types are found to be present in composition ratios of 27.5%, 10.8% and 51.4%, respectively, and it is clear that oligomannose types account for more than 90%. It was.
- Example 23 Interaction analysis with AGEs by biotinylated sRAGE) (Reaction kinetic analysis by surface plasmon resonance using single cycle kinetics)
- Lys which is important for recognition, may be modified, may be inactivated during the immobilization process, and immobilization that maintains orientation
- the recognition ability cannot be maintained due to problems such as impossible, and binding may not be confirmed even if AGEs are added.
- the left diagram of FIG. 56 shows an interaction analysis between silkworm sRAGE and AGEs (F-AGE) when immobilized on a CM5 chip by amine coupling.
- Example 24 Production of single chain antibody by silkworm
- single chain antibody ⁇ + ⁇ 5 was produced in silkworm.
- FIG. 52 Single chain antibody ⁇ + ⁇ 5 was expressed in the middle silk gland of transgenic silkworms.
- a partial silk gland was removed and an extract treated with Triton X-100 / PBS ( ⁇ ) was prepared, 2 ⁇ l / lane And subjected to SDS-PAGE.
- the left figure of FIG. 52 shows the result of Coomassie staining.
- the leftmost lane is a negative control that does not express ⁇ + ⁇ 5, but a strain that expresses an amount of ⁇ + ⁇ 5 that can be clearly confirmed by Coomassie staining could not be confirmed.
- the right figure shows the results of detection with an anti-His antibody, and the expression of the target ⁇ + ⁇ 5 was confirmed in all strains. It was shown that the expression level was higher in the line with the myc tag added to the C-terminal. Soluble ⁇ + ⁇ 5 was obtained by purification utilizing His tag and myc tag. ⁇ + ⁇ 5 expressed in Escherichia coli had the problem that all forms of inclusion bodies required a complicated refolding process, and the purified product was easily re-aggregated. It became possible to produce ⁇ + ⁇ 5 as a protein.
- SEQ ID NO: 1 Sequencing primer M13forward used for plasmid pCR2.1-TOPO
- SEQ ID NO: 2 Sequencing primer M13 reverse used for plasmid pCR2.1-TOPO
- SEQ ID NO: 3 Sequencing primer pCANTAB5-S1 used for plasmid pCANTAB-5E
- SEQ ID NO: 4 Sequencing primer pCANTAB5-S6 used for plasmid pCANTAB-5E
- SEQ ID NO: 6 Sequencing primer T7 terminator used for plasmid pET-22b (+)
- SEQ ID NO: 8 First-strand cDNA synthesis primer HlgM (mu)
- SEQ ID NO: 9 First-strand cDNA synthesis primer Hkappa
- SEQ ID NO: 91 Partial sequence 2 of FIG.
- SEQ ID NO: 92 Partial sequence 3 of FIG.
- SEQ ID NO: 93 Partial sequence 4 of FIG.
- SEQ ID NO: 94 Partial sequence 5 of FIG.
- SEQ ID NO: 95 Partial sequence 6 of FIG.
- SEQ ID NO: 96 Nucleic acid sequence of sRAGE used in the present invention
- SEQ ID NO: 97 Amino nucleic acid sequence of sRAGE used in the present invention
- SEQ ID NO: 98 Biotinylated amino acid sequence “GLNDIFEAAQKIEWHE”
- SEQ ID NO: 99 ⁇ Biotin-RAGE> nucleic acid sequence (including fibroin H chain intron, signal peptide, BioEASE-tag & linker & FLAG and RAGE)
- SEQ ID NO: 100 amino acid sequence of ⁇ Biotin-RAGE>
- SEQ ID NO: 102 RAGE amino nucleic acid sequence
- SEQ ID NO: 103 first partial sequence described in the lower part of FIG.
- SEQ ID NO: 104 FIG. Second partial sequence number 105 in the lower part explanation: Third partial sequence number 106 in the lower part explanation of FIG. 41: Nucleic acid sequence number in biotin ligase (BirA)
- SEQ ID NO: 107 Amino acid sequence number in biotin ligase (BirA) 108: amino acid sequence of ⁇ + ⁇ 5 in which a Myc tag is introduced on the N-terminal side
- SEQ ID NO: 109 amino acid sequence of ⁇ + ⁇ 5 in which a Myc tag is introduced on the C-terminal side
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Abstract
Description
<一本鎖抗体関連(配列、抗体の特性)>
(1)配列番号76~80のいずれかに記載のアミノ酸配列またはその改変体を含む一本鎖抗体またはそのフラグメント。
(2)項目1に記載の一本鎖抗体またはそのフラグメントを含む、LDLおよび変性LDL(LDL修飾物ともいう)を検出するための検出剤であって、該LDLおよび変性LDLは、アセチル化LDLおよび部分酸化LDLからなる群より選択される少なくとも1つの分子を含む、検出剤。
(3)前記LDLおよび変性LDLは、アセチル化LDLおよび部分酸化LDLを含む、項目2に記載の検出剤。
(4)前記LDLおよび変性LDLは、LDL、完全酸化LDL、アルデヒド修飾LDL(例えば、マロンジアルデヒド修飾LDL)、アセチル化LDLおよび部分酸化LDLを含む、項目2または3に記載の検出剤。
(5)前記LDLおよび変性LDLは、100ng/100μlウェル以下の感度で検出可能である、項目2~4のいずれか1項に記載の検出剤。
(6)前記LDLおよび変性LDLは、20ng/100μlウェル以下の感度で検出可能である、項目2~5のいずれか1項に記載の検出剤。
(7)前記LDLおよび変性LDLは、LDL、部分酸化LDLおよび完全酸化LDLであり、10ng/100μlウェル以下の感度で検出可能である、項目2~6のいずれか1項に記載の検出剤。
(8)前記LDLおよび変性LDLは、LDLおよび完全酸化LDLであり、5ng/100μlウェル以下の感度で検出可能である、項目2~7のいずれか1項に記載の検出剤。
<安定供給系>
(A1)項目1に記載の一本鎖抗体またはそのフラグメントを生産する方法であって、該方法は:
A)該一本鎖抗体またはそのフラグメントのアミノ酸配列をコードする核酸を、該核酸が発現される細胞に導入する工程;
B)該細胞を培養し、該細胞から封入体を取り出す工程;
C)該封入体をリフォールディングする工程;
D)リフォールディングで得られた溶液を精製用カラムに接触させ、溶出溶液で溶出する工程;
E)溶出した該一本鎖抗体またはそのフラグメントを透析溶液で透析する工程
を包含する、方法。
(A2)前記リフォールディングは、封入体の量をタンパク質あたり2mg~10mg(例えば、8.6mg)で行うことを特徴とする、項目A1に記載の方法。
(A3)前記アガロースカラムは、Ni-NTAスラリーである、項目A1またはA2に記載の方法。
(A4)前記溶出溶液はイミダゾールを含む溶液である、項目A1~A3のいずれか1項に記載の方法。
(A5)前記溶出溶液はイミダゾールを含むリン酸緩衝化生理食塩水(PBS)である、項目A1~A4のいずれか1項に記載の方法。
(A6)前記透析溶液は、PBS(-)である、項目A1~A5のいずれか1項に記載の方法。
(A7)前記透析は、セルロースエステル膜チューブを用いて行われる、項目A1~A6のいずれか1項に記載の方法。
(A8)前記透析は、セルロースエステル膜チューブを用い、前記透析溶液としてサンプル容量の400倍のPBS(-)を用いて行われる、項目A1~A7のいずれか1項に記載の方法。
(A9)項目1に記載の一本鎖抗体またはそのフラグメントの製造方法であって、
A)カイコまたはカイコと同様の糖鎖を付与する生物に、該一本鎖抗体またはそのフラグメントをコードする核酸分子を発現可能に組み込む工程;
B)該核酸分子が発現する条件下に該カイコまたはカイコと同様の糖鎖を付与する生物を配置し、該一本鎖抗体またはそのフラグメントを発現させる工程;および
C)該一本鎖抗体またはそのフラグメントを得る工程
を包含する、方法。
<LOX-1との複合・間接・サンドイッチ系>
(B1)項目1に記載の一本鎖抗体またはそのフラグメントと、酸化LDL受容体(LOX-1)のリガンド認識領域とを用いる、酸化的修飾を受けたLDLを検出または定量するためのシステム。
(B2)前記システムは、抗原抗体反応を用いる、項目B1に記載のシステム。
(B3)前記システムは、ELISAを行うためのものである、項目B1またはB2に記載のシステム。
(B4)前記システムは、サンドイッチELISAを行うためのものである、項目B1~B3のいずれか1項に記載のシステム。
(B5)前記システムは、ラテラルフローアッセイを行うためのものである、項目B1~B4のいずれか1項に記載のシステム。
(B6)前記システムは、毛管現象による試料の展開を行うメンブレンを備え、前記メンブレンは、サンプルと金属コロイド、シリカ粒子もしくはラテックス粒子で標識された酸化LDL受容体(LOX-1)のリガンド認識領域か、または金属コロイド、シリカ粒子もしくはラテックス粒子で標識された項目1に記載の一本鎖抗体もしくはそのフラグメントを含むコンジュゲート部;酸化LDL受容体(LOX-1)のリガンド認識領域か、または項目1に記載の一本鎖抗体もしくはそのフラグメントを含む検出部;ならびに該酸化LDL受容体(LOX-1)のリガンド認識領域に対する結合分子もしくは一本鎖抗体に対する結合分子を含むコントロール部を含む、項目B5に記載のシステム。
(B7)前記システムは、毛管現象による試料の展開を行うメンブレンを備え、前記メンブレンは、サンプルと金属コロイド、シリカ粒子もしくはラテックス粒子で標識されたCTLD14か、または金属コロイド、シリカ粒子もしくはラテックス粒子で標識された項目1に記載の一本鎖抗体もしくはそのフラグメントを含むコンジュゲート部;CTLD14か、または項目1に記載の一本鎖抗体もしくはそのフラグメントを含む検出部;ならびに該CTLD14に対する結合分子を含むコントロール部を含む、項目B5またはB6に記載のシステム。
(B8)前記CTLD14は、ビオチン化、Hisタグ付加、Mycタグ付加、Flagタグ付加、Eタグ付加、またはStrepタグ付加なされており、それぞれの場合において、前記結合分子は、ストレプトアビジン、抗His抗体、抗Myc抗体、抗Flag抗体、抗Eタグ抗体、またはStrep-Tactinである、項目B7に記載のシステム。
<ニワトリ抗酸化LDL抗体を用いるシステム>
(C1)抗変性LDL抗体、修飾物またはそのフラグメントと、酸化LDL受容体(LOX-1)のリガンド認識領域とをラテラルフローアッセイ形式で用いる、酸化的修飾を受けたLDLを検出または定量するためのシステム。
(C2)抗変性LDL抗体、修飾物またはそのフラグメントと、酸化LDL受容体(LOX-1)のリガンド認識領域とをラテラルフローアッセイ形式で用いる、変性LDLに関連する疾患(例えば、脂質異常、動脈硬化、糖尿病、虚血性心疾患、脳血管障害)の検診のためのシステム。
(C3)前記変性LDLに関連する疾患は、脂質異常、動脈硬化、糖尿病、虚血性心疾患および脳血管障害からなる群より選択される、項目C2に記載のシステム。
(C4)抗変性LDL抗体、修飾物またはそのフラグメントと、酸化LDL受容体(LOX-1)のリガンド認識領域とをラテラルフローアッセイ形式で用いる、食品摂取による変性LDLに関連する疾患(例えば、脂質異常、動脈硬化、糖尿病、虚血性心疾患、脳血管障害)の予防効果の評価のためのシステム。
(C5)前記変性LDLに関連する疾患は、脂質異常、動脈硬化、糖尿病、虚血性心疾患および脳血管障害からなる群より選択される、項目C4に記載のシステム。
<D.改良型CTLD14(カイコ型)>
