CN1067723C - The novel gene transfer system of receptor-mediated target therapy of tumor - Google Patents
The novel gene transfer system of receptor-mediated target therapy of tumor Download PDFInfo
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- CN1067723C CN1067723C CN97191515A CN97191515A CN1067723C CN 1067723 C CN1067723 C CN 1067723C CN 97191515 A CN97191515 A CN 97191515A CN 97191515 A CN97191515 A CN 97191515A CN 1067723 C CN1067723 C CN 1067723C
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Abstract
本发明是一种与生长因子受体结合的基因转移系统。由配体寡肽/多聚阳离子多肽/内吞小体释放寡肽/外源DNA组成的四元复合体基因转移系统或由配体寡肽/多聚阳离子多肽/外源DNA组成的三元复合体基因转移系统。已有的实例为E5、GE7、GV1及GV2系统，能有效地在体内外将外源基因靶向性地导入恶性肿瘤细胞及肿瘤血管内皮细胞。以p15、p16、p21WAF-1为外源基因时可高效抑制肿瘤细胞在动物体内生长。该系统将成为恶性肿瘤靶向性基因治疗的新型导入系统。The present invention is a gene transfer system combined with growth factor receptor. Quaternary complex gene transfer system composed of ligand oligopeptide/polycationic polypeptide/endosome releasing oligopeptide/exogenous DNA or ternary complex composed of ligand oligopeptide/polycationic polypeptide/exogenous DNA Complex gene transfer system. Existing examples are E5, GE7, GV1 and GV2 systems, which can effectively introduce exogenous genes into malignant tumor cells and tumor vascular endothelial cells in vivo and in vitro. When p15, p16, p21 WAF-1 is used as exogenous gene, it can efficiently inhibit the growth of tumor cells in animals. This system will become a new introduction system for malignant tumor targeted gene therapy.
Description
Technical field
The present invention relates to molecular biological field of gene.Specifically,, foreign DNA target ground is imported tumour cell, reach the purpose of treatment tumour by receptor mediated endocytosis about a class and the constructed gene transfer system of growth factor receptors bonded part oligopeptides.
Technical background
Gene therapy be foreign DNA is imported human body specific cells to produce result of treatment, reach the purpose of treatment disease.For this reason, at first need possess gene transfer system safely and effectively.Mainly contain two big class methods at present and can mediate foreign DNA effectively and change human body cell over to, a class is the method for virus vector mediation, and another kind of is the method for non-virus carrier mediation.To belonging to the latter's receptor-mediated gene transfer method, report to some extent in recent years.Receptor-mediated transgenosis can combine the formation carrier by foreign DNA and realize with the respective ligand of acceptor.Part in such carrier can with the corresponding receptors bind on specific cells surface, by receptor mediated endocytosis, foreign DNA is transduceed into specific cells.(J.Biol.Chem. such as Wu.G.Y., 263,14621,1988) report utilizes receptor-mediated transgenosis foreign DNA to be changed over to the liver cell of vitro culture, used part is an asialoglycoprotein glycoprotein in the carrier, it be used for inherited disease gene therapy .MaxL.Birnstiel etc. (P.N.A.S.87,3410-3414.1990) report utilizes Transferrins,iron complexes to combine with the TfR of surface of cell membrane, through the endocytosis of cell, foreign DNA transduceed in the cell and obtain express.
In addition, though this gene transfer system has target, only limiting to does not still have report at normal cell (as liver) for the transgenosis of malignant tumour target.Next has research prompting, by this kind approach by the DNA of cell endocytic in the process that shifts to nucleus because the endocytosis vesicle merges with lysosome, and make the DNA in the carrier may be, thereby make effective transfer efficiency decline by the intravital enzyme liberating of lyase.Know at present, add some virus simultaneously, adenovirus for example can improve the transfer efficiency of foreign DNA.Can escape during the virus infection host cell enzyme system of Cytolysosome to one of reason of dna degradation is: some virus is as influenza virus, adenovirus is in the host cells infected process, after the endocytosis corpusculum forms, by some the proteinic structural domain of outer virionic membrane and the lipid film effect of endocytosis corpusculum, destroy the film system of endocytosis corpusculum, thereby discharge (P.N.A.S.89 such as DNA.Max L.Bimstiel, 6094-6098,1992) reported the adenovirus of utilizing defective type or chemical ablation destruction, thereby strengthened the gene transfering efficiency of TfR mediation the film system of endocytosis corpusculum.
Goal of the invention
For this reason, the purpose of this invention is to provide a kind of novel gene transfer system of receptor-mediated target therapy of tumor, promptly discharge the constructed gene transfer system of oligopeptides with growth factor receptors bonded part oligopeptides and endocytosis corpusculum.This system is that part oligopeptides/polycation polypeptide/endocytosis corpusculum discharges oligopeptides ternary complex carrier and foreign DNA forms quaternary complex body gene transfer system or part oligopeptides/polycation polypeptide binary complex carrier and foreign DNA formation ternary complex gene transfer system.Wherein the part oligopeptides is E5 (14 amino acid), GE7 (16 amino acid), GV1 (32 amino acid) and GV2 (36 amino acid); The polycation polypeptide is poly-lysine, protamine or histone; It is synthetic peptide HA20 with influenza virus adventitia hemagglutinin aminoterminal amino acid sequence homologous that the endocytosis corpusculum discharges oligopeptides.Utilize the growth factor receptors of overexpression on the part oligopeptides tumor cell film,, foreign DNA is optionally imported tumour cell, reach the purpose of treatment tumour by this carrier system through the endocytosis of cell.Summary of the invention
Content of the present invention relates generally to four aspects, and they are respectively: the part oligopeptides with the growth factor receptors specific combination is provided; Provide part oligopeptides/polycation polypeptide binary complex carrier and foreign DNA to form the ternary complex gene transfer system; Provide part oligopeptides/polycation polypeptide/endocytosis corpusculum to discharge oligopeptides ternary complex carrier and foreign DNA formation quaternary complex body gene transfer system; The said gene transfer system is used for the inside and outside genetic treatment of tumor.
A first aspect of the present invention provides with growth factor receptors IGF-II R, IGF-I R (insulin-like growth factor-, I acceptor), EGF R (EGF-R ELISA), part oligopeptides E5, GE7, GV1 and the GV2 of VEGF R (vascular endothelial growth factor receptor) specific combination.
Some tumour such as liver cancer, brain tumor, mammary cancer, ovarian cancer, cancer of the stomach, cervical cancer, adenocarcinoma of lung, nasopharyngeal carcinoma etc., the growth factor receptors on its cancer cells surface is as the VEGF R overexpression of new vessel endotheliocyte in IGF-II R, IGF-I R, EGF R and the knurl.According to the principle of part and acceptor interaction, the present invention adopts the computerized image board design to synthesize a series of and above-mentioned growth factor receptors bonded part oligopeptides.Polypeptide synthesizes on the Peptide synthesizer of American AB I company (Applied Biosystems 430A PeptideSynthesizer) to be finished, and synthesis step carries out according to ABI company polypeptide synthetic operation handbook.Polypeptide is synthetic with after the high pressure liquid chromatography purifying gets the polypeptide powdered product.The gained polypeptide product can be analyzed through hydrolysis and confirm.
Synthetic part oligopeptides screens through following method.At first synthetic part oligopeptides and poly-lysine (or protamine or histone) are generated pyridine two sulphur part oligopeptides and pyridine two sulphur poly-lysines (or protamine or histone) with SPDP (N-Succinimidyl-3-2-pyridyldithio propionate) effect respectively.Form the covalently bound thing of part oligopeptides-poly-lysine (or protamine or histone) that is connected with disulfide linkage with pyridine two sulphur part oligopeptides effects behind pyridine two sulphur poly-lysines (or protamine or histone) sulfhydrylation.Fig. 1, Fig. 2 and Fig. 3 are respectively E5-poly-lysine, GE7-poly-lysine, the covalently bound thing of the GV2-protamine record diagram through Sephadex (sephadex) G-50 column chromatographic isolation and purification, and covalently bound thing is to collect the first peak gained.Fig. 4,5 is respectively the amino acid analysis of components result after E5-poly-lysine, the covalently bound thing hydrolysis of GE7-poly-lysine.Fig. 4 has shown the amino acid that the E5-poly-lysine is contained, and Fig. 5 has shown the amino acid that the GE7-poly-lysine is contained.Form part oligopeptides/polycation polypeptide/endocytosis corpusculum by the electrostatic interaction between positive and negative charge and discharge oligopeptides ternary complex carrier and foreign DNA formation quaternary complex body gene transfer system.Above-mentioned quaternary complex body gene transfer system is used for the inside and outside gene imports experiment, (5-bromo-4-Chloro-3-indolyl-β-D-galactoside) histochemical stain filters out part oligopeptides E5, GE7, GV1, GV2 with the growth factor receptors specific combination of tumour cell and new vessel cell surface through X-gal.Fig. 6,7,8 is respectively 1% agarose gel electrophoresis result of formed E5, GE7, GV2 quaternary complex body gene transfer system, and this result shows that gene DNA is blocked fully, all forms stable quaternary complex body.Fig. 9,10,11,12 shows that respectively E5 quaternary complex body gene transfer system changes external source β-gal gene efficient over to human hepatoma cell strain SMMC-7721 (X-gal staining cell be positive blueness), the PBS control group then shows negative findings (it is blue that the X-gal staining cell is not), and E5 quaternary complex body can not import normal liver cell L02 and primary cultured hepatocyte R02 with β-gal gene.Figure 13 and Figure 14 show that respectively GE7 and GV2 quaternary complex body gene transfer system can go into β-gal gene mediated human hepatoma cell strain BEL-7402 and endothelial cells in tumor neogenetic blood vessels (the X-gal staining cell all presents blueness) respectively.
Synthetic part oligopeptides is through above-mentioned screening, filter out part oligopeptides E5 of the present invention (aminoacid sequence is EPFRS PDLAL ETYG), GE7 (aminoacid sequence is NPVVG YIGER PQYRD L), GV1 (aminoacid sequence is CHPIE TLVDI FQEYP DEIEY IFKPS PVPLM RP), GV2 (aminoacid sequence is PVPTE ESNIT MQIMR IKPHQ GQHIG EMSFL QHNKCE).
