DK2687539T3 - Annexin v-variant, samt fremstilling og anvendelse deraf - Google Patents
Annexin v-variant, samt fremstilling og anvendelse deraf Download PDFInfo
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- DK2687539T3 DK2687539T3 DK11835310.1T DK11835310T DK2687539T3 DK 2687539 T3 DK2687539 T3 DK 2687539T3 DK 11835310 T DK11835310 T DK 11835310T DK 2687539 T3 DK2687539 T3 DK 2687539T3
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- DK
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- Prior art keywords
- annexin
- variant
- bacteria
- amino acid
- labeling
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4721—Lipocortins
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
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Claims (10)
1. En variant af annexin V, hvilken variant, der ved C-terminalen af en annexin V, er tilsat (i) en cysteinrest, eller (iii) et kort peptid med fleksibel struktur, hvor det korte peptid har op til 25 aminosyrerester, hvoraf 1-3 er cystei nrester, og eventuelle resterende aminosyreresterer er glycin-, alanin- og/eller serinrester.
2. Variant af annexin V ifølge krav 1, hvor varianten har aminosyresekvensen SEQ ID NO: 1 eller et derivat deraf, hvori en eller flere aminosyrerester er substitueret, deleteret eller tilsat, idet derivatet har den samme aktivitet som varianten af annexin V med aminosyresekvensen SEQ ID NO: 1.
3. Variant af annexin V ifølge krav 1 eller krav 2, hvor varianten er mærket med et fluorescerende kemikalie eller et radionuklid.
4. Fremgangsmåde til fremstilling af varianten af annexin V ifølge et hvilket som helst af kravene 1 til 3, hvilken fremgangsmåde omfatter: (a) molekylær modellering og design af aminosyresekvensen af det korte peptid, der skal tilsættes C-terminalen af annexin V, udført på basis af krystalstrukturen i annexin V, hvor designet og modelleringen udføres ved hjælp af computerstøttet molekylærdesign; (b) design af primer-par til amplifikation af den kodende nukleotidsekvens af annexin V-protein og udførelse af PCR-amplifikation med annexin V-genet som template; (c) recirkulation af produktet af PCR ved elektroforese, forbindelse af det recirkulerede produkt med en kloningsvektor, overførsel af kloningsvektoren til TOPlO-celler og dyrkning af cellerne; (d) separat fordøjelse af det recirkulerede produkt fra trin (c) og ekspressionsvektoren pET28a med restriktionsenzymerne Nco I og Xho I, forbindelse af de to genfragmenter således opnået ved T4 DNA-ligase, overførsel af de forbundne fragmenter til TOPlO-celler, dyrkning af cellerne og screening for og ekstraktion af det rekombinante ekspressionsplasmid; (e) overførsel af det ekstraherede rekombinante ekspressionsplasmid til Escherichia coli-bakterier, dyrkning af bakterierne, induktion af bakterierne med IPTG og opsamling af bakterierne; (f) ekspansionsdyrkning af bakterierne opnået i trin (e) og opsamling af bakterierne; og (g) knusning og lysering af bakterierne, og rensning og opsamling af varianten af annexin V.
5. Fremgangsmåde ifølge krav 4, hvor primer-parrenes nukleotidsekvenser er: (i) senseprimeren; 5'- gtt cca tgg gcg cac agg tccca gag gca-3 '; og (ii) antisense-primeren; 5'-tcc get egg age agt cat ett ctc cac aga gca-3
6. Fremgangsmåde ifølge krav 4, hvor oprensningen af varianten af annexin V i trin (g) omfatter, i rækkefølge: udfældning, desorption, ammoniumsulfatgraderet udfældning, søjlekromatografi ved Super Q-650M og søjlekromatografi ved SP.
7. Fremgangsmåde ifølge et hvilket som helst af kravene 4 til 6, hvilken fremgangsmåde endvidere omfatter et trin med fluorescerende kemisk mærkning eller radionuklid mærkning af varianten af annexin V.
8. Varianten af annexin V ifølge et hvilket som helst af kravene 1 til 3 til anvendelse som en probe til påvisning eller overvågning af apoptose in vivo.
9. En in v/fro-fremgangsmåde til påvisning eller overvågning af apoptose, hvilken fremgangsmåde omfatter anvendelse af varianten af annexin V ifølge et hvilket som helst af kravene 1 til 3.