(D1)カイコ型糖鎖を有する、配列番号86に示すアミノ酸配列またはその改変体を含むC型レクチン様ドメイン(CTLD)14。
(D2)前記カイコ型糖鎖は、トリマンノシルコア、複合型糖鎖、オリゴマンノース型糖鎖またはハイブリッド型糖鎖の1または複数の種類の糖鎖を含む、項目D1に記載のCTLD14。
(D3)前記カイコ型糖鎖は、トリマンノシルコア(アスパラギン残基からGlcNAc-β1,4-GlcNAc-β1,4-Man(-α1,6-Man)-α1,3-Man)の構造に加え、2分子あたりGlcNAc2分子およびMan2分子のうちの0~4分子が結合した糖鎖を含む、項目D1またはD2に記載のCTLD14。
(D4)前記カイコ型糖鎖は、アスパラギン残基からみて以下の組合せ
1)GlcNAc-β1,4-GlcNAc-β1,4-Man(-α1,3-Man)-α1,6-Man(-α1,3-Man)-α1,6-Manと、GlcNAc-β1,4-GlcNAc-β1,4-Man(-α1,6-Man-β1,2-GlcNAc)-α1,3-Man-β1,2-GlcNAcとの組み合わせ、
2)GlcNAc-β1,4-GlcNAc-β1,4-Man(-α1,3-Man)-α1,6-Man(-α1,3-Man)-α1,6-Man-α1,2-Manと、GlcNAc-β1,4-GlcNAc-β1,4-Man(-α1,6-Man-β1,2-GlcNAc)-α1,3-Manとの組み合わせ、
3)GlcNAc-β1,4-GlcNAc-β1,4-Man(-α1,3-Man)-α1,6-Man(-α1,3-Man)-α1,6-Man-α1,2-Manと、GlcNAc-β1,4-GlcNAc-β1,4-Man(-α1,6-Man-β)-α1,3-Man-β1,2-GlcNAcとの組み合わせ、
の1つまたは複数の組合せを含む、項目D1~D3のいずれか1項に記載のCTLD14。
(D5)前記カイコ型糖鎖は、配列番号86の111位のアスパラギンおよび/または155位のアスパラギンに結合する、項目D1~D4のいずれか1項に記載のCTLD14。
(D6)前記CTLD14は、ビオチン化されたものである、項目D1~D5のいずれか1項に記載のCTLD14。
(D7)項目D1~D6のいずれか1項に記載のCTLD14を含む、変性LDLを検出するための組成物。
(D8)前記組成物は、非変性LDLと変性LDLとを識別して変性LDLを検出するためのものである、項目D7に記載の組成物。
(D9)前記非変性LDLおよび変性LDLはLDLおよび酸化LDLを含む、項目D8に記載の組成物。
(D10)前記非変性LDLおよび変性LDLは複数種の哺乳動物のLDLおよび酸化LDLである、項目D8またはD9に記載の組成物。
(D11)前記哺乳動物は、マウスおよびヒトを含む、項目D10に記載の組成物。
<E.中部絹糸腺にCTLD14を発現させる遺伝子組換えカイコを作出し、CTLD14を可溶性蛋白質として大量発現させ精製した改良型CTLD14の製造方法>
(E1)C型レクチン様ドメイン(CTLD)14の製造方法であって、
A)カイコまたはカイコと同様の糖鎖を付与する生物に、CTLD14をコードする核酸分子を発現可能に組み込む工程;
B)該遺伝子が発現する条件下に該カイコまたはカイコと同様の糖鎖を付与する生物を配置し、該CTLD14を発現させる工程;および
C)該CTLD14を得る工程
を包含する、方法。
(E2)前記発現は、前記カイコまたはカイコと同様の糖鎖を付与する生物の後部絹糸腺、中部絹糸腺または全身において行われる、項目E1に記載の方法。
(E3)前記発現は、前記カイコまたはカイコと同様の糖鎖を付与する生物の中部絹糸腺において行われる、項目E1またはE2に記載の方法。
(E4)前記CTLD14は、ビオチン化された状態またはビオチン化可能な状態で発現される、項目E1~E3のいずれか1項に記載の方法。
(E5)前記生物は、ビオチンリガーゼをコードする核酸配列を含み、前記C)工程において、ビオチンをカイコに経口投与することを含む、項目E1~E4のいずれか1項に記載の方法。
(E6)前記CTLD14は、ビオチン化された状態で発現される、項目E1~E5のいずれか1項に記載の方法。
(E7)前記生物は、ビオチン化を受けるタグ配列を含む、項目E1~E6のいずれかに記載の方法。
(E8)前記ビオチン化を受けるタグ配列は、BioEase.tagおよびAvi.tagのいずれかである、項目E7に記載の方法。
(E9)前記A)工程は、前記CTLD14をコードする核酸分子を含む発現ベクターをマイクロインジェクションすることで達成される、項目E1~E8のいずれか1項に記載の方法。
(E10)前記核酸分子は、配列番号85に示す核酸配列またはその改変体を含む、項目E1~E9のいずれか1項に記載の方法。
(E10-1)前記CTLD14は、項目D1~D6のいずれか1項または複数に記載の特徴を有する、項目E1~E10のいずれか1項に記載の方法。
(E11)CTLD14をコードする核酸分子を発現可能に組み込んだカイコまたはカイコと同様の糖鎖を付与する生物。
(E12)前記核酸分子は、配列番号85に示す核酸配列またはその改変体を含む、項目E11に記載のカイコまたはカイコと同様の糖鎖を付与する生物。
(E13)前記生物は、ビオチンリガーゼをコードする配列を含む、項目E11またはE12に記載の生物。
(E14)前記ビオチンリガーゼはBirA(配列番号116)である、項目E13に記載の生物。
(E15)前記ビオチンリガーゼと前記CTLD14とは中部絹糸腺において共発現される、項目E13またはE14に記載の生物。
(E16)前記生物は、ビオチン化を受けるタグ配列を含む、項目E11~15のいずれかに記載の生物。
(E17)前記ビオチン化を受けるタグ配列は、BioEase.tagおよびAvi.tagのいずれかである、項目E16に記載の生物。
<F.抗LDLニワトリポリクローナルおよびその組合せ>
(F1)抗LDLニワトリ抗体を含む、変性LDLを検出するための組成物。
(F2)前記抗LDLニワトリ抗体は、ポリクローナル抗体である、項目F1に記載の組成物。
(F3)さらに、CTLD14を含む、項目F1またはF2に記載の組成物。
(F4)前記CTLD14は、カイコ型糖鎖を含むCTLD14である、項目F1~F3のいずれかに記載の組成物。
(F5)前記カイコ型糖鎖を含むCTLD14は、項目D1~D6のいずれかに記載のCTLD14である、項目F4に記載の組成物。
(F6)前記組成物は、LDLおよび酸化LDLを検出するためのものである、項目F1~F5のいずれかに記載の組成物。
(F7)前記LDLおよび酸化LDLは、複数種の哺乳動物のLDLおよび酸化LDLである、項目F6に記載の組成物。
(F8)前記哺乳動物は、マウスおよびヒトを含む、項目F7に記載の組成物。
(F9)抗LDLニワトリ抗体と、CTLD14とを含む、変性LDLを検出するためのキット。
(F10)抗LDLニワトリ抗体はポリクローナル抗体である、項目F9に記載の変性LDLを検出するためのキット。
(F11)抗LDLニワトリ抗体は抗酸化LDLニワトリ抗体または抗酸化一本鎖抗体である、項目F8またはF9に記載の変性LDLを検出するためのキット。
(F12)前記CTLD14は、項目D1に記載のCTLD14である、項目F9~F11のいずれか1項に記載のキット。
(F13)LDLおよび酸化LDLを検出するためのものである、項目F10~F12のいずれか1項に記載の組成物。
(F14)前記LDLおよび酸化LDLは、複数種の哺乳動物のLDLおよび酸化LDLである、項目F13に記載のキット。
(F15)前記哺乳動物は、マウスおよびヒトを含む、項目F14に記載のキット。
(G1)カイコ型糖鎖を有する、配列番号97に示すアミノ酸配列またはその改変体を含む再構築終末糖化産物受容体(sRAGE)。
(G2)前記カイコ型糖鎖は、トリマンノシルコア、複合型糖鎖、オリゴマンノース型糖鎖またはハイブリッド型糖鎖の1または複数の種類の糖鎖を含む、項目G1に記載のsRAGE。
(G3-1)前記カイコ型糖鎖は、トリマンノシルコア(アスパラギン残基からGlcNAc-β1,4-GlcNAc-β1,4-Man(-α1,6-Man)-α1,3-Man)の構造に加え、1分子あたりGlcNAc0~2分子およびMan0~4分子のうちの0~4分子が結合した糖鎖を含む、項目G1またはG2に記載のsRAGE。
(G3-2)前記カイコ型糖鎖は、トリマンノシルコア(アスパラギン残基からGlcNAc-β1,4-GlcNAc-β1,4-Man(-α1,6-Man)-α1,3-Man)の構造に加え、2分子あたりGlcNAc0~4分子およびMan0~8分子のうちの0~8分子が結合した糖鎖を含む、項目G1~G2またはG3-1に記載のsRAGE。
(G4-1)前記カイコ型糖鎖の組成比が、オリゴマンノース型:87~97%、複合型:2~8%、ハイブリッド型:1~5%である、項目G1~G2またはG3-1~G3-2のいずれか1項に記載のsRAGE。
(G4-2)前記カイコ型糖鎖の組成比が、オリゴマンノース型:90~94%、複合型:3~6%、ハイブリッド型:2~4%である、項目G1~G2、G3-1~G3-2またはG4-1のいずれか1項に記載のsRAGE。
(G4-3)前記カイコ型糖鎖の組成比が、オリゴマンノース型:92.5%、複合型:4.6%、ハイブリッド型:2.5%である、項目G1~G2、G3-1~G3-2またはG4-1~G4-2のいずれか1項に記載のsRAGE。
(G5-1)前記オリゴマンノース型は、(Man)5(GlcNAc)2、(Man)7(GlcNAc)2、(Man)6(GlcNAc)2および(Man)3(GlcNAc)2を含む、項目G2、G3-1~G3-2またはG4-1~G4-3のいずれか1項に記載のsRAGE。
(G5-2)前記オリゴマンノース型の糖鎖全体における組成比が、(Man)5(GlcNAc)2:45~56%、(Man)7(GlcNAc)2:23~33%、(Man)6(GlcNAc)2:7~15%、および(Man)3(GlcNAc)2:1~5%である、項目G2、G3-1~G3-2、G4-1~G4-3またはG5-1のいずれか1項に記載のsRAGE。
(G5-3)前記オリゴマンノース型の糖鎖全体における組成比が、(Man)5(GlcNAc)2:48~54%、(Man)7(GlcNAc)2:25~31%、(Man)6(GlcNAc)2:9~13%、および(Man)3(GlcNAc)2:2~4%である、項目G2、G3-1~G3-2、G4-1~G4-3またはG5-1~G5-2のいずれか1項に記載のsRAGE。
(G5-4)前記オリゴマンノース型の糖鎖全体における組成比が、(Man)5(GlcNAc)2:51%、(Man)7(GlcNAc)2:27.5%、(Man)6(GlcNAc)2:10.8%、および(Man)3(GlcNAc)2:2.8%である、項目G2、G3-1~G3-2、G4-1~G4-3またはG5-1~G5-3のいずれか1項に記載のsRAGE。
(G6-1)前記複合型が、(Man)3(GlcNAc)3を含む項目G2、G3-1~G3-2、G4-1~G4-3またはG5-1~G5-4のいずれか1項に記載のsRAGE。
(G6-2)前記複合型の糖鎖全体における組成比が、(Man)3(GlcNAc)3:2~8%である項目G2、G3-1~G3-2、G4-1~G4-3、G5-1~G5-4またはG6-1のいずれか1項に記載のsRAGE。
(G6-3)前記複合型の糖鎖全体における組成比が、(Man)3(GlcNAc)3:3~6%である項目G2、G3-1~G3-2、G4-1~G4-3、G5-1~G5-4またはG6-1~G6-2のいずれか1項に記載のsRAGE。
(G6-4)前記複合型の糖鎖全体における組成比が、(Man)3(GlcNAc)3:4.6%である項目G2、G3-1~G3-2、G4-1~G4-3、G5-1~G5-4またはG6-1~G6-3のいずれか1項に記載のsRAGE。
(G7)前記ハイブリッド型が、(Man)4(GlcNAc)3である項目G2、G3-1~G3-2、G4-1~G4-3、G5-1~G5-4またはG6-1~G6-4のいずれか1項に記載のsRAGE。
(G8)前記カイコ型糖鎖は、配列番号97の3位のアスパラギンおよび/または59位のアスパラギンに結合する、項目G1~G2、G3-1~G3-2、G4-1~G4-3、G5-1~G5-4、G6-1~G6-4またはG7のいずれか1項に記載のsRAGE。
(G9)前記sRAGEは、ビオチン化されたものである、項目G1~G2、G3-1~G3-2、G4-1~G4-3、G5-1~G5-4、G6-1~G6-4またはG7~G8のいずれか1項のいずれか1項に記載のsRAGE。
(G10)前記ビオチンを介して前記sRAGEが基板に固定化されている、項目G1~G2、G3-1~G3-2、G4-1~G4-3、G5-1~G5-4、G6-1~G6-4またはG7~G9に記載のsRAGEが固定化された試薬。
(G11)項目G1~G2、G3-1~G3-2、G4-1~G4-3、G5-1~G5-4、G6-1~G6-4またはG7~G9のいずれか1項に記載のsRAGEを含む、終末期糖化生成物(AGEs)を検出するための組成物。
(G12)項目G1~G2、G3-1~G3-2、G4-1~G4-3、G5-1~G5-4、G6-1~G6-4またはG7~G9のいずれか1項に記載のsRAGEを試料に接触させ、AGEsを検出する工程を包含する、AGEsを検出する方法。
(G13)sRAGEの製造方法であって、
A)カイコまたはカイコと同様の糖鎖を付与する生物に、sRAGEをコードする核酸分子を発現可能に組み込む工程;
B)該遺伝子が発現する条件下に該カイコまたはカイコと同様の糖鎖を付与する生物を配置し、該sRAGEを発現させる工程;および
C)該sRAGEを得る工程
を包含する、方法。
(G14)前記発現は、前記カイコまたはカイコと同様の糖鎖を付与する生物の後部絹糸腺、中部絹糸腺または全身において行われる、項目G13に記載の方法。
(G15)前記発現は、前記カイコまたはカイコと同様の糖鎖を付与する生物の中部絹糸腺において行われる、項目G13またはG14に記載の方法。
(G16)前記sRAGEは、ビオチン化された状態またはビオチン化可能な状態で発現される、項目G13~G15のいずれか1項に記載の方法。
(G17)前記sRAGEは、ビオチン化された状態で発現される、項目G13~G16のいずれか1項に記載の方法。
(G18)前記生物は、ビオチンリガーゼをコードする核酸配列を含み、前記C)工程において、ビオチンをカイコに経口投与することを含む、項目G13~G17のいずれか1項に記載の方法。
(G19)前記生物は、ビオチン化を受けるタグ配列を含む、項目G13~G18のいずれか1項に記載の方法。
(G20)前記ビオチン化を受けるタグ配列は、BioEase.tagおよびAvi.tagのいずれかである、項目G19に記載の方法。
(G21)前記A)工程は、前記sRAGEをコードする核酸分子を含む発現ベクターをマイクロインジェクションすることで達成される、項目G13~G20のいずれか1項に記載の方法。
(G22)前記核酸分子は、配列番号96に示す核酸配列またはその改変体を含む、項目G13~21のいずれか1項に記載の方法。
(G22-1)前記sRAGEは、項目G1~G2、G3-1~G3-2、G4-1~G4-3、G5-1~G5-4、G6-1~G6-4またはG7~G9のいずれか1項または複数に記載される特徴を有する、項目G13~22のいずれか1項に記載の方法。
(G23)sRAGEをコードする核酸分子を発現可能に組み込んだカイコまたはカイコと同様の糖鎖を付与する生物。
(G24)前記核酸分子は、配列番号96に示す核酸配列またはその改変体を含む、項目G23に記載のカイコまたはカイコと同様の糖鎖を付与する生物。
(G25)前記生物は、ビオチンリガーゼをコードする配列を含む、項目G23またはG24に記載の生物。
