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CN1323723C - Tie2 receptor mediated gene transfer system for targeted tumor gene therapy - Google Patents

Tie2 receptor mediated gene transfer system for targeted tumor gene therapy Download PDF

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CN1323723C
CN1323723C CNB2003101228303A CN200310122830A CN1323723C CN 1323723 C CN1323723 C CN 1323723C CN B2003101228303 A CNB2003101228303 A CN B2003101228303A CN 200310122830 A CN200310122830 A CN 200310122830A CN 1323723 C CN1323723 C CN 1323723C
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gene
oligopeptide
tie2
transfer system
gene transfer
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CN1634595A (en
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顾健人
吴向华
徐宇虹
李宗海
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SHANGHAI XINSHIJIE GENE TECHN DEVELOPMENT Co Ltd
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SHANGHAI XINSHIJIE GENE TECHN DEVELOPMENT Co Ltd
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Abstract

The present invention provides a gene transfer system mediated by an angiogenin promotion receptor Tie2, which comprises a ligand oligopeptide (a) specifically combined with the receptor Tie2, a polycation polypeptide (b), an optional endocytosis corpuscle released oligopeptide (c) and exogenous a DNA (d). The gene transfer system can effectively lead exogenous gene in endothelial cells of new vessels of tumor in a targeting manner and the tumor cells for expressing the receptor Tie2 in a human body or in vitro so as to inhibit the growth of the tumor.

Description

The gene transfer system of the receptor-mediated targeting therapy of tumor of Tie2
Technical field
The present invention relates to molecular biology and field of gene.Particularly, relate to a kind of based on the gene transfer system of the bonded part oligopeptide of angiogenesis hormone receptor Tie2.This transfer system imports the tumor cell of endothelial cells in tumor neogenetic blood vessels and expression Tie2 receptor by receptor mediated endocytosis with foreign DNA targeting ground, thereby reaches the purpose of treatment tumor.
Background technology
PCT application PCT/CN97/00106 (WO98/18951) discloses the novel receptor-mediated gene transfer system that is used for the targeting therapy of tumor, wherein discloses to comprise at EGFR, IGF I/II R and three kinds of receptor-mediated gene transfer systems of VEGFR and be used for oncotherapy.Yet because the receptor of different tumor cell surfaces is varied, expression has to be had less more, even does not express some receptor, therefore only has three kinds of systems can not solve whole problems.
Partanen J in 1992 etc. find a kind of new receptor type tyrosine kinase at people's endothelial cell surface, called after Tie2 (Tyrosie kinase with Ig and EGF homology domains).The transmembrane protein that this albumen is made up of 1124 amino acid residues.This receptor by extracellular region, stride the film district and intracellular region constitutes.Extracellular region is made up of two immunoglobulin like domain, three EGF homology motifs and three fibronectin III type repetitive sequences; Intracellular region has the catalysis district.Wherein, immunoglobulin like domain is the binding site with part.
Davis in 1996 etc. have found a kind of new albumen-Angiopoietin-1 (Ang-1), and it is the part of Tie2 receptor in the protein tyrosine kinase receptor family, and the Tie2 receptor is almost only expressed on endotheliocyte.Ang-1 is structurally different with the part of known angiogenesis factor or other protein tyrosine kinase receptor, can not directly promote the endothelial cells cultured growth external, so infer that it may be irrelevant with tumor neovasculature startup, and work in the later stage of angiogenesis, make blood vessel network stable.
After finding Ang-1, utilize the homology method for screening, Maisonpierre has found Angiopoietin-2 (Ang2) again, this albumen also can with the Tie2 receptors bind.Can not activate Tie2 receptor on the endotheliocyte at external Ang2, but can activate other cell, as the Tie2 receptor on the NIH-3T3 cell, so, the function of Ang2 may be and the Ang1 antagonism, make vasoganglion be in a kind of unsure state, help the division of endotheliocyte and rebuliding of vasoganglion.
Discover that the protein structure of these parts is made up of three parts: the Coiled-Coil domain of hydrophobicity secreting signal peptide, N-end and the Fibrinogen spline structure territory of C-end.The Coiled-Coil domain of N-end is relevant with its polymerization, and the Fibrinogen spline structure territory mediation part of C-end and combining of receptor.The expression of Tie2 has relative specificity, mainly on vascular endothelial cell, part tumor cell and some hematopoietic cell surfaces.After part and receptors bind, receptor dimerization causes the Tie2 autophosphorylation, can combine with multiple target protein in the born of the same parents, by the different different biological functions of signal transduction pathway performance.Can be by improving the migration of anti-endothelial cell apoptosis of phosphatidylinositols 3 kinase activities and promotion endotheliocyte.Can't survive after the birth of the mice of Tie2 gene knockout, pathologic finding finds, vascularization is arranged in the mice body but can not constitute complicated blood vessel network.Owing to, all increase as Tie2 receptor expression in the tumor tissues blood vessels such as cerebral glioma, pulmonary carcinoma, bladder cancer in kinds of tumors.So the signal conduction by blocking-up Tie2 receptor can suppress the growth of neonate tumour blood vessel and tumor cell simultaneously, reaches the purpose of treatment tumor and the unusual relevant disease of some vascular proliferations.
Bibliographical information is arranged, utilize adenovirus transduction soluble T ie2 receptor extracellular region gene, can suppress mouse breast cancer 4T1 and melanin tumour b16 F10.9 cell in vivo, suppression ratio reaches 64% and 47% respectively, and has suppressed the transfer of tumor effectively.This studies show that the signal transduction that suppresses the Tie2 receptor has certain depression effect to growth of tumor.
In sum, Tie2 can be used as malignant tumor and the new target spot of some aberrant angiogenesis proliferative diseasees treatment.The molecule that can combine and have certain function at present with Tie2 only has the Tie2 antibody of artificial preparation and natural Tie2 part.Though native ligand can efficiently play good targeting effect in conjunction with Tie2, purifying natural Tie2 part is technical sophistication not only, and scale preparation is unrealistic, therefore can not satisfy clinical practice.Only there is a spot of people Tie2 part to be used for scientific experiments at present, and costs an arm and a leg.In addition, the humanized high molecular weight protein of anti-Tie2 antibody right and wrong of artificial preparation is a kind of foreign protein antigen molecule for human body, has stronger immunogenicity, also is difficult to clinical.
Therefore, this area presses for the part oligopeptide of the new targeting Tie2 of exploitation and based on the gene transfer system of the targeting therapy of tumor of Tie2 part oligopeptide.Gene therapy and other targeted therapy measures by targeting Tie2 reach the purpose for the treatment of disease.
Summary of the invention
The objective of the invention is just to provide the gene transfer system of the receptor-mediated targeting therapy of tumor of a kind of Tie2, it is that a kind of part oligopeptide and endocytosis corpusculum with the Tie2 receptors bind discharges the constructed gene transfer system of oligopeptide.
