WO2011072501A1 - 抗EB病毒诱发的肿瘤的融合多肽和大肠菌素Ia突变体 - Google Patents

抗EB病毒诱发的肿瘤的融合多肽和大肠菌素Ia突变体 Download PDF

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WO2011072501A1
WO2011072501A1 PCT/CN2010/070762 CN2010070762W WO2011072501A1 WO 2011072501 A1 WO2011072501 A1 WO 2011072501A1 CN 2010070762 W CN2010070762 W CN 2010070762W WO 2011072501 A1 WO2011072501 A1 WO 2011072501A1
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polypeptide
virus
tumor
colicin
gene
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PCT/CN2010/070762
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English (en)
French (fr)
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丘小庆
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畿晋庆三联（北京）生物技术有限公司
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Priority to ES10836940.6T priority Critical patent/ES2634944T3/es
Priority to EA201270655A priority patent/EA030638B1/ru
Priority to MX2012006952A priority patent/MX2012006952A/es
Priority to AP2012006327A priority patent/AP3535A/xx
Priority to SG2012041786A priority patent/SG181553A1/en
Priority to NZ600729A priority patent/NZ600729A/en
Priority to AU2010333588A priority patent/AU2010333588B2/en
Priority to CA2784784A priority patent/CA2784784C/en
Priority to US13/516,605 priority patent/US8883161B2/en
Priority to JP2012543448A priority patent/JP5683603B2/ja
Application filed by 畿晋庆三联（北京）生物技术有限公司 filed Critical 畿晋庆三联（北京）生物技术有限公司
Priority to BR112012017350-2A priority patent/BR112012017350A2/pt
Priority to EP10836940.6A priority patent/EP2514768B1/en
Priority to DK10836940.6T priority patent/DK2514768T3/en
Priority to UAA201208633A priority patent/UA104933C2/uk
Priority to KR1020127018638A priority patent/KR101464842B1/ko
Priority to MA35062A priority patent/MA33921B1/fr
Publication of WO2011072501A1 publication Critical patent/WO2011072501A1/zh
Priority to TNP2012000253A priority patent/TN2012000253A1/en
Priority to IL220296A priority patent/IL220296A/en
Priority to CU2012000096A priority patent/CU20120096A7/es
Priority to ZA2012/05217A priority patent/ZA201205217B/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
    • C07K16/085—Herpetoviridae, e.g. pseudorabies virus, Epstein-Barr virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)
    • 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
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • 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
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16211—Lymphocryptovirus, e.g. human herpesvirus 4, Epstein-Barr Virus
    • C12N2710/16222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the invention relates to the field of anti-tumor medicines, in particular to a novel anti-EB virus-derived tumor polypeptide and a method and a preparation method thereof.
  • a polypeptide molecule in which a colicin polypeptide is linked with a signal peptide molecule such as a globus globulin pheromone or a staphylococcal pheromone can recognize a target bacterial cell through a pheromone, and guide the colicin to a target bacterial cell membrane to form a transmembrane. Ion channels cause the cell contents to leak dead cells.
  • Malignant tumors are a huge threat to human health. There are about seven million people who die of malignant tumors every year in the world, of which China has one-sixth of them. Malignant tumors are the second cause of death in China. Because its etiology, pathogenesis and clinical manifestations have not yet been elucidated, the control effect is not satisfactory.
  • Existing anti-tumor drugs play an important role in tumor treatment. Although some tumors have achieved certain curative effects, there are still some defects in tumor cells, such as poor selectivity for tumor cells, serious and serious adverse reactions, and drug resistance.
  • Epstein-Barr virus-derived malignant lymphosarcoma in children all have specific Epstein-Barr virus surface antigens. Therefore, Epstein-Barr virus surface antigen can be considered as a specific marker of such tumor cells. .
  • the patent No. ZL200410081446.8 discloses an anti-tumor polypeptide formed by the combination of colicin and a mimetic antibody recognizing the surface antigen of Epstein-Barr virus, capable of specifically killing the Epstein-Barr virus in the body.
  • the cancer cells are caused by no damage to normal cells, and the lethality is several times that of other anticancer drugs, overcoming the anti-tumor used in recent years.
  • the drug has poor selectivity, produces drug resistance, kills cancer cells, and the normal tissues are also seriously damaged.
  • Xiao-Qing Qiu et al., 2007, compared a series of structural mimic antibodies with colicin. tumoricidal capacity antitumor polypeptide constituting found antitumor polypeptide analog antibodies with E.
  • the present invention provides a novel anti-EB virus-derived tumor polypeptide and a method for its application and preparation according to the deficiencies in the above-mentioned fields.
  • a novel anti-EB virus-derived tumor polypeptide which is formed by operatively linking a coliform allosteric polypeptide capable of forming an ion channel with an anti-EB virus antibody polypeptide or an anti-EB virus antibody mimetic antibody polypeptide, which can form an ion channel
  • the colicin allosteric polypeptide is obtained by mutating amino acid residues G11A, H22G, A26G, V31L and H40D from a peptide chain of wild type colicin El, Ia, Ib, A, B, N or an aqueous channel domain thereof,
  • the amino acid sequence of the anti-EBV antibody polypeptide is identical to the antibody polypeptide secreted by the ATCC HB-168 hybridoma.
  • the mock antibody polypeptide refers to a heavy chain CDR1 region of the anti-EB virus antibody, a heavy chain CDR1-CDR2 linking peptide segment, and a linker peptide of the light chain CDR3.
  • the endotoxin allosteric polypeptide that forms an ion channel is mutated from wild-type colicin la.
  • the novel anti-EB virus-derived tumor polypeptide has an amino acid sequence as shown in SEQ ID NO: 9.
  • a gene encoding the novel anti-EB virus-derived tumor polypeptide has the nucleotide sequence as shown in SEQ ID NO.
  • the method for preparing the tumor polypeptide caused by the novel anti-EB virus is as follows: The recombinant plasmid is transformed into an expression system for expression, and the expressed polypeptide is isolated and purified.
  • a gene encoding an allosteric polypeptide of colicin la is a gene encoding an allosteric polypeptide of colicin la.
  • the use of the gene in the preparation of a polypeptide drug means that the gene is operably linked to a gene encoding the polypeptide and cloned into an expression vector, and then the expression vector is transformed into an expression system, and the expressed polypeptide is isolated and purified.
  • the novel anti-EB virus-derived tumor polypeptide provided by the present invention comprises a migradin allosteric polypeptide capable of forming an ion channel and an anti-EB virus antibody polypeptide or an anti-EB virus antibody mimetic antibody polypeptide. Due to the presence of amino acid residues in the wild colicin polypeptide molecule which are likely to cause hypersensitivity reactions, the present invention selectively mutates the hydrophobic segment portion of the polypeptide molecule capable of forming an ion channel structure of the coliform structure to cause hypersensitivity of the body.
  • the polypeptide mutation positions of colicin la are: G11A, H22G, A26G, V31L and H40D
  • experimental data indicate that colicin la polypeptide and allosteric
  • the la peptide was injected into the immunized mice separately.
  • the mice injected with allosteric la had a serum titer that was one to two orders of magnitude lower than the former, ie, the immune response level was low, which proved that the allosteric polypeptide reduced the possibility of causing allergies.
  • Simultaneous; allosteric polypeptides still retain the function of forming transmembrane ion channels on the cell membrane.
  • the recombinant polypeptide of the present invention has no effect on the killing ability of tumor cells, indicating that the mutant amino acid residues have no effect on colicin formation.
  • the anti-EB virus antibody polypeptide or the anti-EB virus antibody mimic polypeptide is recognized by the surface specific antigen of the tumor cell caused by the Epstein-Barr virus, and the colicin is deformed.
  • the polypeptide is directed onto the target cell membrane, and the hydrophobic segment of the transmembrane ion channel domain of the colicin allosteric polypeptide is inserted into the tumor cell membrane to form an ion channel, and the target tumor cell is lethal due to leakage of the content; the anti-EB virus antibody polypeptide
  • the amino acid sequence is completely based on the amino acid sequence of the polypeptide secreting monoclonal antibody of ATCC HB-168 hybridoma cells.
  • the mimetic antibody polypeptide of the above anti-EB virus antibody is operably linked in the embodiment of the present invention.
  • the anti-tumor polypeptide of the present invention having a smaller molecular weight is obtained at the carboxy terminus of the colicin allosteric polypeptide, that is, the small molecule mimetic polypeptide includes only the VH CDR1 region, the VLCDR3 region of the anti-EBV antibody polypeptide, and VH
  • the CDR1-V H CDR2 linking peptide and the light chain VLCDR3 region-ligated peptide chain V H CDR1-V H FR 2 - V L CDR3 gives a mimetic antibody of the novel anti-tumor polypeptide 1 having the amino acid sequence shown in SEQ ID NO.