(G26)前記ビオチンリガーゼはBirA(配列番号106)である、項目G25に記載の生物。
(G27)前記ビオチンリガーゼと前記sRAGEとは中部絹糸腺において共発現される、項目G25または26に記載の生物。
(G28)前記生物は、ビオチン化を受けるタグ配列を含む、項目G23~G27のいずれか1項に記載の生物。
(G29)前記ビオチン化を受けるタグ配列は、BioEase.tagおよびAvi.tagのいずれかである、項目G28に記載の生物。
(H1)標的をコードする核酸分子およびビオチンリガーゼを共発現可能に組み込んだカイコまたはカイコと同様の糖鎖を付与する生物。
(H2)前記ビオチンリガーゼはBirAである、項目H1に記載の生物。
(H3)前記標的は、C型レクチン様ドメイン(CTLD14)または終末糖化産物受容体(sRAGE)あるいはその改変体である、項目H1またはH2に記載の生物。
(H4)前記CTLD14は配列番号85によってコードされ、前記sRAGEは配列番号96によってコードされる、項目H3に記載の生物。
(H5)前記生物は、ビオチン化を受けるタグ配列を含む、項目H1~H4のいずれか1項に記載の生物。
(H6)前記ビオチン化を受けるタグ配列は、BioEase.tagおよびAvi.tagのいずれかである、項目H5に記載の生物。
(H7)ビオチン化タンパク質の製造方法であって、
A)カイコまたはカイコと同様の糖鎖を付与する生物に、ビオチンリガーゼおよびタンパク質をコードする核酸分子を共発現可能に組み込む工程;
B)該核酸分子が発現する条件下に該カイコまたはカイコと同様の糖鎖を付与する生物を配置し、該ビオチンリガーゼおよび該タンパク質を発現させる工程;および
C)該生物にビオチンを投与し、ビオチン化された該タンパク質を得る工程
を包含する、方法。
(H8)前記発現は、前記カイコの中部絹糸腺において行われる、項目H7に記載の方法。
(H9)前記タンパク質は、ビオチン化された状態で発現される、項目H7またはH8に記載の方法。
(H10)前記A)工程は、前記タンパク質をコードする核酸分子を含む発現ベクターをマイクロインジェクションすることで達成される、項目H7~H9のいずれか1項に記載の方法。
(H11)前記タンパク質は、C型レクチン様ドメイン(CTLD14)または終末糖化産物受容体(sRAGE)または配列番号76~80のいずれかに記載のアミノ酸配列もしくはその改変体を含む一本鎖抗体もしくはそのフラグメントあるいはその改変体である、項目H7~H10のいずれか1項に記載の方法。
(H12)前記生物は、ビオチン化を受けるタグ配列を含む、項目H7~H11のいずれか1項に記載の方法。
(H13)前記ビオチン化を受けるタグ配列は、BioEase.tagおよびAvi.tagのいずれかである、項目H12に記載の方法。
以下に本明細書において特に使用される用語の定義を列挙する。
Tm(℃)=81.5+16.6(log[Na+])+0.41(%G+C)-600/N-0.72(%ホルムアミド)
ここで、Nは、形成される二重鎖の長さであり、[Na+]は、ハイブリダイゼーション溶液または洗浄溶液中のナトリウムイオンのモル濃度であり、%G+Cは、ハイブリッド中の(グアニン+シトシン)塩基のパーセンテージである。不完全に一致したハイブリッドに関して、融解温度は、各1%不一致(ミスマッチ)に対して約1℃ずつ減少する。
糖鎖は、糖鎖の結合様式により、アスパラギンと結合する糖鎖(N-グリコシド結合糖鎖という)ならびに、セリン、スレオニンなどと結合する糖鎖(0-グリコシド結合糖鎖という)の2種類に大別される。上述のN-グリコシド結合糖鎖は、様々な構造を有しているが[生物化学実験法23-糖タンパク質糖鎖研究法(学会出版センター)高橋禮子編(1989年)]、いずれの場合も下記に示す基本となる共通のコア構造を有することが好ましい。もっとも、この点は、上述の糖タンパク質が抗体ではない場合にも、同様である。
を含むN-グリコシド結合糖鎖を有する糖タンパク質が含まれることが好ましい。カイコ型の糖鎖では、上述のような複合型糖鎖が含まれていることが知られているからである。本明細書において、糖鎖(4)は、N4-2または糖鎖100.2とも表示される。
本発明のCTLD14が含む前記カイコ型糖鎖は、アスパラギン残基からみて以下の組合せ
1)GlcNAc-β1,4-GlcNAc-β1,4-Man(-α1,3-Man)-α1,6-Man(-α1,3-Man)-α1,6-Manと、GlcNAc-β1,4-GlcNAc-β1,4-Man(-α1,6-Man-β1,2-GlcNAc)-α1,3-Man-β1,2-GlcNAcとの組み合わせ(上記式(5)と(2))、
2)GlcNAc-β1,4-GlcNAc-β1,4-Man(-α1,3-Man)-α1,6-Man(-α1,3-Man)-α1,6-Man-α1,2-Manと、GlcNAc-β1,4-GlcNAc-β1,4-Man(-α1,6-Man-β1,2-GlcNAc)-α1,3-Manとの組み合わせ(上記式(6)と(3))、
3)GlcNAc-β1,4-GlcNAc-β1,4-Man(-α1,3-Man)-α1,6-Man(-α1,3-Man)-α1,6-Man-α1,2-Manと、GlcNAc-β1,4-GlcNAc-β1,4-Man(-α1,6-Man-β)-α1,3-Man-β1,2-GlcNAcとの組み合わせ(上記式(6)と(4))、
の1つまたは複数の組合せを含む。
sRAGEの糖鎖型として、上記(1)~(6)([化1]~[化6])に加えて以下のものも挙げられる。
中性糖・アミノ糖組成分析
糖鎖は、代表的に、ガラクトース、マンノース、フコースなどの中性糖、N-アセチルグルコサミンなどのアミノ糖、シアル酸などの酸性糖から構成されている。糖鎖の組成分析は、トリフルオロ酢酸等で、糖鎖の酸加水分解を行うことにより、中性糖またはアミノ糖を遊離し、その組成比を分析することができる。具体的な方法として、Dionex社製糖組成分析装置(BioLC)を用いる方法が挙げられる。BioLCはHPAEC-PAD(high performance anion-exchange chromatography-pulsed amperometric detection)法[J.Liq.Chromatogr.,6,1577(1983)]によって糖組成を分析する装置である。
糖鎖の構造解析は、2次元糖鎖マップ法[Anal.Biochem.,171,73(1988)、生物化学実験法23-糖タンパク質糖鎖研究法(学会出版センター)高橋禮子編(1989年)]により行うことができる。2次元糖鎖マップ法は、例えば、X軸には逆相クロマトグラフィー糖鎖の保持時間または溶出位置を、Y軸には順相クロマトグラフィーによる糖鎖の保持時間または溶出位置を、それぞれプロットし、既知糖鎖のそれらの結果と比較することにより、糖鎖構造を推定する方法である。
本実施形態では、カイコ等の鱗翅目に分類される昆虫の絹糸腺細胞において、糖タンパク質を発現させる。
以下に好ましい実施形態の説明を記載するが、この実施形態は本発明の例示であり、本発明の範囲はそのような好ましい実施形態に限定されないことが理解されるべきである。当業者はまた、以下のような好ましい実施例を参考にして、本発明の範囲内にある改変、変更などを容易に行うことができることが理解されるべきである。これらの実施形態について、当業者は適宜、任意の実施形態を組み合わせ得る。
本発明の1つの局面において、本発明は、配列番号76~80のいずれかに記載のアミノ酸配列またはその改変体を含む一本鎖抗体またはそのフラグメントあるいはその改変体を提供する。好ましくは、前記一本鎖抗体は、配列番号76または配列番号78のいずれかに記載のアミノ酸配列またはその改変体を含む。さらに好ましくは、前記一本鎖抗体は、配列番号76に記載のアミノ酸配列またはその改変体を含む。本発明の一本鎖抗体またはそのフラグメントは、LDLおよび/またはその修飾物に特異的に結合することができる。
他の局面において、本発明は、配列番号76~80のいずれかに記載のアミノ酸配列またはその改変体を含む一本鎖抗体またはそのフラグメントあるいはその改変体を、LDLおよび/またはその修飾物(変性LDL)を検出する検出剤を提供する。この検出剤は、一本鎖抗体またはそのフラグメントの他に、酸化的修飾を受けたLDLを認識することができる分子をさらに含んでもよい。酸化的修飾を受けたLDLを認識することができる分子としては、CTLD14、抗LDLニワトリ抗体、およびこれらの組み合わせなどが挙げられるが、これらに限定されない。
本発明の他の局面において、前記一本鎖抗体またはそのフラグメントを生産する方法を提供する。この方法は、(A)前記一本鎖抗体またはそのフラグメントのアミノ酸配列をコードする核酸を、前記核酸が発現される大腸菌に導入する工程;(B)前記大腸菌を培養し、前記大腸菌から封入体を取り出す工程;(C)前記一本鎖抗体またはそのフラグメントを前記封入体から抽出し、リフォールディングする工程;(D)リフォールディングで得られた溶液をNi-NTAスラリーに接触させ、イミダゾールを含む溶出溶液で溶出する工程;および(E)溶出した前記一本鎖抗体またはそのフラグメントを透析溶液で透析する工程を包含する。
別の局面において、本発明は、本発明の一本鎖抗体またはそのフラグメントを用いる、LDLまたはその修飾物(変性LDL)を検出または定量するためのシステムまたはキットを提供する。この局面で用いられ得る一本鎖抗体またはそのフラグメントあるいはその改変体、検出または定量の手法およびそれに用いる手段、キット、システム等としては、上述の(一本鎖抗体またはそのフラグメント)の節、(一本鎖抗体またはそのフラグメントを含む検出剤)の節、(一本鎖抗体またはそのフラグメントの安定供給系)の節および他の節において説明された任意の形態を用いることができる。
1つの局面において、本発明は、カイコ型糖鎖を有する、配列番号86に示すアミノ酸配列またはその改変体を含むC型レクチン様ドメイン(CTLD)14を提供する。
1)GlcNAc-β1,4-GlcNAc-β1,4-Man(-α1,3-Man)-α1,6-Man(-α1,3-Man)-α1,6-Manと、GlcNAc-β1,4-GlcNAc-β1,4-Man(-α1,6-Man-β1,2-GlcNAc)-α1,3-Man-β1,2-GlcNAcとの組み合わせ、
2)GlcNAc-β1,4-GlcNAc-β1,4-Man(-α1,3-Man)-α1,6-Man(-α1,3-Man)-α1,6-Man-α1,2-Manと、GlcNAc-β1,4-GlcNAc-β1,4-Man(-α1,6-Man-β1,2-GlcNAc)-α1,3-Manとの組み合わせ、
3)GlcNAc-β1,4-GlcNAc-β1,4-Man(-α1,3-Man)-α1,6-Man(-α1,3-Man)-α1,6-Man-α1,2-Manと、GlcNAc-β1,4-GlcNAc-β1,4-Man(-α1,6-Man-β)-α1,3-Man-β1,2-GlcNAcとの組み合わせ、
の1つまたは複数の組合せを含む(図20を参照)。
B)該遺伝子が発現する条件下に該カイコまたはカイコと同様の糖鎖を付与する生物を配置し、該CTLD14を発現させる工程;およびC)該CTLD14を得る工程を包含する、方法を提供する。
1つの局面において、本発明は、カイコ型糖鎖を有する、配列番号97に示すアミノ酸配列またはその改変体を含む再構築終末糖化産物受容体(sRAGE)を提供する。
1つの局面において、本発明は、終末期糖化生成物(AGEs)を検出することによって、AGE関連の疾患、障害等の検出、予測、予備的な検出または診断を行う方法、キットおよび組成物を提供する。本発明のマーカーについては本明細書上記<改良型sRAGE(カイコ型)およびその製造法>の節を参酌することができ、この節に記載された任意の実施形態を採用することができることが理解される。これらの疾患には、糖尿病、糖尿病性腎症、糖尿病性網膜症、糖尿病性神経症等の糖尿病合併症、アルツハイマー症などの神経変性疾患、関節リュウマチ、後縦靱帯骨化症、骨粗鬆症、非アルコール性脂肪肝炎、歯周症、筋萎縮、加齢黄斑変性、皮膚疾患、皮膚老化、動脈硬化などが挙げられるがそれらに限定されない。なお、理論に束縛されないが、糖尿病性腎症は微小血管系の障害であるが、糖尿病は微小血管障害のみではなく、動脈硬化のような大血管障害の発症にもつながることが知られている。この原因の一つが大動脈血管内皮にも発現しているRAGEを介した血管内皮機能不全誘導にあると考えられる。よって、RAGEの本来機能を抑制することによる血管内皮機能不全誘導(=血管障害)の調整関連に結びつけることができる。ここで、サンプルの取得は、どのような手段で行っても良い。通常、医師以外の担当者が測定に従事する場合は、何らかの形で医師が取得したものであり得る。測定結果から、糖尿病またはその可能性があるかどうかを決定する工程は、正常値と比べて、各々のマーカーに比較して異常であるかどうかを判定することによって実施することができる。理論に束縛されることを望まないが、1つの例示的な実施例に基づいて説明すると、糖尿病モデルとコントロールとの対比実験を行うことによって、これらの疾患等の診断を行うことができる。
1つの局面では、本発明は、抗LDLニワトリ抗体を含む、変性LDLを検出するための組成物を提供する。本発明では、他の動物種(例えば、マウス等)で生産した抗体に比較して、酸化LDL(変性LDL)と未変性LDLとの識別能が格段に上がることが理解される。
(ビオチンリガーゼを共発現するカイコ)
本発明の1つの局面において、標的タンパク質をコードする核酸分子およびビオチンリガーゼを共発現可能に組み込んだカイコまたはカイコと同様の糖鎖を付与する生物を提供する。
本発明の別の局面において、ビオチン化タンパク質の製造方法であって、A)カイコまたはカイコと同様の糖鎖を付与する生物に、ビオチンリガーゼおよびタンパク質をコードする核酸分子を共発現可能に組み込む工程;B)該核酸分子が発現する条件下に該カイコまたはカイコと同様の糖鎖を付与する生物を配置し、該ビオチンリガーゼおよび該タンパク質を発現させる工程;およびC)該生物にビオチンを投与し、ビオチン化された該タンパク質を得る工程を包含する、方法を提供する。本発明のカイコ型ビオチン化タンパク質の製造方法は、目的のタンパク質(例えば、CTLD14、sRAGEなど)を可溶性タンパク質として、高いビオチン化効率でビオチン化された状態で、数日以内に大量に発現させることが可能であるという優れた効果を有する。さらに、カイコまたはカイコと同様の糖鎖を付与する生物を用いて発現された糖鎖付加タンパク質は、糖鎖付加が行われない大腸菌発現系を用いたタンパク質よりも、pH安定性に優れ、脱塩処理によって凝集する率が低く、種々の緩衝液を適用することができるため、活用範囲が広い。得られたビオチン化CTLD14はpH安定性に優れ、金属コロイド修飾し、抗LDLニワトリ抗体、一本鎖抗体、ストレプトアビジンを組み込んだラテラルフローアッセイにより、酸化的修飾を受けた多様な構造のLDLを20分以内に検出可能である。また、得られたsRAGEの糖鎖構造解析により、(Man)5(GlcNAc)2を主とするオリゴマンノース型糖鎖が90%以上を占め、数%のコンプレックス型、および、ハイブリッド型の糖鎖を含むことを明らかにした。さらに糖鎖付加により安定性が向上し(1年近く安定)、ビオチンを介した方向性を維持した固定化により、多様な構造のAGEsなどの検出が可能である。
1つの局面において、本発明は、高い効率でビオチン化されたCTLD14を提供する。高効率のビオチン化に起因して、ストレプトアビジンを組み込んだラテラルフローアッセイにより、酸化的修飾を受けた多様な構造のLDLを20分以内という短時間で検出することが可能である。1つの実施形態において、本発明のCTLD14のビオチン化効率は、約10%以上、約20%以上、約30%以上、約40%以上、または約50%以上である。好ましい実施形態では、本発明のCTLD14のビオチン化効率は、約30%~約40%である。得られたビオチン化CTLD14はpH安定性に優れ、金属コロイド修飾し、抗LDLニワトリ抗体、一本鎖抗体、ストレプトアビジンを組み込んだラテラルフローアッセイにより、酸化的修飾を受けた多様な構造のLDLを20分以内に検出可能である。