10. Fremgangsmåde til fremstilling af en probe til påvisning eller overvågning af apoptose, hvilken fremgangsmåde omfatter tilvejebringelse af varianten af annexin V ifølge krav 1 eller krav 2 og mærkning af varianten med et fluorescerende kemikalie eller et radionuklid.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110072451.2A CN102690345B (zh) | 2011-03-24 | 2011-03-24 | 人膜联蛋白v变体及其表达、制备和应用 |
| PCT/CN2011/001449 WO2012126157A1 (zh) | 2011-03-24 | 2011-08-29 | 膜联蛋白v变体及其表达、制备和应用 |
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| Publication Number | Publication Date |
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| DK2687539T3 true DK2687539T3 (da) | 2017-09-18 |
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| DK11835310.1T DK2687539T3 (da) | 2011-03-24 | 2011-08-29 | Annexin v-variant, samt fremstilling og anvendelse deraf |
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| Country | Link |
|---|---|
| US (1) | US8975380B2 (da) |
| EP (1) | EP2687539B1 (da) |
| JP (1) | JP2014511831A (da) |
| CN (1) | CN102690345B (da) |
| DK (1) | DK2687539T3 (da) |
| ES (1) | ES2638919T3 (da) |
| PT (1) | PT2687539T (da) |
| WO (1) | WO2012126157A1 (da) |
Families Citing this family (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103041411A (zh) * | 2013-01-15 | 2013-04-17 | 南京市第一医院 | 以磷脂酰丝氨酸为靶点的99Tcm-Cys-AnnexinV标记化合物及其标记技术和应用 |
| CN103159841B (zh) * | 2013-03-18 | 2014-07-02 | 江苏省原子医学研究所 | 一种99mTc标记Cys-Annexin V的方法及其应用 |
| CN103159842B (zh) * | 2013-03-18 | 2015-07-01 | 江苏省原子医学研究所 | 一种用于99mTc标记的Cys-Annexin V药盒及其配制方法与应用 |
| CN103665140B (zh) * | 2013-12-20 | 2015-04-22 | 常州千红生化制药股份有限公司 | 大肠杆菌表达的膜联蛋白v或其衍生物的分离纯化方法 |
| GB2542391A (en) | 2015-09-17 | 2017-03-22 | Annexin Pharmaceuticals Ab | Process of manufacture |
| US20190248920A1 (en) | 2016-09-23 | 2019-08-15 | Csl Limited | Coagulation factor binding proteins and uses thereof |
| GB201806581D0 (en) * | 2018-04-23 | 2018-06-06 | Ucl Business Plc | Fluorescent Marker |
| CN109529017B (zh) * | 2018-12-25 | 2022-06-14 | 江苏靶标生物医药研究所有限公司 | 一种膜联蛋白v的应用 |
| CN110172464A (zh) * | 2019-06-05 | 2019-08-27 | 大连博格林生物科技有限公司 | 人膜联蛋白v变体及制备方法 |
| CN114539383B (zh) * | 2022-02-24 | 2024-03-26 | 南京大学 | 一种人源膜联蛋白人源膜联蛋白a5突变体二聚体的晶体结构的制备方法和应用 |
| CN114437197B (zh) * | 2022-02-24 | 2024-11-15 | 江苏靶标生物医药研究所有限公司 | 一种人源膜联蛋白a5突变体二聚体的晶体结构及其解析方法 |
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|---|---|---|---|---|
| US5968477A (en) * | 1994-01-24 | 1999-10-19 | Neorx Corporation | Radiolabeled annexin conjugates with hexose and a chelator |
| US6323313B1 (en) | 1999-06-01 | 2001-11-27 | The University Of Washington | Annexin derivative with endogenous chelation sites |
| WO2002067857A2 (en) * | 2001-02-21 | 2002-09-06 | Surromed, Inc. | Modified annexin proteins and methods for preventing thrombosis |
| DK1755642T3 (da) * | 2004-04-15 | 2010-05-10 | Athera Biotechnologies Ab | Annexin V til forebyggelse af plaksprængning |
| EP2072525A1 (en) * | 2007-12-21 | 2009-06-24 | Affibody AB | New polypeptides having affinity for HER2 |
| US8541549B2 (en) * | 2008-04-01 | 2013-09-24 | University Of Southern California | Annexin-based apoptosis markers |
| EP2349321B1 (en) * | 2008-10-17 | 2020-04-22 | London Health Sciences Centre Research Inc. | Annexin and its use to treat inflammatory disorders |
-
2011
- 2011-03-24 CN CN201110072451.2A patent/CN102690345B/zh active Active
- 2011-08-29 EP EP11835310.1A patent/EP2687539B1/en active Active
- 2011-08-29 JP JP2014500223A patent/JP2014511831A/ja active Pending
- 2011-08-29 DK DK11835310.1T patent/DK2687539T3/da active
- 2011-08-29 US US13/505,454 patent/US8975380B2/en active Active
- 2011-08-29 ES ES11835310.1T patent/ES2638919T3/es active Active
- 2011-08-29 WO PCT/CN2011/001449 patent/WO2012126157A1/zh not_active Ceased
- 2011-08-29 PT PT118353101T patent/PT2687539T/pt unknown
Also Published As
| Publication number | Publication date |
|---|---|
| EP2687539A4 (en) | 2014-10-08 |
| ES2638919T3 (es) | 2017-10-24 |
| EP2687539A1 (en) | 2014-01-22 |
| PT2687539T (pt) | 2017-09-08 |
| CN102690345B (zh) | 2014-03-19 |
| EP2687539B1 (en) | 2017-05-31 |
| JP2014511831A (ja) | 2014-05-19 |
| WO2012126157A1 (zh) | 2012-09-27 |
| US20140127736A1 (en) | 2014-05-08 |
| CN102690345A (zh) | 2012-09-26 |
| US8975380B2 (en) | 2015-03-10 |
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