WO2004009811A1 - A gene entrain system with the core sequence of histone - Google Patents

A gene entrain system with the core sequence of histone Download PDF

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Publication number
WO2004009811A1
WO2004009811A1 PCT/CN2002/000592 CN0200592W WO2004009811A1 WO 2004009811 A1 WO2004009811 A1 WO 2004009811A1 CN 0200592 W CN0200592 W CN 0200592W WO 2004009811 A1 WO2004009811 A1 WO 2004009811A1
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histone
fusion protein
gene
oligopeptide
dna
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PCT/CN2002/000592
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French (fr)
Chinese (zh)
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Jianren Gu
Shengli Yang
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Neworgen Limited
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Priority to AU2002327321A priority Critical patent/AU2002327321A1/en
Publication of WO2004009811A1 publication Critical patent/WO2004009811A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • 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
    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
    • C12N2810/80—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates

Definitions

  • the present invention relates to the fields of molecular biology and gene therapy, and more particularly, to a gene introduction system using a histone as a core sequence, and a preparation method and use thereof. Background technique
  • Gene therapy is to introduce foreign DNA into specific cells of the human body to produce a therapeutic effect and achieve the purpose of treating diseases.
  • a safe and effective gene transfer system is required first.
  • the purpose of the present invention is to provide a targeted gene introduction system which is simple and convenient for manufacturing and suitable for large-scale production, and a method for preparing and using the same.
  • a fusion protein is provided, which contains
  • the fusion protein has a connecting peptide between (a) a histone element and (b) a receptor recognition oligopeptide element.
  • the fusion protein further comprises (c) an endosome-releasing oligopeptide element, and optionally
  • a gene-introduction system for receptor-mediated targeted tumor gene therapy comprising the above-mentioned fusion protein and foreign DNA, and the foreign DNA is selected from the group consisting of Eukaryotic expression vector DNA of genes: antisense oncogene, anti-oncogene, suicide gene, apoptosis gene, cytokine gene, or a combination thereof.
  • the fusion protein is selected from the following group: LOP-Histone Element-HA20, HA20-Histone Element-LOP, Histone Element-LOP, LOP-Histone Element, and the system further contains any Selected HA20-histone element or histone element-HA20 fusion protein.
  • a pharmaceutical composition comprising the above-mentioned fusion protein and a pharmaceutically acceptable Carrier.
  • an isolated DNA encoding the fusion protein an expression vector containing the DNA, and a host cell containing the DNA or the expression vector.
  • a method for producing a fusion protein of the present invention comprising the steps of: culturing the transformed host cell under expression conditions to express the fusion protein; and isolating the fusion protein.
  • Figure 1 is a flowchart of the construction of the GE7-histone H1 ° -HA20 expression sequence.
  • Figure 2 shows the DNA nucleotide sequence and amino acid sequence of histone H1 Q.
  • Figure 3 shows the DNA nucleotide sequence and amino acid sequence of histone H1 Q 97 _193 .
  • Figure 4 shows the DNA nucleotide sequence and amino acid sequence of the fusion protein GE7-Histone H1 Q -HA20.
  • Figure 5 shows the DNA nucleotide sequence and amino acid sequence of the fusion protein GE7 histone Hl ° 97 _ 193 -HA20.
  • Figure 6 shows the DNA nucleotide sequence and amino acid sequence of the fusion protein histone H1 Q- GE7.
  • Figure 7 shows the DNA nucleotide sequence and amino acid sequence of the fusion protein histone H1 Q- HA20.
  • Figure 8 shows the fusion protein histone Hl ° 97 - DNA nucleotide sequence and amino acid sequence of GE7 --193.
  • Figure 9 shows the 97 fusion protein histone H1 Q - 193 - DNA nucleotide sequence and amino acid sequences of HA20.
  • Figure 10 shows the DNA nucleotide sequence and amino acid sequence of the fusion protein HA20-histone H1 Q -GE7.
  • Figure 11 shows the plasmid map of the prokaryotic expression vector PT7450.
  • Figure 12 shows the four proteins purified end product: Lane 1 is GE7- histone H1 Q 97 _ 193 -HA20 (15kD ), lane 2 is histone H1 Q 97 _ 193 (10kD) , lane 3 is GE7 -Histone H1 ° -HA20 (25kD), lane 4 is histone H1 Q (21kD), and M represents a protein low molecular weight marker.
  • Figure 13 shows the final products of three proteins after purification: lane 1 is HA20-Histone-GE7 (25kD), lane 2 is histone Hl ° -GE7 (23. IkD), and lane 3 is histone Hl fl -HA20 (23.4kD), M stands for protein low molecular weight marker.
  • Figure 14 shows the final products of the two proteins after purification: lane 1 is histone Hl fl 97 _ 193 _HA20 (13. IkD), lane 3 is histone Hl fl 97 _ 193 -GE7 (12.8kD), M represents Low molecular weight labeling of proteins.
  • Figure 15 shows the 1% agarose gel electrophoresis of a binary complex gene transfer system formed by GE7-histone H1 Q -HA20 and DNA.
  • M stands for ⁇ / HindIII DNA molecular weight marker;
  • lane 1 is plasmid DNA;
  • lanes 2, 3, 4, 5, 6, 7 respectively represent the DNA: GE7-histone H1 Q -HA20 mass ratio in the gene transfer system of 1: 0.5 , 1: 1, 1: 1.5, 1: 2, 1: 2.5, 1: 3.
  • 1: 3 is the best ratio.
  • Figure 16 shows the 1% agarose gel electrophoresis of a binary complex gene transfer system formed by GE7-histone H1 Q 97 _ 193 -HA20 and DNA.
  • M stands for ⁇ / HindIII DNA molecular weight marker; lane 1 is plasmid DNA; lanes 2, 3, 4, 5, 6 represent 0: 6 £ 7-histone H1 Q 97 193 -HA20 in the gene transfer system, respectively 1: 0.5, 1: 1, 1: 2, 1: 2.5, 1: 3 ⁇ 1: 3 are the best ratios.
  • Figure 17 shows the 1% agarose gel electrophoresis of the binary complex gene transfer system of histone H1 Q and DNA.
  • M stands for ⁇ / HindIII DNA molecular weight marker;
  • lane 1 is plasmid DNA;
  • lanes 2, 3, 4, 5, 6 respectively represent the DNA: histone ⁇ 1 ⁇ mass ratio in the gene transfer system of 1: 0.5, 1: 1, 1 : 2, 1: 2.5, 1: 3.
  • Figure 18 shows the 1% agarose coagulation of the binary complex gene transfer system of histone H1 Q 97 _ 193 and DNA Gel electrophoresis.
  • M represents ⁇ / HindIII DNA molecular weight markers;
  • Lane 1 is the DNA plasmid;
  • lane 2, 3, 4, 5, 6 represent the gene transfer system of DNA: mass Histone Hl ° 97 _ 193 ratio was 1: 0.5, 1: 1, 1: 2, 1: 2.5, 1: 3.
  • 1: 3 is the best ratio.
  • Figure 19 shows the 1% agarose gel electrophoresis of a binary complex gene transfer system formed by other fusion proteins and DNA.
  • M stands for ⁇ / Hindin DNA molecular weight marker; lane 1 is plasmid DNA; lanes 2, 3, 4, 5, and 6 are the fusion protein histone H1 Q- GE7, histone HI 0 -turn, histone H1 ° 97 _ 193 _GE7, Histone Hl ° 97-i93-HA20 HA20-Histone H1 Q -GE7 is a binary complex formed with DNA.
  • the mass ratio of DNA to each fusion protein in the gene transfer system is 1: 3.
  • Figure 20 shows the 1% agarose gel electrophoresis of the ternary complex gene transfer system formed by the combination of different fusion proteins and DNA.
  • M stands for ⁇ / HindIII DNA molecular weight marker; lane 1 is plasmid DNA; lanes 2, 3, and 4 are combinations of different fusion proteins, respectively GE7-Histone H1 G -HA20 / Histone Hl °-GE7 (1: 1 equal amount mixed ) ⁇ Histone H'1 Q -GE7 / Histone Hl °-HA20 (1: 1 equal volume mixing), Histone H1 Q 97 _ 193 -GE7 / Histone H1 ° 97. 193 -HA20 (1: 1 etc.
  • the mass ratio of the combination of DNA and each fusion protein in the gene transfer system is 1: 3.
  • Figure 21 shows the fusion protein combination GE7-Histone-HA20 / Histone H1 Q -GE7, Histone Hl °-
  • GE7 / Histone H1 Q -HA20 results in the introduction of ⁇ -gal gene into BEL-7402 cells but not U20S cells.
  • A, B, and C are U20S cells
  • D, E, and F are BEL-7402 cells.
  • a and D are pure ⁇ -gal genomes
  • B and E are ⁇ -gal genes / histone Hl ° -GE7 / histone Hl ° -HA20 group
  • C and F are ⁇ -gal genes / GE7-histone Hl °- HA20 / Histone H ° -GE7 group.
  • Figure 22 shows the results of X-gal staining of tumor gross and pathological sections of ⁇ -gal gene introduced into human liver cancer BEL-7402 nude mice with several fusion proteins and combinations.
  • A is pure ⁇ -gal gene
  • B is ⁇ -gal gene / histone H1 Q group
  • C is ⁇ -gal gene / histone Hl ° -HA20 group
  • D is ⁇ -gal gene / histone Hl °-GE7 Group
  • E is ⁇ -gal gene / GE7-Histone H1 ° -HA20 group
  • F is ⁇ -gal gene / HA20-Histone H1 ° -GE7 group
  • G is ⁇ -gal gene / Histone Hl ° -GE7 / Histone H1 Q -HA20
  • H is ⁇ -gal gene / GE7-Histone-HA20 / Histone Hl ° -GE7 group.
  • the upper right corner or lower left corner of the pathological section were respectively attached with
  • Figure 23 shows the results of X-gal staining of tumor gross and pathological sections of the ⁇ -gal gene introduced into human ovarian cancer SK0V3 nude mice by several fusion proteins and combinations.
  • A is pure ⁇ -gal gene
  • B is ⁇ -gal gene / histone H1 fl group
  • C is ⁇ -gal gene / histone H1 Q -HA20 group
  • D is ⁇ -gal gene / histone H1 ° -GE7 Group
  • E is ⁇ -gal gene / GE7-Histone Hl ° -HA20 group
  • F is ⁇ -gal gene / HA20-Histone HI 0 -GE7 group
  • G is ⁇ -gal gene / Histone H1 ° -GE7 / Histone H1 ° -HA20
  • H is ⁇ -gal gene / GE7-Histone ⁇ 1 ° -HA20 / Histone ⁇ 1 °-GE7 group.
  • Figure 24 shows that the fusion protein combination GE7-Histone H1 Q -HA20 / Histone H1 Q -GE7 introduced the ⁇ -gal gene into nude mice bearing human ovarian cancer SK0V3, and the expression of the introduced ⁇ -gal gene in tumor tissues followed Changes over time.
  • A is 12 hours after ⁇ -gal gene introduction
  • B is 24 hours after ⁇ -gal gene introduction
  • C is 48 hours after ⁇ -gal gene introduction
  • D is 96 hours after ⁇ -gal gene introduction
  • E is ⁇ -gal gene. Seven days after the introduction, F was 10 days after the ⁇ -gal gene was introduced, G was 14 days after the ⁇ -gal gene was introduced, and H was a control group injected with physiological saline for 24 hours.
  • Figure 25 shows the X-gal staining of tumor tissues injected with different doses of ⁇ -gal gene / GE7-Histone H1 Q -HA20 / Histone H1 Q -GE7 complex.
  • A is 0. 2 ⁇ ⁇ group
  • ⁇ is 0. 5 ⁇ ⁇ group
  • C is 1 ⁇ ⁇ group.
  • histones especially histone HI
  • lysine-rich fragments thereof are very suitable as core sequences of targeted gene introduction systems, and the constructed fusion protein is very suitable Prepared by recombinant methods.
  • the present invention has been completed on this basis.
  • histone H1 Q refers to the histone H1 Q subtype (gene number X03473) found in human end-stage differentiation cells as found by Doenecke D et al. (J. Mol. Biol. 1986, 187, 461). Histone H1 Q is rich in lysine, especially the C-terminal part (Ser 97 -Lys 193 ).
  • Histones are a class of proteins rich in basic amino acids. The proportion of basic amino acids is about 30%. They are positively charged and can be combined with negatively charged DNA by electrostatic attraction to participate in the formation of chromosomal structures.
  • the core histones of the corpuscle H2A, H2B, H3, and H4 are different in that it binds to the connecting DNA between the nucleosomes and is independent of the nucleosomes, so it is also called linker histone; ). Chen J et al. (Hum.
  • HI histones include multiple subtypes, but these subtypes are composed of three regions: the N-terminal region, the central globular domain, and the C-terminal region.
  • the N-terminal region and the central globular domain are the hydrophobic structures of HI histones, while the C-terminal region becomes a hydrophilic region due to the richness of hydrophilic basic amino acids including Lys and Arg (J Mol
  • Histone elements suitable for use in the present invention include Hi Q full-length sequences or lysine-rich fragments thereof.
  • the lysine-rich fragment of m Q contains enough basic amino acids (lysine, arginine) to carry cations.
  • the number of basic amino acids is about 15-85%, preferably about 20-60%, and more preferably about 25-50%.
  • a preferred fragment is a fragment containing amino acids 97-193 in Hl °.
  • Histone elements are used in fusion proteins as part of the core sequence that binds to DNA molecules.
  • a receptor recognition oligopeptide (LOP) can be attached to its N-terminus and / or C-terminus.
  • Histone elements can be derived from human histones, or basic proteins from other natural species, such as other mammalian, microbial, and viral nuclear proteins.
  • Receptor recognition oligopeptide element can be derived from human histones, or basic proteins from other natural species, such as other mammalian, microbial, and viral nuclear proteins.
  • ligand oligopeptide LOP
  • receptor recognition oligopeptide refers to any oligopeptide that recognizes and binds to a target cell.
  • the receptor recognition oligopeptide element suitable for the present invention is a targeting element, which can be any oligopeptide that recognizes and binds to target cells.
  • Representative examples include (but are not limited to): IGF-II R, IGF-IR, EGF R, VEGF R specifically binds E5, GE7, GV1, GV2, or a combination thereof.
  • the amino acid sequence of the receptor recognition oligopeptide E5 is EPFRS PDLAL ETYG (SEQ ID NO: 26)
  • the amino acid sequence of the receptor recognition oligopeptide GE7 is NPVVG YIGER PQYRD L (SEQ ID NO: 27)
  • the receptor recognition oligopeptide GV1 Amino acid sequence is CHPIE TLVDI FQEYP DEIEY IFKPS PVPLM RP (SEQ ID NO: 28)
  • the amino acid sequence of the receptor recognition oligopeptide GV2 is PVPTE ESNIT MQIMR IKPHQ GQHIG EMSFL QHNKC E (SEQ ID NO: 29) a particularly preferred receptor recognition oligopeptide GE7.
  • the ligand oligopeptide functional domain may also have the same immunodeterminant with E5, GE7, GV1, and GV2, can react positively with the corresponding antibodies of the above functional domains, and can interact with IGF-IR, IGF-II R, EGF R, VEGF R binds to the functional domain formed by the polypeptide and oligopeptide.
  • the ligand oligopeptides also include polypeptides that bind to hematopoietic cells, macrophages, lymphocytes, liver cells, kidney cells, endothelial cells, nerve cells, and cardiomyocyte receptors. Endosome release oligopeptide element
  • the fusion protein of the present invention may further contain an endosome-releasing oligopeptide element, which plays a role of preventing the DNA of endocytosis from being degraded.
  • an endosome-releasing oligopeptide element which plays a role of preventing the DNA of endocytosis from being degraded.