別の局面において、本発明は、高い効率でビオチン化されたsRAGEを提供する。高効率のビオチン化に起因して、ビオチンを介した方向性を維持した基板等への固定が可能であり、多様な構造のAGEsの検出が可能である。1つの実施形態において、本発明のsRAGEのビオチン化効率は、約10%以上、約20%以上、約30%以上、約40%以上、約50%以上、または約60%以上である。好ましい実施形態では、本発明のsRAGEのビオチン化効率は、約60%以上である。得られたsRAGEの糖鎖構造解析により、(Man)5(GlcNAc)2を主とするオリゴマンノース型糖鎖が90%以上を占め、数%のコンプレックス型、および、ハイブリッド型の糖鎖を含むことを明らかにした。さらに糖鎖付加により安定性が向上し(1年近く安定)、ビオチンを介した方向性を維持した固定化により、多様な構造のAGEsなどの検出が可能である。
別の局面において、本発明は、一本鎖抗体またはそのフラグメントの製造方法であって、(A)カイコまたはカイコと同様の糖鎖を付与する生物に、該一本鎖抗体またはそのフラグメントをコードする核酸分子を発現可能に組み込む工程;(B)該核酸分子が発現する条件下に該カイコまたはカイコと同様の糖鎖を付与する生物を配置し、該一本鎖抗体またはそのフラグメントを発現させる工程;および(C)該一本鎖抗体またはそのフラグメントを得る工程を包含する、方法を提供する。大腸菌により一本鎖抗体を産生する場合は、発現されたタンパク質が封入体を形成し、リフォールディングの過程を必要としたが、本発明のカイコによる産生においては、可溶性タンパク質として発現されるため、リフォールディングの過程を必要としない。
本明細書において用いられる分子生物学的手法、生化学的手法、微生物学的手法は、当該分野において周知であり慣用されるものであり、例えば、Sambrook J.et al.(1989).Molecular Cloning: A Laboratory Manual,Cold Spring Harborおよびその3rd Ed.(2001);Ausubel,F.M.(1987).Current Protocols in Molecular Biology,Greene Pub.Associates and Wiley-Interscience;Ausubel,F.M.(1989).Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology,Greene Pub.Associates and Wiley-Interscience;Innis,M.A.(1990).PCR Protocols: A Guide to Methods and Applications,Academic Press;Ausubel,F.M.(1992).Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology,Greene Pub.Associates;Ausubel,F.M.(1995).Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology,Greene Pub.Associates;Innis,M.A.et al.(1995).PCR Strategies,Academic Press;Ausubel,F.M.(1999).Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology,Wiley,and annual updates;Sninsky,J.J.et al.(1999).PCR Applications: Protocols for Functional Genomics,Academic Press、別冊実験医学「遺伝子導入&発現解析実験法」羊土社、1997などに記載されており、これらは本明細書において関連する部分(全部であり得る)が参考として援用される。
MarksらとHawkinsらの方法を改変し、一本鎖抗体遺伝子ライブラリーを作成した(Marksら、J. Mol. Biol.222:581-597(1991);Hawkinsら、Blood 83:3279-3288(1994))。配列決定反応は、Bigdye(登録商標)terminator v1.1 cycle sequencing kit(Applied Biosystems)を用い、プロトコールに従った。また、プラスミドDNAの種類によって表1のプライマーを用いた。本明細書では、プライマーの順向きはフォワード(forward、FORWARD、またはFOR)と称し、逆向きはリバース(reverse、REVERSE、またはREV)またはバック(BACK)と称する。リバース(reverse、REVERSE、またはREV)とバック(BACK)とは本明細書においてプライマーに関しては同じ意味を示す。
human spleen poly A+ RNA(BD Biosciences)から、First-strand cDNA synthesis Kit(GE healthcare)を用いて、First-strand cDNAを合成した。抗体遺伝子の種類(γ鎖、μ鎖、κ鎖、λ鎖)に応じて表2のプライマーを用いた。
γ鎖およびμ鎖を、Human VH リバースプライマー(表3)およびHuman JH フォワードプライマー(表3)を混合プライマーとし、PCR法で増幅させた。
γ鎖およびμ鎖にリンカー配列を付加するために、Human VH リバースプライマー(表3)およびHuman JH forward linker primers(表6)を混合プライマーとし、PCR法で増幅させた。κ鎖にリンカー配列を付加するために、Human VΚ back linker primers(表6)およびHuman JΚ フォワードプライマー(表4)を混合プライマーとし、PCR法で増幅させた。λ鎖にリンカー配列を付加するために、Human Vλ リバースリンカープライマー(表6)およびHuman Jλ フォワードプライマー(表5)を混合プライマーとし、PCR法で増幅させた。PCRは、94℃ 3分間、次に94℃ 1分間、60℃ 1分間、72℃ 1分間のサイクルを25サイクル、次に72℃ 10分間で反応させた。
プライマリーPCRおよびセカンダリーPCRの二段階からなるアッセンブリPCRを行った。プライマリーPCRの反応溶液は、表7Aに示すとおりである。反応条件は、94℃ 5分間、次に94℃ 1分間、60℃ 1分間、72℃ 1分間のサイクルを7サイクルとした。
一本鎖抗体遺伝子(γ+κ、μ+λ)に制限酵素サイト(SfiI、NotI)を付加するため、Human リバースSfiIプライマー(表7)およびHuman JΚ フォワードNotIプライマー(表7)またはHuman Jλ フォワードNotプライマー(表7)を混合プライマーとし、PCR法で別々に増幅させた。反応条件は、94℃ 3分間、次に94℃ 1分間、72℃ 2分間のサイクルを25サイクル、次に72℃ 10分間とした。
Recombinant Phage Antibody System(GE healthcare)のプロトコールを改変して、二段階からなる制限酵素処理を行った。
(1.LDLの調製)
調製済みのLDLを定法に従い完全酸化し(Steinbrecher、1984)、抗原となる酸化LDLを調製した。すなわち、1mg/ml LDL(PBSで透析)に最終濃度5μMとなるようにCuSO4を添加して、37℃において無菌条件下で20時間反応させた後、最終濃度が1mMとなるようにEDTAを加えた。次に、PBSで透析し、濾過滅菌した後にNaN3が0.02%となるように加え、4℃で保存した。
調製した、酸化LDLを用いてバイオパニングと呼ばれるスクリーニングを行った。スラントネック培養細胞フラスコ(25ml、BD Falcon)に1μg/ml酸化LDL(PBSで希釈)5mlを添加し、室温で2時間反応後、PBS(30ml)で洗浄した。次に、ブロッキングバッファー(0.25%BSAを含むPBS)を添加し、室温で1時間反応後、PBS(30ml)で3回洗浄した。調製した一本鎖抗体提示ファージ4.5mlに0.01%アジ化ナトリウム含有ブロッキングバッファー4mlを添加し、室温で15分間反応後、スラントネック培養細胞フラスコに添加した。37℃で2時間反応後、40回洗浄し、A600=0.3の大腸菌TG1株2mlを添加して、37℃で1時間振とう培養した。SOBAG寒天培地に菌液100μlを塗布し、30℃で14時間培養後、1mlの2xYTをプレートに加え、スプレッダーで掻き取り菌体を回収した。
ライブラリーごとにコロニーを95個取得し、ELISAを用いて酸化LDLへ結合する一本鎖抗体を有するコロニーを選択した。
(1.使用菌株、プラスミドDNAおよびファージミドDNA)
培養には2×YT培地、LB培地、およびSOBAG培地を用い、固形培地には1.5%の寒天を添加した寒天培地を使用した。薬剤を添加する場合は、終濃度をアンピシリン100μg/ml、カナマイシン15μg/ml、テトラサイクリン12.5μg/ml、ナリジクス酸100μg/ml、およびX-gal30μg/mlとした。またカタボライトリプレッションの目的でグルコースを添加する場合は、終濃度が5%になるよう添加した。
少量調製の場合は、シングルコロニーを2ml LB培地に植菌し、FlexiPrep kit(GE healthcare)を用いてプラスミドDNA、ファージミドDNAを調製した。
DNAのバンドを確認する場合、泳動槽はMupid(登録商標)-3(コスモ・バイオ)を、バッファーは、1×TAEを用いて100V、30分間室温で泳動しエチヂウムブロマイドで染色後、UVで確認した。サイズマーカーはDNA Mass Ladder(TOYOBO)を用いた。
DH5α(invitrogen)、BL21(DE3)(Novagen)、HB2151(GE healthcare)およびBLR(Novagen)を形質転換させる場合は、50μlのコンピテントセルに適当量のプラスミドDANを混合し、氷中で30分間静置後、42℃で45秒間ヒートショックを与えた。さらに氷中で2分間静置し、450μlのSOCを加え37℃で1時間培養後、適当な薬剤を含む寒天培地に塗布し形質転換体を得た。
ミニプロテインII-Dセル(BIORAD)を用いて行った。分子量マーカーは、タンパク質分子量マーカー「第一」・III(第一化学薬品)を用いた。
SDS-PAGEによりタンパク質を分離後、ミニトランスブロットセル(BIORAD)を用いてニトロセルロース膜(BioTrace(登録商標)NT)に転写した。分子量マーカーはprecision plus proteinTM standards(BIORAD)を用いた。
可溶性タンパク質の定量は、標準タンパク質としてBSA(Albumin standard、PIERCE)を用い、BCA Protein Assay Reagent Kitにより行った。不溶性タンパク質の定量は、ローリーの変法(Petersonら、Anal. Biochem.83:6654-6659(1977))を用いた。
NHS-activated sepharose4 Fast Flow(GE healthcare)のプロトコールを参考に抗E抗体をセファロースに固定化した。500μlのNHS-activated sepharoseに7.5mlの氷冷1mM HClを加え混合した後、遠心し(3,000rpm、5分、4℃)、氷中で5分間静置後、50%スラリーとなるように情勢を除き洗浄した。抗E抗体溶液をmicrocon(登録商標)YM-30(MILLIPORE)により限外ろ過(15,000rpm、24分、4℃)し、240μlの0.2M NaHCO3、0.5M NaCl(pH8.3)を加えることによりバッファー交換を行った。精製した抗E抗体を洗浄したNHS-activated sepharoseと混合し、ローテーターに取り付け室温で3時間撹拌させた。次に、遠心し(3,000rpm、5分、4℃)、氷中で、5分間静置し上清を除いた。さらに、750μlの0.1M Tris-HCl(pH8.5)を加え、ローテーターに取り付け室温で2時間撹拌した後、遠心し(3,000rpm、5分、4℃)、氷中で5分間静置し上清を除いた。1.5mlの0.5M NaClを含む0.5M酢酸バッファー(pH4.5)を加え混合後、遠心し(3,000rpm、5分、4℃)、氷中で5分間静置し上清を除いた。さらに、1.5mlの0.1M Tris-HCl(pH8.5)を加え、遠心し(3,000rpm、5分間、4℃)、氷中で5分間静置し上清を除いた。二つのバッファーからなる洗浄操作を3回繰り返した後に0.5mlとなるようTBSを加えた。
いずれのクローンでも誘導区の上清画分にタンパク質が検出され、結果として一本鎖抗体の種類に関わらず、溶菌していることが明らかとなった。
(pET-22b(+)γ+κの構築)
十分量の一本鎖抗体タンパク質を得るために、宿主・ベクター系にpETシステムを用いた。γ+κ5には、g+k リバースプライマー(表8)とg+k aFORプライマー(表8)を用いてPCR法にて5’側にSalIとNdeI、3‘側にXhoI制限酵素サイトを付加した。反応条件は、94℃、3分間、次に94℃、30秒間、55℃、30秒間、72℃、30秒間のサイクルを25サイクルとした。1.5%アガロースゲルで電気泳動後、精製してからNdeIとXhoIを用いて消化した。pET-22b(+)も同様に消化しライゲーションさせ、pET-22b(+)-γ+κ5とpET-22b(+)-γ+κ19を構築した。得られたプラスミドDNAを用いて大腸菌DH5αを形質転換後、2×YT(Amp)寒天培地に塗布し、37℃で16時間培養した。目的の遺伝子が含まれているかコロニーPCRにより確認し、陽性コロニーからプラスミドDNAを調製した。さらに挿入フラグメントのDNA塩基配列を確認した。
CA法(Machida、2000)を用いて、封入体として発現した一本鎖抗体のリフォールディングを行った。封入体懸濁液288μl(タンパク質濃度25mg/ml)を8Mグアニジン塩酸塩900μlおよび4M DTT 12μlと混合して、室温で1時間反応させた。反応産物570μlを2本に分け、70倍容量の最終濃度2mMのDL-シスチンを含む0.1%SB3-14/PBS、または0.1%CTAB/PBSと混合し、室温で1時間反応させた。それぞれに3%シクロアミロース10mlを加え室温で一晩反応させた。反応産物を遠心し(12,000rpm、5分)、上清を回収し、リフォールディング溶液とした。
pET-22b(+)-γ+κ5を用いてBL21(DE3)を形質転換した。得られた形質転換体をLB液体培地(50μg/ml Ampicillin)中にて37℃で一晩培養し、新たな培地に1%になるように植菌した。37℃で培養し、A600が、0.5~0.6の時に終濃度1mMになるようIPTGを添加後、さらに4時間培養した。培養液を6,000×gで10分遠心し、得られた菌体をリン酸バッファー(PBS)で3回洗浄した。菌体をPBSに再懸濁し、超音波破砕後10,000×gで10分遠心し、沈殿をγ+κ5の封入体として得た。
γ+κ5の封入体湿重量50mgを150μlのPBSに懸濁し、8Mグアニジン塩酸塩溶液450μlと4Mジチオスレイトール溶液6μlを加えて混合し室温で1時間以上静置した。次に2mMのDL-シスチン(終濃度)を含む0.1%CTAB/PBS溶液を40ml加えて混合し、室温で1時間静置した。その後3%シクロアミロースを10ml加えて混合し、室温で一晩静置した。反応溶液を12,000rpmで5分遠心し、上清を回収してリフォ-ルディング溶液とした。