  • the mimetic antibody contains less than 30 amino acids and is much smaller than the natural antibody 150 amino acids in molecular weight, which not only meets the requirements for antigen recognition ability, but also greatly reduces the molecular weight of the anti-tumor polypeptide, and is beneficial to enhance the anti-tumor polypeptide of the present invention. Penetration ability in the organization.
  • Another object of the invention is to provide a gene sequence encoding an anti-tumor polypeptide of the invention.
  • the gene of the anti-tumor polypeptide of the present invention is operably linked by a gene encoding a colicin allosteric polypeptide and a gene encoding an anti-EB virus antibody polypeptide or a gene simulating the antibody polypeptide thereof, wherein the colicin polypeptide and the anti-EB virus antibody
  • the gene sequence is known, and the gene of the colicin allosteric polypeptide is obtained by mutation of the corresponding codons of the colicin polypeptide gene as follows: G11A, H22G, A26G, V31L and B H40D. Due to the combinatoriality of the codons, one skilled in the art can adjust the nucleotide sequence encoding the anti-tumor polypeptide of the present invention without changing the amino acid sequence.
  • the recombinant plasmid of the present invention refers to a double-stranded nucleotide point mutation of a primary vector carrying a wild coenzyme gene, and a mutation codon is inserted at a target mutation position to obtain a gene containing a colicin allosteric polypeptide.
  • the mutant vector inserts a mock antibody gene encoding an antibody against Epstein-Barr virus into the carboxy terminus of the colicin allosteric polypeptide gene to obtain a recombinant plasmid of the present invention.
  • the original vector P SELECTTM-1 was purchased from Promega, which was loaded with the colicin la and Immunity protein genes, and the point mutation was performed according to the Stmtegene kit instructions.
  • the present invention is directed to a point mutation to prepare a colicin allosteric polypeptide, wherein the mutation is Five codons were designed, so five pairs of primer sequences (Seq ID No. 1 to 10) were designed; in the examples of the present invention, six pairs of primer sequences (Seq ID No. 11-22) were designed for the mock antibody gene.
  • the invention also provides a method for preparing the anti-tumor polypeptide of the invention, wherein the recombinant plasmid obtained above is transfected into Escherichia coli BL21 (DE3) engineering bacteria, and the positive clone is screened, and the protein expressed by the positive clone is isolated and purified.
  • a tumor polypeptide derived from the novel anti-EB virus of the present invention is obtained.
  • novel anti-EB virus-derived tumor polypeptide provided by the invention can be applied in the preparation of a medicament for treating or preventing tumor caused by Epstein-Barr virus.
  • a clinically applicable drug combination can be prepared by adding a polypeptide of the novel antibiotic obtained in the present invention to a pharmaceutically acceptable carrier or excipient or optionally other ingredients. Things.
  • the present invention also provides an amino acid sequence and a gene sequence of a colicin la allosteric polypeptide, which can be used not only in the present invention, but also can constitute an antibody polypeptide with other guiding polypeptides, and the experimental data of the third embodiment of the present invention is It has been proved that the polypeptide drug containing the allosteric polypeptide has lower antigenicity, and the allosteric polypeptide and other guiding polypeptides are also proved to have bactericidal ability, and the preparation method is a routine experimental operation in the art.
  • the novel anti-tumor polypeptide provided by the invention has the advantages of the anti-tumor polypeptide disclosed by the invention of Patent No. ZL200410081446.8, that is, the high specificity of targeting and the safety to normal cells, and the resistance to chemicals are not easily obtained.
  • the anti-tumor polypeptide of the present invention reduces the immunogenicity of the anti-tumor polypeptide containing the allosteric polypeptide by mutating the amino acid residue which is susceptible to hypersensitivity in the colicin polypeptide, that is, reducing the allergic reaction.
  • FIG. 1 Schematic diagram of the recombinant plasmid pCHCEB11 containing the gene of the mimetic antibody polypeptide V H CDR1-V H FR 2 - V L CDR3 and the colicin la allosteric polypeptide gene
  • FIG. 1 Schematic representation of the recombinant plasmid PCHCEB22 containing the gene for the mimetic antibody polypeptide V H CDR1- V H FR 2 - (Rev) V L CDR3 and the colicin la allosteric polypeptide gene
  • Figure 8 Killing effect of new anti-tumor peptides on EB virus-induced solid tumors grown in Burkitt's human malignant lymphosarcoma cells in nude mice.
  • A control EBV-negative lymphosarcoma sections of mice
  • B control EBV-positive lymphosarcoma sections of control mice
  • C treatment of murine EBV-negative lymphosarcoma sections with novel anti-tumor polypeptide 1
  • D novel anti-tumor Peptide 1 was treated with a mouse EBV virus-positive lymphosarcoma section.
  • Example 1 Construction of a recombinant plasmid containing the gene encoding the mutant colicin la
  • Original plasmid is loaded as the original plasmid pSELECT la colicin polypeptide and TM -l immunity protein gene plasmid (8.3 kb) (purchased from Promega Corporation), the double-stranded oligonucleotide point mutagenesis (QuickChange TM Kit, A gene encoding a mutant amino acid, such as the oligonucleotide primer sequence shown in SEQ ID NO 1 -10, is operably linked to the gene of wild-type colicin la, respectively, to obtain a SEQ ID NO: 3 A gene encoding a coenzyme la allosteric polypeptide is obtained, and a mutant plasmid is obtained; and the gene encoding the mimetic antibody SEQ ID N0.26 or SEQ ID N0.28 is inserted into the gene of the colicin la allosteric polypeptide in the mutant plasmid.
  • pCHCEB ll shown in Figure 1
  • pCHCEB22 shown in Figure 2
  • the six oligonucleotide primer sequences designed for the mimetic antibody gene of the antibody are set forth in SEQ ID N011-22.
  • the recombinant plasmid was transfected into E.
  • SEQ ID N0.25 (referred to as "a novel anti-tumor polypeptide” and SEQ in the following examples) was obtained.
  • ID N0.27 (referred to as “new anti-tumor polypeptide 2" in the following examples) is shown.
  • the double-stranded oligonucleotide point mutation program was carried out according to the Strategene QuickChang Site-Directed Mutagenesis Kit (catalog #200518) kit.
  • the primer sequences designed for point mutation are as follows:
  • Seq ID NO. 3 oligo primer designed by H22G in the mutant colicin gene 5 ' -3 '
  • Seq ID NO. 7 oligo primer designed by V31L in the mutant colicin gene 5' -3'
  • Seq ID NO. 8 The oligomeric primer designed by V31L in the mutant colicin gene 3 ' -5,
  • Seq ID NO. 11 recombinant plasmid pCHCEBl l V CDR1 gene primer 5, -3,
  • Seq ID NO. 14 recombinant plasmid pCHCEBl l V H FR 2 gene primer 3' -5,
  • Gcc tgt ctt ata ttt tat tta GGC CAC CCA CTC CAG ACCT TTT CTC GGG
  • Seq ID NO. 18 Recombinant plasmid pCHCEB22 V CDR1 gene primer 3, -5,
  • Ggt atg cat tgg gtg cgt cag GCC CCC GAG AAA GGT CTG GAG TGG GTG GCC taa ataaaa tat aag aca ggc
  • Seq ID NO. 20 recombinant plasmid pCHCEB22 in V H FR 2 gene primer 3' -5,
  • Gcc tgt ctt ata ttt tat tta GGC CAC CCA CTC CAG ACCT TTT CTC GGG
  • Seq ID NO. 21 recombinant plasmid pCHCEB22 in V L CDR3 gene primer 5' -3
  • Seq ID NO. 22 recombinant plasmid pCHCEB22 in V L CDR3 gene primer 3' -5
  • novel anti-tumor polypeptide 1 prepared by the recombinant plasmids pCHCEB11 and pCHCEB22, the novel anti-tumor polypeptide 2, and the anti-tumor polypeptide 1 and the anti-tumor polypeptide 2 (ZL200410081446.8) in the inventor's prior patent were prepared in the same manner as in the first embodiment.
  • Immunized mice After mixing the above proteins with an adjuvant, the basic amount and the additional amount were intraperitoneally injected once every 50 ⁇ ⁇ (0.5 ml). Two weeks apart, 5 needles were immunized. The ELISA indirect method was used to detect the serum titer of mice.
  • the serum titer of the immunized mice immunized with the novel anti-tumor polypeptides 1 and 2 prepared by the invention was 10 - 3 to 10 - 4 and the anti-tumor polypeptide 1 and the anti-tumor polypeptide 2 were immunosuppressed.
  • the serum titer of the mouse is 10 - 4 to 10 - 5 .
  • novel anti-tumor polypeptides of the present invention are less likely to cause a host sensitization reaction than the anti-tumor polypeptide comprising wild-type coeniform la to be one to two orders of magnitude less likely to cause a host sensitization reaction.