  • Any endosome-releasing oligopeptide can be used in the present invention, and representative examples include (but are not limited to): HA20, VP-1.
  • a linker peptide may optionally be included between (a) a histone element, (b) a receptor recognition oligopeptide element, and (c) an endosome-releasing oligopeptide element, in order to increase The flexibility and stretchability of the spatial structure of the compound peptide allows each element to perform its own function.
  • linker peptide that can be used in the present invention is not particularly limited as long as it serves as a linker.
  • An example of a class of linker peptides is
  • the (a) histone element, (b) receptor-recognizing oligopeptide element, and (c) the endosome-releasing oligopeptide element of the present invention can all be replaced with amino acids of similar properties, as long as the respective similar biological functions are still retained.
  • the ligand is an oligopeptide (L0P) and endosome release oligopeptide (of HA20) elements are connected to histones (e.g., Histone H1 Q, Histone Hl. 97 _ 193) of the N_ or C-
  • histones e.g., Histone H1 Q, Histone Hl. 97 _ 193
  • fusion proteins such as LOP-histone element_HA20, HA20-histone element-L0P, histone element-L0P, and histone element-HA20 are formed. They can be combined with foreign DNA into binary or ternary complexes.
  • the gene transfer system utilizes the specific binding of a ligand oligopeptide to a receptor (eg, GE7 recognizes the EGF receptor overexpressed on tumor cells), and through cell endocytosis, the composite oligopeptide is used to selectively introduce foreign DNA Target cells to achieve the purpose of treatment.
  • a receptor eg, GE7 recognizes the EGF receptor overexpressed on tumor cells
  • the Pi of the fusion protein of the present invention should be greater than 7, usually 8-12. 5, preferably 9-11. 5, and more preferably 10-
  • the exogenous DNA that can be used in the present invention is not particularly limited, and may be various therapeutic or preventive DNA, such as a target gene, an antisense oncogene, an anti-oncogene, a suicide gene, a cell apoptosis gene, a cytokine gene, or A combination thereof, or a eukaryotic expression vector DNA containing the aforementioned genes.
  • Proto-oncogene antisense sequences include proto-oncogenes (ras H , ras K , ras N , c-myc, bcl-2, Akt), antisense DNA sequences of growth factors and their receptor genes, and antisense oligonucleotides And antisense oligodeoxynucleotides; tumor suppressor genes include p53, Rb, PTEN, suicide genes include HSV-TK (herpes simplex virus thymidine kinase) gene and CD (E.
  • proto-oncogenes ras H , ras K , ras N , c-myc, bcl-2, Akt
  • tumor suppressor genes include p53, Rb, PTEN
  • suicide genes include HSV-TK (herpes simplex virus thymidine kinase) gene and CD (E.
  • coli cytosine deaminase gene
  • apoptosis Genes include pl5, pl6 , p21 ffAF - 1
  • cytokine genes include GM-CSF (granulocyte macrophage colony-stimulating factor) gene, TNFa (tumor necrosis factor) gene, INF (interferon) ⁇ , ⁇ gene, IL (interleukin) 2, 3, 4, 12, 18 genes, etc.
  • the gene introduction system of the present invention can be used to treat diseases such as genetic diseases or tumors, especially for gene therapy.
  • the targeted gene introduction system includes a histone, a receptor recognition functional peptide GE7, and an endosome-releasing peptide HA20.
  • the functional peptides GE7 and HA20 also perform their specific functions.
  • the gene introduction system also contains substances that promote endocytosis or stabilize DNA, such as the HA20-histone element or the histone element-HA20 fusion protein. Reorganized production
  • the invention also provides a preparation process for constructing a fusion gene, expressing the fusion protein by using genetic engineering technology, and separating and purifying the fusion protein.
  • the DNA coding sequences of the elements of the present invention can be obtained by PCR using various commercially available cDNA libraries. Or directly obtained by artificial synthesis. LOP, HA20 are connected to the coding sequence of the histone element as the core sequence through several rounds of PCR bypass reactions to form a series of fusion genes expressing each oligopeptide.
  • the polynucleotide sequence of the fusion protein can be inserted into a recombinant expression vector.
  • recombinant expression vector refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, 'retroviruses, or other vectors, which are well known in the art.
  • any plasmid and vector can be used as long as it can be replicated and stabilized in the host.
  • An important feature of expression vectors is that they usually contain origins of replication, promoters, marker genes and translation control elements.
  • E. coli lac or trp promoter eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoters, and other known controllable genes Promoters expressed in prokaryotic or eukaryotic cells or their viruses.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • GFP fluorescent protein
  • tetracycline or ampicillin resistance for E. coli.
  • a vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform an appropriate host cell so that it can express a protein.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a high Eukaryotic cells, such as mammalian cells.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a high Eukaryotic cells such as mammalian cells.
  • Representative examples are: E. coli, Streptomyces; fungal cells such as yeast; plant cells; insect cells of Drosophila S2 or S; animal cells such as CHO and COS cells.
  • a preferred host cell is E. coli.
  • Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote, such as E. coli
  • competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with the CaCl 2 method. The steps used are well known in the art. Another method is to use MgCl 2 . If necessary, transformation can also be performed by electroporation.
  • the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, and liposome packaging.
  • the obtained transformants can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention.
  • the medium used in the culture may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • recombinant polypeptide in the above method can be expressed intracellularly, or on a cell membrane, or secreted extracellularly.
  • recombinant proteins can be isolated and purified by various separation methods using their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation, treatment with a protein precipitant (salting out method), centrifugation, osmotic disruption, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • Pharmaceutical compositions include, but are not limited to: conventional renaturation, treatment with a protein precipitant (salting out method), centrifugation, osmotic disruption, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption
  • the present invention also provides a pharmaceutical composition containing a safe and effective amount of one or more fusion proteins of the present invention and a pharmaceutically acceptable carrier or excipient.
  • a pharmaceutically acceptable carrier or excipient include, but are not limited to: saline, buffers, glucose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should match the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants.
  • the pharmaceutical composition When administered, the pharmaceutical composition also contains one or more of the aforementioned exogenous DNAs.
  • the specific dosage should also consider factors such as the route of administration and the health status of the patient, which are all within the skill of a skilled physician.
  • the main advantage of the invention is that
  • the fusion protein can be ligated to construct an expression plasmid, which can be recombinantly expressed in a large number of host cells such as E. coli, and purified by isolation and purification. It is capable of mass production and is conducive to quality control.
  • the vector system can be used to treat different tumors or non-neoplastic diseases.
  • the present invention is further described below with reference to specific embodiments. It should be understood that these examples are only used to illustrate the present invention It is not intended to limit the scope of the invention.
  • the experimental methods without specific conditions in the following examples are generally based on conventional conditions, for example, Sambrook et al., Molecular Cloning: The conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to manufacturing conditions Conditions recommended by the manufacturer.
  • Example 1 Obtaining the expression sequence of each fusion protein
  • HA20 In HA20, GE7 and HA20 each play independent biological functions.
  • the underlined part is the overlapping sequence in the "bridge reaction", the boxed part is the endonuclease site where the expression sequence is inserted into the expression vector, ATG is the start codon, and TCA is the antisense of the stop codon. sequence. From placenta cDNA library using primers 1 and primer 3 was obtained expressed sequence of histone Hl fl; primer 2 and primer 3 to obtain the PCR protein groups H1 Q 97 - 193 expressed sequences.
  • GE7-linker and linker _HA20 were directly obtained by PCR using primer 4, primer 5, primer 6, and primer 7 PCR.
  • FIG. 1 is a flowchart of constructing a GE7-histone Hl ° -HA20 expression sequence.
  • the expression plasmids were used to transform competent cells expressing the host strain HMS174 (DE3). Competent preparation and transformation conditions were performed according to "Molecular Cloning".
  • the transformed competent bacteria were plated on an ampicillin LB agar plate at 37 ° C overnight. Pick a single colony in 5ml LB containing ampicillin resistance, shake at 37 ° C overnight, dilute 1:50 in 1000ml LB and incubate to a density of 0D600nm to about 0.6, add IPTG to a final concentration of lmM, and induce at 30 ° C 3 hours.
  • Bacteria were collected by centrifugation, and 40 ml of ultrasonic solution (50 mM Tris.Cl, pH 8.0, 10 mM EDTA, 0.5 mM PMSF) was added, and sonication was performed in an ice bath for 1 hour. After sonication, the bacterial solution was centrifuged at high speed, and the supernatant was collected and purified by SP sepharose cation exchange column.
  • ultrasonic solution 50 mM Tris.Cl, pH 8.0, 10 mM EDTA, 0.5 mM PMSF
  • SP sepharose cation exchange column purification protein
  • the SP sepharose cation exchange column was pre-equilibrated with 50 mM Tris-Cl, pH 8.5. The supernatant of the bacterial cells was collected and applied to the cation column. The liquid was naturally drawn out by gravity. The protein to be purified was adsorbed on the SP column because it carried a large positive charge. The effluent was discarded. After exhaustion, add 50 mM Tris_Cl for equilibration to flush the column, so that the liquid remaining in the column is completely replaced by the equilibration buffer. At this time, the UV absorber shows that the absorption curve returns to the baseline.
  • Elution was performed with a gradient of 0- 1M NaCl, 50 mM Tris-Cl, pH 8.5, and the eluent was collected from the appearance of the elution peak. Samples of each elution peak were collected and electrophoresed. The purified protein product was located at the obvious end of the main band. Yifeng.
  • the last eluting peak sample obtained by purifying the SP Sepharose cation exchange column was first concentrated with an ULTRAFREE-15 ultrafiltration device, and then loaded on a Sephadex G-50 molecular sieve.
  • the main peak sample was collected by the absorption curve displayed by an ultraviolet absorber.
  • the molecular sieves were pre-equilibrated with HBS buffer (20 mM HEPES, pH 7. 4, 150 mM NaCl, 20% glycerol), and the collected main peak samples were finally concentrated with a ULTRAFREE-15 ultrafiltration device. Concentrated sample 12 /. SDS electrophoresis was identified and quantified by BCA method. Add protease inhibitor to protein solution (final inhibitor concentration is
  • fusion proteins were expressed, isolated and purified in the same way, including histones H1 Q 97 —lg3 and GE7-histone HI. -HA20, GE7-Histone H1 Q 97 _ 193 -HA20, HA20-Histone H1 Q -GE7, Histone Hl °-GE7, Histone Hl °-HA20, Histone Hl ° 97 _ 193 -GE7, Histone H1 Q 97 _ 193 -HA20.
  • DNA and each fusion protein or a combination of different fusion proteins are mixed at a mass ratio of 1: 3 and left at room temperature for one hour for use in vitro and in vivo biological function tests.
  • the final DNA concentration in the complex was 0.2 ⁇ ⁇ ⁇ 1. See Figure 15-20.
  • Example 4 In vitro introduction test of complex
  • In vitro cell reporter gene introduction experiment trypsinized cells were inoculated into 4xlOV wells in a 24-well plate and incubated at 37 C overnight. The next day, the serum-free culture solution was changed to 1 ml / well, and the prepared complex was added so that the DNA content in the culture solution was 3 g / ml, and incubated overnight. On the third day, the serum-free culture solution containing the complex was removed, and DMEM containing 10% newborn calf serum was replaced. On the fourth day, the culture medium of the test cells was discarded, washed three times with 0.1 M PBS, and fixed at room temperature (fixing solution: 0.2% glutaraldehyde, 2% formaldehyde in 0. M PBS) for 20 minutes.
  • the eukaryotically expressed pSV- ⁇ -gal plasmid DNA is combined with the fusion protein to form a complex (containing ⁇ 0.2 ⁇ ⁇ / 10 ⁇ 1) at an optimal mass ratio of 1: 3, which is used for in vitro cell introduction experiments.
  • a complex containing ⁇ 0.2 ⁇ ⁇ / 10 ⁇ 1 at an optimal mass ratio of 1: 3, which is used for in vitro cell introduction experiments.
  • the expression in cells was used to evaluate the biological activity of the fusion protein. Twenty-four hours after inoculation of BEL-7402 cells, the complex was added to the culture solution, and 1 ml of the culture solution was incubated with DNA for 3 ⁇ overnight. After 24 hours (48 hours after cell transfection), X-gal in situ cell staining analysis was performed.
  • Fusion protein mediated ⁇ -gal gene transfection of U20S cells that do not express EGF receptor as a negative control for in vitro cell transfection experiments fusion protein mediated ⁇ -gal transfection of BEL-7402 cells with positive EGF receptor expression as an experiment group.
  • Figure 21 of the transfection results Protein combination GE7-Histone Hl °-HA20 / Histone H1 Q -GE7 (1: 1), Histone H1 Q -GE7 / Histone ⁇ 1 °-HA20 (1: 1) can introduce ⁇ -gal gene into BEL- In 7402 cells, ⁇ -gal gene was expressed, X-gal staining positive cells (cells turned blue), GE7-histone Hl fl -HA20 / histone
  • H1 Q -GE7 was better than the combination of histone H1 Q -GE7 / histone Hl fl -HA20, and no other X-gal staining cells appeared in other fusion protein groups. U20S cells with negative EGF receptor expression were all negative. Each fusion protein and combination was repeated 10 times, and the results of the in vitro cell introduction experiment are summarized in Table 1.
  • Nude mouse transplantation tumor reporter gene introduction experiment method is as follows: Subcutaneous tumor-bearing nude mice are injected with a complex prepared by the subcutaneous tumor route, the complex is diluted with physiological saline in advance according to the required injection amount, each mouse is disposable The injection volume does not exceed 100 ⁇ l (containing complex 50 ⁇ 1, plasmid pSV- ⁇ -gal 1 ⁇ ⁇ ). Twenty-four hours after tumor injection, sacrifice nude mice, strip tumors, rinse three times in PBS for 15 minutes each. Add freshly prepared fixative solution and fix at 4 ° C for 2 hours. After fixation, wash the PBS three times for 15 minutes each time.
  • Subcutaneously transplanted human liver cancer BEL-7402 tumor tissues in nude mice have positive EGF receptor expression and can be used as target tissues for fusion protein-mediated gene transfer.
  • Subcutaneously in nude mice transplanted with human hepatoma BEL- 7402 the tumor grew to a size of 0. 5cm, peritumoral subcutaneously injected once with various pSV- ⁇ - gal fusion proteins form a complex (containing plasmid pSV-p-gal 1 ⁇ ⁇ ), The complex was diluted with physiological saline to 100 ⁇ 1 before injection. Nude mice were sacrificed 24 hours later, tumors were stripped, and X-gal stained after fixation to observe the introduction of pSV- ⁇ -gal.
  • each fusion protein mediates ⁇ -gal gene transfer in nude mice subcutaneously transplanted with human liver cancer BEL-7402 in each group, the pSV- ⁇ -gal plasmid group alone and the separate group There were no blue-stained cells in the tumor tissues of the protein H1 Q group and the histone H1 Q -HA20 group alone, that is, there was no ⁇ -gal gene expression.
  • the histone H1 Q -GE7 group alone, the GE7-histone Hl °- HA20 Group and HA20-histone H1 Q -GE7 group had a small amount of ⁇ -gal gene expression in tumor tissues, the number of blue-stained cells was small (about 20%), the color was lighter, the histone H1 ° -GE7 and histone H1 After mixing Q -HA20, the number of blue-stained cells increased (approximately 50%) and the blue color was slightly darker.
  • GE7-Histone Hl fl- HA20 and Histone H1 Q- GE7 were mixed in equal amounts to mediate DNA and tumor tissues. There are a large number of ⁇ -gal gene expressions.
  • SK0V3 subcutaneously transplanted tumor has a fast growth rate, and is a good tissue material for in vivo biological activity detection of fusion proteins.
  • the complex prepared by the subcutaneous tumor route injection the DNA content of the complex injected by each animal is 1 ⁇ ⁇ .