溶出後の再凝集を防ぐために、透析速度および効率の条件を最適化した。リフォールディング溶液の1/25量の50%ニッケルアガローススラリーを加え、ローテーターで30分間、4℃で穏やかに撹拌した。その後1,000×gで5分間遠心し、ニッケルアガロースを回収した。ニッケルアガロースは当量のPBSで2-3回洗浄し、2Mイミダゾールを含むPBSを500μl加えて溶出し、1Mイミダゾールを含むPBSでさらに数回溶出した。溶出した画分を、セルロースエステル膜チューブを用いてサンプル容量の400倍のPBSで透析し、精製された一本鎖抗体を得た。得られた一本鎖抗体の純度と濃度はSDS-PAGEにより評価した(図10)。収量は、培養液1Lあたり3~5mgだった。
(1.ダイレクトELISA)
直接ELISA法により、精製した一本鎖抗体の抗原認識活性を評価した。抗原として、LDL、アセチル化LDL、部分酸化LDL、完全酸化LDLを用いた。ポリスチレン製96穴マイクロウェルプレートに0.05-10μg/mlの抗原溶液を100μl/well添加し4℃で一晩吸着させた。プレートをPBS(200μl/well)で3回洗浄し、ブロッキングバッファー(0.25%BSA/PBS)200μl/wellを添加し室温で2時間静置した。プレートをPBS(200μl/well)で3回洗浄した後、2.5μg/ml/PBSの一本鎖抗体100μl/wellを添加し室温で1時間反応させた。プレートをPBS(200μl/well)で5回洗浄し、ブロッキングバッファーで1,000倍に希釈した抗His-HRP抗体100μl/wellを添加し、室温で1時間反応させた。プレートをPBS(200μl/well)で5回洗浄した後、3,3’,5,5’-テトラメチル-ベンジジン(TMB)を50μl/well添加し発色状態を確認し、1NHCl50μ/wellを添加して反応を停止した。その後プレートの各ウェルの450nmにおける吸光度を測定した(図11)。
図11が示すように、一本鎖抗体γ+κ5は、抗原として用いたLDL、アセチル化LDL、部分酸化LDLおよび完全酸化LDLの全てを認識するが、各抗原に対する特異性が異なることが明らかになった(図12)。γ+κ5は、酸化LDLを低濃度でLDLよりも強く認識する特徴を持つと考えられる。また各抗原の検出限界は、LDLが5ng/100μlウェル、アセチル化LDLが10ng/100μlウェル、部分酸化LDLが10ng/100μlウェル、および完全酸化LDLが20ng/100μlウェルであった。(図11)。検出限界は、希釈段階の異なる測定試料の内、検出が可能であった最低濃度を検出限界として算出した。
また酸化LDL受容体であるLOX-1のリガンド認識領域(CTLD14)を用いて、サンドイッチELISA法により酸化LDLの認識能を評価し、間接ELISA法(CTLD14(Hisタグを除去したもの)と一本鎖抗体によるサンドイッチELISA)によりアセチル化LDL、部分酸化LDL、完全酸化LDLの認識評価を行った。ポリスチレン製96穴マイクロウェルプレートに20μg/ml/PBSのCTLD14を50μl/well添加し、一晩吸着させた。プレートをPBS(200μl/well)で3回洗浄し、ブロッキングバッファー(0.25%BSA/PBS)250μl/wellを添加し室温で2時間静置した。プレートをPBS(200μl/well)で3回洗浄し、0.05-10μg/mlの抗原溶液(LDLまたは酸化LDL)を100μl/well添加して室温で2時間反応させた。プレートをPBS(200μl/well)で5回洗浄し、2.5μg/ml/PBSの一本鎖抗体100μl/wellを添加し室温で1時間反応させた。その後は直接法と同様にプレートをPBS(200μl/well)で洗浄し、抗His-HRP抗体反応の後、TMB反応を行い、反応停止後450nmにおける吸光度を測定した(図13)。
次に別の変性LDLの検出が可能であるかどうかを確認するために、他の種類の変性LDLを用いて、同様の実験を行う。
実施例6に準じ、対象として酸化LDL等の代わりにマロンジアルデヒド修飾LDLを用いて同様の実験を行う。
20ng/ウェル程度のマロンジアルデヒド修飾LDL(MDA-LDL)の存在を検出可能であると期待される。
本実施例では、LDLをより簡便に検出するために、ラテラルフローアッセイの原理を用いた検出系を構築する(図14~図16)。
検出試薬として、金属コロイドで標識されたCTLD14または一本鎖抗体を用いた。検出部に、CTLD14または一本鎖抗体を塗布し、コントロール部に、ストレプトアビジン(CTLD14がビオチン化されている場合)、抗His抗体(CTLD14がHisタグを含む場合)を塗布したラテラルフロー用メンブレンを用いる。血清、血漿または全血などの試料をサンプルパッドに添加し、テストランの発色、またはテストライン/コントロールラインの発色の比により、試料中の変性LDL活性を評価する。
代表的に、30分以内に目視で変性LDLの存在を検出できる。
次に、本実施例では、抗LDLニワトリ抗体およびLOX-1をラテラルフロー形式で用いる、酸化的修飾を受けたLDL検出および定量システムを開発する。
抗LDLニワトリ抗体はジャパン・ラム株式会社に依頼して製造した。詳細には以下のとおりである。抗LDLニワトリ抗体は、抗原(LDL)0.3mg(アジュバンドFCA)2週間毎に、6回、ニワトリ皮内に接種後、力価の上昇を5回目以降にチェックし、6回目の追加免疫後に全採血したものから採取した。抗体活性測定は、抗原(LDL)1μg/mlを100μl/wellで固相し、通常のELISAに準じて行った。ここでは、標識2次抗体として、HRP標識抗ニワトリIgY(例えば、Promega等から入手可能)を使用した。
本実施例により、30分以内に目視で変性LDLの存在を検出できることが示される。
次に、本実施例では、抗LDLニワトリ抗体およびLOX-1をラテラルフローアッセイ形式で用いる、脂質異常、動脈硬化、糖尿病、虚血性心疾患、脳血管障害等の変性LDLに関連する疾患の検診のためのシステムを開発する。
実施例9に準じて行うことができる。
本実施例により、30分以内に目視で変性LDLの存在を検出でき、それに基づいて、脂質異常、動脈硬化、糖尿病、虚血性心疾患、脳血管障害等の変性LDLに関連する疾患の検診を短時間で行いうることが示される。
本実施例では、抗LDLニワトリ抗体およびLOX-1をラテラルフローアッセイ形式で用いる、食品摂取による脂質異常、動脈硬化、糖尿病、虚血性心疾患、脳血管障害等の変性LDLに関連する疾患の予防効果の評価システムを開発する。
実施例9に準じて行うことができる。
本実施例により、30分以内に目視で変性LDLの存在を検出でき、それに基づいて、食品摂取による脂質異常、動脈硬化、糖尿病、虚血性心疾患、脳血管障害等の変性LDLに関連する疾患の予防効果を短時間で評価しうることが示される。
本発明の比較として、従来法である大腸菌発現系を用いて封入体を還元条件下で変性する条件でCTLD14を合成した。以下にそのプロトコールを示す。
(透析緩衝液 (1~5) *aの準備)(*a:透析緩衝液1-5とは下記の緩衝液1から5を示し、プレミックスとは、緩衝液1-5の各成分のうちグルタチオン以外のすべての試薬を混合した状態のものを示す。)
・グルタチオン(還元、酸化)以外すべての試薬を混合した。
・pHを各々の緩衝液のために適切なものに調整した
・4℃で貯蔵した。
・タンパク質濃度を1mg/mlに調整した(希釈緩衝液:緩衝液C:緩衝液C: 6M グアニジンHCl, 50mM トリス, pH8.0)
・チューブ(透析チューブ、Spectra/Por 7, RC, MWCO 8,000)を使用した。各々の潮汐緩衝液(1-5)(1~5は下記参照)は、使用前に調製した。
・必要な容積のグルタチオン(還元、酸化)溶液を解凍した。
・透析緩衝液プレミックスおよびグルタチオン溶液を混合した。
・適切な容積に合わせ、十分に混合した。
・タンパク質濃度を1mg/mlに調整した(透析緩衝液:緩衝液C)
・緩衝液1(50mM Tris-Cl, pH 8.5, 4M グアニジンHCl, 0.4M L-アルギニン, 0.4M NaCl, 10%グリセロール、 0.5mM 酸化グルタチオン、5mM還元グルタチオン)に対して4℃で12時間透析した。・緩衝液2(50mM Tris-Cl, pH 8.5, 3M グアニジンHCl, 0.4M L-アルギニン, 0.4M NaCl, 10% Glycerol, 0.5 mM酸化グルタチオン, 5 mM還元グルタチオン)に対して4℃で12時間透析した。
・緩衝液3(50mM Tris-Cl, pH 8.0, 2M グアニジンHCl, 0.4M L-アルギニン, 0.4M NaCl, 10% Glycerol, 0.5mM酸化グルタチオン, 5mM還元グルタチオン)に対して4℃で12時間透析した。
・緩衝液4(50mM Tris-Cl, pH 8.0, 1M グアニジンHCl, 0.4M L-アルギニン, 0.4M NaCl, 10% Glycerol, 0.5mM酸化グルタチオン, 5mM還元グルタチオン) に対して4℃で12時間透析した。(この時点で凝集し始めた)
・緩衝液5(50mM Tris-Cl, pH 8.0, 0.4M L-アルギニン, 0.4M NaCl, 10% グリセロール, 0.5mM酸化グルタチオン, 5mM還元グルタチオン)に対して4℃で12時間透析した。
・TBS(pH7.6)(20mM Tris-Cl, pH 7.6, 150mM NaCl)に対して4℃で48~96時間(3回緩衝液交換する。1回の緩衝液交換は12時間以上間隔をあけた。)透析した。
・チューブ内のタンパク質溶液を収集した。
・これを超遠心分離(100,000×g、60分、4℃)で行い、リフォールドしたタンパク質を収集した。
精密法による精製結果を、図17に示す。
本実施例では、遺伝子組換えカイコによる生産を試みた。
(系統作製、発現等)
ビオチン化タグ(BioEaseTM)が付加されたCTLD14のプラスミド構築は、ライフテクノロジーズ社のpcDNATM6 BioEaseTMGateway(登録商標)Biotinylation Systemを用い、基本的にメーカーのマニュアルに従って行った。
凍結融解処理により中部絹糸腺抽出物からセリシンを除去した溶解液をPBSで平衡化したNi-Agaroseに結合させ、PBS中のイミダゾール濃度を段階的に上げることによりCTLD14を溶出した。250mM-1Mイミダゾール溶出画分を収集しPBSで透析し、さらに精製度をあげる必要がある場合は、TALON(Niの代わりにCoを使用した金属キレートアフィニティー)による精製に供した。200-500mMイミダゾール溶出画分を収集しPBS(-)で透析後に精製CTLD14とした。(図23参照)
(糖ペプチド分析)
PNGaseF処理、無処理の試料を15%SDS-PAGEで泳動後、クマシー染色し、蛋白質バンドを切り出した。切り出したゲル片に含まれるカイコ型CTLD14をアセトアミド化後トリプシン消化し、MALDI-TOFMSLC-ESMSで測定した。PNGaseF処理区および無処理区のマススペクトルを比較し、N結合型糖鎖が付加されると想定される領域を含むアミノ酸配列を観測した。
精製後のCTLD14をβメルカプトエタノール(2ME)を含むSDS-PAGE用サンプルバッファー(+)、あるいは、2MEを含まないSDS-PAGE用サンプルバッファー(-)で処理後に、SDS-PAGEに供し、クマシー染色し、ダイマー形成を確認した。
発現したものの解析結果を図21および22に示す。図21に示されるように、タグの異なるCTLD14(biotinタグ、mycタグ;精製用の共通タグとしてHisタグ)を発現する複数の系統を樹立した。各系統の発現を確認した。図21に示されるように、左:クマシー染色、右:ウェスタンブロットにおいてHisタグによる検出がなされた。図中「←1」で示されるように、Biotin_CTLD14(BioEase タグ付き)の明瞭な発現が観察される。また、分子量も推定分子量(蛋白質部分のみで29kDa、糖鎖が付加されて29kDa以上となる)に一致していた。
中部絹糸腺を1本/1ml(一頭/2ml)のPBS+1% Triton X-100で抽出後、Ni-NTA,TALONの2種類のカラム処理により容易に精製することができた(およそ4日程度以内で可能であった)。また、量的にも0.3mg/頭の供給が可能であった。(図23)
(配列)
本実施例で製造した改良型CTLD14(カイコ型糖鎖付加 ビオチン化CTLD14)のアミノ酸配列および糖鎖修飾に関する情報を図19に示す。
糖鎖については、上記のような分析を行い、カイコ由来CTLD14の糖鎖付加の確認を行い、図24に示されるような結果を得た。図24に示されるように、PNGaseF処理による糖鎖除去により分子量シフトが確認された(図24左)。また、NGaseF処理前後での糖タンパク質染色の結果をみると、PNGaseF処理により糖タンパク質として検出されなくなっていた(図24中)。加えて、PNGaseF処理前後の試料をウェスタンブロット後に抗LOX-1抗体で検出した結果、いずれも抗LOX-1抗体で検出された(図24右)。
電気泳動ゲル片に含まれるカイコ型CTLD14を用いて作成した試料を、還元アルキル化後トリプシン消化し、得られたペプチドをマトリクス支援レーザー脱離イオン化飛行時間型(MALDI-TOF)質量分析装置と液体クロマトグラフ(LC)-エレクトロスプレーイオン化キングドントラップ型(ESI)質量分析装置で測定した。MALDI-TOFMSでは、AB SCIEX社製4800 plus TOF/TOF Analyzerにおいて、マトリクスとしてα-シアノ-4-ヒドロキシ桂皮酸を用い、窒素ガスレーザー337nmを試料に照射してイオン化させ、強度の大きいイオンから順にMS/MSスペクトルを採取した。LC-ESIMSでは、Thermo Fischer Scientific社製Orbitrap Veros Proを用い、Thermo Fischer Scientific社製ナノLC(EASY nLC)によりEASY Sprayカラム(PepMapC18,3μm, 75μmx15cm,Thermo Fisher Scientific)で分離(300nL/min,0.1%ギ酸を含むアセトニトリルの濃度を0%から100%に30分で上昇、10分維持するグラジエントプログラム)したペプチドをイオン化し、それぞれのMS/MSスペクトルを取得した。いずれの手法においても正イオンモードでイオンを検出した。
2-メルカプトエタノール(β-ME)処理によりダイマー位置の蛋白質バンドがモノマー位置にシフトすることが確認されたCTLD14はカイコ体内でもダイマーを形成することが確認された(図25)。
本実施例では、カイコ型CTLD14を用いた評価系の開発を行った。この場合、CTLD14と組み合わせる抗体が必要である。
(抗LDLニワトリポリクローナル抗体)
本実施例で使用した抗LDLニワトリポリクローナル抗体の作製方法は実施例9と同じである。
市販の抗体(ファーマフーズ, HUC20)を購入して使用した。
市販の抗体(Acris BM2149)を購入して使用した。