In a first aspect of the present invention, a kind of gene transfer system of targeting gene therapy of growth factor receptors Tie2 mediation is provided, it comprises the part oligopeptide that (a) specificity is incorporated into the Tie2 receptor, and wherein said part oligopeptide contains SEQ ID NO:1,2 or 3 aminoacid sequence; (b) polycation polypeptide.
Preferably, described gene transfer system also contains (c) endocytosis corpusculum release oligopeptide.
In another preference, described gene transfer system also contains (d) foreign DNA.
In another preference, the aminoacid sequence of described part oligopeptide is SEQ ID NO:1,2 or 3.
In another preference, described polycation polypeptide is selected from down group: poly-D-lysine, poly-acetimide (PEI), protamine, histone, adenovirus pV albumen, adenovirus pVII albumen, m μ albumen, above-mentioned albumen of modifying through Polyethylene Glycol (PEG) and composition thereof.
In another preference, described foreign DNA is selected from down group: the recombinant eukaryotic expression plasmid DNA of antioncogene, apoptosis gene, cytokine gene, oncogene antisense sequences, and composition thereof.More preferably, described foreign DNA is selected from down group:
(i) antioncogene: p53, Rb;
(ii) apoptosis gene: Caspase, p15, p16 and p21 WAF-1
(iii) cytokine gene: GM-CSF (CM-CSF) gene, TNF (tumor necrosis factor) α gene, INF (interferon) α, γ gene, IL (interleukin) 2, IL3, IL4, IL12, IL15, IL18 gene;
The (iv) antisense sequences of oncogene: the antisense sequences of oncogene ras, c-myc, Akt;
(v) recombinant eukaryotic expression plasmid: viral recombinant eukaryotic expression plasmid (retrovirus recombinant expression carrier, adenovirus recombinant expression carrier, adeno-associated virus recombinant expression carrier) and be the non-viral recombinant eukaryotic expression plasmid of cis-regulating element with SV40, CMV promoter.
In another preference, described component (a) part oligopeptide, (b) polycation polypeptide and (c) both or the three that discharge in the oligopeptide of endocytosis corpusculum exist with the fusion rotein form.
In another preference, described endocytosis corpusculum discharges the oligopeptide element and is selected from down group: HA20 or VP-1.
In a second aspect of the present invention, a kind of oligopeptide (length less than 100, more preferably 50 aminoacid) is provided, it is incorporated into the Tie2 receptor, contains following aminoacid sequence: SEQ ID NO:1,2 or 3.
In a third aspect of the present invention, a kind of method of gene transfer system of the targeting gene therapy for preparing growth factor receptors Tie2 mediation is provided, comprise step: the part oligopeptide, (b) polycation polypeptide and optional (c) endocytosis corpusculum release oligopeptide that (a) specificity are incorporated into the Tie2 receptor mix, and form mixture.Preferably, for component (a) and (b), (c), can use any the two formation binary complex.
In a fourth aspect of the present invention, provide a kind of the present invention the antibody of above-mentioned part oligopeptide.
In a fifth aspect of the present invention, a kind of conjugate is provided, it contain the above-mentioned part oligopeptide of the present invention and with the link coupled radionuclide of described part oligopeptide, chemicals and biotoxin.These conjugates can directly apply to the treatment malignant tumor relevant with the Tie2 overexpression, as adenocarcinoma of lung, and osteosarcoma.
In a sixth aspect of the present invention, a kind of pharmaceutical composition is provided, it contains the part oligopeptide of the present invention of safe and effective dosage, and pharmaceutically acceptable excipient, diluent or carrier.Pharmaceutical composition of the present invention can be used for treating the malignant tumor relevant with the Tie2 overexpression, as adenocarcinoma of lung, and osteosarcoma.
Description of drawings
Accompanying drawing 1 is polypeptide GA3 mass spectral analysis figure.
Accompanying drawing 2 is 125I labelling GA3 combines the result of the test sketch map with the SMMC7721 cell surface Tie2 receptor-specific of stably express Tie2.
Accompanying drawing 3 is 125I labelling GA3 combines the result of the test sketch map with rhTie2-Fc fusion rotein specificity.
Accompanying drawing 4 is 125I labelling GA3 combines the dissociation constant curve with the Tie2 receptor-specific, and Kd is 2.5 * 10 -8M.
Accompanying drawing 5 is phage clone and the bonded ELISA result of cell surface Tie2 receptor-specific.
Accompanying drawing 6 is phage clone and the bonded SABC result of cell surface Tie2 receptor-specific.Figure A, B are respectively phage clone and the bonded SABC results of Tie2 (+) SMMC7721 cell who shows GA4 and GA5; Figure C and D are respectively phage clone and the bonded SABC results of Tie2 (-) SMMC7721 cell who shows GA4 and GA5.
Accompanying drawing 7 is that phage clone combines the result of the test sketch map with rhTie2-Fc fusion rotein specificity.
The specific embodiment
The inventor is through extensive and deep research, prepared that specificity is incorporated into the part oligopeptide of Tie2 receptor and based on the gene transfer system of this part oligopeptide.GA3 (SEQ ID NO:1) is applying biological informatics method to the amino acid sequence analysis of angiogenesis hormone-1 and 2 and lives.GA4 and GA5 (SEQID NO:2 and SEQ ID NO:3) are by the phage peptide library triage techniques, directly obtain from the peptide storehouse and the bonded polypeptide of Tie2 acceptor molecule.The ad-hoc location of the specific amino acids of these polypeptide is avtive spots of polypeptide ligand and Tie2 receptors bind, and Tie2 acceptor molecule polypeptide ligand motif has just determined to combine with Tie2 the living features of polypeptide.
Utilize the characteristic of part oligopeptide identification Tie2 receptor of the present invention, endocytosis through cell, foreign DNA is optionally imported the endothelial cells in tumor neogenetic blood vessels or the tumor cell of expressing the Tie2 receptor by this carrier system, thereby angiogenesis inhibiting and tumor cell reach the purpose for the treatment of tumor simultaneously.Finished the present invention on this basis.
The part oligopeptide
The part oligopeptide is the key component of targeting non-virus carrier of the present invention.As used herein, " part oligopeptide (Ligand oligopeptide, LOP) " and " receptor identification oligopeptide " is used interchangeably, and refers to any identification and is incorporated into the oligopeptide that the surface contains the target cell of Tie2 receptor.As mentioned above, but part oligopeptide specific recognition of the present invention and be incorporated into the Tie2 receptor.Particularly, part oligopeptide of the present invention is meant and the bonded polypeptide of surface of cell membrane protein receptor Tie2 extracellular region, perhaps with the bonded polypeptide of soluble T ie2 molecule, comprises pattern of fusion polypeptide and free type polypeptide.