Every have identical immunologic determinants with E5, GE7, GV1, GV2, and can with above-mentioned oligopeptides corresponding antibodies rise positive reaction and and IGF-I R, IGF-II R, EGF R, VEGF R bonded polypeptide and oligopeptides also go for as part oligopeptides provided by the present invention.The polypeptide that forms behind the amino acid with corresponding site in the amino acid replacement of the similar performance part oligopeptides provided by the present invention does not influence its performance basically again, is applicable to part oligopeptides provided by the present invention yet.
Above-mentioned part oligopeptides E5, GE7, GV1, GV2 can be the forms of the DNA of its aminoacid sequence of coding, be used for the structure of part oligopeptides/polycation polypeptide/HA20 oligopeptides recombinant dna plasmid and be used for structure and cytokine and cytotoxic fusion gene recombinant dna plasmid, complex polypeptide is applied to gene therapy and expressed fusion protein is used for the treatment of tumour and other disease to express.
Part also can be to go up associated receptor bonded oligopeptides with hematopoietic cell, scavenger cell, lymphocyte, liver cell, nephrocyte, vascular endothelial cell, neurocyte and myocardial cell, make up the complex polypeptide carrier that is applied to gene therapy with this class part oligopeptides, exogenous gene high-efficient, target ground are imported above-mentioned cell, with treatment tumour and non-tumor disease.
A second aspect of the present invention provides part oligopeptides/polycation polypeptide binary complex carrier and foreign DNA to form the ternary complex gene transfer system.The part oligopeptides is E5, GE7, GV1, GV2.The polycation polypeptide is poly-lysine, protamine or histone.Foreign DNA is the recombinant eukaryotic expression plasmid (comprising that the promotor that retrovirus recombinant expression vector, adenovirus recombinant expression vector reach with SV40, CMV is the recombinant eukaryotic expression plasmid of cis-regulating element) of antisense oncogene, antioncogene, suicide gene, apoptosis gene or cytokine gene.The antisense oncogene comprises oncogene (ras
H, ras
K, ras
N, c-myc, bcl-2), antisense dna sequence, antisense oligonucleotide and the antisense oligodeoxyribonucleotide of somatomedin and acceptor gene thereof, antioncogene comprises p53 and Rb, suicide gene comprises HSV-TK (herpes simplex virus thymidine kinase) gene and CD (coli cytosine deaminase) gene, and apoptosis gene comprises p15, p16, p21
WAF-1, cytokine gene comprises GM-CSF (rHuGM-CSF) gene, TNF (tumour necrosis factor) α gene, INF (Interferon, rabbit) α, γ gene, IL (interleukin-) 2,3,4,12,15 genes.The building process of this complex body gene transfer system is similar to employed method in the above-mentioned part oligopeptides screening.At first part oligopeptides and poly-lysine (protamine or histone) are combined into the covalently bound thing of part oligopeptides-poly-lysine (protamine or histone), again with electronegative foreign DNA formation ternary complex gene transfer system.
A third aspect of the present invention provides part oligopeptides/polycation polypeptide/endocytosis corpusculum to discharge oligopeptides ternary complex carrier and foreign DNA forms quaternary complex body gene transfer system.The existence of endocytosis corpusculum release oligopeptides can improve the transduction efficiency of such carrier.The endocytosis corpusculum discharges the synthetic peptide HA20 of oligopeptides employing and influenza virus adventitia hemagglutinin aminoterminal amino acid sequence homologous.The synthetic basic reference literature of HA20 (Midonx P. etc., Nuc.Aci.Res., 21,871,1993).HA20 is one and contains 20 amino acid whose polypeptide that its aminoacid sequence is GLFEAIAEFI EGGWE ELIEG.
The construction process of such quaternary complex body gene transfer system equally also is at first with part oligopeptides and poly-lysine (or protamine or histone) covalently bound (passing through SPDP).Equally HA20 and poly-lysine (or protamine or histone) are combined into the covalently bound thing of HA20-poly-lysine (protamine or histone) again.Common and the electronegative by a certain percentage foreign DNA mat electrostatic interaction of these two covalently bound things is combined into part oligopeptides/poly-lysine (or protamine or histone)/HA20 ternary complex carrier and foreign DNA forms quaternary complex body gene transfer system.Fig. 6,7,8 is respectively E5 quaternary complex body gene transfer system, GE7 quaternary complex body gene transfer system, GV1 and GV2 quaternary complex body gene transfer system agarose gel electrophoresis result, and this result shows and formed stable quaternary complex body gene transfer system.
HA20 is except existing in quaternary complex body carrier with combined as mentioned above, can also exist with non-binding attitude with part oligopeptides/polycation polypeptide binary complex carrier and the ternary complex gene transfer system that foreign DNA forms with the covalently bound thing of HA20-poly-lysine (or protamine or histone), can also exist with non-binding attitude with the ternary complex gene transfer system with independent HA20, can both improve the transduction efficiency of gene transfer system of the present invention.
Quaternary complex body gene transfer system of the present invention, be equally applicable to use two or more different part oligopeptides and polycation polypeptide and the formed complex polypeptide of HA20 and the complex body that forms with a kind of foreign gene DNA or virus, or be applied to form complex body with a kind of part oligopeptides/polycation polypeptide/HA20 foreign gene DNA different with two or more or the virus that contains different foreign genes, be used for therapy of tumor.
Above-mentioned ternary complex gene transfer system also can combine the gene transfer system that is applied to gene therapy with recombinant virus, and recombinant virus is recombinant retrovirus and recombinant adenovirus.Recombinant virus can non-binding attitude or combined exist with the ternary complex gene transfer system.The genetic material that this recombinant virus wrapped up can comprise following foreign gene: suicide gene HSV-TK, CD; Apoptosis gene p15, p16, p21
WAF-1Cancer suppressor gene p53, Rb; Cytokine gene GM-CSF, TNF α, INF α, γ, IL2,3,4,12,15 or do not comprise the defective virus of any foreign gene.This system is equally applicable to the antisense oligonucleotide and the antisense oligodeoxyribonucleotide of oncogene, growth factor receptor gene.
For ease of Quality Control, above-mentioned part oligopeptides/polycation polypeptide/endocytosis corpusculum discharges oligopeptides ternary complex carrier, and the Nucleotide series of the described polypeptide of available code is set up into recombinant dna plasmid, prepares described ternary complex carrier with genetically engineered.
A fourth aspect of the present invention is that part oligopeptides/polycation polypeptide/endocytosis corpusculum discharges oligopeptides ternary complex carrier and foreign DNA formation quaternary complex body gene transfer system is applied to inside and outside gene transfer and genetic treatment of tumor.
In vitro tests: (I) outer-gene shifts test: E5/ poly-lysine/HA20/ β-gal quaternary complex body gene transfer system is used for hepatoma cell strain SMMC-7721 and the normal former liver cell R02 that is commissioned to train foster, the normal liver cell strain L02 of transfection vitro culture.The result show quaternary complex body gene transfer system only can be efficiently, target ground imports liver cancer cell (Fig. 9-12) with foreign gene.And setting is with the control group of simple β-gal gene DNA, E5/ poly-lysine/β-gal ternary complex, HA20/ poly-lysine/β-gal ternary complex, poly-lysine/β-gal binary complex body and PBS difference transfection hepatoma cell strain SMMC-7721.Detect efficient and the target of above-mentioned carrier system with the X-gal histochemical stain to IGF-II R, the external transduction foreign gene of IGF-I R male hepatoma cell strain.The results are summarized in down tabulation 1～table 3.Table 1 shows the transfer efficiency of different composite body control group and quaternary complex body gene transfer system transduction foreign gene.Table 2 shows that E5 quaternary complex body gene transfer system only can import the SMMC-7721 liver cancer cell with β-gal, and β-gal gene can not be imported normal liver cell L02 and primary cultured hepatocyte R02.Table 3 shows that E5 quaternary complex body gene transfer system mediation β-gal gene goes into the SMMC-7721 cell, and β-gal gene began to express after transfection in 48 hours, expresses paramount peak in 72 hours.
(or GE7/ protamine/HA20/ β-gal) quaternary complex body gene transfer system is used for hepatoma cell strain SMMC-7721, the BEL-7402 of transfection vitro culture to GE7/ poly-lysine/HA20/ β-gal, glioma cell line U87, breast carcinoma cell strain Bcap-37, ovarian cancer cell strain 3AO, AO, lung adenocarcinoma cell line SPC-A1 and normal liver cell strain L02, l cell strain NIH/3T3.Figure 13, Figure 15-22 shows that respectively GE7 quaternary complex body gene transfer system can import EGFR with external source β-gal gene in target ground
The different mixture of table 1 shifts the transfer efficiency of pSV-β-gal gene
Grouping PBS β-gal Pp Ep Hp EpH
β-gal positive cell percentage ratio (%) 00 0.1 10 0.2 60 is annotated: (1) PBS: negative control group
(2)β-gal:pSV-β-gal?DNA
(3) Pp: poly-lysine/pSV-β-gal
(4) Ep:E5/ poly-lysine/pSV-β-gal
(5) Hp:HA20/ poly-lysine/pSV-β-gal
(6) EpH:E5/ poly-lysine/pSV-β-gal/HA20-poly-lysine
(7) target cell: human liver cancer cell SMMC-7721
Table 2 liver cancer cell SMMC-7721 and liver cell (L02, R02) importing of middle β-gal gene
Cell type quiding gene β-gal positive cell percentage ratio (%)
SMMC-7721 (PBS) contrast 0
SMMC-7721 pSV-β-gal 60
L02 pSV-β-gal 0.1
R02 pSV-β-gal 0.1
Time after the time course transfection that table 3 β-gal genosome outer (SMMC-7721) is expressed (hour) 24 48 72 96
β-gal expression of gene-++++-positive cells strain BEL-7402, SMMC-7721, U87, Bcap-37,3AO, AO, SPC-Al (the X-gal staining cell presents blueness), and almost external source β-gal gene mediated can not be gone into cell strain L02, the NIH/3T3 (X-gal dyeing does not almost have cell to present blueness) of EGF R feminine gender.Figure 23-25 shows that respectively ternary complex HA20/ poly-lysine/β-gal, binary complex body poly-lysine/β-gal and β-gal gene DNA almost can not import foreign gene EGF R positive cells strain BEL-7402 (X-gal dyeing does not almost have cell to present blueness).Figure 26 shows that GE7 ternary complex gene transfer system GE7/ poly-lysine/β-gal only can import BEL-7402 cell (the X-gal staining cell seldom presents blueness) with β-gal gene to be lower than 10% efficient.And be provided with the control group of transfection hepatoma cell strain BEL-7402 respectively with simple β-gal gene DNA, GE7/ poly-lysine/β-gal ternary complex, HA20/ poly-lysine/β-gal ternary complex, poly-lysine/β-gal binary complex body and PBS, detect efficient and the target of this carrier system with the X-gal histochemical stain, the results are summarized in down tabulation 4 and table 5 the external transduction foreign gene of EGFR male tumour cell.Table 4 shows that GE7 quaternary complex body gene transfer system is to different cell strains and the different composite body efficient to hepatoma cell strain BEL-7402 transduction foreign gene.Table 5 shows that GE7 quaternary complex body gene transfer system almost can not import foreign gene people's normal liver cell L02 and l cell NIH/3T3.Table 6 shows the expression level of the β-gal gene different time of GE7 quaternary complex body gene transfer system mediation, and transgenosis β after 24 hours-gal gene begins to express, and expresses paramount peak to the transfection in 168 hours.
