人组织因子基因RNA干扰载体的构建与鉴定

Construction and identification of RNA interference vector for human tissue factor gene

目的构建人组织因子基因RNA干扰载体,为达到阻止凝血过程的启动打下基础。方法利用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,再制备双链寡核苷酸(dsoligo),退火后形成的dsoligo双链克隆到pENTRTM/U6载体的粘性末端,连接产物转化到感受态细胞,增菌,质粒DNA小提、测序。将构建的载体转染到脐静脉内皮细胞,应用RT-PCR和免疫荧光验证载体对目的基因的干扰情况。结果测序证实阳性克隆为pENTRTM/U6-TF-shRNA,转染到脐静脉内皮细胞后组织因子的表达受到明显抑制。结论成功构建出凝血途径的人组织因子基因RNA干扰载体,为研究组织因子基因RNA干扰载体在凝血疾病中的应用提供了稳定的转染细胞载体。

Objective To construct a RNA interference vector for human tissue factor （TF） gene. Methods Human TF short hairpin RNA （shRNA） sequence was designed using online design software （lnvitrogen） and synthesized into double-strand oligonucleotide （ds oligo）, which was cloned into the pENTRTM/U6 plasmid, followed by transformation of the product into competent Top 10 E. coli cells. After expansion of the transformed bacteria, the plasmid was extracted and sequenced, which was subsequently transfected into human umbilical vein endothelial cells （HUVECs）, The interference effect of the vector on the target gene expression was detected by RT-PCR and immunofluorescence assay, Results The sequencing result indicated that the plasmid pENTRTM/U6-RelB-shRNA was constructed correctly, which resulted in effective inhibition of TF expression in HUVECs after transfection. Conclusion The RNA interference vector against human TF gene has been constructed successfully, which may provide a stable transfection vector for potential treatment of blood coagulation abnormalities.