【摘要】 目的构建转录端粒酶逆转录酶(hTERT)基因小发夹RNA(hTERT-shRNA)的条件增殖腺病毒。方法设计能转录hTERT-shRNA的模板DNA序列,煺火后克隆至pCA13质粒,构建重组质粒pCA13-hTERT。用Bg1Ⅱ从pCA13-hTERT酶切出包含CMV启动子及hTERT-shRNA模板的表达框,将表达框克隆入条件增殖腺病毒质粒pZD55,构建重组质粒pZD55-hTERT。将pZD55-hTERT与腺病毒右臂质粒pBHGE3共转染293细胞,9~12天后出现病毒空斑。提取重组腺病毒的DNA,PCR鉴定正确者即为条件增殖腺病毒ZD55-hTERT。大量扩增,氯化铯梯度离心纯化,测滴度。感染人肝癌细胞株(BEL-7404),通过荧光显微镜、结晶紫染色法观察细胞病作用,MTT法检测细胞存活情况。结果酶切分析、测序鉴定表明pCA13-hTERT构建成功;PCR、酶切分析、测序鉴定表明pZD55-hTERT构建成功;PCR鉴定表明ZD55-hTERT包含目的基因且无野生型腺病毒的污染;ZD55-hTERT滴度为1×1011PFU/m l;ZD55-hTERT在肝癌细胞株BEL-7404中可导致明显细胞病变效应。结论成功构建的ZD55-hTERT为利用hTERT-shRNA靶向肿瘤治疗奠定了基础。更多还原

【Abstract】 Objective To construct conditionally replicative adenovirus(CRAds) expressing short hairpin RNA targeting hTERT gene(hTERT-shRNA).Methods hTERT-shRNA template DNA sequences were designed and synthesized.The annealed shRNA template was inserted into pCA13 plasmid to construct the recombinant plasmid(pCA13-hTERT).It was identified that vector had been constructed successfully.The pCA13-hTERT plasmid was cut with Bg1Ⅱto get the expression cassette of hTERT-shRNA,then the expression cassette was cloned to pZD55 which had been cut with Bg1Ⅱ and dephosphated to form pZD55-hTERT plasmid.It was identified that the pZD55-hTERT had been constructed successfully.The plasmid pZD55-hTERT shRNA and pBHGE3 were transfected in 293 cells together to construct the recombined CRAds.The viral plaques of CRAds appeared 9～12 days after infection.The DNA extracted from CRAds was verified by PCR.Viruses were plaque purified,propagated on HEK293 cells and purified by CsCI gradient according to standard techniques,and functional PFU titers were determined by plaque assay on 293 cells.The anti-tumor potential to BEL-7404 was evaluated by fluorescence microscopy,MTT assay and crystal violet dye method.Results The successful cloning of hTERT-siRNA template DNA sequences into pCA13 was confirmed by the analysis of restriction enzyme digestion and nucleotides sequencing technique.pZD55-hTERT was formed successfully by the analysis of PCR,restriction enzyme digestion and nucleotides sequencing technique.The analysis of PCR indicated the recombinant adenovirus ZD55-hTERT contained hTERT-siRNA gene without wild adenovirus.Functional PFU titers of ZD55-hTERT were 1×10(11)PFU/ml.In vitro,ZD55-hTERT had anti-tumour effect on hepatic carcinoma cells(BEL-7404).Conclusion CRAds expressing hTERT-shRNA may be used for further research on Gene-Viro therapy of cancer.更多还原