人组织因子基因RNAi载体pENTR^TM/U6-TF-shRNA的构建与鉴定

Construction and identification of RNAi vector pENTR^(TM)/U6-TF-shRNA vector on human tissue factor

目的:RNAi效应与基因转移有密切关系。拟通过构建组织因子基因RNAi载体,为达到阻止凝血过程的启动打下基础。方法:实验于2006-11/2007-05在南方医科大学中医药学院中医症候实验室完成。利用Invitrogen公司在线软件设计人组织因子基因(NM_001993)干扰片段,合成shRNA序列,再行寡核苷酸双链的合成,退火后形成的寡核苷酸双链克隆到pENTRTM/U6载体的黏性末端,连接产物转化到感受态细胞,增菌,质粒感受态扩增,质粒DNA小提、测序。结果:合成的寡核苷酸双链退火处理后,经4%琼脂糖凝胶电泳,紫外观察可见ds oligo和ss oligo清晰可见,双链寡核苷酸与pENTRTM/U6的连接反应,转化到质粒感受态扩增后,提取得到的质粒DNA经过测序结果证实pENTRTM/U6-TF-shRNA为阳性克隆。结论:成功构建出凝血途径的人组织因子基因pENTRTM/U6-TF-shRNA载体,为研究组织因子基因RNAi载体在出凝血疾病中的应用提供了稳定的转染细胞工具。

AIM： RNAi effect is closely correlated with gene transfer. In this study, we constructed pENTR^TM/U6- TF-shRNA vector on human tissue factor, so as to lay a foundation for treating blood clotting diseases. METHODS： The experiment was performed at Laboratory of Symptomatology, College of Traditional Chinese Medicine, Southern Medical University from November 2006 to May 2007. Human tissue factor （NM-001993） was designed by on-line designer software on Corp. Invitrogen, and shRNA sequence was synthesized, then double strands oligonucleotide （ds oligo） was synthesized and cloned into the pENTR^TM/U6 plasmid. Connection product was inverted competent cells and multiplied. Plasmid was extracted and sequenced. RESULTS： After renaturation, clear ds oligo and ss oligo were observed under ultraviolet after 4% agarose gel electrophoresis in the synthesized double strands oligonucleotide. Sequencing results indicated that pENTR^TM/U6-RelB-shRNA plasmid was positive clone. CONCLUSION： Human tissue factor gene pENTR^TM/U6-TF-shRNA vector is constructed successfully. It develops stable transfection vector for the application of tissue factor gene RNAi vector in treatment blood clotting diseases.