WO2003008594A1 - Construction du plasmide pour l'expression de la proteine de fusion thrombolytique ciblant de thrombus - Google Patents

Construction du plasmide pour l'expression de la proteine de fusion thrombolytique ciblant de thrombus Download PDF

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Publication number
WO2003008594A1
WO2003008594A1 PCT/CN2001/001301 CN0101301W WO03008594A1 WO 2003008594 A1 WO2003008594 A1 WO 2003008594A1 CN 0101301 W CN0101301 W CN 0101301W WO 03008594 A1 WO03008594 A1 WO 03008594A1
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WIPO (PCT)
Prior art keywords
anx32
scupa
gene
sequence
plasmid
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PCT/CN2001/001301
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English (en)
Chinese (zh)
Inventor
Shuhan Sun
Hongli Yan
Ruiwen Chen
Dexter H. H. Chun
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Gene-Life Biotechnology (Shanghai) Limited
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Publication of WO2003008594A1 publication Critical patent/WO2003008594A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6456—Plasminogen activators
    • C12N9/6462—Plasminogen activators u-Plasminogen activator (3.4.21.73), i.e. urokinase
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21073—Serine endopeptidases (3.4.21) u-Plasminogen activator (3.4.21.73), i.e. urokinase
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to the field of medical biotechnology, and in particular to the preparation of a thrombus-targeting thrombolytic protein expression plasmid by a genetic engineering method. Background technique
  • Thrombosis such as acute myocardial infarction (AMI) and venous thromboembolism
  • AMD acute myocardial infarction
  • thromboembolism is a type of cardiovascular disease that seriously endangers human health and life.
  • deaths due to thrombosis already account for the largest proportion of total deaths in the population.
  • thrombolytic drugs commonly used in clinical practice are mainly plasminogen activators, such as streptokinase (SK), urokinase (UK), single-chain urokinase (scu-PA), and tissue-type plasminogen activator (t -PA), etc.
  • scu-PA Low-molecular-weight single-chain urokinase
  • scu-PA 144 ⁇ 411
  • scu-PA Single-chain urokinase
  • It is composed of amino acid residues 144 ⁇ 411 of scu-PA.
  • -PA is similar, with less bleeding side effects, but lacks specific affinity for fibrin, so the efficiency of thrombolysis is not high, and its clinical application is limited.
  • Annexin 32 (Anx 32) gene, cloned from the cysticercus cerevisiae cDNA library by the inventors (Sun Shuhan et al., Molecular cloning of cDNA encoding antigens for cysticercosis, Chinese Parasitology and Parasitology Journal of Insects, 1997, 15 (1): 15-20).
  • the gene encodes a 347 amino acid protein with a molecular weight of 38KDa.
  • Annexin 32 can have high affinity, Specifically binds acidic phospholipids.
  • the blood coagulation process can be divided into three stages: (1) Platelet activation stage.
  • the present inventors conducted a fusion expression study of annexin 32 and scu-PA (144 to 411), and completed the present invention.
  • the present invention provides a fusion gene anx32- SC uPA, the fusion gene comprising a gene encoding annexin 32 shown in sequence 1 of the sequence listing and a gene encoding amino acids 144 to 411 of single-chain urokinase shown in sequence 3 Fusion.
  • the present invention also provides a thermally-inducible expression plasmid pJM-anx32-scuPA, which is a circular double-stranded chain of 6.8 Kb in length and cloned with a protein encoding annexin 32 and single-chain urokinase amino acids 144 to 411. Gene sequence and contains? ⁇ ! ⁇ Promoter.
  • the invention further provides an engineering bacterium K802 (pJM- anx32- scuPA), which is E. coli K802
  • the fusion gene anx32- SC uPA of the present invention is prepared by the following steps: The gene and sequence encoding annexin 32 shown in sequence 1
  • the genes encoding single-stranded urokinase amino acids 144 to 411 shown in Fig. 3 were amplified by PCR respectively; then they were ligated by overlapping region extension.
  • the expression plasmid pJM_anx32-scuPA of the present invention is constructed by double-digesting the fusion gene anx32-scuPA with EcoR I and Sal I, and then ligating the vector pJM with EcoR I and Sal I.
  • the fusion expression plasmid of annexin 32 and scu-PA (144 ⁇ 411) constructed by the inventors can fuse and express annexin 32 and scu-PA (144 ⁇ 411), and the expression products can retain their respective biological activities, thereby A new thrombolytic drug with dual functions of anticoagulation and thrombolysis can be obtained.
  • existing thrombolytic drugs mainly dissolve thrombus by activating plasmin, and platelets are not sensitive to the action of plasmin, so these drugs have a poor therapeutic effect on platelet-rich thrombosis.