  • Example 3 Low sensitization effect of colicin la allosteric polypeptide constituting novel antitumor polypeptide
  • the S. aureus pheromone AgrDl (YSTCDFIM) was ligated to prepare two antibacterial polypeptides.
  • the polypeptide prepared by linking AgrDl to the carboxyl terminus of allosteric colicin is named as anti-Golden Peptide 1, and AgrDl is ligated to the amino terminus of allosteric laminin.
  • the polypeptide is named as Pseudomonas aeruginosa.
  • a plasmid of wild-type colicin la was prepared, and a S. aureus pheromone AgrD1 was ligated to the amino terminus thereof to prepare an anti-Pseudomonas aeruginosa polypeptide 2.
  • Experiment 1 A batch of Kunming mice were injected intraperitoneally with a lethal dose of methicillin-resistant Staphylococcus aureus (ATCC BAA42) randomly divided into (1) control group, (2) ampicillin group, and (3) anti-S. aureus polypeptide. group,
  • Anti-S. aureus polypeptide 1 group There were 10 mice in each group.
  • Anti-S. aureus polypeptide group Intravenous injection of the anti-S. aureus polypeptide invented by the inventors (ZL 01128836.1) 6 mg/kg once;
  • Anti-S. aureus polypeptide group 1 Intravenous injection of anti-S. aureus peptide 1 6 mg / kg - times.
  • Experiment 2 After the 14th day of experiment 1, a batch of Kunming mice were used as control and ampicillin group, anti-S. aureus polypeptide group and anti-S. aureus polypeptide group 1 surviving mice as anti-S. aureus polypeptide group and anti-gold The above test was repeated for the Glucopolypeptide 1 group. The control group and the ampicillin group all died within two days. In the anti-S. aureus peptide group, 75 % of the mice survived, and 90% of the anti-S. aureus polypeptide group 1 survived.
  • Experiment 3 41 days after experiment 1, a batch of Kunming mice were used as a control group, levofloxacin group and ceftriaxone sodium group, anti-S. aureus polypeptide group and anti-S. aureus polypeptide group 1 surviving mice as Pseudomonas aeruginosa Group 2 of polypeptide and group 1 against Pseudomonas aeruginosa.
  • control group was injected with 0.5 ml of 0.3 M NaCl + 50 mM boric acid buffer once in the tail vein.
  • Levofloxacin group was injected with levofloxacin 5 mg/kg once in the levofloxacin group.
  • Ceftriaxone sodium 30 mg/kg-time in the ceftriaxone sodium group
  • Pseudomonas aeruginosa peptide 2 group was injected intravenously with Pseudomonas aeruginosa peptide 2 8 mg/kg once,
  • Anti-Pseudomonas aeruginosa polypeptide 1 group was injected intravenously with Pseudomonas aeruginosa peptide 1 8 mg/kg once. The control group and the levofloxacin group all died in one day. In the ceftriaxone sodium group, 25% of the mice survived. 60% of the mice in the anti-P. aeruginosa polypeptide group survived. All of the mice in the group 1 survived, indicating that the interference of the host antibody on the killing effect of the allosteric polypeptide was lower than that of the wild type polypeptide.
  • the sera of the above-mentioned anti-S. aureus polypeptide/anti-Pseudomonas aerugin polypeptide 2 group and anti-S. aureus polypeptide 1/anti-Pseudomonas aerugin polypeptide group 1 were subjected to ELISA at the first week, the second week and the 7th week of the test, respectively.
  • the indirect method was used to detect the antibodies in the blood.
  • the wells of the microplate were coated with wild type coenzyme la and colicin la allosteric polypeptide, 100 ng/well, and the primary antibody was anti-S. aureus polypeptide/anti-green. Pseudomonas polypeptide 2 group and anti-S.
  • aureus polypeptide 1 / anti-Pseudomonas aerugin polypeptide 1 group of surviving mouse serum, 2 anti-goat anti-mouse labeled antibody, negative control with 5% milk-PBS as primary antibody, 1:50,000 drops The results obtained are as follows (see Figure 4)
  • A (anti-Golden Peptide/Anti-Phosin Peptide 2) (Anti-Golden Peptide 1 / Anti-Phospy Peptide 1) 1 (1st week) 0.914 0.254
  • colicin la allosteric polypeptide prepared by the present invention is less likely to cause a host sensitization reaction than the wild type colicin la to cause a host sensitization reaction;
  • Example 4 In vitro killing effect of a novel anti-tumor polypeptide against EBV-induced human lytic lymphoma (Burkitt's lymphoma)
  • Epstein-Barr virus positive and negative cell lines American ATCC standard cell line.
  • test cells were divided into 3 groups, the first group was a blank group, that is, the anti-tumor polypeptide blank preservation solution was added.
  • the second group was added 20 (Vg/ml of novel anti-tumor polypeptide 1 (plasmid was pCHCEBll, preservation solution was lOmMPB + 0.2M NaCI phosphate buffer pH 7.4).
  • the third group was added 20 (Vg/ml of novel anti-tumor polypeptide 2 (plasmid was pCHCEB22, preservation solution was lOmMPB + 0.2 M NaCI phosphate buffer pH 7.4).
  • EBV-positive cell line ATCC CCL-86 (Raji cells, Burkitt's malignant lymphosarcoma cells); ATCC CRL-2230, 46-year-old male AIDS patient (AIDS) malignant lymphosarcoma cell line, prion and Kaposi sarcoma virus (HHV8) was positive; EBV-negative cell line: ATCC CRL-1648 (CA-46, cells isolated from ascites from US-type Burkitt's patients with malignant lymphosarcoma).
  • ATCC CCL-86 Raj cells, Burkitt's malignant lymphosarcoma cells
  • ATCC CRL-2230 46-year-old male AIDS patient (AIDS) malignant lymphosarcoma cell line, prion and Kaposi sarcoma virus (HHV8) was positive
  • EBV-negative cell line ATCC CRL-1648 (CA-46, cells isolated from ascites from US-type Burkitt's patients with malignant lymphosarcoma).
  • the first group was a new anti-tumor polypeptide blank preservation solution (10mMPB+0.2M NaCI phosphate buffer (pH7.4).
  • the second group was added with 20 (Vg/ml new anti-antibody).
  • Tumor polypeptide 1 (plasmid is pCHCEBll)
  • the preservation solution is lOmMPB + 0.2M NaCI phosphate buffer ⁇ 7 ⁇ 4.
  • Epstein-Barr virus-negative tumor cells grew well, indicating that the novel anti-tumor polypeptide does not attack cells that do not carry Epstein-Barr virus surface antigen on the cell membrane. This suggests that the novel anti-tumor polypeptides of the invention have desirable targeting specificities and safety.
  • Example 6 Killing effect of a novel anti-tumor polypeptide on solid tumors grown in nude mice by Burk et's human malignant lymphosarcoma cells induced by Epstein-Barr virus
  • SCID immunodeficient mice were purchased from the Shanghai Experimental Animal Center of the Chinese Academy of Sciences. The rats were fed as standard. The water, bedding and the feeder were sterilized by high temperature or ultraviolet light. The animals were incubated for a week under relatively sterile conditions and inoculated without abnormalities.
  • Group B New anti-tumor polypeptide 1 (plasmid is pCHCEBll) treatment group, 300 ⁇ ⁇ / mouse (calculated in 25 g) / day, for 20 consecutive days;
  • Each group of 10 tumor-bearing mice was intraperitoneally injected twice a day, 0.5 ml each time, and continued for 20 days. .
  • the activity of the rats was observed daily and recorded, and the tumor size was accurately measured and photographed the next day.
  • Example 6 The mice were sacrificed after the experiment was over, and the tumors were removed and fixed in 10% formalin, paraffin sections, and HE staining were observed by conventional light microscopy.
  • the solid tumors of the control group were proliferated vigorously; the EBV virus-positive solid tumor cells of the new anti-tumor peptide group were significantly reduced, and the tumor cell clusters in the specimens were almost necrotic tumor cells, and a large number of lymphocytes were observed in the periphery. infiltration. Histopathological results suggest that the new anti-tumor polypeptide almost killed all tumor cells in solid tumors during the 20-day treatment period (see Figure 8, D).