Ligand polypeptide of the present invention also comprises variant form identical function, SEQ IDNO.1-3 sequence that has with the Tie2 receptors bind.These variant forms comprise (but being not limited to): several (are generally 1-5, preferably 1-3, more preferably 1-2,1 best) amino acid whose disappearance, insertion and/or replacement, and add one or several at C-terminal and/or N-terminal and (be generally in 20, preferably being in 10, more preferably is in 5) aminoacid.
One skilled in the art will appreciate that nonpolar (hydrophobicity) aminoacid comprises alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine.Polar neutral amino acid comprises glycine, serine, threonine, tyrosine, agedoite and glutamine.Basic amino acid comprises lysine, histidine and arginine.Acidic amino acid comprises glutamic acid and aspartic acid.In the art, when replacing, can not change the function of polypeptide usually with the close or similar aminoacid of performance.In addition, add one or several aminoacid at C-terminal and/or N-terminal and also can not change proteinic function usually.
As used herein, term " part oligopeptide " comprise can with active fragment and the reactive derivative of the bonded GA3 of Tie2, GA4 and GA5.
As used herein, term " part oligopeptide " also comprises the polypeptide of amide or ester or its salt form.Both comprised free type polypeptide, pattern of fusion polypeptide and mosaic type polypeptide, comprised also that with polypeptide be monomeric various polymer.For example, contain protein molecule, chimeric protein molecule or polymers a kind of among SEQ ID NO:1, SEQ ID NO:2 and the SEQ ID NO:3, two kinds or three seed amino acid sequences.
In the application's description and accompanying drawing, be used to represent that base, amino acid whose abbreviation are to be recommended by biochemistry name IUPCA-IUB committee.Wherein aminoacid is generally the L type, unless other explanation is arranged.Polypeptide described in this description, by convention, left distal end is amino terminal (a N-end), right end is carboxyl terminal (a C-end).Usually when the C-end be carboxyl (COOH) or carboxylate (COO-) time, it can be amide (COONH2) or ester (COOR) form.Ester residue R comprises C1-6 alkyl (as methyl, ethyl, n-propyl group, isopropyl, n-butyl etc.), C3-8 cycloalkyl (as cyclopenta, cyclohexyl etc.), C6-12 aryl (as phenyl, a-naphthyl etc.), C7-14 alkyl (as benzyl, phenethyl etc.).
The particularly preferred part oligopeptide of one class is that length is 15-30 aminoacid, and contains the oligopeptide of the elementary cell sequence that is selected from down group:
WTIIQRREDG?SVDFQRTWKE?YK(SEQ?ID?NO：1)；
HATGTHGLSL?SH(SEQ?ID?NO：2)；
NSLSNASQFR?AP(SEQ?ID?NO：3)。
More preferred example is following oligopeptide:
Oligopeptide GA3, its aminoacid sequence are WTIIQRREDG SVDFQRTWKE YK (SEQ IDNO:1);
Oligopeptide GA4, its aminoacid sequence are HATGTHGLSL SH (SEQ ID NO:2);
Oligopeptide GA5, its aminoacid sequence are NSLSNASQFR AP (SEQ ID NO:3);
The aminoacid sequence of GA3, GA4 and GA5 is to draw according to the bioinformatic analysis to the aminoacid sequence of angiogenesis hormone-1 and 2.
In addition, part oligopeptide of the present invention comprises that also the aminoacid in one or several (1-8 usually, preferably 1-5) site can replace formed part oligopeptide with the aminoacid of similar performance.
Part oligopeptide of the present invention can contain single elementary cell sequence, or a plurality of (as 2-10) elementary cell sequence.
Part oligopeptide of the present invention can be easily with synthetic or genetic engineering recombinant methods.About these synthetic oligopeptide method for makings, can be referring to such as WO98/18951 many documents such as (PCT/CN97/00106).
The present invention also provides the coded sequence of Tie2 part oligopeptide such as GA3, GA4, GA5, and they contain the nucleotide sequence shown in the SEQ ID NO:5,6 and 7 respectively.These coded sequences can obtain with conventional artificial synthesis or recombination method.
The polycation polypeptide
The polycation polypeptide be targeting non-virus carrier of the present invention another key component.
As used herein, " polycation polypeptide " refers to that a class is rich in the polypeptide of basic amino acid, and its basic amino acid (lysine, histidine and arginine) ratio is about more than 20%, thereby positively charged, can combine with electronegative DNA mat electrostatic attraction, make DNA more stable.
Can be used for polycation polypeptide of the present invention and be not particularly limited, as long as its basic amino acid (lysine and arginine) ratio is about more than 20%, preferably 30%, more preferably more than 40%, thereby positively charged getting final product.Representational example comprises: poly-D-lysine, poly-acetimide, protamine, histone, adenovirus pV albumen, adenovirus pVII albumen, m μ albumen, above-mentioned albumen of modifying through Polyethylene Glycol (PEG) and composition thereof.
The endocytosis corpusculum discharges the oligopeptide element
Targeting non-virus carrier of the present invention also can contain the endocytosis corpusculum and discharge the oligopeptide element, swallows the effect that the DNA of cell is degraded in it plays and prevents.
Any endocytosis corpusculum discharges oligopeptide and all can be used for the present invention, and representational example comprises (but being not limited to): HA20, VP-1.
The targeting non-virus carrier
As used herein, " targeting non-virus carrier " of the present invention refers to contain following component (a) and (b) or contain component (a) and (b) and mixture (c) or complex: (a) the specificity part oligopeptide, (b) polycation polypeptide, (c) endocytosis corpusculum that are incorporated into the Tie2 receptor discharges oligopeptide.
In the present invention, component (a) and (b), (c) can be non-types of attachment, also can be any both or three link together by mode covalently or non-covalently.Particularly preferred form comprises: part oligopeptide-polycation polypeptide binary complex, polycation polypeptide-endocytosis corpusculum discharge the oligopeptide binary complex, part oligopeptide-polycation polypeptide-endocytosis corpusculum discharges the oligopeptide ternary complex.For example HA20-poly-D-lysine, HA20-protamine, the covalently bound thing of HA20-histone etc.
Connection can be passed through chemical method (as coupling agents such as use SPDP), also can pass through recombination method, for example forms fusion rotein.
For fusion rotein, at (a) part oligopeptide, (b) polycation polypeptide, and (c) the endocytosis corpusculum discharges between the oligopeptide element, can randomly contain connection peptides, so that increase the flexibility and the stretching of complex polypeptide space structure, allow each element performance function separately.Can be used for connection peptides of the present invention and be not particularly limited, needed only interconnect function.One class connection peptides example is (Gly) 2-6Ser, for example (Gly) 4Ser.Connection peptides can be used in a plurality of series connection.
