TW200920843A - Design and preparation of annexin-staphylokinase fusion protein with enhanced specificity of thrombolytic activity - Google Patents

Abstract

Novel fusion proteins comprising annexins and staphylokinase (SAK) are provided. These fusion proteins have increased thrombolytic activities. The fusion proteins are useful in treating diseases related to injured endothelial cells, activated platelets, and apoptotic cells such as myocardial infarction and Ischemic Stroke … etc.

Description

200920843 IX. Description of the invention [Technical field to which the invention belongs] The main invention relates to a novel embedded 厶艚 厶艚 迎 迎 迎 迎 迎 迎 迎 迎 迎 迎 迎 迎 迎 迎 迎 迎 迎 迎 迎 迎 迎 迎 迎 迎 迎 迎 迎 迎 迎 迎 迎 迎 迎 迎 迎 迎 迎 迎 迎 迎 迎 迎application. [Prior Art 1 ^^gLCThrombolytic丄 treatment, in recent years, used in the left, muscle infarction, ischemic;

Stroke} these two important diseases. ^ Care, give people unlimited hope, but the existing side effects of blood stasis treatment or the existence of platelet-rich secondary blood clots, the main side effect is the unintended 3 to solve the bleeding problem and enhance the effectiveness Price, many improvements have been made, of which targeting therapy is the best choice for solving the above problems. The present inventors have devoted themselves to the research of target therapy for many years, first at the seminar, using the concept of binding to annexin and the concept of thrombolytic ability of staphylokinase, FEBS J^RNAL 272: 407-407 Suppl. 1 JUL 2005, the inventor of the present invention at the seminar will reveal the concept of linking annexin to staphylokinase with a long-chain linker, but at the time, and other things like In CN1343777, long chain (water worm to macromolecule) is used as the connection method, and no good effect can be achieved. After the inventors continued to improve the far-reaching method, they finally succeeded in discovering that the frog i (linker) with 铕 has a particularly good effect. The results are published in bi〇science BIOTECHNOLOGY AND BIOCHEMISTRY 71 (5): 1122-1129 7th MAY 2007. [Summary] 10 200920843 ^^Althrombolyticj treatment, in recent years, has been applied to myocardial infarction and ischemic stroke (ischemi strol! ei, two important diseases, although it brings hope to humans, it will Causes serious side effects such as blood stasis or effect on the blood platelet-rich secondary blood clots = acid is to improve the financial effect and reduce the side of the film 1 egg = (annexins) is a calcium ion-dependent phospholipid-binding egg Shell: Lin linings such as phospholipidylserine (Phosphatidylserine), production, injured injured endothelial cells, sputum blood, (ac^vated platelets) and apoptotic cells (apopt〇tic ceUs) f On the surface of ten cells. This annexin (inducin) and imperfect ability can be used as a guiding tool to guide the drug to no, and the (staphylokinase) is known to be fibrin-soluble blood stasis.丨.

