WO2010066090A1 - 凋亡素-ec-sod羧基末端蛋白转导域融合蛋白 - Google Patents

凋亡素-ec-sod羧基末端蛋白转导域融合蛋白 Download PDF

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WO2010066090A1
WO2010066090A1 PCT/CN2009/000379 CN2009000379W WO2010066090A1 WO 2010066090 A1 WO2010066090 A1 WO 2010066090A1 CN 2009000379 W CN2009000379 W CN 2009000379W WO 2010066090 A1 WO2010066090 A1 WO 2010066090A1
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sod
apoptin
transduction domain
carboxy terminal
protein
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PCT/CN2009/000379
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English (en)
French (fr)
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赵健
王富军
傅龙云
张涛铸
单含文
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浙江日升昌药业有限公司
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Priority to EP09831367A priority Critical patent/EP2380914A4/en
Priority to US13/130,904 priority patent/US20110230421A1/en
Priority to JP2011539871A priority patent/JP2012511309A/ja
Publication of WO2010066090A1 publication Critical patent/WO2010066090A1/zh

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    • 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
    • A61K38/162—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
    • 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
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/10011—Circoviridae
    • C12N2750/10022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to the field of biotechnology, and particularly relates to an apoptin-EC-SOD carboxy-terminal egg ⁇ transduction domain fusion protein, gene, recombinant vector, transformant, use thereof and preparation method thereof. Background technique
  • Superoxide dismutase is an enzyme that catalyzes the disproportionation of superoxide anion (0 2_ ).
  • SOD superoxide dismutase
  • EC-SOD [Marklund SL, Bjelle A, Elmqvist LG, et al. Ann Rheum Dis.
  • N-terminal is a signal peptide of 18 amino acid residues
  • 95-domain domain related to EC-SOD formation of tetramer followed by 98 residues of SOD activity Domain
  • 29 residues of protein transduction domains rich in basic amino acid residues HTDalmarsson K, Marklund SL, Engstrom A, et al. Proc Natl Acad Sci USA. 1987 , 84: 6340-6344.
  • This domain binds to heparin-like polysaccharide tissue on the cell surface [Karlsson K, Marklund SL. Lab Invest. 1989, 60: 659-666] and is capable of transmembrane transduction by endocytosis, localizing it to the nucleus.
  • apoptotic protein derived from chicken anemia virus, consisting of 121 amino acid residues, which induces a p53-independent and Bcl- in human tumor cells. 2 Insensitive to apoptosis, but no effect on normal human cells, is a very promising drug candidate for selective treatment of tumors [Zhuang SM, Shvarts A, van Ormondt H, etal. Cancer Res. 1995, 55 ( 3): 486-489]. Studies have shown that apoptin-induced apoptosis requires apoptocin itself to be transported into the cell, and then rely on the nuclear localization sequence (NLS) of apoptin itself to accumulate in the nucleus.
  • NLS nuclear localization sequence
  • Apoptosis-induced apoptosis is related to the phosphorylation process of its own specific amino acid residues, and its apoptosis process is closely related to the Caspase-3 pathway [Notebom MH. Vet Microbiol. 2004, 98(2): 89-94 ].
  • PTDs transmembrane domains
  • Typical are TAT domains derived from human HIV virus, Drosophila melanogaster's contact peptide (ATP), and herpes simplex virus transcription.
  • Regulatory protein (VP22) polyarginine sequence
  • carboxy terminal protein transduction domain of EC-SOD and the like.
  • PTDs are generally composed of a sequence of less than 20 amino acids in length, containing a highly positively charged region and a helical structure.
  • PTD can carry a variety of substances, including hydrophilic proteins, peptides, DNA and even particulate matter for intercellular/internal transport, and is not restricted by cell type [Schwarze SR, Dowdy SF. Trends Pharmaeol Sci. 2000, 21 (2): 45-48].
  • its transport efficiency and PTD It is closely related to the nature of the protein itself and its fusion. Therefore, to achieve a transmembrane transfer of a protein molecule, it is important to choose a suitable PTD.
  • the technical problem to be solved by the present invention provides an apoptin fusion protein, so that it can be rapidly and efficiently transported into the interior of the cell to exert its apoptosis effect, thereby achieving the purpose of controlling tumor cell growth and treating diseases.
  • an aspect of the invention discloses an apoptin-EC-SOD carboxy terminal protein transduction domain fusion protein, characterized in that the fusion protein comprises an EC-SOD carboxy terminus as described in SEQ ID NO.
  • the amino acid sequence to which the protein transduction domain or its mutant is fused is an apoptin or a mutant thereof as set forth in SEQ ID NO.
  • the inventive idea of the present invention is to artificially construct a fusion protein of an EC-SOD carboxy terminal protein transduction domain or a mutant thereof with apoptin or a mutant thereof by genetic engineering, through an EC-SOD carboxy terminal protein transduction domain or The mutant realizes the translocation of transmembrane protein molecules, thereby exerting the apoptosis-inducing effect of apoptin or its mutant, and achieving the purpose of controlling tumor cell growth and treating diseases.
  • the scope of protection of the present invention includes, but is not limited to, the above fusion proteins.
  • the apoptin-EC-SOD carboxy terminal protein transduction domain fusion protein can have the formula: R2-R1, Rl-R2, Rl-L-R2-L-R1, Rl-L- R2, R2-L-R1 or R2-L-R1-L-R1, wherein R1 is an amino acid sequence such as the EC-SOD carboxy terminal protein transduction domain of SEQ ID NO. 1, or a mutant thereof, and L is a linker peptide, R2 is at least one amino acid sequence such as apoptin as described in SEQ ID NO: 2 or a mutant thereof, but need not be the same therapeutic protein.
  • Linking peptides include, but are not limited to, for example: (GGGGS) N or (GGGS)! ⁇ or (GGS) N , wherein N is an integer greater than or equal to 1, G represents glycine, and S represents serine.
  • the amino acid sequence of the EC-SOD carboxy terminal protein transduction domain or a mutant thereof as set forth in SEQ ID NO. The basal ends of the fusion protein.
  • the apoptin-EC-SOD carboxy terminal protein transduction domain fusion protein is a protein of (a) or (b):
  • the invention features a polynucleotide molecule encoding a protein of the apoptin-EC-SOD carboxy terminal protein transduction domain of the invention.
  • nucleic acid sequence of the polynucleotide molecule is as described in SEQ ID NO.
  • the invention features a recombinant expression vector comprising a polynucleotide molecule of the invention.
  • the invention features a transformant comprising the recombinant expression vector of the invention.
  • the transformant is Escherichia coli.
  • the present invention discloses a fusion of apoptin-EC-SOD carboxy terminal protein transduction domain of the present invention.
  • the method for preparing the protein comprises the following steps:
  • the invention features a composition comprising an apoptin-EC-SOD carboxy terminal protein transduction domain fusion protein of the invention and a pharmaceutically acceptable carrier.
  • the present invention discloses the use of the apoptin-EC-SOD carboxy terminal protein transduction domain fusion protein of the present invention for the preparation of a medicament for treating a disease caused by excessive cell proliferation.
  • the disease caused by excessive cell proliferation is a tumor.
  • the invention encompasses a method of preventing, treating or ameliorating a related disease or disorder caused by excessive cell proliferation, the method comprising administering to a mammal in need of such prevention, treatment or amelioration an effective amount of apoptin-EC -SOD carboxy terminal protein transduction domain fusion protein to treat, prevent or ameliorate disease or disorders.
  • the apoptotic-EC-SOD carboxy terminal protein transduction domain fusion protein prepared by the invention has strong ability to induce tumor cell apoptosis by the tumor cell apoptosis test and the mouse ascites tumor inhibition test in vitro. It is used to prepare drugs for treating tumors.
  • FIG. 1 PCR-amplified EC-S0D carboxy-terminal protein transduction domain gene fragment 2% agarose gel electrophoresis map;
  • Figure 2 Schematic diagram of the expression plasmid C- pET28a construction process;
  • FIG. 3 PCR-amplified apoptin gene fragment 1. 5 % agarose gel electrophoresis map;
  • FIG. 4 Schematic diagram of the construction process of the cloning plasmid Apop-pMD18T
  • FIG. 5 Schematic diagram of the construction process of the expression plasmid ApopC_pET28a
  • Figure 6 Colony PCR detection of recombinant inserts of 1. 5 % agarose gel electrophoresis
  • Figure 8 Effect of different concentrations of recombinant Apoptin-EC-S0D-PTD on the survival rate of HeLa cells;
  • Figure 9 Effect of recombinant Apoptin-EC-SOD-PTD on the survival rate of human normal hepatocytes cultured in vitro.
  • mutant refers to a mutant of an apoptin in an amino acid sequence such as the EC-SOD carboxy terminal protein transduction domain or the amino acid sequence of SEQ ID NO. Compared to the native EC-SOD carboxy terminal protein transduction domain or the apoptin protein, the mutants have enhanced activity and/or altered stereospecificity compared to their wild type.
  • Amino acid sequence mutants of the native protein can be prepared by introducing appropriate nucleotide changes into the nucleotides of the invention, or by synthesizing the desired polypeptide in vitro. These mutants include, for example, deletions, insertions or substitutions of residues in the amino acid sequence. Can be obtained by a combination of missing, inserting and replacing To the final construct, the final protein product is provided.
  • GAP Needleman and Wunsh, 1970 analysis
  • the parameter gap creation penalty 5
  • gap extension penalty 0.3.
  • the GAP analysis was tested in the region of at least 15 amino acids of the two sequences involved in the test. More preferably, when the sequence being analyzed is at least 50 amino acids in length, the GAP analysis is tested in a region of at least 50 amino acids of the two sequences involved in the assay. More preferably, when the sequence being analyzed is at least 100 amino acids in length, the GAP analysis is tested in the region of at least 100 amino acids of the two sequences involved in the assay.
  • the GAP analysis is tested in a region of at least 250 amino acids of the two sequences involved in the assay. Even more preferably, when the sequence being analyzed is at least 500 amino acids in length, the GAP assay is tested in a region of at least 500 amino acids of the two sequences involved in the assay.
  • aspects of the present invention also include apoptin-EC-SOD carboxy terminal protein transduction domain fusion protein analogs, which are modified during or after their synthesis, for example, by biotinylation, benzylation, sugar Substitution, acetylation, phosphorylation, derivatization by known protecting/blocking groups, cleavage by proteolysis, attachment to antibody molecules or other cellular ligands, and the like. These modifications can be used to increase the stability and/or biological activity of the apoptin-EC-SOD carboxy terminal protein domain fusion protein of the invention.
