CN1927402A - Construction and preparation of tumor target gene transmission non-viral vector - Google Patents

Abstract

The invention relates to a non-virus carrier used to target guide the external gene into special part of cancer, and relative production. Wherein, said carrier is characterized in that: it has several cancer local target properties, nucleic acid combine properties and liposome recombine properties; it builds the anchor expression carrier of eucaryon cell, to anchor and express the target peptide, anode peptide on the surface of cancer cell, to be decomposed and extract cell membrane, to obtain the non-virus gene target transfer carrier with nucleic acid combine properties and liposome recombine properties. The invention is characterized in that: said carrier can use the cancer new blood vessel and several cancer cell target ligands to target the external gene to special part of cancer, and can use penetrate peptide, and phagocytose release agent to make said carrier increase the transmission of external gene and avoid degradation.

Description

The structure of tumor target gene transmission non-viral vector and preparation

Technical field

The invention belongs to the molecular biology therapy of tumor and use the carrier field, relate to a kind of structure and preparation that supplies the non-virus carrier of exogenous gene targeting importing tumor by local.

Background technology

Genophore is the transmission system of using in gene therapy that exogenous gene is imported body cell.Allogenic gene enters target cell 3 kinds of obstacles: 1. cell membrane; 2. intracytoplasmic phagocytic vacuole-lysosome; 3. nuclear membrane.For the structure by this several roads barrier genophore generally comprises carrier framework, targeting part, phagocytic vacuole releasing agent and nuclear localization signal peptide.At first carrier framework is used for carrying and the protection dna molecular; Secondly the targeting part can combine with the cell-membrane receptor specificity, strengthen endocytosis, form phagocytic vacuole, the pH value of the phagocytic vacuole formation lysosome that descends gradually, exogenous gene will be degraded in lysosome, the amphiphatic molecule peptide of viral source (amphipathic peptides) is a kind of phagocytic vacuole releasing agent at present commonly used, plays to help gene composite to escape phagocytic vacuole to enter endochylema, avoid the effect of lysosome degraded; Last nuclear localization signal peptide can help genes of interest to change nucleus over to from endochylema, finishes whole gene importing process.If the cancerous cell targeting, exogenous gene can utilize frequent cell division to enter chromosome, and it is just inessential to appraise and decide the position so.

The genophore of research can be divided into two big classes at present: viral vector and non-virus carrier.Viral vector is by the natural infection ability of viral pair cell exogenous origin gene integrator to be advanced host cell, has higher transfection efficiency, obtains easily the long time of exogenous gene is expressed.Its shortcoming is to produce inconvenience, and toxicity especially immunogenicity is big, should not use repeatedly and restricted etc. to the pulsating size of entrained exogenous gene.Though non-virus carrier is being not so good as viral vector aspect transgene efficiency and the expression of acquisition long time, but there are not above-mentioned these shortcomings, through novel non-virus carrier that modification to transform in safety, effectively, aspect such as targeting all has breakthrough, and therefore becomes the research focus of gene therapy vector.The non-virus carrier system that this invention relates to has possessed above several features simultaneously, in the hope of accomplishing safety, effective, targeting several respects.

The targeting problem of gene therapy directly influences its safety and effectiveness, and now normally carrier to be carried out targeting ligand modified for the method that solves.

Tumor vessel is keeping a kind of continuous new life's state, and there is the molecular marker relevant with hypertrophy on its surface, and whole tumor body is exactly a cambium, and he relies on neovascularity and supplies nutrient, keeps metabolism for it.(v) beta3, alpha v beta5 cross expression [ErkkiRuoslahti at the vascular system endotheliocyte of tumor to integrate plain alpha, Drug targeting to specific vascular sites, DDT Vol.7, No.22 November2002], relevant with the effect of tumor locus angiogenesis promoting, at some tumor cell surface also high expressed, domestic and international many researcheres are studied it for a long time as the target of tumor.The RGD peptide that finds by the display technique of bacteriophage people be α v integrin in conjunction with the territory, with the peptide that contains RGD as ligand modified carrier, gene target α v can be integrated plain position [the Erkki Ruoslahti that expresses, Targeting tumorvasculature with homing peptides from phage display, seminars in CANCERBIOLOGY, Vol.10,2000:pp.435-442].For example expressing on the Mewo cell of integrating element, the PEI carrier of modifying with the RGDC tetrapeptide improves 1-2 the order of magnitude [Klaus Kunath than the PEI vector gene transfection efficiency that does not have to modify, Thomas Merdanl, Oliver Hegener, Hanns H aberlein, Thomas Kissel, Integrin targeting using RGD-PEI conjugates in vitro genetransfer, J Gene Med 2003; 5:588-599].And for example the liposome of modifying with the RGD peptide improves doubly [Scott ES of 10-200 in the gene expression rate of people's bronchial epithelial cell strain (16HBE), Wiseman JW, Evans MJ, Colledge WH.Enhanced gene delivery to human airway epithelial cellsusing an integrin-targeting lipoplex.J Gene Med 2001 Mar-Apr; 3 (2): 125-34].Discover in addition, lipid-the protamine of PEGization-DNA complex (LPD-PEG) makes the gene transfection rate of LPD descend, estimation is because the PEG chain has hindered due to the contacting of particle and cell surface, but after modifying LPD-PEG with RGD-4C peptide (CDCRGDCFCG), combination rate to the tumor cell of expressing integrin receptor has raised 5-15 doubly, transfection efficiency improves 100 times at the MDA-MB-231 cell, does not see then that for integrin alpha v beta 3 and all low Huh7 cell strain of expressing of α v β 5 transfection strengthens.And transfection strengthens and can be blocked [Harvie P by free RGD peptide, Dutzar B, Galbraith T, Cudmore S, O ' Mahony D, Anklesaria P, Paul R.Targeting of lipid-protamine-DNA (LPD) lipopolyplexes using RGD motifs.J Liposome Res.2003Nov; 13 (3-4): 231-47].Tumor mouse also shows in the body experiment: synthetic peptide CDCRGDCFC can suppress by the cell adhesion of integrating plain alpha v beta 5 and alpha v beta3 mediation, suppressing effect has only the similar peptide of a sulfide framing structure strong at least 20 times than it, linear fully RGD peptide suppresses strong nearly 200 times of effect, should synthesize effect [the Koivunen E that peptide has target tumor new vessels and tumor, Wang B, Ruoslahti E Phage libraries displaying cyclic peptides with different ring sizes:ligand specificities of the RGD-directed integrins.Biotechnology (N Y) .1995Mar; 13 (3): 265-70].