In contrast, a chimera that binds to the ability of annexin to direct the ability of platin and tapl^o^se), seems to be able to improve the ability of the drug; In fact, however, the inventors have intensively studied and developed a series of membrane annexins; and chimeras with staphylokinase; and found that annexin XI and staphylokinase chimera ( In addition to its 1, it is also found that the linker and the chain length of the annexin, and the chain are also affected. The linker of the present invention and the annexin χι have a special „ (SAK-) NXXI ) 'Not only has effect on platelet-rich blood = nch p asma clots, but also on platelet-poor cells. τ 200920843 , 〒 〒, plasminc gen aetivati〇n Reaction) 'Compared with the clot lysis assay to measure the efficacy of annexin and staphyl〇kinase chimeras, the results indicate that these chimeras are industrially useful. The highly probable (thrombolytic treatment) is also expected to be used in two important diseases, the myocardial infarction and the ischemic stroke (Ischemic Stroke). The invention is characterized by a chimeric body of the annexin kinase streptokinase short chain, and the following genetic engineering technique illustrates the present invention, but is not limited thereto. First, the annexin gene and the staphylokinase gene are selected. Embed in the appropriate 1 vector' followed by PCR (polymerase chain reaction) to replicate the gluco-suppressor gene, and insert the replicated staphylokinase gene into the vector containing the annexin gene. The above-mentioned staphylokinase gene and annexin gene are included. The vector expresses its protein (SAK-ANXXI) under appropriate conditions. ” Expression of the chimeric protein (SAK-AMXI), which is measured after purification by a column. Activity measurement includes plasminogen activation (plasmin〇gen activation) And the results of the clot lysis assay, the results of which are respectively described in the drawings. The following examples are intended to illustrate the invention but should not be used to limit the scope of the patent application of the invention. Example 1, genetic selection, construction Expression system, and purification of staphylokinase (SAK) gene: the staphylokinase () gene obtained by chemical synthesis (411bps Sta Phylococcus arueus subsp. Aureus N315, A17531 at NCBI) plus Nde I and Xhol two restriction enzyme sites (purchased from MDBio, lnc., Taipei, Taiwan) were cloned into the vector pET24a by conventional molecular biology routine methods. (+) (Novagen, Madison, WI) 'ie pET24-SAK. Annexin gene (ANXV and ANXXI) gene selection: The guanine protein gene (ANXVand ANXXI) is derived from human T-cell leukemia 12 200920843 cell line MOLT-4 cells, which is colonized by conventional molecular biology methods, first using Trizole RNA was isolated (Invitrogen, Carlsbad, CA), and the PCR primers used in RT-PCR system (Invitrogen) °ANXV were 5'-GGAATTCCATATGGCACAGGTTCTCAGA-3'. The antisense primer was 5' =CCGCTCGAGGTCATCTTCTCCAGAGAG-3', used by ANXXI. The PCR primers are 5'-GGAATTCCATATGAGCTACCCTGGCTAT and its antisense primer is 5'-CCGCTCGAGGTCATTGCCACCACAGAT. The ANXV and ANXXI PCR products are inserted into the Ndel and Xhol sites of pET24a(+), respectively called pET24=ANXV and pET24-ANXXI. (SAK) and annexin gene (ANXV and ANXXI) gene selection: Design PCR primers and create Ndel sites for PCR on pET24-SAK. Primer: 5'-GGAATTCCATATGTCAAGTTCATTCGAC-3' antisense primer: primer 5' -GGAATTCCATATGTTTCTTTTCTATAAC-3'. The staphylokinase (SAK) gene with Ndel site was inserted into pET24-ANXV and PET24-ANXXI by PCR, and was called pET24-SAK-ANXV and PET24-SAK-ANXXI, respectively. The sequence was sequenced to confirm its sequence as shown by the sequence identification number. The vector pET24a(+) expressed its protein using BL21, and the protein was purified by Ni-NTA column, and subsequent activity tests were performed. Sequence Identification No. 1 (SAK-ANXV DNA sequence): latgtcaagtt cattcgacaa aggaaaatat