  • the present invention includes DNA encoding the apoptin-EC-SOD carboxy terminal protein transduction domain fusion protein of the present invention, and vectors and transformants containing the same.
  • transf ormant i.e., a host cell carrying a heterologous DNA molecule.
  • the present invention also encompasses a method of producing the apoptin-EC-SOD carboxy terminal protein transduction domain 3 ⁇ 4 protein of the present invention by synthetic and recombinant techniques.
  • Polynucleotides DNA or RNA
  • vectors, transformants and organisms can be isolated and purified by methods known in the art.
  • the vector used in the present invention may be, for example, a bacteriophage, a plasmid, a cosmid, a minichromosome, a virus or a retroviral vector.
  • a vector useful for cloning and/or expressing a polynucleotide of the present invention is a vector capable of replicating and/or expressing a polynucleotide in a host cell in which a polynucleotide is to be replicated and/or expressed.
  • polynucleotides and/or vectors can be used in any eukaryotic or prokaryotic cell, including mammalian cells (eg, human (eg, HeLa), monkey (eg, Cos), rabbit (eg, rabbit reticulocyte), rat, Hamsters (such as CH0, NS0 and baby hamster kidney cells) or mouse cells (such as L cells), plant cells, yeast cells, insect cells or bacterial cells (such as E. coli).
  • mammalian cells eg, human (eg, HeLa), monkey (eg, Cos), rabbit (eg, rabbit reticulocyte), rat, Hamsters (such as CH0, NS0 and baby hamster kidney cells) or mouse cells (such as L cells), plant cells, yeast cells, insect cells or bacterial cells (such as E. coli).
  • suitable vectors for various types of host cells see, for example, F. Ausubel et al., Current Protocols in Molecular Biology. Greene Publishing Associates and Wiley-Interscience (19
  • a variety of methods have been developed for operably linking polynucleotides to vectors via complementary cohesive ends. For example, a complementary homomeric sequence fragment can be added to a DNA segment to be inserted into the vector DNA. Complementary homopolymerization The hydrogen bond between the body ends connects the vector and the DNA segment to form a recombinant DNA molecule.
  • a synthetic linker containing one or more restriction sites provides another means of joining the DNA segment to the vector.
  • the DNA segment produced by endonuclease restriction digestion is treated with phage T4 DNA polymerase or E. coli DNA polymerase I, and the two polymerases are excised by their 3', 5'-nucleic acid exonuclease activity. Y-single-stranded end, and using its polymerization activity to fill the 3'-concave end. Thus, the combination of these activities produces a blunt-ended DNA segment.
  • the blunt-ended segment is then incubated with a large molar excess of linker molecule in the presence of an enzyme that catalyzes the ligation of blunt-ended DNA molecules, such as phage T4 DNA ligase.
  • an enzyme that catalyzes the ligation of blunt-ended DNA molecules such as phage T4 DNA ligase.
  • the reaction product is a DNA segment carrying a polymeric linker sequence at the end.
  • These DNA segments are then cleaved with appropriate restriction enzymes and ligated into an expression vector that has been cleaved with an enzyme that produces a terminus that is compatible with the DNA segment.
  • Synthetic linkers containing multiple restriction endonuclease sites are available from a variety of merchants.
  • the polynucleotide insert should be operably linked to a suitable promoter compatible with the host cell expressing the polynucleotide.
  • the promoter may be a strong promoter and/or an inducible promoter. Some examples include promoters include phage ⁇ PL promoter, the E. coli lac, trP, P hoA, tac promoter, the SV40 early and late promoters and promoters of retroviral LTR. Other suitable promoters are known to those skilled in the art.
  • the expression recombinant vector further contains a transcription initiation and termination site, and contains a ribosome binding site for translation in the transcribed region.
  • the coding portion of the transcript expressed by the recombinant vector can include a translation initiation codon at the start and a stop codon (UAA, UGA or UAG) suitably located at the end of the translated polypeptide.
  • the expression vector can include at least one selectable marker.
  • the marker includes dihydrofolate reductase, G418, glutamine synthase or neomycin resistance for eukaryotic cell culture; and tetracycline, kanamycin for culture of Enterobacter and other bacteria Or an ampicillin resistance gene.
  • suitable hosts include, but are not limited to, bacterial cells, such as E.
  • yeast cells such as Saccharomyces cerevisiae or Pichia pastoris
  • insect cells if flies S2 and Noctuida SF9 cells
  • animal cells such as CH0, COS, NS0, 293 and Bowes melanoma cells
  • plant cells Suitable media and culture conditions for the above host cells are known in the art.
  • Tag a tag protein or a tag polypeptide
  • GST glutathione S-transferase
  • His.Tag Protein A
  • cellulose binding domain a tag protein or a tag polypeptide
  • GST glutathione S-transferase
  • His.Tag Protein A
  • cellulose binding domain a tag protein or a tag polypeptide
  • GST glutathione S-transferase
  • His.Tag specifically binds to the M-Chelating Sepharose column.
  • the tagged protein or tag polypeptide can be purified by site-specific protease digestion to remove the fusion sequence, such as thrombin, enterokinase and factor Xa, to obtain the target protein.
  • the invention also encompasses host cells comprising a nucleotide sequence of the invention operably linked to one or more heterologous control regions (e.g., promoters and/or enhancers) by techniques known in the art. Connected.
  • a host strain capable of modulating the expression of the inserted gene sequence or modifying and processing the gene product in a specific manner as desired can be selected. In the presence of certain inducers, expression of certain promoters is elevated; therefore, expression of the genetically engineered polypeptide can be controlled.
  • different host cells have characteristic and special translation, post-translational processing and modification. The mechanism of protein (such as phosphorylation, cleavage). Appropriate cell lines can be selected to ensure desirable modification and processing of the expressed foreign protein.
  • the nucleic acid and nucleic acid recombinant vector of the present invention can be introduced into a host by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection or other methods. cell.
  • the method is described in a number of standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986).
  • a polynucleotide encoding the fusion protein of the present invention can be ligated to a vector containing a selectable marker for propagation in a host.
  • a plasmid vector can be introduced into a precipitate, such as a calcium phosphate precipitate or a complex thereof with a charged lipid. If the vector is a virus, it can be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.
  • Successfully transformed cells i.e., cells containing the DNA recombinant vector of the present invention
  • cells obtained by expression of a recombinant vector can be cultured to produce a desired polypeptide.
  • the cells are collected and lysed, and the presence of DNA in the DNA content is detected using a method such as Southern (1975) J. Mol. Biol. 95, 503 or Berent et al (1985) Biotech. 3, 208.
  • antibodies are used to detect the presence of proteins in the supernatant.
  • fusion protein of the present invention It is advantageous to recover and purify the fusion protein of the present invention from recombinant cell culture by well-known methods, including sulfuric acid or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic Action chromatography, affinity chromatography, hydroxyapatite chromatography, hydrophobic charge chromatography and lectin chromatography.
  • purification can be performed using one-shot liquid chromatography (HPLC).
  • the fusion protein of the invention can be purified using one or more of the chromatographic methods described above. In other embodiments, the fusion protein of the invention may be purified using one or more of the following columns, a Q sepharose FF column, a SP sepharose FF column, a Q sepharose High Performance column. , Blue sepharose FF column, Blue column, Phenyl Sepharose FF column, DEAE Sepharose FF, Ni-Chelating Sepharose FF column or Methyl column.
  • fusion proteins of the invention can be purified using the methods described in International Publication No. W000/44772, which is incorporated herein by reference in its entirety.
  • One of skill in the art can readily modify the methods described therein for use in purifying the fusion proteins of the present invention.
  • the fusion proteins of the invention can be recovered from products produced by recombinant techniques, including prokaryotic or eukaryotic hosts of, for example, bacteria, yeast, higher plant, insect and mammalian cells.
  • the apoptin-EC-S0D carboxy terminal protein transduction domain fusion protein preparation method comprises the following steps:
  • ApopC-pET28a was cloned with the apoptin-EC-SOD carboxy-terminal protein transduction domain fusion protein gene ;
  • the expression plasmid C-pET28a described in the above a. is constructed by: designing the 5' and 3' primers, PCR method
  • the EC-SOD carboxy terminal protein transduction domain (amino acid residues 196-222) was cloned from the EC-SOD-pSK plasmid harboring the full-length EC-SOD gene, and the N-terminus of this fragment was ligated.
  • the ligation peptide of 8 amino acid residues in length at the upper end was cloned into the Hind III and Xho I sites of pET28a to form the expression plasmid C- pET28a of the EC-SOD carboxy-terminal protein transduction domain gene.
  • the construction method of the cloning plasmid Apop- P MD18T described in b. above is: designing the 5'-end and 3'-end primers, and adopting PCR method from the Apoptin- P VK plasmid cloned with the full-length apoptin gene Cloning of the full-length gene (amino acid residues 1 - 121) of the gene, the Nde I and BamH I restriction sites were ligated to the 5' and 3' ends of the gene, respectively, and the fragment was cloned into pMD - 1ST plasmid, which constitutes the cloned plasmid Apop-pMD18T cloned with the apoptin gene.
  • the expression plasmid ApopC- pET28a described in the above c. is constructed by: using the cloning plasmid Apop-pMD18T to isolate the target fragment, and then inserting the fragment into the C-type which has been digested with Nde I and BamH I as well.
  • an expression plasmid ApopC- pET28a was cloned with the apoptin-EC-S0D carboxy terminal protein transduction domain fusion protein gene.
  • the method for expressing and purifying the apoptin-EC-S0D carboxy terminal protein transduction domain fusion protein described in the above d. is: transferring the constructed ApopC- P ET28a recombinant plasmid into the large intestine fused with the T7 RNA polymerase gene In the bacillus host.
  • the transferred host can be E. coli BL21 (DE3) , BL21 (DE3) pLysS, JM109 (DE3) ,
  • the fusion protein of the present invention can be used as an active ingredient to treat various diseases caused by excessive cell proliferation, such as tumors, including but not limited to: bone cancer, including: Ewing sarcoma, osteosarcoma, chondrosarcoma, etc.; CNS tumors, including: acoustic neuroma, neuroblastoma, neutrophils and other brain tumors, spinal cord tumors, breast cancer, colorectal cancer, advanced colorectal adenocarcinoma; endocrine cancers, including: adrenocortical carcinoma, pancreatic cancer, Pituitary cancer, thyroid cancer, parathyroid cancer, thymus cancer, multiple endocrine neoplasms; gastrointestinal cancer, including: gastric cancer, esophageal cancer, small intestine cancer, liver cancer, extrahepatic cholangiocarcinoma, gastrointestinal carcinoid tumor, gallbladder cancer Genitourinary cancer, including: Cui Pill cancer, penile cancer, prostate cancer
  • the cancers which can be used for the treatment of the fusion proteins disclosed in the present invention are firstly liver cancer or lung cancer.
