NIPSNAP3A短发夹RNA腺病毒载体的构建及病毒包装  

Construction of Adenovirus Vector and Virus Packaging of shRNA Against NIPSNAP3A

作　　者：俞华吉[1] 闫永红[2] 唐刘君[2] 杨晓明[2] 王晓辉[2] 汪思应[1] 

机构地区：[1]安徽医科大学病理生理学教研室,安徽合肥230032 [2]军事医学科学院放射与辐射医学研究所,北京蛋白质组研究中心,北京102206

出　　处：《生物技术通讯》2012年第4期485-487,492,共4页Letters in Biotechnology

基　　金：国家自然科学青年基金(30900546);国家重点基础研究发展计划(2011CB910600)

摘　　要：目的:构建靶向NIPSNAP3A的短发夹RNA(shRNA)腺病毒载体,干扰HEK293A细胞中NIPSNAP3A的表达。方法:设计靶向NIPSNAP3A的shRNA,插入穿梭质粒pENTRY,通过Gateway法获得腺病毒颗粒pAd-NIPSNAP3A-shRNA,转染HEK293A细胞,在细胞内包装获得腺病毒。结果:重组腺病毒载体经酶切鉴定正确,制备的病毒感染效率高,能显著抑制NIPSNP3A蛋白的表达。结论:干扰HEK293A细胞NIPSNAP3A表达的shRNA重组腺病毒载体构建成功。Objective： To construct the recombinant adenovirus vector of short hairpin RNA（shRNA） against NIPSNAP3A and explore the knocking-down expression of NIPSNAP3A in HEK293A cell. Methods： The short hairpin oligonucleotide and complementary strands targeting the consensus sequences of NIPSNAP3A were designed and inserted in the shuttle plasmid pENTRY, then adenovirus particles pAd/PL-DEST-NIPSNAP3A-shRNA through the Gateway method was obtained. The adenovirus particles was transfected into HEK293A cells via liposome mediation to package and amplify pAd/PL-DEST-NIPSNAP3A-shRNA adenovirus. Results： The adenovirus vectors were verified by enzyme digestion and DNA sequencing, the infection efficiency of constructed adenovirus was high, and could obviously inhibit the expression of NIPSNAP3A. Conclusion： The recombinant adenovirus pAd/ PL-DEST-NIPSNAP3A-shRNA was successfully constructed to knockdown the expression of NIPSNAP3A in 293A cell. This result lays the foundation for further study to investigate the function of NIPSNAP3A.

关 键 词：NIPSNAP3A 短发夹RNA 腺病毒载体 

分 类 号：Q78[生物学—分子生物学]