(II) external mediation causes apoptotic gene p21
WAF-1, p16, p15 efficiency assay: E5/ poly-lysine/HA20/p21
WAF-1The hepatoma cell strain of quaternary complex body gene transfer system transfection vitro culture was cultivated after 72 hours, used the acetone fixed cell, carried out fluorescence staining with fluorescent dye DAPI pair cell again,
Table 4GE7 quaternary complex body gene transfer system and different composite body transduction β-gal gene efficient
Cell strain
The different composite body
BEL-7402 SMMC-7721 SPC-Al Bcap-37 3AO U87 AOPBS 0 ------β-gal 0 ------P.L/β-gal ＜0.1 ------ GE7-P.L/β-gal ＜10 ------HA20-P.L/β-gal ＜0.1 ------
Annotate β-gal＞90＞80＞80＞80＞65＞30＞15: 1. transfection efficiency is the average transfection foreign gene percentage efficiency mean value of three independent experiments.
2. "-" is " not experimentizing ".
3.PBS: negative control.
4.P.L: poly-lysine.
5. β-gal: the plasmid pSV-β-gal that contains the LacZ gene.Table 5GE7 quaternary complex body gene transfer system is to the efficient of L02, NIH/3T3 transduction foreign gene
Cell strain L02 NIH/3T3
Transduction efficiency＜0.1＜0.1
Annotate: gene transfering efficiency is the mean value of three independent experiment transduction foreign gene efficiencies (%).
β-gal the gene of table 6 GE7 quaternary complex body gene transfer system mediation is in the BEL-7402 cell
The different time expression level
Annotate: "+" is gene expression dose.To show the nucleus of transfected cell.Under luminescence microscope, the characteristic of observing the programmed cell death in the nucleus of liver cancer cell changes nuclear fragment and nuclear concentrates, and the liver cancer cell nuclear of empty carrier transfection then is kept perfectly.Shown in Figure 27 is the caryclastic DAPI coloration result of liver cancer cell of this gene transfer system in-vitro transfection, and this result shows that gene transfer system of the present invention has good liver cancer treatment effect.E5/ poly-lysine/HA20/p21
WAF-1Quaternary complex body gene transfer system is used for the hepatoma cell strain SMMC-7721 of transfection vitro culture, carry out end mark with Bao Lingman (Boehringer Mannheim) company " In situCell Death Detection Kit (AP) " pair cell after 96 hours, Figure 28,29 shows the p21 of E5 quaternary complex body gene transfer system mediation respectively
WAF-1Gene induced percentage of cell apoptosis reaches 15%, and empty carrier transfection group coloration result is negative.E5/ poly-lysine/HA20/p21
WAF-1Quaternary complex body gene transfer system, E5/ poly-lysine/HA20/p16 quaternary complex body gene transfer system, E5/ poly-lysine/HA20/p15 quaternary complex body gene transfer system are respectively applied for transfection hepatoma cell strain SMMC-7721, and are provided with the control group of empty carrier transfection group.Cell cultures is in 24 well culture plates, after the transfection every two days countings once and make corresponding growth curve (Figure 30).
GE7/ poly-lysine/HA20/p21
WAF-1Quaternary complex body gene transfer system is used for the hepatoma cell strain BEL-7402 of transfection vitro culture.After the transfection 5,7 days, the showy cell of extracting, attached cell reach the DNA of float cell and attached cell mixture respectively, and through 1.5% agarose gel electrophoresis identification of dna gradient situation, the result shows the dna ladder degree and occurs, and control group does not then have.Illustrate that this carrier system will cause apoptotic gene p21
WAF-1Import the apoptosis that causes liver cancer cell in the liver cancer cell.The BEL-7402 cell inoculation in 6 porocyte culture plates, is used GE7/ poly-lysine/HA20/p21
WAF-1Quaternary complex body gene transfer system transfection BEL-7402 cell, respectively at 1,2,3,4,5,6,7,8 day counting cells sum, the result showed that this carrier system can be efficiently with p21
WAF-1Gene imports in the liver cancer cell, cell growth inhibiting, and growth curve is seen Figure 31.Figure 31 shows GE7 quaternary complex body gene transfer system mediation p21
WAF-1Gene suppresses the growth curve of BEL-7402 cell growth and the cell growth curve of PBS control group.
In vivo test: the test of (I) vivo gene transfer: E5/ poly-lysine/HA20/ β-gal quaternary complex body gene transfer system is used for transgenosis in the knurl.With the knurl week of this gene transfer system subcutaneous injection in lotus people liver cancer SMMC-7721 nude mice, put to death animal respectively at 12,24,48,60,72,96 hours and get the knurl piece, the knurl piece is carried out the X-gal histochemical stain.The result shows that β-gal gene began at 12 hours to express, and reaches the peak in 24 hours, and 48 hours expression levels begin to descend, and lasts till to still have expression in 96 hours, and X-gal dyeing knurl piece presents blueness (Figure 32).
GE7/ poly-lysine/HA20/ β-gal quaternary complex body gene transfer system is used for transgenosis in the knurl.With this carrier system subcutaneous injection in lotus people liver cancer SMMC-7721, BEL-7402, human glioma U251, human breast carcinoma Bcap-37, human ovarian cancer 3AO, human lung adenocarcinoma SPC-Al, human small cell lung carcinoma H128, people's enteraden cancer LOVO, the knurl week of people's adenocarcinoma of stomach SGC and human cervical carcinoma's transplanted tumor nude mice, put to death animal respectively at 4,8,12,24,48,96 hours and 7,15,20,30,40 days and get the knurl piece, the knurl piece is carried out the X-gal histochemical stain.The result shows that this carrier system can import exogenous gene high-efficient, target ground following tumour: SMMC-7721, BEL-7402, U251, Bcap-37,3AO, SPC-Al, LOVO, SGC and cervical cancer transplanted tumor (X-gal dyeing knurl piece presents blueness), and this carrier system can not import foreign gene human small cell lung carcinoma H128 (X-gal dyeing knurl piece does not present blueness, sees Figure 33).Figure 34-41 is respectively GE7 quaternary complex body β-gal gene is imported related neoplasms pathological section X-gal histochemical stain result after the tumour.Figure 42 shows that GE7 quaternary complex body gene transfer system can not import β-gal gene the tumour pathological section X-gal coloration result (tumour cell does not present blueness) of human small cell lung carcinoma H128.
GV2/ protamine or histone/HA20/ β-gal quaternary complex body gene transfer system goes into to transplant the nude mice animal model in the high-transfer human liver cancer liver by the hepatic vein microinjection.After 14 days, put to death animal, the knurl piece is through the X-gal histochemical stain.Frozen section, is observed in microscopically after fast red redyes through bush or nuclear.At new vessel, particularly in the endotheliocyte of capillary vessel and little blood vessel, the foreign gene that especially can observe greater efficiency in the necrotic area on every side imports.In part wetting property tumour cell, also can observe foreign gene and import (cell that β in the section-gal gene imports presents blue Figure 43), then can only observe inefficient foreign gene and import in the endotheliocyte of great vessels, then not seeing in normal hepatocytes has foreign gene to import.
GV2/ protamine/HA20/ β-gal gene transfer system is through being injected into the high liver cancer nude mice animal model that shifts of subcutaneous transplantation knurl week.After 2 days, put to death animal, the knurl piece is through the X-gal histochemical stain.Frozen section is observed down in light microscopic after redying, and (cell that β in the section-gal gene imports presents blueness, Figure 44), and does not see have foreign gene to import in normal hepatocytes in visible foreign gene importing in the endotheliocyte of capillary vessel and little blood vessel.
Mediation causes apoptotic gene p21 in (II) body
WAF-1Efficiency assay: the nude mice with lotus people liver cancer SMMC-7721 knurl piece is a laboratory animal, at nude mice subcutaneous vaccination knurl piece, treat that the knurl block length is when diameter is about 0.5cm, random packet, every group of 6 nude mices, subcutaneous intratumor injection quaternary complex body of the present invention gene transfer system E5/ poly-lysine/HA20/p21 respectively
WAF-1Or E5/ poly-lysine/HA20, per week injection secondary, totally four times, per injection dosage is for containing 20 μ gp21
WAF-1Plasmid DNA reaches and does not contain p21
WAF-1The carrier system consumption of plasmid DNA.Injection three weeks of back, put to death animal, measurement knurl block is long-pending and claim its weight, the results are summarized in table 7.Figure 45 shows that tumor growth is subjected to remarkable inhibition.This result shows, E5/ poly-lysine/HA20/p21
WAF-1Can suppress the growth (seeing Table 7) of liver cancer cell in vivo significantly.Table 7 shows that from tumor size and weight ratio E5 quaternary complex body can suppress the growth of people's liver cancer SMMC-7721 significantly than E5 ternary complex carrier.
