昆虫细胞制备AAV-ITR基因表达微载体

Preparation of a novel AAV-ITR gene expression mini vector in Sf9 insect cells via baculovirus

摘要:

AAV-ITR基因表达微载体是只含有腺相关病毒 (Adeno-associated virus, AAV) 倒置末端重复序列 (Inverted terminal repeats, ITR)、基因表达顺式元件和目的基因，而不含有其他外源DNA序列的双链或单链DNA。本研究利用杆状病毒表达系统，制备得到两种重组杆状病毒Bac-ITR-EGFP和Bac-inrep，并将二者的P3代病毒共同感染昆虫细胞Spodoptera frugiperda (Sf9)，抽提小分子量DNA，获得AAV-ITR-EGFP基因表达微载体，2×107的Sf9细胞抽提可以得到100 μg AAV-ITR-EGFP基因表达微载体，核酸电泳显示AAV-ITR-EGFP基因表达微载体主要以单体和二聚体的形式存在。将AAV-ITR-EGFP基因表达微载体通过polyethylenimine (PEI) 转染HEK 293T细胞，24 h后荧光显微镜观察有EGFP表达，48 h后达到高峰，转化效率达到65%。

Abstract:

AAV-ITR gene expression mini vector is a double-strand or single-strand DNA that only contains inverted terminal repeats of adeno-associated virus, cis-elements and gene of interest and does not contain any other foreign DNA sequences. We prepared Bac-ITR-EGFP and Bac-inrep. Spodoptera frugiperda cells were infected with Bac-ITR-EGFP (P3) and Bac-inrep (P3). Up to 100 μg of AAV-ITR-EGFP gene expression mini vectors were extracted from 2×107 cells of Sf9 72 h after infection. The gel electrophoresis analysis shows that most forms of AAV-ITR-EGFP gene expression mini vector were monomer and dimer. The mini vector expression efficacy was examined in vitro with HEK 293T cells. The EGFP expression was observed at 24 h after transfection, and the positive ratio reached 65% at 48 h after transfection.