aaaaaaggcg atgacgcgag ttattttgaa 61ccaacaggcc cgtatttgat ggtaaatgtg actggagttg atagtaaagg aaatgaattg 121 ctatcccctc attatgtcga gtttcctatt aaacctggga ctacacttac aaaagaaaaa 181 attgaatact atgtcgaatgggcattagat gcgacagcat ataaagagtt tagagtagtt 241 gaattagatc caagcgcaaa gatcgaagtc acttattatg ataagaataa gaaaaaagaa 301 gaaacgaagt ctttccctat aacagaaaaa ggttttgttg tcccagattt atcagagcat 361 attaaaaacc ctggattcaa cttaattaca aaggttgtta tagaaaagaa a lcatatggcacagg ttctcagagg cactgtgact gacttccctg gatttgatga gcgggctgat 64 gcagaaactc ttcggaaggc tatgaaaggc ttgggcacag atgaggagag catcctgact 124 ctgttgacat cccgaagtaa tgctcagcgc caggaaatct ctgcagcttt taagactctg 184 tttggcaggg atcttctgga tgacctgaaa tcagaactaa ctggaaaatt tgaaaaatta 244 attgtggctc tgatgaaacc ctctcggctt tatgatgctt atgaactgaa acatgccttg 304 aagggagctg gaacaaatga aaaagtactg acagaaatta ttgcttcaag gacacctgaa 13 200920843 364 gaactgagag ccatcaaaca agtttatgaa gaagaatatg gctcaagcct ggaagatgac 424 gtggtggggg acacttcagg gtactaccag cggatgttgg tggttctcct tcaggctaac 484 agagaccctg atgctggaat tgatgaagct caagttgaac aagatgctca ggctttattt 544 caggctggag aacttaaatg ggggacagat gaagaaaagt ttatcaccat ctttggaaca 604 cgaagtgtgt ctcatttgag aaaggtgttt gacaagtaca tgactatatc aggatttcaa 664 at tgaggaaa ccat tgaccg cgagact tct ggcaat t tag agcaactact cct tgctgt t 724 gtgaaatcta ttcgaagtat acctgcctac cttgcagaga ccctctatta tgctatgaag 784 ggagctggga cagatgatca taccctcatc agagtcatgg tttccaggag tgagattgat 844 ctgtttaaca tcaggaagga gtttaggaag aattttgcca cctctcttta ttccatgatt 904 aagggagata catctgggga ctataagaaa gctcttctgc tgctctgtgg agaaga t gacctcgagcaccaccaccaccaccac sequence identification number 2 (SAK-ANXXI the DM sequence) atgtcaagtt cattcgacaa aggaaaatat aaaaaaggcg atgacgcgag ttattttgaa 61 ccaacaggcc cgtatttgat ggtaaatgtg actggagttg atagtaaagg aaatgaattg 121 Ctatcccctc attatgtcga gtttcctatt aaacctggga ctacacttac aaaagaaaaa 181 attgaatact atgtcgaatgggcattagat gcgacagcat ataaagagtt tagagtagtt 241 gaattagatc caagcgcaaa gatcgaagtc acttattatg ataagaataa gaaaaaagaa 301 gaaacgaagt ctttccctat aacagaaaaa ggttttgttg tcccagattt atcagagcat 361 attaaaaacc ctggattcaa cttaattaca aaggttgtta tagaaaagaa acat latgagctacc ctggctatcc cccgccccca ggtggctacc caccagctgc accaggtggt 61 ggtccctggg gaggtgctgc ctaccctcct ccgcccagca tgccccccat cgggctggat 121 aacgtggcca cctatgcggg gcagttcaac caggactatc tctcgggaat ggcggccaac 181 atgtctggga catttggagg agccaacatg cccaacctgt accctggggc ccctggggct 241 ggctacccac cagtgccccc tggcggcttt gggcagcccc cctctgccca gcagcctgtt 301 cctccctatg ggatgtatcc acccccagga ggaaacccac cctccaggat gccctcatat 361 ccgccatacc caggggcccc tgtgccgggc cagcccatgc caccccccgg acagcagccc 421 ccaggggcct accctgggca gccaccagtg acctaccctg gtcagcctcc agtgccactc 481 cctgggcagc agcagccagt gccgagctac ccaggatacc cggggtctgg gactgtcacc 541 cccgctgtgc ccccaaccca gtttggaagc cgaggcacca tcactgatgc tcccggcttt 601 gaccccctgc gagatgccga ggtcctgcgg aaggccatga aaggcttcgg gacggatgag 661 caggccatca ttgactgcct Ggg gagtcgc tccaacaagc agcggcagca gatcctactt 721 tccttcaaga cggcttacgg caaggatttg atcaaagatc tgaaatctga actgtcagga 781 aactttgaga agacaatctt ggctctgatg aagaccccag tcctctttga catttatgag 841 ataaaggaag ccatcaaggg ggttggcact gatgaagcct gcctgattga gatcctcgct 901 tcccgcagca atgagcacat ccgagaatta aacagagcct acaaagcaga attcaaaaag 961 accctggaag aggccattcg aagcgacaca tcagggcact tccagcggct cctcatctct 1021 ctctctcagg gaaaccgtga tgaaagcaca aacgtggaca tgtcactcgc ccagagagat 1081 gcccaggagc tgtatgcggc cggggagaac cgcctgggaa cagacgagtc caagttcaat 1141 gcggttctgt gctcccggag ccgggcccac ctggtagcag ttttcaatga gtaccagaga 14 200920843 1201 atgacaggcc gggacattga gaagagcatc tgccgggaga tgtccgggga cctggaggag 1261 ggcatgctgg ccgtggtgaa atgtctcaag aataccccag ccttctttgc ggagaggctc 1321 aacaaggcca tgaggggggc aggaacaaag gaccggaccc tgattcgcat catggtgtct 1381 cgcagcgaga ccgacctcct ggacatcaga tcagagtata agcggatgta cggcaagtcg 1441 ctgtaccacg acatctcggg agatacttca ggggattacc ggaagattct gctgaagatc 1501 tgtggtggcaatgacctcgagca Ccaccacccaccaccac SEQ ID NO: 3 (SAK-ANXV protein sequence)