In numerous angiogenesis factors, VEGF (VEGF) has played crucial effects [Napoleone Ferrara.Vascular endothelial growth facor.Trends Cardiovasc Med 1993 in neonate tumour blood vessel; 3:244-50], VEGF is mainly by tumor cells expression, relevant [Erika Hatva with angiogenesis and neoplasm metastasis, Arta Kaipainen, Panu Mentula et al.Expression of endothelial cell specific receptor tyrosine kinases and growthfactors in human brain tumors, American Journal of Pathology.1995; 146 (2): 368-78], there are some researches show, in many solid tumors, follow the rise of tumor cell vegf expression, also corresponding rise [the Kunio Suzuki of the expression of vegf receptor in the tumor vascular endothelial cell, NorioHayashi, Yasuhide Miyamoto et al.Expression of vascular permeabilityfactor/vascular endothelial growth factor in human hepatocellular carcinoma, Cancer Res 1996; 56:3004-9], vegf receptor has an expression [Christine A Boocock in the cancerous cell that some has spread, D Stepjen Charnock-Jones, Andrew M Sharkey et al.Expression of vascular endothelial growth factor and its receptors flt and KDR inovarian carcinoma.J Nat Cancer Inst 1995; 87:506-16.].And in normal structure, angiogenesis remains static, vegf receptor is not expressed or utmost point low expression level [Adrian L Harris, Huatang zhang, Amir Moghaddam et al.Breast cancer angiogenesis-newapproaches to therapy via antigiogenesis, hypoxic activated drugs and vasculartargeting, Breast Cancer Reasearch and Treament.1996; 38:97-108.], so people can be delivered to the tumor cell of tumor vascular endothelial cell or some express VEGF receptors with the therapeutic gene targeting, and not influence Normocellular growth with vegf receptor as the tumor target spot.By the Shanghai tumor national oncogene and related gene research department people such as research worker Li Yunmin according to the VEGF-VEGF receptor of generally acknowledging in conjunction with the territory, computer homology mould is built and has been designed and synthesized two oligopeptide GV1 and GV2 in addition.

GV1(CHPIETLVDIFQEYPDEIEYIFKPSPVPLMRP)

GV2(PVPTEESNITMQIMRIKPHQGQHIGEMSFLQHNKCE)

Wish and gene target can be delivered to tumor vascular endothelial cell.These can combine on the cation skeleton of DNA by electrostatic adsorption in the experiment GV1 or GV2 and phagocytic vacuole releasing agent HA20 to be connected to poly-D-lysine or protamine, with pSV2-galactosidase (tilactase) as reporter gene, in vitro tests confirms behind endotheliocyte (ABAE) that the transfection of external source reporter gene is originated to the bovine aortic bow and the people's malignant melanoma cell (A375) expression to be arranged, in vivo test shows, exogenous gene has expression at the tumor cell of the nude mice of tumor vascular endothelial cell and subcutaneous transplantation tumor cell, and graft comprises human colon cancer cell (LOVO), people's malignant melanoma cell (A375) and human liver cancer cell.Part small peptide GV1, GV2 that the targeting vegf receptor is described successfully also express exogenous gene complex targeted delivery to tumor vascular endothelial cell and multiple solid tumor cell.

