WO2017028558A1 - sDR5-Fc融合蛋白突变体及其应用 - Google Patents

sDR5-Fc融合蛋白突变体及其应用 Download PDF

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WO2017028558A1
WO2017028558A1 PCT/CN2016/081552 CN2016081552W WO2017028558A1 WO 2017028558 A1 WO2017028558 A1 WO 2017028558A1 CN 2016081552 W CN2016081552 W CN 2016081552W WO 2017028558 A1 WO2017028558 A1 WO 2017028558A1
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sdr5
amino acid
acid sequence
fusion protein
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马远方
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河南大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/1774—Immunoglobulin superfamily (e.g. CD2, CD4, CD8, ICAM molecules, B7 molecules, Fc-receptors, MHC-molecules)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/1793—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the invention belongs to the technical field of genetic engineering and protein engineering, relates to sDR5-Fc fusion protein and application thereof, and particularly relates to sDR5-Fc fusion protein and application thereof in preparing medicine for treating myocardial infarction.
  • Myocardial ischemia refers to a pathological state in which the blood supply to the heart is reduced due to the decrease of coronary blood perfusion, and the myocardial energy metabolism is disordered. Coronary stenosis or occlusion is the most common and most common cause of myocardial ischemia.
  • ischemia-reperfusion injury After myocardial ischemia, thrombolysis, coronary artery bypass grafting or percutaneous coronary angioplasty is required to restore the blood supply to the ischemic myocardium. Reperfusion can save the myocardium in the ischemic area, and it will further aggravate the dying cardiomyocyte injury. This phenomenon of tissue damage after the recovery of blood flow is aggravated, and even irreversible cell death is called ischemia-reperfusion injury.
  • the mechanism of reperfusion injury involves many cell and molecular biological events such as ROS production, intracellular calcium overload, mitochondrial dysfunction, cellular inflammation, autophagy, apoptosis, etc., which cross each other into a complex network, which ultimately leads to the death of cardiomyocytes.
  • ROS production ROS production
  • intracellular calcium overload mitochondrial dysfunction
  • mitochondrial dysfunction mitochondrial dysfunction
  • cellular inflammation autophagy
  • apoptosis etc.
  • DR5 Death receptor
  • TRAIL tumor necrosis factor-related apoptosis-inducing ligand
  • the full-length DR5 is a type I transmembrane glycoprotein containing 411 amino acids. Soluble death receptor 5 (soluble DR5, sDR5) is secreted outside the cell due to the lack of transmembrane regions that cannot be expressed on the cell membrane.
  • sDR5 is expressed in normal human peripheral blood because it has an intact structure of extracellular domain that binds to TRAIL ligand and competes with death receptors on the cell membrane for binding to TRAIL molecules, thereby blocking TRAIL-induced apoptosis. .
  • sDR5 can attenuate hepatocyte injury caused by hepatitis B virus (HBV) infection by blocking TRAIL-induced apoptosis, and can also inhibit brain cell apoptosis in cerebral ischemia-reperfusion model.
  • HBV hepatitis B virus
  • the invention has been experimentally screened to prove that the fusion protein composed of the N-terminal 1-182 amino acids of the extracellular region of DR5 and the human anti-Fc fragment can substantially avoid the Fc-mediated ADCC effect, and can significantly reduce the myocardial ischemia-reperfusion model rat. Myocardial infarct size.
  • the present invention provides a fusion protein consisting of an sDR5 fragment and a human antibody Fc fragment.
  • the sDR5 fragment is the entire amino acid sequence of the extracellular region of DR5 or a partial amino acid sequence thereof as long as it retains the activity of binding to the TRIL ligand.
  • This fusion protein was named sDR5-Fc.
  • amino acid sequence of the sDR5 fragment is any one of the following groups:
  • amino acid sequence of the sDR5 fragment is set forth in SEQ ID NO.
  • the number of substitutions and/or deletions and/or additions of one or several amino acids in the amino acid sequence set forth in SEQ ID NO. 1 is no more than ten.
  • human antibody Fc fragment in the present invention means "human immunoglobulin heavy chain constant region" and can be derived from antibodies belonging to each immunoglobulin class called IgA, IgD, IgE, IgG and IgM. Furthermore, immunoglobulin heavy chain constant regions are contemplated to be derived from any of the IgG antibody subclasses known in the art as IgGl, IgG2, IgG3 and IgG4.
  • the human antibody Fc fragment of the invention is derived from the human antibody IgGl.
  • amino acid sequence of the human antibody Fc segment of the invention is any one of the following groups:
  • amino acid sequence of the human antibody Fc fragment is set forth in SEQ ID NO.
  • amino acid sequence of the above fusion protein sDR5-Fc of the present invention is any one of the following groups:
  • amino acid sequence of the fusion protein sDR5-Fc of the present invention is shown in SEQ ID NO.
  • a wild-type sDR5-Fc sequence that differs by one or more conservative amino acid substitutions or by one or more non-conservative amino acid substitutions, deletions or insertions, wherein such substitutions, deletions or insertions do not abolish the wild-type sequence Biological activity.
  • Conservative substitutions typically include the substitution of one amino acid by another amino acid having similar characteristics, such as substitutions in the following groups: proline, glycine; glycine, alanine; valine, isoleucine, leucine; Aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
  • Other conservative amino acid substitutions are known in the art and are included herein.
  • Non-conservative substitutions such as replacement of basic amino acids by hydrophobic amino acids, are also well known in the art.
  • Modifications to increase the stability of a protein or peptide of the fusion protein sDR5-Fc of the present invention are also included in the scope of the present invention; such modifications include, for example, one or more non-peptide bonds in the protein or peptide sequence (for Instead of a peptide bond); such modifications also include amino acids comprising D-amino acids or non-naturally occurring or synthetic, such as beta or gamma amino acids.
  • the present invention provides a DNA molecule encoding the aforementioned fusion protein sDR5-Fc.
  • nucleotide sequence of the DNA molecule is any one of the following groups:
  • nucleotide sequence shown in SEQ ID NO. 4 (2) a nucleotide sequence obtained by substituting and/or deleting and/or adding one or several nucleotides in the nucleotide sequence shown in SEQ ID NO. 4; the nucleotide sequence of SEQ ID NO The nucleotide sequence shown in .4 or a degenerate sequence thereof encodes a protein having the same function or similarity;
  • nucleotide sequence of the DNA molecule encoding the fusion protein sDR5-Fc described above is set forth in SEQ ID NO.
  • the "stringent conditions" of the present invention may specifically hybridize to the nucleotide sequence shown in SEQ ID NO. 4 at 65 ° C in a 6 x SSC, 0.5% SDS solution, and then use 2 x SSC, 0.1. %SDS and 1 ⁇ SSC, 0.1% SDS were each washed once.
  • a nucleotide sequence which hybridizes under stringent conditions to the nucleotide sequence defined by SEQ ID NO. 4 and which has the same function is at least about 40%-50% homologous to the sequence set forth in SEQ ID NO. 4, about 60%, 65% or 70% homologous, even at least 98% or more homologous.