検出対象として、LDL、アセチル化(Ac)LDL、マロンジアルデヒド化(MDA)LDL、部分酸化(moOx)LDL、完全酸化(fuOx)LDL、を用いた。LDL(バイオメディカルテクノロジーズ,BT-903)とAcLDL(バイオメディカルテクノロジーズ,BT-906)は市販されているものを使用した。MDA-LDLは、市販のLDLをマロンジ化アルデヒドと反応させて調製した。酸化LDLは、市販のLDLを酸化銅と反応させて調製した。37℃で4時間反応させたものを部分酸化とし、37℃で20時間反応させたものを完全酸化とした。抗ApoBニワトリモノクローナル抗体は市販のもの(ファーマフーズ、HUC20)を用いた。二次抗体には抗ニワトリ IgY-HRP コンジュゲート(プロメガ、G1351)を用いた。抗ApoBマウスモノクローナル抗体は市販のもの(Acris、BM2149)を用いた。二次抗体にはHRP標識抗マウスIgG(ミリポア、AP192P)を用いた。
検出対象のLDL類(LDL、アセチル化LDL、部分酸化LDL、完全酸化LDL)をポリスチレン製96穴マイクロウェルプレートに0.2,1,5μg/mlの濃度で100μl/well添加し4℃で一晩吸着させた。プレートをPBS(200μl/well)で3回洗浄し、ブロッキングバッファー(0.25%BSA/PBS)250μl/wellを添加し室温で2時間静置した。プレートをPBS(200μl/well)で3回洗浄した後、ブロッキングバッファーで3,000倍希釈した抗ApoBニワトリポリクローナル抗体、またはブロッキングバッファーで8,000倍希釈した抗ApoBニワトリモノクローナル抗体100μl/wellを添加し、室温で1時間反応させた。プレートをPBS(200μl/well)で5回洗浄し、ブロッキングバッファーで2,000倍希釈したHRP標識抗ニワトリIgY(プロメガ,G1351)100μl/wellを添加し、室温で1時間反応させた。プレートをPBS(200μl/well)で5回洗浄した後、3,3’,5,5’-Tetramethyl-benzidine(TMB)を50μl/well添加し発色状態を確認し、1NHCl50μ/wellを添加して反応を停止した。その後プレートの各ウェルの450nmにおける吸光度を測定した。
結果を図26に示す。左には、抗LDLニワトリポリクローナル抗体によるLDLの検出を示す。完全酸化LDL(fuOxLDL)、MDA-LDL、AcLDL、未変性LDLの全てを認識可能であることがわかる。図26中では、抗ApoBニワトリモノクローナル抗体によるLDLの検出を示す。完全酸化LDL(FoxLDL)認識化能は高いが、修飾の種類によって認識強度が一定しないことがわかる。図26右には、抗ApoBマウスモノクローナル抗体によるLDLの検出を示す。全般に認識能が低く、酸化LDLと未変性LDLの認識能に大差がないことがわかる。
次に、実施例13の結果を踏まえ、抗LDLニワトリポリクローナル抗体とCTLD14の組合せにより、動脈硬化発症の真の危険因子の検出系の開発を試みた。
大腸菌型CTLD14は比較例1に記載のように製造した。
(材料)
ダイレクト検出法に加え、上述のように比較例1および実施例12に記載される方法に基づき、カイコ及び大腸菌で発現・精製したCTLD14を用いた。
(CTLD14とニワトリ抗体のサンドイッチ法)
ポリスチレン製96穴マイクロウェルプレートに20μg/ml/PBSのCTLD14(カイコ発現、大腸菌発現)を50μl/well添加し、一晩吸着させた。プレートをPBS(200 μl/well)で3回洗浄し、ブロッキングバッファー(0.25% BSA/PBS)250μl/wellを添加し室温で2時間静置した。プレートをPBS(200μl/well)で3回洗浄し、0.05-8μg/mlのLDL類(LDL、アセチル化LDL、部分酸化LDL、完全酸化LDL)を100μl/well添加して室温で2時間反応させた。プレートをPBS(200μl/well)で5回洗浄し、ブロッキングバッファーで6,000倍希釈した抗LDLニワトリポリクローナル抗体、またはブロッキングバッファーで8,000倍希釈した抗ApoBニワトリモノクローナル抗体100μl/wellを添加し、室温で1時間反応させた。プレートをPBS(200μl/well)で5回洗浄し、ブロッキングバッファーで2,000倍希釈したHRP標識抗ニワトリIgY(プロメガ,G1351)100μl/wellを添加し、室温で1時間反応させた。プレートをPBS(200μl/well)で5回洗浄した後TMBを50μl/well添加し発色状態を確認し、1NHCl 50μl/wellを添加して反応を停止した。その後プレートの各ウェルの450nmにおける吸光度を測定した。
大腸菌型CTLD14と、本出願抗LDLニワトリ抗体の組み合わせの結果を図27に示す。図27左には、大腸菌型CTLD14と抗LDLニワトリポリクローナル抗体によるサンドイッチ法による検出を示す。低濃度領域(0.1μg/well、溶液濃度にして1μg/ml以下)では、酸化的修飾を受けたLDLの特異的な検出が可能であることがわかる(moLDL:部分酸化LDLに関しては、LDLとの有意差無し)。図27右には大腸菌型CTLD14と抗apoBニワトリモノクローナル抗体によるサンドイッチ法による検出を示す。fuOxLDL:完全酸化LDLの検出は良好であるが、MDA-LDL、AcLDL、moOxLDLの認識が弱い。代表的なLDLであるMDA-LDLの検出も困難であることがわかる。
実施例14と同様の手法を用いて、対象として、糖化LDLを用いた実験を行う。
比較対象として、従来の技術であるE.coli発現系でsRAGEの発現を行った。以下の詳細を示す。
宿主細菌細胞としてOrigamiB(DE3)を用いた。目的RAGE遺伝子クローニングサイトとして、5’側はNdeI,3’側にはXhoI(BamHI,SmaI,HindIIIもまた使用可能)を使用する。その結果、目的タンパク質のC末側にビオチンリガーゼによって認識されるビオチン化アミノ酸配列が付加される。RAGEの場合、N末側が細胞外領域でありAGEの認識に必須な領域であるため、チップ化などに際してC末側で固定化が可能なように、C末側にビオチン化アミノ酸配列を付加した。この手順を用いて、配列番号97のアミノ酸配列のC末端側にビオチン化アミノ酸配列「GLNDIFEAQKIEWHE」(配列番号98)をコードする核酸配列を連結し、T7プロモーターの制御下に配置したプラスミドを作製した。このプラスミドと、ビオチンリガーゼ遺伝子を発現するプラスミド(ColE1系であるpETベクターと不和合性グループが異なるため同一細胞内で安定に共存しうるp15A系プラスミドであるpACベクターにビオチンリガーゼ遺伝子を挿入したプラスミド)の両方を用いて宿主細菌細胞を形質転換した。この形質転換した宿主細胞の培養に使用した培地は、50μg/mlアンピシリン、34μg/mlクロラムフェニコール、15μg/mlカナマイシン、12.5μg/mlテトラサイクリンを添加した、改変LB培地(0.5%NaCl、0.5%酵母エキス、1%トリプトン)である。25℃で一晩培養した前培養液を、5%の最終濃度になるように培地に添加し、25℃で攪拌培養した。なお、本培養への添加量を5%以上にして、誘導開始までの培養時間を短縮することも可能である。また、前培養を37℃で行なった場合、添加量が5%を越えると可溶性として得られる割合が減少した。培養後に、A600での吸光度が0.5~0.7に達した段階で、1mMIPTG、50μM d-biotin(D型[(+)-cis-ヘキサヒドロ-2-オキソ-1H-チエノ-(3,4)-イミダゾール-4-吉草酸)](いずれも最終濃度)を添加し、目的タンパク質の誘導とビオチン化を開始し、さらに18時間攪拌培養をすることにより、目的とするタンパク質を可溶性ビオチン化タンパク質として発現させることができた。なお、培養温度37℃では、目的タンパク質は、100%近く不溶性となるが、培養温度を25℃に下げることにより、発現した目的タンパク質の内、80%程度を可溶性タンパク質として発現させることが可能であった。
菌体を収菌後、溶解緩衝液(プロテアーゼ阻害剤を添加した20mM Tris,150mM NaCl,10mMイミダゾール、pH7.6)に懸濁し、超音波破砕した後、遠心した上清を租溶解液(可溶性画分)とした。N末端に存在するHisタグを利用しNiアガロースカラムに結合させ、FPLCシステムによるイミダゾール濃度勾配により溶出を行なった。可溶性ビオチン化RAGEを含む画分を回収し溶解緩衝液にて透析後、AviTag(C末)(「GLNDIFEAQKIEWHE」(配列番号98)を利用しストレプトアビジンムテインマトリックス(Roche社製)カラムに結合させた。3mM d-biotinにて溶出することにより精製可溶性ビオチン化RAGEを得た。1L当りの培養から、最終的に5mgの精製可溶性ビオチン化RAGEを得ることが可能であった。
E.coli発現系でsRAGEの発現は可能だが、非常に不安定で、1.5ヶ月程度で100%近くが断片化し、認識能を失う。断片化部位のアミノ酸変異、認識に必須な部位のみを安定化したminiRAGEの作出にも行ったが、細胞外領域全長(sRAGE)に比べ、発現量が低く、また認識能も劣る。(図38)。
そこで、本実施例では、本発明において着目した遺伝子組換えカイコによるsRAGEの生産を試みた。手短に述べると、sRAGEベクターをカイコ卵にマイクロインジェクションを行い、RAGE発現カイコを得た。さらに、中部絹糸腺で発現可能な系統と交配し、中部絹糸腺(抽出が容易で純度も高い)にRAGEを発現する遺伝子組換えカイコを作出した。模式図を図33に示し、以下に詳細なプロトコールを提示する。
(系統作製、発現等)
pBac[Ser-UAS/3xP3EGFP](Ken-ichiro Tatematsu, Isao Kobayashi, Keiro Uchino, Hideki Sezutsu, Tetsuya Iizuka, Naoyuki Yonemura, Toshiki Tamura, Transgenic Research, 19, 473 (2010))のUAS(Upstream Activation Sequence)配列の下流に、PCRで増幅したフィブロインH鎖のシグナルペプチドをコードする断片およびsRAGEをコードする断片を挿入することによりUASベクターを構築した。この発現ベクターを用いて遺伝子組換えカイコを作出した。農業生物資源研究所で維持されている白眼・白卵・非休眠系統のw1-pnd系統を宿主系統として用いた。得られた遺伝子組換えカイコを中部絹糸腺でGAL4を発現する系統(Ken-ichiro Tatematsu, Isao Kobayashi, Keiro Uchino, Hideki Sezutsu, Tetsuya Iizuka, Naoyuki Yonemura, Toshiki Tamura, Transgenic Research, 19, 473 (2010))のUAS(Upstream Activation Sequence)と交配した。得られた次世代カイコのうち、GAL4コンストラクトとUASコンストラクトを共に持つ個体を選抜マーカーにより選抜した。5齢6日目の幼虫を解剖し、中部絹糸腺を摘出した。1本当たり1mLのPBS+1% Triton X-100の抽出液で、4℃、2時間振とうすることにより、タンパク質を抽出した。
凍結融解処理により中部絹糸腺抽出物からセリシンを除去した溶解液をPBSで平衡化したNi-Agaroseに結合させ、PBS中のイミダゾール濃度を段階的に上げることによりsRAGEを溶出した。250mM -1Mイミダゾール溶出画分を収集しPBSで透析し、さらに精製度をあげる必要がある場合は、TALON(Niの代わりにCoを使用した金属キレートアフィニティー)による精製に供した。100mM-1Mイミダゾール溶出画分を収集しPBS(-)で透析後に精製sRAGEとした(図36参照)。
PNGaseF処理、無処理の試料を15%SDS-PAGEで泳動後、クマシー染色し、蛋白質バンドを切り出した。切り出したゲル片に含まれるカイコ型sRAGEをアセトアミド化後トリプシン消化し、MALDI-TOFMS LC-ESMSで測定した。PNGaseF処理区および無処理区のマススペクトルを比較し、N結合型糖鎖が付加されると想定される領域を含むアミノ酸配列を観測した。
発現したものの解析結果を図35に示した。遺伝子組換えカイコ4系統(1-4)、および、ネガティブコントロール(中部絹糸腺GALA4のみ、UAS無し:sRAGEを発現していない)から中部絹糸腺を取り出し、続いて、中部絹糸腺1本に1%Triton X-100を含むPBS 1mlを添加し4℃で2時間転倒混和し調製した。左は、4~12%グラジエントのSDS-ポリアクリルアミドゲルにより試料を電気泳動後に、クマシー・ブリリアント・ブルーによる蛋白質染色した結果である。各レーンへの試料の添加量は左側が5μl/lane, 右側が、10μl/laneである。M分子量マーカー(ベンチマーク:インビトロジェン)、1-4は、作出された遺伝子組換えカイコの系統番号を、Nega:ネガティブコントロールを示す。右はウェスタンブロットの結果であり、4~12%グラジエントのSDS-PAGEにより試料を展開後にPVDF膜に転写し、一次抗体として抗Hisウサギ抗体(1000倍希釈)+抗Hisマウス抗体(3000倍希釈)を用い、続いて二次抗体であるHRP標識抗ウサギIgG&抗マウスIgG抗体(200,000倍希釈)と反応させ、最後にECL Primeと反応させ生じる発光を検出し、sRAGEの発現を確認した。また、分子量も推定分子量(タンパク質部分のみで44kDa、糖鎖が付加されて、44kDa以上の分子量となる)に一致していた。
本実施例で製造したsRAGEの配列を図34に示す。カイコの中部絹糸腺発現タンパク質のN結合型糖鎖の構造は、以下の通りであり、sRAGEはカイコ中部絹糸腺に特異的に発現させている。中部絹糸腺に発現させた糖タンパク質の糖鎖は、中部絹糸腺以外では認められるバウチマンノース型(フコースあり)糖鎖や高マンノース型糖鎖は殆ど無く、複合型糖鎖、ハイブリッド型糖鎖もしくは、オリゴマンノース型糖鎖である。糖鎖解析の結果、糖鎖付加部位は、N-I-T、および、N-G-Sのアスパラギン酸であると決定された。カイコの中部絹糸腺発現タンパク質のN結合型糖鎖の構造の例を、図20および図54に示す。
糖鎖については、上記のような分析を行い、カイコ由来sRAGEの糖鎖付加の確認を行い、図39に示されるような結果を得た。図39に示されるように、PNGaseF処理による糖鎖除去により分子量シフトが確認された(図39左)。また、NGaseF処理前後での糖蛋白染色の結果。PNGaseF処理により糖蛋白として検出されなくなっていた(図39中央)。加えて、PNGaseF処理前後の試料をwestern blot後に抗RAGE抗体で検出した結果。いずれも抗RAGE抗体で検出された(図39右)。
電気泳動ゲル片に含まれる、カイコ型糖鎖を有するsRAGEをPNGaseF処理したものとしないものについて、還元アルキル化後トリプシン消化した。得られたペプチドをマトリクス支援レーザー脱離イオン化飛行時間型(MALDI-TOF)質量分析装置と液体クロマトグラフ(LC)-エレクトロスプレーイオン化キングドントラップ型(ESI)質量分析装置で測定した。
装置:AB SCIEX社製4800 plus TOF/TOF Analyzer
マトリクス:α-シアノ-4-ヒドロキシ桂皮酸
レーザー:窒素ガスレーザー337 nm
MS/MS:強度の大きいイオンから順にMS/MSスペクトルを採取した。
装置(MS): Thermo Fischer Scientific社製Orbitrap Veros Pro
装置(LC): Thermo Fischer Scientific社製ナノLC(EASY nLC)
カラム:EASY Sprayカラム(PepMapC18, 3μm, 75μmx15cm, Thermo Fisher Scientific)
溶離液:300 nL/min, A) 0.1% ギ酸、B)0.1%ギ酸を含むアセトニトリル、Bの濃度を 0%から100%に30分で上昇、10分間維持するグラジエントプログラムを使用した。