Table 7 E5 quaternary complex body gene transfer system mediation p21
WAF-1The knurl growth-inhibiting of gene pairs lotus people liver cancer nude mice
V(cm
3) W(g)
E5/ poly-lysine/HA20 0.85 ± 0.05 0.23 ± 0.06
E5/ poly-lysine/HA20/p21
WAF-10.21 ± 0.06 0.055 ± 0.02
Inhibiting rate 75.3% 76.1%
P value＜0.05＜0.02
Do not contain ternary complex E5/ poly-lysine/p21 that the endocytosis corpusculum discharges oligopeptides HA20
WAF-1Reach and do not contain p21
WAF-1The complex carrier E5/ poly-lysine/HA20 of gene DNA, ternary complex HA20/ poly-lysine/p21
WAF-1, binary complex body poly-lysine/p21
WAF-1, p21
WAF-1Gene DNA all shows the function of unrestraint liver cancer cell growth.This test shows E5/ poly-lysine/HA20/p21
WAF-1The cell conjugate gene transfer system can target ground mediate foreign gene into liver cancer cell in body, reaches the purpose that suppresses liver cancer cell growth.
Nude mice with lotus people liver cancer transplanted tumor is a laboratory animal, at nude mice subcutaneous vaccination knurl piece, treats the knurl block length when diameter is about 0.5cm, beginning subcutaneous tumors week injection quaternary complex body gene transfer system GE7/ poly-lysine/HA20/p21 provided by the invention
WAF-1Treat.Only inject once, injected dose is for containing 0.2 μ gp21
WAF-1The carrier consumption of gene DNA.And be provided with physiological saline, simple p21
WAF-1The control group of gene DNA, ternary complex carrier GE7/ poly-lysine/HA20.Administration two week, the back put to death animals, and it is long-pending and claim its weight to measure the knurl block.Figure 46 shows that tumor growth is subjected to remarkable inhibition.The results are summarized in table 8, table 9. table 8 show from the gross tumor volume size relatively GE7 quaternary complex body can suppress growth of tumor significantly, table 9 shows also can suppress growth of tumor significantly from tumor weight comparison GE7 quaternary complex body.This is table as a result
Table 8 different composite body is to growth inhibition ratio (V/V) group V ± SD (cm of people's liver cancer transplanted tumor
3) inhibiting rate (%) P value physiological saline 1.253 ± 0.497p21
WAF-1DNA 1.000 ± 0.280 20.19＞0.1GE7/ poly-lysine/HA20,2.253 ± 1.406-102.808＞0.05GE7/ poly-lysine/HA20/p21
WAF-10.164 ± 0.091 86.91＜0.02 annotates: inhibiting rate is the volume growth inhibition percentage ratio with respect to the physiological saline group.
Table 9 different composite body is to growth inhibition ratio (W/W) group W ± SD (g) inhibiting rate (%) the P value physiological saline 0.663 ± 0.160p21 of people's liver cancer transplanted tumor
WAF-1DNA 0.556 ± 0.130 16.22＞0.1GE7/ poly-lysine/HA20,0.169 ± 0.501-76.24＞0.05GE7/ poly-lysine/HA20/p21
WAF-10.129 ± 0.354 80.64＜0.001
Annotate: inhibiting rate is the weight growth-inhibiting percentage ratio with respect to the physiological saline group.Bright GE7 quaternary complex body gene transfer system can import foreign gene in the liver cancer transplanted tumor on target ground in body, reaches the purpose that suppresses liver cancer cell growth.
Because recombinant virus can wrap up above-mentioned various foreign gene, therefore, makes gene transfer system of the present invention also can be used for polygene combined treatment, and great potential using value is arranged.
Experiment confirm to sum up, gene transfer system of the present invention has broad application prospects in therapy of tumor, and it can import the inside and outside tumour cell with foreign gene DNA target ground, reach the purpose of treatment tumour.The present invention also lays a good foundation for further clinical study.
Advantage of the present invention: the overexpression that has some growth factor receptors according to new vessel endotheliocyte in tumour cell and the tumour, part oligopeptides among the present invention in binary and the ternary complex carrier, with foreign gene target importing tumour cell, this still belongs to the first time in the world.Use protamine among the present invention as the polycation polypeptide and as the skeleton of complex carrier, and with the integral part of HA20 as the ternary complex carrier, do not appear in the newspapers in the world.Because above three's compound composition is succeedd in animal body to the targeting gene therapy of part malignant tumour first.
Provide a class can with part oligopeptides E5, GE7, GV1 and the GV2 of surface of cell membrane growth factor receptors specific combination, and then made up that part oligopeptides/polycation polypeptide binary complex carrier and foreign DNA form the ternary complex gene transfer system and part oligopeptides/polycation polypeptide/endocytosis corpusculum discharges oligopeptides ternary complex carrier and foreign DNA formation quaternary complex body gene transfer system.The existence of endocytosis corpusculum release oligopeptides more can improve the transduction efficiency of such carrier.The endocytosis corpusculum discharges the synthetic peptide HA20 of oligopeptides employing and influenza virus adventitia hemagglutinin aminoterminal amino acid sequence homologous, and HA20 can exist with combined or non-binding attitude form.
Transgenosis non-virus carrier of the present invention can import external and interior tumor cell with foreign DNA target ground, reaches to suppress to kill this tumour cell, and normal cell is then unaffected on every side.Utilize this carrier high-efficiency, target ground that the function that foreign DNA is transferred to tumour cell is reached the purpose for the treatment of tumour.
The magnitude range of the foreign DNA of transgenosis non-virus carrier target of the present invention transfer can be from tens bases to several ten thousand bases, and this has just alleviated the size restriction of the foreign gene that virus vector delivered commonly used.To the controllability gene therapy, the difficult problem that the virus vector that polygene combined treatment and long segment gene run into when being used for the treatment of is difficult to pack can be resolved, can wrap up above-mentioned various foreign gene and unite use owing to recombinant virus again with ternary complex gene transfer system of the present invention, therefore make gene transfer system of the present invention both can enter tumour cell with gene of complex polypeptide delivery of a plurality of oligopeptides parts, can deliver a plurality of genes simultaneously to relevant tumour cell in target ground again, play polygene combined result of treatment, clinical efficacy will be better than the single-gene treatment greatly.
Part oligopeptides among the present invention/polycation polypeptide/endocytosis corpusculum discharges oligopeptides ternary complex carrier, it can set up recombinant dna plasmid by the prokaryotic cell prokaryocyte promoters driven according to the nucleotide sequence of coding aforementioned polypeptides, pass through gene engineering expression, obtain above-mentioned ternary complex carrier, be used for mass preparation and production.
The complex polypeptide system of the part oligopeptides among the present invention, be equally applicable to use part oligopeptides establishment ternary complex polypeptide at the acceptor of hematopoietic cell, lymphocyte, scavenger cell, endotheliocyte, liver cell, nephrocyte, myocardial cell and neuronal cell surface, foreign gene target ground is imported above-mentioned various cells, treatment tumour and non-tumor disease.Therefore has great potential application foreground.
Description of drawings
Fig. 1 is the record diagram of the Sephadex-G50 column chromatographic isolation and purification of the covalently bound thing of E5-poly-lysine.
Fig. 2 is the record diagram of the Sephadex-G50 column chromatographic isolation and purification of the covalently bound thing of GE7-poly-lysine.
Fig. 3 is the record diagram of the Sephadex-G50 column chromatographic isolation and purification of the covalently bound thing of GV2-protamine.
Fig. 4 is amino acid analysis of components result after the covalently bound thing hydrolysis of E5-poly-lysine.
Fig. 5 is amino acid analysis of components result after the covalently bound thing hydrolysis of GE7-poly-lysine.
Fig. 6 is the 1% agarose gel electrophoresis retardance experiment of E5 quaternary complex body gene transfer system.M represents λ/Hind III dna molecular amount mark among the figure; 0 expression plasmid DNA; 1,2,3,4,5 to represent the mass ratio of part oligopeptides-polycation polypeptide: DNA in the E5 quaternary complex body gene transfer system respectively be 1: 2.4,1: 2,1: 1.4,1: 1,1: 0.5.
Fig. 7 is the 1% agarose gel electrophoresis retardance experiment of GE7 quaternary complex body gene transfer system.1,2,3,4,5,6 to represent the mass ratio of part oligopeptides-polycation polypeptide: DNA in the GE7 quaternary complex body gene transfer system respectively be 1: 2.2,1: 2.0,1: 1.8,1: 1.7,1: 1.6,1: 1.5 among the figure; 7 represent plasmid DNA; 8 representatives are gone into/Hind III dna molecular amount mark.
Fig. 8 is the 1% agarose gel electrophoresis retardance experiment of GV1 and GV2 quaternary complex body gene transfer system.M represents into/Hind III dna molecular amount mark among the figure; 1,2,3,4,5 to represent the mass ratio of part oligopeptides-polycation polypeptide: DNA in the GV1 quaternary complex body gene transfer system respectively be 2: 1,3: 1,4: 1,5: 1,6: 1; It is 2: 1,3: 1,4: 1,5: 1,6: 1 that A, B, C, D, E represent the mass ratio of part oligopeptides-polycation polypeptide: DNA in the GV2 quaternary complex body gene transfer system respectively.
Fig. 9 is the X-gal coloration result that E5 quaternary complex body gene transfer system is led β-gal gene in the SMMC-7721 cell.
Figure 10 is the PBS control group X-gal coloration result of Fig. 9.
Figure 11 is the X-gal coloration result that E5 quaternary complex body gene transfer system can not import β-gal gene the L02 cell.
Figure 12 is the X-gal coloration result that E5 quaternary complex body gene transfer system can not import β-gal gene the R02 cell.
Figure 13 is the X-gal coloration result that GE7 quaternary complex body gene transfer system imports β-gal gene in the BEL-7402 cell.
Figure 14 is the X-gal coloration result that GV2 quaternary complex body gene transfer system imports β-gal gene the tumor neogenetic blood vessels intradermal cell.
Figure 15 is the X-gal coloration result that GE7 quaternary complex body gene transfer system imports β-gal gene in the SMMC-7721 cell.
Figure 16 is the X-gal coloration result that GE7 quaternary complex body gene transfer system imports β-gal gene in the U87 cell.
Figure 17 is the X-gal coloration result that GE7 quaternary complex body gene transfer system imports β-gal gene in the Bcap-37 cell.
Figure 18 is the X-gal coloration result that GE7 quaternary complex body gene transfer system imports β-gal gene in the 3AO cell.
Figure 19 is the X-gal coloration result that GE7 quaternary complex body gene transfer system imports β-gal gene in the AO cell.