  • Preferred tumors which can be treated by the fusion proteins of the invention are solid tumors and hematological malignancies.
  • tumor generally refers to a wide range of conditions characterized by uncontrolled abnormal growth of cells.
  • the effective dose of the active ingredient employed will vary with the mode of administration and the severity of the condition being treated. 01 ⁇ 1000 ⁇ For most large mammals, the total dose of active ingredients per day is about 0.01-1000mg. Usually, the clinical dose of the adult is in the range of from 0.01 to 200 mg/day, preferably from 0.05 to 100 mg/day.
  • Effective dose refers to an amount sufficient to produce a therapeutic effect.
  • An effective amount can be administered in one or more divided doses. Generally, an effective amount is sufficient to alleviate, improve, stabilize, slow or delay further progression of the disease.
  • a composition for use in the invention or comprising the fusion protein of the invention may be mixed with one or more pharmaceutically acceptable carriers or excipients to prepare pharmaceutical dosage forms of different administration routes, such as tablets and capsules. , powders, granules, syrups, solutions, oral liquids, elixirs, tinctures, aerosols, powders, injections, sterile powders for injection, suppositories, etc.
  • a “pharmaceutically acceptable” ingredient is one which is suitable for use in humans and/or animals without excessive adverse side effects (e.g., toxicity, irritation, and allergies), i.e., having a reasonable benefit/risk ratio.
  • a “pharmaceutically acceptable carrier” is a pharmaceutically or food acceptable solvent, suspending agent or excipient for delivering the fusion protein of the invention to an animal or human.
  • the carrier can be a liquid or a solid.
  • the fusion proteins of the invention can be administered orally, intravenously, intramuscularly or subcutaneously.
  • the dosage forms which can be orally administered in the above dosage forms are: tablets, capsules, powders, granules, syrups, solutions, elixirs.
  • Solid carriers include: starch, lactose, calcium hydrogen phosphate, microcrystalline cellulose, sucrose, kaolin, micronized silica gel, talc, low-substituted hydroxypropylcellulose, sodium carboxymethyl starch, polyvinylpyrrolidone.
  • Liquid carriers include: sterile water, ethanol, polyethylene glycol, nonionic surfactants, and edible oils such as corn oil, peanut oil, and sesame oil.
  • Adjuvants commonly used in the preparation of pharmaceutical compositions include: flavoring agents, coloring agents, preservatives (such as hydroxyalkyl butyl acrylate, sodium benzoate, sorbic acid) and antioxidants (such as vitamin E, vitamin C, coke) Sodium sulfite and dibutylhydroxytoluene).
  • the dosage forms of the above dosage forms which can be administered by the injection route include: injections, sterile powder for injection, which are in the form of a mixture of the drug and one or more pharmaceutically acceptable excipients for administration by injection.
  • Solvents include: sterile water, ethanol, glycerol, propylene glycol, polyethylene glycol.
  • bacteriostatic agents such as benzyl alcohol, hydroxyphenyl butyl ketone, thimerosal
  • isotonicity regulators such as sodium chloride, glucose
  • suspending agents such as sodium carboxymethyl cellulose, methyl cellulose.
  • compositions are solid compositions, especially lyophilized powders. Intravenous administration is preferred.
  • Example 1 Construction of an expression plasmid cloned with the EC-SOD carboxy terminal protein transduction domain gene C-pET28a
  • reaction conditions for PCR amplification are: first 94 ° C, denaturation 5 min, then 94 ° C, denaturation 30 s ⁇ 60 ° C, renaturation 30 s ⁇ 72 ° C, extension lmin, cycle 30 times, finally 72 ° C , 5min.
  • the PCR-amplified and digested insert was ligated with the pET28a plasmid DNA which had been hydrolyzed and recovered by the same enzyme, transformed into E. c ⁇ ' DH5a strain, cultured, and the recombinant plasmid DNA was recovered and verified by sequencing.
  • Target fragment obtained by PCR amplification The 2% agarose electropherogram is shown in Figure 1.
  • M is a standard molecular weight DNA marker
  • 1 is an approximately 120 bp EC-SOD carboxy terminal protein transduction domain gene fragment obtained by amplification.
  • reaction conditions for PCR amplification are: first 94 ° C, denaturation 5 min, then 94 ° C, denaturation 30 s ⁇ 55 ° C, renaturation 30 s ⁇ 72 ° C, extension 45 s, cycle 30 times, finally 72 ° C , 5min.
  • the apoptotic full-length gene fragment obtained by PCR amplification is shown in Figure 3 for the 1.5% agarose gel electrophoresis pattern.
  • 1 is a full-length apoptotic gene of about 370 bp amplified by PCR; M is a standard molecular weight DNA marker.
  • the Apop-pMD18T plasmid DNA prepared and sequenced was digested with Nde I and BamH I, and the fragment was digested by 1% agarose gel electrophoresis, and the target fragment DNA was recovered by a gel recovery kit.
  • the C-pET28a plasmid DNA which was constructed and amplified was also digested with Nde I and BamH I, and the digested fragments were separated by 1% agarose gel electrophoresis, and the plasmid DNA was recovered using a gel recovery kit.
  • the recovered apoptin insert DNA was ligated with the C-pET28a plasmid DNA, transformed into E. coli BL21 (DE3) strain, cultured at 37 ° C, and the plasmid DNA was recovered and verified by sequencing.
  • Recombinant colony PCR detection The 1.5% agarose electropherogram is shown in Figure 6.
  • 1 is a negative control using pET28a empty plasmid DNA as a template
  • 2 is the result of PCR detection of ApopC-pET28a, and the primers used are primer P3 and primer P2, and the fragment size is about 500 bp
  • M is a standard molecular weight DNA marker .
  • Example 4 Expression and purification of apoptin-EC-SOD carboxy terminal protein transduction domain fusion protein
  • the induced cells were disrupted by ultrasonication and analyzed by 15% SDS-PAGE electrophoresis, see Figure 7.
  • 1 is the supernatant of the cell disruption before induction
  • 2, 3, 4, 5, and 6 are the cell disrupted supernatants at 1, 2, 3, 4, and 5 hours after induction
  • 7, 8 for induction.
  • M was a standard molecular weight protein.
  • HeLa cells were cultured in a 96-well cell culture plate at 37 ° C until the cell density reached about 60%.
  • the recombinant Apoptin-EC-SOD-PTD isolated and purified after expression was subjected to multiple dialysis to remove the imidazole. Then sterile filtration, dilute into different volumes of DMEM cell culture medium to dilute to different protein concentrations, from the highest protein concentration, gradually reduce the concentration to half, to zero protein concentration, as a negative control, each group of protein concentration Take 3-4 wells as a parallel control.
  • the recombinant Apoptin protein isolated and purified from the prokaryotic expression system was similarly used as a control sample for the same experimental procedure.
  • the medium was aspirated, added to DMEM containing 5 ug/ml MTT, and incubated at 37 ° C for 4 hours.
  • the medium was aspirated, and the purple crystals were dissolved in ⁇ DMSO and incubated at 37 ° C for 10 minutes.
  • the absorbance was measured at 490 nm.
  • concentration of recombinant Apoptin-EC-SOD-PTD 100 g/ml
  • recombinant Apoptin as a control group
  • Fig. 8 The results of apoptosis induction by cultured HeLa cells using recombinant Apoptin-EC-SOD-PTD and recombinant Apoptin are shown in Fig. 8. The survival rate of HeLa cells treated with different concentrations of samples was detected by MTT method.
  • Example 6 Effect of apoptin-EC-SOD carboxy terminal protein transduction domain fusion protein on normal animal cells cultured in vitro.
  • Normal human liver cell L02 was cultured in RPMI 1640 medium (containing 10% fetal bovine serum). After trypsinization, a single cell suspension was prepared in 1640 medium, and seeded into a 96-well cell culture plate at a cell concentration of lxlO 5 /ml, each well having a volume of 100 ⁇ l. Incubate for 4 h at 37 ° C in a 5% C0 2 incubator.
  • test drug Prepared with a final concentration of 200, 100, 50, 25, ( ⁇ g/ml, 100 ⁇ l per well, set 3 parallel holes per set, set 37 ° C, 5% C0 2 and saturate The humidity incubator was continued for 48 h.
  • MTT solution was added to each culture well for detection, and the viability of the cells was determined by a colorimetric assay. Test data are expressed as mean ⁇ standard deviation.
  • C C57BL/6 mouse Lewis lung cancer model was used to verify the inhibitory effect of recombinant Apoptin-EC-SOD-PTD on tumor proliferation.
  • the experiment set blank control group, positive control group, recombinant Apoptin-EC-SOD-PTD test group, recombinant polyarginine There were four groups of acid-apoptosis test group (PolyArg-Apoptin), 8 animals in each group.
  • the blank control group was intraperitoneally injected with physiological saline after inoculation of the tumor.
  • the positive control group was injected with cyclophosphamide solution (20 mg/kg BW/day).
  • the experimental group was injected with purified dialysis, sterile filtered recombinant Apoptin-EC-SOD-PTD (10 mg/kg BW/day) and recombinant PolyArg-Apoptin (40 mg/kg BW/day).
  • Tumor At 6 weeks of age, C57BL/6 (male) mice weighing approximately 20 g were inoculated with 0.2 ml of Lewis lung cancer cell suspension, and after 3-4 days of feeding, the tumor growth of the infraorbital mouse was observed. The administration test can be started when the tumor to be injected is as long as several millimeters in diameter.
  • Administration Administration by intraperitoneal injection, continuous administration for 7-8 days. Body weight measurements were taken before daily dosing.
  • Tumor inhibition rate (average tumor weight in the blank group - mean tumor weight in the drug group) / average tumor weight in the blank group X100%
  • Recombinant Apoptin-EC-SOD-PTD had a tumor inhibition rate of 37.8% for Lewis lung cancer, which was higher than that of the recombinant Ploy Arg-Apoptin group, which was close to the positive control group (the positive control group had a tumor inhibition rate of 47.4%). It is indicated that apoptin has greatly enhanced its transmembrane transport effect after EC-SOD-PTD, and it has a good inhibitory effect on tumor growth.

