中文摘要：

目的构建并包装以人端粒酶逆转录酶（humantelomerasereversetranscriptase，hTERT）启动子调控E1A（CR2区缺失）蛋白表达的肿瘤细胞双重靶向条件复制型腺病毒（conditionallyreplicatingadenovirus，cRAd），并探讨其对肿瘤细胞的抑制作用。方法采用PCR技术扩增hTERT启动子序列，将其克隆至pShuttle—EIA—E1Bp—E1855K载体（E1A基因CR2区缺失）中，构建重组腺病毒质粒，并转染HEK293细胞进行病毒包装，PCR鉴定；CCK-8法检测重组腺病毒（CRAd．p-hTERT-E1A-EIBp-E1855K，简称CRAd．p）对肿瘤细胞的抑制作用。结果hTERT启动子序列克隆正确；pshuttle-hTERT—E1A—E1Bp—EIB55K经KpnI单酶切可见1542bp和6775bp两条电泳带，PCR鉴定得到250、1148bp和298bp基因片段；CRAd．P经PCR扩增得到250、1148bp和298bp基因片段，鉴定结果与预期完全相符；CRAd．P感染MCF-7、MDA—MB-231、HepG2和HTC-8细胞72h后与各自对照组比较差异均有统计学意义（P〈O．01）；CRAd．P感染MDA-MB-231细胞后10～250MOI组细胞吸光度值与对照组比较差异均有统计学意义（P〈O．05或P〈O．01），随着感染时间延长，抑制作用越发明显。结论成功获得对肿瘤细胞具有双重靶向和溶瘤作用的条件复制型腺病毒，为基因治疗的靶向性和有效性提供了可能。

英文摘要：

Objective To construct and package double-targeted conditionally replicative adenovirus （CRAd. p） regulated by hTERT promoter E1A protein （CR2 missing） expression and to study its inhibitory effect on tumor cells. Methods The hTERT promoter was amplified by RT-PCR and cloned into the shuttle vector of pShuttle- hTERT-E1A-E1Bp-E1B55K. Then it was transfected into HEK293 to package CRAd. p. Anti-tumor effect of CRAd. p was detected by CCK-8 assay. Results The sequence of amplified hTERT was consistent with that published on GenBank, indicating that the hTERT was cloned successfully. Furthermore, the recombinant plasmid pShuttle-hTERT-ElA-E1Bp-E1B55K was identified by endonuclease digestion and PCR, which could be digested into two fagments of Kpn I . One was 1 542 bp and the other was 6 775 bp. The length of fragments amplified by PCR was 250 bp, 1 148 bp and 298 bp, which were consistent with those amplified fragments of recombinant adenovirus. CRAd. p was identified by PCR. These results were in agreement with the plan. The results of CCK-8 assay showed that the inhibitory effects of CRAd. p on the MCF-7, MDA-MB-231 and HepG2 cells were significantly different from their control groups （P〈0.01）, especially on MDA-MB-231 cells. The inhibition of therecombinant adenovirus on HCT-8 recombinant adenovirus at 24, ,18 adenovirus doses, the inhibition of virus is packed successfully, which ceils was irregular. The MDA-MB-231 and 72 hours after infection. Their cells were infected with different titers of results showed that with the increase of cellular growth increased gradually. Oonclusion A double-targeted oncolytic may make gene therapy targeting and effective.