  • sequence identity is distributed at least about 40%-50%, about 60%, 65%, or 70% homology, even at least about 75%, 80%, 85%, 90%, 91%, 92%, 93. Sequence homology of %, 94%, 95%, 96%, 97%, 98%, 99% or greater.
  • the present invention provides a recombinant vector comprising the DNA molecule encoding the fusion protein sDR5-Fc as described above.
  • vector is understood to mean a nucleotide sequence comprising a nucleotide sequence capable of being incorporated into a host cell and recombined with the host cell genome and integrated into the host cell genome, or autonomously replicating as a free body. Any nucleic acid.
  • vectors include linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors, and the like.
  • Vectors suitable for use in the present invention include, but are not limited to, expression vectors, cloning vectors, for example, for prokaryotic (such as bacteria such as Escherichia coli), lower eukaryotic cells (such as yeast), insect cells, plant cells, mammalian cells. a.
  • prokaryotic such as bacteria such as Escherichia coli
  • lower eukaryotic cells such as yeast
  • insect cells such as bacteria such as Escherichia coli
  • plant cells mammalian cells.
  • mammalian cells mammalian cells.
  • any plasmid and vector can be used as long as it can replicate and stabilize in the host.
  • the expression vector may also contain a marker gene and other translational control elements.
  • Viral vectors useful in the present invention include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, herpes simplex virus vectors (herpes simplex virus- Based vectors), lentiviral vectors.
  • the vector of the invention uses the eukaryotic expression vector pGS-Fc.
  • the invention also provides host cells comprising the recombinant vectors described above.
  • the host cell may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a mammalian cell.
  • Representative examples are: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium: fungal cells such as yeast; plant cells; insect cells of Drosophila S2 or Sf9; CHO, COS, 293 cells, or Bowes melanoma cells Animal cells, etc.
  • the invention also provides a preparation method of the aforementioned fusion protein sDR5-Fc:
  • fusion proteins there are various expression systems that can be used to express these fusion proteins, and may be eukaryotic cells or prokaryotic cells including, but not limited to, mammalian cells, bacteria, yeast, insect cells, and the like. Since the present invention optimizes the amino acid sequence of the fusion protein to comprise a glycosylated amino acid, the mammalian cell is the preferred system for expression of the protein.
  • mammalian cells that can be used for large-scale expression of proteins, such as CHO cells, 293 cells, NSO cells, COS cells, BHK cells, etc. Many other cells can also be used for protein expression, and thus are included in the present invention. The list of cells used.
  • Expression systems other than mammalian cells such as bacteria, yeast, insect cells, and the like, can also be used to express the optimized fusion proteins of the invention, which are also included in the host cells that can be used in the present invention.
  • the protein production of these expression systems is higher than that of mammalian cells, but the expressed proteins lack glycosylation or form a sugar chain structure that is different from mammalian cells.
  • a recombinant plasmid containing the above fusion protein gene can be transfected into a host cell, and the method of transfecting the cell can be carried out by various methods including, but not limited to, electroporation, lipofection, and calcium phosphate transfection. .
  • fusion proteins contain an immunoglobulin Fc
  • protein A affinity chromatography can be used to purify the expressed fusion protein.
  • the fusion protein of the present invention can be further purified in combination with other protein purification methods such as ion exchange chromatography and the like.
  • the present invention also provides a fusion protein sDR5-Fc as described above, a DNA molecule encoding the fusion protein sDR5-Fc described above, a recombinant vector as described above, or a host cell comprising the recombinant vector described above for preparing a myocardial Application of infarct treatment drugs.
  • the present invention also provides a pharmaceutical composition comprising the fusion protein sDR5-Fc described above, the aforementioned DNA molecule encoding the fusion protein sDR5-Fc, the aforementioned recombinant vector, or the former Said host cell comprising a recombinant vector.
  • the pharmaceutical composition of the present invention further comprises a carrier that is to be scientifically acceptable, including but not limited to any adjuvant or form that has been approved by the U.S. Food and Drug Administration for use in humans or animals.
  • a carrier that is to be scientifically acceptable, including but not limited to any adjuvant or form that has been approved by the U.S. Food and Drug Administration for use in humans or animals.
  • Agents, glidants, sweeteners, diluents, preservatives, dyes/colorants, flavor enhancers, surfactants, wetting agents, dispersing agents, suspending agents, stabilizers, isotonic agents, solvents or Emulsifiers and the like have various forms of carriers which do not have side effects in constituting the pharmaceutical composition.
  • composition of the present invention can be administered by various means such as by oral, sublingual, buccal, parenteral, nasal, topical, rectal, transdermal, transmucosal and the like.
  • the pharmaceutical compositions may be prepared in a variety of dosage forms including, but not limited to, tablets, solutions, granules, patches, ointments, capsules, aerosols, suppositories. .
  • compositions of the invention may be administered, for example, in a form suitable for immediate or sustained release. Immediate release or sustained release can be achieved by the use of a suitable pharmaceutical composition, or particularly in the case of sustained release, by the use of devices such as subcutaneous implants or osmotic pumps.
  • the subject to be treated is a patient undergoing myocardial ischemic injury, including but not limited to mammals such as humans, dogs, cats and the like.
  • the particular dosage level and dosage frequency for any particular subject may vary and will depend on a variety of factors, including the activity of the pharmaceutical composition employed; the metabolic stability and duration of action of the pharmaceutical composition; Species' age, age, weight, general health status, gender and diet; mode and timing of administration; excretion rate and clearance rate; drug combination; and severity of specific conditions.
  • compositions of the invention may be used alone or in combination with other suitable therapeutic agents useful for the treatment of myocardial ischemic injury. Or use with other surgical methods for treating myocardial ischemic injury.
  • Treatment includes a complete cure for a condition and also includes amelioration of the condition.
  • gene expression or “expression” is understood to mean the transcription of a DNA sequence, the translation of an mRNA transcript, and the secretion of an Fc fusion protein product.
  • mutant and wild type as used in the context of the present invention are directed to the sequence of the Fc fragment.
  • the mutation site of the "fusion protein mutant” is on the Fc segment.
  • the present invention constructs the sDR5(1-182)-Fc fusion protein mutant for the first time, and the use of the fusion protein reduces the ADCC effect due to mutation of the Fc segment.
  • the sDR5 (1-182) fragment selected from the sDR5(1-182)-Fc fusion protein mutant of the present invention has the following advantages as compared with other fragments of the extracellular region of DR5: high expression, easy acquisition, and The TRIL ligand binding ability is strong, the ADCC effect is weak, and the effect of inhibiting myocardial damage is stronger.