  • the expressions of introduced genes were observed at 24 hours, 72 hours, 96 hours, 7 days, 10 days, and 14 days, respectively.
  • Peritumoral injection through subcutaneous routes composite preparation each animal was injected DNA complexes respectively containing an amount of 0. 2 g, 0. 5 ⁇ ⁇ , 1 ⁇ ⁇ , nude mice were sacrificed after 24 hours stripping tumors, fixed, stained Observe the situation of gene introduction and expression.
  • ⁇ -gal gene mediated by the protein combination GE7-Histone Hl °- ⁇ 20 / Histone ⁇ 1 ⁇ -GE7 in SK0V3 tumors
  • the expression of tissues changed at different time points.
  • the reporter gene was expressed 12 hours after injection. The expression reached a peak at 24 hours. The expression began to decrease at 48 hours. The expression continued to decrease at 96 hours, 7 days, and 10 days, and remained at 14 days. There is a small amount of expression. See Figure 24.

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Abstract

The invention relates to a gene entrain system with the core sequence of histone, its method and use. The gene entrain system include the fusion protein with the core sequence of histone, comprising (a) histone component, it choose from H1° full length sequence or the fragment of enrichment lysine; (b) receptor recognition oligopeptide component LOP; (c) any coated vesicle release oligopeptide EROP. The invention relates to a gene entrain system can not only highly efficiency transfer foreign DNA, but also preparation by gene engineering technical.

Description

以组蛋白为核心序列的靶向性基因导入*** 技术领域  Targeted gene introduction system using histone as core sequence Technical field
本发明涉及分子生物学和基因治疗领域， 更具体地， 涉及以组蛋白为核心序列的基 因导入***、 及其制法和用途。 背景技术  The present invention relates to the fields of molecular biology and gene therapy, and more particularly, to a gene introduction system using a histone as a core sequence, and a preparation method and use thereof. Background technique
基因治疗是将外源 DNA导入人体的特定细胞以产生治疗效果， 达到治疗疾病的目 的。 为此,首先需具备安全有效的基因转移***.目前主要有两大类方法可以有效地介导 外源 DNA转入人体细胞，一类是病毒载体介导的方法,另一类是非病毒载体介导的方法。  Gene therapy is to introduce foreign DNA into specific cells of the human body to produce a therapeutic effect and achieve the purpose of treating diseases. For this purpose, a safe and effective gene transfer system is required first. Currently, there are two main types of methods that can effectively transfer foreign DNA into human cells. One is a viral vector-mediated method and the other is a non-viral vector Leading method.
Fritz等人曾在大肠杆菌体内表达了组蛋白 Hl°的 C -末端区， 但必须在脂质体与氯 喹的参与下， 才能将 DNA导入了真核细胞（Hum. Gene Ther. 1996, 7： 1395-1404) 0 此 夕卜， 该***无靶向性。 Fritz et al. Once expressed the C-terminal region of the histone Hl ° in E. coli, but the liposome and chloroquine must be involved in order to introduce DNA into eukaryotic cells (Hum. Gene Ther. 1996, 7: 1395-1404) 0 Bu this evening, the system has no targeting.
国际出版物 W098/18951 (PCT/CN97/00106) 公开了一种由配体寡肽 /多聚阳离子 多肽 /内吞小体释放寡肽 /外源 DNA组成的四元复合体基因转移***， 或由配体寡肽 /多 聚阳离子多肽 /外源 DNA组成的三元复合体基因转移***。该***对肿瘤细胞有靶向性， 可高效抑制肿瘤细胞生长。 然而， 该专利申请的缺点是， 多聚阳离子多肽需化学合成， 然后再与配体寡肽、 内吞小体释放寡肽等连接， 因此制备工艺复杂， 不适合大规模生产。  International publication W098 / 18951 (PCT / CN97 / 00106) discloses a quaternary complex gene transfer system consisting of ligand oligopeptides / polycationic peptides / endosome release oligopeptides / foreign DNA, or Ternary complex gene transfer system consisting of ligand oligopeptide / polycationic polypeptide / foreign DNA. The system is targeted to tumor cells and can effectively inhibit tumor cell growth. However, the patent application has the disadvantage that the polycationic polypeptide needs to be chemically synthesized and then linked with a ligand oligopeptide, an endosome-released oligopeptide, etc., so the preparation process is complicated and is not suitable for large-scale production.
因此，本领域迫切需要开发新的制法简便，适合大规模生产的靶向性基因导入***。 发明内容  Therefore, there is an urgent need in the art to develop a targeted gene introduction system that is simple and suitable for large-scale production. Summary of the Invention
本发明的目的就是提供一种制法简便，适合大规模生产的靶向性基因导入***及其 制法和用途。 在本发明的第一方面， 提供了一种融合蛋白， 它含有  The purpose of the present invention is to provide a targeted gene introduction system which is simple and convenient for manufacturing and suitable for large-scale production, and a method for preparing and using the same. In a first aspect of the present invention, a fusion protein is provided, which contains
(a)组蛋白元件， 所述的组蛋白元件选自 Hl°全长序列或其富含赖氨酸的片段； (a) a histone element selected from the Hl ° full-length sequence or a lysine-rich fragment thereof;
(b) 受体识别寡肽元件 LOP。 (b) Receptor recognition oligopeptide element LOP.
在一优选例中，所述的融合蛋白在 (a)组蛋白元件和 (b) 受体识别寡肽元件之间有连 接肽。  In a preferred example, the fusion protein has a connecting peptide between (a) a histone element and (b) a receptor recognition oligopeptide element.
在另一优选例中，所述的融合蛋白还含有 (c)内吞小体释放寡肽元件，和任选的位于 In another preferred example, the fusion protein further comprises (c) an endosome-releasing oligopeptide element, and optionally
(a)组蛋白元件， (b) 受体识别寡肽元和 (c)内吞小体释放寡肽元件之间的连接肽。 (a) a histone element, (b) a linker peptide between a receptor-recognizing oligopeptide and (c) an endosome-releasing oligopeptide element.
在本发明的第二方面， 提供了一种受体介导的靶向性肿瘤基因治疗的基因导入系 统， 包含上述的融合蛋白和外源 DNA， 所述的外源 DNA是含选自下组基因的真核表达 载体 DNA: 反义癌基因、 抗癌基因、 ***基因、 细胞调亡基因、 细胞因子基因、 或其组 合。  In a second aspect of the present invention, a gene-introduction system for receptor-mediated targeted tumor gene therapy is provided, comprising the above-mentioned fusion protein and foreign DNA, and the foreign DNA is selected from the group consisting of Eukaryotic expression vector DNA of genes: antisense oncogene, anti-oncogene, suicide gene, apoptosis gene, cytokine gene, or a combination thereof.
在一优选例中， 所述的融合蛋白选自下组： LOP-组蛋白元件 -HA20、 HA20-组蛋白 元件 -LOP、 组蛋白元件 -LOP、 LOP-组蛋白元件， 并且该***还含有任选的 HA20-组蛋 白元件或组蛋白元件 -HA20融合蛋白。  In a preferred example, the fusion protein is selected from the following group: LOP-Histone Element-HA20, HA20-Histone Element-LOP, Histone Element-LOP, LOP-Histone Element, and the system further contains any Selected HA20-histone element or histone element-HA20 fusion protein.
在本发明第三方面，提供了一种药物组合物， 它上述的融合蛋白和药学上可接受的 载体。 In a third aspect of the present invention, there is provided a pharmaceutical composition comprising the above-mentioned fusion protein and a pharmaceutically acceptable Carrier.
在本发明第四方面， 提供了一种分离的、 编码上述融合蛋白的 DNA， 含所述 DNA 的表达载体， 以及含所述 DNA或表达载体的宿主细胞。  In a fourth aspect of the present invention, there is provided an isolated DNA encoding the fusion protein, an expression vector containing the DNA, and a host cell containing the DNA or the expression vector.
在本发明的第五方面， 提供了一种产生本发明融合蛋白的方法， 包括步骤： 在表达 条件下，培养上述转化的宿主细胞从而表达出所述的融合蛋白；和分离所述的融合蛋白。 附图说明  In a fifth aspect of the present invention, there is provided a method for producing a fusion protein of the present invention, comprising the steps of: culturing the transformed host cell under expression conditions to express the fusion protein; and isolating the fusion protein. . BRIEF DESCRIPTION OF THE DRAWINGS
图 1是构建 GE7-组蛋白 H1°-HA20表达序列的流程图。  Figure 1 is a flowchart of the construction of the GE7-histone H1 ° -HA20 expression sequence.
图 2显示了组蛋白 H1Q的 DNA核苷酸序列及氨基酸序列。 Figure 2 shows the DNA nucleotide sequence and amino acid sequence of histone H1 Q.
图 3显示了组蛋白 H1Q 97_193的 DNA核苷酸序列及氨基酸序列。 Figure 3 shows the DNA nucleotide sequence and amino acid sequence of histone H1 Q 97 _193 .
图 4显示了融合蛋白 GE7-组蛋白 H1Q- HA20的 DNA核苷酸序列及氨基酸序列。 Figure 4 shows the DNA nucleotide sequence and amino acid sequence of the fusion protein GE7-Histone H1 Q -HA20.
图 5显示了融合蛋白 GE7 组蛋白 Hl°97_193- HA20的 DNA核苷酸序列及氨基酸序列。 图 6显示了融合蛋白组蛋白 H1Q-GE7的 DNA核苷酸序列及氨基酸序列。 Figure 5 shows the DNA nucleotide sequence and amino acid sequence of the fusion protein GE7 histone Hl ° 97 _ 193 -HA20. Figure 6 shows the DNA nucleotide sequence and amino acid sequence of the fusion protein histone H1 Q- GE7.
图 7显示了融合蛋白组蛋白 H1Q-HA20的 DNA核苷酸序列及氨基酸序列。 Figure 7 shows the DNA nucleotide sequence and amino acid sequence of the fusion protein histone H1 Q- HA20.
图 8显示了融合蛋白组蛋白 Hl°97—193- GE7的 DNA核苷酸序列及氨基酸序列。 Figure 8 shows the fusion protein histone Hl ° 97 - DNA nucleotide sequence and amino acid sequence of GE7 --193.
图 9显示了融合蛋白组蛋白 H1Q 97― 193- HA20的 DNA核苷酸序列及氨基酸序列。 Figure 9 shows the 97 fusion protein histone H1 Q - 193 - DNA nucleotide sequence and amino acid sequences of HA20.
图 10显示了融合蛋白 HA20 -组蛋白 H1Q- GE7的 DNA核苷酸序列及氨基酸序列。 图 11显示了原核表达载体 PT7450的质粒图谱。 Figure 10 shows the DNA nucleotide sequence and amino acid sequence of the fusion protein HA20-histone H1 Q -GE7. Figure 11 shows the plasmid map of the prokaryotic expression vector PT7450.
图 12显示了经纯化后 4个蛋白的终产物： 泳道 1是 GE7-组蛋白 H1Q 97_193-HA20 (15kD) ， 泳道 2是组蛋白 H1Q 97_193 (10kD) ， 泳道 3是 GE7-组蛋白 H1°-HA20 (25kD)， 泳道 4是组蛋白 H1Q (21kD) ， M代表蛋白低分子量标记。 Figure 12 shows the four proteins purified end product: Lane 1 is GE7- histone H1 Q 97 _ 193 -HA20 (15kD ), lane 2 is histone H1 Q 97 _ 193 (10kD) , lane 3 is GE7 -Histone H1 ° -HA20 (25kD), lane 4 is histone H1 Q (21kD), and M represents a protein low molecular weight marker.
图 13显示了经纯化后 3个蛋白的终产物：泳道 1是 HA20-组蛋白 Η - GE7(25kD)， 泳道 2是组蛋白 Hl°- GE7 (23. IkD) ， 泳道 3是组蛋白 Hlfl-HA20 (23.4kD) ， M代表蛋 白低分子量标记。 Figure 13 shows the final products of three proteins after purification: lane 1 is HA20-Histone-GE7 (25kD), lane 2 is histone Hl ° -GE7 (23. IkD), and lane 3 is histone Hl fl -HA20 (23.4kD), M stands for protein low molecular weight marker.
图 14显示了经纯化后 2个蛋白的终产物：泳道 1是组蛋白 Hlfl 97_193_HA20(13. IkD), 泳道 3是组蛋白 Hlfl 97_193-GE7 (12.8kD) ， M代表蛋白低分子量标记。 Figure 14 shows the final products of the two proteins after purification: lane 1 is histone Hl fl 97 _ 193 _HA20 (13. IkD), lane 3 is histone Hl fl 97 _ 193 -GE7 (12.8kD), M represents Low molecular weight labeling of proteins.
图 15显示了 GE7-组蛋白 H1Q- HA20与 DNA形成的二元复合体基因转移***的 1% 琼脂糖凝胶电泳情况。 M代表 λ/HindIII DNA分子量标记； 泳道 1是质粒 DNA; 泳道 2, 3， 4, 5， 6， 7分别代表基因转移***中 DNA:GE7-组蛋白 H1Q- HA20的质量比为 1： 0.5， 1:1， 1:1.5， 1:2， 1:2.5, 1:3。 1:3是最佳配比。 Figure 15 shows the 1% agarose gel electrophoresis of a binary complex gene transfer system formed by GE7-histone H1 Q -HA20 and DNA. M stands for λ / HindIII DNA molecular weight marker; lane 1 is plasmid DNA; lanes 2, 3, 4, 5, 6, 7 respectively represent the DNA: GE7-histone H1 Q -HA20 mass ratio in the gene transfer system of 1: 0.5 , 1: 1, 1: 1.5, 1: 2, 1: 2.5, 1: 3. 1: 3 is the best ratio.
图 16显示了 GE7-组蛋白 H1Q 97_193-HA20与 DNA形成的二元复合体基因转移***的 1%琼脂糖凝胶电泳情况。 M代表 λ/HindIII DNA分子量标记； 泳道 1是质粒 DNA; 泳道 2，3，4，5，6分别代表基因转移***中0 :6£7-组蛋白 H1Q 97_193- HA20的质量比为 1:0.5， 1:1， 1:2， 1:2.5， 1:3ο 1:3是最佳配比。 Figure 16 shows the 1% agarose gel electrophoresis of a binary complex gene transfer system formed by GE7-histone H1 Q 97 _ 193 -HA20 and DNA. M stands for λ / HindIII DNA molecular weight marker; lane 1 is plasmid DNA; lanes 2, 3, 4, 5, 6 represent 0: 6 £ 7-histone H1 Q 97 193 -HA20 in the gene transfer system, respectively 1: 0.5, 1: 1, 1: 2, 1: 2.5, 1: 3ο 1: 3 are the best ratios.
图 17显示了组蛋白 H1Q与 DNA形成的二元复合体基因转移***的 1%琼脂糖凝胶电 泳情况。 M代表 λ/HindIII DNA分子量标记； 泳道 1是质粒 DNA; 泳道 2， 3, 4， 5， 6 分别代表基因转移***中 DNA:组蛋白 Η1β的质量比为 1:0.5， 1:1， 1:2， 1:2.5, 1:3。 Figure 17 shows the 1% agarose gel electrophoresis of the binary complex gene transfer system of histone H1 Q and DNA. M stands for λ / HindIII DNA molecular weight marker; lane 1 is plasmid DNA; lanes 2, 3, 4, 5, 6 respectively represent the DNA: histoneΗ1 β mass ratio in the gene transfer system of 1: 0.5, 1: 1, 1 : 2, 1: 2.5, 1: 3.
1:3是最佳配比。 1: 3 is the best ratio.
