靶向ADAM17基因RNA干扰慢病毒载体的构建及慢病毒包装  

Construction and identification of ADAM17-targeting RNA interference lentivirus expressive vectors

作　　者：李亚清[1] 严建平[1] 许武林[1] 王宏[1] 任卓超[1] 陈瑜[1] 

机构地区：[1]浙江省人民医院呼吸内科

出　　处：《中国微生态学杂志》2012年第5期389-393,399,共6页Chinese Journal of Microecology

基　　金：国家自然科学基金(81000016);浙江省医药卫生科学研究基金(2008B007)

摘　　要：目的构建靶向ADAM17基因RNA干扰(RNAi)慢病毒载体及包装慢病毒。方法根据人ADAM17mRNA序列设计4个靶序列,合成4对寡核苷酸序列,同时合成1对阴性对照寡核苷酸序列;将以上5对寡核苷酸序列退火后连入pLVTHM质粒,经酶切和测序鉴定。将重组慢病毒质粒转染至A549细胞,以Real-time PCR检测A549细胞中ADAM17 mRNA表达。将干扰效果最佳的质粒载体和包装质粒共转染至293T细胞,包装产生病毒颗粒。以流式细胞术检测重组慢病毒的滴度。结果酶切和测序证实干扰靶序列已被准确克隆到pLVTHM质粒载体。pLVTHM-ADAM17-siRNA1-4均可显著抑制A549细胞ADAM17 mRNA的表达,其中pLVTHM-ADAM17-siRNA4的抑制效果最佳。LV-ADAM17-siRNA4重组慢病毒的滴度为2.16×108TU/ml。结论成功构建了靶向人ADAM17基因RNAi慢病毒载体及包装了重组慢病毒。Objective To construct recombinant lentiviral vectors for RNA interference （ RNAi ） targeting a disintegrin and metalloproteinase 17 （ADAM17） gene and package recombinant lentivirus. Method Four pairs of short hairpin RNA （shRNA） sequences were designed according to the sequence of human ADAM17 gene. After synthesis and annealing, the double-stranded oligonucleotides were cloned into the pLVTHM vectors, which were confirmed using DNA sequencing analysis. The silencing effects of the ADAM17 siRNA expression vectors were analyzed. The most effective ADAM17 RNAi expression vector was packaged to recombinant lentivirus in 293T cells. The titer of lentivirus was detected by flow cytometry. Result DNA sequencing demonstrated that the recombinant lentiviral vectors were constructed successfully. The most effective AD- AM17 siRNA expression vector was selected successfully. Then the recombinant lenfivirus containing human ADAM17 gene was packaged successfully and its titer was 2.16 ×10^8 TU/mL. Conclusion The recombinant lentiviral vector targeting human ADAM17 gene was constructed, and the recombinant lentivirus was packaged successfully.

关 键 词：解整合素-金属蛋白酶17 RNA干扰 慢病毒 

分 类 号：Q78[生物学—分子生物学]