中文摘要：

目的：设计靶向人端粒酶反转录酶的RNAi序列，构建shRNA表达载体质粒和hTERT真核表达质粒，采用共转染工具细胞筛选出有效靶点。方法：首先以EASY^TM siRNA软件针对hTERT设计5个靶点siRNA序列和1条通用阴性对照序列．合成shRNA oligo表达框架，将其分别连接至经酶切消化的载体质粒、pGCsi—U6／Neo／GFP、pGCL—GFP，重组质粒转化DH5α，阳性克隆进行PCR与测序鉴定；然后从cDNA文库中利用PCR钓取hTERT基因片段，与表达载体pEGFP—C1分别进行双酶切、纯化及定向连接、重组、转化，阳性克隆行PCR与测序鉴定，荧光镜检GFP表达；最后上述2个质粒系统共转染293T细胞，Western印迹检测hTERT蛋白表达水平，筛选最有效序列。应用SPSS16．0软件包对所得数据进行单因素方差分析。结果：BLAST检索5条hTERTsiRNA序列均能100％命中，且不与其他基因同源：PCR鉴定及阳性克隆测序验证shRNAoligo与载体质粒成功连接、重组。hTERT基因PCR钓取片段产物大小1．4Kp，经酶切与质粒连接，形成pEGFP—C1-hTERT表达载体，阳性克隆PCR、测序鉴定确定成功重组；转染293T细胞48h时荧光镜下可观察到GFP标记的hTERT融合蛋白。hTERT表达质粒和shRNA载体质粒共转染293T细胞．48h时Western印迹检测结果显示：实验各组hTERT蛋白量均有不同程度减少，而内参对照β-aetin和GAPDH无明显变化。经β—actin校正，得到各组hTERT蛋白相对表达量：KDNo．1-5实验组与NC组相比，hTERT基因表达均得到不同程度的敲减（54．61％．76．84％，P〈0．05），其中No.4敲减效能最高。结论：5条RNAi设计序列均可有效、特异性地沉默hTERT基因的转录和表达水平，其中以5’-GCAAGTTGCAAAGCATTGGAA-3’敲减效能最优；构建外源基因的过表达载体质粒转染工具细胞，可作为RNAi敲减效率筛选的验绩标靶。

英文摘要：

PURPOSE： To design RNAi sequences targeting hTERT and to construct hTERT-shRNA vector plasmids and hTERT eukaryotic expression plasmid. To screen the most efficient knock-down sequence by co-transfection of two plasmids above to 293T cells. METHODS： First, five siRNA sequences targeting hTERT and one general negative control sequence were designed by EASyTMsiRNA. The 6 shRNA oligo expression frames were synthesized and linked into vector plasmids, pGCsi-U6/Neo/GFP and pGCL-GFP, which contained U6 promoter, respectively. The recombinant vectors were transformed into competent cells, DH5α, and were identified by PCR and sequencing of positive clones. Then, hTERT gene segment was obtained by PCR from cDNA library. Double digested hTERT gene segment was inserted into pEGFP- C1 vector and was confirmed by PCR and sequencing after transformed into competent cell. GFP expression was detected by fluoroscope at 48 hour when pEGFP-C1-hTERT was transfected into 293T cells. Finally, total proteins were extracted from 293T cells co-transfected by shRNA expression and hTERT expression vector plasmids at 48 hour. HTERT protein was detected by Western blot. hTERT protein gray scale values were calibrated by β-actin, SPSSTM v16.0 software package was used for one-way ANOVA to judge different knock-down efficiency of different target sites in which the most potent one was chosen as the consequent experiments＇ sequence. RESULTS： In BLAST searching, all five designed siRNA sequences were 100% specially targeted hTERT transcript variant 1 and had no homology with any other genes. The general negative control sequence had no homology with any genes. The six synthesized shRNA double strains frames with cohesive ends were correctly inserted and recombinated into vectors above, respectively, indentified by PCR and sequencing of Amp-screened positive clones picked from competent cell transformation. Agarose gel electrophoresis results affirmed double digested hTERT gene segment, 1.4 kp, and pEGFP-C1, 4.7 kb, both concorded to ex