摘要
目的构建特异性组蛋白甲基转移酶G9a基因的siRNA表达载体并检测QBC939细胞对G9a表达的抑制作用。方法设计、合成G9a特异性的短链寡核苷酸,经退火形成双链DNA片段.克隆到pSilencer neo^TM2.1-U6载体中,构建G9a特异性siRNA的表达载体。EcoRⅠ+BamHⅠ和EcoRⅠ+HindⅢ双酶切及测序鉴定重组体,并转染QBC_(939)细胞,G418筛选出稳定表达细胞株,半定量RT-PCR方法和Western印迹法检测G9a mRNA和蛋白的表达水平。结果双酶切与测序鉴定证实成功构建了G9a-siRNA表达载体,转染人胆管癌细胞株QBC939后,在mRNA和蛋白表达水平对G9a的抑制率分别为55.2%和54.8%。结论运用pSileneer neo^TM2.1-U6载体构建的G9a-siRNA表达载体可有效抑制G9a在胆管癌细胞株QBC939中的表达。
Objective To construct histone methyhransferase Gga siRNA expressing vector and to exam its inhibiting effect on Gga expression in a cholangiocarcinoma QBC939 cell lines. Method G9a specific oligonucleotides were designed and synthesized. These oligonucleotides were annealed to form a double strand DNA fragments and this fragment was cloned into pSilencer neo^TM 2. 1-U6 vector. The recombinant Gga-siRNA expressing product was confirmed by using EcoR + BamH Ⅰ and EcoR + Hind Ⅲ double digestion and by sequencing. The Gga-siRNA was transfected into QBC939 cell and G418 was used for selecting stable cell line. The inhibitory effect of Gga-siRNA construct was examined with semiquantitative RT-PCR and Western blot. Result Gga-siRNA expression vector was successfully constructed, and it can effectively reduce the mRNA (55. 2% ) and protein (54, 8% ) levels of Gga in stable transfected QBC939. Conclusion The Gga-siRNA expression vector can effectively inhibit the expression of Gga in cholangiocarcinoma cell line QBC939.
出处
《中华普通外科杂志》
CSCD
北大核心
2007年第7期530-532,共3页
Chinese Journal of General Surgery
基金
国家高技术研究发展计划(863计划)资金资助项目(2002AA214061)