Preparation method and application of CBX3-containing L-arginine amination polyamine polymer gene vector
A gene carrier and polymer technology, applied in the fields of new formulations of biological preparations, gene carriers, and new excipients for pharmaceutical preparations, can solve the problems of non-degradability, high toxicity, inhibition, etc., and achieve the effect of good nuclear localization effect.
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Embodiment 1A
[0036] Embodiment 1 The preparation method of ARG-CBA polymer.
[0037] Take 0.11g of L-arginine guanidinium reagent, 0.105g of CBA, put it in a 50ml round bottom flask, add 2.5ml of water and 6ml of DMF as solvent, 45-60℃, avoid light, and react under nitrogen protection and magnetic stirring conditions for 3 1 day; TLC monitors the reaction progress, adds about 10% excess L-arginine guanidinium reagent (0.013g) after 72 hours, continues to react for 24 hours; Stops the reaction, adjusts the pH of the system to be 2-5 with 4mol / L hydrochloric acid , collect the whole reaction system solution, move it into a dialysis bag, dialyze and purify the reaction product, the dialysis medium is distilled water, the molecular weight cut off (MWCO) of the dialysis bag is 1000, dialyze for three days, and change the dialysis medium at least once a day; collect the solution in the dialysis bag, put In a dry petri dish, the polymer product was collected by lyophilization, and the structure w...
Embodiment 2
[0038] Example 2 Preparation of CBX3 / target gene / ARG-CBA gene carrier complex.
[0039] (1) Preparation of CBX3 / target gene complex.
[0040] pcDNA3.1-EGFP-cDNA plasmid, pSliencer TM 4.1 CMV-FANCF-shRNA plasmid, FANCF-shRNA, 328-microRNA were diluted to 0.016mg / ml with 50mM HEPES buffer solution with pH 7.4, and CBX3 powder was prepared with deionized water to a concentration of 5×10 -3 For the stock solution of μg / μl, the concentration will be 5×10 -3 The μg / μl CBX3 stock solution was diluted to different concentrations with HEPES, and then mixed according to the mass ratio of CBX3 and plasmid gene at 1:50-1:10000, and the mixture was vortexed in the EP tube for 5 seconds, and kept at room temperature. Incubate for 10-30min to obtain different ratios of CBX3 / pDNA complexes.
[0041] (2) Prepare CBX3 / target gene / ARG-CBA gene carrier complex.
[0042] The polymer gene carrier as described in Example 1 was dissolved in HEPES buffer at 0.9 mg / ml, and diluted with HEPES buffe...
Embodiment 3
[0046] Example 3 Evaluation of in vitro transfection efficiency of CBX3 / target gene / ARG-CBA gene vector.
[0047] (1) Study on in vitro transfection efficiency of pcDNA3.1-EGFP-cDNA plasmid carrying CBX3.
[0048] After preparing complexes with different N / P values (1 / 12, 1 / 24, 1 / 48) according to the method described in Example 2, the three complexes were added to six wells of normally cultured MCF10A cells at a concentration of 500 μl per well In the plate, the medium contained serum (BSA); 48 hours after transfection, the expression of green fluorescent protein was detected by flow cytometry, and the transfection efficiency was determined by flow cytometry software. Select commercial transfection reagent Lipofectamine2000 and PEI as controls, the results are as follows figure 2 shown.
[0049] (2) Study on in vitro transfection efficiency of pSliencer TM 4.1CMV-FANCF-shRNA plasmid carrying CBX3.
[0050] After preparing complexes with different N / P values (1 / 12, 1 / 2...
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