摘要
目的构建骨桥蛋白(osteopontin,OPN)基因RNA干扰慢病毒载体并在小鼠体内鉴定其抑制效果。方法参照OPN GenBank上的序列,对OPN基因沉默位点进行分析,设计针对OPN信使RNA的小发夹RNA(shorthairpin RNA,shRNA)OPN-shRNA及阴性对照(negative control-shRNA,NC-shRNA)序列,合成pSicoR-GFP-shRNA重组体。经鉴定序列无误后,与pCMV-VSV-G和pCMV-dR8.91辅助质粒包装并转染293FT细胞,得到有效滴度为5×109 TU/mL的病毒原液。经尾静脉将构建的慢病毒载体注射入小鼠体内,qRT-PCR及Western blot检测小鼠肝脏OPN mRNA及蛋白的抑制效果。结果成功构建针对OPN的shRNA慢病毒载体,经纯化、浓缩得到滴度为5×109 TU/mL病毒原液。与正常对照组、阴性对照组相比,实验组OPN mRNA及蛋白表达水平明显下降;正常对照组、实验组OPN mRNA及蛋白表达水平无明显差别。结论利用RNA干扰技术,借助慢病毒载体,成功构建小鼠肝脏OPN基因低表达模型。为进一步研究OPN在胆石症等疾病中的作用提供了有力工具。
Objective To construct short hairpin （shRNA） lentiviral vector targeting mouse osteopontin （OPN） gene, and to detect its efficacy of gene silencing in vivo. Methods Referring to the sequence of OPN in GenBank, we analyzed the silencing sites of OPN,then designed the specific OPN-shRNA sequence targeting OPN messenger RNA （mRNA） and the negative control NC-shRNA sequence. Next,pSicoR-GFP- shRNA recombinant was constructed. After identification of the sequence of each recombinant, they were all packaged with pCMV-VSV-G and pCMV-dRS. 91 auxiliary plasmid,and transmitted into 293FT ceils. Virus was successfully packaged with a titer of 5 x 109 TU/mL. Mice were injected with this virus through tail vein for in vivo study. OPN mRNA and protein levels were detected by qRT-PCR and Western blot for the detection of its inhibition efficacy. Results The recombinant lentiviral vector of OPN-shRNA were successfully constructed, purified and concentrated, the virus reached a titer of 5 × 10^9 TU/mL. Compared with normal group and negative control group, the expressions of OPN mRNA and protein in experimental group were obviously decreased; and there was no obvious difference between normal group and negative control group. Conclusions Using RNA interference technique, with the help of lentiviral vector, the low liver OPN gene expression mouse model was successfully constructed. This provide a useful tool for further research on the function of OPN in cholelithiasis.
出处
《复旦学报（医学版）》
CAS
CSCD
北大核心
2012年第5期496-501,共6页
Fudan University Journal of Medical Sciences
基金
国家自然科学基金项目(30801105)~~