  • Figure 1 shows the expression of purified 8 human sDR5-Fc fusion proteins verified by Western Blot, wherein #1: new signal peptide (sp)-sDR5(56-133)-Fc mutant; #2:sDR5(1 -182)-Fc mutant; #3: sDR5(1-133)-Fc mutant; #4: new signal peptide (sp)-sDR5(56-182)-Fc mutant; #5: sDR5(1- 182)-Fc wild type; #6: sDR5(1-133)-Fc wild type; #7: new signal peptide (sp)-sDR5(56-133)-Fc wild type; #8: new signal peptide (sp )-sDR5(56-182)-Fc wild type;
  • Figure 2 shows the expression of human sDR5-Fc in CHO-K1 supernatant by SDS-PAGE Coomassie blue staining, wherein #1: new signal peptide (sp)-sDR5(56-133)-Fc mutant;# 2: sDR5(1-182)-Fc mutant; #3: sDR5(1-133)-Fc mutant; #4: new signal peptide (sp)-sDR5(56-182)-Fc mutant; #5 : sDR5(1-182)-Fc wild type;
  • Figure 3 shows the affinity of human sDR5-Fc fusion protein to TRAIL as determined by ELISA, wherein #1: new signal peptide (sp)-sDR5(56-133)-Fc mutant; #2:sDR5(1-182)- Fc mutant; #3: sDR5(1-133)-Fc mutant; #4: new signal peptide (sp)-sDR5(56-182)-Fc mutant; #5: sDR5(1-182)-Fc Wild type;
  • Figure 4 shows the effect of human sDR5-Fc fusion protein on TRAIL-induced apoptosis; #1: ⁇ Signal peptide (sp)-sDR5(56-133)-Fc mutant; #2: sDR5(1-182)-Fc mutant; #3: sDR5(1-133)-Fc mutant; #4: new signal Peptide (sp)-sDR5(56-182)-Fc mutant; #5: sDR5(1-182)-Fc wild type;
  • Figure 5 shows the affinities of sDR5(1-182)-Fc mutant and sDR5(1-182)-Fc wild type and Fc receptor by ELISA; wherein #2:sDR5(1-182)-Fc mutant;# 5: sDR5(1-182)-Fc wild type;
  • Figure 6 shows the ADCC effect of the sDR5(1-182)-Fc mutant detected by the LDH method
  • Figure 7 shows the protective effect of human sDR5(1-182)-Fc mutant on cardiomyocytes.
  • Pichia pastoris GS115 strain containing pGAPZ ⁇ A-sDR5 (see: Song K, Chen Y, Goke R, et al. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an inhibitor of autoimmune inflammation and cell cycle Progression. JExp Med, 2000; 191(7): 1095-1104) is a PCR template for amplification of sDR5 (1-133), sDR5 (1-182), new signal peptide (sp)-sDR5 (56-182), New signal peptide (sp)-sDR5 (56-133) encodes a gene fragment
  • step (1) The gene fragment of step (1) was cloned into the eukaryotic expression vector pGS-Fc carrying the human IgG Fc fragment.
  • the pGS-Fc multiple cloning site was inserted into the double restriction enzyme of EcoR I using Hind III.
  • Amplification of the recombinant expression plasmid, and the recombinant expression plasmid constructed in the step 1 is introduced into a prokaryotic host cell for amplification.
  • the high purity plasmid (OMEGA Plasmid Midi Kit) was extracted and adjusted to a concentration of 500 ng/ ⁇ l.
  • the plasmid was transiently transfected into 293T cells. After 48 hours, the supernatant and cells were collected, and the supernatant and cell lysate were adsorbed by ProteinA/G beads. Western blot was used to detect the expression of fusion protein.
  • the cell state was adjusted to the optimal, transient electrotransformation (300 v, interval 0.125 s, duration 0.1 ms, electroporation three times), and after 24 hours, the supernatant was collected and purified by affinity chromatography to obtain a human sDR5-Fc fusion protein.
  • the purified fusion protein was confirmed by Coomassie blue staining after SDS-PAGE, and the yield of the fusion protein of the present invention was 30 mg/L.
  • the eight sDR5-Fc fusion proteins constructed were expressed in 293T cells.
  • #1 new signal peptide (sp)-sDR5(56-133)-Fc mutant
  • #2 sDR5(1-182)-Fc mutant
  • #3 sDR5(1-133)-Fc mutant
  • #4 New signal peptide (sp)-sDR5(56-182)-Fc mutant
  • #5 sDR5(1-182)-Fc wild type
  • #6 sDR5(1-133)-Fc wild type
  • #7 New signal peptide (sp)-sDR5(56-133)-Fc wild type
  • #8 New signal peptide (sp)-sDR5(56-182)-Fc wild type
  • the selected 5 sDR5-Fc fusion proteins were expressed in the supernatant of CHO/K1 cells.
  • #1 new signal peptide (sp)-sDR5(56-133)-Fc mutant
  • #2 sDR5(1-182)-Fc mutant
  • #3 sDR5(1-133)-Fc mutant
  • #4 New signal peptide (sp)-sDR5(56-182)-Fc mutant
  • #5 sDR5(1-182)-Fc;
  • the affinity of human sDR5-Fc to TRAIL was determined using an ELISA method.
  • the coated TRAIL was diluted with CBS from 2 ⁇ g/mL (2, 1, 0.5 ⁇ g/mL), 100 ⁇ L/well, and coated at 4 ° C overnight;
  • the solution in the plate was taken out, patted dry, and washed 3 times with the washing solution for 2 min each time;
  • Stop solution was added, 50 ⁇ L per well to terminate the color reaction, and then the OD450 value of each well was measured with a microplate reader.
  • Step 1 Jurkat cells were cultured in a 100 mm cell culture dish.
  • the culture medium was 1640 medium containing 10% serum.
  • the cells of the 3-4th generation were collected, centrifuged at 500g for 10 minutes, counted, and the cells were plated at 96.
  • each treatment factor is 4 holes.
  • the treatment of blocking the TRAIL group was performed by pre-mixing TRAIL and blocking proteins for 30 min.
  • the working concentration of TRAIL was 250 ng/ml and the working concentration of blocking protein was 5 ⁇ g/ml.
  • the #2:sDR5(1-182)-Fc mutant had lower affinity with the three types of Fc receptors FcgammaR1, FcgammaRllb, and FcgammaRll than the #5:sDR5(1-182)-Fc wild type.
  • step 6 Add 10 ml of the cell washing solution to the obtained centrifuge tube in step 5 and mix.
  • Steps 8, 9, and 10 Discard the supernatant. Steps 8, 9, and 10 were repeated, after which the cells were resuspended in 1 ml of 1640 medium containing 10% FBS and counted.
  • the sDR5(1-182)-Fc mutant had almost no ADCC effect.
  • Example 6 sDR5(1-182)-Fc mutant protects cardiomyocytes from myocardial infarction rats
  • Ligation/release of the rat for anterior descending coronary artery to establish an ischemia-reperfusion model clean-grade male Wistar rats (body weight 200-250 g), 10% chloral hydrate peritoneal injection anesthesia (3-4 ml/kg), trachea Intubate and connect to the ventilator before opening the intercostal space, adjust the parameter suction ratio 1:2, respiratory rate 90-120 beats / min, tidal volume 2-4ml; sternal left margin 3-4 intercostal transverse incision, cut happy bag
  • the exposed heart was marked with the left coronary vein at the junction of the pulmonary artery cone and the left atrial appendage.