NG2 is adjustable film chondroitin sulfate glycoprotein of striding, and is the homologous melanoma glycoprotein of Mus people, is also referred to as the high molecular melanic related antigen.Human melanoma glycoprotein h MP (humanmelanoma proteoglycan) is homology with it.NG2 is expressed in the perithelial cells of new vessels widely, belongs to the vascular stroma part, comprises the new vessels in the normal development tissue, new vessels substrate in coarse wound in the reparation and the tumor stroma, and immobilized blood vessel is not then found.At adults, NG2 only is expressed in to limitation tumor cell and newborn tumor vessel substrate position, and NG2/HMP is expressed in multiple different tumor widely, comprises on colloid blastoma, chondrosarcoma, melanoma and some the leukemic cell.People such as Michael A found two small peptide molecules that targeting NG2 effect is arranged in 1999 by display technique of bacteriophage, TAASGVRSMH (TAA) and LTLRWVGLMS (LTL), two peptides are the male tumor vessel substrate of energy targeting xenotransplantation melanoma mice NG2 position [Michael A.Burg all, Renata Pasqualini, Wadih Arap, Erkki Ruoslahti, and William B.Stallcup, NG2 Proteoglycan-binding Peptides Target Tumor Neovasculature, CANCERRESEARCH 59,2869-2874, June 15,1999].It is presently believed that perithelial cells is to take place and stablize capillary wall to influence the blood vessel generation by hypertrophy, the blood capillary of regulating endotheliocyte.We think that NG2 not only is expressed in kinds of tumor cells but also be expressed in the tumor vessel perithelial cells, and when only becoming big in the endothelial cells in tumor neogenetic blood vessels gap, perithelial cells is just exposed, therefore the part small peptide of NG2 target tumor cell and tumor neogenetic blood vessels substrate simultaneously, and targeting is more special than the part of targeting new vessels endothelium.

EGFR is one of the member with receptor superfamily of endogenous Lip river propylhomoserin kinase activity, and it is distributed in many cell types widely, comprises the cell in an epidermis and a leaf source, crosses expression at some tumor cell.The activation of EGFR can increase ability of cell proliferation, and motor capacity.HA20 (GLFEAIAEFIEGGWEGLEG) be one section with influenza virus capsid protein hemagglutinin district N-hold homologous both sexes peptide, be synthesized as phagocytic vacuole and discharge oligopeptide endosome-releasing oligopeptide (EROP), it is to targeting and to wear membrane process nonsensical, its role is to the allogenic material that enters cell by the formation phagosome is discharged in time, the lysosome of avoiding being formed is gradually degraded, play effect [the Masayuki Murata of protection exogenous gene, Satoshi Kagiwada, Sho Takahashi et al.Membrane Fusion induced by mutual interaction of the two charge-reversedamphiphilic peptides at neutral pH.J Biol Chem 1991; 266 (22): 14353-8.].People such as Liu Xiang by computer software design be targeted to the EGF of EGFR in conjunction with territory small peptide GE7 (NPVVGYIGERPQYRDL), modify poly-D-lysine by chemical method respectively with two peptide GE7 and HA20, poly-D-lysine with modified mixes with a certain amount of DNA as skeleton then, form complex GE7-PLL/DNA/HA20-PLL by electrostatic adsorption, DNA is plasmid p21WAF-1/pGM-CSF, set up the hepatoma transplantation model of Mus, complex is weekly in all drug administration by injection of tumor, continue one month, effective [the Xiang Liu of experiment confirm three part Combined Ration two parts, Jianren Gu, Enhanced antitumor effect of EGF R-targeted p21WAF-1 andGM-CSF gene transfer in the established murine hepatoma by peritumoralinjection, Cancer Gene Therapy (2002) 9,100-108].

Wear film peptide (cell-penetrating peptides) and be that Recent study finds that some are rich in alkaline amino acid residue and participate in nuclear translocation and the small peptide of cell adhesion, can pass eukaryotic plasma membrane by a kind of nothing power consumption, no receptor-mediated mode.The small peptide that has kind more than ten to derive from Tat demonstrates the function that can pass the different cytoplasm film, and the internalization process only finished in several minutes, when temperature is reduced to 4 ℃, this process does not have significant change, and [Wang Li wears film peptide research prospect, Journal of Immunology, the 17th volume, the 3rd June calendar year 2001 phase].Below be several confirmed film peptide sequences of wearing:

Tat?fragment(48-60)：GRKKRRQRRRPPQ

Tat fragment (49-57): RKKRRQRRR (it is disconnected that tool is worn the short-movie of film effect in the Tat peptide)

Signal-sequence-based

peptides?I：GALFLGWLGAAGSTMGAWSQPKKKRKV

Signal-sequence-based?peptides?II：AAVALLPAVLLALLAP

Derive from the protamine short type of protamine, be one section and contain 28 amino acid whose little peptides, it is rich in positive charge, can combine [Li S, et al Characterization ofcationic lipid-protamine-DNA (LPD) complexes for intravenous gene deliveryGene Ther. 1998 with the plasmid DNA high-affinity; 5 (7): 930-7.], therefore can be used as the carrier framework composition of gene.Glycosylation phosphatidylinositols (GPI), it is a kind of fat structure of protein being carried out post translational modification, protein can be anchored to surface of cell membrane by the GPI structure, and do not cross over its immobilized artificial membrane double-decker [Cimino AM, et alCancer vaccine development:protein transfer of membrane-anchored cytokinesand immunostimulatory molecules.Immunol Res.2004; 29 (1-3): 231-40].We imagine the gene of the compound targeted molecular of cancer target and the gene fusion of protamine short type, by the GPI structure fusion gene anchor ingot is expressed on the cell membrane then, smudge cells, separate and obtain cell membrane, because cell membrane itself contains fat materials such as abundant phospholipid and cholesterol, under certain buffer condition, can self assembly form lipid granule [Westerman LE, Jensen PE.Liposomes composed ofreconstituted membranes for induction of tumor-specific immunity.MethodsEnzymol.2003; 373:118-27], thus the lipid granule in this cell membrane source have simultaneously tumor cell internal target tropism, with plasmid DNA Electrostatic Absorption and lipid self regroup, be to transmit carrier in a kind of non-viral gene cell with tumor-targeting.