(a) of the present invention part oligopeptide, (b) the polycation polypeptide and (c) the endocytosis corpusculum discharge the oligopeptide element can be with the amino acid replacement of similar performance, as long as still keep separately similarly biological function.
The preparation method of targeting non-virus carrier
Usually, above-mentioned (a) and (b), (c) component or its complex are mixed, can obtain targeting non-virus carrier of the present invention.Wherein, the mixed proportion of (a) and (b), (c) component is generally 0.5-1: 1-2: 0-1 preferably is 0.8-1: 1-1.5: 0.5-1.
In addition, when with (a)-(b) complex with (b)-(c) when the form of complex is used, (a)-(b) complex: (b)-(c) mixed proportion of complex is generally 0.8-1.2: 0.8-1.2 preferably is 0.9-1.1: 0.9: 1.1.
Foreign DNA
Can be used for foreign DNA of the present invention is not particularly limited, can be various therapeutic or preventative DNA, for example genes of interest, antisense oncogene, antioncogene, suicide gene, natural death of cerebral cells gene, cytokine gene or its combination perhaps contains the carrier for expression of eukaryon DNA of said gene.The proto-oncogene antisense sequences comprises proto-oncogene (ras H, ras K, ras N, c-myc, bcl-2, Akt), antisense dna sequence, antisense oligonucleotide and the antisense oligodeoxyribonucleotide of somatomedin and acceptor gene thereof; Antioncogene comprises p53, Rb, PTEN, and suicide gene comprises HSV-TK (herpes simplex virus thymidine kinase) gene and CD (coli cytosine deaminase) gene; Apoptosis gene comprises p15, p16, p21 WAF-1Cytokine gene comprises GM-CSF (granulocyte macrophage colony stimulating factor) gene, TNF α (tumor necrosis factor) gene, INF (interferon) α, γ gene, IL (interleukin) 2,3,4,12,15,18 genes etc.
Target gene entraining system
Targeting non-virus carrier of the present invention is mixed with foreign DNA, just constitute the gene import system of the receptor-mediated targeting therapy of tumor of Tie2.Depend on used foreign DNA, gene import system of the present invention can be used for treating diseases such as genetic diseases or tumor, in particular for gene therapy.
Pharmaceutical composition
The present invention also provides a kind of pharmaceutical composition, and it contains one or more targeting non-virus carrier of the present invention and pharmaceutically acceptable carrier or excipient of the present invention of safe and effective amount.This class carrier comprises (but being not limited to): saline, buffer, glucose, water, glycerol, ethanol and combination thereof.Pharmaceutical preparation should be complementary with administering mode.Pharmaceutical composition of the present invention can be made into the injection form, for example is prepared by conventional method with normal saline or the aqueous solution that contains glucose and other adjuvant.
When using, also contain one or more above-mentioned foreign DNAs in the pharmaceutical composition.
Concrete dosage also should be considered factors such as route of administration, patient health situation, and these all are within the skilled practitioners skill.
Major advantage of the present invention is,
(1) the present invention combines Tie2 with the binding site of Tie2 with the natural Tie2 part of micromolecule polypeptide anthropomorphic dummy from the interactional mechanism of protein molecular.Compare with monoclonal antibody, the micromolecule polypeptide structure is clear, with Tie2 binding site limitation, is difficult for the space steric effect between generation polypeptide and the polypeptide, does not also have the interference of Fc section.
(2) micromolecule polypeptide does not almost have antigenicity, and side effect is little, easily preparation and large-scale production and characteristics such as easy to use.
(3) the relevant polypeptide of the present invention is owing to can combine with cell surface Tie2 receptor protein and may reach the function of regulating angiogenesis by activating or block the Tie2 signal transduction pathway, thereby can be used as precursor or the pharmaceutical composition that clinical medicine is developed, it comprises code book and invents the DNA of relevant polypeptide and the antibody of the relevant polypeptide of the present invention.
(4) the relevant polypeptide of the present invention can be used as the targeting peptide and is used for gene transfer system, carries out at the endothelial cells in tumor neogenetic blood vessels of Tie2 receptor high expressed and the target gene therapy of tumor cell.
(5) the relevant polypeptide of the present invention can be used as the targeting peptide and connects the targeted therapy effect that radionuclide, chemicals, biotoxin are used for malignant tumor and Tie2 systemic-function dysfunctional disease.
(6) DNA sequence of the relevant polypeptide of the present invention and this polypeptide of encoding thereof also can be used as and detects or diagnostic reagent.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1 chemically synthesized polypeptide and mass spectral analysis thereof
In the present embodiment, prepare GA3, GA4 and GA5 with artificial synthesis.
With polypeptide GA3[SEQ ID NO:1] be example, its synthetic solid-phase synthesis that adopts carries out on Peptide synthesizer.Used vector resin is a HMP amino resins, and amino acid whose amount is 1 mM, i.e. aminoacid: resin=4: 1.According to polypeptide GA3[SEQ ID NO:1] aminoacid sequence, add each aminoacid ingredient successively and carry out solid phase synthesis.After polypeptide is synthetic, cut according to the step that PE company is recommended, reacted mixed liquor is filtered resin with the G4 glass sand hourglass, with filtrate at normal temperatures low pressure be evaporated to 1ml, the ether sedimentation polypeptide that adds the 50ml pre-cooling, 4 ℃ are spent the night, and filter through the G6 glass sand hourglass, and vacuum lyophilization obtains the polypeptide crude product.Adopt Sephadex G10 column purification,, collect first peak, concentrate with 0.1N glacial acetic acid eluting, disacidify, with the low amounts of water dissolving, vacuum lyophilization gets dry powder.Carry out mass spectral analysis, the molecular weight size of gained polypeptide is close with theoretical value, is 2842, shows this polypeptide GA3[SEQ ID NO:1 just] (Fig. 1).
With the GA4 of same procedure system, the molecular weight of mensuration is 1380, and is close with theoretical value, shows this polypeptide GA4[SEQ ID NO:2 just].
With the GA5 of same procedure system, the molecular weight of mensuration is 1456, and is close with theoretical value, shows this polypeptide GA5[SEQ ID NO:3 just].
Embodiment 2 GA3 combine test with the SMMC7721 cell surface Tie2 receptor-specific of stably express Tie2
(1) foundation of stably express Tie2 extracellular region cell line
(1) the Western blotting by routine filters out the negative human cell line SMMC7721 that expresses of Tie2, and method is as follows:
Each collects the well-grown different tumor cell lines of a plate (9cm), and it is inferior to give a baby a bath on the third day after its birth with PBS, adds 1mlPBS, with curet cell is scraped, collect the 1.5ml centrifuge tube, 8000rpm, 1min, remove most supernatant, add protein lysate 100 μ l (containing protease inhibitor 1 μ g), put cracking on ice 30 minutes, 10000rpm, centrifugal 10min changes supernatant in new pipe, is total protein of cell.Get 20 μ l protein solutions respectively and add 4 μ l sample loading buffers, boil 10min, 10000rpm, centrifugal 10min gets supernatant and is used for protein electrophoresis, changes film, 0.25% skim milk/PBS sealing adds 1: 2000 Tie2 extracellular region antibody (Santa Cruzbiotechnology), and 4 ℃ are spent the night, PBS gives a baby a bath on the third day after its birth inferior, and 5 minutes/time, 1: 5,000 two anti-(Santa Cruzbiotechnology), PBS gives a baby a bath on the third day after its birth inferior, and 5 minutes/time, ECL reaction 5 minutes, tabletting develops photographic fixing.The result shows that the expression of SMMC7721 cell strain is negative.