次に、比較例で製造した、E.coli型のsRAGEおよび実施例16で製造したカイコ型糖鎖を含むsRAGEの安定性を確認した。以下に手順を示す。
(材料)
精製直後のE.coli型、および、カイコ型sRAGE、並びに、精製後4℃に40日間保存したE.coli型sRAGE,精製後4℃に90日間保存したカイコ型sRAGEを用いた。
精製直後のE.coli型、および、カイコ型sRAGE 10μg(10μl)をSDS-PAGE用サンプルバッファー5μlで処理し、マイナス20℃で保管した。精製後4℃に40日間保存したE.coli型sRAGE,精製後4℃に90日間保存したカイコ型sRAGEも同様に処理し、12%SDS-PAGEにて試料を泳動後にクマシー染色を行った。
結果を図38に示す。カイコ型sRAGEの安定性が示されている。左はE.coli型を示し、右は本発明のカイコ型を示す。それぞれ、1日、40日保存および1日、90日保存を示す。カイコ型糖鎖を含むsRAGEは非常に安定な分子であり、E.coli型が40日で断片化するのに対して、4℃での保存で長期にわたり(出願時点で半年まで確認)完全長を維持していた。
次に、本発明のsRAGEを用いて各種AGEsの検出が可能かどうかをサンドイッチELISAを用いて確認した。以下にその手順を示す。
(材料)
検出対象であるAGEsは、リボース(R),フルクトース(F),グルコース(G)存在下でBSAを無菌的にpHを一定に保ちつつ37℃で12週間保温することで調製した。コントロールとなるBSA(ctlBSA)は、糖非存在下で同様に反応させることで調製した。サンドイッチ様の検出システムに使用する抗体として、抗BSAマウスモノクローナル抗体(アブカム ab3781)を用い、二次抗体としてHRP標識抗マウスIgG抗体(メルクミリポア AP192P)を用いた。
ポリスチレン製96穴マイクロウェルプレートに5μg/ml/TBSのsRAGE(カイコ型、もしくは、E.coli型)を50μl/well添加し、一晩吸着させた。プレートを200μl/wellのTBST(TBS,0.05%Tween20)で3回洗浄し、Protein free blocking buffer(サーモサイエンティフィック)250μl/wellを添加し室温で2時間静置した。プレートをTBST(200μl/well)で3回洗浄し、0.01-8μg/mlのAGEs類(R-AGEs、F-AGEs、G-AGEs、Ctl-AGEs)を100μl/well添加して室温で2時間反応させた。プレートをTBST(200μl/well)で5回洗浄し、Protein free blocking bufferで6,000倍希釈した抗BSAマウスモノクローナル抗体100μl/wellを添加し、室温で1時間反応させた。プレートをTBST(200μl/well)で5回洗浄し、Protein free blocking bufferで5,000倍希釈したHRP標識抗マウスIgG抗体100μl/wellを添加し、室温で1時間反応させた。プレートをTBST(200μl/well)で5回洗浄した後TMBを50μl/well添加し発色状態を確認し、1N HCl50μ/wellを添加して反応を停止した。その後プレートの各ウェルの450nmにおける吸光度を測定した。
結果を図40に示す。左は、精製後1週間保存後のE.coli型sRAGEの認識活性を示し、右は精製後3ヶ月保存後のカイコ型sRAGEの認識活性を示す。E.coli型は40日程度で断片化するため、40日後には、AGEs認識能が失われ、左図で観察されるAGEs検出は不可能になる。他方、右図は、精製後90日後のカイコ型sRAGEによるAGEs検出結果である。90日を経過しても認識能を維持しており、精製後1週間目のE.coli型と変わらない検出が可能であることが確認された。このように、本発明のsRAGEは、きわめて安定に維持することができ、かつ、感度よく検出することもできることが理解される。
本実施例では、中部絹糸腺特異的に、ビオチンリガーゼ(BirA)を発現するカイコによるカイコ型CTLD14およびカイコ型sRAGEの産生を試みた。
標的配列USA下流にBioEase tag CTLD14(またはBioEase tag sRAGE)を挿入したUSA-Biotin tag CTLD14(またはUSA-Biotin tag sRAGE)、標的配列USA下流にビオチンリガーゼ(BirA)を挿入したUSA-BirA、ならびにGAL4を中部絹糸腺特異的に発現させるセリシン1プロモーター(Ser1)-GALの3つのコンストラクトを導入した遺伝子組換えカイコを作出した。このカイコは、中部絹糸腺特異的に、Biotin tag CTLD14(またはBiotin tag sRAGE)およびBirAを発現する。さらに、5令幼虫に1g当たり20μgのビオチンを添加した餌を与え、CTLD14またはsRAGEのビオチン化に必要なビオチンを供給した。繭を作る直前に中部絹糸腺を摘出し、Triton X-100/PBS(-)によりタンパク質を抽出した(図43を参照のこと)。
図44に、CTLD14およびsRAGEの発現をSDS-PAGE後のクマシー染色にて確認した結果を示す。「-」は、BirAを発現しない系統であり、各系統について、普通餌とビオチン餌の実験区を設け、ビオチン餌区では1g当たり20μgのビオチンを添加した餌を与えた。繭を作る直前に中部絹糸腺を摘出し、Triton X-100/PBS(-)により処理した抽出液を調製しSDS-PAGEに供した。Line 72は、Ser1-GAL4のみを導入してあり、USB-CTLD14(またはUSB-sRAGE)を導入していないネガティブコントロールである。sRAGEに関しては、line 72に対して、sRAGEを導入した系統では、sRAGEの分子量(約50kDa)の位置に発現が確認された。CTLD14は、sRAGEほど発現量が高くなかった。
次に、ウェスタンブロットにより各系統のビオチン化を確認した(図45)。SDS-PAGEにて抽出液を展開後にPVDF膜に転写し、HRP標識ストレプトアビジンと反応後に、ECLによる発光により、ビオチン化を確認した。通常、シグナルは黒で得られるが、白く抜けて見えるのは発光シグナルが強すぎる(すなわち、ビオチン化が大過剰である)ためである。ネガティブコントロール(line 72)ではシグナル検出できないが、(-)BirAのlineでは極めて弱いシグナルであるが、BirAの共発現(line 1~4)でシグナルが確認可能になる。分子量の大きなシグナルも同時に検出されるが、これは、CTLD14のダイマーであると思われる。さらに、ビオチン餌の投与により、極めて強いシグナルが観察された。BirA共発現とビオチン添加により、ビオチン化効率が劇的に向上することが確認された。
ビオチンと特異的に結合するMutein matrixと抽出液とを反応させ、処理前のLysate(L)、処理後のflow through画分(F)、Resin binding画分(R)をSDS-PAGEに供し、クマシー染色によるタンパク質バンド(矢印がsRAGEの位置)の濃さから、ビオチン化効率を見積もった(図46)。sRAGEのビオチン化効率は60%であると判断される(BirAの共発現無しでは、1%未満)。ビオチン化効率は、flow through画分(F)/Resin binding画分(R)比により計算した。
lysateをTALONによる精製を行った(図47左)。続いて、TALONにより精製されたCTLD14をMutein matrixに吸着させた後、3mMd-Biotinで溶出した。(sRAGEでも同様の処理を行った)。MUTEIN処理後のflow through画分(F)、Resin binding画分(R)の比からビオチン化効率は、30~40%と見積もられた。さらに、TALON,MUTEINカラム処理により、カイコ1頭当たり0.04mgのビオチン化CTLD14が、0.1mgのビオチン化sRAGEが得られることが明らかとなった。右図は、得られた精製タンパク質のウェスタンブロットの結果である。SDS-PAGEで展開後に、PVDF膜に転写し、抗LOX-1抗体(右上)、または、抗RAGE抗体(右下)と反応させた後、2次抗体であるHRP標識抗ウサギIgG抗体と反応させ、ECLによる化学発光により、得られたタンパク質が、CTLD14、RAGEで間違いないことを確認した。さらに、CTLD14に関しては、SDS-PAGEで安定して検出されていたダイマー位置の分子量に観察されるタンパク質がCTLD14のダイマーであることが確認され、カイコ型CTLD14はダイマーとして安定に存在することも示された。
E.coli型CTLD14は、脱塩処理により沈殿する、pH変化により沈殿するなど、金属コロイド修飾を困難にする特性を有している。一方、カイコ型CTLD14は、PBS(-)から塩を含まないリン酸バッファー(PB)に変換しても沈殿せずに活性を維持していた。さらに、カイコ型CTLD14は、E.coli型では沈殿を生じやすいTrisバッファーでも安定であった。また、使用可能なpH範囲も7.4~9.2と広かった。E.coli型CTLD14は、Trisバッファー(7.4~9.0)では安定した活性が得られなかった。したがって、カイコ型CTLD14は、塩の存在/非存在、pHの範囲、バッファーの種類にかかわらず安定であり(図48)、検出・評価系に活用する際の選択の幅が広いことが明らかになった。
図53に示す通り、E.coli型sRAGEは、1.5ヶ月以内に断片化し認識能を失うが、カイコ型は、4℃で1年近く保存しても安定であった。
本実施例では、カイコ型CTLD14により、ヒト血漿中の微量酸化LDLの検出が可能かどうかを検討した。
試料2:1 ug/ml OxLDL
試料3:3 ug/ml OxLDL
試料4:6 ug/ml OxLDL
試料5:12 μg/ml OxLDL
それぞれ、PBS(-)で1/1000に希釈した。
本実施例では、ビオチン化CTLD14を用いたラテラルフローアッセイの原理に基づく検出系の検討を行った(図50)。
検出試薬として、白金コロイド標識ビオチン化CTLD14を使用した。ハーフストリップは、ニトロセルロース膜の一方に濾紙を貼付して作製した。さらに、テストラインに抗ApoBニワトリモノクローナル抗体、または抗LDLニワトリポリクローナル抗体を塗布し、コントロールラインにストレプトアビジンを塗布し、37℃で2時間乾燥して常温保存した。
本実施例では、カイコ型sRAGEの糖鎖構造の同定を行った。
(シングルサイクルカイネティックスを用いた表面プラズモン共鳴による反応速度論解析)
ビオチン化されていない場合、アミンカップリングによる固定化が一般的であるが、認識に重要なLysが修飾される可能性、固定化の過程で失活する可能性、方向性を維持した固定化は不可能などの問題により、認識能を維持できず、AGEsを添加しても結合が確認できないことがある。図56の左図は、アミンカップリングによりCM5チップ上に固定化した場合の、カイコ型sRAGEとAGEs(F-AGE)との相互作用解析を示す。矢印で示した時点でF-AGEsを添加しているが、結合(縦軸:RUの増大)が弱く、解離速度も速かった。本来の結合能を維持しているのか不明であり、相互作用解析は不可能であった。一方、ビオチン化sRAGEのビオチンを介してストレプトアビジンチップに固定する場合、ビオチンは、C末端側の1箇所のみに導入されているため、方向性を維持したまま固定化が可能である。また、固定化は中性条件下でsRAGE溶液を添加するのみでsRAGEの活性を維持したまま行える。図56の右図に示す通り、AGEs添加(添加濃度は0.25μg、0.5μg、1μg、2μg、4μgのように徐々に上げている)により、結合-解離のサイクルが観察された。得られたセンサーグラムからBIA evaluation Softwareにより速度論的解析を行った。その結果、カイコ型sRAGE_F-AGEsに関して、結合速度定数:7.25E+03、解離速度定数:1.64E-03、解離定数:2.2e-7であった。一方、E.coli型の場合、結合速度定数:8.61E+03、解離速度定数:2.41E-03、解離定数:2.8e-7であり、AGEsとの相互作用においてカイコ型とE.coli型に大きな差は無いことが示された。さらに、この解離定数は、動物細胞上でのRAGE_F-AGEの解離定数とも近く、細胞上でのRAGEの認識能をカイコ型sARGEも再現していることが示された。
本実施例では、一本鎖抗体γ+κ5のカイコにおける生産を行った。
配列番号2:プラスミドpCR2.1-TOPOに用いる配列決定用プライマーM13reverse
配列番号3:プラスミドpCANTAB-5Eに用いる配列決定用プライマーpCANTAB5-S1
配列番号4:プラスミドpCANTAB-5Eに用いる配列決定用プライマーpCANTAB5-S6
配列番号5:プラスミドpET-22b(+)に用いる配列決定用プライマーT7promoter
配列番号6:プラスミドpET-22b(+)に用いる配列決定用プライマーT7terminator
配列番号7:First-strand cDNA合成用プライマーHIgG(gamma)
配列番号8:First-strand cDNA合成用プライマーHlgM(mu)
配列番号9:First-strand cDNA合成用プライマーHkappa
配列番号10:First-strand cDNA合成用プライマーHlambda
配列番号11:γ鎖およびμ鎖を増幅するためのリバースプライマーHVH1REV
配列番号12:γ鎖およびμ鎖を増幅するためのリバースプライマーHVH2REV
配列番号13:γ鎖およびμ鎖を増幅するためのリバースプライマーHVH3REV
配列番号14:γ鎖およびμ鎖を増幅するためのリバースプライマーHVH4REV
配列番号15:γ鎖およびμ鎖を増幅するためのリバースプライマーHVH5REV
配列番号16:γ鎖およびμ鎖を増幅するためのリバースプライマーHVH6REV
配列番号17:γ鎖およびμ鎖を増幅するためのフォワードプライマーHJH12FOR配列番号18:γ鎖およびμ鎖を増幅するためのフォワードプライマーHJH3
配列番号19:γ鎖およびμ鎖を増幅するためのフォワードプライマーHJH45FOR配列番号20:γ鎖およびμ鎖を増幅するためのフォワードプライマーHJH6 FOR配列番号21:κ鎖を増幅するためのリバースプライマーHVΚ1REV
配列番号22:κ鎖を増幅するためのリバースプライマーHVΚ2REV
配列番号23:κ鎖を増幅するためのリバースプライマーHVΚ3REV
配列番号24:κ鎖を増幅するためのリバースプライマーHVΚ4REV
配列番号25:κ鎖を増幅するためのリバースプライマーHVΚ5REV
配列番号26:κ鎖を増幅するためのリバースプライマーHVΚ6REV
配列番号27:κ鎖を増幅するためのフォワードプライマーHJΚ1FOR
配列番号28:κ鎖を増幅するためのフォワードプライマーHJΚ2FOR
配列番号29:κ鎖を増幅するためのフォワードプライマーHJΚ3FOR
配列番号30:κ鎖を増幅するためのフォワードプライマーHJΚ4FOR
配列番号31:κ鎖を増幅するためのフォワードプライマーHJΚ5FOR
配列番号32:λ鎖を増幅するためのリバースプライマーHVλ1REV
配列番号33:λ鎖を増幅するためのリバースプライマーHVλ2REV
配列番号34:λ鎖を増幅するためのリバースプライマーHVλ3aREV
配列番号35:λ鎖を増幅するためのリバースプライマーHVλ3bREV
配列番号36:λ鎖を増幅するためのリバースプライマーHVλ4REV
配列番号37:λ鎖を増幅するためのリバースプライマーHVλ5REV
配列番号38:λ鎖を増幅するためのリバースプライマーHVλ6REV
配列番号39:λ鎖を増幅するためのフォワードプライマーHJλ1FOR
配列番号40:λ鎖を増幅するためのフォワードプライマーHJλ2-3FOR
配列番号41:λ鎖を増幅するためのフォワードプライマーHJλ4-5FOR