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Description

说明书 抗 EB病毒诱发的肿瘤的融合多肽和大肠菌素 la突变体 技术领域
本发明涉及抗肿瘤药物领域， 特别涉及一种新型抗 EB病毒所致肿瘤多肽及 其应用与制备方法。
背景技术
在抗生素研究领域，人们一直在致力于开发模仿同种异株细菌之间互相杀伤 的工作方式来开发新型抗生素，自然界中有不少细菌毒素直接在细菌胞膜上形成 离子通道来杀死细菌， 其模式标本就是大肠杆菌分泌的一种细菌毒素一大肠菌 素。 其中大肠菌素 la自 1952年被 Jacob发现之后， 经过数代人的努力， 1996年 Qiu et al (Major transmemebrane movement soociated with colicin la channel gating. J. Gen. Physiology, 107:313-328 (1996) ) 终于揭示了大肠菌素 la在人工脂质双分 子膜上所形成离子通道开放和关闭时的跨膜立体结构，为在分子水平上设计和制 备新型的抗菌素奠定了理论基础。 并相继有大肠菌素多肽与白色连珠球菌信息 素、葡萄球菌信息素等信号肽分子连接而成的多肽分子能够通过信息素识别靶细 菌细胞，将大肠菌素引导到靶细菌细胞膜上形成跨膜离子通道导致胞容物外泄致 死细胞。
恶性肿瘤是对人类健康的巨大威胁。全世界每年死于恶性肿瘤的患者约有七 百万人， 其中中国就占有六分之一， 恶性肿瘤已是我国第二位的死亡原因。 由于 其病因、 发病机制、 临床表现尚未阐明， 故防治效果不理想。 现有抗肿瘤药物在 肿瘤治疗中占重要地位， 虽然对一些肿瘤已取得一定的疗效， 但仍存在着对肿瘤 细胞的选择性差， 免疫抑制及不良反应多而严重， 产生耐药性等缺陷。
EB病毒所致的非洲儿童恶性淋巴肉瘤， Hodgkin's淋巴肉瘤和鼻咽癌细胞表 面均带有特异的 EB病毒表面抗原， 因此， EB病毒表面抗原可以认为是该类肿 瘤细胞的一种特异性标志物。 在 EB 病毒所致的肿瘤药物上， 专利号为 ZL200410081446.8的发明，公开了大肠菌素与识别 EB病毒表面抗原的模拟抗体 结合形成的抗肿瘤多肽， 能够特异性杀死机体中的 EB病毒所致癌细胞， 而对正 常细胞没有伤害， 杀伤力是其他抗癌药物的若干倍， 克服了近年来使用的抗肿瘤 药物中存在的药物选择性差、产生耐药性、杀死癌细胞的同时正常组织也受到严 重损伤等问题， Xiao-Qing Qiu等， 2007的文章中对比了一系列结构的模拟抗体 与大肠菌素构成的抗肿瘤多肽的杀肿瘤的能力， 发现 VHCDR1- VHFR2-VLCDR3 及 VLCDRI- VHFR2-VHCDR3两种结构的模拟抗体与大肠菌素构成的抗肿瘤多肽 具有较好的杀伤能力 （Xiao-Qing Qiu et al.， 2007, Small antibody mimetics comprising two complementarity-determining regions and a framework region for tumor targeting, Nature Biotechnology 25, 921-929, 1 August 2007 ) ,这为进一步制 备抗 EB病毒所致肿瘤多肽提供了更多的候选模拟抗体。
但是在上述抗肿瘤多肽中，由于大肠菌素多肽的熟睡端存在较易引起超敏反 应的氨基酸残基， 有可能导致含有大肠杆菌多肽的药物较易引起机体异常的免 疫应答； 有研究报道， 很多癌症患者由于其代谢机制受到癌细胞干扰而不正常， 对多肽类药物容易产生过敏反应， 而导致不能用药， 因此有必要对大肠菌素多肽 进行改进， 以获得更安全适合更多患者的抗癌药物。
发明内容
本发明根据上述领域存在的不足， 提供一种新型抗 EB病毒所致肿瘤多肽及 其应用与制备方法。 为治疗 EB病毒所致肿瘤提供高杀伤力、 高特异性， 且致敏 可能性较低的药物。
一种新型抗 EB病毒所致肿瘤多肽， 由可形成离子通道的大肠菌素变构多肽 与抗 EB病毒抗体多肽或者抗 EB病毒抗体的模拟抗体多肽可操作地连接构成， 所述可形成离子通道的大肠菌素变构多肽由野生型大肠菌素 El、 Ia、 Ib、 A、 B、 N或其水性孔道结构域的肽链突变了氨基酸残基 G11A、 H22G、 A26G、 V31L和 H40D而获得，所述抗 EB病毒抗体多肽的氨基酸序列与 ATCC HB-168杂交瘤分 泌的抗体多肽相同。
所述模拟抗体多肽指所述抗 EB病毒抗体的重链 CDR1区、重链 CDR1-CDR2 连接肽段和轻链 CDR3的连接肽。
所述可形成离子通道的大肠菌素变构多肽由野生型大肠菌素 la突变而来。 所述新型抗 EB病毒所致肿瘤多肽， 具有如 SEQ ID N0.29所示的氨基酸序 列。
编码所述新型抗 EB病毒所致肿瘤多肽的基因。 所述基因， 具有如 SEQ ID NO.30所示的核苷酸序列。
含有上述基因的重组质粒。
上述新型抗 EB病毒所致肿瘤多肽的制备方法， 步骤如下： 将上述重组质粒 转化到表达系统中进行表达， 分离纯化表达的多肽。
上述新型抗 EB病毒所致肿瘤多肽在制备治疗和预防 EB病毒所致肿瘤的药 物中的应用。
一种大肠菌素 la的变构多肽， 其氨基酸序列如 SEQ ID N0.24所示。
编码大肠菌素 la的变构多肽的基因。
所述基因在制备多肽药物中的应用，指将所述基因与诱导多肽的基因可操作 地连接并克隆到表达载体中，然后将表达载体转化到表达系统中， 分离纯化表达 的多肽
本发明提供的新型抗 EB病毒所致肿瘤多肽， 由可形成离子通道的大肠菌素 变构多肽与抗 EB病毒的抗体多肽或者抗 EB病毒的抗体的模拟抗体多肽构成。 由于野生大肠菌素多肽分子中存在容易引起超敏反应的氨基酸残基，本发明在可 形成离子通道结构的大肠菌素的多肽分子中，选择性地突变了疏水区段部分容易 引起机体超敏反应的氨基酸残基， 如本发明的一个优选实施例中， 大肠菌素 la 的多肽突变位置为： G11A、 H22G、 A26G、 V31L和 H40D， 实验数据表明， 采 用大肠菌素 la多肽和变构的 la多肽分别注射免疫小鼠，注射变构的 la的小鼠产 生的血清效价要比前者低 1~2个数量级， 即免疫应答水平较低，证明变构的多肽 降低了引起机体过敏的可能性；同时变构多肽仍然保留了在细胞膜上形成跨膜离 子通道的功能， 实验证明， 本发明的重组多肽对肿瘤细胞的杀伤能力未受影响， 说明突变的氨基酸残基没有影响大肠菌素形成离子通道的功能。本发明提供的新 型抗 EB病毒所致肿瘤多肽中， 抗 EB病毒的抗体多肽或者抗 EB病毒的抗体的 模拟多肽通过与 EB病毒所致的肿瘤细胞的表面特异性抗原识别， 大肠菌素变构 多肽引导至靶细胞膜上，大肠菌素变构多肽的跨膜离子通道结构域疏水区段插入 肿瘤细胞膜中， 进而形成离子通道， 靶肿瘤细胞因内容物泄漏而被致死； 抗 EB 病毒的抗体多肽的氨基酸序列完全参照 ATCC HB-168杂交瘤细胞分泌单克隆抗 体的多肽的氨基酸序列。
本发明的实施例中优选采用上述抗 EB病毒抗体的模拟抗体多肽可操作地连 接在大肠菌素变构多肽的羧基端而获得分子量较小的本发明的抗肿瘤多肽，即这 些小分子模拟多肽仅包括该抗 EB病毒的抗体多肽的 VHCDR1区、 VLCDR3区， VHCDR1-VHCDR2 连接肽段和轻链 VLCDR3 区连接的肽链 VHCDR1- VHFR2- VLCDR3得到氨基酸序列如 SEQ ID N0.25所示的新型抗肿瘤多肽 1的模拟抗体， 模拟抗体仅包含不到 30个氨基酸，在分子量上远远小于天然抗体 150个氨基酸， 既达到了对抗原识别能力的要求又大幅度消减了抗肿瘤多肽的分子量，有利于提 高本发明抗肿瘤多肽在组织中的穿透能力。
本发明的另一目的是提供编码本发明所述抗肿瘤多肽的基因序列。本发明抗 肿瘤多肽的基因由编码大肠菌素变构多肽的基因与编码抗 EB病毒的抗体多肽的 基因或者其模拟抗体多肽的基因可操作地连接构成， 其中大肠菌素多肽和抗 EB 病毒抗体的基因序列是已知的，大肠菌素变构多肽的基因是在大肠菌素多肽基因 的相应密码子上进行如下点突变而得： G11A、 H22G、 A26G、 V31L禾 B H40D。 由于密码子的兼并性，本领域技术人员可在不改变氨基酸序列的前提下对编码本 发明所述的抗肿瘤多肽的核苷酸序列进行调整。