(2) structure of PCDNA3.0-ExTie2 plasmid
Forward primer: 5 '-CCG GAA TTC ATG GAC TCT TTA GCC AGC TT-3 ' (SEQ ID NO:8)
Downstream primer: 5 '-CCG CTC GAG CTA GAT GGC TAT AAG CAG CAT-3 ' (SEQ ID NO:9)
Oligonucleotide with top a pair of introducing EcoR I and Xho I restriction enzyme site is a primer, is template with pAdvtie2 plasmid (providing available from the Shanghai Inst. of Tumor), obtains the dna fragmentation of coding Tie2 extracellular region (ExTie2) through PCR.Equally PCR product and PCDNA3.0 vector plasmid (available from Invitrogen company) are carried out EcoR I and the reaction of Xho I double digestion, gel electrophoresis is cut glue and is reclaimed carrier segments and insert fragment.Carrier segments behind the enzyme action is connected through the T4 ligase with the insertion fragment, and 16 ℃ are spent the night.Transformed competence colibacillus Bacillus coli cells (DH5 α) (available from Invitrogen company), coated plate (Amp resistance), 37 ℃ of overnight incubation.Choose the monoclonal amplification, a small amount of extracting plasmid, enzyme action identifies that order-checking obtains the PCDNA3.0-ExTie2 plasmid.
(3) with the liposome transfection method PCDNA3.0-ExTie2 plasmid is imported above-mentioned SMMC7721 cell, with the G418 screening, choose the monoclonal amplification, extract total protein, Western blot identifies, obtains the ExTie2 stable expression cell line.
(2), 125I GA3 combines with the SMMC7721 cell surface Tie2 receptor-specific of stably express Tie2
The SMMC7721 cell of inoculation stably express Tie2 is in 96 orifice plates, 5 * 10 3Cells/well, overnight incubation is treated cell attachment, wash 3 times with PBST (PBS+0.1%Tween20), the competition group adds the usefulness binding buffer liquid PBS+20mM HEPES+2.5mg/ml BSA of pre-cooling, pH 7.4) dilution the every hole 20 μ l (containing GA320pmol) of GA3, non-competing group adds 20 μ l binding buffer liquid, every group of parallel three holes; Iodine mark GA3 is diluted to 1 milliliter with binding buffer liquid simultaneously, and every hole adds 20 μ l and (contains 125I GA3 0.02pmol).4 ℃ in conjunction with 3 hours.PBST washes 3 times, adds the pancreatin of 100 μ l 0.25%, digests 10 minutes, transfers to and measures in the pipe, washes with 100 μ l PBST, equally washing liquid is added in the corresponding mensuration pipe gamma counting.
Experimental result is seen Fig. 2.Show that GA3 can combine with the SMMC7721 cell surface Tie2 receptor generation specificity of stably express Tie2.
Embodiment 3 125I labelling GA3 combines with rhTie2-Fc fusion rotein specificity
Every hole 0.1M NaHCO 3(PH 8.6) 50 μ l wrap by 1 μ g rhTie2-Fc fusion rotein (available from R﹠amp; D company) in 96 orifice plate, 4 ℃ are spent the night, and PBST washes 3 times, and the competition group adds the every hole 20 μ l (containing GA320pmol) of GA3 with binding buffer liquid (PBS+20mMHEPES+2.5mg/ml BSA PH 7.4) dilution of pre-cooling, non-competing group adds 20 μ l binding buffer liquid, every group of parallel three holes; Iodine mark GA3 is diluted to 1 milliliter with binding buffer liquid simultaneously, and every hole adds 20 μ l and (contains 125I GA3 0.02pmol).Under the room temperature in conjunction with 1 hour.PBST washes 3 times, adds 100 μ l Glysine-HCl (PH 2.2) eluting 15 minutes, transfers to and measures in the pipe, and reuse 100 μ l Glysine-HCl wash, and equally washing liquid is added in the corresponding mensuration pipe gamma counting.
The results are shown in Figure 3.Show that GA3 can combine with rhTie2-Fc fusion rotein generation specificity.
Embodiment 4 GA3 and Tie2 receptor affinity detect
The SMMC7721 cell of inoculation stably express Tie2 is in 96 orifice plates, 1 * 10 4Cells/well, overnight incubation is treated cell attachment, washes 3 times with PBST (PBS+0.1%Tween20), with sealing buffer (BlockingBuffer) (0.05mM PB+5mg/ml BSA) 100 μ l, 4 ℃ were sealed 1 hour, and PBST washes 3 times, added corresponding according to the every hole of following Concentraton gradient 125The GA3 of I labelling, each concentration group is established three parallel holes, and every hole reaction system is 100 μ l.4 ℃ in conjunction with 3 hours, and PBST washes 3 times, adds the pancreatin of 100 μ l 0.25%, digests 10 minutes, transfers to and measures in the pipe, with 100 μ l PBST washing, equally washing liquid is added in the corresponding mensuration pipe, and gamma is counted.According to experimental result, the dissociation constant Kd[Concentraton gradient that application Scatcard plot calculates reaction GA3 and Tie2 receptor affinity size is: 6.13nM, 12.5nM, 25nM, 50nM, 100nM, 200nM, 300nM, 500nM, 750nM].
The results are shown in Figure 4.The GA3 that measures and the Kd of Tie2 receptors bind are 2.5 * 10 -8M is far above present existing Tie2 part oligopeptide.
Embodiment 5 phage clones combine with cell surface Tie2 receptor-specific
(1) elisa assay
Take the logarithm SMMC 7721 cell inoculations of the Tie2 (+) of trophophase and Tie2 (-) to 96 orifice plates, 1 * 10 4Cells/well, overnight incubation is treated cell attachment, PBS washes 3 times, simultaneously with rhTie2, BSA and skim milk bag by to 96 orifice plates (500ng/ul), with the BSA sealing, PBS washes 3 times, every hole adds 1 * 10 12Pfu is through the amplification and the phage of purification, 37 ℃ 1 hour.After PBST washes 6 times, add HRP-goat-anti M13 polyclonal antibody 100 μ l/ holes, 37 ℃ 1 hour.After the PBST rinsing, add enzyme reaction substrate (ABST) solution and develop the color, enzyme connection instrument detects OD405nm.Blank is set up in experiment, comprises each clone's acellular hole and does not have the segmental wild phage of insertion.Set up 3 parallel holes for every group.