图 18显示了组蛋白 H1Q 97_193与 DNA形成的二元复合体基因转移***的 1%琼脂糖凝 胶电泳情况。 M代表 λ/HindIII DNA分子量标记； 泳道 1是质粒 DNA; 泳道 2， 3， 4, 5， 6分别代表基因转移***中 DNA:组蛋白 Hl°97_193的质量比为 1 : 0. 5， 1 : 1 , 1 : 2， 1 : 2. 5， 1 : 3。 1 : 3是最佳配比。 Figure 18 shows the 1% agarose coagulation of the binary complex gene transfer system of histone H1 Q 97 _ 193 and DNA Gel electrophoresis. M represents λ / HindIII DNA molecular weight markers; Lane 1 is the DNA plasmid; lane 2, 3, 4, 5, 6 represent the gene transfer system of DNA: mass Histone Hl ° 97 _ 193 ratio was 1: 0.5, 1: 1, 1: 2, 1: 2.5, 1: 3. 1: 3 is the best ratio.
图 19显示了其他融合蛋白与 DNA形成的二元复合体基因转移***的 1%琼脂糖凝胶 电泳情况。 M代表 λ/Hindin DNA分子量标记； 泳道 1是质粒 DNA; 泳道 2、 3、 4、 5、 6分别为融合蛋白组蛋白 H1Q-GE7、 组蛋白 HI0-翻、 组蛋白 H1°97_193_GE7、 组蛋白 Hl°97-i93-HA20 HA20-组蛋白 H1Q- GE7与 DNA形成的二元复合体， 基因转移***中 DNA与 各融合蛋白的质量比均为 1 : 3。 Figure 19 shows the 1% agarose gel electrophoresis of a binary complex gene transfer system formed by other fusion proteins and DNA. M stands for λ / Hindin DNA molecular weight marker; lane 1 is plasmid DNA; lanes 2, 3, 4, 5, and 6 are the fusion protein histone H1 Q- GE7, histone HI 0 -turn, histone H1 ° 97 _ 193 _GE7, Histone Hl ° 97-i93-HA20 HA20-Histone H1 Q -GE7 is a binary complex formed with DNA. The mass ratio of DNA to each fusion protein in the gene transfer system is 1: 3.
图 20显示了不同融合蛋白的组合与 DNA形成的三元复合体基因转移***的 1%琼脂 糖凝胶电泳情况。 M代表 λ/HindIII DNA分子量标记； 泳道 1是质粒 DNA; 泳道 2、 3、 4分别为不同融合蛋白的组合 GE7-组蛋白 H1G- HA20/组蛋白 Hl°- GE7 ( 1 : 1等量混合） 、 组蛋白 H'1Q-GE7/组蛋白 Hl°- HA20 ( 1 : 1等量混合） 、 组蛋白 H1Q 97_193 - GE7/组蛋白 H1°97.193-HA20 ( 1 : 1等量混合）与 DNA形成的三元复合体， 基因转移***中 DNA与各融合 蛋白组合的质量比均为 1 : 3。 Figure 20 shows the 1% agarose gel electrophoresis of the ternary complex gene transfer system formed by the combination of different fusion proteins and DNA. M stands for λ / HindIII DNA molecular weight marker; lane 1 is plasmid DNA; lanes 2, 3, and 4 are combinations of different fusion proteins, respectively GE7-Histone H1 G -HA20 / Histone Hl °-GE7 (1: 1 equal amount mixed ) 、 Histone H'1 Q -GE7 / Histone Hl °-HA20 (1: 1 equal volume mixing), Histone H1 Q 97 _ 193 -GE7 / Histone H1 ° 97. 193 -HA20 (1: 1 etc. In the ternary complex formed with DNA, the mass ratio of the combination of DNA and each fusion protein in the gene transfer system is 1: 3.
图 21 显示了融合蛋白组合 GE7-组蛋白 Η - HA20/组蛋白 H1Q - GE7、 组蛋白 Hl°-Figure 21 shows the fusion protein combination GE7-Histone-HA20 / Histone H1 Q -GE7, Histone Hl °-
GE7/组蛋白 H1Q- HA20将 β- gal基因导入 BEL- 7402细胞而不能导入 U20S细胞的结果。 A、 B、 C均为 U20S细胞， D、 E、 F均为 BEL- 7402细胞。 A、 D是单纯 β- gal基因组， B、 E 是 β-gal基因 /组蛋白 Hl°- GE7/组蛋白 Hl°- HA20组， C、 F是 β - gal基因 /GE7-组蛋白 Hl°- HA20/组蛋白 Hl°- GE7组。 GE7 / Histone H1 Q -HA20 results in the introduction of β-gal gene into BEL-7402 cells but not U20S cells. A, B, and C are U20S cells, and D, E, and F are BEL-7402 cells. A and D are pure β-gal genomes, B and E are β-gal genes / histone Hl ° -GE7 / histone Hl ° -HA20 group, C and F are β-gal genes / GE7-histone Hl °- HA20 / Histone H ° -GE7 group.
图 22显示了几种融合蛋白及组合将 β- gal基因导入荷人肝癌 BEL- 7402裸鼠的肿瘤 大体及病理切片的 X- gal染色结果。 A是单纯的 β- gal基因， B是 β-gal基因 /组蛋白 H1Q 组， C是 β - gal基因 /组蛋白 Hl°- HA20组， D是 β- gal基因 /组蛋白 Hl°- GE7组， E是 β- gal 基因 /GE7-组蛋白 H1°-HA20组， F是 β-gal基因 /HA20-组蛋白 H1°-GE7组， G是 β-gal基 因 /组蛋白 Hl°- GE7/组蛋白 H1Q- HA20， H是 β- gal基因 /GE7 -组蛋白 Η - HA20/组蛋白 Hl°- GE7组。 病理切片的右上角或左下角， 分别附有各肿瘤大体的 X-gal染色情况。 Figure 22 shows the results of X-gal staining of tumor gross and pathological sections of β-gal gene introduced into human liver cancer BEL-7402 nude mice with several fusion proteins and combinations. A is pure β-gal gene, B is β-gal gene / histone H1 Q group, C is β-gal gene / histone Hl ° -HA20 group, D is β-gal gene / histone Hl °-GE7 Group, E is β-gal gene / GE7-Histone H1 ° -HA20 group, F is β-gal gene / HA20-Histone H1 ° -GE7 group, G is β-gal gene / Histone Hl ° -GE7 / Histone H1 Q -HA20, H is β-gal gene / GE7-Histone-HA20 / Histone Hl ° -GE7 group. The upper right corner or lower left corner of the pathological section were respectively attached with the X-gal staining of each tumor.
图 23显示了几种融合蛋白及组合将 β- gal基因导入荷人卵巢癌 SK0V3裸鼠的肿瘤 大体及病理切片的 X-gal染色结果。 A是单纯的 β-gal基因， B是 β-gal基因 /组蛋白 Hlfl 组， C是 β- gal基因 /组蛋白 H1Q- HA20组， D是 β- gal基因 /组蛋白 H1°-GE7组， E是 β-gal 基因 /GE7-组蛋白 Hl°- HA20组， F是 β-gal基因 /HA20-组蛋白 HI0- GE7组， G是 β-gal基 因 /组蛋白 H1°-GE7/组蛋白 H1°-HA20, H是 β-gal基因 /GE7-组蛋白 Η1°- HA20/组蛋白 Η1° - GE7组。 病理切片的右上角或左下角， 分别附有各肿瘤大体的 X- gal染色情况。 Figure 23 shows the results of X-gal staining of tumor gross and pathological sections of the β-gal gene introduced into human ovarian cancer SK0V3 nude mice by several fusion proteins and combinations. A is pure β-gal gene, B is β-gal gene / histone H1 fl group, C is β-gal gene / histone H1 Q -HA20 group, D is β-gal gene / histone H1 ° -GE7 Group, E is β-gal gene / GE7-Histone Hl ° -HA20 group, F is β-gal gene / HA20-Histone HI 0 -GE7 group, G is β-gal gene / Histone H1 ° -GE7 / Histone H1 ° -HA20, H is β-gal gene / GE7-Histone Η1 ° -HA20 / Histone Η1 °-GE7 group. The upper right corner or lower left corner of the pathological section were respectively attached with the X-gal staining of each tumor.
图 24显示了融合蛋白组合 GE7-组蛋白 H1Q- HA20/组蛋白 H1Q- GE7将 β- gal基因导 入荷人卵巢癌 SK0V3裸鼠后， 导入的 β- gal基因在肿瘤组织中的表达随时间的推移而变 化的情况。 A是 β-gal基因导入后 12小时， B是 β- gal基因导入后 24小时， C是 β- gal 基因导入后 48小时， D是 β- gal基因导入后 96小时， E是 β- gal基因导入后 7天， F是 β - gal基因导入后 10天， G是 β-gal基因导入后 14天， H是注射生理盐水 24小时的对 照组。 Figure 24 shows that the fusion protein combination GE7-Histone H1 Q -HA20 / Histone H1 Q -GE7 introduced the β-gal gene into nude mice bearing human ovarian cancer SK0V3, and the expression of the introduced β-gal gene in tumor tissues followed Changes over time. A is 12 hours after β-gal gene introduction, B is 24 hours after β-gal gene introduction, C is 48 hours after β-gal gene introduction, D is 96 hours after β-gal gene introduction, and E is β-gal gene. Seven days after the introduction, F was 10 days after the β-gal gene was introduced, G was 14 days after the β-gal gene was introduced, and H was a control group injected with physiological saline for 24 hours.
图 25显示了注射不同剂量的 β- gal基因 / GE7-组蛋白 H1Q- HA20/组蛋白 H1Q- GE7 复合体肿瘤组织的 X- gal染色情况。 A是 0. 2μ§组， Β是 0. 5μ§组， C是 1μ§组。 具体实施方式 Figure 25 shows the X-gal staining of tumor tissues injected with different doses of β-gal gene / GE7-Histone H1 Q -HA20 / Histone H1 Q -GE7 complex. A is 0. 2μ § group, Β is 0. 5μ § group, C is 1μ § group. detailed description
本发明人经过广泛而深入的研究， 发现组蛋白（尤其是组蛋白 HI)及其富含赖氨酸 的片段非常适合作为靶向性基因导入***中的核心序列， 而且构建的融合蛋白非常适合 用重组方法制备。 在此基础上完成了本发明。 组蛋白元件  After extensive and in-depth research, the present inventors found that histones (especially histone HI) and lysine-rich fragments thereof are very suitable as core sequences of targeted gene introduction systems, and the constructed fusion protein is very suitable Prepared by recombinant methods. The present invention has been completed on this basis. Histone element
如本文所用， "组蛋白 H1Q"指 Doenecke D 等 ( J. Mol. Biol. 1986, 187， 461 ) 发现的存在于人分化终末期细胞中的组蛋白 H1Q亚型 （基因号 X03473 ) 。 组蛋白 H1Q富 含赖氨酸， 尤其是 C-末端部分 （Ser97- Lys193) 。 As used herein, "histone H1 Q " refers to the histone H1 Q subtype (gene number X03473) found in human end-stage differentiation cells as found by Doenecke D et al. (J. Mol. Biol. 1986, 187, 461). Histone H1 Q is rich in lysine, especially the C-terminal part (Ser 97 -Lys 193 ).
如本文所用， "组蛋白 H1Q 97— 193 "指组蛋白 111°的 C -末端部分 （Ser97- Lys193) 。 组蛋白是一类富含碱性氨基酸的蛋白， 碱性氨基酸比例约 30%以上， 带正电荷， 能 与带负电荷的 DNA藉静电引力结合， 参与染色体结构的形成， HI组蛋白与形成核小体的 核心组蛋白 （core histone) H2A、 H2B、 H3、 H4不同， 在于它与核小体之间的连接 DNA 结合，独立于核小体之外，因此也叫连接组蛋白（linker histone；)。 Chen J等（Hum. Gene Ther, 5， 429, 1994) 曾经通过化学偶联的方法将提纯的五种组蛋白 （Hl、 H2A、 H2B、 H3、 H4) 分别半乳糖基化作为靶向于去唾液酸糖蛋白受体的导入***， 转染 HepG2细胞 (表面表达去唾液酸糖蛋白受体）后， 发现 HI组蛋白组报告基因的表达高于其他组蛋白 亚型组。 As used herein, "histone H1 Q 97 - 193" C 111 ° histone - terminal portion (Ser 97 - Lys 193). Histones are a class of proteins rich in basic amino acids. The proportion of basic amino acids is about 30%. They are positively charged and can be combined with negatively charged DNA by electrostatic attraction to participate in the formation of chromosomal structures. The core histones of the corpuscle H2A, H2B, H3, and H4 are different in that it binds to the connecting DNA between the nucleosomes and is independent of the nucleosomes, so it is also called linker histone; ). Chen J et al. (Hum. Gene Ther, 5, 429, 1994) used chemical coupling methods to target five purified histones (Hl, H2A, H2B, H3, H4) to galactosylation as targets for The sialic glycoprotein receptor introduction system, after transfection of HepG2 cells (surface expressing asialoglycoprotein receptor), found that the expression of the HI histone group reporter gene was higher than other histone subtype groups.
HI组蛋白包括了多个亚型， 但这些亚型都由 3个区域组成： N-末端区， 中心的球 形结构域及 C-末端区。 N-末端区和中心的球形结构域是 HI组蛋白的疏水性结构， C -末 端区则因富含亲水性的碱性氨基酸包括 Lys、 Arg而成为亲水区（J Mol  HI histones include multiple subtypes, but these subtypes are composed of three regions: the N-terminal region, the central globular domain, and the C-terminal region. The N-terminal region and the central globular domain are the hydrophobic structures of HI histones, while the C-terminal region becomes a hydrophilic region due to the richness of hydrophilic basic amino acids including Lys and Arg (J Mol
Biol. 1986， 187 :461)。 Biol. 1986, 187: 461).
适用于本发明的组蛋白元件包括 HiQ全长序列或其富含赖氨酸的片段。对于 mQ 的富 含赖氨酸的片段没有特别限制， 只要含有足够的碱性氨基酸（赖氨酸、 精氨酸） ， 从而带有 阳离子即可。通常， 碱性氨基酸数量为约 15-85%， 较佳地为约 20-60%， 更佳地约 25-50%。 一种优选的片段是含 Hl°中第 97-193位氨基酸的片段。组蛋白元件在融合蛋白中作为与 DNA 分子结合的核心序列部分。 在其 N-末端和 /或 C-末端可连接受体识别寡肽 （L0P) 。 Histone elements suitable for use in the present invention include Hi Q full-length sequences or lysine-rich fragments thereof. There is no particular limitation on the lysine-rich fragment of m Q , as long as it contains enough basic amino acids (lysine, arginine) to carry cations. Generally, the number of basic amino acids is about 15-85%, preferably about 20-60%, and more preferably about 25-50%. A preferred fragment is a fragment containing amino acids 97-193 in Hl °. Histone elements are used in fusion proteins as part of the core sequence that binds to DNA molecules. A receptor recognition oligopeptide (LOP) can be attached to its N-terminus and / or C-terminus.
组蛋白元件可以源自人的组蛋白， 也可以其他天然物种的碱性蛋白，例如其他哺乳 动物、 微生物、 病毒的核内蛋白。 受体识别寡肽元件  Histone elements can be derived from human histones, or basic proteins from other natural species, such as other mammalian, microbial, and viral nuclear proteins. Receptor recognition oligopeptide element
如本文所用， "配体寡肽 （Ligand oligopeptide, LOP) "和 "受体识别寡肽"可 互换使用， 指任何识别并结合于靶细胞的寡肽。  As used herein, "ligand oligopeptide (LOP)" and "receptor recognition oligopeptide" are used interchangeably and refer to any oligopeptide that recognizes and binds to a target cell.