  • the left anterior descending artery was ligated about 0.1 cm in the middle of the lower atrial appendage, and the sham operation group was used as a control (only threaded without ligation). After the routine injection of penicillin 400,000 U / only.
  • Rats with coronary artery ligation were randomly divided into PBS group and sDR5(1-182)-Fc mutant group. Each group was divided into three groups according to different doses. Each group of 6 mice was repeated three times. With The experimental methods and results are described as follows:
  • the human sDR5-Fc group was injected with human sDR5(1-182)-Fc mutant through the tail vein of the rat; the PBS group was injected with the corresponding dose of sterile PBS through the tail vein.
  • the rats were anesthetized with chloral hydrate, the thoracic artery was opened and the aorta was ligated, and 5 ml of 1% Evans Blue was injected from the left ventricle after re-ligating the left anterior descending coronary artery. The speed was moderate; then the left ventricle was quickly cut and pre-cooled. Rinse three times in physiological saline and freeze at -20 ° C for at least 1 h. The tissue was taken out from the refrigerator before TTC staining, and quickly cut into 1-2 mm thick sections on tin foil paper and placed in a 12-well plate containing TTC dye solution, incubated at 37 ° C for 15 min in the dark, and then rinsed with tissue.
  • TTC Dyeing Kit Nanjing Jiancheng Technology Co., Ltd. washed away the excess dye on the surface of the tissue, observed and photographed, in which white is the infarct zone, blue is the normal myocardium, and red and white are the danger zones.
  • the infarct area was expressed as IS
  • the risk area was expressed as AAR
  • the left ventricle was expressed as LV
  • the myocardial infarct size was expressed as IS/AAR ratio
  • the myocardial ischemic area was expressed as AAR/LV.
  • Fig. 7 In the rat model of ischemia 1 h reperfusion for 3 h, the sDR5(1-182)-Fc mutant 15 mg/kg was injected into the tail vein for 5 min before reperfusion, compared with the control group. Can significantly reduce myocardial infarct size, reducing more than 40%.

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Abstract

提供了一种人sDR5-Fc融合蛋白及其在制备治疗心肌梗死药物中的应用。所述融合蛋白是将sDR5胞外区的182个氨基酸与人抗体Fc片段连接制备而成，其可基本避免Fc介导的ADCC效应，并显著减少心肌缺血-再灌注模型大鼠的心肌梗死面积。

Description

sDR5-Fc融合蛋白突变体及其应用 技术领域
本发明属于基因工程和蛋白质工程技术领域，涉及sDR5-Fc融合蛋白及其应用，具体涉及sDR5-Fc融合蛋白及其在制备心肌梗死治疗药物的应用。
背景技术
心肌缺血是指因冠状动脉血液灌注减少，导致心脏供氧不足，心肌能量代谢紊乱，不能支持心脏正常工作的一种病理状态。冠脉狭窄或闭塞是引起心肌缺血最主要、最常见的原因。
心肌缺血后须进行溶栓、冠脉搭桥或经皮冠脉血管成形术等使缺血心肌恢复血液供应。再灌注能挽救缺血区心肌，同时也会进一步加重濒临死亡的心肌细胞损伤。这种在恢复血流后组织损伤反而加重，甚至发生不可逆性细胞死亡的现象称为缺血-再灌注损伤。
再灌注损伤机制涉及ROS生成、细胞内钙超载、线粒体功能紊乱、细胞炎症、自噬、凋亡等诸多细胞与分子生物学事件，相互之间交叉成一个复杂的网络，最终导致心肌细胞死亡。怎样通过详细的机制研究，开发有针对性的药物有效避免可能发生的再灌注损伤是目前科研及临床工作的重点和难点。坏死曾一度被认为是心肌缺血-再灌注损伤的主导因素。近年来，凋亡在其中的作用日益受到重视。死亡受体5(Death receptor，DR5)与肿瘤坏死因子相关凋亡诱导配体(TNF-related apoptosis-inducing ligand，TRAIL)结合所激活的死亡受体通路，可通过招募相关蛋白形成DISC(death-inducing signaling complex)，进而级联式活化Caspase-8、3等，引起细胞凋亡。全长的DR5是含有411个氨基酸的I型跨膜糖蛋白。可溶性死亡受体5(soluble DR5，sDR5)因缺乏跨膜区不能在细胞膜上表达而被分泌至细胞外。sDR5在正常人外周血中表达较低，因其具有与TRAIL配体相结合的胞外段完整结构，可与细胞膜上的死亡受体竞争性结合TRAIL分子，从而阻断TRAIL诱导的细胞凋亡。