ELISA the results are shown in accompanying drawing 5.Show that the phage of showing GA5 combines with the SMMC7721 cell of rh-Tie2 and stably express Tie2.
(2) immunohistochemical analysis
(a) preparation of the phage of displaying GA4 and GA5
Show the preparation of the phage of GA4:
Use 0.1M NaHCO respectively 3(PH 8.6) 50 μ l bag is put in the wet box 4 ℃ by 5 μ g recombined human Tie2 (rh-Tie2) in 96 orifice plates and is spent the night.PBST washes 6 times, sealing buffer (0.1M NaHCO 3(PH 8.6) 100 μ l+5mg/ml BSA PH 7.5) sealing, 4 ℃ 3 hours.TBST washes 10 times, takes out 10 μ l (4 * 10 simultaneously 10Pfuphage) former storehouse phage (New England BioLabs Inc.) adds Tissue Culture Plate, decolorization swinging table jog 1 hour with 1 milliliter of TBST dilution back.Back-off is removed unconjugated phage on clean filter paper.TBST washes 10 times, add 100 μ l Glysine-HCl (PH 2.2) eluting 8 minutes, and eluent was transferred to measured in the pipe, add 15 μ l Tris-HCl (PH 9.1) neutralization, get the eluent that 1 μ l contains phage then and carry out titration, all the other join in the ER2537 bacterium and increase.Repeat preceding step, carry out second and third, the four-wheel screening.The single bacterium colony of ER2537 of the new recovery of picking is inoculated in 2mlLB, and it is standby to be expanded to mid-log phase.Get 1 μ l four-wheel eluent and carry out titration, choose single blue colonies adds 20 μ l mid-log phases simultaneously in 2ml LB ER2537 bacterium, 37 ℃ of shaking tables shook 4.5 hours.Collect bacterium liquid to the 1.5ml centrifuge tube, 4 ℃ of centrifugal 10min of 10000rpm, carefully get 500 μ l supernatants and in new pipe, (take a morsel in 4 ℃ of preservations), add 200 μ lPEG/NaCl in order to follow-up amplification, mixing, room temperature is placed 10min, and the centrifugal 10min of 10000rpm removes supernatant, heavy centrifugal, carefully remove most supernatant.Add 100 μ l lodide buffer mixings, add 250 μ l ethanol simultaneously, room temperature is placed 10min, and the centrifugal 10min of 10000rpm removes supernatant, washes once with 70% ethanol, and precipitation is dissolved in 30 μ lTE and is used for order-checking.Sequencing result finds that obvious enrichment appears in the phage of showing GA4.Get the phage supernatant 10 μ l that show GA4 that contain that preserved and be added to the ER2537 bacterium that adds 200 μ l mid-log phases among the 20ml LB simultaneously, 37 ℃ of shaking tables shook 4.5 hours.Collect bacterium liquid to centrifuge tube, 4 ℃ of centrifugal 10min of 10000rpm shift supernatant in new pipe, and weight is centrifugal, and the centrifugal 10min of 10000rpm gets 80% supernatant in new pipe, adds the PEG/NaCl of 1/6 volume, and 4 ℃ are spent the night.The centrifugal 15min of 10000rpm removes supernatant, and is heavy centrifugal, removes most remaining supernatant.1ml TBS is resuspended, transfers to the 1.5ml centrifuge tube, and centrifugal 10 minutes of 10000rpm gets 80% of supernatant and puts in the new pipe, adds the PEG/NaCl of 1/6 volume, ice bath 1 hour; Centrifugal 10 minutes of 10000rpm removes most supernatant, with 200 μ l TBS/0.02%NaN 3Resuspended, centrifugal 1 minute of 10000rpm shifts supernatant in new pipe, is the phage of the displaying GA4 of amplification.
The preparation of showing the phage of GA5 is with 1 * 10 4The SMMC7721-ExTie2 cell is the screening target, with the TBS eluting that contains Ang-2 (100 μ g/ml).All the other steps are with the preparation of showing the GA4 phage.
(b) binding analysis
Take the logarithm the Tie2 (+) of trophophase and Tie2 (-) SMMC 7721 cell inoculations to 96 orifice plates, 1 * 10 4Cells/well, overnight incubation is treated cell attachment, PBST washes 3 times, 37 ℃ of 10% formaldehyde fixed 30 minutes, PBST washes 3 times, and 5 minutes/time, added 37 ℃ of 0.3% hydrogen peroxide (methanol configuration) 30 minutes, PBST washes 3 times, adds 10 11Pfu shows 37 ℃ of warm baths 1 hour of phage (containing 0.1%BSA) of GA4 and GA5.PBST washes 6 times, adds HRP-goat-anti M13 polyclonal antibody 100 μ l/ holes, 37 ℃ 1 hour.After the PBST rinsing, add the colour developing of DAB working solution black out, cessation reaction when suitable of waiting to develop the color.
The results are shown in Figure 6.Figure A is phage and the bonded SABC figure of Tie2 (+) SMMC 7721 cells that shows GA4, figure B is phage and the bonded SABC figure of Tie2 (+) SMMC 7721 cells that shows GA5, figure C is phage and the bonded SABC figure of Tie2 (-) SMMC 7721 cells that shows GA4, and figure D is phage and the bonded SABC figure of Tie2 (-) SMMC 7721 cells that shows GA5.
Experimental result confirms that all the pattern of fusion polypeptide relevant with the present invention (GA4 and GA5) can specially combine with cell surface Tie2 receptor protein.
Embodiment 6 phage clones combine with rhTie2-Fc fusion rotein specificity
Every hole 0.1M NaHCO 3(PH 8.6) 50 μ l bag is by 1 μ g rhTie2-Fc fusion rotein (R﹠amp; D company) in 96 orifice plates, 4 ℃ are spent the night, and PBST washes 3 times, sealing buffer (PBS+5mg/ml BSA PH 7.5) sealing, 4 ℃ 3 hours, set up negative control (bag by 1 μ g/50 μ l BSA) and blank simultaneously.PBST washes 3 times, adds 2 * 10 1037 ℃ of temperature of pfu phage (containing 0.1%BSA) were bathed 1 hour.PBST washes 6 times, adds 100 μ l Glysine-HCl (PH 2.2) eluting 8 minutes, eluent is transferred to measured in the pipe, adds 15 μ lTris-HCl (PH 9.1) neutralization, then the phage that reclaims is carried out titration, calculations incorporated efficient.
Experimental result is seen Fig. 7.Show that the phage of showing GA4 and GA5 all can combine with rh-Tie2.