Figure 20 is the X-gal coloration result that GE7 quaternary complex body gene transfer system imports β-gal gene in the SPC-A1 cell.
Figure 21 is the X-gal coloration result that GE7 quaternary complex body gene transfer system can not import β-gal gene the L02 cell.
Figure 22 is the X-gal coloration result that GE7 quaternary complex body gene transfer system can not import β-gal gene the NIH/3T3 cell.
Figure 23 is the X-gal coloration result that ternary complex HA20/ poly-lysine/β-gal can not import β-gal gene the BEL-7402 cell.
Figure 24 is the X-gal coloration result that binary complex body poly-lysine/β-gal can not import β-gal gene the BEL-7402 cell.
Figure 25 is the X-gal coloration result that β-gal gene DNA can not import β-gal gene the BEL-7402 cell.
Figure 26 is the X-gal coloration result that GE7 ternary complex gene transfer system GE7/ poly-lysine/β-gal imports β-gal gene in the BEL-7402 cell.
Figure 27 is that E5 quaternary complex body gene transfer system is with p21
WAF-1Gene imports liver cancer cell and causes apoptotic DAPI coloration result.
Figure 28 is that E5 quaternary complex body gene transfer system is with p21
WAF-1Gene imports liver cancer cell and causes apoptotic end mark result.
Figure 29 does not contain p21
WAF-1The empty carrier transfection group can not cause apoptotic end mark result.
Figure 30 is that E5 quaternary complex body gene transfer system is respectively with p15, p16, p21
WAF-1Gene imports the cytostatic growth curve chart of SMMC-7721 cell.
Figure 31 is that GE7 quaternary complex body gene transfer system is with p21
WAF-1Gene imports the growth curve chart of cytostatic growth curve of BEL-7402 cell and PBS control group.
Figure 32 is the cancer piece X-gal coloration result that E5 quaternary complex body gene transfer system imports β-gal gene lotus people liver cancer SMMC-7721 nude mice.
Figure 33 is that GE7 quaternary complex body gene transfer system imports lotus people liver cancer SMMC-7721, BEL-7402 with β-gal gene, human lung adenocarcinoma SPC-A1, human ovarian cancer 3A0, people's enteraden cancer LOVO, people's adenocarcinoma of stomach SGC, human breast carcinoma Bcap-37, human neuroglia cancer U251, human cervical carcinoma's transplanted tumor, and can not be with the X-gal coloration result of β-gal gene importing small cell lung cancer H128 knurl piece.
Figure 34 is the pathological section X-gal coloration result that GE7 quaternary complex body gene transfer system imports β-gal gene people's liver cancer SMMC-7721.
Figure 35 is the pathological section X-gal coloration result that GE7 quaternary complex body gene transfer system imports β-gal gene people's liver cancer BEL-7402.
Figure 36 is the pathological section X-gal coloration result that GE7 quaternary complex body gene transfer system imports β-gal gene human glioma U251.
Figure 37 is the pathological section X-gal coloration result that GE7 quaternary complex body gene transfer system imports β-gal gene human ovarian cancer 3AO.
Figure 38 is the pathological section X-gal coloration result that GE7 quaternary complex body gene transfer system imports β-gal gene human lung adenocarcinoma SPC-A1.
Figure 39 is that GE7 quaternary complex body gene transfer system imports people's enteraden cancer LOVO pathological section X-gal coloration result with β-gal gene.
Figure 40 is the pathological section X-gal coloration result that GE7 quaternary complex body gene transfer system imports β-gal gene people's adenocarcinoma of stomach SGC.
Figure 41 is the pathological section X-gal coloration result that GE7 quaternary complex body gene transfer system imports β-gal gene human cervical carcinoma's transplanted tumor.
Figure 42 is the pathological section X-gal coloration result that GE7 quaternary complex body gene transfer system can not import β-gal gene human small cell lung carcinoma H128.
Figure 43 is GV2 quaternary complex body gene transfer system soaks into β-gal gene importing endothelial cells in tumor neogenetic blood vessels and part tumour cell in body a pathological section X-gal coloration result.
Figure 44 is GV2 quaternary complex body gene transfer system imports β-gal gene people's tumor vascular endothelial cell in body a pathological section X-gal coloration result.
Figure 45 is E5 quaternary complex body gene transfer system mediation p21
WAF-1With of the comparison of E5 ternary complex carrier to the knurl piece inhibition growth result of lotus people liver cancer SMMC-7721 nude mice.
Figure 46 is GE7 quaternary complex body gene transfer system mediation p21
WAF-1With of the comparison of GE7 ternary complex carrier to the knurl piece inhibition growth result of lotus people liver cancer transplanted tumor nude mice.
Most preferred embodiment
Adopt the Peptide synthesizer of American AB I company to reach according to its polypeptide synthetic operation handbook, carry out polypeptide E5 and synthesize, step is summarized as follows:
1. the solid-phase synthetic peptide synthesizer is to be means with the solid phase synthesis, and used vector resin is the PAM amino-acid resin that ABI company provides, and the consumption of synthetic amino acid needed resin is 0.5 mmole at every turn, and the each consumption of amino acid is 2 mmoles.Press the amino acid whose implementation sequence of part oligopeptides E5, add each amino acid whose composition successively and carry out solid phase synthesis.
2. the resin cracking is after solid phase synthesis obtains peptide-resin, carry out the deresination reaction, 1 milliliter of TFMSA (trifluoromethane sulfonic acid) lysate that adopts that ABI company provides and 10%TFA (trifluoracetic acid), stirred 2 hours under the room temperature, cross the elimination resin, 250ml is settled out crude product with anhydrous diethyl ether, puts 4 ℃ and spends the night.Centrifugal, remove supernatant, drying.
3. contain a certain amount of salt in the crude product that the desalination cracking gets, adopt Sephadex G10 column purification, use 0.1M Glacial acetic acid wash-out, collect first peak, concentrate, remove disacidify, with less water dissolving, freezing, the dry dry powder that gets.
4. the high pressure liquid chromatography (HPLC) separation is carried out at Beckman 420 class Ultraviolet Detector with the C8 reversed-phase column (250mm is long, the 10mm diameter) of ABI company, collects required peak, and lyophilize gets dry powder product oligopeptides E5.
Press the amino acid whose implementation sequence of part oligopeptides GE7 except adding the amino-acid sequence step, all the other operation stepss get dry powder product part oligopeptides GE7 with embodiment 1.
Press part oligopeptides GV1, the amino acid whose implementation sequence of GV2 except that adding the amino-acid sequence step, all the other operation stepss get dry powder product part oligopeptides GV1, GV2 with embodiment 1.
Embodiment 4 (LOP)
2-PCP, (EROP)
2-PCP and (LOP)
2-PCP-(EROP)
2The polypeptide systems produce
Be called for short representative: LOP (Ligand oligopeptide, part oligopeptides) respectively, PCP (Poly-cationic polypeptide, polycation polypeptide), EROP (endosome release oligo-peptide, the endocytosis corpusculum discharges oligopeptides).The concrete operations step is as follows:
4-1 (LOP)
2The preparation of-PCP:
Utilize albumen coupling agent SPDP (N-succinimidyl-3-(2-Pyridyl-dithio)-propionate), preparation (LOP)
2-PCP.Synthesis step is as follows:
4-1-1 prepares part oligopeptides two thiopyridines (LOP-PDP, Ligand oligopeptide 3-(2-pyri-dyldithio)-propiorate)
The mol ratio of LOP and SPDP is 1: 8, and 25 ℃ of reactions 60 minutes, dissimilar LOP was dissolved in different Laemmli buffer system Laemmlis, and SPDP is dissolved in the dehydrated alcohol.Remove the SPDP that does not react by dialysis and get product LOP-PDP.
4-1-2 prepares sulfhydrylation polycation polypeptide (Polycationic polypeptide-(SH)
2, PCP-(SH)
2).
The mol ratio of PCP and SPDP is 1: 2, generates PCP-(PDP) in 60 minutes 25 ℃ of reactions
2PCP-(PDP)
2Be polycation peptide-3-(2-pyridyldithio propionate)
2, polycation polypeptide-two thiopyridines.PCP-(PDP)
2Generated PCP-(SH) with dithiothreitol (DTT) (DTT) in 60 minutes in 37 ℃ of effects
2
Different PCP are dissolved in the corresponding Laemmli buffer system Laemmli, with mixture to corresponding Laemmli buffer system Laemmli dialyse respectively elementary product P CP-(PDP)
2With product P CP-(SH)
2The final concentration of DTT is 25mM in the reaction mixture.
4-1-3 (LOP)
2The preparation of-PCP (the covalently bound thing of polycation polypeptide one part oligopeptides)
LOP-PDP and PCP-(SH)
2Mol ratio be 10: 1, in 37 ℃ the reaction 12 hours.Be reflected in the corresponding Laemmli buffer system Laemmli and carry out, end product PCP-(LOP)
2Be to collect the first peak gained with Sephadex G50 post separate reacted mixture.(LOP) to gained
2-PCP product carries out the aminoacid component analysis further to determine, confirms the purpose product.
4-2. (EROP)
2The preparation of-PCP
Utilize albumen coupling agent SPDP to synthesize EROP-PDP, step is as follows:
4-2-1 prepares the endocytosis corpusculum and discharges oligopeptides two thiopyridines (Endosome release oligopeptide 3-(2-Pyridyldithio)-Propionate)
The mol ratio of EROP and SPDP was 10: 1, in 25 ℃ of reactions 60 minutes.Different EROP is dissolved in the corresponding damping fluid, reaction mixture to corresponding Laemmli buffer system Laemmli dialyse product EROP-PDP.
4-2-2 PCP-(SH)
2Preparation
Method is with present embodiment 4-1-2.
4-2-3 (EROP)
2The preparation of-PCP (polycation one endocytosis corpusculum discharges oligopeptides)
EROP-PDP and PCP-(SH)
2Mol ratio be 10: 1, in 37 ℃ the reaction 12 hours.Be reflected in the corresponding Laemmli buffer system Laemmli and carry out, end product PCP-(EROP)
2Be to collect the first peak gained with Sephadex G50 post separate reacted mixture.To products therefrom (EROP)
2-PCP carries out the amino acid analysis of components further to determine, to confirm the purpose product.