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Description

凋亡素 -EC-SOD羧基末端蛋白转导域融合蛋白 技术领域
本发明涉及生物技术领域， 具体涉及本发明涉及的一种凋亡素 -EC- SOD羧基末端蛋 ' 甶转导域融合蛋白、 基因、 重组载体、 转化体及其用途和制备方法。 背景技术
超氧化歧化酶 (superoxide dismutase简称 SOD)是一种催化超氧物阴离子 (02_)歧化反 应的酶。 人和哺乳动物的 SOD 有 3 种同工酶： 胞内 SOD(Cu， Zn-SOD)、 线粒体 SOD(Mn-SOD)和胞外 SOD(EC-SOD，也是 Cu， Zn-SOD)。 EC-SOD[Marklund SL, Bjelle A， Elmqvist LG, et al. Ann Rheum Dis. 1986， 45： 847-851]在人体内的含量最少， 主 要存在于组织的胞外基质和细胞表面， 其氨基酸序列按功能可分成四个结构域： N端 先是一个 18个氨基酸残基的信号肽； 然后是 95个 基的与 EC-SOD形成四聚体有关 的结构域； 后面是 98个残基的 SOD活性结构域； 最后是 29个残基的富含碱性氨基酸 残基的细胞转导结构域 ( PTD, protein transduction domains) [Hjalmarsson K, Marklund SL, Engstrom A, et al. Proc Natl Acad Sci USA. 1987， 84： 6340-6344]。 这个结构域 能与细胞表面的肝素类多糖组织结合 [Karlsson K， Marklund SL. Lab Invest. 1989， 60： 659-666], 并能以内吞作用实现跨膜转导， 将其定位到细胞核。
源于鸡贫血病毒的凋亡素（Apoptin)， 或称凋亡蛋白， 由 121个氨基酸残基的组成， 它能够在人的各种肿瘤细胞中诱导一种不依赖于 p53，并且对 Bcl-2不敏感的细胞凋亡， 但对正常人体细胞没有影响， 是一个非常有应用前景的肿瘤选择性治疗的候选药物 [Zhuang SM, Shvarts A, van Ormondt H, etal. Cancer Res. 1995， 55(3)： 486-489]。 研究表明， 凋亡素诱导的细胞凋亡需要凋亡素本身被转运到细胞内， 然后依靠凋亡素 本身所具有的核定位序列（NLS， nuclear localization sequence)在细胞核中聚集， 以目 前尚不太清楚的机制， 导致细胞的凋亡。 凋亡素诱导的细胞凋亡与其本身的特殊氨基 酸残基的磷酸化过程有关，其凋亡过程与 Caspase-3通路有密切联系 [Notebom MH. Vet Microbiol. 2004， 98(2)： 89-94]。
由上可知， 由凋亡素诱导的肿瘤细胞凋亡的关键点是如何将凋亡素快速、 高效的转 运入肿瘤细胞， 实现跨膜蛋白质分子的转运。 目前， 已经有许多具有蛋白质转运功能 的跨膜结构域（PTD) 被研究发现， 典型的有来自于人 HIV病毒的 TAT结构域， 黑腹 果蝇的触足肽（ Antp)，纯疱疹病毒转录调节蛋白（ VP22)，多聚精氨酸序列以及 EC-SOD 的羧基末端蛋白转导域等等。 PTD—般由长度多数小于 20个氨基酸序列组成， 含有一 个高度正电荷区和一个螺旋结构。 PTD可以携带多种物质， 包括亲水性蛋白、 多肽、 DNA甚至颗粒性物质等进行细胞间 /内的传输， 且不受细胞类型的限制 [Schwarze SR， Dowdy SF. Trends Pharmaeol Sci. 2000, 21(2)： 45-48]。 不过， 其转运的效率与 PTD 本身以及与其融合的蛋白质性质有密切关系。 因此， 要实现一个蛋白质分子的髙效跨 膜转移， 选择一个合适的 PTD至关重要。 对于加强凋亡素诱导肿瘤细胞凋亡的效应来 说， 就是需要寻找合适的 PTD, 帮助将凋亡素快速、 高效的转运入细胞内部， 发挥其 凋亡作用， 达到控制肿瘤细胞生长， 治疗疾病的目的。 发明内容
本发明所要解决的技术问题提供了一种凋亡素融合蛋白， 以便于其能快速、 高效的 转运入细胞内部， 发挥其凋亡作用， 达到控制肿瘤细胞生长， 治疗疾病的目的。
为此本发明一方面公开了， 一种凋亡素 -EC-SOD羧基末端蛋白转导域融合蛋白， 其 特征在于该融合蛋白含有与氨基酸序列如 SEQ ID NO.l所述 EC-SOD羧基末端蛋白转 导域或其突变体融合的氨基酸序列如 SEQ ID N0.2所述凋亡素或其突变体。
• 本发明的发明思路为通过基因工程技术人为将 EC-SOD羧基末端蛋白转导域或其 突变体与凋亡素或其突变体构成融合蛋白， 通过 EC-SOD羧基末端蛋白转导域或其突 变体实现跨膜蛋白质分子的转运， 从而发挥凋亡素或其突变体的诱导凋亡作用， 达到 控制肿瘤细胞生长， 治疗疾病的目的。 在这一思路下， 本发明所保护范围包括但不局 限于上述融合蛋白。
在一实施方式中， 所述凋亡素 -EC-SOD羧基末端蛋白转导域融合蛋白可具有下式： R2-R1、 Rl-R2、 Rl-L- R2-L-R1、 Rl-L-R2、 R2-L-R1 或 R2-L-R1-L-R1 , 其中 Rl是 氨基酸序列如 SEQ ID NO.l所述 EC-SOD羧基末端蛋白转导域或其突变体， L 是连接 肽， R2是至少一个氨基酸序列如 SEQ ID N0.2所述凋亡素或其突变体，但不必是相同 的治疗性蛋白质。连接肽包括但不限于， 例如： （GGGGS )N或 ( GGGS)！^或（GGS) N, 其中 N是大于或等于 1的整数， G表示甘氨酸， S 表示丝氨酸。
在一实施方式中， 所述凋亡素 -EC-SOD羧基末端蛋白转导域融合蛋白中， 氨基酸序 列如 SEQ ID NO.l所述 EC-SOD羧基末端蛋白转导域或其突变体位于所述融合蛋白的 幾基末端。
在一实施方式中， 所述凋亡素 -EC-SOD羧基末端蛋白转导域融合蛋白， 为如下（a) 或 (b)的蛋白：
- (a) 其氨基酸序列如 SEQ ID NO. 3所述的蛋白；（b) 至少与（a)中的氨基酸序列 60 % 同源性且具有诱导细胞凋亡作用活性的蛋白。
• 一方面，本发明公开了一种编码本发明所述凋亡素 -EC-SOD羧基末端蛋白转导域融 合蛋白的多核苷酸分子。
在一实施方式中， 所述多核苷酸分子的核酸序列如 SEQ ID N0. 4所述。
一方面， 本发明公开了一种包含本发明所述多核苷酸分子的重组表达载体。
一方面， 本发明公开了一种包含本发明所述重组表达载体的转化体。
在一实施方式中， 所述转化体为大肠杆菌。
另一方面，本发明公开了一种本发明所述凋亡素 -EC-SOD羧基末端蛋白转导域融合 蛋白的制备方法， 包括如下步骤：
① 凋亡素 -EC-SOD羧基末端蛋白转导域融合蛋白表达载体的构建；
② 凋亡素 -EC-SOD羧基末端蛋白转导域融合蛋白转化体的制备；
③ 凋亡素 -EC-SOD羧基末端蛋白转导域融合蛋白的表达；
④ 凋亡素 -EC-SOD羧基末端蛋白转导域融合蛋白的纯化。
另一方面，本发明公开了一种组合物，包括本发明所述凋亡素 -EC-SOD羧基末端蛋 白转导域融合蛋白和药学上可接受的载体。
另一方面，本发明公开了本发明所述凋亡素 -EC-SOD羧基末端蛋白转导域融合蛋白 在制备治疗由细胞增殖过度引起的疾病药物中应用。
在一实施方式中， 所述由细胞增殖过度引起的疾病为肿瘤。
另一方面，本发明包括预防、治疗或改善由细胞增殖过度引起的相关疾病或失调的 方法， 所述方法包括给需要所述预防、 治疗或改善的哺乳动物施用含有有效量凋亡素 -EC-SOD羧基末端蛋白转导域融合蛋白， 以治疗、 预防或改善疾病或失调。 通过体外培养的肿瘤细胞凋亡试验和小鼠腹水瘤抑制试验证明， 本发明制备的凋亡 素- EC- SOD羧基末端蛋白转导域融合蛋白具有很强的诱导肿瘤细胞凋亡的能力，可以用 来制备治疗肿瘤的药物。 附图说明
图 1 PCR扩增的 EC-S0D羧基末端蛋白转导域基因片段 2 %琼脂糖凝胶电泳图谱； 图 2 表达质粒 C- pET28a构建过程示意图；
图 3 PCR扩增的凋亡素基因片段 1. 5 %琼脂糖凝胶电泳图谱；
图 4 克隆质粒 Apop- pMD18T构建过程示意图；
图 5 表达质粒 ApopC_pET28a构建过程示意图；
. 图 6 菌落 PCR检测重组菌插入片段的 1. 5 %琼脂糖凝胶电泳图谱；
图 7 纯化的重组 Apoptin- EC- SOD- PTD SDS— PAGE电泳分析图谱；
图 8 不同浓度重组 Apoptin- EC-S0D-PTD对 HeLa细胞的存活率的影响； 图 9 重组 Apoptin-EC-SOD- PTD对体外培养的人正常肝细胞的存活率的影响。 具体实施方式
在本文， 术语 "突变体"是指氨基酸序列如 SEQ ID N0.1所述 EC-SOD羧基末端蛋 白转导域或氨基酸序列如 SEQ ID N0.2所述凋亡素的突变体。 相比于天然的 EC-SOD 羧基末端蛋白转导域或所述凋亡素蛋白， 该突变体与它们野生型相比， 具有增强的活 性和 /或改变了的立体专一性。 天然蛋白的氨基酸序列突变体可通过向本发明的核苷 酸中引入适当的核苷酸变化、 或通过体外合成所需多肽来制备。 这些突变体包括， 例 如缺失、 插入或替换该氨基酸序列中的残基。 可以通过缺失、 插入和替换的组合以得 到最终的构建体， 提供最终的蛋白产品。