Summary of the invention

The objective of the invention is to design and synthesize a kind of novel non-viral gene vector with tumor-targeting, aspect tumor-targeting, the present invention proposes the short peptide sequence that has the tumor by local targeting with a plurality of, and the gene order of wearing film peptide and phagocytic vacuole releasing agent is fused to together, form a compound targeted molecular, flexibly connect arm by each small peptide of computer-aided design, make composition target each small peptide conformation in fusogenic peptide constant, thereby guaranteed the function of each small peptide in the compound targeted molecular.Hope is transmitted in target cell by increasing exogenous gene in the scope of application that increases cancer target, avoids its degraded, improves the effect that exogenous gene transmits in the tumor by local cell.

The said a plurality of short peptide sequences of the present invention with tumor by local targeting, comprise all be targeted to tumor endothelial and tumor cell surface some integrate the plain part small peptide that contains RGD, be targeted to tumor cell and new vessels endothelial cell VEGF receptor the part small peptide, be targeted to new vessels perithelial cells surface NG2 small peptide and be targeted to the part small peptide of kinds of tumor cells EGF receptor.

The present invention is said to wear the short peptide sequence that the film peptide comprises all the suitable native systems with membrane penetration effect.

The said phagocytic vacuole releasing agent of the present invention comprises the short peptide sequence of all the suitable native systems with phagocytic vacuole release action.

The present invention is with the gene of above-mentioned compound targeted molecular and the gene fusion of protamine short type, by the GPI structure fusion gene anchor ingot is expressed on the eukaryotic cell membrane then, obtain cell membrane by certain technology, this film is assembled into lipid granule voluntarily under certain condition.The lipid granule in this cell membrane source have simultaneously tumor cell internal target tropism, with plasmid DNA Electrostatic Absorption and lipid self regroup, be to transmit carrier in a kind of non-viral gene cell with tumor-targeting.

The said protamine short type of the present invention gene comprise all positively chargeds of deriving out by the protamine molecule can with the bonded short peptide sequence of nucleic acid static.

The said certain technology separation and Extraction cell membrane of the present invention comprises the method for the laboratory separation and Extraction cell membrane that all are suitable for.

The present invention combines together targeting, electropositive and lipids form are ingenious as the carrier of gene transmission, has the function of good gene delivery vector.Wherein targeting integrates target tumor new vessels and kinds of tumor cells again, makes this carrier have the exogenous gene of increasing to transport the effect of avoiding exogenous gene to be degraded in tumor cell by wearing film peptide, phagocytic vacuole releasing agent in lysosome simultaneously.Simultaneously, the present invention on technology ingenious avoided conventional biotech drug the inevitable technology of complex and expensive such as protein purification, help carrying out industrialization development and application.

Description of drawings

Fig. 1 is the stereochemical structure of independent L1

Fig. 2 is the stereochemical structure of independent L2

Fig. 3 is the stereochemical structure of independent L3

Fig. 4 is the stereochemical structure of independent L4

Fig. 5 is the stereochemical structure of independent P

Fig. 6 is the stereochemical structure of independent R

Fig. 7 is the stereochemical structure of simulation fusion molecule

Fig. 8 is the synthetic evaluation of P6

1.P1 2.P2 3.P3 4.P4

5.P5 6.P6 7.DL2000

Fig. 9 identifies for the pCI-P6-pr+-GPI double digestion

1.DL2000 2.pCI-P6-pr+-GPI double digestion 3.pCI-P6-pr+-GPI

The specific embodiment

The structure of the tumor target gene transmission non-viral vector that the present invention proposes is as follows with the preparation embodiment:

At first, by the flexible small peptide linking arm of computer-aided design, with a plurality of short peptide sequences with tumor by local targeting, and the gene order of wearing film peptide and phagocytic vacuole releasing agent is fused to together, form a compound targeted molecular, and guarantee that each small peptide is consistent with its native conformation in the conformation of composition target in fusogenic peptide.

Then, with the gene of above-mentioned compound targeted molecular and the gene fusion of protamine short type, utilize anchor ingot Protein G PI that fusion gene anchor ingot is expressed on the eukaryotic cell membrane, obtain cell membrane by certain technology, this film is assembled into lipid granule voluntarily under certain condition.

The more detailed implementation method of the present invention can be referring to embodiment, and present embodiment is to be used for explaining rather than limiting by any way the present invention.

Embodiment one, each part small peptide linking arm of computer-aided design

By computer-aided design small peptide linking arm, the conformation of each part small peptide is remained unchanged in fusogenic peptide, thereby guarantee its function.

