pH敏感型SOD1-DNA-CPP聚集体的构建

发布时间：2018-06-28 06:05

本文选题：SOD1 + DNA　；参考：《华中师范大学》2015年硕士论文

【摘要】：基因治疗为治疗疾病提供了新手段,如何将有治疗作用的基因导入体内并进入靶细胞表达,是基因治疗研究中急需解决的问题；另一方面将活性蛋白质载入靶细胞释放,治疗因蛋白质异常而引起的疾病正越来越受到人们的关注。这些设想的实现都需要高效的载体。不同细胞、不同细胞器具有不同的pH,这使得pH敏感型载体在基因和活性蛋白质释放方面具有天然的优势。为了实现对基因和活性超氧化物歧化酶(SOD1)的共释放,我们尝试构建pH敏感的SOD1-DNA-CPP三元聚集体。在一定的条件下,SOD1以二聚体形式围绕DNA模板,由一种协同的过程自组装成含DNA的聚集体,可用于组装仿病毒的基因和SOD1共释放平台,CPP的引入可增加聚集体进入细胞的能力。当pH改变时聚集体解聚,释放出DNA和SOD1。我们首先研究了酸性条件下DNA和SOD1的组装,发现在pH4.0的环境中可以形成不同粒径的聚集体；它们可在pH 7.4的环境中解聚,将SOD1和质粒DNA释放出来,释放的SOD1的活性和DNA的结构几乎没有改变。引入少量的CPP可以通过非特异性的静电相互作用与SOD1-DNA形成SOD1-DNA-CPP三元聚集体,提高聚集体进入细胞的能力。延长反应时间可以增加聚集体的稳定性,但对SOD1的活性和DNA的结构有一定的影响。其次,我们发现在近生理条件下,CPP、SOD1、质粒DNA可通过非特异性的静电相互作用形成不同粒径的SOD1-DNA-CPP三元聚集体,聚集过程对SOD1的活性和DNA二级结构的影响较小。与在酸性条件中构建的聚集体相比,中性条件下形成的聚集体是通过带正电荷的CPP与带负电荷的SOD1、质粒DNA同时作用形成的,所以需要加入过量的CPP。胞内pH的改变会使SOD1所带电荷发生变化从而可能使聚集体解聚,将目的基因和活性SOD1释放出来。
[Abstract]:Gene therapy provides a new method for the treatment of disease. How to transfer the therapeutic gene into the body and enter the target cell expression is an urgent problem in gene therapy research; on the other hand, the active protein is loaded into the target cell and released. People pay more and more attention to the treatment of diseases caused by abnormal protein. The realization of these ideas requires efficient carriers. Different cell and organelle have different pH, which makes pH sensitive vector have natural advantages in gene and active protein release. In order to realize the co-release of genes and active superoxide dismutase (SOD1), we try to construct the pH sensitive SOD1-DNA-CPP ternary aggregates. Under certain conditions, the DNA template is surrounded by dimer and self-assembled into DNA-containing aggregates by a synergistic process, which can be used to assemble virus-like genes and SOD1 co-release platform-CPP can increase the ability of aggregates to enter cells. When pH changed, the aggregates depolymerized, releasing DNA and SOD1. We first studied the assembly of DNA and SOD1 under acidic conditions, and found that aggregates with different particle sizes could be formed in pH 4.0, and they could be depolymerized in pH 7.4 to release SOD1 and plasmid DNA. The activity of the released SOD1 and the structure of DNA were almost unchanged. The introduction of a small amount of CPP can form SOD1-DNA-CPP ternary aggregates through non-specific electrostatic interaction with SOD1-DNA, and improve the ability of aggregates to enter cells. Prolonging reaction time can increase the stability of aggregates, but it has a certain effect on the activity of SOD1 and the structure of DNA. Secondly, we found that under near physiological conditions, plasmid DNA can form SOD1-DNA-CPP ternary aggregates with different particle sizes through non-specific electrostatic interaction, and the aggregation process has little effect on the activity of SOD1 and the secondary structure of DNA. Compared with the aggregates constructed in acidic conditions, the aggregates formed under neutral conditions are formed by the interaction of positively charged CPP and negatively charged SOD1, plasmid DNA, so excessive CPP is needed. The change of intracellular pH will change the charge of SOD1, which may depolymerize the aggregates and release the target gene and active SOD1.
【学位授予单位】：华中师范大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R450

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