有研究表明：sDR5可通过阻断TRAIL诱导的细胞凋亡减轻乙型肝炎病毒(HBV)感染引起的肝细胞损伤，也可抑制脑缺血-再灌注模型中脑细胞的凋亡。目前，sDR5是否可以通过抑制凋亡改善心肌缺血-再灌注损伤，国内外尚无报道。
发明内容：
本发明的目的在于提供一种可以用于治疗心肌缺血-再灌注损伤的sDR5-Fc融合蛋白。本发明经过试验筛选，证明DR5胞外区N端1-182个氨基酸与人抗Fc片段构成的融合蛋白可基本避免Fc介导的ADCC效应，并能显著减少心肌缺血-再灌注模型大鼠心肌梗死面积。
为了实现上述目的，本发明采用了如下技术方案：
本发明提供了一种融合蛋白，所述融合蛋白是由sDR5片段和人抗体Fc段构成。所述sDR5片段为DR5胞外区的全部氨基酸序列或其部分氨基酸序列，只要其保留与TRIL配体结合的活性即可。将该融合蛋白命名为sDR5-Fc。
优选地，所述sDR5片段的氨基酸序列为以下组中的任意一个：
(1)如SEQ ID NO.1所示的氨基酸序列；
(2)在SEQ ID NO.1所示的氨基酸序列中经过取代和/或缺失和/或添加一个或几个氨基酸所得功能相同或相似的氨基酸序列。
(3)与(1)或(2)限定的氨基酸序列至少具有99％同源性且具有相同功能的氨基酸序列。
更优选地，所述sDR5片段的氨基酸序列如SEQ ID NO.1所示。
优选地，在SEQ ID NO.1所示的氨基酸序列中经过取代和/或缺失和/或添加一个或几个氨基酸的个数不超过10个。
本发明中的“人抗体Fc段”是指“人免疫球蛋白重链恒定区”，可以来源于属于称为IgA、IgD、IgE、IgG及IgM的各免疫球蛋白类别的抗体。此外，预期免疫球蛋白重链恒定区可以来源于本领域中称为IgG1、IgG2、IgG3和IgG4的任一IgG抗体子类。
优选地，本发明的人抗体Fc段来自于人抗体IgG1。
优选地，本发明的所述人抗体Fc段的氨基酸序列为以下组中的任意一个：
(1)如SEQ ID NO.2所示的氨基酸序列；
(2)在SEQ ID NO.2所示的氨基酸序列中经过取代和/或缺失和/或添加一个或几个氨基酸所得功能相同或相似的氨基酸序列；
(3)与(1)或(2)限定的氨基酸列至少具有98％同源性且具有相同功能的氨基酸序列。
更优选地，所述人抗体Fc段的氨基酸序列如SEQ ID NO.2所示。
本发明的上述融合蛋白sDR5-Fc的氨基酸序列为以下组中的任意一个：
(1)如SEQ ID NO.3所示的氨基酸序列；
(2)在SEQ ID NO.3所示的氨基酸序列中经过取代和/或缺失和/或添加一个或几个氨基酸所得功能相同或相似的氨基酸序列；
(3)与(1)或(2)限定的氨基酸列至少具有98％同源性且具有相同功能的氨基酸序列。
优选地，本发明的融合蛋白sDR5-Fc的氨基酸序列如SEQ ID NO.3所示。
因一个或多个保守氨基酸取代或因一个或多个非保守性氨基酸取代、缺失或插入而不同于的野生型的sDR5-Fc序列，其中这些取代、缺失或插入不会废除该野生型序列的生物活性。保守取代通常包括一个氨基酸被具有类似特征的另一氨基酸取代，例如在以下各组内的取代：缬氨酸、甘氨酸；甘氨酸、丙氨酸；缬氨酸、异亮氨酸、亮氨酸；天冬氨酸、谷氨酸；天冬酰胺、谷氨酰胺；丝氨酸、苏氨酸；赖氨酸、精氨酸；及苯丙氨酸、酪氨酸。其它保守氨基酸取代是本领域中已知的并且包括在本文中。非保守性取代，如碱性氨基酸被疏水性氨基酸置换，也是本领域中众所周知的。
对本发明的融合蛋白sDR5-Fc进行增加蛋白质或肽稳定性的修饰也包括在本发明的保护范围之内；这样的修饰包括在蛋白质或肽序列中含有例如一个或多个非肽键(用以代替肽键)；这样的修饰还包括包含D-氨基酸或非天然存在或合成的氨基酸，例如β或γ氨基酸。
本发明提供了一种编码前面所述融合蛋白sDR5-Fc的DNA分子。
进一步，所述DNA分子的核苷酸序列为以下组中的任意一个：
(1)如SEQ ID NO.4所示的核苷酸序列或其简并序列；
(2)在SEQ ID NO.4所示的核苷酸序列中经过取代和/或缺失和/或添加一个或几个核苷酸所得的核苷酸序列；所述核苷酸序列SEQ ID NO.4所示的核苷酸序列或其简并序列编码功能相同或相似蛋白质；
(3)在严格条件下与(1)限定的核苷酸序列杂交且具有相同功能的核苷酸序列；
(4)与(1)限定的核苷酸序列至少具有98％同源性且具有相同功能的核苷酸序列。
优选地，编码前面所述融合蛋白sDR5-Fc的DNA分子的核苷酸序列如SEQ ID NO.4所示。
本发明所述“严格条件”可为在6×SSC、0.5％SDS溶液中，在65℃下与SEQ ID NO.4所示的核苷酸序列发生特异性杂交，然后用2×SSC，0.1％SDS和1×SSC，0.1％SDS各洗膜一次。在严格条件下与SEQ ID NO.4限定的核苷酸序列杂交且具有相同功能的核苷酸序列与SEQ ID NO.4所示的序列至少大约40％-50％同源，大约60％、65％或70％同源，甚至至少98％或更多同源。即序列同一性的范围分布在至少大约40％-50％、大约60％、65％或70％同源，甚至至少大约75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更大的序列同源性。
本发明提供了一种包含前面所述的编码所述融合蛋白sDR5-Fc的DNA分子的重组载体。
如本文中所使用，术语“载体”应理解为意指包含了能够并入到宿主细胞中并与宿主细胞基因组重组并整合到宿主细胞基因组中，或作为游离体自主复制的核苷酸序列的任何核酸。此类载体包括线性核酸、质粒、噬菌粒、粘粒(cosmid)、RNA载体、病毒载体等。
在本发明中适用的载体包括但不限于：表达载体、克隆载体，例如适用于原核(如大肠杆菌等细菌)、低等真核细胞(如酵母)、昆虫细胞、植物细胞、哺乳动物细胞中的载体。总之，只要能在宿主体内复制和稳定，任何质粒和载体都可以用。在表达载体中，除了含有复制起点外，还可含有标记基因和其他翻译控制元件。
可用于本发明的病毒载体包括但不限于腺病毒载体(adenoviral vectors)、腺相关病毒载体(adeno-associated viral vectors)、反转录病毒载体(retroviral vectors)、单纯疱疹病毒载体(herpes simplex virus-based vectors)、慢病毒载体(lentiviral vectors)。
优选地，本发明的载体使用真核表达载体pGS-Fc。
本发明还提供了包含前面所述的重组载体的宿主细胞。
进一步，宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。代表性例子有：大肠杆菌，链霉菌属；鼠伤寒沙门氏菌的细菌细胞：真菌细胞如酵母；植物细胞；果蝇S2或Sf9的昆虫细胞；CHO，COS，293细胞、或Bowes黑素瘤细胞的动物细胞等。
本发明还提供了前面所述的融合蛋白sDR5-Fc的制备方法：