  • annexin 32 specifically binds to activated platelets, so it can specifically bind scu-PA (144 ⁇ 411) to the thrombus site, which can greatly improve the Therapeutic effect of platelet-containing arterial thrombosis.
  • the present invention uses a heat-inducible expression vector, which can reduce the cost during large-scale cultivation, and is conducive to large-scale production. Because the biological activities of Annexin 32 and single-chain urokinase are not affected by glycosylation Therefore, the present invention may use a prokaryotic expression system, and a yeast expression system or a mammalian cell expression system may also be used as required. BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 Schematic diagram of plasmid pJM- anx32- scuPA constructed by overlapping region extension method
  • Lane 1 DNA Marker
  • Lane 2 anx32 gene amplified by PCR # 1
  • Lane 3 PCR # 2 amplified low-molecular-weight single-chain urokinase gene
  • Lane 4 Plasmid pJM- anx32_scuPA was double digested with EcoR I and Sal I Figure 3 Expression and purification of fusion protein ANX32-SCUPA
  • Lane 1 Uninduced bacterial protein
  • Lane 3 bacterial protein 2 hours after induction
  • Lane 4 bacterial protein 3 hours after induction
  • Lane 5 Supernatant after bacterial sonication (almost no protein of interest)
  • Lane 6 Cells precipitate after sonication (with large amounts of protein of interest)
  • Lane 9 Western blot indicates that the purified protein is a fusion protein containing single-chain urokinase
  • the anx32 full-length gene-containing plasmid P UC19-anx32 used in the present invention is preserved by our laboratory (see Sun Shuhan et al., Molecular cloning of cDNA encoding antigens for cysticercosis diagnosis, Chinese Journal of Parasitology and Parasites, 1997, 15 (1 ): 15-20); Low-molecular-weight single-chain urokinase (scu-PA (144 ⁇ 411)) Gene refers to the human urokinase cDNA sequence (this sequence can be obtained in the gene database (Genbank), sequence number: M15476), where Some amino acids have been changed to E. coli preferred codons, which were artificially synthesized by Shanghai Shenggong Company.
  • the gene encoding annexin 32 and the gene encoding single-stranded urokinase amino acids 144 to 411 were amplified by PCR respectively; then, the fusion gene anx32-scu-PA (144 ⁇ 41 1). Specific steps are as follows:
  • primers a and b Amplify all the genes encoding Annexin 32 gene with primers a and b.
  • the primer a is 5, TG GAATTC ATGGCCTACTGTCGCTCCC3 ', the primer is consistent with the 5 end of the anx32 coding sequence, and an EcoR I restriction enzyme site is introduced.
  • Primer b is: 5 'GCCACACTGAAATTTTAA / TGCAGGGCCGATGAG3', in which the 18 'end of the 5' end is identical to the 5 end of the single-chain urokinase coding sequence, and the latter 15 bp is complementary to the 3 'end of the anx32 gene.
  • High-fidelity pfu DNA polymerase was used for the PCR reaction.
  • the concentration of dNTP was 20M, the concentration of Mg 2+ was 1.5 mM, and the denaturation, annealing, and extension temperatures were 94 ° C, 55 ° C, and 72 ° C, respectively, and the time was 45 seconds. , 4 seconds and 1 minute, a total of 30 cycles.
  • primers c and d were used to amplify a coding sequence fragment of a low molecular weight single-chain urokinase (scu-PA (144 to 411)).
  • Primer c is: 5, CTCATCGGCCCTGCA / TTAAAATTTCAGTGTGGC3 '.
  • the 5' end of the primer is 15 bp consistent with the 3 'end of anx32, and the latter 18 bp is consistent with the sequence of the 5' end of the low molecular weight single-chain urokinase.
  • Primer d is: 5'TTGTCGAC GCA GAG GGC CAG GCC3, which is complementary to the 3 'end sequence of the low molecular weight single-chain urokinase coding sequence.
  • a Sal I restriction enzyme site was also introduced. The PCR reaction procedure is the same as (1), and a total of 30 cycles are performed.
  • a fusion gene encoding annexin 32 and a low-molecular-weight single-chain urokinase was obtained by the overlapping region extension method.
  • the products obtained by the above two-step PCR reactions were separated by 1.0% agarose electrophoresis, and then recovered by gel.
  • the recovered product was denatured at 94 ° C for 5 minutes, and the products of the two PCRs were mixed and annealed at 50 ° C. Because primers b and c have overlapping portions, the single strand of the anx32 gene can be paired with the single strand of scu-PA (14: ⁇ 411) after annealing.
  • the primers a and d were used to sequence the fusion gene anx32-scu-PA (144 ⁇ 411) amplified by the above method, and the sequencing reaction was completed by Shanghai Ji Kang Company. After the sequencing is correct, it is double-digested with EcoR I and Sai l and ligated to the vector pJM double-digested with EcoR I and Sal I. Restriction enzymes and T 4 ligases for enzyme digestion and ligation reactions were purchased from TaKaRa Company, and the reaction was performed using the system recommended in the enzyme instructions.