适用于本发明的受体识别寡肽元件是提供靶向性的元件，它可以是任何识别并结合 于靶细胞的寡肽，代表性的例子包括 (但并不限于)： 与生长因子受体 IGF- II R、 IGF- I R、 EGF R、 VEGF R特异结合的 E5， GE7、 GV1、 GV2、 或其组合。 受体识别寡肽 E5的氨基 酸序列为 EPFRS PDLAL ETYG (SEQ ID NO : 26) , 受体识别寡肽 GE7的氨基酸序列为 NPVVG YIGER PQYRD L (SEQ ID NO : 27) , 受体识别寡肽 GV1的氨基酸序列为 CHPIE TLVDI FQEYP DEIEY IFKPS PVPLM RP (SEQ ID NO : 28)， 受体识别寡肽 GV2的氨基酸序列为 PVPTE ESNIT MQIMR IKPHQ GQHIG EMSFL QHNKC E (SEQ ID NO : 29) a 特别优选的受体识别寡肽的 GE7。 The receptor recognition oligopeptide element suitable for the present invention is a targeting element, which can be any oligopeptide that recognizes and binds to target cells. Representative examples include (but are not limited to): IGF-II R, IGF-IR, EGF R, VEGF R specifically binds E5, GE7, GV1, GV2, or a combination thereof. The amino acid sequence of the receptor recognition oligopeptide E5 is EPFRS PDLAL ETYG (SEQ ID NO: 26), the amino acid sequence of the receptor recognition oligopeptide GE7 is NPVVG YIGER PQYRD L (SEQ ID NO: 27), and the receptor recognition oligopeptide GV1 Amino acid sequence is CHPIE TLVDI FQEYP DEIEY IFKPS PVPLM RP (SEQ ID NO: 28), the amino acid sequence of the receptor recognition oligopeptide GV2 is PVPTE ESNIT MQIMR IKPHQ GQHIG EMSFL QHNKC E (SEQ ID NO: 29) a particularly preferred receptor recognition oligopeptide GE7.
关于这些合成寡肽制法和一般情况， 在 W098/18951 (PCT/CN97/00106)中已有详 细阐述。  These synthetic oligopeptide preparation methods and general conditions are described in detail in W098 / 18951 (PCT / CN97 / 00106).
此外， 所述的配体寡肽功能域也可以是与 E5、 GE7、 GV1、 GV2具有相同免疫决 定簇， 能与上述功能域相应抗体起阳性反应， 并能与 IGF-I R、 IGF-II R, EGF R、 VEGF R结合的多肽与寡肽所形成的功能域。  In addition, the ligand oligopeptide functional domain may also have the same immunodeterminant with E5, GE7, GV1, and GV2, can react positively with the corresponding antibodies of the above functional domains, and can interact with IGF-IR, IGF-II R, EGF R, VEGF R binds to the functional domain formed by the polypeptide and oligopeptide.
所述的配体寡肽还包括与造血细胞、 巨噬细胞、 淋巴细胞、 肝细胞、 肾细胞、 内皮 细胞、 神经细胞、 和心肌细胞受体结合的多肽。 内吞小体释放寡肽元件  The ligand oligopeptides also include polypeptides that bind to hematopoietic cells, macrophages, lymphocytes, liver cells, kidney cells, endothelial cells, nerve cells, and cardiomyocyte receptors. Endosome release oligopeptide element
本发明的融合蛋白还可含有内吞小体释放寡肽元件， 它起到防止内吞入细胞的 DNA 被降解的作用。 任何内吞小体释放寡肽都可用于本发明， 代表性的例子包括 (但并不限 于）： HA20、 VP-1。 连接肽  The fusion protein of the present invention may further contain an endosome-releasing oligopeptide element, which plays a role of preventing the DNA of endocytosis from being degraded. Any endosome-releasing oligopeptide can be used in the present invention, and representative examples include (but are not limited to): HA20, VP-1. Linker
在本发明融合蛋白中， 在 (a)组蛋白元件， (b) 受体识别寡肽元件， 和 (c)内吞小体释 放寡肽元件之间， 可任选地含有连接肽， 以便增加复合多肽空间结构的柔性和舒展性， 让各元件发挥各自的功能。  In the fusion protein of the present invention, a linker peptide may optionally be included between (a) a histone element, (b) a receptor recognition oligopeptide element, and (c) an endosome-releasing oligopeptide element, in order to increase The flexibility and stretchability of the spatial structure of the compound peptide allows each element to perform its own function.
可用于本发明的连接肽没有特别限制， 只要起连接作用即可。 一类连接肽例子是 The linker peptide that can be used in the present invention is not particularly limited as long as it serves as a linker. An example of a class of linker peptides is
(Gly)2.6Ser, 例如 (Gly)4Ser。 连接肽可多个串联使用。 (Gly) 2. 6 Ser, for example (Gly) 4 Ser. Multiple linkers can be used in series.
本发明的 (a) 组蛋白元件， (b) 受体识别寡肽元件，和 (c)内吞小体释放寡肽元件都可 以用类似性能的氨基酸替代， 只要仍保留各自类似的生物功能。  The (a) histone element, (b) receptor-recognizing oligopeptide element, and (c) the endosome-releasing oligopeptide element of the present invention can all be replaced with amino acids of similar properties, as long as the respective similar biological functions are still retained.
在本发明中， 将配体寡肽 （L0P) 和内吞小体释放寡肽 ( HA20) 分别连接于组蛋白 元件 （如 Histone H1Q、 Histone Hl。97_193) 的 N_末端或 C-末端， 就形成 LOP-组蛋白元件 _HA20、 HA20 -组蛋白元件 -L0P、 组蛋白元件- L0P、 组蛋白元件 -HA20等融合蛋白。 它们可 与外源 DNA结合成为二元或三元复合体。 该基因转移***利用配体寡肽与受体的特异性 结合（如 GE7识别肿瘤细胞上过量表达的 EGF受体） ， 经细胞内吞作用， 通过该复合寡 肽将外源 DNA靶向性导入靶细胞， 达到治疗的目的。 In the present invention, the ligand is an oligopeptide (L0P) and endosome release oligopeptide (of HA20) elements are connected to histones (e.g., Histone H1 Q, Histone Hl. 97 _ 193) of the N_ or C- At the end, fusion proteins such as LOP-histone element_HA20, HA20-histone element-L0P, histone element-L0P, and histone element-HA20 are formed. They can be combined with foreign DNA into binary or ternary complexes. The gene transfer system utilizes the specific binding of a ligand oligopeptide to a receptor (eg, GE7 recognizes the EGF receptor overexpressed on tumor cells), and through cell endocytosis, the composite oligopeptide is used to selectively introduce foreign DNA Target cells to achieve the purpose of treatment.
本发明融合蛋白的 Pi应大于 7， 通常为 8-12. 5， 较佳地为 9-11. 5， 更佳地为 10- The Pi of the fusion protein of the present invention should be greater than 7, usually 8-12. 5, preferably 9-11. 5, and more preferably 10-
11。 外源 DNA 11. Foreign DNA
可用于本发明的外源 DNA没有特别限制， 可以是各种治疗性或预防性的 DNA, 例如 目的基因、 反义癌基因、 抗癌基因、 ***基因、 细胞调亡基因、 细胞因子基因、 或其组 合， 或者含有上述基因的真核表达载体 DNA。 原癌基因反义序列包括原癌基因 （rasH、 rasK、 rasN、 c- myc、 bcl- 2、 Akt) 、 生长因子及其受体基因的反义 DNA序列、 反义寡核 苷酸及反义寡脱氧核苷酸； 抑癌基因包括 p53、 Rb、 PTEN, ***基因包括 HSV- TK (单纯 疱疹病毒胸苷激酶）基因及 CD (大肠杆菌胞嘧啶脱氨酶）基因； 细胞凋亡基因包括 pl5、 pl6、 p21ffAF-1 ; 细胞因子基因包括 GM-CSF (粒细胞巨噬细胞集落刺激因子） 基因， TNFa (肿瘤坏死因子） 基因， INF (干扰素） α、 γ基因， IL (白细胞介素） 2、 3、 4、 12、 18 基因等。 靶向性基因导入*** The exogenous DNA that can be used in the present invention is not particularly limited, and may be various therapeutic or preventive DNA, such as a target gene, an antisense oncogene, an anti-oncogene, a suicide gene, a cell apoptosis gene, a cytokine gene, or A combination thereof, or a eukaryotic expression vector DNA containing the aforementioned genes. Proto-oncogene antisense sequences include proto-oncogenes (ras H , ras K , ras N , c-myc, bcl-2, Akt), antisense DNA sequences of growth factors and their receptor genes, and antisense oligonucleotides And antisense oligodeoxynucleotides; tumor suppressor genes include p53, Rb, PTEN, suicide genes include HSV-TK (herpes simplex virus thymidine kinase) gene and CD (E. coli cytosine deaminase) gene; apoptosis Genes include pl5, pl6 , p21 ffAF - 1 ; cytokine genes include GM-CSF (granulocyte macrophage colony-stimulating factor) gene, TNFa (tumor necrosis factor) gene, INF (interferon) α, γ gene, IL (interleukin) 2, 3, 4, 12, 18 genes, etc. Targeted gene introduction system
将本发明的融合蛋白与外源 DNA混合，就构成受体介导的靶向性肿瘤基因治疗的基 因导入***。取决于所用的外源 DNA，本发明的基因导入***可用于治疗遗传疾病或肿 瘤等疾病， 尤其是用于基因治疗。  Mixing the fusion protein of the present invention with foreign DNA constitutes a gene introduction system for receptor-mediated targeted tumor gene therapy. Depending on the exogenous DNA used, the gene introduction system of the present invention can be used to treat diseases such as genetic diseases or tumors, especially for gene therapy.
在本发明的一个实施例中，靶向性基因导入***包括组蛋白、受体识别功能肽 GE7、 内吞小体释放肽 HA20。当在结合 DNA的同时，功能肽 GE7、 HA20也发挥各自的特定功能。 此外， 在基因导入***中还含有促进内吞或稳定 DNA的物质， 例如 HA20-组蛋白元件 或组蛋白元件 -HA20融合蛋白。 重组生产  In one embodiment of the present invention, the targeted gene introduction system includes a histone, a receptor recognition functional peptide GE7, and an endosome-releasing peptide HA20. When binding DNA, the functional peptides GE7 and HA20 also perform their specific functions. In addition, the gene introduction system also contains substances that promote endocytosis or stabilize DNA, such as the HA20-histone element or the histone element-HA20 fusion protein. Reorganized production
本发明还提供了构建融合基因，用基因工程技术表达融合蛋白， 并对融合蛋白的分 离、 纯化的制备工艺。  The invention also provides a preparation process for constructing a fusion gene, expressing the fusion protein by using genetic engineering technology, and separating and purifying the fusion protein.
本发明的各元件 DNA编码序列， 可以用市售的各种 cDNA文库， 通过 PCR法而获得。 或者直接通过人工合成的方法获得。 L0P、 HA20经数轮 PCR搭桥反应与作为核心序列的 组蛋白元件的编码序列连接， 就形成一系列表达各寡肽的融合基因。  The DNA coding sequences of the elements of the present invention can be obtained by PCR using various commercially available cDNA libraries. Or directly obtained by artificial synthesis. LOP, HA20 are connected to the coding sequence of the histone element as the core sequence through several rounds of PCR bypass reactions to form a series of fusion genes expressing each oligopeptide.
由于遗传密码的兼并性，因此能够编码本发明融合蛋白的所有多核苷酸序列均包括 在本发明范围内。  Due to the annexation of the genetic code, all polynucleotide sequences capable of encoding the fusion protein of the present invention are included within the scope of the present invention.
本发明中， 融合蛋白的多核苷酸序列可***到重组表达载体中。 术语 "重组表达载 体"指本领域熟知的细菌质粒、 噬菌体、 酵母质粒、 植物细胞病毒、 哺乳动物细胞病毒 如腺病毒、'逆转录病毒或其他载体。 总之， 只要能在宿主体内复制和稳定， 任何质粒和 载体都可以用。 表达载体的一个重要特征是通常含有复制起点、 启动子、 标记基因和翻 译控制元件。  In the present invention, the polynucleotide sequence of the fusion protein can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, 'retroviruses, or other vectors, which are well known in the art. In short, any plasmid and vector can be used as long as it can be replicated and stabilized in the host. An important feature of expression vectors is that they usually contain origins of replication, promoters, marker genes and translation control elements.
本领域的技术人员熟知的方法能用于构建含融合蛋白编码 DNA序列和合适的转录 / 翻译控制信号的表达载体。 这些方法包括体外重组 DNA技术、 DNA合成技术、 体内重 组技术等。 所述的 DNA序列可有效连接到表达载体中的适当启动子上， 以指导 mRNA 合成。 这些启动子的代表性例子有： 大肠杆菌的 lac或 trp启动子真核启动子包括 CMV 立即早期启动子、 HSV胸苷激酶启动子、 早期和晚期 SV40启动子和其他一些已知的可 控制基因在原核或真核细胞或其病毒中表达的启动子。 表达载体还包括翻译起始用的核 糖体结合位点和转录终止子。  Methods well known to those skilled in the art can be used to construct expression vectors containing fusion protein-encoding DNA sequences and appropriate transcription / translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology. The DNA sequence can be operably linked to an appropriate promoter in an expression vector to guide mRNA synthesis. Representative examples of these promoters are: E. coli lac or trp promoter eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoters, and other known controllable genes Promoters expressed in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
此外， 表达载体优选地包含一个或多个选择性标记基因， 以提供用于选择转化的宿 主细胞的表型性状， 如真核细胞培养用的二氢叶酸还原酶、 新霉素抗性以及绿色荧光蛋 白 (GFP)， 或用于大肠杆菌的四环素或氨苄青霉素抗性。  In addition, the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture. Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
包含上述的适当 DNA序列以及适当启动子或者控制序列的载体， 可以用于转化适 当的宿主细胞， 以使其能够表达蛋白质。  A vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform an appropriate host cell so that it can express a protein.
宿主细胞可以是原核细胞， 如细菌细胞； 或是低等真核细胞， 如酵母细胞； 或是高 等真核细胞， 如哺乳动物细胞。 代表性例子有： 大肠杆菌， 链霉菌属； 真菌细胞如酵母； 植物细胞； 果蝇 S2或 S 的昆虫细胞； CHO、 COS细胞等动物细胞。一种优选的宿主 细胞是大肠杆菌。 The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a high Eukaryotic cells, such as mammalian cells. Representative examples are: E. coli, Streptomyces; fungal cells such as yeast; plant cells; insect cells of Drosophila S2 or S; animal cells such as CHO and COS cells. A preferred host cell is E. coli.
用重组 DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。 当宿主为原 核生物如大肠杆菌时， 能吸收 DNA的感受态细胞可在指数生长期后收获， 用 CaCl2法处 理， 所用的步骤在本领域众所周知。 另一种方法是使用 MgCl2。 如果需要， 转化也可用 电穿孔的方法进行。当宿主是真核生物，可选用如下的 DNA转染方法：磷酸钙共沉淀法， 常规机械方法如显微注射、 电穿孔、 脂质体包装等。 Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote, such as E. coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with the CaCl 2 method. The steps used are well known in the art. Another method is to use MgCl 2 . If necessary, transformation can also be performed by electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, and liposome packaging.
