靶向端粒酶反转录酶基因短发夹RNA载体的构建

Construction of targeting short hairpin RNA plasmid vector expressing TERT gene

背景:端粒酶反转录酶对端粒酶活化起重要作用。目的:利用pLentilox3.7.U6载体构建靶向大鼠脊髓星形胶质细胞端粒酶反转录酶基因的短发夹RNA干扰分子表达载体并鉴定其作用。方法:选择端粒酶反转录酶基因上两段序列体外合成RNA干扰分子的正义链和反义链模板序列,经退火成互补双链,与线性化pLentilox3.7.U6载体连接、转化大肠杆菌和序列测定,应用蛋白质印迹和免疫荧光技术,在体外培养的大鼠脊髓星形胶质细胞模型上验证构建的干扰载体抑制端粒酶反转录酶基因表达的效果。结果与结论:蛋白质印迹和免疫荧光检测结果表明,重组质粒干扰组中星形胶质细胞端粒酶反转录酶均呈低表达。结果证实,实验成功构建了针对大鼠脊髓星形胶质细胞端粒酶反转录酶基因的短发夹RNA质粒表达载体,此载体能有效抑制体外培养的大鼠脊髓星形胶质细胞端粒酶反转录酶的表达。

BACKGROUND：Telomerase reverse transcriptase （TERT） plays an important role in telomerase activation. OBJECTIVE：To construct the targeting short hairpin RNA plasmid vector expressing TERT gene from astrocytes by using pLentilox3.7.U6. METHODS：By using two sequences from TERT gene, we synthetized sense and antisense strand template sequences of RNA interference molecular in vitro, and then obtained the complementary strands through annealing procedure. We connected the strands with pLentilox3.7.U6 that was sequenced and transfected into the Escherichia coli. In the end, we tested its effect of reducing the TERT gene expressing by using cultured astrocytes from rat spinal cord in vitro through western blot and immunofluorescence technique. RESULTS AND CONCLUSION：Western blot and immunofluorescence assay showed that, compared with the control group, the interference groups had a lower TERT expression in astrocytes. The targeting short hairpin RNA plasmid vector expressing TERT gene is useful to reduce the TERT gene expression. The targeting short hairpin RNA plasmid vector expressing TERT gene is valid for us to do the further test learning the mechanism of astrocytes in spinal cord injury.