Gene (the R)-EcoRV-3 ' of the gene (P) of the part small peptide gene (L4) of the part small peptide gene (L3) of part small peptide gene (the L2)-targeting NG2 of part small peptide gene (the L1)-targeting vegf receptor of compound targeted molecular P6:5 '-XhoI-targeted integration element-targeting EGF receptor-cell penetrating peptide Tat (48-60)-phagocytic vacuole releasing agent (HA20)

L1：CDCRGDCFC

L2：PVPTEESNITMQIMRIKPHQGQHIGEMSFLQHNKCE

L3：TAASGVRSMH

L4：NPVVGYIGERPQYRDL

P：GRKKRRQRRRPPQ

R：GLFEAIAEFIEGGWEGLEG

L1---GGGGS---L2---GGGSAAA---L3---GSPT---L4---GGGSA---P---GGGSGA---R

As can be seen from Figure 7:, can reach and make six small peptides keep the purpose of its original conformation by these five linking arms that computer-aided design is come out.The space conformation of fusion molecule is unfolded, and the stereochemical structure of six small peptides is fully represented.

Embodiment 2 complete synthesis compound targeted molecular P6

2.1 peptide sequence is translated into gene order with mammal preference codon

Code?and?Peptide

C D C R G D C F C G G G G S P V P T E E S

TGC?GAC?TGT?CGC?GGC?GAC?TGT?TTC?TGC?GGC?GGA?GGC?GGG?AGC?CCT?GTG?CCC?ACC?GAG?GAA?TCC

N I T M Q I M R I K P H Q G Q H I G E M S

AAC?ATC?ACC?ATG?CAG?ATC?ATG?AGG?ATC?AAG?CCC?CAC?CAG?GGC?CAG?CAC?ATT?GGC?GAG?ATG?AGC

F L Q H N K C E G G G S A A A T A A S G V

TTC?CTG?CAG?CAC?AAC?AAG?TGC?GAG?GGC?GGG?GGA?TCC?GCT?GCC?GCA?ACA?GCC?GCT?AGC?GGC?GTG

R S M H G S P T N P V V G Y I G E R P Q Y

CGC?TCC?ATG?CAC?GGC?AGC?CCA?ACA?AAC?CCC?GTC?GTG?GGC?TAC?ATC?GGC?GAG?CGG?CCC?CAG?TAC

R D L G G G S A G R K K R R Q R R R P P Q

AGG?GAC?CTG?GGG?GGC?GGA?TCC?GCT?GGC?CGC?AAG?AAA?CGC?AGG?CAG?AGG?CGG?CGC?CCA?CCT?CAG

G G G S G A G L F E A I A E F I E G G W E

GGA?GGC?GGC?AGC?GGC?GCT?GGC?CTG?TTC?GAG?GCC?ATC?GCC?GAG?TTC?ATT?GAG?GGA?GGG?TGG?GAG

G L E G

GGC?CTG?GAG?GGC

The gene order of P6:

TGC?GAC?TGT?CGC?GGC?GAC?TGT?TTC?TGC?GGC?GGA?GGC?GGG?AGC?CCT?GTG?CCC

ACC?GAG?GAA?TCC?AAC?ATC?ACC?ATG?CAG?ATC?ATG?AGG?ATC?AAG?CCC?CAC?CAG

GGC?CAG?CAC?ATT?GGC?GAG?ATG?AGC?TTC?CTG?CAG?CAC?AAC?AAG?TGC?GAG?GGC

GGG?GGA?TCC?GCT?GCC?GCA?ACA?GCC?GCT?AGC?GGC?GTG?CGC?TCC?ATG?CAC?GGC

AGC?CCA?ACA?AAC?CCC?GTC?GTG?GGC?TAC?ATC?GGC?GAG?CGG?CCC?CAG?TAC?AGG

GAC?CTG?GGG?GGC?GGA?TCC?GCT?GGC?CGC?AAG?AAA?CGC?AGG?CAG?AGG?CGG

CGC?CCA?CCT?CAG?GGA?GGC?GGC?AGC?GGC?GCT?GGC?CTG?TTC?GAG?GCC?ATC?GCC

GAG?TTC?ATT?GAG?GGA?GGG?TGG?GAG?GGC?CTG?GAG?GGC

2.2, please Bo Ya company synthesize with 12 primers of center die way design.

(1)GGAGGTTCTGCTGCCGCAACAGCAGCTTCTGGAGTCAGGAGTATGCACG

(2)CCAATATATCCGACAACGGGGTTGGTAGGGCTCCCGTGCATACTCCTGA

(3)GATGTCATTCCTGCAGCACAACAAGTGCGAGGGAGGAGGTTCTGCTGCC

(4)TCCGCCACCGAGGTCCCTGTACTGCGGCCTCTCTCCAATATATCCGACA

(5)GCATCAAGCCCCATCAAGGACAGCACATTGGCGAGATGTCATTCCTGCA

(6)GTCTCCTTTGCCTTCTTTTCTTACGCCCGGCAGATCCGCCACCGAGGTC

(7)ACCGAAGAGTCAAACATCACCATGCAGATCATGCGCATCAAGCCCCATC

(8)AGTCCAGCACCACTACCTCCGCCTTGAGGTGGTCGTCTCCTTTGCCTTC

(9)TTGCTTCTGTGGTGGCGGTGGGTCTCCAGTGCCTACCGAAGAGTCAAAC

(10)GCCTCCCTCAATAAACTCGGCAATGGCTTCGAACAGTCCAGCACCACTA

(11)GCCTCGAGTGCGACTGTAGAGGAGATTGCTTCTGTGGTGG

(12)GCGATATCGCCCTCCAACCCTTCCCAGCCTCCCTCAATAA

Obtain the P6 gene 2.3 take turns overlapping extension PCR, be cloned into the order-checking of pMD 18-T carrier correctly by six.