  • the heat-inducible expression vector pJM present invention utilized the full length 4. 9Kb (kilobase pairs), containing the phage P R PL promoter, heat-inducible gene CI1857, plasmid origin of replication (ori) and ampicillin resistance gene (AP r ).
  • the specific reaction process is as follows: EcoR I and Sal I are used to double-digest the above PCR products and vectors.
  • the reaction uses a high-salt (H) buffer system and 37 ° C for 3 hours. It was then separated by 1.0% agarose electrophoresis and recovered as a gel.
  • the enzyme fragments and the carrier were mixed at a molar ratio of 5: 1, and the corresponding ligation buffer solution and ligase were added, and the ligation was performed at 16 ° C for 12 hours.
  • H high-salt
  • the enzyme fragments and the carrier were mixed at a molar ratio of 5: 1, and the corresponding ligation buffer solution and ligase were added, and the ligation was performed at 16 ° C for 12 hours.
  • the ligated product was transformed into E. coli strain K802 to obtain an engineered strain K802 (pJM-anx32-scuPA).
  • the specific steps are as follows: The host bacteria K802 is first cultured in 50ml LB medium (containing 1% tryptone, 0.5% yeast extract and 1% sodium chloride, PH7.0) at 37 ° C with shaking to 0D. At 0.4 o'clock, centrifuge at 4000 rpm at 4 ° C to collect the host bacteria. After removing the supernatant, resuspend it in 1-2 ml of ice-cold 100 mmol / L calcium chloride solution to obtain the competent state. bacterial. 100 ng of the constructed plasmid and competent bacteria were cooled in an ice bath for 30 minutes, heat-shocked at 42 ° C for 90 seconds, and then cooled in an ice bath for 10 minutes.
  • 50ml LB medium containing 1% tryptone, 0.5% yeast extract and 1% sodium chloride, PH7.0
  • Engineering bacteria K802 can express the fusion protein ANX32-SCUPA (lane 2, 3, 4) of fusion protein Annexin 32 and low-molecular-weight single-chain urokinase by way of temperature induction. Most proteins are in the form of inclusion bodies (lanes 5, 6), and their expression can account for more than 20% of the bacterial protein. After protein purification, 10% SDS-PAGE electrophoresis and staining with Coomassie Brilliant Blue showed a single band with a molecular weight of 68 kDa (lane 7).
  • the inclusion body prepared by the above method was added with 5 ml of lysate (8 M urea, 0.5 M NH 4 C1, 500 mM Tris-HCl, pH 8. 5), and shaken at room temperature for 4 hours. The 7700 g was then centrifuged to remove the insoluble fraction. This method can be used to prepare about 70% crude protein.
  • the crude protein was further purified by a Sephacryl S-400 gel chromatography column (Pharmacia). Equilibrium and eluent were 5 M urea, 0.5 NH 4 C1, 50 mM Tris-HCl, pH 8. 0, 0.5 mM EDTA, 10 mM DTT o SDS-PAGE analysis, and collected the wash containing the protein of interest Decant, store at -20 ° C.
  • the sample was dialyzed for 12 hours.
  • the dialysate was 10 times the volume of 5 M urea, 0.5 NH 4 C1, 50 mM Tris-HCl, pH 8.5.
  • Urokinase contains 12 pairs of disulfide bonds, so care should be taken to remove oxygen from the dialysate during dialysis.
  • the dialysate was first degassed by ultrasound for 10 minutes, then passed through for 5 minutes, and finally the dialyzed Erlenmeyer flask was sealed with a parafilm membrane.
  • the dialyzed sample was added dropwise to a 100-fold volume of buffer containing 2 M urea, 0.5 NH 4 C1, 50 mM Tris-HCl, 0.5 mM EDTA, 1.
  • the above samples were then subjected to ion exchange chromatography using a weak anion exchange column (the chromatography medium was diethylaminoethyl agarose, DEAE Sepharose Fast Flow, Pharmacia).
  • the column was equilibrated with 50 mM Tris ⁇ HCl, pH 8.0.
  • the eluted salt concentration was eluted at a concentration gradient from 0 to 1 mol / L NaCl at a flow rate of 1 ml / min.
  • Polyacrylamide gel electrophoresis (SDS-P AGE) was used to determine the peak of the recombinant protein, and it was found that the recombinant protein was eluted at a salt concentration of 0.4 M.
  • the expression of recombinant protein was detected by Western blot.
  • the specific method is: After the expressed recombinant protein is separated by 10% SDS-PAGE, it is electrically transferred to a nitrocellulose membrane with a current of 0.65 mA / cm 2 and a time of 2 hours. Add TBST buffer (10 mM Tris-HC1, 150 mM NaCl, 0.05% Tween-20, pH 8.3) and wash three times. Add TBST + 5% skimmed milk powder and shake at 37 ° C for 1 hour. Then the blocking solution was discarded, and an antibody against single-chain urokinase was added at a ratio of 1:50, and shaken at 37 ° C for 1 hour.
  • Coomassie brilliant blue staining (Bradford method) was used to determine the concentration of purified protein and the recovery was calculated. The resulting protein was freeze-dried and divided into aliquots and stored at -20 ° C.

