WO2004009811A1 - Systeme d'entrainement de genes comprenant une sequence de noyau d'histone - Google Patents

Systeme d'entrainement de genes comprenant une sequence de noyau d'histone Download PDF

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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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English (en)
Chinese (zh)
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Jianren Gu
Shengli Yang
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Neworgen Limited
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Publication of WO2004009811A1 publication Critical patent/WO2004009811A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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

L'invention concerne un système d'entraînement de gènes comprenant une séquence de noyau d'histone, un procédé et une méthode d'utilisation correspondants. Le système d'entraînement de gènes comprend la protéine de fusion avec la séquence de noyau d'histone, qui comprend (a) un composant d'histone choisi dans une séquence pleine longueur H1° du fragment de lysine d'enrichissement; (b) le composant d'oligopeptide de reconnaissance de récepteur LOP; n'importe quel oligopeptide EROP à vésicule de libération à revêtement . L'invention concerne un système d'entraînement de gènes qui permet non seulement de transférer l'ADN étranger avec une efficacité élevée mais aussi de le préparer par une technique de génie génétique.
PCT/CN2002/000592 2002-07-24 2002-08-26 Systeme d'entrainement de genes comprenant une sequence de noyau d'histone WO2004009811A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998018951A1 (fr) * 1996-10-31 1998-05-07 Shanghai Cancer Institute Systeme de transfert de genes par l'intermediaire de recepteurs pour therapie genique ciblee de tumeurs

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998018951A1 (fr) * 1996-10-31 1998-05-07 Shanghai Cancer Institute Systeme de transfert de genes par l'intermediaire de recepteurs pour therapie genique ciblee de tumeurs

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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