SSGEEFYIK K MATVIETPI K

SSFDKGKYKKGDD

YFEPTGPYLMVNV

VDSKGNELLSPHY

FPIKPGTTLTKEK

YYVEWALDATAYK

RVVELDPSAKIEV

YDKNKKKEETKSF

TEKGFVVPDLSEH

NPGFNLITKVVIE

-H-MAQVLRGTVTDFPGFDERADAETLRKAMKGIjGTDEESILTLLTS

RSNAQRQEISAAFKILFGRDLLDDLKSELTGKFEKLIVALMKPSRLYDAYELKHALKG

AGTOEKVLTEIIASRTPEELRAIKQVYEEEYGSSLEDDWGDTSGYYQRMLWLLQAN

RDPDAGIDEAQVEQDAQALFQAGELKWGTDEEKFITIFGTRSVSHLRKVFDKYMnSG

FQIEETIDRETSGNLEQLLLAWKSIRSIPAYLAErLYYAMKGAGTDDHTLIRVMVSR

SEIDLFNIRKEFRKNFATSLYSMIKGOTSGDYKKALLLIjCGEDD-LGHHHHHH Sequence f> White eggs n X X Μ I κ· SA c' No. 4 identification sequence

D D G K K Y K G K D F S S S

Y Y N Η VP Ms L L Y L PE G N T G p K E s F D Y V s G AT

K K E Y K A T T T T T T T G G A p w K E IV p Y F Y EE VI

V F E s 1—IK K T A E s E p K D K L K E N V K V D R Y F Y E T

HE El s V L V D K p T V I V L F N G F K G E p T N IK p I K Κ-Η-

MSYPGYPPPPGGYPPAAPGGGPWGGAAYPPPPSMPPIGLDNVAT

YAGQFNQDYLSGMAANMSGTFGGANMPNLYPGAPGAGYPPVPPGGFGQPPSAQQPVPP 15 200920843

YGMYPPPGGNPPSRMPSYPPYPGAPVPGQPMPPPGQQPPGAYPGQPPVTYPGQPPVPL

PGQQQPVPSYPGYPGSGIYTPAVPPTQFGSRGTITDAPGFDPLRDAEVLRKAMKGFGT

DEQAIIDCLGSRSNKQRQQILLSFKTAYGKDLIKDLKSELSGNFEKTILALMKTPVLF

DIYEIKEAIKGVGTDEACLIEILASRSNEHIRELNRAYKAEFKKTLEEAIRSDTSGHF

QRLLISLSQGNRDESTOVDMSLAQRDAQELYMGENRLGTDESKFNAVLCSRSRAHLV

AVFNEYQRMTGRDIEKS ICRMSGDLEEGMLAmaKmPAFFAERLNKAMRGAGTK

DRTLIRIMVSRSETDLLDIRSEYKRMYGKSLYHDISGDTSGDYRKILLKICGGND-LGHH

HHHH Example 2, Activity test plasminogen activation reaction: Protoplasmin activation reagents include: 1//M human plasminogen (Chromogenix, Milano, Italy), protein (3nM) in 100 mM Tris- HCl, pH 7.5, 37 ° C Reaction. 2, 4, and 6 min, 5-//1 aliquots Dilute at 35// 1 (700 mM NaCl, 100 mM Tris-HCl, pH 7.5). Finally add the substrate Np- tosyl-Gly-Pro-Lys nitroanilide (Chromogenix). Measured price plus / must be 0. 019pM-1S_1 (SAK-ANXV),

