靶向人端粒酶逆转录酶基因小片段干涉RNA慢病毒表达载体的构建与鉴定

The construction of lentivirus-mediated RNAi vector containing hTERT

目的通过构建靶向人端粒酶催化亚基人端粒酶逆转录酶基因（human telomerase reverse transcriptase,hTERT）RNA干涉（RNA interference，RNAi）慢病毒载体，获得可供转染的滴度，为进一步研究该基因缺陷在真核细胞中的影响提供物质基础。方法根据hTERT设计的两条互补的单链寡核苷酸退火后形成双链插入到pLVTHM质粒，生成含RNAi盒的载体，然后与pCMV—dR8．74和pMD2G病毒包装所必须的元件经脂质体导入T293细胞，包装成慢病毒，收集上清浓缩病毒并检测其滴度。所得病毒感染U87胶质瘤细胞，逆转录PCR和端粒重复序列扩增法（telomeric repeat amplification protocol, TRAP）检测干扰后细胞内hTERT表达变化及端粒酶表达变化，流式细胞仪检测细胞凋亡。结果将目的序列成功连接到载体上，并经测序分析证实载体构建成功；成功包装成高滴度的慢病毒。逆转录PER和TRAP检测结果证实构建的hTERT-小片段干涉RNARNA （small interference RNA,siRNA）慢病毒表达载体可显著抑制hTERT基因的表达和端粒酶活性并导致细胞凋亡。结论成功构建了携带靶向hTERT基因的RNAi慢病毒载体。该载体能够有效抑制hTERT的表达和端粒酶活性并导致细胞凋亡，为以hTERT为靶点的胶质瘤基因治疗的后续研究奠定基础。

Objective To construct a recombinant lentivirus RNA interference （RNAi） vector carrying hTERT gene, and to obtain the titer of the lentiviral stock for investigating the expression in the eukaryotic cells and the effect on the hTERT gene silencing in the eukaryotic cells. Methods Two complimentary oligos of small interference RNA （ siRNA） with hairpin structures targeting the hTERT gene and a negative control were synthesized, then ligated with pLVTHM vector and sequenced. The recombinant vectors were then transfected with viral packaging mix into T293 cells, viral supernatant was harvested to detemfine the titer. U87 cells infected by virus were harvested and the expression of hTERT, telomerase activity and apaptosis were detected by reverse transcription-PCR（RT-PCR）, TRAP assay and flow cytometry separately. Results Sequencing data showed that the constructed plasmids contained the correct sequences of hTERT siRNA transcript templates. A vector producing cell line T293 was established, and the titer for transfection was obtained. RT-PCR and TRAP flow cytometry analyses demonstrated that hTERT shRNA expression construct could suppress the expression of hTERT and telomerase activity and induce apaptosis. Condusion A lentivirus RNAi vector targeting hTERT gene was successfully constructed, which decreased the expression of hTERT and telomerase activity effectively and induced apaptosis. It has set up a research platform for the gene therapy of tumors which take hTERT as the target.