hTERT核心启动子调控的hNIS重组腺病毒的构建与鉴定

Construction and verification of adenovirus containing the hNIS gene under the control of the hTERT core promoter

目的:构建含人端粒酶逆转录酶(hTERT)核心启动子调控的人钠碘转运体(hNIS)基因重组腺病毒,并初步鉴定。方法:将hTERT核心启动子从hTERT/pcDNA3.1(+)质粒中切出,亚克隆至含全长hNIS cDNA序列的质粒载体FL*-hNIS/pcDNA3,应用限制性内切酶将hTERT-hNIS片段切出后连入穿梭质粒载体pAdTrack,应用AdEasy系统构建出重组腺病毒Ad-hTERT-hNIS。同时,构建CMV启动子调控的hNIS重组腺病毒Ad-CMV-hNIS作为阳性对照。应用RT-PCR方法验证hTERT在转染肿瘤细胞中的转录活性。结果:成功构建重组腺病毒Ad-hTERT-hNIS、Ad-CMV-hNIS并经PCR验证正确。RT-PCR证实hNIScDNA能从Ad-hTERT-hNIS转染的细胞中扩增出来。结论:成功构建重组腺病毒Ad-hTERT-hNIS;hTERT核心启动子在转染的肺癌A549细胞中具有转录活性。为下一步的碘治疗实验提供了实验依据。

Objective： To construct a recombinant adenovirus harboring the human sodium iodide symporter （hNIS） under control of the human telomerase reverse transcriptase （hTERT） core promoter. Methods： The hTERT core promoter was cut from plasmid hTERT/pcDNA3.1 （ ＋ ）, and then subcloned into plasmid vector FL ＊ -hNIS/pcDNA3 with the full-length hNIS cDNA, and then the hTERT-hNIS sequence was subcloned into the shuttle plasmid pAdTrack. The recombinant adenovirus Ad-hTERT-hNIS was constructed by AdEasy system. In addition, a positive control of adenovirus Ad-CMV-hNIS containing the CMV promoter followed with hNIS gene was created by AdEasy system. The tran scriptional activity of hTERT was examined by RT-PCR in transiently transfected A549 cell lines. Results： Recombinant Ad-hTERT-hNIS was correctly constructed and coni＇mned by restriction enzyme analysis and PCR. RT-PCR showed that hNIS cDNA could be amplified from transfected cells. Conclusion： The recombinant adenovirus Ad-hTERT-hNIS is constructed. This study demonstrates that hTERT core promoter has transcriptional activity in transfected A549 cells, and it provides the foundation for further research.