【摘要】 目的:构建负载人端粒酶逆转录酶(hTERT)基因的复制缺陷型腺病毒.方法:将克隆有正义hTERTcDNA的腺病毒穿梭质粒pDC315-hTERT与腺病毒包装质粒pBHGlox-DeltaE1,3Cre共转染人胚肾293细胞(HEK293),包装负载hTERT基因的复制缺陷型腺病毒表达载体(Ad-hTERT).对Ad-hTERT扩增、纯化并浓缩后,进一步行感染性鉴定、电镜鉴定及双引物PCR鉴定.结果:纯化浓缩后的Ad-hTERT滴度可达5×1012pfu/L.电镜下可见在HEK293细胞中形成的病毒颗粒,PCR双引物鉴定Ad-hTERT可扩增出Ad及hTERT特异片段,而对照则不能扩出hTERT片段.结论:成功构建了负载hTERT基因的复制缺陷型腺病毒.更多还原

【Abstract】 AIM: To construct a replication-deficient recombinant adenovirus expression vector of hTERT （Ad-hTERT）. METHODS: The hTERT gene was cloned at the downstream of CMV promoter of the adenoviral shuttle plasmid pDC315 in sense direction and the resultant recombinant plasmid pDC315-hTERT was cotransfected into HEK 293 cells together with plasmid pBHGlox （deltaE1, 3） containing adenoviral genome. The adenovirus expression vector was then obtained and identified by infection test, electron microscopic observation and PCR co-amplification. RESULTS: After purification and concintration, the titer of Ad-hTERT reached 5×10¹² pfu/L. Virus particles could be found in HEK 293 cells under transmission electron microscope. Both adenovirus and hTERT special fragments could be amplified from Ad-hTERT by PCR, whereas hTERT special fragment could not be amplified from the control. CONCLUSION: The replication-deficient recombinant adenovirus expression vector of hTERT is successfully constructed.更多还原