本发明所述的重组质粒，是指将装载了野生大肠菌素基因的原始载体进行双 链核苷酸点突变，在目标突变位置上插入突变密码子， 获得含有大肠菌素变构多 肽的基因的突变载体， 相同的点突变技术将编码抗 EB病毒的抗体的模拟抗体基 因经插入到大肠菌素变构多肽基因的羧基端，得到本发明的重组质粒。原始载体 PSELECT™-1购于 Promega公司， 其中装载了大肠菌素 la和 Immunity蛋白基 因， 点突变操作按照 Stmtegene公司试剂盒说明书； 本发明针对点突变制备大肠 菌素变构多肽， 其中突变了 5 个密码子， 因此设计了 5 对引物序列 （Seq ID No.l~10 )； 本发明实施例中针对模拟抗体基因设计了 6 对引物序列 （Seq ID No.ll~22 )。
本发明还提供了制备本发明所述抗肿瘤多肽的方法， 是将上述获得的重组质 粒转染入大肠杆菌 BL21(DE3)工程菌中，筛选出阳性克隆， 分离纯化阳性克隆表 达的蛋白即能得到为本发明新型抗 EB病毒所致肿瘤多肽。
本发明提供的新型抗 EB病毒所致肿瘤多肽， 可在制备治疗或预防 EB病毒 所致肿瘤的药物中应用。可以通过将本发明中获得的新型抗生素的多肽添加到药 学上可接受的载体或者赋形剂或可选的其它成分而制成临床上适用的药物组合 物。
本发明还提供了大肠菌素 la变构多肽的氨基酸序列和基因序列， 该变构多 肽不仅可用于本发明中， 还可以与其他引导多肽构成抗体多肽， 本发明实施例 3 的实验数据一方面证明了含有该变构多肽的多肽药物具有较低的抗原性，也证明 该变构多肽与其他引导多肽构成抗体多肽具有杀菌能力，而制备方法是本领域的 常规实验操作。
本发明提供的新型抗肿瘤多肽， 具有专利号 ZL200410081446.8的发明所公 开的抗肿瘤多肽的优点， 即高特异性的靶向性和对正常细胞的安全性， 不易产生 耐药性等优点，同时本发明的抗肿瘤多肽通过对大肠菌素多肽中易引起超敏反应 的氨基酸残基进行了突变， 降低了含该变构多肽的抗肿瘤多肽的免疫原性， 即降 低了引起过敏反应的可能性，改进了现有该类多肽的药物使用安全性和杀肿瘤效 果， 还为其他含大肠菌素多肽的药物提供了改进的参考。 附图说明
图 1.含有模拟抗体多肽 VHCDR1- VHFR2- VLCDR3的基因和大肠菌素 la变构多 肽基因的重组质粒 pCHCEBll的结构示意图
图 2.含有模拟抗体多肽 VHCDR1- VHFR2- (Rev) VLCDR3的基因和大肠菌素 la 变构多肽基因的重组质粒 PCHCEB22的结构示意图
图 3 .大肠菌素 la变构多肽的致敏效果实验 1
(A) 昆明小鼠腹腔注射致死剂量的耐甲氧西林金葡菌 （ATCC BAA42) , 随 机分组为 （1 ) 对照组， （2) 氨苄青霉素组， （3) 抗金葡菌多肽 (ZL 01128836.1) 组， （4) 抗金葡菌多肽 1组。
(B) 14天后， 再取昆明小鼠一批作为对照和氨苄青霉素组， 抗金葡菌多肽组 及抗金葡菌多肽 1组存活鼠作为抗金葡菌多肽组及抗金葡菌多肽 1组重复上述试 验。
(C) 41 天后， 再取昆明小鼠一批作为 （1) 对照组、 （2) 左氧氟沙星组和 （3) 头孢曲松钠组，（4) 抗金葡菌多肽组及 （5) 抗金葡菌多肽 1组存活鼠作为抗绿脓 杆菌多肽 2组及抗绿脓杆菌多肽 1组。。
图 4.大肠菌素 la变构多肽的低致敏效果实验 2 (A) 抗金葡菌多肽 /抗绿脓杆菌多肽 2组血清， 1:50,000 滴度；
(B) 抗金葡菌多肽 1/抗绿脓杆菌多肽 1组血清， 1:50,000 滴度。
( 1 ) 第一周， （2) 第二周， （3) 第 7周血清。 （4) 阴性对照。 图 5. 新型抗肿瘤多肽对 EB病毒所致人恶性淋巴肉瘤 (Burkitt's lymphoma)体外 杀伤效果比较
(A) 对照组， （B) 新型抗肿瘤多肽 1处理组， （C) 新型抗肿瘤多肽 2处理组。 图 6. 新型抗肿瘤多肽对 EB病毒所致 Burkitt's人恶性淋巴肉瘤细胞和其它肿瘤 细胞的体外杀伤作用
(A) EBV病毒阳性的 Burkitt's 人恶性淋巴肉瘤细胞，
(B) EBV病毒阴性的 Burkitt's 人恶性淋巴肉瘤细胞，
(C) EBV病毒阳性的爱滋病人 （AIDS) 恶性淋巴肉瘤细胞。
( 1 ) 对照组， （2) 新型抗肿瘤多肽 1处理组。。 图 7 新型抗肿瘤多肽对 EB病毒所致 Burkitt's人恶性淋巴肉瘤细胞种植到裸鼠体 内生长出的实体肿瘤的杀伤作用
(A)对照组 。
(B) 新型抗肿瘤多肽 1处理组的 SCID免疫缺陷鼠，鼠双腋下均接种 Burkitt's 人恶性淋巴肉瘤细胞。 左边箭头， EBV病毒阴性的淋巴肉瘤， 右边箭头， EBV 病毒阳性的淋巴肉瘤。
图 8 新型抗肿瘤多肽对 EB病毒所致 Burkitt's人恶性淋巴肉瘤细胞种植到裸鼠体 内生长出的实体肿瘤的杀伤作用
(A)对照鼠 EBV病毒阴性的淋巴肉瘤切片， （B) 对照鼠 EBV病毒阳性的淋巴 肉瘤切片， (C) 新型抗肿瘤多肽 1处理鼠 EBV病毒阴性的淋巴肉瘤切片， (D) 新 型抗肿瘤多肽 1处理鼠 EBV病毒阳性的淋巴肉瘤切片。 具体实施方式
结合附图， 通过对本发明较佳实施例的描述具体说明本发明。 本发明所用的原始质粒： pSELECTTM-l购于 Promega公司。
工程菌： E.coW BL-21(DE3) 工程菌， 购于 Novagen公司。
实施例 1. 构建含编码突变大肠菌素 la的基因的重组质粒
原始质粒为原始质粒为装载了大肠菌素多肽 la和 immunity 蛋白基因的 pSELECTTM-l质粒 （8.3 kb ) (购于 Promega公司）， 经双链寡聚核苷酸点突变技 术（QuickChange™Kit， Strategene公司）分别将编码突变氨基酸的基因如： SEQ ID NO 1 -10所示的寡聚核苷酸引物序列与野生型大肠菌素 la的基因可操作地连 接， 获得如 SEQ ID N0.23所示编码大肠菌素 la变构多肽的基因， 同时得到突变 质粒； 再将编码模拟抗体的基因 SEQ ID N0.26或 SEQ ID N0.28插入到突变质 粒中大肠菌素 la变构多肽的基因的 1626位密码子之后， 制备出到新型抗 EB病 毒所致肿瘤多肽的两种重组质粒 pCHCEB ll (如图 1所示）， pCHCEB22 (如图 2所示），为制备重组质粒中编码抗 EB病毒抗体的模拟抗体基因所设计的 6条寡 聚核苷酸引物序列如 SEQ ID N011-22所示。 重组质粒转染入 E.coli BL21(DE3) 工程菌 （购于 Novagen公司） 里制备多肽， 获得序列表中 SEQ ID N0.25 (在后 面的实施例中称为"新型抗肿瘤多肽 )和 SEQ ID N0.27 (在后面的实施例中称 为"新型抗肿瘤多肽 2" ) 所示。
双链寡聚核苷酸点突变程序按 Strategene QuickChang Site-Directed Mutagenesis Kit(catalog#200518)试剂盒进行。
1 . 准备点突变反应物：
5ul 10X buffer
2ul (10 ng) 装载了野生型大肠菌素变构多肽和 immunity 蛋白基因的原始质粒 pSELECT™- l o
1.25 ul (125 ng)设计的 5'-3'寡聚核苷酸引物
1.25 ul (125 ng)设计的 3'-5'寡聚核苷酸引物
1 ul dNTP
双蒸水 50 ul
1 ul pfu
(除质粒、 引物和双蒸水外， 均为试剂盒所备试剂）
2. 进行 PCR扩增， 扩增条件： 变性 95°C, 35秒， 退火 53 °C, 70秒， 延伸 68 °C, 17分， 共 20个循环;
3. 加入 Dpn 1内切酶 1 ul 消化母体 DNA链 （37 °C, 1小时）， 取 1 ul反应物 与 XLl-Blue感受态细胞 50 ul冰孵 30分钟， 热冲击 42 °C， 45秒， 再置 入冰中 2分钟；
4. 加入 NZY培基 0.5 ml, 220 rpm, 37 °C摇菌 1小时， 取 50-100 ul 反应物铺 板 （LB培基加 1 %琼脂加 50 ug/ml氨苄青霉素， 37 °C过夜）；
5. 18 小时后挑菌， 提取质粒后测序确定突变成功；
6. 将完成多个位点突变的最终获得的重组质粒 50 ng 与制备的 E.coli BL-21(DE3)工程菌感受态细胞 40 ul冰孵 5分钟， 42 °C热冲击 30秒， 再置 入冰中 2分钟， 加入 Novagen公司 SOC培养液 160 ul后 220 rpm, 37 °C 摇菌 1小时后铺板 （LB培基加 1 %琼脂， 加 50 Ug/ml氨苄青霉素） 37 °C 过夜。