The structure of embodiment 7 non-virus carriers
A. the acquisition of each component
(1) part oligopeptide GA's is synthetic:
Press the identical method of embodiment 1, adopt the Peptide synthesizer of American AB I company and synthesize according to its polypeptide synthetic operation handbook, column chromatography for separation obtains oligopeptide GA3, GA4 and GA5, and its aminoacid sequence is respectively:
WTIIQRREDG?SVDFQRTWKE?YK(SEQ?ID?NO：1)；
HATGTHGLSL?SH(SEQ?ID?NO：2)；
NSLSNASQFR?AP(SEQ?ID?NO：3)。
(2) poly-L-lysine (Poly-L-lysine is abbreviated as P.L.)
Available from U.S. SIGMA company, molecular weight distribution is 15000-30000 dalton.
(3)HA20
Adopt the Peptide synthesizer of American AB I company and synthesize according to its polypeptide synthetic operation handbook, column chromatography for separation obtains oligopeptide HA20, and its aminoacid sequence is GLFEA IAEFI EGGWE ELIEG (SEQ IDNO:4).
B. the preparation of binary complex
At first, GA (GA3 or GA4 or GA5), HA20 and poly-L-lysine (Poly-L-lysine is abbreviated as P.L.) are dissolved among the PSB (0.1M sodium phosphate buffer, pH7.4 contain 0.1M NaCl), then, follow these steps to react.
(1)P.L.+SPDP→P.L.-PDP
Annotate: SPDP is N-butanimide-3-(2-pyridine dithio)-propionic ester.
P.L. the mol ratio with SPDP (N-) is 1: 5, and the response time is 2 hours, and temperature is 25 ℃.Reaction finishes the back by the unreacted SPDP of dialysis removal.The P.L.-PDP lyophilization.
(2) P.L.-PDP dry powder is dissolved among the PSB
P.L.-PDP+DTT→P.L.-SH
Annotate: DTT is a dithiothreitol, DTT.
DTT is excessive in the reaction, and the response time is 1 hour, and temperature is 25 ℃.Reaction finishes the back by the unreacted DTT of dialysis removal.The product lyophilization.
(3)GA+SPDP→GA-PDP
HA20+SPDP→HA20-PDP
The mol ratio of GA, HA20 and SPDP is 1: 5, and the response time is 2 hours, and temperature is 25 ℃.Unreacted SPDP is removed in bag filter dialysis by MWCO1000 after reaction finishes.The product lyophilization.
(4) GA-PDP, HA20-PDP and P.L.-SH dry powder all are dissolved among the PSB.
GA-PDP+P.L.-SH→P.L.-GA
HA20-PDP+P.L.-SH→P.L.-HA20
The mol ratio of GA-PDP, HA20-PDP and P.L.-SH is 3: 1, and the response time is 24 hours, and temperature is 25 ℃.Reaction finishes the back by dialysis unreacted GA-PDP of removal and HA20-PDP.
C. the preparation of targeting non-virus carrier
Mix P.L.-GA and P.L.-HA20 in 1: 1,0.8: 1.2,1.2: 0.8 with mass ratio respectively, promptly get required non-virus carrier.
Perhaps, GA, polylysine and HA20 are mixed, form required non-virus carrier by mass ratio 1: 1.5: 1.
The extracting and the purification of embodiment 8 plasmid DNA
The isolation and purification of used recombinant eukaryon expression vector plasmid DNA all utilizes the Maxi plasmid kit of QIAGEN company and the pure product of DNA that obtain in the present embodiment.
Select monoclonal from the LB flat board and be inoculated in the 5mlLB culture medium that contains ampicillin, 37 ℃, the 250rpm jolting is spent the night.Transfer at 1: 1000 in the 200ml LB culture medium that contains ampicillin, 37 ℃, 250rpm jolting 16 hours.Shift bacterium liquid in the 500ml centrifuge tube, 4,000rpm, 4 ℃ centrifugal 10 minutes, abandon supernatant, collect thalline.Precipitation is resuspended in 10ml Buffer P1 (50mM TrisCl, pH8.0,10mM EDTA pH8.0,100 μ g/ml Rnase A).(200mM NaOH puts upside down 4-6 time after 1%SDS) gently, and mixing left standstill under the room temperature 5 minutes to add 10ml Buffer P2.Add 10ml Buffer P3 (3.0M NaAc, pH5.5), put upside down mixing after, be transferred to the 50ml centrifuge tube, 10,000rpm, 4 ℃ centrifugal 30 minutes, get supernatant.Supernatant is aseptically filled in the QIAGEN Maxi plasmid extraction post through four layers and (uses 10ml Buffer QBT balance in advance), allow under its spontaneous current, enter in the post fully Deng supernatant, add Buffer QC 30ml washing secondary again, add BufferQF 15ml at last, collect effluent, add 0.7 times of volume isopropyl alcohol, 10,000rpm, 4 ℃ centrifugal 30 minutes, abandon supernatant, 75% washing with alcohol precipitation 1 time, natural drying under the room temperature is dissolved among the TE (pH 8.0) of an amount of volume the quality of the firm DNA of 1% agarose gel electrophoresis, its purity of UV spectrophotometer measuring is also quantitative ,-20 ℃ of preservations.
The structure of embodiment 9 quaternary complex gene import systems
1) after .GA-P.L. and HA20-P.L. were dissolved in water, through 0.22 μ m filter filtration sterilization ,-20 ℃ stored for future use.All plasmids extract and purification through the QIAGEN post, the ethanol of 2 times of volumes of reuse and the 3M NaAc of 1/3 volume precipitation, and after 75% ethanol is washed once, air drying.With an amount of MilliQ water dissolution, make concentration become 0.2 μ g/ μ l.
2). the optimum quality ratio when non-virus carrier and DNA form complex between the two.
0.2 μ g CMV-β-gal plasmid DNA was mixed with mass ratio with non-virus carrier in 1: 0.5,1: 1,1: 1.5,1: 2,25 ℃ left standstill 30 minutes, with the retardance situation of 1% agarose gel electrophoresis identification of dna, determined optimum quality ratio.
3). prepare quaternary complex gene transfer system by above-mentioned definite proportioning.
Embodiment 10 GA gene import systems transduction CMV-β-gal gene in vitro imports experiment
1). cell culture
The SPC-A1 passage is cultivated: the SPC-A1 cell with 0.25% trypsinization after, be inoculated in the Tissue Culture Flask, adding contains the DMEM complete culture solution of 10% calf serum, at 37 ℃, 5%CO 2Cultivate in the incubator.