2.3.1 synthetic P6 gene

More than GeneAmp PCR System 2400 type PCR, carry out following reaction:

First round PCR synthesizes P1:

Reaction system 50 μ l:10 * PCR buffer (without MgCL2) 5 μ l, MgCL2 (25mmol/L) 5 μ l, dNTPs (10mmol/L) 4 μ l, primer (1) (20 μ mol/L) 1 μ l, primer (2) (20 μ mol/L) 1 μ l, Taq DNA Polymerase (2.5U/ μ l) 1 μ l, sterile purified water 33 μ l (Taq archaeal dna polymerase, Buffer, dNTPs all available from Tacara Bioisystech Co., Ltd).

Reaction condition: 95 ℃ 1 minute (95 ℃ 1 minute, 56 ℃ 1 minute, 72 ℃ 30 seconds) circulation 5 times, 72 ℃ 5 minutes, 4 ℃ of ∞

Second takes turns the synthetic P2 of PCR:

Reaction system 50 μ l:10 * PCR buffer (without MgCL2) 5 μ l, MgCL2 (25mmol/L) 5 μ l, dNTPs (10mmol/L) 4 μ l, primer (3) (20 μ mol/L) 1 μ l, primer (4) (20 μ mol/L) 1 μ l, Taq DNA Polymerase (2.5U/ μ l) 1 μ l, template (10 times of P1 dilutions) 1 μ l, sterile purified water 32 μ l

Reaction condition: 95 ℃ 1 minute (95 ℃ 1 minute, 56 ℃ 1 minute, 72 ℃ 1 minute) circulation 32 times, 72 ℃ 10 minutes, 4 ℃ of ∞

Third round to the six is taken turns PCR reaction synthetic respectively P3, P4, P5, P6:

Reaction system: except primer is used instead respectively: (5,6) (7,8) (9,10) (11,12) in addition, other with second take turns identical.

Reaction condition: with second take turns identical.

Product is identified: description of drawings is seen Fig. 8

2.3.2 purification P6

Get 5 μ l P6 and run 1.2% agarose gel electrophoresis and make molecular weight identification, will all remain P6 afterwards and run 1.2% preparative agarose gel electrophoresis, cut glue, reclaim test kit, reclaim P6 with the PCR product glue of vast Imtech.Step: uviol lamp downcuts the P6 bright band down, put into the 1.5ml centrifuge tube, add sol solutions 700 μ l/100 μ g glue, the centrifugal 30s of 12000rpm, repeated centrifugation once, add 500 μ l washing buffer, the centrifugal 30s of 12000rpm repeats empty once centrifugal, add 35 μ l sterile purified waters, placed 5 minutes for 37 ℃, 12000rpm is centrifugal, both the P6 solution behind the purification.

Connect pMD 18-T carrier 2.3.3 insert

Get sterilization PCR pipe and add Ligation Solution I 5 μ l, sterilization distilled water 3 μ l, the P61 μ l behind the purification, pMD 18-T carrier 1 μ l is put 16 ℃ and is spent the night.

2.3.4 transform

Asepticly get 100 μ l competent cells (DH5 α bacterial strain is by " molecular cloning "-Science Press, the CaCl2 method preparation of second edition), in ice bath, add and connect product 10 μ l, rotate gently, in ice bath, placed 30 minutes with the mixing content, transfer to immediately in 42 ℃ of water-baths and left standstill 2 minutes, every then pipe adds the not LB culture medium of added with antibiotic of 0.5ml, and 37 ℃ of water-baths are after 15 minutes, 37 ℃ of shaking table jogs 45 minutes; Get 200 μ l and transform thalline, evenly coat on the Agar Plating that contains 100mg/ml ammonia benzyl, dry up about 10 minutes, be inverted overnight incubation for 37 ℃ at superclean bench.Picking colony is about 10 from flat board, is inoculated in respectively to contain in the 100mg/ml ammonia benzyl LB culture medium (5ml/ pipe) 37 ℃ of shaking table overnight incubation.

2.3.5 bacterium colony PCR Preliminary Identification P6

Get 10 times of diluents of 0.5 μ l incubated overnight bacterium and carry out Preliminary Identification with the PCR method, primer is selected P6 two terminal sequences 1.1 and 1.2 for use.Carry out pcr amplification with the PCR instrument, amplification system is: 10 * PCR Buffer (without MgCL2), 2.5 μ l, MgCL2 (25mmol/L) 2.5 μ l, 1.1, each 0.5 μ l of 1.2 (being 20 μ mol/L), dNTPs (2.5mM) 2 μ l, Taq archaeal dna polymerase (2.5U/ μ l) 0.5 μ l adds water to 25 μ l.(Taq archaeal dna polymerase, Buffer, dNTPs all available from Tacara Bioisystech Co., Ltd).Amplification condition: 95 ℃ 1 minute (95 ℃ 1 minute, 56 ℃ 1 minute, 72 ℃ 1 minute) circulation 30 times, 72 ℃ 10 minutes, 4 ℃ of ∞

2.3.6 choose two bacterium order-checkings

No. 2, picking and No. 6 bacterium go the order-checking of three rich companies, and both all contain the gene of correct compound targeted molecular as a result.