7. 挑取单克隆菌落大量增菌， 8-16升 FB培基， 250 rpm, 30 °C， 4-5小时， 热冲 击 250 rpm, 42 °C, 30分钟， 250 rpm, 37 °C, 2小时；离心沉淀菌体， 4 °C， 6000g, 20分钟， 取 4°C， 50mM硼酸缓冲液 （2mM EDTA +2mM DTT) 50-80ml悬浮 菌体， 加入 0.2M PMSF 250毫升后， 使用超声破碎（4 °C， 400W, 2分钟，）， 高速离心沉淀破碎的菌体 （4 °C， 75,000g, 90分钟）， 取上清加入硫酸链霉素 500万单位沉淀 DNA， 15000g, 4 °C, 10分钟离心沉淀后， 取上清装入分子 量 15,000透析袋于 4 °C， 50 mM硼酸缓冲液 4升透析过夜后， 再次 15000g, 4 °C， 10分钟离心沉淀， 取上清上样于 CM离子交换柱， 经 0.1-0.3 M NaCl + 50 mM硼酸缓冲液梯度洗脱即可得到重组抗肿瘤多肽。
点突变设计的引物序列如下：
Seq ID NO. 1 突变大肠菌素基因中 G11A所设计的寡聚引物 5 ' -3 '
cgt att aca aat ccc GCA gca gaa tcg ctg ggg
Seq ID NO. 2 突变大肠菌素基因中 G11A所设计的寡聚引物 3 ' _5 '
ccc cag cga ttc tgc TGC ggg att tgt aat acg
Seq ID NO. 3 突变大肠菌素基因中 H22G所设计的寡聚引物 5 ' -3 '
gat tea gat ggc GGT aaa tta tgg gtg
Seq ID NO. 4 突变大肠菌素基因中 H22G所设计的寡聚引物 3 ' -5 '
cac cca taa ttt ACC gcc ate tga ate
Seq ID NO. 5 突变大肠菌素基因中 A26G所设计的寡聚引物 5 ' -3 '
gaaa ttatgGGTgt tgatatttat
Seq ID NO. 6 突变大肠菌素基因中 A26G所设计的寡聚引物 3 ' -5 ' ataaatatacaacACCcataatttc
Seq ID NO. 7 突变大肠菌素基因中 V31L所设计的寡聚引物 5' -3'
gt t gat att tat CTC aaccctc cacgtgtc
Seq ID NO. 8 突变大肠菌素基因中 V31L所设计的寡聚引物 3 ' -5，
gacacgtggagggttGAGataaatatcaac
Seq ID NO. 9 突变大肠菌素基因中 H40D所设计的寡聚引物 5' -3'
cgtgtcga tgtctttGATggtaccccgc ctgcat
Seq ID NO. 10 突变大肠菌素基因中 H40D所设计的寡聚引物 3' -5'
atgcaggcggggtaccATCaaagacatcgacacg
Seq ID NO. 11重组质粒 pCHCEBl l中 V„CDR1基因引物 5， -3，
gcg aat aag ttc tgg ggt att TCC TTC GGT ATG CAT TGG GTG CGTCAGtaa ata aaa tat aag aca ggc
Seq ID NO. 12重组质粒 pCHCEBl l中 VHCDR1基因引物 3' -5，
gcc tgt ctt ata ttt tat tta CTG ACG CAC CCA ATG CAT ACC GAA GGA aat acc cca gaa ctt att cgc
Seq ID NO. 13重组质粒 pCHCEBl 1 中 VHFR2基因引物 5 ' _3'
8'8't atg cat tgg gtg cgt cag GCC CCC GAG AAA GGT CTG GAG TGG GTG GCC taa ata aaa tat aag aca ggc
Seq ID NO. 14重组质粒 pCHCEBl l中 VHFR2基因引物 3' -5，
gcc tgt ctt ata ttt tat tta GGC CAC CCA CTC CAG ACCT TTT CTC GGG GGC ctg acg cac cca atg cat acc
Seq ID NO. 15重组质粒 pCHCEBl 1 中 （Rev) V^DRS 基因引物 5' -3，
aaa ggt ctg gag tgg gtg gcc ACC TAC CCC TAC TCC TAC GGT CAG GGT taa ata aaa tat aag aca ggc
Seq ID NO. 16重组质粒 pCHCEBl l中 (Rev) VLCDR3 基因引物 3' -5，
gcc tgt ctt ata ttt tat tta ACC CTG ACC GTA GGA GTA GGG GGT ggc cac cca etc cag acc ttt
Seq ID NO. 17重组质粒 pCHCEB22中 V„CDR1基因引物 5' -3，
gcg aat aag ttc tgg ggt att TCC TTC GGT ATG CAT TGG GTG CGT CAG taa ata aaa tat aag aca ggc
Seq ID NO. 18重组质粒 pCHCEB22中 V„CDR1基因引物 3， -5，
gcc tgt ctt ata ttt tat tta CTG ACG CAC CCA ATG CAT ACC GAA GGA aat acc cca gaa ctt att cgc
Seq ID NO. 19重组质粒 pCHCEB22中 VHFR2基因引物 5' _3'
ggt atg cat tgg gtg cgt cag GCC CCC GAG AAA GGT CTG GAG TGG GTG GCC taa ataaaa tat aag aca ggc
Seq ID NO. 20重组质粒 pCHCEB22 中 VHFR2基因引物 3' -5，
gcc tgt ctt ata ttt tat tta GGC CAC CCA CTC CAG ACCT TTT CTC GGG GGC ctg acg cac cca atg cat acc
Seq ID NO. 21重组质粒 pCHCEB22 中 VLCDR3基因引物 5' -3，
aaa ggt ctg gag tgg gtg gcc GGT CAG GGT TAC TCC TAC CCC TAC ACC taa ata aaa tat aag aca ggc
Seq ID NO. 22重组质粒 pCHCEB22中 VLCDR3基因引物 3' -5，
gcc tgt ctt ata ttt tat tta GGT GTA GGG GTA GGA GTA ACC CTG ACC ggc cac cca etc cag acc ttt 实施例 2.重组质粒 pCHCEBll、 pCHCEB22制备的新型抗肿瘤多肽的免疫效果观 察
取实施例 1制备获得重组质粒 pCHCEBll、 pCHCEB22制备得到的新型抗 肿瘤多肽 1， 新型抗肿瘤多肽 2， 发明人的前期专利中的抗肿瘤多肽 1、 抗肿瘤 多肽 2 (ZL200410081446.8)各一份免疫小鼠： 上述各蛋白与佐剂混合后， 基础 量和追加量为腹腔注射每只 50 μ§ (0.5 ml) 1 次。间隔两周，免疫 5 针。 ELISA 间接法检测小鼠血清效价，本发明制得的新型抗肿瘤多肽 1和 2所免疫小鼠血清 效价为 10—3 至 10—4 而抗肿瘤多肽 1、抗肿瘤多肽 2所免疫小鼠的血清效价为 10— 4 至 10—5。
可见本发明新型抗肿瘤多肽造成宿主致敏反应的可能性要比含野生型大肠 菌素 la的抗肿瘤多肽造成宿主致敏反应的可能性低一至两个数量级。 实施例 3.构成新型抗肿瘤多肽的大肠菌素 la变构多肽的低致敏效果实验
取实施例 1中大肠菌素 la变构多肽 （其水性孔道结构域的肽链中突变了氨 基酸残基 G11A、 H22G、 A26G、 V31L和 H40D) 的突变质粒， 在其氨基端或 羧基端可操作地连接金黄色葡萄球菌信息素 AgrDl( YSTCDFIM )， 制备出两种 抗菌多肽。 AgrDl连接在变构的大肠菌素 la羧基端制备而得的多肽命名为抗金 葡多肽 1， AgrDl连接在变构的大肠菌素 la氨基端制备而得的多肽命名为抗绿脓 杆菌多肽 1。取野生型大肠菌素 la的质粒，在其氨基端连接金黄色葡萄球菌信息 素 AgrDl制备出抗绿脓杆菌多肽 2。
实验 1 : 取昆明小鼠一批， 腹腔注射致死剂量耐甲氧西林金葡菌 （ATCC BAA42) , 随机分组为 （1 )对照组， （2)氨苄青霉素组， （3)抗金葡菌多肽组，