获得的转化子可以用常规方法培养， 表达本发明的基因所编码的多肽。 根据所用的 宿主细胞， 培养中所用的培养基可选自各种常规培养基。 在适于宿主细胞生长的条件下 进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法 (如温度转换或化学诱导) 诱导选择的启动子， 将细胞再培养一段时间。  The obtained transformants can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the medium used in the culture may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
在上面的方法中的重组多肽可在细胞内、 或在细胞膜上表达、 或分泌到细胞外。 如 果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。 这些方法是本领域技术人员所熟知的。 这些方法的例子包括但并不限于： 常规的复性处 理、 用蛋白沉淀剂处理 (盐析方法)、 离心、 渗透破菌、 超处理、 超离心、 分子筛层析 (凝 胶过滤)、 吸附层析、离子交换层析、高效液相层析 (HPLC)和其它各种液相层析技术及这 些方法的结合。 药物组合物  The recombinant polypeptide in the above method can be expressed intracellularly, or on a cell membrane, or secreted extracellularly. If desired, recombinant proteins can be isolated and purified by various separation methods using their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation, treatment with a protein precipitant (salting out method), centrifugation, osmotic disruption, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods. Pharmaceutical composition
本发明还提供了一种药物组合物， 它含有安全有效量的本发明的一种或多种融合蛋 白以及药学上可接受的载体或赋形剂。这类载体包括 (但并不限于)： 盐水、 缓冲液、葡萄 糖、 水、 甘油、 乙醇、 及其组合。 药物制剂应与给药方式相匹配。 本发明的药物组合物 可以被制成针剂形式， 例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法 进行制备。  The present invention also provides a pharmaceutical composition containing a safe and effective amount of one or more fusion proteins of the present invention and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to: saline, buffers, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants.
施用时， 药物组合物中还含有一种或多种上述的外源 DNA。  When administered, the pharmaceutical composition also contains one or more of the aforementioned exogenous DNAs.
具体剂量还应考虑给药途径、 病人健康状况等因素， 这些都是熟练医师技能范围之 内的。 本发明的主要优点在于，  The specific dosage should also consider factors such as the route of administration and the health status of the patient, which are all within the skill of a skilled physician. The main advantage of the invention is that
(1)本融合蛋白基因导入载体***不需脂质体或氯喹的参与。  (1) The introduction of the fusion protein gene into the vector system does not require the participation of liposomes or chloroquine.
(2)本融合蛋白可连接构建成表达质粒， 在大肠杆菌等宿主细胞中大量重组表达并 分离纯化获得融合蛋白。 能进行大规模生产， 有利于质控。  (2) The fusion protein can be ligated to construct an expression plasmid, which can be recombinantly expressed in a large number of host cells such as E. coli, and purified by isolation and purification. It is capable of mass production and is conducive to quality control.
(3)体内、 体外导入实验以及免疫组化分析证实， 本基因导入载体***可有效地将 外源 DNA导入靶细胞。  (3) In vivo and in vitro introduction experiments and immunohistochemical analysis confirmed that the gene introduction vector system can effectively introduce foreign DNA into target cells.
(4)基于导入载体***中所用的不同的外源基因， 该载体***可用于治疗不同的肿 瘤或非肿瘤性疾病。 下面结合具体实施例， 进一步阐述本发明。应理解， 这些实施例仅用于说明本发明 而不用于限制本发明的范围。 下列实施例中未注明具体条件的实验方法， 通常按照常规 条件， 例如 Sambrook等人， 分子克隆： 实验室手册（New York : Cold Spring Harbor Laboratory Press, 1989)中所述的条件， 或按照制造厂商所建议的条件。 实施例 1 各融合蛋白的表达序列的获得 (4) Based on the different foreign genes used in the introduction of the vector system, the vector system can be used to treat different tumors or non-neoplastic diseases. The present invention is further described below with reference to specific embodiments. It should be understood that these examples are only used to illustrate the present invention It is not intended to limit the scope of the invention. The experimental methods without specific conditions in the following examples are generally based on conventional conditions, for example, Sambrook et al., Molecular Cloning: The conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to manufacturing conditions Conditions recommended by the manufacturer. Example 1 Obtaining the expression sequence of each fusion protein
以构建 GE7-组蛋白 Hl°- HA20和 GE7 - 组蛋白 Hlc 97-193 -HA20的表达序列为例: 1、 接头-组蛋白 H1Q-接头和接头-组蛋白 Hl°97_193 -接头表达序列的获得： To construct GE7- histone Hl ° - HA20 GE7 and - expressed sequence 97-193 -HA20 histone Hl c Case: 1, the linker - Histone H1 Q - linker and linker - Histone Hl ° 97 _ 193 - linker Obtaining the expressed sequence:
为保证在基因工程表达的融合蛋白 GE7-组蛋白 H1°-HA20和 GE7 -组蛋白 Hl( 97-193In order to ensure the genetically engineered fusion proteins GE7-Histone H1 ° -HA20 and GE7-Histone Hl ( 97-193
HA20中， GE7和 HA20各自发挥独立的生物功能， 在它们与组蛋白 Hl°和组蛋白 Hlc 0 之间设计了由（Gly) 4Ser组成的寡肽作为柔性结构 (以下称为接头) 以维持 GE7、 HA20 功能肽的结构舒展性。 In HA20, GE7 and HA20 each play independent biological functions. An oligopeptide (Gly) 4 Ser composed of a flexible structure (hereinafter referred to as a linker) was designed between them and histone Hl ° and histone Hl c 0 to Maintain structural stretch of functional peptides of GE7 and HA20.
设计并合成表 A所示的引物。  Design and synthesize the primers shown in Table A.
表 A引物  Table A Primers
引物 序列 SEQ ID NO: 引物 1-组蛋白 L 5 '-TGGTGGCGGTTCTACCGAGAATTCCACGTCC-3 ' 19 引物 2-组蛋白 97_ -L 5 -TGGTGGCGGTTCTAGCGACGAACCCAAGAAG-3 ' 20 引物 3-组蛋白 -R 5 -ACCGCCACCACCCTTCTTCTTGCCGGCCCT-3 ' 21 引物 4- GE7-L 5 '-GGAATTC|CATBAACCCGGTTGTTGGTTAC-3 ' 22 引物 5- GE7- R -GAACCGCCACCACCCAGGTCACGGTACTG-3' 23 引物 6- HA20-L 5 -TGGTGGCGGTTCTGGTCTGTTCGAAGCTATC-3 ' 24 引物 7- HA20- R 5 '-CCC|AAGCTTHACCTTCGATCAGACCTTC-3 ' 25 Primer sequence SEQ ID NO: Primer 1-Histone L 5 '-TGGTGGCGGTTCTACCGAGAATTCCACGTCC-3' 19 Primer 2-Histone 97 _ -L 5 -TGGTGGCGGTTCTAGCGACGAACCCAAGAAG-3 '20 Primer 3-Histone-R 5 -ACCGCCACCACCCTTCTTCTTGCCGGCCCT-3' 21 Primer 4- GE7-L 5 '-GGAATTC | CATBAACCCGGTTGTTGGTTAC-3' 22 Primer 5- GE7- R -GAACCGCCACCACCCAGGTCACGGTACTG-3 '23 Primer 6-HA20-L 5 -TGGTGGCGGTTCTGGTCTGTTCGAAGCTATC-3' 24 Primer 7- HA20- R 5 '- CCC | AAGCTTHACCTTCGATCAGACCTTC-3 '25
下划线所标为在 "搭桥反应" 中的重叠 （overlap) 序列部分， 加框所标为表 达序列***表达载体的内切酶位点， ATG为起始密码子， TCA为终止密码子的反义序列。 从胎盘 cDNA文库中用引物 1和引物 3获得组蛋白 Hlfl的表达序列； 用引物 2和引 物 3 PCR获得组蛋白 H1Q 97— 193的表达序列。 The underlined part is the overlapping sequence in the "bridge reaction", the boxed part is the endonuclease site where the expression sequence is inserted into the expression vector, ATG is the start codon, and TCA is the antisense of the stop codon. sequence. From placenta cDNA library using primers 1 and primer 3 was obtained expressed sequence of histone Hl fl; primer 2 and primer 3 to obtain the PCR protein groups H1 Q 97 - 193 expressed sequences.
2、 GE7-组蛋白 H1Q- HA20和 GE7-组蛋白 Η1°97_193- HA20表达序列的获得： 2. Ge7-Histone H1 Q -HA20 and GE7-Histone Η1 ° 97 _ 193 -HA20 expression sequence obtained:
分别以质粒 pBluescripl; SK (-) -GE7- protamine和 pBluescript SK (-) - HA20- protamine为模板， 利用上述引物 4、 引物 5、 引物 6、 引物 7 PCR直接获得 GE7-接头和 接头 _HA20。  Using plasmids pBluescripl; SK (-)-GE7-protamine and pBluescript SK (-)-HA20-protamine as templates, GE7-linker and linker _HA20 were directly obtained by PCR using primer 4, primer 5, primer 6, and primer 7 PCR.
通过两轮 PCR进行"搭桥"反应： 先不加引物，模板 GE7-接头与接头-组蛋白 H1D- 接头变性后 55Ό退火延伸， 得到少量模板 GE7-组蛋白 Hl°-接头， 加入引物 4、 引物 3 扩增 GE7-组蛋白 Hl°-接头。 不加引物， GE7-组蛋白 H1Q-接头和接头 -HA20退火延伸， 得到少量模板 GE7-接头-组蛋白 H1Q-接头- HA20 (以下简称 GE7 -组蛋白 H1Q- HA20) ， 加 入引物 4、 引物 7， 大量扩增得到 GE7-组蛋白 H1D- HA20的表达序列。 "搭桥 "反应可以 将两条具有部分相同顺序的 DNA连接起来，这部分相同的序列即为连接两条 DNA的桥梁。 按同样方法获得 GE7-组蛋白 H1Q 97_193_HA20。附图 1为构建 GE7-组蛋白 Hl°- HA20表达序 列的流程图。 "Bypass" reaction was performed by two rounds of PCR: without primers, template GE7-linker and linker-Histone H1 D -linker were annealed and extended after 55Ό annealing. Primer 3 amplifies the GE7-histone Hl ° -linker. Without primers, GE7-Histone H1 Q -linker and Linker-HA20 are annealed and extended to obtain a small amount of template GE7-Linker-Histone H1 Q -Linker-HA20 (hereinafter referred to as GE7-Histone H1 Q -HA20), and primer 4 is added , Primer 7, a large amount of amplification to obtain the expression sequence of GE7-histone H1 D -HA20. "Bridge" reaction can connect two DNAs with the same sequence, and this same sequence is the bridge connecting the two DNAs. GE7-Histone H1 Q 97 _ 193 _HA20 was obtained in the same way. FIG. 1 is a flowchart of constructing a GE7-histone Hl ° -HA20 expression sequence.
同样的 PCR方法获得其他融合蛋白的表达序列。 所有融合蛋白的表达序列见附图 N2002/000592 The same PCR method was used to obtain the expressed sequences of other fusion proteins. The expression sequences of all fusion proteins are shown in the attached figure N2002 / 000592
2-10。 2-10.
所有表达序列用 Ndel/Hindlll双酶切后与原核表达载体 pT7450 Ndel/Hindlll双 酶切后回收的大片段连接，连接产物转化 DH50C感受态，转化细菌涂布于含氨苄青霉素的 LB平板， 挑取单克隆， 小量抽提质粒， 酶切鉴定、 测序鉴定， 成为含有融合基因的原核 表达质粒。 PT7450原核表达载体（得自上海生物工程中心）的图谱见附图 11。 实施例 2 组蛋白 Hl°为骨架的系列融合蛋白的表达与分离、 纯化：  All expressed sequences were digested with Ndel / Hindlll and double-digested with the prokaryotic expression vector pT7450 Ndel / Hindlll, and the recovered large fragments were ligated. The ligated products were transformed into DH50C competent cells. The transformed bacteria were coated on LB plates containing ampicillin and picked Monoclonal, small-scale extraction plasmids, enzyme digestion identification, sequencing identification, become prokaryotic expression plasmids containing fusion genes. See Figure 11 for a map of the PT7450 prokaryotic expression vector (obtained from Shanghai Biotechnology Center). Example 2 Expression, isolation and purification of a series of fusion proteins with a histone Hl ° as a backbone:
1.诱导、 表达：  1. Induction and expression:
表达质粒分别转化表达宿主菌 HMS174 (DE3 ) 的感受态细胞， 感受态的制备和转化 条件按照 《分子克隆》 进行。 转化后的感受态细菌涂布于氨苄 LB琼脂板， 37°C过夜。 挑取单菌落于含氨苄抗性的 5ml LB中， 37°C震荡过夜， 1 : 50稀释于 1000ml LB中培养 至菌密度 0D600nm约为 0. 6,加入 IPTG至终浓度 lmM， 30°C诱导 3小时。离心收集菌体， 加入超声液 40ml ( 50mM Tris.Cl, pH8. 0, lOmM EDTA, 0. 5mM PMSF) ,冰浴超声 1小时。 超声后的菌液高速离心， 收集上清， 准备过 SP sepharose阳离子交换柱纯化。  The expression plasmids were used to transform competent cells expressing the host strain HMS174 (DE3). Competent preparation and transformation conditions were performed according to "Molecular Cloning". The transformed competent bacteria were plated on an ampicillin LB agar plate at 37 ° C overnight. Pick a single colony in 5ml LB containing ampicillin resistance, shake at 37 ° C overnight, dilute 1:50 in 1000ml LB and incubate to a density of 0D600nm to about 0.6, add IPTG to a final concentration of lmM, and induce at 30 ° C 3 hours. Bacteria were collected by centrifugation, and 40 ml of ultrasonic solution (50 mM Tris.Cl, pH 8.0, 10 mM EDTA, 0.5 mM PMSF) was added, and sonication was performed in an ice bath for 1 hour. After sonication, the bacterial solution was centrifuged at high speed, and the supernatant was collected and purified by SP sepharose cation exchange column.
2. SP sepharose阳离子交换柱纯化蛋白： 2. SP sepharose cation exchange column purification protein:
2. 1 SP sepharose阳离子交换柱纯化组蛋白：  2.1 SP sepharose cation exchange column to purify histones:
SP sepharose阳离子交换柱预先用 50 mM Tris-Cl , pH 8. 5平衡处理。 收集菌体超 声后的上清加样于阳离子柱， 液体靠重力吸引自然流出， 需纯化的蛋白因携带大量正电 荷而吸附于 SP柱， 弃流出液， 待阳离子交换柱表面的上样液即将流尽， 加入平衡用的 50 mM Tris_Cl冲洗柱， 使残留在柱中的液体完全被平衡缓冲液替代， 此时紫外吸收仪 显示吸收曲线回到基线。 以 0- lM NaCl , 50 mM Tris-Cl , pH 8. 5梯度洗脱， 自洗脱峰出 现开始收集洗脱液， 收集各洗脱峰样品， 电泳， 纯化的蛋白质产物位于主带明显的最后 一峰。  The SP sepharose cation exchange column was pre-equilibrated with 50 mM Tris-Cl, pH 8.5. The supernatant of the bacterial cells was collected and applied to the cation column. The liquid was naturally drawn out by gravity. The protein to be purified was adsorbed on the SP column because it carried a large positive charge. The effluent was discarded. After exhaustion, add 50 mM Tris_Cl for equilibration to flush the column, so that the liquid remaining in the column is completely replaced by the equilibration buffer. At this time, the UV absorber shows that the absorption curve returns to the baseline. Elution was performed with a gradient of 0- 1M NaCl, 50 mM Tris-Cl, pH 8.5, and the eluent was collected from the appearance of the elution peak. Samples of each elution peak were collected and electrophoresed. The purified protein product was located at the obvious end of the main band. Yifeng.