(1)构建含有sDR5-Fc片段重组载体；
(2)将重组载体导入宿主细胞表达sDR5-Fc融合蛋白；
(3)分离纯化sDR5-Fc融合蛋白。
进一步，能够用于表达这些融合蛋白的表达系统有多种，可以是真核细胞，也可以是原核细胞，它们包括(但不限于)哺乳动物细胞、细菌、酵母、昆虫细胞等。由于本发明优化融合蛋白的氨基酸序列中包含可糖基化的氨基酸，因此哺哺乳动物细胞是表达该蛋白的优选系统。可用于大规模表达蛋白质的哺乳动物细胞有多种，例如CHO细胞、293细胞、NS0细胞、COS细胞、BHK细胞等，其它许多细胞也可以用于蛋白的表达，因此都包括在本发明所能使用的细胞之列。哺乳动物细胞以外的其他表达系统，例如细菌、酵母、昆虫细胞等也可以用于表达本发明的优化融合蛋白，它们也被包含本发明所能使用的宿主细胞之列。这些表达系统的蛋白质产量比哺乳动物细胞的较高，但是所表达的蛋白质缺乏糖基化或形成的糖链结构与哺乳动物细胞有所不同。
含有编码上述融合蛋白基因的重组质粒可经转染进入宿主细胞，转染细胞的方法有多种，其中包括(但不限于)电穿孔法、脂质体转染法和磷酸钙转染法等。
由于这些融合蛋白含有免疫球蛋白Fc，因此可以用蛋白A亲和层析法来纯化所表达的融合蛋白。此外，与其它蛋白纯化方法如离子交换层析等联合使用，可进一步纯化本发明的融合蛋白。
本发明还提供了前面所述的融合蛋白sDR5-Fc、前面所述的编码融合蛋白sDR5-Fc的DNA分子、前面所述的重组载体、或前面所述的包含重组载体的宿主细胞在制备心肌梗死治疗药物的应用。
本发明还提供了一种药物组合物，所述药物组合物包括前面所述的融合蛋白sDR5-Fc、前面所述的编码融合蛋白sDR5-Fc的DNA分子、前面所述的重组载体、或前面所述的包含重组载体的宿主细胞。
进一步，本发明的所述药物组合物还包括要学上可接受的载体，所述载体包括但不限于已经被美国食品与药品管理局认可的而可用于人类或动物的任何佐剂、赋形剂、助流剂、甜味剂、稀释剂、防腐剂、染料/着色剂、香味增强剂、表面活性剂、润湿剂、分散剂、助悬剂、稳定剂、等渗压剂、溶剂或乳化剂等对组成药物组合物无副作用的各种形式的载体。
本发明的药物组合物可以通过多种手段施用，所述手段例如通过口服、舌下、口腔、肠胃外、经鼻、经局部、经直肠、经皮、经粘膜等。
根据药物组合物的施用形式，可将药物组合物制备成相应的各种剂型，所述剂型包括但不限于片剂、溶液剂、颗粒剂、贴剂、膏剂、胶囊剂、气雾剂、栓剂。
本发明的组合物可以例如呈适于立即释放或持续释放的形式施用。立即释放或持续释放可以通过使用适合的药物组合物，或特别是在持续释放情形中，通过使用如皮下植入物或渗透泵等装置实现。
供治疗的受试者为经历心肌缺血损伤的患者，受试者包括但不限于哺乳动物，如人、狗、猫等。
应了解，用于任何特定受试者的具体剂量水平和剂量频率可以变化，并且将取决于多种因素，包括所用药物组合物的活性；该药物组合物的代谢稳定性和作用时间长度；受试者的物种、年龄、体重、一般健康状况、性别和饮食；施用的模式和时间；排泄速率和清除率；药物组合；及特定病状的严重程度。
本发明的药物组合物可以单独使用，或与可用于治疗心肌缺血损伤的其它适合的治疗剂组合使用。或者与其他治疗心肌缺血损伤手术方法一起使用。
本发明所述的“治疗”包括对病症的彻底治愈，还包括对病症的缓解。
如本文中所使用，术语“基因表达”或“表达”应理解为意指DNA序列的转录、mRNA转录物的翻译和Fc融合蛋白产物的分泌。
本发明的上下文中使用的“突变体”和“野生型”均针对Fc片段的序列而言。“融合蛋白突变体”的突变位点在Fc段上。
本发明的优点和有益效果如下：
(1)本发明首次构建了sDR5(1-182)-Fc融合蛋白突变体，由于Fc段突变导致该融合蛋白的使用降低了ADCC效应。
(2)本发明的sDR5(1-182)-Fc融合蛋白突变体中选择的sDR5(1-182)片段与DR5胞外区其他片段相比，存在以下优势：表达量高、容易获取、与TRIL配体结合能力强、ADCC效应弱、抑制心肌损伤的效果更强。
附图说明
图1显示利用Western Blot验证纯化的8种人sDR5-Fc融合蛋白的表达情况，其中，#1：新信号肽(sp)-sDR5(56-133)-Fc突变体；#2：sDR5(1-182)-Fc突变体；#3：sDR5(1-133)-Fc突变体；#4：新信号肽(sp)-sDR5(56-182)-Fc突变体；#5：sDR5(1-182)-Fc野生型；#6：sDR5(1-133)-Fc野生型；#7：新信号肽(sp)-sDR5(56-133)-Fc野生型；#8：新信号肽(sp)-sDR5(56-182)-Fc野生型；
图2显示利用SDS-PAGE考马斯亮蓝染色鉴定人sDR5-Fc在CHO-K1上清中的表达情况，其中，#1：新信号肽(sp)-sDR5(56-133)-Fc突变体；#2：sDR5(1-182)-Fc突变体；#3：sDR5(1-133)-Fc突变体；#4：新信号肽(sp)-sDR5(56-182)-Fc突变体；#5：sDR5(1-182)-Fc野生型；
图3显示利用ELISA测定人sDR5-Fc融合蛋白与TRAIL的亲和力，其中，#1：新信号肽(sp)-sDR5(56-133)-Fc突变体；#2：sDR5(1-182)-Fc突变体；#3：sDR5(1-133)-Fc突变体；#4：新信号肽(sp)-sDR5(56-182)-Fc突变体；#5：sDR5(1-182)-Fc野生型；
图4显示人sDR5-Fc融合蛋白对TRAIL诱导的细胞凋亡的影响；其中，#1：新 信号肽(sp)-sDR5(56-133)-Fc突变体；#2：sDR5(1-182)-Fc突变体；#3：sDR5(1-133)-Fc突变体；#4：新信号肽(sp)-sDR5(56-182)-Fc突变体；#5：sDR5(1-182)-Fc野生型；
图5显示利用ELISA测定sDR5(1-182)-Fc突变体和sDR5(1-182)-Fc野生型与Fc受体的亲和力；其中#2：sDR5(1-182)-Fc突变体；#5：sDR5(1-182)-Fc野生型；
图6显示利用LDH法检测sDR5(1-182)-Fc突变体的ADCC效应；
图7显示人sDR5(1-182)-Fc突变体对心肌细胞的保护作用。
具体实施方式
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室指南(New York：Cold Spring Harbor LaboratoryPress，1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。除非另行定义，文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本发明中。文中所述的较佳实施方法与材料仅作示范之用。
实施例1 sDR5-Fc融合蛋白的表达、纯化和鉴定
1、构建sDR5-Fc真核表达质粒
1.1 sDR5-Fc融合蛋白野生型表达质粒的构建