4-3 (LOP)
2-PCP-(EROP)
2Preparation
4-3-1 prepares (LOP)
2-PCP
Method is with present embodiment 4-1
4-3-2 prepares part oligopeptides-polycation polypeptide-(pyridine two sulphur) 2, [(LOP)
2-PCP-(PDP)
2, (Ligand oligopeptide)
2-polycation peptide-(3-(2-Pyridyldithio)-propionat-e)
2]
PCP-(LOP)
2With the mol ratio of SPDP be 1: 2, in 25 ℃ the reaction 60 minutes.Be reflected in the corresponding Laemmli buffer system Laemmli and carry out, reaction mixture is dialysed to corresponding Laemmli buffer system Laemmli, to remove small molecular weight impurity, gets product (LOP)
2-PCP-(PDP)
2
4-3-3 prepares EROP-SH (Endosome release oligopeptide-SH)
The preparation of EROP-PDP is with present embodiment 4-2-1
EROP-PDP and DTT were in 37C effect 60 minutes.Be reflected in the corresponding Laemmli buffer system Laemmli and carry out, reaction mixture to corresponding Laemmli buffer system Laemmli dialyse product EROP-SH.The final concentration of reaction DTT is 25mM.
4-3-4 preparation (part oligopeptides) 2-polycation polypeptide-(the endocytosis corpusculum discharges oligopeptides)
2[(LOP)
2-PCP-(EROP)
2]
EROP-SH with (LOP)
2-PCP-(PDP)
2Mol ratio be 10: 1, in 25 ℃ the reaction 12 hours.Be reflected in the corresponding Laemmli buffer system Laemmli and carry out, end product (LOP)
2-PCP-(EROP)
2Be with SephadexG50 separated and collected first peak gained.To products therefrom (LOP)
2-PCP-(EROP)
2Carry out the amino acid analysis of components to determine, to confirm the purpose product.
The isolation and purification of used recombinant eukaryon expression vector plasmid DNA all utilizes the Maxi plasmid kit of QIAGEN company and obtains the pure product of DNA among the present invention.According to the description of product of QIAGEN company, now will separate, the step of plasmid DNA purification is summarized as follows.
Contain corresponding antibiotic 5ml LB substratum and insert a single bacterium colony, overnight incubation under 37 ℃ of violent joltings is transferred in containing corresponding antibiotic 200ml LB substratum, overnight incubation under 37 ℃ of violent joltings.The 200ml culture is added in the 500ml plastic centrifuge tube, and 4 ℃, centrifugal 5 minutes kinds of 2500rpm are abandoned supernatant liquor, make precipitation dry as far as possible.Bacterial precipitation is resuspended in 10ml damping fluid P1 (50mM TrisHCl, pH8.0; 10mM EDTA, 100 μ g/ml RNaseA) with the dropper pressure-vaccum until there not being granular substance to exist; Be transferred to the 50ml plastic centrifuge tube and add 10ml damping fluid P2 that (200mM NaOH 1%SDS) reverses mixing 4-6 time, room temperature 5 minutes gently; (the 3.0M Potassium ethanoate pH5.5), reverses 5-6 time immediately gently, puts ice bath 20 minutes, and 12000rpm4 ℃ from 1 heart 20 minutes, and supernatant refilters once to add 10ml damping fluid P3.10ml damping fluid QBT balance QAGEN-tip500 allows under its spontaneous current; Add the supernatant liquor of having filtered, under the spontaneous current.Damping fluid QC30ml washes 2 times, adds damping fluid QF15ml, collects effluent liquid; Add the Virahol under 0.7 times of volume room temperature, immediately 12000rpm4 ℃ centrifugal, 30 minutes, abandon supernatant, 5ml70% washing with alcohol DNA precipitation, dry air 5 minutes is dissolved in the TE solution of an amount of volume, ultraviolet spectrophotometer is checked purity, quantitatively.
Embodiment 6 preparation polypeptide-dna complexs
6-1 prepares quaternary complex body gene transfer system LOP/PCP/EROP/DNA (with (LOP)
2-PCP with (EROP)
2-PCP and DNA form complex body)
With (LOP)
2-PCP and (EROP)
2-PCP is to mix at 1: 1 with mol ratio, and with filter type to mixed solution sterilize ((LOP)
2-PCP, (EROP)
2-PCP all is dissolved in the physiological saline).DNA is also with filter type sterilize (DNA is dissolved in the physiological saline).With DNA with (LOP)
2-PCP/ (EROP)
2-PCP mixture is to mix at 1.5: 1 with mass ratio, and when dripping dna solution, constantly mixes solution.Mixture is respectively observed DNA blocked and quaternary complex body formed granular size with 1% agarose gel electrophoresis and Electronic Speculum in 25 ℃ of insulations after 0.5 hour.
6-2 prepares quaternary complex body gene transfer system (LOP)
2-PCP-(EROP)
2/ DNA is (with (LOP)
2-PCP-(EROP)
2Form complex body with DNA)
To be dissolved in (LOP) in the physiological saline
2-PCP-(EROP)
2With DNA with after the filter type sterilization, with (LOP)
2-PCP-(EROP)
2With the mass ratio of DNA 1: 1.5, drip DNA and constantly mix solution.Mixture is observed DNA blocked and quaternary complex body formed granular size with 1% agarose gel electrophoresis and electronic microscope photos in 25 ℃ of insulations after 0.5 hour.
The enforcement of embodiment 7 external transfered cells
5 * 10
4Cell inoculation changes after 1 day with the fresh liquid (DMEM that contains 10% (v/v) calf serum) of training fully in Costar six well culture plates.Add the quaternary complex body gene transfer system that contains preparation among the embodiment 6 in second day and train liquid fully.The amount that adds quaternary complex body gene transfer system in every hole 1ml training liquid is the complex body (i.e. the compound scale of construction of the formed non-virus of 0.2 μ g DNA) that is equivalent to 0.2 μ g DNA.Remove the training liquid that contains non-viral complex body after 12 hours, change with fresh training liquid.Import efficient and target respectively at 24,48,72,96,120,144,168 hours analyzing genes.Gene imports efficient and analyzes with the mediation pSV-β-galactosidase of this complex body system, promptly carries out histochemical stain respectively at above-mentioned time pair cell.Step is: after removing training liquid, with PBS rinsing cell, use 4% formaldehyde in 4 ℃ of fixed cells 5 minutes then, then with PBS rinsing cell, add the staining fluid that contains X-gal at last in 37 ℃ of insulations observations after 24 hours.(staining fluid is formed: X-gal, K
4[Fe (CN)
6], K
3[Fe (CN)
6], MgCl
2Final concentration be respectively: 1mg/ml, 5mM, 5mM, 2mM).
The LOP and the relative growth factor are competed the inhibition experiment and are mediated about causing that the concrete operations of the gene of apoptosis or other function see the following examples for details in order to confirm target and carrier system that gene imports.
Import experiment in embodiment 8 bodies
In the knurl of tumor bearing nude mice or lotus knurl SCID mouse, concrete steps are as follows with the quaternary complex body gene transfer system direct injection of embodiment 6 preparation:
8-1 presses embodiment 6 preparation quaternary complex body gene transfer systems.
8-2 is injected in the knurl a certain amount of above-mentioned complex body is disposable, observes, analyzes efficient and target that import system imports foreign gene respectively at different time sections.Concrete operations are: at first take out the knurl piece, get half and do the pathology section, second half after the PBS rinsing with 4% formaldehyde in 4 ℃ of fixing 15min, then with PBS rinsing 3 times, each 10 minutes, at last in 37 ℃ with the tissue staining liquid that contains X-gal in 37 ℃ of insulations observations (tissue staining liquid is formed with embodiment 5) after 12 hours.The dyeing course of pathological section is, in 4% formaldehyde fixed, fixing 5min, then with the PBS rinsing be placed on for 3 times in the tissue staining liquid in 37 ℃ be incubated 12 hours after observations (tissue staining liquid is formed with embodiment 7).
The target that imports foreign gene in this complex body system body carries out so that relevant control group to be set.
The mediation of this import system of 8-3 causes apoptotic genes involved or its its functional gene, is to be purpose with the shown oncotherapy effect of gene of observing this carrier system mediation.The lead-in mode of import system imports dosage and is determined on a case-by-case basis with intratumor injection or corresponding vascular drug delivery.The following examples are seen in the importing concrete operations of apoptogene or other functional gene.
Embodiment 9 E5 quaternary complex body gene transfer systems import being imbued with IGF-I acceptor or IGF-II recipient cell targets tropism gene
The composition of 9-1 E5 quaternary complex body gene transfer system:
The amino-acid sequence of LOP:E5 part oligopeptides is EPFRS POLAL ETYG
PCP: the molecular weight of poly-lysine is 26,000
The amino-acid sequence of EROP:HA20 is GLFEAIAEFI EGGWE GLIEG
DNA: β-gal gene, CKI gene p21
WAF-1, p15, p16.
The preparation method of 9-2 E5 poly-lysine and HA20-poly-lysine and embodiment 4 are same, and the damping fluid that is used for E5 is: 0.1M PBS/0.1M NaCl, pH7.5; The damping fluid that is used for HA20 is: 0.1MPBS/0.1M NaCl, pH7.8.
9-3 adopts E5-poly-lysine and HA20-poly-lysine and DNA to prepare E5 quaternary complex body gene transfer system with reference to embodiment 6, and the best proportioning of DNA and polypeptide is: 1.5: 1 (W/W), corresponding mol ratio is 1: 75-80.
The external transfer beta-galactosidase gene of 9-4 is to human hepatoma cell strain SMMC-7721 and normal liver cell strain L02.
Gene transfer method is with reference to embodiment 7, the result is presented at table 1,2,3 and Fig. 9-12, table 1 confirms to have only E5-poly-lysine and HA20-poly-lysine pSV-β-gal DNA could be transferred to human liver cancer cell SMMC-7721 efficiently, efficient reaches 60%, and E5/ poly-lysine/pSV-β-gal transfer efficiency very low (＜10%) may be because lysosomal Degradation.Table 2 shows that E5 quaternary complex body gene transfer system only can import the SMMC-7721 liver cancer cell with β-gal gene, and β-gal gene can not be led normal liver cell L02 of AE and primary cultured hepatocyte R02.Table 3 confirms that β-gal gene began to express in 48 hours after transfection, 72 hour meters peak.