蛋白的同源性百分比由 GAP ( Needleman和 Wunsh , 1 970)分析 ( GCG程序）确 定， 其中参数 gap creation penalty = 5， gap extension penalty = 0.3。 被分析的序歹 ϋ长度 至少为 15个氨基酸时， GAP 分析就在参与测试的两个序列的至少为 15个氨基酸的区 域进行测试。 更优选地， 被分析的序列长度至少为 50个氨基酸时， GAP 分析就在参 与测试的两个序列的至少为 50 个氨基酸的区域进行测试。 更优选地， 被分析的序列 长度至少为 100个氨基酸时， GAP 分析就在参与测试的两个序列的至少为 100 个氨 基酸的区域进行测试。更优选地，被分析的序列长度至少为 250 个氨基酸时， GAP 分 析就在参与测试的两个序列的至少为 250 个氨基酸的区域进行测试。 甚至更优选地， 被分析的序列长度至少为 500 个氨基酸时， GAP 分析就在参与测试的两个序列的至 少为 500 个氨基酸的区域进行测试。
本发明涉及的方面还包括凋亡素 -EC-SOD羧基末端蛋白转导域融合蛋白类似物， 在 它们合成期间或合成后进行了不同的修饰， 例如， 通过生物素化、 苄基化、 糖基化、 乙酰化、 磷酸化、 由已知保护 /封闭基团的衍生作用、 蛋白水解的切割作用、 连接到 抗体分子或其它细胞配体上等。 这些修饰可以用来增加本发明凋亡素 -EC-SOD羧基末 端蛋白转导域融合蛋白的稳定性和 /或生物活性。
融合蛋白的表达
本发明包括编码本发明的凋亡素 -EC-SOD羧基末端蛋白转导域融合蛋白的 DNA以 及含有这些 DNA的载体、 转化体。
本发明中， 使用的术语 "转化体" （transf ormant)， 即带有异源 DNA分子的宿主细 胞。
本发明还包括通过合成和重组技术生产本发明的凋亡素 -EC-SOD羧基末端蛋白转导 域 ¾合蛋白的方法。通过本领域已知的方法可以分离和纯化多核苷酸（DNA或 RNA)、 载体、 转化体和生物体。
用于本发明的载体可以是如噬菌体、 质粒、 粘粒、 微型染色体、 病毒或逆转录病毒 载体。 可用于克隆和 /或表达本发明的多核苷酸的载体是能在需复制和 /或表达多核 苷酸的宿主细胞中复制和 /或表达多核苷酸的载体。 一般说来， 多核苷酸和 /或载体 可用于任何真核或原核细胞， 包括哺乳动物细胞 （如人 （如 HeLa) 、 猴 （如 Cos ) 、 兔（如兔网织红细胞） 、 大鼠、 仓鼠（如 CH0、 NS0 和幼仓鼠肾细胞）或小鼠细胞（如 L 细胞） ） 、 植物细胞、 酵母细胞、 昆虫细胞或细菌细胞 （如大肠杆菌） 。 有关适用 于多种类型宿主细胞的适当载体的例子可参见例如 F. Ausubel et al. , Current Protocols in Molecular Biology. Greene Publishing Associates and Wiley-Interscience (1992)和 Sambrook et al. (1989)。 可以使用含有这些多核苷酸 的宿主细胞来大量表达可用于例如药物、 诊断试剂、 疫苗和治疗剂的蛋白质。
已开发出多种方法用于经由互补的粘性末端使多核苷酸与载体可操作相连。 例如， 可在欲插入载体 DNA 内的 DNA 区段添加互补的同聚体序列片段。 然后通过互补同聚 体尾之间的氢键连接载体和 DNA 区段以形成重组 DNA分子。
含有一或多种限制性位点的合成接头提供了另一种连接 DNA 区段与载体的方法。用 噬菌体 T4 DNA 聚合酶或大肠杆菌 DNA聚合酶 I 处理通过内切核酸酶限制性消化产 生的 DNA 区段， 所述的两种聚合酶用其 3'，5 ' -核酸外切活性除去突出的 Y -单链末 端， 并用其聚合活性补平 3 ' -凹端。 因此， 这些活性的联合产生了平端 DNA 区段。 然后在能催化平端 DNA 分子连接的酶， 如噬菌体 T4 DNA 连接酶的存在下将平端区段 与大摩尔过量的接头分子一起保温。 因此， 反应产物是末端携有聚合接头序列的 DNA 区段。然后用适当的限制性酶裂解这些 DNA 区段，并连接至已用酶裂解的表达载体中， 所述酶能产生与所述 DNA 区段相容的末端。从多个商家可以买到含有多个限制性内切 核酸酶位点的合成接头。
多核苷酸插入物应该可操作地连接于同表达多核苷酸的宿主细胞相容的适当启动子 上。 启动子可以是强启动子和 /或诱导型启动子。 列举的一些启动子的例子包括噬菌 体 λ PL 启动子、 大肠杆菌 lac、 trP 、 PhoA、 tac 启动子、 SV40 早期和晚期启动子 以及逆转录病毒 LTR 启动子。其它适当启动子是本领域技术人员已知的。 表达重组载 体进一步含有转录起始、 终止位点， 并在转录区含有用于翻译的核糖体结合位点。 重 组载体表达的转录物的编码部分可包括位于起点处的翻译起始密码子和适当地位于被 翻译多肽的末端的终止密码子 （UAA, UGA或 UAG) 。
如上所述， 表达载体可包括至少一个选择标记。 所述标记包括对真核细胞培养物而 言的二氢叶酸还原酶、 G418 、 谷氨酰胺合酶或新霉素抗性； 以及用于^:肠杆菌和其 它细菌培养的四环素、 卡那霉素或氨卞青霉素抗性基因。 适当宿主的代表性例子包括 但不限于： 细菌细胞， 如大肠杆菌、 链霉菌和鼠伤寒沙门氏菌细胞； 真菌细胞， 如酵 母细胞 （如酿酒酵母或巴斯德毕赤酵母） ； 昆虫细胞， 如果蝇 S2 和夜蛾 SF9细胞； 动物细胞， 如 CH0 , COS , NS0 , 293 和 Bowes 黑素瘤细胞； 和植物细胞。 上述宿 主细胞的适当培养基和培养条件是本领域已知的。
为了有效地分离纯化或分泌目标蛋白， 常常还可利用便于分离纯化的标签蛋白或标 签多肽 （Tag) 。 常用的有谷胱甘肽 -S-转移酶 （glutathione S-transferase, GST) 、 六聚 组氨酸肽（His.Tag)、蛋白质 A(protein A)和纤维素结合位点（cellulose binding domain) 等。 通过特殊性蛋白或多肽与目标蛋白构成融合蛋白的形式， 表达后利用所述的标签 蛋白或标签多肽的特殊性质可对目标蛋白进行分离和纯化。 如 His.Tag与 M-Chelating Sepharose柱特异性结合。 所述的标签蛋白或标签多肽可以在纯化后用位点特异性蛋白 酶消化去除融合序列， 如可用凝血酶、 肠激酶和 Xa因子等， 以获得目标蛋白。
本发明还包括含有本发明的核苷酸序列的宿主细胞， 所述核苷酸序列经本领域已知 的技术与一或多种异源控制区 （如启动子和 /或增强子） 可操作相连。 可以选择能调 节插入的基因序列的表达， 或能按照所需的特殊方式修饰和加工基因产物的宿主菌株。 在某些诱导物的存在下， 某些启动子启动的表达会升髙； 因此， 可以控制经基因改造 的多肽的表达。 另外， 不同宿主细胞具有特征性的和特殊的翻译、 翻译后加工和修饰 (如磷酸化、 裂解） 蛋白质的机制。 可以选择适当的细胞系以确保对表达的外源蛋白 质进行合乎需要的修饰和加工。
通过磷酸钙转染、 DEAE-葡聚糖介导的转染、 阳离子脂质介导的转染、 电穿孔、 转 导、 感染或其它方法， 即可将本发明的核酸和核酸重组载体导入宿主细胞。 所述方法 描述于多个标准的实验室手册中， 如 Davis et al. , Basic Methods In Molecular Biology (1986)。
编码本发明的所述融合蛋白的多核苷酸可以与含有选择标记的载体连接以在宿主中 增殖。 一般说来， 可在沉淀物， 如磷酸钙沉淀物或其与带电脂质的复合物中导入质粒 载体。 如果载体是病毒， 可使用适当的包装细胞系在体外对其进行包装， 再转导至宿 主细胞。
通过众所周知的技术可以鉴定出被成功转化的细胞， 即含有本发明的 DNA重组载体 的细胞。 例如， 可培养导入表达重组载体所得的细胞以产生所需多肽。 收集并裂解细 胞， 使用如 Southern ( 1975 ) J. Mol. Biol. 95, 503或 Berent et al (1985) Biotech. 3, 208 所述的方法， 检测其 DNA内容物中 DNA的存在。 或者， 使用抗体检测上清液中 蛋白质的存在。
通过众所周知的方法从重组细胞培养物中回收和纯化本发明的所述融合蛋白较为有 利， 所述方法包括硫酸按或乙醇沉淀、 酸提取、 阴离子或阳离子交换层析、 磷酸纤维 素层析、 疏水作用层析、 亲和层析、 羟基磷灰石层析、 疏水电荷作用层析和凝集素层 析。 在一些实施方案中， 可使用髙效液相层析 （HPLC) 进行纯化。
在一些实施方案中，可使用上述的一种或多种层析方法纯化本发明的所述融合蛋白。 在其它实施方案中， 可使用下述的一种或多种层析柱纯化本发明的所述融合蛋白， 所 述层析柱有 - Q sepharose FF柱、 SP sepharose FF柱、 Q sepharose High Performance 柱、 Blue sepharose FF柱、 Blue柱、 Phenyl Sepharose FF柱、 DEAE Sepharose FF、 Ni- Chelating Sepharose FF柱或 Methyl 柱等。
另外， 可使用国际公开号 W000/44772 (全文列入本文作为参考） 中描述的方法纯 化本发明的融合蛋白。 本领域技术人员可以容易地改动其中所述的方法以用于纯化本 发明的融合体蛋白。 可以从包括例如细菌、 酵母、 高等植物、 昆虫和哺乳动物细胞的 原核或真核宿主经重组技术产生的产物中回收本发明的融合蛋白。
. 在一较优实施例中， 本发明提供的凋亡素 -EC-S0D羧基末端蛋白转导域融合蛋白制 备方法包括以下步骤：
a. 构建克隆有 EC- S0D羧基末端蛋白转导域基因的表达质粒 C- PET28a;
b. 构建克隆有凋亡素基因的克隆质粒 Apop- PMD18T;
c. 构建克隆有凋亡素- EC- SOD羧基末端蛋白转导域融合蛋白基因的表达质粒 ApopC-pET28a;
d. 凋亡素 -EC-SOD羧基末端蛋白转导域融合蛋白的表达和纯化；
上述 a.中所述的表达质粒 C- pET28a的构建方法是： 设计 5' 端和 3' 端引物， 采用 PCR的方法从克隆有 EC- SOD全长基因的 EC-SOD- pSK质粒中将 EC-SOD羧基末端蛋白转 导域 （第 196- 222位氨基酸残基） 克隆， 在此片段的 N-末端接上一端 8个氨基酸残基 长度的连接肽， 然后将此片段克隆入 pET28a的 Hind III和 Xho I位点， 构成 EC- SOD 羧基末端蛋白转导域基因的表达质粒 C- pET28a。