2.3.7 extract the correct plasmid pMD 18-T-P6 of order-checking with alkaline denaturation

Description operation according to vast Imtech plasmid rapid extraction test kit: (1) collects bacterium liquid in 1.5ml sterilization Eppendorf pipe, and 12000rpm is centrifugal, removes supernatant; (2) add 150 μ l solution I, vibration is to thoroughly suspending; (3) add 150 μ l solution II, softly put upside down immediately for several times, make cellular lysate, room temperature was placed 1-2 minute; (4) add 300 μ l solution III, softly put upside down immediately for several times, room temperature was placed 5 minutes, centrifugal 12 minutes of 12000rpm; (5) in centrifugal adsorption column, add 400 μ l binding buffer liquid in advance, add then and go up centrifugal supernatant of step, mixing, centrifugal 30 seconds of 12000rpm, waste liquid discards; (6) add the centrifugal rinsing liquid of 750 μ l in centrifugal adsorption column, centrifugal 30 seconds of 12000rpm, waste liquid discards; (7) repeat 6 steps an of order, empty then centrifugal 2 minutes, thoroughly remove rinsing liquid; (8) carefully take out centrifugal adsorption column, put it in the 1.5ml sterilization Eppendorf pipe of getting ready, add 35 μ l sterile purified waters, placed 2-5 minute for 37 ℃, centrifugal 30 seconds of 12000rpm takes down adsorption column, and the plasmid solution that obtains is in-20 ℃ of preservations.

Embodiment 3 makes up tumor-targeting eukaryotic cell anchor ingot expression vector pCI-P6-pr+-GPI

I have made up the pCI-pr+-GPI universal support in the chamber on pCI-neo carrier for expression of eukaryon basis, be between former multiple clone site NheI of eukaryon expression plasmid pCI-neo and XhoI, to insert signal peptide (sig) sequence, between EcoRV and EcoRI, insert the gene order of 5 '-GGGSSGGG-protamine short type-3 ', between EcoRI and XbaI, insert 5 '-IgGFc-GPI-3 ' anchor ingot albumen and related gene sequence.Therefore, as long as between XhoI and EcoRV, insert the gene order of compound targeted molecular P6, just can obtain tumor-targeting eukaryotic cell anchor ingot expression vector pCI-P6-pr+-GPI.

Double digestion: above-mentioned with alkaline denaturation plasmid pMD 18-T-P6 that extracts and the pCI-pr+-GPI universal support plasmid that my chamber makes up, use XhoI and EcoRV double digestion respectively.The enzyme action system: 10 * NEBbuffer3 of 5 μ l, the XhoI 2 μ l of 10u/ μ l, the EcoRV 2 μ l of 10u/ μ l, 100 * BSA0.5 μ l, plasmid 30 μ l add water to cumulative volume 50 μ l, 37 ℃ of water-baths 8 hours.The purpose fragment is reclaimed in the rubber tapping of enzyme action afterproduct, and concrete grammar is the same referring to one of 2.3.2 alcoholization P6.

Insert and connect: get plasmid pCI-pr+-GPI fragment and each 1 μ l of P6 genetic fragment after sterilization PCR pipe adds the double digestion purification, 10 * T4DNA connects buffer 1 μ l, and T4DNA ligase (12u/ μ l) 1 μ l adds sterilization distilled water to 10 μ l, and 16 ℃ are spent the night.

Transform: concrete steps are the same to be transformed referring to 2.3.4

Identify: concrete steps are the same referring to 2.3.5 bacterium colony PCR, and 2.3.6

Double digestion is identified: the plasmid pCI-P6-pr+-GPI with above-mentioned alkaline denaturation extracts, use XhoI and EcoRV double digestion.Enzyme action system: 10 * NEBbuffer3 of 2 μ l, the XhoI 1 μ l of 20u/ μ I, the EcoRV 1 μ l of 20u/ μ l, 10 * BSA, 2 μ I, plasmid 14 μ l, cumulative volume 20 μ l, 37 ℃ of water-baths 6 hours.Product is got 15 μ l and is carried out the agarose gel electrophoresis evaluation, the results are shown in shown in Figure 9.

Gene sequencing is identified: the gene sequencing proof has correct P6 gene to insert in the pCI-pr+-GPI universal support, so far, has obtained correct pCI-P6-pr+-GPI.Plasmid extraction kit Wizard  PureFection Plasmid DNA Purification System with promega company extracts transfection level plasmid pCI-P6-pr+-GPI.

Embodiment 4 obtains the tumor cell line of stably express targeting compound molecule

The Renca cell is the renal carcinoma cell line in Balb/c mice source.With above-mentioned carrier pCI-P6-pr+-GPI transfection tumor cell, screening obtains the tumor cell line of stably express targeting compound molecule.In Renca cell transfecting forward pass generation, be inoculated on six orifice plates, makes the next day cell can be paved with ground, bottom surface, hole 80%, and the transfection step is undertaken by the Lipofectamine2000Reagent description of Invitrogen company.Get 1 μ g plasmid respectively, be diluted to 250 μ l, be A liquid with unparalleled anti-DMEM culture medium; Other gets 7 μ l Lipofectamine2000 and is diluted to 250 μ l with unparalleled anti-DMEM culture medium, is B liquid, B liquid chamber temperature is placed 5 minutes after, with A liquid B liquid mixing gently, be C liquid immediately, room temperature was placed 20 minutes.After taking out six orifice plates and washing once with fresh culture, every hole adds the 2.5ml fresh culture.C liquid is joined respectively in each hole, and mixing is put the CO2 incubator and is cultivated.After the transfection 48 hours, when cell density reaches 50-70% and merges, abandon culture fluid, use instead and contain G-418 (400 μ g/ml) culture medium and carry out resistance screening, the cell with untransfected compares simultaneously.When control cells was almost all dead, the concentration of G-418 was reduced to 200 μ g/ml to keep the screening effect.When treating positive cell clone to occur about 2wk, amplification culture is carried out subsequent experimental.

