基因治疗微链载体的构建及表达分析

Construction and Expression Analysis of Micro-linear Vector as a New General Gene Therapy Vector

摘要:

基因载体的转染、表达效率低和存在安全问题是基因治疗的难点。由于传统病毒及质粒载体含有大量外源基因, 其表达有可能引发较严重免疫副反应。本课题旨在新的设计思路上开发高效安全基因治疗载体。 微链载体利用设计好的Cap序列封闭基因表达框两端, 起到防止细胞内核酸外切酶降解的作用。从pEGFP-N3质粒中分离纯化得到GFP基因作为微链载体的报告基因。将微链载体与原质粒载体(pEGFP-N3质粒)分别转染入真核细胞, 利用荧光显微镜和流式细胞仪检测并比较其转染效率。结果显示微链载体在293、CNE2、3T3、B95-8等真核细胞中的转染、表达效率较高, 并具有较小的细胞毒性。初步证实了微链载体在真核细胞中转染、表达效率及安全性等方面具有一定的优越性。

Abstract:

The most difficult field in gene therapy is that vector system should offer both a means of successful transfection and a maximum of safety for the patient. Viral vectors and plasmid vectors are traditional vectors; they may cause unwanted immunological side effects resulting from the expression of nontherapeutic genes. Our aim is to develop a new general gene therapy vector which is suggested to be called as Micro-Linear Vector. The gene expression cassette is capped by our designed cap, including promoter, enhancer, objective gene, and RNA-stabilizing sequence, so it can defend the exnuclease in the eukaryotic cell, at the same time, DNA not encoding the objective gene is reduced to a minimum. The GFP gene is separated from the pEGFP-N3 plasmid, and acts as a reporter gene to construct the Micro-Linear Vector, then both the new vector and the plasmid are transfected to cells, the results are tested by fluorescence microscope and flow cytometry. The results show that the Micro-Linear Vector has a high effective of transfection and safety in 293, 3T3, CNE2 and B95-8 cell lines, at the same time it is less toxicity than the plasmid. We can get the rudiments of conclusion that Micro-Linear Vector has high effection of the transfection and more safety than tradition plasmid in eukaryotic cell.