2. 2 Sephadex G- 50分子筛分离组蛋白：  2. Sephadex G-50 molecular sieve to separate histones:
将 SP Sepharose阳离子交换柱纯化所得的最后一个洗脱峰样品，先用 ULTRAFREE - 15 超滤装置浓缩， 然后上样于 Sephadex G-50分子筛， 通过紫外吸收仪显示的吸收曲线情 况， 收集主峰样品。 分子筛预先用 HBS缓冲液 （20mM HEPES, pH 7. 4, 150mM NaCl, 20% 甘油）平衡，收集的主峰样品最后用 ULTRAFREE- 15超滤装置浓缩。浓缩后的样品 12/。SDS 电泳鉴定， 用 BCA方法定量。 蛋白溶液中加入蛋白酶抑制剂 （抑制剂终浓度为  The last eluting peak sample obtained by purifying the SP Sepharose cation exchange column was first concentrated with an ULTRAFREE-15 ultrafiltration device, and then loaded on a Sephadex G-50 molecular sieve. The main peak sample was collected by the absorption curve displayed by an ultraviolet absorber. The molecular sieves were pre-equilibrated with HBS buffer (20 mM HEPES, pH 7. 4, 150 mM NaCl, 20% glycerol), and the collected main peak samples were finally concentrated with a ULTRAFREE-15 ultrafiltration device. Concentrated sample 12 /. SDS electrophoresis was identified and quantified by BCA method. Add protease inhibitor to protein solution (final inhibitor concentration is
1μ1/500μ1 ) ， - 20°C保存。 1μ1 / 500μ1), stored at -20 ° C.
2. 3 含组蛋白骨架的各种融合蛋白的表达与分离：  2.3 Expression and isolation of various fusion proteins containing histone backbone:
按同样方法表达、 分离与纯化各种融合蛋白， 包括组蛋白 H1Q 97— lg3、 GE7-组蛋白 HI。- HA20、 GE7-组蛋白 H1Q 97_193-HA20、 HA20-组蛋白 H1Q- GE7、 组蛋白 Hl°- GE7、 组蛋白 Hl°- HA20、 组蛋白 Hl°97_193- GE7、 组蛋白 H1Q 97_193- HA20。 Various fusion proteins were expressed, isolated and purified in the same way, including histones H1 Q 97 —lg3 and GE7-histone HI. -HA20, GE7-Histone H1 Q 97 _ 193 -HA20, HA20-Histone H1 Q -GE7, Histone Hl °-GE7, Histone Hl °-HA20, Histone Hl ° 97 _ 193 -GE7, Histone H1 Q 97 _ 193 -HA20.
表 B 融合蛋白  Table B Fusion proteins
DNA序列 氨基酸序列 附图 分子量 i DNA sequence Amino acid sequence Figure molecular weight i
(SEQ ID NO :) (SEQ ID NO :) (kDa) (SEQ ID NO :) (SEQ ID NO :) (kDa)
组蛋白 HI0 1 2 2 21 10. 84 组蛋白 Hl°97-193 3 4 3 10. 686 11. 09Histone HI 0 1 2 2 21 10. 84 Histone Hl ° 97 -193 3 4 3 10. 686 11. 09
GE7-组蛋白 Hl。- HA20 5 6 4 25. 69 10. 52GE7-Histone Hl. -HA20 5 6 4 25. 69 10. 52
GE7—组蛋白 Hl°97_193- HA20 7 8 5 15. 156 10. 57 组蛋白 Hl°- GE7 9 10 6 23. 035 10. 75 组蛋白 Hl°- HA20 11 12 7 23. 296 10. 59 组蛋白 m°97_193-GE7 13 14 8 12. 721 10. 87 组蛋白 Hl°97_i93-HA20 15 16 9 12. 982 10. 61GE7—Histone Hl ° 97 _ 193 -HA20 7 8 5 15. 156 10. 57 Histone Hl °-GE7 9 10 6 23. 035 10. 75 Histone Hl °-HA20 11 12 7 23. 296 10. 59 Histone m ° 97 _ 193 -GE7 13 14 8 12. 721 10. 87 Histone Hl ° 97 _i93-HA20 15 16 9 12. 982 10. 61
HA20-组蛋白 H1°-GE7 17 18 10 25. 469 10. 52HA20-Histone H1 ° -GE7 17 18 10 25. 469 10. 52
12%SDS-PAGE鉴定各融合蛋白见附图 12 - 14。 实施例 3 组蛋白 H1Q为骨架的复合多肽与 DNA形成复合体 12% SDS-PAGE identification of each fusion protein is shown in Figures 12-14. Example 3 A histone H1 Q complex peptide forms a complex with DNA
DNA与各融合蛋白或不同融合蛋白的组合， 按质量比 1 : 3混勾， 室温放置一小时， 以备体外、 体内生物功能试验所用。 取含 0. 2μ§ 质粒 DNA的复合体用于 1%琼脂糖电泳 鉴定， 以确定质粒 DNA已被完全阻遏在加样孔中。 复合体中 DNA的终浓度为 0. 2μ^μ1。 见附图 15-20。 实施例 4 复合体体外导入试验 DNA and each fusion protein or a combination of different fusion proteins are mixed at a mass ratio of 1: 3 and left at room temperature for one hour for use in vitro and in vivo biological function tests. Take the complex containing 0.2 μ § plasmid DNA for identification by 1% agarose electrophoresis to confirm that the plasmid DNA has been completely suppressed in the loading well. The final DNA concentration in the complex was 0.2 μ ^ μ1. See Figure 15-20. Example 4 In vitro introduction test of complex
体外细胞报告基因导入实验：胰酶消化细胞后接种 24孔板 4xlOV孔， 37 C过夜。第 二天换无血清培液 lml/孔， 加入制备的复合体， 使在培液中的 DNA含量为 3 g/ml， 温 育过夜。 第三天去除含复合体的无血清培液， 更换含 10%新生小牛血清的 DMEM。 第四天 弃检测细胞的培养液， 用 0. 1M PBS洗涤三次， 室温固定 （固定液： 0. 2%戊二醛， 2%甲 醛于 0. M PBS中） 20分钟。 PBS洗涤三次， 每次 10分钟。 加入新鲜配制的 X- gal染色 液 ( 5mM K3Fe (CN) 6, 5mM K4Fe (CN) 6, 2mM MgCl2， lmg/ral X-gal , 用 PBS配制） ， 37 °C 温育显色过夜， 光镜下观察外源基因的导入情况。 In vitro cell reporter gene introduction experiment: trypsinized cells were inoculated into 4xlOV wells in a 24-well plate and incubated at 37 C overnight. The next day, the serum-free culture solution was changed to 1 ml / well, and the prepared complex was added so that the DNA content in the culture solution was 3 g / ml, and incubated overnight. On the third day, the serum-free culture solution containing the complex was removed, and DMEM containing 10% newborn calf serum was replaced. On the fourth day, the culture medium of the test cells was discarded, washed three times with 0.1 M PBS, and fixed at room temperature (fixing solution: 0.2% glutaraldehyde, 2% formaldehyde in 0. M PBS) for 20 minutes. Wash three times in PBS for 10 minutes each. Add freshly prepared X-gal staining solution (5mM K 3 Fe (CN) 6 , 5mM K 4 Fe (CN) 6 , 2mM MgCl 2 , 1mg / ral X-gal, prepared with PBS)), and incubate at 37 ° C for overnight. The introduction of foreign genes was observed under a light microscope.
真核表达的 pSV-β- gal质粒 DNA以最佳的质量比例 1： 3与融合蛋白结合形成复合体 (含 ϋΝΑ 0. 2μ§/10μ1 ) ， 用于体外细胞导入实验， 根据 β- gal基因在细胞内的表达情况 评价融合蛋白的生物学活性。 BEL- 7402细胞接种后 24小时， 复合体加入培液中， 1 ml 培液中含 DNA 3μ 温育过夜， 24小时后 （细胞转染后 48小时） 进行 X-gal原位细胞 染色分析。融合蛋白介导 β- gal基因转染不表达 EGF受体的 U20S细胞作为体外细胞转染 实验的阴性对照，融合蛋白介导 β- gal 因转染 EGF受体表达阳性的 BEL- 7402细胞作为 实验组。 转染结果附图 21。 蛋白组合 GE7-组蛋白 Hl°- HA20/组蛋白 H1Q- GE7 (1： 1)， 组 蛋白 H1Q- GE7/组蛋白 Η1°- HA20 (1 : 1)能将 β- gal基因导入 BEL- 7402细胞中， β-gal基因 得到表达， 出现 X- gal染色阳性的细胞（细胞变蓝）， GE7-组蛋白 Hlfl- HA20/组蛋白The eukaryotically expressed pSV-β-gal plasmid DNA is combined with the fusion protein to form a complex (containing ϋΝΑ 0.2 μ § / 10μ1) at an optimal mass ratio of 1: 3, which is used for in vitro cell introduction experiments. According to the β-gal gene The expression in cells was used to evaluate the biological activity of the fusion protein. Twenty-four hours after inoculation of BEL-7402 cells, the complex was added to the culture solution, and 1 ml of the culture solution was incubated with DNA for 3 μ overnight. After 24 hours (48 hours after cell transfection), X-gal in situ cell staining analysis was performed. Fusion protein mediated β-gal gene transfection of U20S cells that do not express EGF receptor as a negative control for in vitro cell transfection experiments, fusion protein mediated β-gal transfection of BEL-7402 cells with positive EGF receptor expression as an experiment group. Figure 21 of the transfection results. Protein combination GE7-Histone Hl °-HA20 / Histone H1 Q -GE7 (1: 1), Histone H1 Q -GE7 / Histone Η1 °-HA20 (1: 1) can introduce β-gal gene into BEL- In 7402 cells, β-gal gene was expressed, X-gal staining positive cells (cells turned blue), GE7-histone Hl fl -HA20 / histone
H1Q- GE7组合比组蛋白 H1Q- GE7/组蛋白 Hlfl- HA20组合效果更好， 其他融合蛋白组都没 有出现 X-gal染色阳性的细胞。 EGF受体表达阴性的 U20S细胞的导入结果均为阴性。每 种融合蛋白及组合均重复了 10次， 体外细胞导入实验结果汇总如表 1。 The combination of H1 Q -GE7 was better than the combination of histone H1 Q -GE7 / histone Hl fl -HA20, and no other X-gal staining cells appeared in other fusion protein groups. U20S cells with negative EGF receptor expression were all negative. Each fusion protein and combination was repeated 10 times, and the results of the in vitro cell introduction experiment are summarized in Table 1.
就体外转染实验结果而言， 组蛋白 H1Q- GE7及组蛋白 H1°_HA20 ( 1 : 1 ) 混合组有 少量导入。 但将 GE7-组蛋白 Hl°- HA20与组蛋白 Hl°- GE7两者按 1： 1混合， 可见 β- gal 基因的导入。 细胞转染实验可初步筛选出能将外源 β-gal基因通过 EGF受体的介导转入肿瘤细胞 的最佳融合蛋白组合： GE7-组蛋白 H1Q- HA20/组蛋白 Hl°- GE7和组蛋白 H1°-GE7/组蛋白 H1Q-HA20。 用筛选出的蛋白组合进行深入的体内导入研究。 As for the results of in vitro transfection experiments, a small amount of the histone H1 Q -GE7 and histone H1 ° _HA20 (1: 1) mixed groups were introduced. However, mixing GE7-histone Hl ° -HA20 and histone Hl ° -GE7 at a ratio of 1: 1 shows the introduction of β-gal gene. Cell transfection experiments can preliminary screen the best fusion protein combination that can transfer exogenous β-gal gene into tumor cells via EGF receptor: GE7-Histone H1 Q -HA20 / Histone Hl °-GE7 and Histone H1 ° -GE7 / Histone H1 Q -HA20. Use the selected protein combinations for in-vivo introduction studies.
Figure imgf000012_0001
实施例 5 复合体体内导入试验
Figure imgf000012_0001
Example 5 Complex introduction test in vivo
裸小鼠移植瘤报告基因导入实验的方法如下：皮下荷瘤裸小鼠通过皮下瘤周途径注 射制备的复合体， 复合体预先用生理盐水按所需的注射量稀释， 每只小鼠一次性注射体 积不超过 ΙΟΟμΙ (内含复合体 50μ1， 质粒 pSV- β-gal 1μ§ ) 。 瘤周注射后 24小时， 牺 牲裸鼠， 剥取肿瘤， PBS漂洗三次， 每次 15分钟。 加入新鲜配制的固定液， 4°C固定 2 小时。 固定后 PBS洗涤三次， 每次 15分钟， 滤纸吸净瘤块及组织块上的 PBS， 加入新鲜 配制的染色液于 37 °C显色 24小时（染色液配制同上）。染色后的组织用 PBS洗净， 0. C. T 包埋， 冰冻切片， 中性红复染， 光镜下观察外源基因的导入情况。 Nude mouse transplantation tumor reporter gene introduction experiment method is as follows: Subcutaneous tumor-bearing nude mice are injected with a complex prepared by the subcutaneous tumor route, the complex is diluted with physiological saline in advance according to the required injection amount, each mouse is disposable The injection volume does not exceed 100 μl (containing complex 50 μ1, plasmid pSV- β-gal 1 μ § ). Twenty-four hours after tumor injection, sacrifice nude mice, strip tumors, rinse three times in PBS for 15 minutes each. Add freshly prepared fixative solution and fix at 4 ° C for 2 hours. After fixation, wash the PBS three times for 15 minutes each time. Filter the PBS on the tumor block and tissue block with filter paper, add freshly prepared staining solution and develop at 37 ° C for 24 hours (staining solution preparation is the same as above). The stained tissues were washed with PBS, embedded in 0. C. T, frozen sections, counterstained with neutral red, and the introduction of foreign genes was observed under a light microscope.
裸鼠皮下移植人肝癌 BEL- 7402肿瘤组织中 EGF受体表达阳性， 可用于融合蛋白介 导基因转移的靶组织。 裸小鼠皮下移植人肝癌 BEL- 7402， 待肿瘤长至 0. 5cm大小， 皮下 瘤周一次性注射 pSV-β- gal与各种融合蛋白形成的复合体 （含 pSV-p-gal质粒 1μ§) ， 注射前复合体用生理盐水稀释至 100μ1。 24小时后牺牲裸鼠， 剥取肿瘤， 固定后 X- gal 染色， 观察 pSV- β- gal的导入情况。 根据大体标本和冰冻切片显微镜下的 X-gal染色情 况， 各融合蛋白介导 β- gal基因转移裸鼠皮下移植人肝癌 BEL-7402的各组中， 单独 pSV- β- gal质粒组和单独组蛋白 H1Q组、单独组蛋白 H1Q- HA20组的肿瘤组织中未见蓝染 的细胞， 即无 β - gal基因的表达， 单独组蛋白 H1Q- GE7组、 单独 GE7-组蛋白 Hl°- HA20 组和 HA20-组蛋白 H1Q- GE7组的肿瘤组织中有少量 β- gal基因的表达， 蓝染细胞数量少 (约 20%) ， 颜色较浅， 将组蛋白 H1°-GE7和组蛋白 H1Q- HA20混合后， 蓝染细胞数目 有所增加 （约 50%) ， 蓝颜色略深， 将 GE7-组蛋白 Hlfl-HA20和组蛋白 H1Q-GE7等量混 合后介导 DNA，肿瘤组织中有大量 β- gal基因的表达，绝大多数细胞 X-gal染色阳性（约 90%) ， 阳性细胞显示的蓝颜色也更深， 说明基因导入效率高， 表达水平也高。 BEL-7402 肿瘤的体内导入实验比体外细胞转染实验的效果更显著，相同的有效蛋白组合 GE7-组蛋 白 Hl°- HA20/组蛋白 H1D-GE7在体内的导入效率比体外的效率髙， 但就各融合蛋白及组 合导入 β- gal基因的情况而言， 相对的趋势在体外与体内实验中是相似的。 见附图 22。 Subcutaneously transplanted human liver cancer BEL-7402 tumor tissues in nude mice have positive EGF receptor expression and can be used as target tissues for fusion protein-mediated gene transfer. Subcutaneously in nude mice transplanted with human hepatoma BEL- 7402, the tumor grew to a size of 0. 5cm, peritumoral subcutaneously injected once with various pSV-β- gal fusion proteins form a complex (containing plasmid pSV-p-gal 1μ § ), The complex was diluted with physiological saline to 100 μ1 before injection. Nude mice were sacrificed 24 hours later, tumors were stripped, and X-gal stained after fixation to observe the introduction of pSV-β-gal. According to the X-gal staining of gross specimens and frozen section microscopes, each fusion protein mediates β-gal gene transfer in nude mice subcutaneously transplanted with human liver cancer BEL-7402 in each group, the pSV-β-gal plasmid group alone and the separate group There were no blue-stained cells in the tumor tissues of the protein H1 Q group and the histone H1 Q -HA20 group alone, that is, there was no β-gal gene expression. The histone H1 Q -GE7 group alone, the GE7-histone Hl °- HA20 Group and HA20-histone H1 Q -GE7 group had a small amount of β-gal gene expression in tumor tissues, the number of blue-stained cells was small (about 20%), the color was lighter, the histone H1 ° -GE7 and histone H1 After mixing Q -HA20, the number of blue-stained cells increased (approximately 50%) and the blue color was slightly darker. GE7-Histone Hl fl- HA20 and Histone H1 Q- GE7 were mixed in equal amounts to mediate DNA and tumor tissues. There are a large number of β-gal gene expressions. Most cells are positive for X-gal staining (about 90%), and the positive cells show a darker blue color, indicating that the gene introduction efficiency is high and the expression level is also high. BEL-7402 tumor in vivo introduction experiment is more effective than in vitro cell transfection experiment. The same effective protein combination, GE7-Histone Hl ° -HA20 / Histone H1 D- GE7, is more efficient in vivo than in vitro. However, in the case of β-gal gene introduced by each fusion protein and combination, the relative trend is similar in vitro and in vivo experiments. See Figure 22.