0. 020 μΜ—ΡθΑΚ-ΑΝΧΧΙ), 0. 028 μΜ-it 1 (SAK) was further analyzed by SDS-PAGE. Clot lysis assay: Platelet rich plasma PRP was obtained from the General Hospital of the Three Armies and centrifuged (5 000 xg for 5 min) to obtain platelet poor plasma (PPP). . Human thrombin (0.8 NIH units/ml, Sigma, St. Louis, MI) and CaCl2 (20 mM) in Hepes-buffered saline (HBS, 20 mM) HEPES, 130 mM NaCl, pH 7.4) were first added to form a blood clot. The clot lysis test (ci〇t iysis assay) was carried out at room temperature, adding 200 nM and human plasminogen (1 " M) in HBS-CaCl2 (100 from 1). The gold nugget dissolution system is based on turbidity reduction and is 4 〇 5 nm wavelength Light supervision 16 200920843 control, measuring its 0D value. The result is shown in the figure. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1A shows an SDS-PAGE diagram of the results of plasminogen activation reaction of chimeric proteins. Column 1 is unreacted SAK-ΑΝΠΙ (70KD); Column 2 is unreacted ANXXI (54KD); Column 3 is unreacted SAK (15.5KD); Columns 4-10 are plasminogen (90. 5KD) As a result of reaction with SAK-AMXI (70KD), columns 4-10 reacted with 〇, 2, 4, 8, 16, 32, and 64 minutes, respectively; column 11 was plasminogen reacted with SAK for 10 minutes. Figure 1B SDS-PAGE of the results of the piasmin〇 activation reaction of the chimeric protein. Column 1 is unreacted SAK-ANXV (51.5 kD); column 2 is unreacted 2 ANXV (36KD); columns 3-9 are reactive plasminogen (90.5KD) and SAK-ANXV (51.5KD) As a result of the reaction, columns 3-9 were reacted for 0, 2, 4, 8, 16, 32, and 64 minutes, respectively. Figure 2A shows less platelet poor plasma (PPP) blood, the white circle of the clot lysis assay represents SAK; the white triangle represents SAK-ANXV; the black square represents SAK-ΑΝΠΙ; the diamond represents the control group. Figure 2B shows platelet-containing blood polyphosphate (piateiet rich piasma pRp) blood. The clot iySis assay has a white circle representing sak; a white triangle representing SAK-ANXV; a black square representing SAK-ΑΝΠΙ; and a diamond representing the control group. 17

Claims (1)

200920843 X. Patent application scope: L's it membrane 5fi!annexin) and staphylokinase-e) knot 43⁄4 然=ίϊ; 激 • 之间 之间 ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ 专利 ^ The protein is annexin 3. J°. The chimera of claim 1 of the patent scope, the capsid protein is annexin complex 'co-linked protein and staphylokinase embedded 5. U please SSSSii body, wherein annexin and staphylokinase embedded 6' body' with annexin and Portugal excited ride 7. Second? (annexin) and staphylokinase tiS IS is characterized by the fact that the membrane-linked egg is self-intercalated with the enzyme enzyme, and it is short-lived by its _ egg-to-thief enzyme-embedded correction. The chimera of item 7, wherein the annexin is a reading 11. The chimera of the seventh aspect of the patent application wherein the annexin is a surface such as 200920843 v° 12 chimera, which is annexin (annexin), Portuguese (staphylokinase) and a short linker. 13. The chimera of claim 12, wherein the membrane is intercalated between the membranes via an amino acid. Liao protein and Portuguese _ 14. 士 申请 彻 彻 彻 彻 彻 彻 彻 彻 彻 彻 彻 彻 彻 彻 彻 彻 彻 彻 彻 彻 彻 彻 彻 彻 彻 彻 彻 彻 彻 彻 彻 彻 彻The chimera from the Portuguese version 15. = Patent Application No. 12, wherein the link between the ligated egg and the occlusion is a _2 touch link. The chimera of the 12th patent of the invention, wherein the annexin is a chimera of the patent item 12, wherein the annexin is a xin body egg from f coding niacin, money Nucleic acid f-encoded nucleic acid, which is identified by the sequence identification number 2 20. A nucleic acid vector, including the sequence of the sequence identification number 2 or No. 2. The 21 acid-free chimeric protein is a chimeric protein isolated from the amino acid 22 acid group contained in SEQ ID NO: 3, which is identified by the amino acid group 23 of the sequence number 4, and the range of interest is 1'. a method for producing a chimera of 17 and 2, which comprises binding an annexin to a staphylokinase, and the chimera 19 200920843 of the scope of the patent application is linked by a short linker. . 24. A pharmaceutical composition for suppository lysis comprising annexin and staphylokinase. 25. The pharmaceutical composition according to claim 24, wherein the annexin intercalating enzyme is linked via two amino acids. 〃 Portugal 26. For example, the pharmaceutical composition of the scope of the patent application, wherein annexin XI is annexin XI. ..., 27· A pharmaceutical composition for the treatment of sputum, which comprises a chimera as claimed in the first item. ~ Offense 28. If the pharmaceutical composition of claim 22 or 25 or 27 is applied, it is used for the treatment of myocardial infarction. , 糸 for 29. The pharmaceutical composition of claim 22, 25 or 27, for the treatment of ischemic stroke. 30. A pharmaceutical composition according to claim 22 or 25 or 27, which is used for the treatment of injured endothelial cells, activated platelets and apoptotic cells (叩印仂七^ cells) related diseases. 31. Use of a compound according to claim 1 or a therapeutically acceptable salt thereof for the manufacture of thrombolytic therapy: $ 竿 丨.