靶向人STAT3基因的shRNA慢病毒载体的构建  

Construction of lentivirus-mediated short hairpin RNA targeting human STAT3 gene

作　　者：党微旗[1] 唐浩[1] 曹红[1] 王林[1] 陈婷梅[1] 

机构地区：[1]重庆医科大学教育部临床检验诊断学重点实验室,重庆400016

出　　处：《细胞与分子免疫学杂志》2012年第11期1204-1207,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(30971131)

摘　　要：目的:构建并鉴定靶向人转录因子STAT3基因的shRNA慢病毒载体。方法:设计合成针对人STAT3基因的shRNA序列,运用基因重组技术将其插入到含U6启动子及绿色荧光蛋白基因的慢病毒表达载体pSicoR中,构建shRNA重组质粒pSicoR-STAT3-shRNA。经双酶切及DNA测序鉴定后,利用转染试剂X-tremeGENE HP将第3代慢病毒载体共转染至HEK293细胞进行病毒包装。以阴性载体为对照,用RT-PCR和Western blot分别检测STAT3基因和蛋白表达水平。结果:双酶切及测序证实载体构建正确,在HEK293中成功包装出高滴度慢病毒颗粒,感染HEK293后STAT3基因表达和蛋白质表达水平均较对照组明显降低(P<0.05)。结论:成功构建了靶向人STAT3基因的shRNA慢病毒载体。AIM： To construct and identify a lentivirus- mediated short hairpin RNA （shRNA） targeting human signal transducer and activator of transcription 3 （STAT3） gene. METHODS： The shRNA chains targeting to human STAT3 gene were designed and synthesized, and then inserted into lentivirus expression vector pSicoR containing U6 promoter and green fluorescent protein （GFP） gene by gene recombination technique. The constructed recombinant plasmid pSicoR-STAT3-shRNA was identified by double restriction enzyme digestion and DNA sequencing, and then mixed with the 3rd generation of lentiviral packaging system and co-transfected to HEK293 cells using new generation of Roche X-tremeGENE HP DNA Transfection Reagent media- ted transfection method. RT-PCR and Western blotting were employed to detect the expressions o{ STAT3 at mRNA and protein levels, respectively. Negative plasmid transfected into the same cell line was used as a control group. ~JLTS; RestricUon analysis and sequencing proved that the recombinant plasmid pSicoR-STAT3-shRNA was construc- ted correctly. Lentivirus particles were successfully pack- aged in HEK293 cells with high titer. The expressions of STAT3 at mRNA and protein levels in the transfected HEK293 cells were weaker than those of the control group （ P 〈 0.0.5 ）. CONCLUSION： The lentivirus-mediated shRNA targeting human STAI3 gene is successfully constructed.

关 键 词：STAT3 RNA干扰 慢病毒 乳腺癌 

分 类 号：R392-33[医药卫生—免疫学]