经免疫组化分析， 裸鼠皮下移植人卵巢癌 SK0V3肿瘤组织 EGF受体表达也阳性， 而 且 SK0V3皮下移植瘤，生长速度快，是进行融合蛋白体内生物活性检测的良好组织材料。 裸小鼠荷人卵巢癌 SK0V3, 待皮下移植瘤长至 0. 5cm， 皮下瘤周一次性注射 pSV- β-gal 与各种融合蛋白形成的复合体 （含 pSV- β- gal质粒 1μ§) ， 注射前复合体用生理盐水稀 释至 100μ1。 24小时后， 牺牲裸鼠， 剥取肿瘤， 固定后进行 X- gal染色。 经 X- gal染色 后的肿瘤组织冰冻切片， 中性红复染， 显微镜下观察 β- gal基因的导入情况。 从肿瘤组 织的 X- gal染色后的大体标本、冰冻切片来看，各组融合蛋白介导 β-gal基因转移 SK0V3 肿瘤组织情况与 BEL- 7402肿瘤组织类似， 见附图 23。 体内导入试验重复三次。 融合蛋 白介导的 β- gal基因在两种不同的皮下移植瘤的表达情况归纳如表 2。 表 2、 融合蛋白介导 β-gal基因的体内细胞实验结果一览 Immunohistochemical analysis showed that the expression of EGF receptor in human ovarian cancer SK0V3 tumor tissue subcutaneously transplanted in nude mice was also positive, and SK0V3 subcutaneously transplanted tumor has a fast growth rate, and is a good tissue material for in vivo biological activity detection of fusion proteins. Nude mice bearing human ovarian cancer SK0V3, to be subcutaneously transplanted to a length of 0.5 cm, and a single injection of pSV-β-gal and various fusion proteins (including pSV-β-gal plasmid 1μ § ) around the subcutaneous tumor. Before injection, the complex was diluted to 100 μl with physiological saline. After 24 hours, nude mice were sacrificed, tumors were stripped, and X-gal stained after fixation. Frozen sections of tumor tissues after X-gal staining were counterstained with neutral red, and the introduction of β-gal gene was observed under a microscope. From X-gal stained specimens and frozen sections of tumor tissues, the fusion protein-mediated β-gal gene transfer of SK0V3 tumor tissue in each group is similar to that of BEL-7402 tumor tissue, see FIG. 23. The in vivo introduction test was repeated three times. The expression of fusion protein-mediated β-gal gene in two different subcutaneous xenografts is summarized in Table 2. Table 2. List of results of in vivo cell experiments with fusion proteins mediated β-gal gene
Figure imgf000013_0001
实施例 6 裸鼠移植瘤报告基因导入时效与剂量的观察
Figure imgf000013_0001
Example 6 Observation of the Time-effect and Dose of the Transplanted Tumor Reporter Gene in Nude Mice
通过皮下瘤周途径注射制备的复合体， 每只动物注射的复合体含 DNA量 1μ§。 分别 观察 24小时、 72小时、 96小时、 7天、 10天、 14天导入基因的表达情况。 通过皮下瘤 周途径注射预备的复合体， 每只动物注射的复合体分别含 DNA量 0. 2 g、 0. 5μ§, 1μ§, 24小时后牺牲裸小鼠， 剥取肿瘤， 固定、 染色， 观察基因导入与表达的情况。 The complex prepared by the subcutaneous tumor route injection, the DNA content of the complex injected by each animal is 1 μ § . The expressions of introduced genes were observed at 24 hours, 72 hours, 96 hours, 7 days, 10 days, and 14 days, respectively. Peritumoral injection through subcutaneous routes composite preparation, each animal was injected DNA complexes respectively containing an amount of 0. 2 g, 0. 5μ §, 1μ §, nude mice were sacrificed after 24 hours stripping tumors, fixed, stained Observe the situation of gene introduction and expression.
由蛋白组合 GE7-组蛋白 Hl°- ΗΑ20/组蛋白 Η1β- GE7介导的 β-gal基因在 SK0V3肿瘤 组织中的表达在不同时间点有变化， 注射 12小时后即有报告基因的表达， 24小时表达 达到髙峰， 48小时表达开始下降， 96小时、 7天、 10天表达继续下降， 14天时仍有少 量表达。 见附图 24。 Β-gal gene mediated by the protein combination GE7-Histone Hl °-ΗΑ20 / Histone Η1 β -GE7 in SK0V3 tumors The expression of tissues changed at different time points. The reporter gene was expressed 12 hours after injection. The expression reached a peak at 24 hours. The expression began to decrease at 48 hours. The expression continued to decrease at 96 hours, 7 days, and 10 days, and remained at 14 days. There is a small amount of expression. See Figure 24.
由蛋白组合 GE7-组蛋白 Hlfl- HA20/组蛋白 Hl°- GE7介导的 β- gal基因皮下瘤周注射 不同剂量的复合体（分别含 DNA 0. 2 g、 0. 5μ§, 1μ§ ) ， 24小时后牺牲裸鼠， 剥取肿瘤， 固定后 X- gal 染色分析 β- gal基因的导入情况， 发现随注射剂量的增加， β- gal基因导 入和表达增加， 在所选的 3个剂量中， 0. 2μ§组 β- gal基因导入和表达最少， 仅少数肿 瘤细胞染色阳性， 而且颜色较浅； 0. 5μ§组 β- gal基因导入和表达有所增加， 染色阳性 的细胞增多， 蓝色也比 0. 2μ§组略深； 1μβ组的 β- gal基因导入和表达最多， 蓝染的细 胞较普遍， 颜色也最深。 1μ§的导入效果最好， 是所选择的三个剂量中最佳的剂量。 参考文献 The protein composition GE7- histone Hl fl - HA20 / Histone Hl ° - injection of different doses of the complexes under β- gal peritumoral GE7 mediated Jiyin Pi (respectively containing DNA 0. 2 g, 0. 5μ § , 1μ § ), Nude mice were sacrificed after 24 hours, tumors were stripped, and the introduction of β-gal gene was analyzed by X-gal staining after fixation. It was found that as the injection dose increased, β-gal gene introduction and expression increased. dose, 0. 2μ § group β- gal gene expression and introducing a minimum, only a few positive staining of tumor cells, and lighter in color; [beta] gene introduction and expression of gal 0. 5μ § group increased, an increase in positive cells stained The blue color is also slightly darker than the 0.2 μ § group; the β -gal gene is introduced and expressed the most in the 1 μ β group. Blue-stained cells are more common and the color is the darkest. The introduction effect of 1μ § is the best, which is the best of the three selected doses. references
1. Xiang Liu, Peikun Tian, Jianren Gu, et al. Enhanced antitumor effect of EGF R - targeted p21 AF~1 and GM - CSF gene transfer in the established murine hepatoma ' by peritumoral injection. Cancer Gene Therapy, 2002； 9： 100-108 1. Xiang Liu, Peikun Tian, Jianren Gu, et al. Enhanced antitumor effect of EGF R-targeted p21 AF ~ 1 and GM-CSF gene transfer in the established murine hepatoma 'by peritumoral injection. Cancer Gene Therapy, 2002; 9: 100-108
2. Fritz J. D, Herwei jer H. , Zhang G. , et al. Gene transfer into mammalian cells using histone - condensed plasmid DNA. Hum. Gene Ther. , 1996, 7： 1395-1404 2. Fritz J. D, Herwei jer H., Zhang G., et al. Gene transfer into mammalian cells using histone-condensed plasmid DNA. Hum. Gene Ther., 1996, 7: 1395-1404
3. Jian Chen, Robert J, Katherine A, et al. Galactosylated histone- mediated gene transfer and expression. Human Gene Ther. , 1994, 5： 429-435 3. Jian Chen, Robert J, Katherine A, et al. Galactosylated histone- mediated gene transfer and expression. Human Gene Ther., 1994, 5: 429-435
4. Doenecke D, Tonjes R. Differential distribution of lysine and arginine residues in the closely related histories HI and H5. J Mol Biol, 1986, 187 (3)： 461-464  4. Doenecke D, Tonjes R. Differential distribution of lysine and arginine residues in the closely related histories HI and H5. J Mol Biol, 1986, 187 (3): 461-464
5. W098/18951  5. W098 / 18951

Claims

权 利 要 求 书 Claim
1. 一种融合蛋白， 其特征在于， 它含有 1. A fusion protein, characterized in that it contains
(a)组蛋白元件， 所述的组蛋白元件选自 Hl°全长序列或其富含赖氨酸的片段； (b) 受体识别寡肽元件 LOP。  (a) a histone element selected from the Hl ° full-length sequence or a lysine-rich fragment thereof; (b) a receptor recognition oligopeptide element LOP.
2. 如权利要求 1所述的融合蛋白， 其特征在于， 它含有 (c)内吞小体释放寡肽元件， 和任选的位于 (a)组蛋白元件， (b) 受体识别寡肽元和 (c)内吞小体释放寡肽元件之间的连 接肽。  2. The fusion protein according to claim 1, further comprising (c) an endosome-releasing oligopeptide element, and optionally located in (a) a histone element, (b) a receptor recognition oligopeptide (C) Endosome releases a linker peptide between oligopeptide elements.
3. 如权利要求 1所述的融合蛋白， 其特征在于， 在 (a)组蛋白元件和 (b) 受体识别 寡肽元件之间有连接肽。  3. The fusion protein according to claim 1, wherein a linker peptide is provided between (a) a histone element and (b) a receptor recognition oligopeptide element.
4. 如权利要求 1所述的融合蛋白， 其特征在于， 所述的受体识别寡肽元件选自下 组： E5， GE7、 GV1、 GV2、 或其组合；  4. The fusion protein according to claim 1, wherein the receptor recognition oligopeptide element is selected from the group consisting of: E5, GE7, GV1, GV2, or a combination thereof;
所述的内吞小体释放寡肽元件选自下组： HA20或 VP-1 ;  The endosome-releasing oligopeptide element is selected from the group consisting of: HA20 or VP-1;
所述的 H1Q 的富含赖氨酸的片段包括含 Hl°中第 97-193位氨基酸的片段。 The lysine-rich fragment of H1 Q includes a fragment containing amino acids 97-193 in H1 °.
5. 如权利要求 4所述的融合蛋白， 其特征在于，.受体识别寡肽 E5的氨基酸序列为 5. The fusion protein according to claim 4, wherein the amino acid sequence of the receptor recognition oligopeptide E5 is
EPFRS PDLAL ETYG, EPFRS PDLAL ETYG,
受体识别寡肽 GE7的氨基酸序列为 NPWG YIGER PQYRD L,  The amino acid sequence of the receptor recognition oligopeptide GE7 is NPWG YIGER PQYRD L,
受体识别寡肽 GV1的氨基酸序列为 CHPIE TLVDI FQEYP DEIEY IFKPS PVPLM RP, 受体识别寡肽 GV2的氨基酸序列为 PVPTE ESNIT MQIMR IKPHQ GQHIG EMSFL QHNKC E。  The amino acid sequence of the receptor recognition oligopeptide GV1 is CHPIE TLVDI FQEYP DEIEY IFKPS PVPLM RP, and the amino acid sequence of the receptor recognition oligopeptide GV2 is PVPTE ESNIT MQIMR IKPHQ GQHIG EMSFL QHNKC E.
6. 一种受体介导的靶向性肿瘤基因治疗的基因导入***， 其特征在于， 包含权利 要求 1所述的融合蛋白和外源 DNA， 所述的外源 DNA是含选自下组基因的真核表达载 体 DNA: 反义癌基因、抗癌基因、 ***基因、细胞调亡基因、细胞因子基因、或其组合。  6. A gene introduction system for receptor-mediated targeted tumor gene therapy, characterized in that it comprises the fusion protein of claim 1 and exogenous DNA, and the exogenous DNA is selected from the group consisting of Eukaryotic expression vector DNA of genes: antisense oncogene, anti-oncogene, suicide gene, apoptosis gene, cytokine gene, or a combination thereof.
7. 如权利要求 6所述的基因导入***， 其特征在于， 所述的融合蛋白选自下组： LOP-组蛋白元件 -HA20、 HA20-组蛋白元件 -LOP、 组蛋白元件 -LOP、 LOP-组蛋白元件， 并且还含有任选的 HA20-组蛋白元件或组蛋白元件 -HA20融合蛋白。  7. The gene introduction system according to claim 6, wherein the fusion protein is selected from the group consisting of: LOP-Histone Element-HA20, HA20-Histone Element-LOP, Histone Element-LOP, LOP A histone element, and also contains an optional HA20-histone element or a histone element-HA20 fusion protein.
8. —种药物组合物， 其特征在于， 它含有权利要求 1所述的融合蛋白和药学上可 接受的载体。  A pharmaceutical composition comprising the fusion protein according to claim 1 and a pharmaceutically acceptable carrier.
9. 一种分离的 DNA， 其特征在于， 它编码权利要求 1所述的融合蛋白。  9. An isolated DNA, characterized in that it encodes the fusion protein according to claim 1.
10. 一种表达载体， 其特征在于， 它含有权利要求 9所述的 DNA分子。  10. An expression vector, comprising the DNA molecule according to claim 9.
11. 一种宿主细胞， 其特征在于， 它含有权利要求 9所述的 DNA分子或权利要求 10所述的载体。  A host cell comprising the DNA molecule according to claim 9 or the vector according to claim 10.
12. 一种产生权利要求 1所述的融合蛋白的方法， 其特征在于， 包括步骤： 在表达条件下， 培养权利要求 11所述的宿主细胞从而表达出所述的融合蛋白； 和 分离所述的融合蛋白。  12. A method for producing a fusion protein according to claim 1, comprising the steps of: culturing the host cell according to claim 11 to express the fusion protein under expression conditions; and isolating the fusion protein; Fusion protein.
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998018951A1 (en) * 1996-10-31 1998-05-07 Shanghai Cancer Institute Receptor-mediated gene transfer system for targeting tumor gene therapy

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998018951A1 (en) * 1996-10-31 1998-05-07 Shanghai Cancer Institute Receptor-mediated gene transfer system for targeting tumor gene therapy

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
J. BIOL. CHEM., vol. 273, no. 21, 1998, pages 13324 - 13330 *
J. MOL. BIOL., vol. 187, no. 3, 1986, pages 461 - 464 *

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