核定位信号肽提高核糖体区打靶载体转染效率的研究(英文)

The Transfection Efficiency Improvement of hrDNA Targeting Vectors With NLS Peptide

核糖体区打靶载体(10～14kb)是中南大学医学遗传学国家重点实验室构建的一种具有定点整合能力的非病毒载体,具有安全性好及长期稳定表达的特点,但是较低的转染率成为其应用于临床的主要障碍.核定位信号肽可以促进非病毒载体进入细胞核,从而提高其转染效率.但是核定位信号肽提高外源基因表达的能力却严重受到其与DNA偶联方式及偶联剂的影响.采用偶联剂SPB可以通过静电作用将核糖体区打靶载体与SV40核定位信号肽有效结合,并且可以防止其被DNase降解.应用聚乙烯亚胺转染人原代皮肤成纤维细胞后,激光共聚焦显微镜观察显示,核定位信号肽可以在60min内携带12kb的质粒DNA进入细胞核.转染后48h,流式细胞仪检测GFP表达,结果显示转染率提高了4～5倍.聚乙烯亚胺是一种毒性小,而且价格低廉的高分子转染试剂,广泛被应用于体内基因治疗的研究中,上述研究将会促进核糖体区打靶载体在临床基因治疗中的应用.

Human ribosomal gene （hrDNA） targeting vectors （ 10- 14 kb） constructed by the group are novel non-viral vectors which could specifically integrate into the ribosomal loci and characterized by their security and stable expression of therapeutic genes. However, the low transfection efficiency handicapped their clinical application. Although nuclear localization signals （NLS） could facilitate the nuclear entry of non-viral vectors and improve the transfection efficiency, the expression of therapeutic gene varied dramatically with the coupling methods and the type of chemistry used. The hrDNA vectors were conjugated by succinimidyl- [4-（psoralen-8-yloxy）]butyrate （SPB） with Simian Virus 40 NLS （SV 40 NLS） peptide through electrostatic interaction efficiently, which could protect the plasmid DNA（pDNA） from degradation of DNase. The polyethylenimine （PEI）, which is an economical and low toxic polymer and wildly used in vivo gene therapy, was employed to transfect the primary human dermal fibroblasts （HDF）. When conjugated with NLS peptide, the 12 kb hrDNA emerged in the nucleus within 60 min under the view of confocal microscopy. The GFP fluorescence analysis by flow cytometry showed that the transfection efficiency was increased to 4 -5 folds. In conclusion, an effective procedure was developed to improve the non-viral transfection efficiency and promise the preclinical trial ofhrDNA vectors.