上述 b.中所述的克隆质粒 Apop-PMD18T的构建方法是： 设计 5' 端和 3' 端引物， 采用 PCR的方法从克隆有凋亡素全长基因的 Apoptin- PVK质粒中将凋亡素全长基因（第 1 - 121位氨基酸残基） 克隆， 此基因的 5' 端和 3 ' 端两侧分别接上了 Nde I和 BamH I 酶切位点， 然后将此片段克隆入 pMD- 1ST质粒， 构成克隆有凋亡素基因的克隆质粒 Apop - pMD18T。
上述 c.中所述的表达质粒 ApopC- pET28a的构建方法是： 将克隆质粒 Apop- pMD18T 分别用， 分离得目标片段， 然后将此片段插入已同样经 Nde I和 BamH I双酶切的 C-pET28a表达载体中， 构建成克隆有凋亡素 -EC-S0D羧基末端蛋白转导域融合蛋白基 因的表达质粒 ApopC- pET28a。
上述 d.中所述的凋亡素 -EC-S0D羧基末端蛋白转导域融合蛋白的表达和纯化的方法 是： 将构建的 ApopC-PET28a重组质粒转入融合有 T7 RNA聚合酶基因的大肠杆菌宿主 中。 转入的宿主可以是 E. coli BL21 (DE3) , BL21 (DE3) pLysS, JM109 (DE3) ,
Rosetta (DE3)， Rosetta DE3 pLysS等。 将构建的工程菌在添加有 50ug/ml终浓度卡那 霉素的 LB培养基 37°C振荡培养至 OD600nm达到 0. 3-0. 6时， 加入 IPTG至终浓度为 0. 5-3mM， 诱导培养 3-5hr， 收集菌体进行超声破碎， 离心收集包涵体， 经 SDS- PAGE 电泳检测后， 采用 8M尿素变性溶解包涵体， 上镍柱进行亲和层析分离， 分段收集目的 蛋白组分， 进行稀释法复性， 浓缩。 除菌过滤后进行蛋白含量测定， 保存待用。
用途
本发明所示的融合蛋白作为活性成分可用以治疗各种由细胞增殖过度引起的疾病， 如肿瘤， 包括但不限于： 骨癌类， 包括：尤因肉瘤、 骨肉瘤、 软骨肉瘤等； 脑和 CNS肿 瘤， 包括： 听神经瘤、 神经母细胞瘤、 神经胶瘤和其他脑肿瘤， 脊髓肿瘤、 乳癌、 结 肠直肠癌、 进展期结肠直肠腺癌； 内分泌癌类， 包括： 肾上腺皮质癌、 胰癌、 脑垂体 癌、 甲状腺癌、 副甲状腺癌、 胸腺癌、 多发性内分泌肿瘤； 胃肠癌类， 包括： 胃癌、 食道癌、 小肠癌、 肝癌、 肝外胆管癌、 胃肠类癌性肿瘤、 胆囊癌； 泌尿生殖器癌类， 包括： 翠丸癌、 阴茎癌、 前列腺癌； 妇科癌类， 包括： 子宫颈癌、 卵巢癌、 阴道癌、 子宫 /子宫内膜癌、 阴部癌、 妊娠滋养细胞肿瘤、 输卵管癌、 子宫肉瘤； 头和颈部肿瘤 类， 包括： 口腔癌、 唇癌、 唾腺癌、 喉头癌、 下咽癌、 正咽癌、 鼻癌、 鼻窦癌、 鼻咽 癌； 血癌类， 包括： 儿童白血病、 急性淋巴性白血病、 急性骨髓性白血病、 慢性淋巴 性白血病、 慢性骨髓性白血病、 发状细胞性白血病、 急性早幼粒细胞白血病、 血浆细 胞性白血病； 骨髓癌血液病症， 包括： 骨髓分化不良症候群、 骨髓增生性病症、 再生 障碍性贫血、 范可尼贫血、 特发性巨球蛋白血症； 肺癌类， 包括： 小细胞肺癌、 非小 细胞肺癌； 淋巴癌类， 包括： 霍奇金病、 非霍奇金氏淋巴瘤、 皮肤型 T-细胞淋巴瘤、 周围 T-细胞林巴瘤、 AIDS相关性淋巴瘤； 眼癌类， 包括： 视网膜母细胞瘤、 葡萄膜黑 色素瘤； 皮肤癌类， 包括：黑色素瘤、 非黑色素瘤皮肤癌、 梅克尔细胞癌； 软组织肉瘤 类， 例如：儿童软组织肉瘤、 成人软组织肉瘤、 卡波希肉瘤； 泌尿系统癌症， 包括：肾 癌维尔姆斯肿瘤、 膀肤癌、 尿道癌或转移性细胞癌。
本发明所公开的融合蛋白， 可以用来治疗的癌类首先为肝癌或肺癌。
可藉由本发明的融合蛋白加以治疗的优选肿瘤类为固态肿瘤和血液恶性疾病。
本文所使用的术语 "肿瘤"一般是指广泛的以细胞的失控性异常生长为特征的病 症。
所用的活性成分的有效剂量可随给药模式和待治疗疾病的严重程度而变化。 对大部 分大型哺乳动物而言， 每天施以有效成分的总剂量约为 0. 01-1000mg。通常， 成人临床 给药量的范围为 0. 01- 200mg/日, 优选为 0. 05- lOOmg/日。
"有效剂量"或 "治疗量"均是指足以产生疗效的量。 有效量可分一或多次给药。 通常， 有效量足以缓和、 改善、 稳定、 减慢或延迟疾病的进一步发展。
组合物
用于本发明或者含有本发明所述融合蛋白的组合物。 通常， 当本发明组合物用于上 述用途时， 所述融合蛋白可与一种或多种药学上可接受的载体或赋形剂混合制成不同 给药途径的药物剂型， 如片剂、 胶囊、 散剂、 颗粒剂、 糖浆剂、 溶液剂、 口服液、 醑 剂、 酊剂、 气雾剂、 粉雾剂、 注射剂、 注射用无菌粉末、 栓剂等。
"药学上可接受的"成分是适用于人和 /或动物而无过度不良副反应（如毒性、刺激 和变态反应） 即有合理的效益 /风险比的物质。 "药学上可接受的载体"是用于将本发 明的融合体蛋白传送给动物或人的药学上或食品上可接受的溶剂、 悬浮剂或赋形剂。 载体可以是液体或固体。
本发明的融合蛋白可经过口服、 静脉内、 肌内或皮下途径给药。
上述剂型中可经口服给药的剂型为： 片剂、 胶囊、 散剂、 颗粒剂、 糖桨剂、 溶液剂、 醑剂。 固态载体包括： 淀粉、 乳糖、 磷酸氢钙、 微晶纤维素、 蔗糖、 白陶土、 微粉硅 胶、 滑石粉、 低取代羟丙基纤维素、 羧甲基淀粉钠、 聚乙烯吡咯垸酮。 而液态载体包 括： 无菌水、 乙醇、 聚乙二醇、 非离子型表面活性剂和食用油（如玉米油、 花生油和芝 麻油）。在制备药物组合物的过程中通常使用的佐剂包括： 调味剂、着色剂、防腐剂 (如 羟苯烷基丁酯、 苯甲酸钠、 山梨酸）和抗氧化剂（如维生素 E、 维生素 C、 焦亚硫酸钠和 二丁基羟基甲苯）。
上述剂型中可用于注射途径给药的剂型包括： 注射剂、 注射用无菌粉末， 它们是将 药物与一种或多种药学上可接受的赋形剂混合制成以供注射给药的形式。 溶剂包括： 无菌水、 乙醇、 甘油、 丙二醇、 聚乙二醇。 此外， 还需加入抑菌剂（如苯甲醇、 羟苯丁 酯、 硫柳汞）、 等渗调节剂（如氯化钠、 葡萄糖）、 助悬剂 (如羧甲基纤维素钠、 甲基纤 维素）、 增溶剂（吐温 -80、 卵磷酯）、 抗氧化剂（如维生素 E、 维生素 C、 焦亚硫酸钠）和 填充剂（如乳糖、 甘露醇）。 从易于制备和给药的立场看，优选的药物组合物是固态组合物，尤其是冻干粉针剂。 优选静脉给药。
以下结合具体实施例， 进一步阐明本发明。 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， 或按照制造厂商所建议的条件。 比例和百分比基于重量， 除非特别说明。 实施例 1: 构建克隆有 EC-SOD羧基末端蛋白转导域基因的表达质粒 C-pET28a
1. EC-SOD羧基末端蛋白转导域基因的 PCR扩增
( 1 )根据 Genbank公布的人 EC-SOD的基因序列， 设计亚克隆 EC-SOD羧基末端蛋白 转导域基因的正向和反向引物：
P1： 5，— TAATA GCTTCCGCTGGAGGCGGTGGAAGCGGGCCCGGGCTC -3，
P2: 5，- ATCJCGL4GGGCGGCCTTGCACTCGCTCT -3，
在正向引物 P1 中， 导入了一个 Hind III酶切位点和一段 8个氨基酸残基的连接肽 (AlaSerAlaGlyGlyGlyGlySer) ； 在反向引物 P2中， 导入了一个 Xho I酶切位点。
(2) PCR扩增的反应条件为： 先 94°C， 变性 5min， 然后 94°C， 变性 30s→60°C， 复性 30s→72°C , 延伸 lmin， 循环 30次， 最后 72°C， 5min。
(3 )将 PCR产物回收后用 Hind III和 Xho I双酶切待用。
2.重组表达质粒 C-pET28a的构建
将 PCR扩增、酶切得到的插入片段，与已经用同样的酶水解、回收的 pET28a质粒 DNA. 连接， 转化 E. c^' DH5a菌株， 培养， 回收重组质粒 DNA， 测序验证。
3. PCR扩增所得的目标片段 2%琼脂糖电泳图谱见图 1。 图中， M为标准分子量 DNA Marker; 1为扩增得到的约 120bp的 EC-SOD羧基末端蛋白转导域基因片段。
4.表达质粒构建过程示意图见图 2。 实施例 2: 构建克隆有凋亡素基因的克隆质粒 Apop-pMD18T
1.凋亡素基因的 PCR扩增
( 1 )根据 Genbank公布的鸡贫血病毒 VP3蛋白的基因序列， 设计亚克隆凋亡素全长基 因的正向和反向引物：
P3： 5，- GAC4Z4JGATGAACGCTCTCCAAGAAGA -3'
P4: 5'- TGGG4JC TTACAGTCTTATACGCCTTTTTG -3，
在正向引物 P3中， 导入了一个 Nde l酶切位点； 在反向引物 P2中， 导入了一个 BamH I 酶切位点和终止密码子。
(2) PCR扩增的反应条件为： 先 94°C， 变性 5min， 然后 94°C , 变性 30s→55°C， 复性 30s→72°C， 延伸 45s， 循环 30次， 最后 72°C， 5min。
(3 )将 PCR产物回收待用。
2.重组克隆质粒 Ap0p-pMD18T的构建 将 PCR扩增回收得到 '的插入片段与市售 pMD18T载体（购自宝生物工程公司） DNA连 接，转化 E. C0/ DH5cc菌株， 培养， 通过蓝白斑筛选阳性克隆， 再经液体培养基培养后回收 重组质粒 DNA, 测序验证。
3. PCR扩增所得的凋亡素全长基因片段 1.5 %琼脂糖电泳图谱见图 3。 图中 1为 PCR扩 增得的约 370bp的凋亡素全长基因； M为标准分子量 DNA Marker。