2). external importing experiment
Cultured cell with 0.25% trypsinization after, be inoculated in six orifice plates every hole 1.5 * 10 5, the cell full scale was about 60% o'clock in second day, added the quaternary complex gene transfer system be equivalent to 2 μ g DNA (promptly respectively based on three kinds of quaternary complex gene transfer systems of GA3, GA4, GA5) in the 1ml culture fluid.After the transfection 24 hours, renew the bright DMEM complete culture solution that contains 10% calf serum, continue to cultivate 24 hours.Cell is washed 2 times with PBS then, add freshly prepared fixative and fix 10 minutes in room temperature, reuse PBS rinsing 3 times, each 15 minutes, with thorough removal fixative, blot PBS on tumor piece and the internal organs with filter paper, add freshly prepared dyeing liquor in 37 ℃ of insulations after 24 hours, the situation that the observation exogenous gene imports (form: 1mg/ml X-gal, 5mM K by dyeing liquor 4[Fe (CN) 6], 5mMK 3[Fe (CN) 6], 2mM MgCl 2).
The result shows, the cell of transduction β-gal gene of the present invention is blue, the matched group that adds normal saline and the simple plasmid respectively importing of source gene of then not regarding sb. as an outsider.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
＜110〉Shanghai Xinshijie Gene Techn Development Co., Ltd.
＜120〉gene transfer system of the receptor-mediated targeting therapy of tumor of Tie2
<130>034757
<160>9
<170>PatentIn?version?3.1
<210>1
<211>22
<212>PRT
＜213〉artificial sequence
<220>
<221>MISC_FEATURE
＜223〉be incorporated into the part oligopeptide GA3 of Tie2 receptor
<400>1
Trp?Thr?Ile?Ile?Gln?Arg?Arg?Glu?Asp?Gly?Ser?Val?Asp?Phe?Gln?Arg
1 5 10 15
Thr?Trp?Lys?Glu?Tyr?Lys
20
<210>2
<211>12
<212>PRT
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉be incorporated into the part oligopeptide GA4 of Tie2 receptor
<400>2
His?Ala?Thr?Gly?Thr?His?Gly?Leu?Ser?Leu?Ser?His
1 5 10
<210>3
<211>12
<212>PRT
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉be incorporated into the part oligopeptide GA5 of Tie2 receptor
<400>3
Asn?Ser?Leu?Ser?Asn?Ala?Ser?Gln?Phe?Arg?Ala?Pro
1 5 10
<210>4
<211>20
<212>PRT
＜213〉artificial sequence
<220>
<221>MISC_FEATURE
<222>(1)..(20)
＜223〉the endocytosis corpusculum discharges oligopeptide HA20
<400>4
Gly?Leu?Phe?Glu?Ala?Ile?Ala?Glu?Phe?Ile?Glu?Gly?Gly?Trp?Glu?Glu
1 5 10 15
Leu?Ile?Glu?Gly
20
<210>5
<211>66
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉coded sequence of part oligopeptide GA3
<400>5
tgtacaatta?ttcagcgacg?tgaggatggc?agcgttgatt?ttcagaggac?ttggaaagaa 60
tataaa 66
<210>6
<211>36
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉coded sequence of part oligopeptide GA4
<400>6
catgctacgg?gtactcatgg?gctttcgctt?tctcat 36
<210>7
<211>36
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉coded sequence of part oligopeptide GA5
<400>7
aattcgctgt?cgaatgcttc?ggagtttcgg?gcgccg 36
<210>8
<211>29
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>8
ccggaattca?tggactcttt?agccagctt 29
<210>9
<211>30
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>9
ccgctcgagc?tagatggcta?taagcagcat 30

Claims (9)

1. the gene transfer system of the targeting gene therapy of a growth factor receptors Tie2 mediation is characterized in that it comprises the part oligopeptide that (a) specificity is incorporated into the Tie2 receptor, and the aminoacid sequence of wherein said part oligopeptide is shown in SEQ ID NO:1; (b) polycation polypeptide; (d) foreign DNA.
2. gene transfer system as claimed in claim 1 is characterized in that, it also contains (c) endocytosis corpusculum and discharges oligopeptide.
3. gene transfer system as claimed in claim 2 is characterized in that, the mixed proportion of (a) and (b), (c) component is 0.5-1: 1-2: 0-1.
4. gene transfer system as claimed in claim 1, it is characterized in that, the polycation polypeptide is selected from down group: poly-D-lysine, poly-acetimide, protamine, histone, adenovirus pV albumen, adenovirus pVII albumen, m μ albumen, and through polyethyleneglycol modified above-mentioned albumen and composition thereof.
5. gene transfer system as claimed in claim 1 is characterized in that, described foreign DNA is selected from down group: the recombinant eukaryotic expression plasmid DNA of antioncogene, apoptosis gene, cytokine gene, oncogene antisense sequences, and composition thereof.
6. gene transfer system as claimed in claim 1 is characterized in that, described foreign DNA is selected from down group:
(i) antioncogene: p53, Rb;
(ii) apoptosis gene: Caspase, p15, p16 and p21 WAF-1
(iii) cytokine gene: GM-CSF gene, TNF α gene, INF α, γ gene, IL2, IL3, IL4, IL12, IL15, IL18 gene;
The (iv) antisense sequences of oncogene: the antisense sequences of oncogene ras, c-myc, Akt;
(v) recombinant eukaryotic expression plasmid: viral recombinant eukaryotic expression plasmid and be the non-viral recombinant eukaryotic expression plasmid of cis-regulating element with SV40, CMV promoter.
7. gene transfer system as claimed in claim 2 is characterized in that, described component (a) part oligopeptide, (b) polycation polypeptide and (c) both or the three that discharge in the oligopeptide of endocytosis corpusculum exist with the fusion rotein form.
8. gene transfer system as claimed in claim 2 is characterized in that, described endocytosis corpusculum discharges the oligopeptide element and is selected from down group: HA20 or VP-1.
9. an oligopeptide is characterized in that, it is incorporated into the Tie2 receptor, and its aminoacid sequence is shown in SEQ IDNO:1.
CNB2003101228303A 2003-12-26 2003-12-26 Tie2 receptor mediated gene transfer system for targeted tumor gene therapy Expired - Fee Related CN1323723C (en)

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US9464114B2 (en) * 2014-02-13 2016-10-11 Arch Cancer Therapeutics, Inc. Peptides that block leukocyte recruitment and methods of use
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Publication number Priority date Publication date Assignee Title
WO1996011269A2 (en) * 1994-10-07 1996-04-18 Regeneron Pharmaceuticals, Inc. Tie-2 ligands, methods of making and uses thereof
WO1998005779A1 (en) * 1996-08-02 1998-02-12 Regeneron Pharmaceuticals, Inc. Modified tie-2-receptor ligands

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WO1996011269A2 (en) * 1994-10-07 1996-04-18 Regeneron Pharmaceuticals, Inc. Tie-2 ligands, methods of making and uses thereof
WO1998005779A1 (en) * 1996-08-02 1998-02-12 Regeneron Pharmaceuticals, Inc. Modified tie-2-receptor ligands

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