Fig. 9-12 shows that E5 carrier system energy high-level efficiency, target ground import human liver cancer cell SMMC-7721 with β-gal gene, and foreign gene can not be imported normal liver cell L02 and primary cultured hepatocyte R02.This figure carries out coloration result after 72 hours after being shown as transfection.
Import the cell nude mice of pSV-β-gal gene in the body of 9-5 E5 carrier system to lotus people liver cancer SMMC-7721.
100 μ l contain quaternary complex body E5-poly-lysine/HA20-poly-lysine/subcutaneous intratumor injection tumor bearing nude mice of pSV-β-gal dna direct of 20 μ g β-gal gene plasmid DNA, beta-galactosidase enzymes dyeing shows that pSV-β-gal gene began to have expression and continues to 96 hours at 12 hours, and injecting pSV-β-gal DNA group does not merely then have the expression (seeing Figure 32) of beta-galactosidase gene.
9-6 uses the growth of the external importing of E5-poly-lysine/HA20-poly-lysine CKI gene inhibition tumour cell and induces its apoptosis
Use E5-poly-lysine/HA20 poly-lysine with CKI gene (p21
WAF-1, p15, p16) external importing human hepatoma cell strain (SMMC-7721) is seen Figure 30 to the restraining effect of growth of tumour cell.
1～2 * 10
5Human liver cancer cell SMMC-7721 contains the CKI gene with 0.2 μ g or does not contain the plasmid DNA transfection of CKI gene in 1ml training liquid, and after the transfection 24 hours, renew bright training liquid, 48 hours, peptic cell pressed 1 * 10
4Plant 24 orifice plates, per then 48 hours peptic cells are also counted, and several 4 times altogether, get three hole mean values and make cell growth inhibiting curve (Figure 30).
9-7 uses E5-poly-lysine and HA20-poly-lysine and p21
WAF-1Gene DNA forms the quaternary complex body, and lotus people liver cancer nude mice is observed in the body restraining effect to liver cancer cell growth.Above-mentioned complex body 100 μ l are injected in the nude mice subcutaneous transplantation people SMMC-7721 liver cancer knurl, and complex body contains 20 μ gDNA.Fortnight is injected in one week injection twice altogether.Inject and put to death animal in back 17 days, get its Subcutaneous tumor, tumour inhibiting rate sees Table 7 and Figure 45.
GE7 quaternary complex body gene transfer system imports the target gene that is imbued with EGF acceptor or erbB family member cell
The composition of 10-1 GE7 gene transfer system
LOP: the aminoacid sequence of part oligopeptides GE7 is NPVVG YIGER PQTRD L
PCP: poly-lysine, molecular weight are 26,000; Protamine, molecular weight are 7000
EROP: with EROP among the embodiment 9-1
DNA: β-gal gene, p21
WAF-1Gene.
10-2 is according to embodiment 4 preparation GE7-poly-lysine (or protamine) and HA20-poly-lysines (protamine), and the damping fluid that is used for GE7 is 0.1M PBS/0.1M NaCl, pH7.4.The damping fluid of HA20 is 0.1M PBS/0.1M NaCl, pH7.8.
10-3 adopts GE7-poly-lysine and HA20-poly-lysine (or GE7-protamine and HA20-protamine) to prepare quaternary complex body gene transfer system with DNA with reference to embodiment 6-1.
To the poly-lysine system, the optimum proportion of DNA and polypeptide is 1.5: 1 (W/W), is equivalent to 1: the mol ratio of 75-80.And the protamine system is 1: 1 (W/W).。
10-4 is external to import human liver cancer cell SMMC-7721, BEL-7402 with β-gal gene, neuroglial cytoma U251, ovarian cancer cell 3AO, AO, breast cancer cell Bcap-37, lung adenocarcinoma cell Spc-A1 and normal liver cell L02, l cell NIH/3T3.
Press the gene transfer method of embodiment 7, carry out transgenosis.The results are shown in Table 4,5,6 and Figure 13,15-26.As can be seen from Table 4 GE7-poly-lysine and HA20-poly-lysine and β-gal be built into quaternary complex body gene transfer system can be with foreign gene respectively to import in BEL-7402, SMMC-7721, Bcap-37, SPC-A1,3AO, U87, the AO cell greater than 90%, 80%, 80%, 80%, 65%, 30%, 15% transduction efficiency.GE7-poly-lysine (or GE7-protamine) is lower than 10% to the efficient of BEL-7402 cell transduction foreign gene when no HA20 exists.And HA20-poly-lysine (or HA20-protamine), poly-lysine and simple plasmid DNA are lower than 0.1% to the efficient of BEL-7402 cell transduction foreign gene.
Table 5 shows normal liver cell L02, l cell NIH/3T3, gene transfering efficiency very low (＜0.1%).Table 6 shows β-gal gene time dependent situation of expression level in the BEL-7402 cell.After the transgenosis 24 hours, β-gal gene begins to express, and peaks to 168 hours.
GE7 quaternary complex body gene transfer system mediation β-gal gene imports people's liver cancer BEL-7402, the SMMC-7721 of the subcutaneous lotus of nude mice in the 10-5 body, neurospongioma U251, mammary cancer Bcap-37, ovarian cancer 3AO, adenocarcinoma of lung SPC-A1, small cell lung cancer H128, adenocarcinoma of stomach SGC, the tumour cell of enteraden cancer LOVO and cervical cancer transplanted tumor.
Figure 34-41 shows that 100 μ l GE7 quaternary complex body gene transfer systems (containing 0.2 μ g β-gal plasmid DNA) import following tumour cell: BEL-7402, SMMC-7721, U251, Bcap-37,3AO, SPC-A1, SGC, LOVO, cervical cancer transplanted tumor with β-gal gene efficient, target ground.But this carrier system can not import foreign gene H128 (small cell lung cancer) tumour cell.
Expression of exogenous gene respectively at transgenosis after 4,8,12,24,48,96 hours, observed in 7,15,20,30,40 days.Foreign gene just had expression in 4 hours, express and can continue until 40 days, and peak expression is 24,48 hours.Only there is the constructed quaternary complex body gene transfer system of GE7-poly-lysine and HA20-poly-lysine (or GE7-protamine and HA20-protamine) and β-gal efficiently foreign gene to be imported tumour cell, and simple GE7-poly-lysine, HA20-poly-lysine, poly-lysine and simple plasmid DNA almost can not import tumour cell with foreign gene.
The CKI gene p21 of 10-6 GE7-poly-lysine and the mediation of HA20-poly-lysine
WAF-1Can in body, suppress the growth of transplantability people liver cancer in the nude mice.With GE7-poly-lysine and HA20-poly-lysine and p21
WAF-1The quaternary complex body that gene forms is injected in people's liver cancer transplanted tumor of the subcutaneous institute of nude mice lotus, is provided with physiological saline, simple p21 simultaneously
WAF-1Gene DNA, and do not contain the complex polypeptide of gene, every group of tumor bearing nude mice is 6.The consumption of DNA is that 0.2 μ g contains p21
WAF-1The plasmid DNA of gene, carrier polypeptide are that 0.13 μ g injection was put to death animal after 14 days, and measurement knurl volume size is also weighed, and calculates the generation inhibiting rate, the results are summarized in table 8,9, and Figure 46 shows that tumor growth is subjected to remarkable inhibition.
Embodiment 11
GV1 (or GV2) quaternary complex body gene transfer system imports the target gene that is imbued with the vegf receptor cell
The composition of 11-1 GV1 (or GV2) quaternary complex body gene transfer system:
The amino-acid sequence of LOP:GV1 oligopeptides is:
CHPIE?TLVDI?FQEYP?DEIEYIFKPS?PVPLM?RP
The amino-acid sequence of GV2 oligopeptides is:
PVPTE?ESNIT?MQIMRIKPHQ?GQHIG?EMSFL?QHNKCE
PCP: protamine, molecular weight are 7000
The amino-acid sequence of EROP:HA20 oligopeptides is with embodiment 9-1.
DNA: β-gal gene
The preparation of 11-2 GV1-protamine or (GV2-protamine) and HA20-protamine polypeptide system:
Utilize isodigeranyl functional protein coupling agent SPDP to form bridging between the polypeptide by embodiment 4 described methods.
The preparation of 11-2-1 polycation two thiopyridines protamine-PDP:
Protamine mutually mixes with mol ratio with SPDP at 1: 5, be diluted in pH7.4, contain in the 0.1M sodium phosphate buffer of 0.1M NaCl to protamine concentration be 1mg/ml, 25 ℃ of reactions are after 2 hours, generate protamine-PDP, (3cm * 50cm) remove uses above-mentioned buffer solution elution to residual SPDP by Sephadex G-25 column chromatography.
The preparation of 11-2-2 part oligopeptides-polycation complex body (GV1-protamine, GV2-protamine):
GV1 (or GV2) and protamine-PDP mutually mix with mol ratio at 1: 1, and 25 ℃ of reactions generate GV1-protamine (or GV2-protamine) after 24 hours, and (purifying of 1.8cm * 70cm) is with H through Sephadex G-50 column chromatography
2O wash-out, purified product are quantitative by ultraviolet after concentrating.
(the preparation of protamine-SH) of the polycation of 11-2-3 sulfhydrylation
Protamine-PDP and excessive DTT (20mM) generate PCP-(SH) 25 ℃ of reactions after 40 minutes
2(3cm * 50cm) remove free DTT contains the 0.1M phosphate buffered saline buffer wash-out of 0.1M NaCl with pH7.4 through Sephadex G-25 column chromatography.
11-2-4 endocytosis corpusculum discharges the preparation of oligopeptides two thiopyridines (HA20-PDP)
HA20 mixes with mol ratio with SPDP at 1: 5, generates HA20-PDP in 2 hours in 25 ℃ of reactions.The same 11-2-1 of all the other methods.
The preparation of 11-2-5 HA20-protamine
HA20-PDP mixes with mol ratio with protamine-SH at 1: 1, and 25 ℃ of reactions generated HA20-protamine, the same 11-2-2 of all the other methods in 72 hours.
The preparation of 11-3 GV1-protamine/H 20-protamine/β-gal and GV2-protamine/HA20-protamine/β-gal quaternary complex body gene transfer system:
11-3-1 β-gal separation and purification method is referring to embodiment 5.