1)以含pGAPZαA-sDR5的毕赤酵母GS115菌株(参见：Song K，ChenY，Goke R，et al.Tumor necrosis factor-related apoptosis-inducing ligand(TRAIL)is an inhibitor of autoimmune inflammation and cell cycle progression.JExp Med，2000；191(7)：1095-1104)为PCR模板，扩增sDR5(1-133)、sDR5(1-182)、新信号肽(sp)-sDR5(56-182)、新信号肽(sp)-sDR5(56-133)编码基因片段
设计扩增引物如下：
sDR5(1-133)：
正向引物：
Figure PCTCN2016081552-appb-000001
反向引物：
Figure PCTCN2016081552-appb-000002
Figure PCTCN2016081552-appb-000003
正向引物：
Figure PCTCN2016081552-appb-000004
反向引物：
Figure PCTCN2016081552-appb-000005
新信号肽(sp)-sDR5(56-182)：
正向引物：
Figure PCTCN2016081552-appb-000006
反向引物：
Figure PCTCN2016081552-appb-000007
新信号肽(sp)-sDR5(56-133)：
正向引物：
Figure PCTCN2016081552-appb-000008
反向引物：
Figure PCTCN2016081552-appb-000009
2)将步骤(1)的基因片段克隆入带人IgG Fc段的真核表达载体pGS-Fc中。利用Hind III，EcoR I双酶切插入pGS-Fc多克隆位点。
1.2利用点突变试剂盒构建sDR5-Fc融合蛋白突变体表达质粒
按照试剂盒说明书(Toyobo，SMK-101)进行操作，Fc段的突变引物：
正义引物：
Figure PCTCN2016081552-appb-000010
反义引物：
Figure PCTCN2016081552-appb-000011
2、扩增重组表达质粒，将步骤1构建的重组表达质粒导入原核宿主细胞扩增。
3、提取高纯度质粒(OMEGA Plasmid Midi Kit)并调整浓度为500 ng/μl，将质粒瞬时转染293T细胞，48h后收集上清和细胞，用ProteinA/G珠子对上清和细胞裂解液进行吸附，Western blot检测融合蛋白表达。
4、选择5个融合蛋白表达质粒转染CHO/K1细胞
调整细胞状态至最佳，瞬时电转(300v，间隔时间0.125s，持续时间0.1ms，电击三次)，24小时后收集上清并经亲和层析柱纯化得到人sDR5-Fc融合蛋白。SDS-PAGE后考马斯亮蓝染色确定纯化的融合蛋白，本发明融合蛋白产量可达到30mg/L。
5、结果
如图1所示，构建的8个sDR5-Fc融合蛋白在293T细胞中均可表达。其中，#1：新信号肽(sp)-sDR5(56-133)-Fc突变体；#2：sDR5(1-182)-Fc突变体；#3：sDR5(1-133)-Fc突变体；#4：新信号肽(sp)-sDR5(56-182)-Fc突变体；#5：sDR5(1-182)-Fc野生型；#6：sDR5(1-133)-Fc野生型；#7：新信号肽(sp)-sDR5(56-133)-Fc野生型；#8：新信号肽(sp)-sDR5(56-182)-Fc野生型；
如图2所示，选择的5个sDR5-Fc融合蛋白在CHO/K1细胞的上清中均表达。其中，#1：新信号肽(sp)-sDR5(56-133)-Fc突变体；#2：sDR5(1-182)-Fc突变体；#3：sDR5(1-133)-Fc突变体；#4：新信号肽(sp)-sDR5(56-182)-Fc突变体；#5：sDR5(1-182)-Fc；
实施例2人sDR5-Fc与TRAIL的亲和力测定
使用ELISA方法测定人sDR5-Fc与TRAIL的亲和力。
1、步骤：
(1)包被
将包被原TRAIL用CBS从2μg/mL开始倍比稀释(2、1、0.5μg/mL)，100μL/孔，4℃包被过夜；
(2)洗涤
将板内溶液甩出，拍干，并用洗涤液清洗3次，每次2min；
(3)封闭
甩去洗液并于不掉纤维的吸水纸上拍干，然后加入每孔250μL封闭液，37℃温箱中反应1.5h；烘干备用；
(4)加样
将sDR5-Fc从1∶500开始稀释，直到1∶40000，并加入到不同稀释度的包被 孔中，每孔100μL，37℃温箱中反应1h；充分洗涤后，加入用酶稀液1∶5000稀释的二抗HRP-羊抗人IgG，每孔100μL，37℃温箱中反应1h；
(5)显色
取出酶标板，重复第2步进行洗涤，然后每孔加入各加入50μL显色液A和B，室温避光反应15min；
(6)终止和测值
加入终止液，每孔50μL以终止显色反应，然后用酶标仪测定各孔的OD450值。
(7)结果判读
以测定的OD450值大于或等于阴性孔的2.1倍(即P/N≥2.1)判定为阳性。
2、结果：
结果如图3所示，#2：sDR5(1-182)-Fc突变体，#4：新信号肽(sp)-sDR5(56-182)-Fc突变体，以及#5：sDR5(1-182)-Fc野生型与TRAIL的亲和力较高。
实施例3人sDR5-Fc融合蛋白对TRAIL诱导的细胞凋亡的影响
1、步骤：Jurkat细胞悬浮培养于100mm细胞培养皿中，培养液为含10％血清的1640培养基，收集第3-4代生长状态良好的细胞，500g离心10min，计数，将细胞铺于96孔板中，每个处理因素4个孔。空白对照孔，TRAIL单独作用孔，5种挑选出来的sDR5-Fc阻断组，hIgG-Fc同型对照组。阻断TRAIL组的处理方法均为将TRAIL和阻断蛋白预混孵育30min。TRAIL的工作浓度为250ng/ml，阻断蛋白的工作浓度为5μg/ml。
2、结果：
结果如图4显示，#2：sDR5(1-182)-Fc突变体、#4：新信号肽(sp)-sDR5(56-182)-Fc突变体、以及#5：sDR5(1-182)-Fc野生型的阻断效果较好，证明sDR5-Fc融合蛋白可以很好的阻断TRAIL诱导的Jurkat细胞凋亡。
实施例4 sDR5(1-182)-Fc突变体和sDR5(1-182)-Fc野生型与Fc受体的亲和力测定
考虑几种sDR5-Fc融合蛋白的蛋白表达、与TRAIL的亲和力以及对TRAIL诱导的细胞凋亡的阻断能力，选择sDR5(1-182)-Fc突变体和sDR5(1-182)-Fc野生型进行以下实验。
1、步骤：利用Fc受体包板，进行ELISA检测，操作步骤同实施例2。
2、结果
结果如图5所示，#2：sDR5(1-182)-Fc突变体较#5：sDR5(1-182)-Fc野生型与三类Fc受体FcgammaRl、FcgammaRllb、FcgammaRlll的亲和力较低。
实施例5sDR5(1-182)-Fc突变体的ADCC效应检测
1、淋巴细胞的获取
1)从两名健康人的外周血中分别抽取10ml肝素抗凝血，于18-22℃以250g离心10分钟，弃去血浆，补充添加全血及组织稀释液，添加量为所弃去血浆体积的1.5-2倍，混匀备用。
2)取一支适当的离心管，加入分离液(与稀释后的血液样本体积相等)，置于18-22℃.
