SHARP-2特异的RNA干扰腺病毒载体的构建及功能鉴定

Silencing of SHARP-2 gene using adenoviral vector-based short harpin RNA interfering

目的 构建转录SHARP-2 基因特异性的小发夹RNA（SHARP-2-shRNA）的重组腺病毒（Rad-hSHARP）,并观察其对正常大鼠肾细胞（NRK cell）SHARP-2基因的表达影响.方法 设计带有SHAPR-2特异性干扰序列的PDC316-SHARP-shRNA穿梭质粒与腺病毒骨架质粒pBHGloxdelE13cre 共转染293 细胞,并在293 细胞内进行同源重组,构建Rad-hSHARP.并使用实时定量RT-PCR在NRK细胞中对重组腺病毒干扰效果进行鉴定.结果 成功构建了特异性SHARP-2基因RNA 干扰的腺病毒载体系统,并有效抑制NRK 细胞中SHARP-2 基因的表达.结论 成功构建SHARP-2-shRNA 的Rad-hSHARP,为利用特异性沉默SHARP-2基因的腺病毒载体抑制T细胞增殖活化研究奠定基础.

Objective To construct recombinant adenovirus （Rad-hSHARP） expressing short hairpin RNA （SHARP-2-shRNA）. Methods The annealed shRNA templates were inserted into PDC316-EGFP-U6 shuttle plasmid to construct the recombinant Plasmids （PDC316-SHARP-shRNA）.Both the shuttle plasmid and the adenovirus backbone plasmid pB- HGIoxdelE13cre were co-transfected into 293 cells, to construct the recombined adenovirus （Rad-hSHARP）. The SHARP-2 gene silencing with vector of recombinant adenovirus was determined by realtime RT-PCR in NRK cells. Results The adenovirus vector expressing small interfering RNA targeting SHARP-2 gene could specifically shutdown the SHARP-2 expression in NRK cells. Conclusion The recombinant adenovirus which expressing short hairpin RNA targeting SHARP-2 gene had been constructed successfully, which may be used for further investigation of the function of SHARP-2 gene silencing with adenoviral vector in proliferation and differentiation of T cells.