4.表达质粒构建过程示意图见图 4。 实施例 3: 构建克隆有凋亡素 -EC-SOD羧基末端蛋白转导域融合蛋白基因的表达质粒 ApopC-pET28a
1.凋亡素基因插入片段的获取
将制备并经测序验证的 Apop-pMD18T质粒 DNA用 Nde I和 BamH I双酶切， 通过 1 % 琼脂糖凝胶电泳分离酶切片段， 采用胶回收试剂盒回收目标片段 DNA。
2.载体的准备
将构建并扩增制备的 C-pET28a质粒 DNA同样也用 Nde I和 BamH I双酶切， 通过 1 % 琼脂糖凝胶电泳分离酶切片段， 采用胶回收试剂盒回收质粒 DNA。
3.重组表达质粒 ApopC-pET28a的构建
将回收的凋亡素插入片段 DNA和 C-pET28a质粒 DNA连接， 转化 E. coli BL21 (DE3 ) 菌株， 37°C培养， 回收质粒 DNA, 测序验证。
4.表达质粒构建过程示意图见图 5。
5.重组菌菌落 PCR检测 1.5 %琼脂糖电泳图谱见图 6。 图中， 1为以 pET28a空载质粒 DNA为模板的阴性对照； 2为构建的 ApopC-pET28aPCR检测的结果， 采用的引物为引物 P3和引物 P2, 片段大小约为 500bp; M为标准分子量 DNA Marker。
实施例 4: 凋亡素 -EC-SOD羧基末端蛋白转导域融合蛋白的表达和纯化
1.将带有 ApopC-pET28a的 R coli BL21 (DE3 )重组工程菌在含卡那霉素的 LB固体平 皿上培养， 挑取单克隆， 在 37Ό采用含卡那霉素 50ug/ml的 LB培养基进行过夜培养。 然 后以此培养液按 1%接种量转接新的含卡那霉素 50ug/ml 的 LB 培养基 37Ό培养至 OD600=0.3-0.6, 加入终浓度为 0.5-3mM的 IPTG进行诱导， 于 37°C继续培养 3-5小时， 收 集菌体， 超声波破碎， 离心收集包涵体。
2.诱导表达的菌体经超声波破碎， 进行 15%SDS-PAGE电泳分析， 见图 7。 图中， 1为 诱导前菌体破碎上清液； 2, 3， 4, 5， 6分别为诱导后 1， 2, 3 , 4， 5小时的菌体破碎上 清液； 7, 8为诱导后 4， 5小时菌体破碎沉淀部分； M为标准分子量蛋白质。
3.将经洗涤纯化的融合蛋白包涵体用 8M尿素溶液溶解 l-2hr， 离心去除沉淀。调节 pH 至 7.4— 9.0， 加入镍亲和层析凝胶进行柱外吸附 l-2hr。 吸附完毕后， 将凝胶装入玻璃层析 柱中经洗涤后用 20-500mM咪唑分段洗脱， 收集纯化的重组凋亡素 -EC-SOD羧基末端蛋白 转导域融合蛋白 （Apoptin-EC-SOD-PTD)。
4.洗脱的目标蛋白 SDS— PAGE电泳分析结果见图 7。 图中， 1为上样液， 2为穿出液， 3、 4、 5、 6为 20mM咪唑洗脱分段收集液， M为标准分子量蛋白质， 7、 8、 9为 lOOmM 眯唑洗脱分段收集液， 10、 11、 12为 500mM咪唑洗脱分段收集液。 实施例 5： 凋亡素 -EC-SOD羧基末端蛋白转导域融合蛋白对体外培养的肿瘤细胞的诱导凋 亡作用。
1.将 HeLa细胞在 96孔细胞培养板中于 37°C培养至细胞密度达到 60%左右。 将表达后 分离纯化得到的重组 Apoptin-EC-SOD-PTD经多次透析去除咪唑。 然后进行无菌过滤， 力口 入不同体积的 DMEM细胞培养基稀释成不同蛋白浓度，从最高蛋白浓度起，逐步对半降低 浓度， 至蛋白浓度为零， 以此作为阴性对照， 每组蛋白浓度取 3-4个孔作为平行对照。 同 时， 将采用类似的方法由原核表达系统表达后分离纯化的重组 Apoptin蛋白作为对照样品 进行相同的实验操作。两组样品都在培养 24小时后， 吸取培养基， 加入 ΙΟΟμΙ含有 5ug/ml MTT的 DMEM， 37°C孵育 4小时， 吸去培养基， 加入 ΙΟΟμΙ DMSO溶解紫色结晶， 37°C孵 育 10分钟， 于酶标仪 490nm测定吸光值。 在重组 Apoptin- EC-SOD-PTD浓度在 lOO g/ml 时， HeLa细胞的存活率为 47% (IC5()=105 g/ml)。而同样情况下，作为对照组的重组 Apoptin
( lOf^g/ml)和空白对照的缓冲液对 HeLa细胞生长影响不明显。
2.采用重组 Apoptin-EC-SOD-PTD及重组 Apoptin对培养的 HeLa细胞诱导凋亡处理的 结果见图 8。 以 MTT法检测经不同浓度样品处理后的 HeLa细胞存活率。 实施例 6： 凋亡素 -EC-SOD羧基末端蛋白转导域融合蛋白对体外培养的正常动物细胞的影 响。
采用 MTT法测定重组 Apoptin-EC-SOD-PTD对体外培养的正常人体肝细胞 L02的 IC50 值， 观察给药剂量与药物细胞毒性的关系。
1.将正常人肝细胞 L02以 RPMI 1640培养基（含 10%胎牛血清）培养。 用胰酶消化后， 以 1640培养液配成单细胞悬液， 按 lxl05/ml细胞浓度接种到 96孔细胞培养板中， 每孔体 积 100μ1。 37°C， 5% C02培养箱中培养 4h。
2.用培养液以终浓度分别为 200、 100、 50、 25、 （^g/ml配制试验药物， 每孔 100μ1， 每 组设 3个平行孔， 置 37°C， 5% C02及饱和湿度的培养箱继续培养 48h。
3.在每个培养孔中加入 MTT溶液进行检测，采用酶标仪比色法测定、计算细胞的存活率。 试验数据以平均值±标准偏差表示。
4.凋亡素 -EC-SOD羧基末端蛋白转导域融合蛋白对体外培养的正常动物细胞的毒性影 响见图 9。 表明， 重组 Apoptin-EC-SOD-PTD对体外培养的人正常肝细胞无明显毒性影响。 实施例 7: 凋亡素 -EC-SOD羧基末端蛋白转导域融合蛋白对小鼠腹水瘤动物模型的抑瘤作 用
釆用 C57BL/6小鼠 Lewis肺癌模型验证重组 Apoptin-EC-SOD-PTD对肿瘤的增殖抑制作 用。 实验设空白对照组， 阳性对照组， 重组 Apoptin-EC-SOD-PTD试验组， 重组多聚精氨 酸-凋亡素试验组（PolyArg-Apoptin)四个组别，每组 8只动物。空白对照组在接种肿瘤后， 以生理盐水腹腔注射。 阳性对照组则以注射环磷酰胺溶液 （20mg/kgBW/天)。 试验组分别 注射经纯化透析、无菌过滤的重组 Apoptin-EC-SOD-PTD ( 10mg/kgBW/天）和重组 PolyArg- Apoptin (40mg/kgBW/天）。
1.接瘤： 在 6周龄， 体重约 20g的 C57BL/6(雄性） 小鼠腋下接种 0.2ml的 Lewis肺癌细 胞悬液， 喂养 3-4天后， 观察小鼠腋下肿瘤生长情况。 待注射部位的肿瘤长至直径数毫米 大小时即可开始给药试验。
2.给药： 采用腹腔注射的方式给药， 连续给药 7-8天。 每天给药前进行体重测量。
3.抑瘤率测定： 停止给药 2天后， 采用断颈法处死动物， 将小鼠体内的肿瘤组织剥离， 取出称重， 计算抑瘤率。
抑瘤率 = (空白组平均瘤重 -药物组平均瘤重） /空白组平均瘤重 X100%
4. 重组 Apoptin-EC-SOD-PTD 对 Lewis 肺癌的抑瘤率为 37.8%， 高于重组 Ploy Arg-Apoptin组， 与阳性对照组接近 （阳性对照组抑瘤率为 47.4%)。 说明凋亡素在接有 EC-SOD-PTD后， 大大提高了其跨膜转运效果， 对肿始开瘤细胞具有很好的抑制生长作用。 最后
凋亡素 -EC-SOD羧基末端蛋白转导域融合蛋白对实体瘤的抑制作用
剂量 动物数 瘤体重:
抑制 组别 (mg/kgBW
方式 X±SD
/d)
2.3士 -- 对照组 -- i.p 8 7
0.32
1.21士 47.4 环磷酰胺 20 i-P 8 8
0.119 %
1.90±0.4 17.4
Poly Arg-Apoptin 40 i.p 8 8
18 %
Apoptin-EC-SOD 1.43±0·2 37.8
10 i-P 8 8
-PTD 81 % 本发明的范围不受所述具体实施方案的限制， 所述实施方案只欲作为阐明本发明各个方 面的单个例子， 本发明范围内还包括功能等同的方法和组分。 实际上， 除了本文所述的内 容外， 本领域技术人员参照上文的描述和附图可以容易地掌握对本发明的多种改进。 所述 改进也落入所附权利要求书的范围之内。 上文提及的每篇参考文献皆全文列入本文作为参 考。

Claims

权 利 要 求
1、 一种凋亡素 -EC-SOD羧基末端蛋白转导域 合蛋白， 其特征在于该融合蛋白含 有与氨基酸序列如 SEQ ID N0.1所述 EC-SOD羧基末端蛋白转导域或其突变体融合的 氨基酸序列如 SEQ ID N0.2所述凋亡素或其突变体。
2、 如权利要求 1所述的凋亡素 -EC-SOD羧基末端蛋白转导域融合蛋白， 其特征在 于所述凋亡素 -EC-SOD羧基末端蛋白转导域融合蛋白可具有下式： R2-R1、 Rl-R2、 R1-L- R2-L-R1、 Rl-L-R2、 R2-L-R1 或 R2-L-R1-L-R1， 其中 R1是氨基酸序列如 SEQ ID N0.1所述 EC-SOD羧基末端蛋白转导域或其突变体， L是连接肽， R2是至少一个 氨基酸序列如 SEQ ID N0.2所述凋亡素或其突变体。
3、 如权利要求 1所述的凋亡素 -EC-SOD羧基末端蛋白转导域融合蛋白， 其特征在 于所述凋亡素 -EC-SOD羧基末端蛋白转导域融合蛋白中， 氨基酸序列如 SEQ ID N0.1 所述 EC-SOD羧基末端蛋白转导域或其突变体位于所述融合蛋白的羧基末端。
4、 如权利要求 1所述的凋亡素 -EC-SOD羧基末端蛋白转导域融合蛋白， 其特征在 于所述凋亡素 -EC-SOD羧基末端蛋白转导域融合蛋白， 为如下（a)或（b)的蛋白：
(a) 其氨基酸序列如 SEQ ID NO. 3所述的蛋白；.