3)将经稀释处理的血液样本小心铺在分离液面上，18-22℃，400g离心20分钟。
4)离心后，用移液枪小心吸出分离液上层0.5cm以上的上清液部分，弃去。
5)用移液枪小心吸取分离液层及淋巴细胞层至另一新离心管内。
6)在步骤5中所得离心管中加入10ml细胞洗涤液混匀。
7)250g离心10分钟。
8)弃上清。
9)用移液枪以5ml细胞洗涤液重悬所得细胞。
10)250g离心10分钟。
11)弃上清。重复步骤8、9、10，之后用1ml含有10％FBS的1640培养基重悬细胞，并计数。
2、ADCC检测
1)取一96孔板，每空加入50μl处于对数生长期靶细胞(H9C2和Jurkat)(1×105/ml)，之后再加入50μl重组蛋白sDR5(1-182)-Fc突变体(200μg/ml)，37℃、5％CO2培养2h。
2)向对应孔中分别加入100μl不同浓度步骤一中所获得的细胞悬液，使其细胞个数分别为2×105/孔(靶细胞∶淋巴细胞＝1∶40)和5×105/孔(靶细胞∶淋巴细胞＝1∶100)。同时设置空培养基、单独淋巴细胞和单独靶细胞三组阴性对照，另外设置靶细胞裂解液的阳性对照。
3)37℃、5％CO2培养48h。
4)按照LDH试剂盒(Roche，11644793001)的指示，加入相应的试剂，之后490nm处检测吸光度值。
3、结果：
结果如图6所示，sDR5(1-182)-Fc突变体几乎没有ADCC效应。
实施例6sDR5(1-182)-Fc突变体对心肌梗死大鼠心肌细胞的保护作用
1、大鼠心肌缺血1h再灌注3h模型的制备
结扎/松开大鼠做冠状动脉前降支建立缺血-再灌注模型：清洁级雄性Wistar大鼠(体重200-250g)，10％水合氯醛腹腔注射麻醉(3-4ml/kg)，气管插管并在打开肋间隙之前接呼吸机，调参数吸呼比1∶2，呼吸频率90-120次/分，潮气量2-4ml；胸骨左缘3-4肋间横行切口，剪开心包，暴露心脏，以肺动脉圆锥与左心耳交界处的左冠状静脉为标志，于左心耳下缘正中约0.1cm处绕左前降支穿线结扎，同时设假手术组作为对照(只穿线不结扎)。术后常规注射青霉素40万U/只。
将进行冠状动脉结扎的大鼠随机分组，分为PBS组、sDR5(1-182)-Fc突变体组，每组根据剂量不同再分为三小组，每小组6只老鼠，实验均重复三次，具 体实验方法及结果叙述如下：
在冠状动脉结扎后1h，人sDR5-Fc组通过大鼠尾静脉注射人sDR5(1-182)-Fc突变体；PBS组则通过尾静脉注射相应剂量的无菌PBS。
2、大鼠心肌组织TTC-EvansBlue染色
用水合氯醛将大鼠麻醉，开胸并结扎主动脉，重新结扎左冠状动脉前降支后将5ml 1％Evans Blue从左心室注入，速度适中；然后快速剪下左心室，在预冷的生理盐水中漂洗三次，置-20℃冷冻至少1h。TTC染色前从冰箱中取出组织，在锡箔纸上迅速切成1-2mm厚的切片并置于装有TTC染液的12孔板中，37℃避光孵育15min，然后用组织冲洗应用液(TTC染液试剂盒，南京建成科技有限公司)将组织表面多余的染液冲掉，观察并拍照，其中白色为梗死区，蓝色为正常心肌，红色和白色为危险区。
与假手术组相比，注射PBS的大鼠心肌出现大面积灰白区(即梗死区)，而注射sDR5-Fc的大鼠梗死区缩小，心肌梗死面积计算方法：梗死区心肌重量/危险区心肌重量×100％；心肌梗死面积减少率计算方法：(对照组梗死面积-用药组梗死面积)/对照组梗死面积×100％。梗死区表示为IS，危险区表示为AAR、左心室表示为LV、心肌梗死面积以IS/AAR比值表示，心肌缺血面积以AAR/LV表示。
3、结果
结果如图7所示，在缺血1h再灌注3h的大鼠模型里，再灌注的前5min，单次尾静脉注射sDR5(1-182)-Fc突变体15mg/kg，与对照组相比，可明显减少心肌梗死面积，减少超过40％。
以上内容是结合具体的优选实施方式对本发明所作的进一步详细说明，不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干简单推演或替换，都应当视为属于本发明的保护范围。

Claims (10)

  1. 一种融合蛋白，其特征在于，所述融合蛋白是由sDR5片段和人抗体Fc段构成；其中，所述sDR5片段的氨基酸序列为以下组中的任意一个：
    (1)如SEQ ID NO.1所示的氨基酸序列；
    (2)在SEQ ID NO.1所示的氨基酸序列中经过取代和/或缺失和/或添加一个或几个氨基酸所得功能相同或相似的氨基酸序列。
    (3)与(1)或(2)限定的氨基酸序列至少具有99％同源性且具有相同功能的氨基酸序列。
  2. 根据权利要求1所述的融合蛋白，其特征在于，所述人抗体Fc段的氨基酸序列为以下组中的任意一个：
    (1)如SEQ ID NO.2所示的氨基酸序列；
    (2)在SEQ ID NO.2所示的氨基酸序列中经过取代和/或缺失和/或添加一个或几个氨基酸所得功能相同或相似的氨基酸序列；
    (3)与(1)或(2)限定的氨基酸序列至少具有98％同源性且具有相同功能的氨基酸序列。
  3. 根据权利要求1所述的融合蛋白，其特征在于，所述融合蛋白的氨基酸序列为以下组中的任意一个：
    (1)如SEQ ID NO.3所示的氨基酸序列；
    (2)在SEQ ID NO.3所示的氨基酸序列中经过取代和/或缺失和/或添加一个或几个氨基酸所得功能相同或相似的氨基酸序列；
    (3)与(1)或(2)限定的氨基酸序列至少具有98％或同源性且具有相同功能的氨基酸序列。
  4. 编码权利要求1或3中任一项所述的融合蛋白的DNA分子。
  5. 根据权利要求4所述的DNA分子，其特征在于，所述DNA分子的核苷酸序列为以下组中的任意一个：
    (1)如SEQ ID NO.4所示的核苷酸序列或其简并序列；
    (2)在SEQ ID NO.4所示的核苷酸序列中经过取代和/或缺失和/或添加一个或几个核苷酸所得的核苷酸序列；所述核苷酸序列SEQ ID NO.4所示的核苷酸序列或其简并序列编码功能相同或相似蛋白质；
    (3)在严格条件下与(1)限定的核苷酸序列杂交且具有相同功能的核苷酸序列；
    (4)与(1)限定的核苷酸序列至少具有98％同源性且具有相同功能的核苷酸序列。
  6. 包含权利要求4或5所述的DNA分子的重组载体。
  7. 根据权利要求6所述的重组载体，其特征在于，所述重组载体是非病毒载体。
  8. 包含权利要求6或7所述的重组载体的宿主细胞。
  9. 权利要求1-3中任一项所述的融合蛋白、权利要求4或5所述的DNA分子、权利要求6或7所述的重组载体、或权利要求7所述的宿主细胞在制备心肌梗死治疗药物的应用。
  10. 一种药物组合物，其特征在于，所述药物组合物包含权利要求1-3中任一项所述的融合蛋白、权利要求4或5所述的DNA分子、权利要求6或7所述的重组载体、或权利要求7所述的宿主细胞。
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