配列番号42:γ鎖およびμ鎖にリンカー配列を付加するためのフォワードプライマーHJH12FORLK
配列番号43:γ鎖およびμ鎖にリンカー配列を付加するためのフォワードプライマーHJH3 FORLK
配列番号44:γ鎖およびμ鎖にリンカー配列を付加するためのフォワードプライマーHJH45FORLK
配列番号45:γ鎖およびμ鎖にリンカー配列を付加するためのフォワードプライマーHJH6 FORLK
配列番号46:κ鎖にリンカー配列を付加するためのリバースプライマーHVΚ1REVLK
配列番号47:κ鎖にリンカー配列を付加するためのリバースプライマーHVΚ2REVLK
配列番号48:κ鎖にリンカー配列を付加するためのリバースプライマーHVΚ3REVLK
配列番号49:κ鎖にリンカー配列を付加するためのリバースプライマーHVΚ4REVLK
配列番号50:κ鎖にリンカー配列を付加するためのリバースプライマーHVΚ5REVLK
配列番号51:κ鎖にリンカー配列を付加するためのリバースプライマーHVΚ6REVLK
配列番号52:λ鎖にリンカー配列を付加するためのリバースプライマーHVλ1REVLK
配列番号53:λ鎖にリンカー配列を付加するためのリバースプライマーHVλ2REVLK
配列番号54:λ鎖にリンカー配列を付加するためのリバースプライマーHVλ3aREVLK
配列番号55:λ鎖にリンカー配列を付加するためのリバースプライマーHVλ3bREVLK
配列番号56:λ鎖にリンカー配列を付加するためのリバースプライマーHVλ4REVLK
配列番号57:λ鎖にリンカー配列を付加するためのリバースプライマーHVλ5REVLK
配列番号58:λ鎖にリンカー配列を付加するためのリバースプライマーHVλ6REVLK
配列番号59:制限酵素サイトを付加するためのHumanリバースSfiIプライマーHVH1REVSfiI
配列番号60:制限酵素サイトを付加するためのHumanリバースSfiIプライマーHVH2REVSfiI
配列番号61:制限酵素サイトを付加するためのHumanリバースSfiIプライマーHVH3REVSfiI
配列番号62:制限酵素サイトを付加するためのHumanリバースSfiIプライマーHVH4REVSfiI
配列番号63:制限酵素サイトを付加するためのHumanリバースSfiIプライマーHVH5REVSfiI
配列番号64:制限酵素サイトを付加するためのHumanリバースSfiIプライマーHVH6REVSfiI
配列番号65:制限酵素サイトを付加するためのHuman JΚフォワードNotIプライマーHJΚ1FORNotI
配列番号66:制限酵素サイトを付加するためのHuman JΚフォワードNotIプライマーHJΚ2FORNotI
配列番号67:制限酵素サイトを付加するためのHuman JΚフォワードNotIプライマーHJΚ3FORNotI
配列番号68:制限酵素サイトを付加するためのHuman JΚフォワードNotIプライマーHJΚ4FORNotI
配列番号69:制限酵素サイトを付加するためのHuman JΚフォワードNotIプライマーHJΚ5FORNotI
配列番号70:制限酵素サイトを付加するためのHuman JλフォワードNotIプライマーHJλ1FORNotI
配列番号71:制限酵素サイトを付加するためのHuman JλフォワードNotIプライマーHJλ2-3FORNotI
配列番号72:制限酵素サイトを付加するためのHuman JλフォワードNotIプライマーHJλ4-5FORNotI
配列番号73:g+k aリバースプライマー
配列番号74:g+k bリバースプライマー
配列番号75:g+k aフォワードプライマー
配列番号76:一本鎖抗体γ+κ5のアミノ酸配列
配列番号77:一本鎖抗体γ+κ12のアミノ酸配列
配列番号78:一本鎖抗体γ+κ19のアミノ酸配列
配列番号79:一本鎖抗体γ+κ40のアミノ酸配列
配列番号80:一本鎖抗体γ+κ96のアミノ酸配列
配列番号81:LOX-1またはLOX-1をコードする核酸配列
配列番号82:LOX-1またはLOX-1のアミノ酸配列
配列番号83:CTLD14をコードする核酸配列
配列番号84:CTLD14をコードするアミノ酸配列
配列番号85:カイコ型に用いたCTLD14をコードする核酸配列
配列番号86:カイコ型に用いたCTLD14をコードするアミノ酸配列
配列番号87:プライマーCTLD14-F: 5’- AATCTCCAAGAAACACTGAAG -3’
配列番号88:プライマーCTLD14s-R: 5’- TCACTGTGCTCTTAGGTTTGC -3’
配列番号89:エンテロキナーセ認識部位(DDDDK)
配列番号90:図29の部分配列1
配列番号91:図29の部分配列2
配列番号92:図29の部分配列3
配列番号93:図29の部分配列4
配列番号94:図29の部分配列5
配列番号95:図29の部分配列6
配列番号96:本発明で使用されるsRAGEの核酸配列
配列番号97:本発明で使用されるsRAGEのアミノ核酸配列
配列番号98:ビオチン化アミノ酸配列「GLNDIFEAQKIEWHE」
配列番号99:<Biotin-RAGE>の核酸配列(フィブロインH鎖イントロン、シグナルペプチド、BioEASE-tag&linker & FLAGおよびRAGEを含む)
配列番号100:<Biotin-RAGE>のアミノ酸配列
配列番号101:RAGEの核酸配列
配列番号102:RAGEのアミノ核酸配列
配列番号103:図41下段説明の1つ目の部分配列
配列番号104:図41下段説明の2つ目の部分配列
配列番号105:図41下段説明の3つ目の部分配列
配列番号106:ビオチンリガーゼ(BirA)の核酸配列
配列番号107:ビオチンリガーゼ(BirA)のアミノ酸配列
配列番号108:N末端側にMycタグが導入されているγ+κ5のアミノ酸配列
配列番号109:C末端側にMycタグが導入されているγ+κ5のアミノ酸配列
Claims (33)
- 標的をコードする核酸分子およびビオチンリガーゼを共発現可能に組み込んだカイコまたはカイコと同様の糖鎖を付与する生物。
- 前記ビオチンリガーゼはBirAである、請求項1に記載の生物。
- 前記標的は、C型レクチン様ドメイン(CTLD14)または終末糖化産物受容体(sRAGE)あるいはその改変体である、請求項1または2に記載の生物。
- 前記CTLD14は配列番号85によってコードされ、前記sRAGEは配列番号96によってコードされる、請求項3に記載の生物。
- 前記生物は、ビオチン化を受けるタグ配列を含む、請求項1~4のいずれか1項に記載の生物。
- 前記ビオチン化を受けるタグ配列は、BioEase.tagおよびAvi.tagのいずれかである、請求項5に記載の生物。
- ビオチン化タンパク質の製造方法であって、
A)カイコまたはカイコと同様の糖鎖を付与する生物に、ビオチンリガーゼおよびタンパク質をコードする核酸分子を共発現可能に組み込む工程;
B)該核酸分子が発現する条件下に該カイコまたはカイコと同様の糖鎖を付与する生物を配置し、該ビオチンリガーゼおよび該タンパク質を発現させる工程;および
C)該生物にビオチンを投与し、ビオチン化された該タンパク質を得る工程
を包含する、方法。 - 前記発現は、前記カイコの中部絹糸腺において行われる、請求項7に記載の方法。
- 前記タンパク質は、ビオチン化された状態で発現される、請求項7または8に記載の方法。
- 前記A)工程は、前記タンパク質をコードする核酸分子を含む発現ベクターをマイクロインジェクションすることで達成される、請求項7~9のいずれか1項に記載の方法。
- 前記タンパク質は、C型レクチン様ドメイン(CTLD14)または終末糖化産物受容体(sRAGE)または配列番号76~80のいずれかに記載のアミノ酸配列もしくはその改変体を含む一本鎖抗体もしくはそのフラグメントあるいはその改変体である、請求項7~10のいずれか1項に記載の方法。
- 前記生物は、ビオチン化を受けるタグ配列を含む、請求項7から11のいずれか1項に記載の方法。
- 前記ビオチン化を受けるタグ配列は、BioEase.tagおよびAvi.tagのいずれかである、請求項12に記載の方法。
- 配列番号76~80のいずれかに記載のアミノ酸配列またはその改変体を含む一本鎖抗体またはそのフラグメント。
- 請求項14に記載の一本鎖抗体またはそのフラグメントを含む、LDLおよび変性LDLを検出するための検出剤であって、該LDLおよび変性LDLは、アセチル化LDLおよび部分酸化LDLからなる群より選択される少なくとも1つの分子を含む、検出剤。
- 請求項14に記載の一本鎖抗体またはそのフラグメントを生産する方法であって、該方法は:
A)該一本鎖抗体またはそのフラグメントのアミノ酸配列をコードする核酸を、該核酸が発現される細胞に導入する工程;
B)該細胞を培養し、該細胞から封入体を取り出す工程;
C)該封入体をリフォールディングする工程;
D)リフォールディングで得られた溶液を精製用カラムに接触させ、溶出溶液で溶出する工程;
E)溶出した該一本鎖抗体またはそのフラグメントを透析溶液で透析する工程
を包含する、方法。 - 請求項14に記載の一本鎖抗体またはそのフラグメントの製造方法であって、
A)カイコまたはカイコと同様の糖鎖を付与する生物に、該一本鎖抗体またはそのフラグメントをコードする核酸分子を発現可能に組み込む工程;
B)該核酸分子が発現する条件下に該カイコまたはカイコと同様の糖鎖を付与する生物を配置し、該一本鎖抗体またはそのフラグメントを発現させる工程;および
C)該一本鎖抗体またはそのフラグメントを得る工程
を包含する、方法。 - 請求項14に記載の一本鎖抗体またはそのフラグメントと、酸化LDL受容体(LOX-1)のリガンド認識領域とを用いる、酸化的修飾を受けたLDLを検出または定量するためのシステム。
- 抗変性LDL抗体、その改変体またはそのフラグメントと、酸化LDL受容体(LOX-1)のリガンド認識領域とをラテラルフローアッセイ形式で用いる、変性LDLに関連する疾患の検診のためのシステム。
- 抗変性LDL抗体、その改変体またはそのフラグメントと、酸化LDL受容体(LOX-1)のリガンド認識領域とをラテラルフローアッセイ形式で用いる、食品摂取による変性LDLに関連する疾患の予防効果の評価のためのシステム。
- カイコ型糖鎖を有する、配列番号86に示すアミノ酸配列またはその改変体を含むC型レクチン様ドメイン(CTLD)14。
- 請求項21に記載のCTLD14を含む、変性LDLを検出するための組成物。
- C型レクチン様ドメイン(CTLD)14の製造方法であって、
A)カイコまたはカイコと同様の糖鎖を付与する生物に、CTLD14をコードする核酸分子を発現可能に組み込む工程;
B)該核酸分子が発現する条件下に該カイコまたはカイコと同様の糖鎖を付与する生物を配置し、該CTLD14を発現させる工程;および
C)該CTLD14を得る工程
を包含する、方法。 - CTLD14をコードする核酸分子を発現可能に組み込んだカイコまたはカイコと同様の糖鎖を付与する生物。
- 抗LDLニワトリ抗体を含む、変性LDLを検出するための組成物。
- 抗LDLニワトリ抗体と、CTLD14とを含む、変性LDLを検出するためのキット。
- カイコ型糖鎖を有する、配列番号97に示すアミノ酸配列またはその改変体を含む再構築終末糖化産物受容体(sRAGE)。
- 前記sRAGEは、ビオチン化されたものである、請求項27に記載のsRAGE。
- 前記ビオチンを介して前記sRAGEが基板に固定化されている、請求項28に記載のsRAGEが固定化された試薬。
- 請求項27または28に記載のsRAGEあるいは請求項29に記載の試薬を含む、終末期糖化生成物(AGEs)を検出するための組成物。
- 前記sRAGEを試料に接触させ、AGEsを検出する工程を包含する、AGEsを検出する方法。
- sRAGEの製造方法であって、
A)カイコまたはカイコと同様の糖鎖を付与する生物に、sRAGEをコードする核酸分子を発現可能に組み込む工程;
B)該遺伝子が発現する条件下に該カイコまたはカイコと同様の糖鎖を付与する生物を配置し、該sRAGEを発現させる工程;および
C)該sRAGEを得る工程
を包含する、方法。 - sRAGEをコードする核酸分子を発現可能に組み込んだカイコまたはカイコと同様の糖鎖を付与する生物。
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JP2016551559A JP6617110B2 (ja) | 2014-10-01 | 2015-10-01 | 遺伝子組換えカイコにより生産したビオチン化酸化ldl受容体・終末糖化産物受容体 |
US15/515,998 US11311002B2 (en) | 2014-10-01 | 2015-10-01 | Biotinylated and oxidized LDL receptor and advanced glycation end product receptor produced using genetically engineered silkworm |
HK18106800.7A HK1247043A1 (zh) | 2014-10-01 | 2018-05-25 | 使用基因修飾的家蠶生產的生物素化和氧化ldl受體和晚期糖化終末產物受體 |
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JP2020134368A (ja) * | 2019-02-21 | 2020-08-31 | 国立研究開発法人農業・食品産業技術総合研究機構 | AGEsを検出するためのアッセイ系 |
CN111781371A (zh) * | 2020-06-18 | 2020-10-16 | 中国计量大学 | 一种基于磁珠载体双抗夹心elisa的考古现场快速检测痕量丝素蛋白的方法 |
WO2021090922A1 (ja) * | 2019-11-08 | 2021-05-14 | 国立研究開発法人農業・食品産業技術総合研究機構 | 変性LDL及び刺激性AGEs簡易迅速定量法 |
WO2021090923A1 (ja) * | 2019-11-08 | 2021-05-14 | 国立研究開発法人農業・食品産業技術総合研究機構 | 変性LDLまたは刺激性AGEs目視検出法 |
KR20210103962A (ko) | 2020-02-14 | 2021-08-24 | 충북대학교 산학협력단 | B세포 성숙화 항원을 표적으로 하는 키메라 항원 수용체 및 이의 용도 |
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US10898860B2 (en) | 2015-03-24 | 2021-01-26 | Arstroma Co., Ltd. | Fluid separation apparatus comprising fluid separation membrane, and fluid separation membrane module |
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HK1247043A1 (zh) | 2018-09-21 |
US11311002B2 (en) | 2022-04-26 |
JPWO2016051808A1 (ja) | 2017-08-24 |
US20170354129A1 (en) | 2017-12-14 |
CN107404863B (zh) | 2021-04-02 |
CN107404863A (zh) | 2017-11-28 |
JP6617110B2 (ja) | 2019-12-04 |
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