Collect the Renca cell of pCI-P6-pr+-GPI and empty carrier pCI-neo transfection, PBS (containing the 20ml/L calf serum) with the 4oC pre-cooling washs 3 times, add fluorescently-labeled mouse-anti human IgG (dilution in 1: 1000), 45min is hatched in concussion in ice bath, and reuse PBS washes 3 times.Add the 5g/L paraformaldehyde fixing after, observe down and take a picture in fluorescence microscope, the result proves that transfection success P6-pr+p is illustrated on the host cell membrane well by GPI.

The extraction of embodiment 5 cell membrane components and the particulate formation of composite membrane fat

Collect composition target to the Renca of molecular modification cell totally 10 ⁸Individual, with hypotonic buffer (25mmol/L Tris-HCl.PH7.4) cell lysis that contains protease inhibitor, aspirate more than 3 times with No. 27 syringe needles (No. 4.5), mixing 2, (2,000g) centrifugal 10min gets supernatant (containing film component) to 300rpm.25, (22,000g) centrifugal 30min gets precipitation to 400rpm.

With solution I (0.15mol/LNaCl, 10mmol/L Tris, pH8.0) wash twice, with solution II (0.14mol/L NaCl, 25mmol/L Tris-HCl, pH7.4 wherein contains 2%1-S-OctylBeta-D-thioglucopyranoside) dissolving, reach the suitable 108/mL of concentration, 4 ℃ of concussion 30min.The centrifuging and taking supernatant, with dialysis solution (0.14mol/L NaCl, 25mmol/L Tris-HCl, pH7.4) the saturating suction spent the night, change three times dialysis solution, collect the film fat granule that forms, be tumor target gene transmission non-viral vector, can carry out the research and development of tumor target gene therapy biotech drug by the cationic peptide bind nucleic acid composition in the complex.

Claims (8)

1. one kind is used for the non-virus carrier that the tumor-targeting gene transmits, and it is characterized in that can be used as the carrier of tumor target gene therapy by cancer target complex and cell membrane embedding and the common film fat granule that forms of film fat.

2. be meant the tumor-targeting small peptide, wear film peptide, phagocytic vacuole releasing agent, cationic protein and connect the complex that obtains successively according to claim 1 cancer target complex, by the embedding of GPI structure with to cell membrane.

3. be by each linking arm of computer-aided design according to the complex that claim 2 connects the targeting small peptide successively, wears the film peptide, phagocytic vacuole releasing agent, cationic protein obtain, make that the space multistory conformation of each composition in complex is consistent with conformation own, thereby guarantee its function.

According to claim 2 in the cancer target complex, play the small peptide that is targeted to tumor by local and tumor cell effect comprise be targeted to some integrate plain part small peptide that contains RGD, be targeted to tumor cell and new vessels endothelial cell VEGF receptor the part small peptide, be targeted to new vessels perithelial cells surface NG2 small peptide and be targeted to the part small peptide of kinds of tumor cells EGF receptor.

According to claim 2 in the cancer target complex, play wear comprising of film peptide effect multiple have to need not to consume energy need not the polypeptide that acceptor molecule mediation just can import polypeptide, protein molecule, dna segment multiple mammalian cell function.

6. play in the cancer target complex according to claim 2 and multiplely have the dna segment that to enter the cytophagy corpusculum and discharge comprising of phagocytic vacuole releasing agent effect, enter cytoplasm, avoid the polypeptide of lysosomal Degradation.

7. comprise the multiple positive electricity that has according to the cationic protein of claim 2 in the cancer target complex, can be by the cationic polypeptide of electrostatic interaction in conjunction with plasmid.

8. according to the preparation method of claim 1 tumor target gene transmission non-viral vector, it is characterized in that:

1) by computer-aided design, connect the targeting small peptide successively, wear the film peptide, phagocytic vacuole releasing agent, cationic protein, obtain cancer target complex P6, and the protein molecular sequence is converted to gene order.

2), obtain complete cancer target complex gene by the synthetic method of full gene.

3) cancer target complex gene is inserted in the pCI-pr+-GPI carrier, makes up grappling carrier for expression of eukaryon pCI-P6-pr+-GPI.

4) the corresponding tumor cell line of transfection, and the grappling expression of the surface of cell membrane of evaluation cancer target complex molecule.

5) gather in the crops above-mentioned cell, with the cell membrane cracking, the regroup self assembly by lipid under certain buffer condition forms film fat granule.This film fat granule has tumor-targeting and nucleic acid associativity simultaneously, is that novel tumor-targeting non-viral gene transmits carrier.

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