Tie2 receptor-induced gene transfer system for treating target tumor genes
A gene transfer and targeting technology, applied in the fields of molecular biology and gene therapy, can solve problems such as non-expression
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[0060] Preparation method of targeted non-viral vector
[0061] Usually, the above-mentioned (a), (b), (c) components or their complexes are mixed together to obtain the targeting non-viral vector of the present invention. Wherein, the mixing ratio of (a), (b) and (c) components is usually 0.5-1:1-2:0-1, preferably 0.8-1:1-1.5:0.5-1.
[0062] In addition, when administered in the form of (a)-(b) complex and (b)-(c) complex, the mixing ratio of (a)-(b) complex: (b)-(c) complex Usually 0.8-1.2:0.8-1.2, preferably 0.9-1.1:0.9:1.1.
[0063] exogenous DNA
[0064] The exogenous DNA that can be used in the present invention is not particularly limited, and can be various therapeutic or preventive DNAs, such as target genes, antisense oncogenes, anticancer genes, suicide genes, apoptosis genes, cytokine genes, or A combination thereof, or a eukaryotic expression vector DNA containing the above genes. Proto-oncogene antisense sequences include proto-oncogene (ras H 、ras K 、ras ...
Embodiment 1
[0076] The construction of embodiment 1 non-viral vector
[0077] A. Obtaining of each component
[0078] (1) Synthesis of ligand oligopeptide GA1:
[0079] The peptide synthesizer of ABI Company in the United States was used to synthesize according to its peptide synthesis operation manual, and the oligopeptide GA1 was separated by column chromatography, and its amino acid sequence was WKEYK VGFGN PSGEY WLGN (SEQ ID NO: 1).
[0080] (2) Poly-L-lysine (Poly-L-lysine, abbreviated as P.L.)
[0081] Purchased from SIGMA Company of the United States, the molecular weight distribution is 15000-30000 Daltons.
[0082] (3) HA20
[0083] The peptide synthesizer of ABI Company in the United States was used to synthesize according to its peptide synthesis operation manual, and the oligopeptide HA20 was obtained by column chromatography separation, and its amino acid sequence was GLFEA IAEFI EGGWE ELIEG (SEQ ID NO: 2).
[0084] B. Preparation of Binary Complexes
[0085] First, GA1,...
Embodiment 2
[0103] Extraction and purification of embodiment 2 plasmid DNA
[0104] The separation and purification of recombinant eukaryotic expression vector plasmid DNA used in this example were pure DNA obtained by using the Maxi plasmid kit of QIAGEN Company.
[0105] Pick a single clone from the LB plate and inoculate it in 5ml LB medium containing ampicillin, shake overnight at 250rpm at 37°C. 1:1000 transfer to 200ml LB medium containing ampicillin, shake at 250rpm for 16 hours at 37°C. Transfer the bacterial solution to a 500ml centrifuge tube, centrifuge at 4,000 rpm at 4°C for 10 minutes, discard the supernatant, and collect the bacterial cells. The pellet was resuspended in 10ml Buffer P1 (50mM Tris.Cl, pH8.0, 10mM EDTA pH8.0, 100μg / ml Rnase A). After adding 10ml Buffer P2 (200mM NaOH, 1% SDS), gently invert 4-6 times, mix well, and let stand at room temperature for 5 minutes. Add 10ml of Buffer P3 (3.0M NaAc, pH5.5), invert and mix well, transfer to a 50ml centrifuge tube,...
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