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Abstract

La présente invention propose un gène de fusion anx-32-scuPA fusionné par un gène codant pour l'annexine 32 and un autre codant pour le site 144 à 411 de la séquence d'acides aminés du pro-urokinase. La longueur du plasmide cyclique bicaténaire thermiquement induit (pJM-anx32-scuPA) du gène de fusion est de 6.8 Kb. La transformation de E.coli au moyen du plasmide (pJM-anx32-scuPA) produit la bactérie d'ingénierie génétique K802 de l'invention. Le gène de fusion de l'invention est capable d'exprimer la protéine de fusion complexée par l'annexine 32 et le site 144 à 411 de la séquence d'acides aminés de la pro-urokinase. La protéine hybride possède la double fonction d'anticoagulothérapie et de traitement thrombolytique. Elle présente une haute efficacité dans le traitement de troubles thrombolytiques et n'entraîne pas d'effet secondaire d'hémorragie.
PCT/CN2001/001301 2001-07-20 2001-09-03 Construction du plasmide pour l'expression de la proteine de fusion thrombolytique ciblant de thrombus WO2003008594A1 (fr)

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CN01126298.2 2001-07-20
CN 01126298 CN1188522C (zh) 2001-07-20 2001-07-20 一种血栓靶向性溶栓蛋白表达质粒及其构建

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2856069A1 (fr) * 2003-06-10 2004-12-17 Bionexis Molecules de ciblage et de liberation de composes therapeutiques et leur utilisation
WO2006094536A1 (fr) * 2005-03-04 2006-09-14 Paion Deutschland Gmbh Proteines de fusion activatrices du plasminogene cible utilisees en tant qu’agents thrombolytiques

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1480466A (zh) * 2002-09-03 2004-03-10 �й������ž�����ҽѧ��ѧԺ����ҽ 一类溶栓抗凝双功能融合蛋白及应用
CN100519585C (zh) * 2007-02-06 2009-07-29 中国人民解放军军事医学科学院基础医学研究所 P11与sak的融合蛋白及其制备方法和用途

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998025641A1 (fr) * 1996-12-09 1998-06-18 Hadasit Medical Research Services & Development Company Ltd. UTILISATIONS EN MEDECINE DE COMPLEXE scuPA/suPAR
WO1999005277A1 (fr) * 1997-07-25 1999-02-04 Mogam Biotechnology Research Institute Vecteur d'expression de recombinaison et parathormone humaine utilisant phosphoribulokinase comme partenaire de fusion
CN1247195A (zh) * 1999-03-12 2000-03-15 中国科学院上海生物化学研究所 一种血栓靶向性溶栓融合蛋白

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998025641A1 (fr) * 1996-12-09 1998-06-18 Hadasit Medical Research Services & Development Company Ltd. UTILISATIONS EN MEDECINE DE COMPLEXE scuPA/suPAR
WO1999005277A1 (fr) * 1997-07-25 1999-02-04 Mogam Biotechnology Research Institute Vecteur d'expression de recombinaison et parathormone humaine utilisant phosphoribulokinase comme partenaire de fusion
CN1247195A (zh) * 1999-03-12 2000-03-15 中国科学院上海生物化学研究所 一种血栓靶向性溶栓融合蛋白

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2856069A1 (fr) * 2003-06-10 2004-12-17 Bionexis Molecules de ciblage et de liberation de composes therapeutiques et leur utilisation
WO2004111090A2 (fr) * 2003-06-10 2004-12-23 Commissariat A L'energie Atomique Molecules de ciblage et de liberation de composes therapeutiques et leur utilisation
WO2004111090A3 (fr) * 2003-06-10 2005-09-09 Bionexis Molecules de ciblage et de liberation de composes therapeutiques et leur utilisation
WO2006094536A1 (fr) * 2005-03-04 2006-09-14 Paion Deutschland Gmbh Proteines de fusion activatrices du plasminogene cible utilisees en tant qu’agents thrombolytiques

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CN1398972A (zh) 2003-02-26

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