(4) 抗金葡菌多肽 1组。 每组小鼠均为 10只。
处理方法：
腹腔注射致死剂量耐甲氧西林金葡菌 （ATCC BAA42) —小时后， 对照组： 尾静脉注射 0.5毫升 0.3 M NaCl + 50 mM硼酸缓冲液一次； 氨苄青霉素组： 尾静脉注射氨苄青霉素 2.5 mg/kg—次；
抗金葡菌多肽组： 尾静脉注射发明人发明的抗金葡菌多肽 （ZL 01128836.1) 6 mg/kg一次；
抗金葡菌多肽 1组： 尾静脉注射抗金葡菌多肽 1 6 mg/kg—次。
结果： 对照组及氨苄青霉素组鼠在两天内全部死亡。 抗金葡菌多肽组及抗 金葡菌多肽 1组有 85 %的鼠存活。
实验 2: 实验 1的第 14天后， 再取昆明小鼠一批作为对照和氨苄青霉素组， 抗金葡菌多肽组及抗金葡菌多肽 1 组存活鼠作为抗金葡菌多肽组及抗金葡菌多 肽 1组重复上述试验。对照组及氨苄青霉素组鼠在两天内全部死亡。抗金葡菌多 肽组有 75 %的鼠存活， 抗金葡菌多肽 1组有 90 %的鼠存活。
实验 3: 实验 1的 41天后， 再取昆明小鼠一批作为对照组、左氧氟沙星组和 头孢曲松钠组，抗金葡菌多肽组及抗金葡菌多肽 1组存活鼠作为抗绿脓杆菌多肽 2组及抗绿脓杆菌多肽 1组。
小鼠腹腔注射致死剂量多重耐药绿脓杆菌 （四川大学华西医院试验医学系临 床分离株 13578) —小时后，
对照组尾静脉注射 0.5毫升 0.3 M NaCl + 50 mM硼酸缓冲液一次，
左氧氟沙星组尾静脉注射左氧氟沙星 5 mg/kg一次，
头孢曲松钠组尾静脉注射头孢曲松钠 30 mg/kg—次，
抗绿脓杆菌多肽 2组尾静脉注射绿脓杆菌多肽 2 8 mg/kg一次，
抗绿脓杆菌多肽 1组尾静脉注射抗绿脓杆菌多肽 1 8 mg/kg一次。 对照组和 左氧氟沙星组鼠在一天内全部死亡。 头孢曲松钠组有 25 %的鼠存活。 抗绿脓杆菌 多肽 2组有 60%的鼠存活。 抗绿脓杆菌多肽 1组鼠全部存活， 说明宿主抗体对变 构多肽杀伤效用的干扰比对野生型多肽的干扰要低。
见附图 3 。
在试验第一周、第二周和第 7周分别采取上述抗金葡菌多肽 /抗绿脓杆菌多肽 2组和抗金葡菌多肽 1/抗绿脓杆菌多肽 1组存活鼠的血清进行 ELISA间接法检 测其血中的抗体， 酶标板的孔中分别包被野生型大肠菌素 la和大肠菌素 la变构 多肽， 100 ng/ well, 一抗分别为抗金葡菌多肽 /抗绿脓杆菌多肽 2组和抗金葡菌 多肽 1/抗绿脓杆菌多肽 1组存活鼠血清， 2抗为山羊抗小鼠标记抗体， 阴性对照 以 5%牛奶 -PBS为一抗， 1:50,000滴度的所得结果如下 （见附图 4)
A (抗金葡多肽 /抗绿脓多肽 2组) (抗金葡多肽 1/抗绿脓多肽 1 ) 1 (第 1周） 0.914 0.254
2 (第 2周） 1.623 0.598
3 (第 7周） 2.911 1.41
4 (对照） 0.065 0.069
可见本发明所制备的大肠菌素 la变构多肽造成宿主致敏反应的可能性要比 野生型大肠菌素 la造成宿主致敏反应的可能性低；
实施例 4 新型抗肿瘤多肽对 EBV所致人恶性淋巴肉瘤 (Burkitt's lymphoma)体 外杀伤效果
EB病毒阳性及阴性细胞株： 为美国 ATCC标准细胞株。
细胞培养： 取出 0.1 ml复苏培养的 Raji细胞悬浮液， 缓缓加入含 3 ml 1640液 体培养基 (加 10%血清)的培养皿中 (稀释比例为 1:30)， 混合均匀， 放入 37°C C02 培养箱中培养。 实验使用一株 EB病毒阳性细胞株： ATCC CCL-86 (即全世界细 胞培养室使用的标准人 Burkitt's恶性淋巴肉瘤细胞, Raji细胞， 1963年分离自 12 岁非洲男性患儿）。
受试细胞共分 3组， 第一组为空白组， 即加入抗肿瘤多肽空白保存液
(10mMPB+0.2M NaCI磷酸盐缓冲液 （ρΗ7·4) ；)。
第二组为加入 20(Vg/ml的新型抗肿瘤多肽 1 (质粒为 pCHCEBll, 保存液为 lOmMPB + 0.2M NaCI磷酸缓冲液 pH7.4)。
第三组为加入 20(Vg/ml的新型抗肿瘤多肽 2 (质粒为 pCHCEB22, 保存液为 lOmMPB + 0.2M NaCI磷酸缓冲液 pH7.4)。
细胞在培养 24小时后， 分别加入上述各组处理物于培养皿中， 在加入处理 物后的第 72小时加入 100 uMol碘化丙啶 （Propidium Iodide, PI) 20 ul, 10分钟 后在荧光显微镜下观察。 结果显示空白组细胞生长良好， 唯新型抗肿瘤多肽 1 组中绝大部分细胞被 PI染成了红色， 提示这些细胞膜已被抗肿瘤多肽破坏,造成 肿瘤细胞死亡。 从细胞死亡数量来比较， 两种新型抗肿瘤多肽中新型抗肿瘤多 肽 2效果较差， 见附图 5。 实施例 5. 新型抗肿瘤多肽对 EB病毒所致 Burkitt's人恶性淋巴肉瘤细胞和其它 肿瘤细胞的体外杀伤作用之多重荧光染色观察。 细胞培养条件同实施例 2。实验共使用三株细胞， EB病毒阳性细胞株： ATCC CCL-86 (Raji 细胞， Burkitt's 恶性淋巴肉瘤细胞）； ATCC CRL-2230, 46岁男性 爱滋病人 （AIDS) 恶性淋巴肉瘤细胞株 , ΕΒ病毒和 Kaposi肉瘤病毒 （HHV8) 均 阳性； EB病毒阴性细胞株： ATCC CRL-1648 (CA-46, 细胞分离自美国型 Burkitt's 恶性淋巴肉瘤病人的腹水）。
每株细胞分为二个实验组， 第一组为加入新型抗肿瘤多肽空白保存液 (10mMPB+0.2M NaCI磷酸盐缓冲液（pH7.4) 第二组为加入 20(Vg/ml的新型抗 肿瘤多肽 1(质粒为 pCHCEBll), 保存液为 lOmMPB + 0.2M NaCI磷酸缓冲液 ρΗ7·4。
细胞在培养 24小时后， 分别加入上述各组处理物于培养皿中， 在加入处理 物后的第 72小时分别加入 50 uMol FITC 20ul及 50 uMol Rodamin-123 20ul两种 荧光染料。 10分钟后在 Olympus IX-71荧光显微镜下观察。
结果显示： EB病毒阴性的肿瘤细胞株被新型抗肿瘤多肽 1处理后仍生长良 好。 而 EB病毒阳性的各肿瘤细胞株被新型抗肿瘤多肽 1处理后， 绝大多数细胞 出现线粒体及胞核结构消失， 明显肿胀和坏死， 细胞几乎全部死亡。 显然多重荧 光染色的结果比实施例 4碘化丙啶染色的结果更为精确的展示了新型抗肿瘤多 肽 1对 EB病毒阳性肿瘤细胞的强大杀伤作用， 见图 6。
EB病毒阴性的肿瘤细胞生长良好， 表明新型抗肿瘤多肽并不攻击细胞膜上 不带有 EB病毒表面抗原的细胞。这提示本发明新型抗肿瘤多肽具有理想的靶向 特异性和安全性。 实施例 6.新型抗肿瘤多肽对 EB病毒所致 Burkitt's人恶性淋巴肉瘤细胞种植到裸 鼠体内生长出的实体肿瘤的杀伤作用
SCID 免疫缺陷小鼠购自中国科学院上海实验动物中心，鼠喂养按标准伺养 要求， 水、 垫草和伺料均经高温或紫外线灭菌。 在相对无菌条件下伺养一周， 无 异常后进行接种实验。
收集指数生长期的 Raji (ATCC CCL-86) 禾卩 1648 (ATCC CRL-1648)细胞悬 液于 50毫升离心管中， 4°C离心， 弃上清后， 用 1640液体培养基（加小牛血清） 重悬细胞， 使之达到 1.0 X 107/ml个细胞。 在鼠左腋处皮下注射 Raji细胞悬液 0.1ml, 在鼠右腋处皮下注射 1648细胞悬液 0.1ml 。
注射细胞后 3— 4日肿瘤即长到约 2 x 2 mm， 将荷瘤裸鼠随机分为： ( A 组） 抗肿瘤多肽空白保存液 (10mM PBS+0.2M NaCI 磷酸盐缓冲液 (pH7.4) )为对照组；
(B组） 新型抗肿瘤多肽 1(质粒为 pCHCEBll)治疗组， 300 μ§ /鼠 (以 25克 重计算 )/日， 连续 20日；
每组 10只荷瘤鼠， 给药方式为腹腔注射， 每天注射 2次， 每次 0.5 ml ， 连 续给药 20天。。每天观察鼠活动情况，并作记录，隔天精确测量肿瘤大小并照相。
结果显示： （见图 7) B 组新型抗肿瘤多肽组的鼠肿瘤生长明显受到抑制， 其中 7只鼠的瘤块消失，其余 3只的瘤块明显小于对照组。新型抗肿瘤多肽在鼠 体内可以有效地抑制 EBV病毒阳性淋巴肉瘤细胞所致的实体瘤块生长。 但对同 体鼠内接种的 EBV病毒阴性淋巴肉瘤细胞所致的实体瘤块却无抑制作用。
实施例 7.体内实体肿瘤消除实验的病理观察.
