甲胎蛋白特异性小干扰RNA重组腺病毒载体的构建

背景：RNA干扰技术的应用，关键在于能够采用1个有效的基因转移系统将小干扰RNA转入至靶细胞，目前广泛应用的是构建小干扰RNA表达载体。 目的：利用AdMax腺病毒载体系统构建表达人甲胎蛋白-小干扰RNA的腺病毒载体。 设计、时间及地点：开放性实验，于2007-03/10在山东大学附属济南市中心医院中心实验室完成。 材料：穿梭质粒pDC316-EGFP-U6为本元正阳基因技术公司产品；AdMax KitD试剂盒与低代数HEK293细胞为Microbix Biosystems Inc.（Canada）公司产品。 方法：选择针对甲胎蛋白 mRNA的特异性小干扰RNA靶序列，设计合成为相应的双链DNA，并将其与酶切线性化的pDC316-EGFP-U6载体片段连接，构建好的穿梭质粒pDC316-EGFP-U6-AFP-siRNA和腺病毒骨架质粒pBHGlox_E1,3Cre共转染HEK293细胞，同源重组产生重组腺病毒。 主要观察指标：对重组腺病毒进行聚合酶链反应鉴定及扩增、纯化、滴度测定。 结果：构建的穿梭质粒载体经聚合酶链反应鉴定和测序分析，证实与设计一致。重组腺病毒Ad-AFP-siRNA经聚合酶链反应和绿色荧光蛋白表达检测证实构建成功，测定滴度为1.4×109 nfu/L。 结论：实验成功构建了Ad-EGFP-U6-AFP-siRNA重组腺病毒。

Constructing recombinant adenovirus vector of small interfering RNA specific for alpha-fetoprotein

BACKGROUND: The key of applying RNA intervention technique is transfection of small interfering RNA (siRNA) into target cells by an effective gene transfer system. Constructing siRNA expression vector is presently widely used. OBJECTIVE: To construct the adenovirus vector expressing the siRNA specific for alpha-fetoprotein by AdMax adenovirus vector system. DESIGN, TIME AND SETTING: An open experiment was conducted at the Central Laboratory of Jinan Central Hospital Affiliated to Shandong University from March to October 2007. MATERIALS: Shuttle plasmid pDC316-EGFP-U6 (Vector Gene Technology Company, China) and AdMax KitD kits and HEK293 cell (Microbix Biosystems Inc., Canada) were used in this study. METHODS: To choose a specific siRNA sequence targeting alpha-fetoprotein mRNA, design and synthesize the homologous double-strand DNA. Ligating the DNA into linearized pDC316-EGFP-U6 vector. After having been conformed, the pDC316-EGFP-U6 -AFP-siRNA plasmid was cotransfected with genomic plasmid pBHGlox_E1, 3Cre into HEK293 cells to amplify the recombinant adenovirus. MAIN OUTCOME MEASURES: Recombinant adenovirus was tested by polymerase chain reaction (PCR), amplified, purified and titer was detected. RESULTS: The pDC316-EGFP-U6-AFP-siRNA plasmid was identified with the method of PCR and sequencing. Recombinant adenovirus Ad-EGFP-U6-siRNA-AFP was constructed, which was confirmed by PCR and enhanced green fluorescent protein (EGFP) expression. The titer was 1.4×109 nfu/L. CONCLUSION: The recombinant adenovirus vector Ad-EGFP-U6 -AFP-siRNA is successfully constructed.