(b) 至少与（a)中的氨基酸序列 60%同源性且具有诱导细胞凋亡作用活性的蛋白。
5、一种编码权利要求 1一 4任一项所述凋亡素 -EC-SOD羧基末端蛋白转导域融合蛋 白的多核苷酸分子。
6、 如权利要求 5所述的多核苷酸分子， 其特征在于所述多核苷酸分子的核酸序列 如 SEQ ID N0.4所述。
7、 一种包含权利要求 5所述多核苷酸分子的重组表达载体。
8、 如权利要求 7所述的重组表达载体， 其特征在于所述多核苷酸分子的核酸序列 如 SEQ ID N0.4所述 、
9、 一种包含权利要求 7所述重组表达载体的转化体。
10、 如权利要求 9所述的转化体， 其特征在于所述转化体为大肠杆菌。
11、 一种权利要求 1一 4任一项所述凋亡素 -EC-SOD羧基末端蛋白转导域融合蛋白 的制备方法， 包括如下步骤： '
① 权利要求 1所述凋亡素 -EC-SOD羧基末端蛋白转导域融合蛋白表达载 体的构建；
② 权利要求 1所述凋亡素 -EC-SOD羧基末端蛋白转导域融合蛋白转化体 的制备；
③ 权利要求 1所述凋亡素 -EC-SOD羧基末端蛋白转导域融合蛋白的表达；
④ 权利要求 1所述凋亡素 -EC-SOD羧基末端蛋白转导域融合蛋白的纯化。
12、 如权利要求 11所述的制备方法， 其特征在于所述凋亡素 -EC-SOD羧基末端蛋 白转导域融合蛋白的转化体可为原核基因表达系统或真核基因表达系统。
13、 一种组合物， 其特征在于其包括权利要求 1一 4任一项所述凋亡素 -EC-SOD羧 基末端蛋白转导域融合蛋白和可药学上接受的载体。
14、 权利要求 1一 4任一项所述凋亡素 -EC-SOD羧基末端蛋白转导域融合蛋白在制 备治疗由细胞增殖过度引起的疾病药物中的应用。
15、 如权利要求 14中所述的应用， 其特征在于所述由细胞增殖过度引起的疾病为 肿瘤。
16、 如权利要求 15中所述的应用， 其特征在于所述肿瘤可为子宫颈癌、 肝癌、 或 肺癌。
17、一种预防、 治疗或改善由细胞增殖过度引起的疾病或失调的方法， 所述方法包 括给需要所述预防、治疗或改善的哺乳动物施用患者有效剂量的权利要求 1一 4任一项 所述凋亡素 -EC-SOD羧基末端蛋白转导域融合蛋白。
18、 如权利要求 17中所述的方法， 其特征在于所述由细胞增殖过度引起的疾病为 肿瘤。
19、 如权利要求 18中所述的方法， 其特征在于所述肿瘤可为子宫颈癌、 肝癌、 或 肺癌。
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CN103897032A (zh) * 2012-12-28 2014-07-02 浙江日升昌药业有限公司 一种新型细胞穿膜肽
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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000044772A2 (en) 1999-01-30 2000-08-03 Delta Biotechnology Limited Human serum albumin
CN1683415A (zh) * 2004-04-13 2005-10-19 中国农业科学院蔬菜花卉研究所 转导肽－杀虫晶体蛋白融合蛋白及其对应序列与应用
CN1763093A (zh) * 2005-06-16 2006-04-26 华东理工大学 含有hiv转导结构域的生存素突变体及制备方法和应用
CN1908016A (zh) * 2006-08-24 2007-02-07 复旦大学 一类具有蛋白转导结构域tat－ptd的融合蛋白及其应用
CN101081871A (zh) * 2006-05-31 2007-12-05 华中科技大学 蛋白转导域4－凋亡素融合蛋白(PTD4－Apoptin)及其制法和应用
CN101081870A (zh) * 2006-05-31 2007-12-05 华中科技大学 一种抗肿瘤生物药物PTD4－GFP－Apoptin融合蛋白及其制备方法
CN101232895A (zh) * 2005-04-15 2008-07-30 亚利桑那生物医学研究委员会 用于治疗转移性癌症的治疗肽

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6162461A (en) * 1990-09-12 2000-12-19 Leadd B.V. Chicken anemia virus mutants and vaccines and uses based on the viral proteins VP1, VP2 and VP3 or sequences of that virus coding therefor
US6962696B1 (en) * 1999-10-04 2005-11-08 Vion Pharmaceuticals Inc. Compositions and methods for tumor-targeted delivery of effector molecules
WO2002085305A2 (en) * 2001-04-24 2002-10-31 Washington University Compositions and methods for inducing cancer cell death

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000044772A2 (en) 1999-01-30 2000-08-03 Delta Biotechnology Limited Human serum albumin
CN1683415A (zh) * 2004-04-13 2005-10-19 中国农业科学院蔬菜花卉研究所 转导肽－杀虫晶体蛋白融合蛋白及其对应序列与应用
CN101232895A (zh) * 2005-04-15 2008-07-30 亚利桑那生物医学研究委员会 用于治疗转移性癌症的治疗肽
CN1763093A (zh) * 2005-06-16 2006-04-26 华东理工大学 含有hiv转导结构域的生存素突变体及制备方法和应用
CN101081871A (zh) * 2006-05-31 2007-12-05 华中科技大学 蛋白转导域4－凋亡素融合蛋白(PTD4－Apoptin)及其制法和应用
CN101081870A (zh) * 2006-05-31 2007-12-05 华中科技大学 一种抗肿瘤生物药物PTD4－GFP－Apoptin融合蛋白及其制备方法
CN1908016A (zh) * 2006-08-24 2007-02-07 复旦大学 一类具有蛋白转导结构域tat－ptd的融合蛋白及其应用

Non-Patent Citations (10)

* Cited by examiner, † Cited by third party
Title
HJALMARSSON K, MARKLUND SL, ENGSTROM A ET AL., PROC NATL ACAD SCI USA., vol. 84, 1987, pages 6340 - 6344
HJALMARSSON, K. ET AL.: "Isolation and sequence of complementary DNA encoding human extracellular superoxide dismutase.", PROC. NATL. ACAD. SCI. USA., vol. 84, no. 18, 1987, pages 6340 - 6344, XP008148538 *
KARLSSON K, MARKLUND SL., LAB INVEST., vol. 60, 1989, pages 659 - 666
LARS, G ET AL.: "TAT-apoptin is efficiently delivered and induces apoptosis in cancer cells.", ONCOGENE, vol. 23, no. 5, 5 February 2004 (2004-02-05), pages 1153 - 1165, XP008148721 *
MARKLUND SL, BJELLE A, ELMQVIST LG ET AL., ANN RHEUM DIS., vol. 45, 1986, pages 847 - 851
NOTEBORN MH., VET MICROBIOL., vol. 98, no. 2, 2004, pages 89 - 94
SCHWARZE SR, DOWDY SF., TRENDS PHARMAEOL SCI., vol. 21, no. 2, 2000, pages 45 - 48
See also references of EP2380914A4
ZHU, X.Q. ET AL.: "A review of EC-SOD studies.", FOOD AND DRUG., vol. 7, no. 4A, 2005, pages 5 - 10 *
ZHUANG SM, SHVARTS A, VAN ORMONDT H, CANCER RES., vol. 55, no. 3, 1995, pages 486 - 489

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CN101747437A (zh) 2010-06-23
EP2380914A1 (en) 2011-10-26

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