瘤体病理组织学观察： 实施例 6实验到期后处死小鼠， 取出瘤体于 10%福 尔马林中固定， 石蜡切片， HE染色常规光镜观察。
镜下观察对照组鼠的实体肿瘤增殖旺盛； 新型抗肿瘤多肽组鼠的 EBV病毒 阳性实体肿瘤细胞显著减小，标本中縮小的肿瘤细胞团中几乎均为坏死的肿瘤细 胞， 周边可见大量淋巴细胞浸润。 病理组织学结果提示在 20 日处理期限中， 新 型抗肿瘤多肽几乎杀灭了实体肿瘤中的所有肿瘤细胞 （见图 8、 D)。

Claims

权利要求书
1一种新型抗 EB病毒所致肿瘤多肽， 由可形成离子通道的大肠菌素变构多肽与 抗 EB病毒抗体多肽或者抗 EB病毒抗体的模拟抗体多肽可操作地连接构成，所 述可形成离子通道的大肠菌素变构多肽由野生型大肠菌素 El、 Ia、 Ib、 A、 B、 N或其水性孔道结构域的肽链突变了氨基酸残基 G11A、 H22G、 A26G、 V31L和 H40D而获得， 所述抗 EB病毒抗体的氨基酸序列与 ATCC HB-168杂交瘤分泌 的单克隆抗体相同。
2.根据权利要求 1所述的新型抗 EB病毒所致肿瘤多肽， 所述模拟抗体多肽指所 述抗 EB病毒抗体的重链 CDR1区、 重链 CDR1-CDR2连接肽段和轻链 CDR3 的连接肽。
3.根据权利要求 2所述的新型抗 EB病毒所致肿瘤多肽， 所述可形成离子通道的 大肠菌素变构多肽由野生型大肠菌素 la突变而来。
4根据权利要求 3所述的新型抗 EB病毒所致肿瘤多肽， 所述新型抗 EB病毒所 致肿瘤的多肽， 具有如 SEQ ID N0.29所示的氨基酸序列。
5.编码权利要求 1~4任一所述新型抗 EB病毒所致肿瘤多肽的基因。
6. 根据权利要求 5所述的基因， 具有如 SEQ ID NO.30所示的核苷酸序列。
7.含有权利要求 5所述基因的重组质粒。
8.权利要求 1~4任一所述新型抗 EB病毒所致肿瘤多肽的制备方法， 步骤如下： 将权利要求 7所述的重组质粒转化到表达系统中进行表达，分离纯化表达的多 肽。
9.权利要求 1~4任一所述新型抗 EB病毒所致肿瘤多肽在制备治疗和预防 EB病 毒所致肿瘤的药物中的应用。
10.—种大肠菌素 la的变构多肽， 其氨基酸序列如 SEQ ID N0.24所示。
11.编码权利要求 10所述的大肠菌素 la的变构多肽的基因。
12. 权利要求 11 所述的基因在制备多肽药物中的应用， 指将所述基因与诱导多 肽的基因可操作地连接并克隆到表达载体中，然后将表达载体转化到表达系统 中， 分离纯化表达的多肽。
PCT/CN2010/070762 2009-12-17 2010-02-26 抗EB病毒诱发的肿瘤的融合多肽和大肠菌素Ia突变体 WO2011072501A1 (zh)

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BR112012017350-2A BR112012017350A2 (pt) 2009-12-17 2010-02-26 "polipeptídeo de fusão contra tumor induzido por vírus eb e mutante de colicina ia".
EA201270655A EA030638B1 (ru) 2009-12-17 2010-02-26 Полипептид для лечения и профилактики опухоли, вызванной вирусом эпштейна-барр, способ его получения и применение
AP2012006327A AP3535A (en) 2009-12-17 2010-02-26 Fusion polypeptide against eb virus-induced tumor and colicin ia mutant
SG2012041786A SG181553A1 (en) 2009-12-17 2010-02-26 Fusion polypeptide against eb virus-induced tumor and colicin ia mutant
NZ600729A NZ600729A (en) 2009-12-17 2010-02-26 Fusion polypeptide against eb virus-induced tumor and colicin ia mutant
AU2010333588A AU2010333588B2 (en) 2009-12-17 2010-02-26 Fusion polypeptide against EB virus-induced tumor and colicin Ia mutant
CA2784784A CA2784784C (en) 2009-12-17 2010-02-26 Fusion polypeptide against eb virus-induced tumor and colicin ia mutant
EP10836940.6A EP2514768B1 (en) 2009-12-17 2010-02-26 Fusion polypeptide against eb virus-induced tumor and colicin ia mutant
JP2012543448A JP5683603B2 (ja) 2009-12-17 2010-02-26 Ｅｂウィルスにより誘発される腫瘍に対する融合ポリペプチドとコリシンｉａ変異体
ES10836940.6T ES2634944T3 (es) 2009-12-17 2010-02-26 Polipéptido de fusión frente a un tumor inducido por el virus de EB y un mutante de la colicina Ia
MX2012006952A MX2012006952A (es) 2009-12-17 2010-02-26 Polipeptido de fusion contra tumor inducido por virus eb y mutante de colicina ia.
US13/516,605 US8883161B2 (en) 2009-12-17 2010-02-26 Fusion polypeptide against EB virus-induced tumor and colicin Ia mutant
DK10836940.6T DK2514768T3 (en) 2009-12-17 2010-02-26 Fusion polypeptide against EB virus-induced tumor and colicin Ia mutant
UAA201208633A UA104933C2 (uk) 2009-12-17 2010-02-26 ЗЛИТИЙ ПОЛІПЕПТИД ПРОТИ ПУХЛИНИ, ІНДУКОВАНОЇ ВІРУСОМ EB, І МУТАНТ КОЛІЦИНУ Ia
KR1020127018638A KR101464842B1 (ko) 2009-12-17 2010-02-26 ＥＢ 바이러스에 의해 유도된 종양에 대한 융합 폴리펩티드 및 콜리신 Ｉａ 돌연변이체
MA35062A MA33921B1 (fr) 2009-12-17 2010-02-26 Polypeptide de fusion dirigé contre une tumeur induite par le virus eb et mutant de la colicine ia
TNP2012000253A TN2012000253A1 (en) 2009-12-17 2012-05-24 Fusion polypeptide against eb virus-induced tumor and colicin ia mutant
IL220296A IL220296A (en) 2009-12-17 2012-06-11 Polypeptide Protected against Epstein-Bar Virus Virus and Colicin ia Mutation
CU2012000096A CU20120096A7 (es) 2009-12-17 2012-06-12 Polipéptido de fusión contra tumor inducido por virus eb y mutante de colicina ia
ZA2012/05217A ZA201205217B (en) 2009-12-17 2012-07-13 Fusion polypeptide against eb virus-induced tumor and colicin ia mutant

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