The preparation of 11-3-2 GV1-protamine/HA20-protamine/β-gal and GV2-protamine/HA20-protamine/β-gal quaternary complex body gene transfer system:
GV1-protamine, GV2-protamine, HA20-protamine and β-gal are separately by 0.22 μ m membrane filtration degerming, β-gal is dissolved in a small amount of sterilized water, the HA20-protamine dropped among β-gal 25 ℃ of reactions after 5 minutes, drip GV1-protamine or GV2-protamine again, the limit edged shakes, behind the mixture reaction 30 minutes, be diluted to finite concentration with physiological saline.The mass ratio of β-gal and HA20-protamine and GV1-protamine (or GV2-protamine) is 1: 5, and corresponding mol ratio is 1: 686, and the evaluation of mixture is with embodiment 6 (referring to Fig. 8).
11-4 pSV-β-gal gene is transferred to tumor bearing nude mice by GV2 quaternary complex body gene transfer system:
(contain 0.5 μ g β-gal) goes into high the transfer by the hepatic vein microinjection and transplants the nude mice animal model in the liver cancer liver 50 μ l GV2 gene transfer system/β-gal mixtures.After 14 days, nude mice is sacrificed the back tissue through X-gal dyeing, and frozen section is observed down in light microscopic after bush and nuclear fast red are redyed.At new vessel, particularly in the endotheliocyte of capillary vessel and little blood vessel, especially can observe high betagalactosidase activity on every side in the necrotic area; In part wetting property tumor cell group, also can be observed external source betagalactosidase activity (seeing Figure 43); In the endotheliocyte of great vessels, then observe lower betagalactosidase activity; Then do not see betagalactosidase activity in the normal hepatocytes.
100 μ l GV2 gene transfer system/β-gal mixtures (contain 1 μ g β-gal), by being injected into the high liver cancer animal model that shifts of subcutaneous transplantation knurl week.After 2 days, nude mice is sacrificed the back tissue through X-gal dyeing, and frozen section is observed down in light microscopic after redying, visible betagalactosidase activity (seeing Figure 44) is not then seen betagalactosidase activity in the normal hepatocytes in the endotheliocyte of capillary vessel and little blood vessel.
Claims (31)
1. the gene transfer system of the target therapy of tumor of growth factor receptors mediation, form by part/polycation/endocytosis corpusculum releaser/foreign gene, it is characterized in that described gene transfer system is the quaternary complex body gene transfer system that is formed by part oligopeptides/polycation polypeptide/endocytosis corpusculum release oligopeptides HA20 ternary complex carrier and foreign DNA.
2. gene transfer system as claimed in claim 1 is characterized in that it being that the endocytosis corpusculum discharges oligopeptides HA20 and do not exist under the situation, the ternary complex gene transfer system of being made up of part oligopeptides/polycation polypeptide binary complex carrier and foreign DNA.
3. gene transfer system as claimed in claim 1 is characterized in that described part oligopeptides is and growth factor receptors IGF-II R, IGF-I R, EGF R, the part oligopeptides E5 of VEGF R specific combination, GE7, GV1, GV2.
4. gene transfer system as claimed in claim 2 is characterized in that described part oligopeptides is and growth factor receptors IGF-II R, IGF-I R, EGF R, the part oligopeptides E5 of VEGF R specific combination, GE7, GV1, GV2.
5. gene transfer system as claimed in claim 3, the aminoacid sequence that it is characterized in that described part oligopeptides E5 is EPFRS PDLAL ETYG, the aminoacid sequence of part oligopeptides GE7 is NPVVG YIGERPQYRD L, the aminoacid sequence of part oligopeptides GV1 is CHPIE TLVDI FQEYP DEIEYIFKPS PVPLM RP, and the aminoacid sequence of part oligopeptides GV2 is PVPTE ESNIT MQIMRIKPHQ GQHIG EMSFL QHNKCE.
6. gene transfer system as claimed in claim 4, the aminoacid sequence that it is characterized in that described part oligopeptides E5 is EPFRS PDLAL ETYG, the aminoacid sequence of part oligopeptides GE7 is NPWG YIGERPQYRD L, the aminoacid sequence of part oligopeptides GV1 is CHPIE TLVDI FQEYP DEIEYIFKPSPVPLM RP, and the aminoacid sequence of part oligopeptides GV2 is PVPTE ESNIT MQIMRIKPHQ GQHIG EMSFL QHNKC E.
7. as claim 1,2,3,4,5,6 described gene transfer systems, it is characterized in that described part oligopeptides also can be to have identical immunologic determinants with E5, GE7, GV1, GV2, can play positive reaction with above-mentioned oligopeptides corresponding antibodies, and can with IGF-I R, IGF-II R, EGF R, VEGF R bonded polypeptide and oligopeptides.
8. as claim 1,2,3,4,5,6 described gene transfer systems, it is characterized in that the amino acid in corresponding site in the described part oligopeptides, can use the amino acid replacement of similar performance.
9. as claim 1,2 described gene transfer systems, it is characterized in that described polycation polypeptide is poly-lysine, protamine or histone.
10. as claim 1,2 described gene transfer systems, it is characterized in that described foreign DNA comprises the antisense oncogene, antioncogene, suicide gene, the recombinant eukaryotic expression plasmid DNA of apoptosis gene or cytokine gene.
11. gene transfer system as claimed in claim 10 is characterized in that described antisense oncogene comprises oncogene ras
H, ras
K, ras
N, c-myc, bcl-2, the antisense dna sequence of somatomedin and acceptor gene thereof, antisense oligonucleotide and antisense oligodeoxyribonucleotide.
12. gene transfer system as claimed in claim 10 is characterized in that described antioncogene comprises p53 and Rb.
13. gene transfer system as claimed in claim 10 is characterized in that described suicide gene comprises HSV-TK (herpes simplex virus thymidine kinase) gene and CD (coli cytosine deaminase) gene.
14. gene transfer system as claimed in claim 10 is characterized in that described apoptosis gene comprises p15, p16 and p21
WAF-1
15. gene transfer system as claimed in claim 10, it is characterized in that described cytokine gene comprises GM-CSF (CM-CSF) gene, TNF (tumour necrosis factor) α gene, INF (Interferon, rabbit) α, γ gene, IL (interleukin-) 2,3,4,12,15 genes.
16. gene transfer system as claimed in claim 10 is characterized in that described recombinant eukaryotic expression plasmid comprises viral recombinant eukaryotic expression plasmid: the promotor that retrovirus recombinant expression vector, adenovirus recombinant expression vector reach with SV40, CMV is the recombinant eukaryotic expression plasmid of cis-regulating element.
17., it is characterized in that it is synthetic peptide with influenza virus adventitia hemagglutinin aminoterminal amino acid sequence homologous that described endocytosis corpusculum discharges oligopeptides HA20 as claim 1,2 described gene transfer systems.
18. gene transfer system as claimed in claim 17 is characterized in that described endocytosis corpusculum discharges oligopeptides HA20 and exists with the combined that is combined into part oligopeptides/polycation polypeptide/HA20/ foreign DNA quaternary complex body gene transfer system.
19. gene transfer system as claimed in claim 17 is characterized in that described endocytosis corpusculum discharges oligopeptides HA20 and exists with non-binding attitude with part oligopeptides/polycation polypeptide/foreign DNA ternary complex gene transfer system with HA20-poly-lysine, HA20-protamine or the covalently bound thing of HA20-histone.
20. gene transfer system as claimed in claim 17 is characterized in that described endocytosis corpusculum discharges oligopeptides HA20 and exists with non-binding attitude with part oligopeptides/polycation polypeptide/foreign DNA ternary complex gene transfer system with independent HA20.
21. gene transfer system as claimed in claim 2 is characterized in that described ternary complex gene transfer system can combine the gene transfer system that is applied to gene therapy with recombinant virus.
22. gene transfer system as claimed in claim 21 is characterized in that described recombinant virus comprises recombinant retrovirus, recombinant adenovirus.
23. gene transfer system as claimed in claim 22 is characterized in that the genetic material that described recombinant virus wraps up can comprise following foreign gene: suicide gene HSV-TK, CD, apoptosis gene p15, p16, p21
WAF-1Antioncogene p53, Rb, cytokine gene comprises GM-CSF (CM-CSF) gene, TNF (tumour necrosis factor) α gene, INF (Interferon, rabbit) α, γ gene, IL (interleukin-) 2,3,4,12,15 genes or do not comprise the defective virus of any foreign gene.
24., it is characterized in that described part oligopeptides can be the polypeptide with hematopoietic cell, scavenger cell, lymphocyte, liver cell, nephrocyte, endotheliocyte, neurocyte and myocardial cell's receptors bind as claim 1,2,21 described gene transfer systems.
25., it is characterized in that to use two or more different part oligopeptides and polycation polypeptide, the formed complex carrier of HA20 and form complex body with a kind of foreign gene DNA or virus as claim 1,3,5 described gene transfer systems; Or use with a kind of part oligopeptides/polycation polypeptide/HA20 oligopeptides and two kinds and two or more different foreign gene DNA or contain the virus formation complex body of different foreign genes.
26. gene transfer system as claimed in claim 1, it is characterized in that wherein said part oligopeptides/polycation polypeptide/endocytosis corpusculum discharges oligopeptides ternary complex carrier and can use the nucleotide sequence of coding said polypeptide and set up into recombinant dna plasmid, obtain by gene engineering expression.
27., it is characterized in that they are the gene transfer systems that foreign DNA gene target ground imported the inside and outside tumour cell that are applied to make up therapy of tumor as claim 1,2 described gene transfer systems.
28., it is characterized in that this quaternary gene transfer system is the gene transfer system that is applied to make up therapy of tumor as claim 21 and 22 described gene transfer systems.
29. gene transfer system as claimed in claim 24 is characterized in that described part oligopeptides is applied to make up exogenous gene high-efficient, the tumour of the various cells of target ground importing and the complex polypeptide carrier of non-neoplastic disease gene therapy.
30. gene transfer system as claimed in claim 25 is characterized in that described complex body is applied to make up the gene transfer system of therapy of tumor.
31. gene transfer system as claimed in claim 26 is characterized in that described ternary complex carrier can be applicable to the biological products of genetically engineered producer gene treatment.
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| CN102010461B (en) * | 2010-10-11 | 2014-02-12 | 华南理工大学 | A kind of alpha helical cationic polypeptide molecule and its preparation method and application |
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