硬脂酰鱼精蛋白的合成及用作非病毒基因载体的研究

发布时间：2018-05-15 16:32

本文选题：非病毒基因载体 + 鱼精蛋白　；参考：《上海交通大学》2007年硕士论文

【摘要】： 本文研究了硬脂酰鱼精蛋白的合成,并将其作为非病毒基因载体,考察了与质粒DNA的复合性能,复合物的体外基因转染效果。主要内容包括以下三部分: 1)通过N-羟基琥珀酰亚胺(NHS)与硬脂酸形成的活泼酯,在水性反应环境中与鱼精蛋白进行酯交换反应,从而将疏水性的硬脂酰基团偶联到鱼精蛋白上,成功合成了三种硬脂酰鱼精蛋白(stearyl protamine,简称SP),分别为SP1,SP2,SP3。将SP在HCl中降解后回收硬脂酸,并用酸碱微量滴定的方法测定含量。SP中硬脂酰基团的含量(质量百分比)分别为2.5%、3.0%、5.5%。通过动态光散射(DLS)对SP以及硫酸鱼精蛋白(PS)的研究发现,SP3可以在水中自组装形粒径110nm左右的稳定聚集体,此聚集体的粒径不随SP3浓度的提高而发生变化,说明是一种热力学稳定的自组装聚集体。 2)通过工程菌扩增,碱法抽提,获得编码萤火虫荧光素酶的质粒DNA pGL3-control,并以此作为后继实验中采用的DNA材料。凝胶电泳阻滞实验检验SP对DNA的复合能力,结果表明当载体/DNA质量比超过1.0时,DNA均能被三种SP完全包装起来。与未修饰的鱼精蛋白相比,随着SP中硬脂酰基团含量的升高,其对DNA的复合能力略有减弱。对鱼
[Abstract]:In this paper, the synthesis of stearyl protamine was studied, and it was used as a non-viral gene vector. The compound with plasmid DNA and the effect of gene transfection in vitro were investigated. The main contents include the following three parts: 1) the hydrophobic stearyl group was coupled to protamine by transesterification of active ester formed by N-hydroxysuccinimide (NHS) with stearic acid. Three stearyl protamine (SPN) stearyl protamines were successfully synthesized. The stearic acid was recovered after the degradation of SP in HCl, and the content (mass percentage) of stearyl group in SP was determined by acid-base microtitration. The content of stearyl group (mass percentage) was 2.5% and 3.5% respectively. The study of SP and protamine sulfate PSs by dynamic light scattering (DLS) showed that SP3 could be used to form stable aggregates with self-assembled particle size (110nm) in water, and the particle size of the aggregates did not change with the increase of SP3 concentration. It shows that it is a thermodynamically stable self-assembly aggregate. 2) the plasmids DNA pGL3-control encoding luciferase of firefly were obtained by amplification and alkali extraction, which were used as DNA materials in subsequent experiments. The results of gel electrophoresis block test showed that when the mass ratio of vector / DNA was more than 1.0, the DNA could be completely packaged by three kinds of SP. Compared with unmodified protamine, the compound ability of SP to DNA decreased slightly with the increase of stearyl group content in SP. Yeah, fish.
【学位授予单位】：上海交通大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R346

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