| 摘要: |
| 【目的】构建能够表达人端粒酶逆转录酶（hTERT）基因的腺病毒。【方法】通过对PCI-neo-hTERT质粒的改造，依次构建出JMB293-hTERT、pAdTrack-hTERT载体，将pAdTrack-hTERT经PmeⅠ线性化后与pAdEasy-1载体共转化到 BJ5183 细菌中进行重组，经卡那霉素抗性筛选获得重组腺病毒载体pAdEasy-hTERT。将pAdEasy-hTERT PacⅠ酶切线性化并回收后，转染HEK293人胚肾细胞，培养7 d后，收集细胞并反复冻融，收集细胞裂解液；反复扩增3次后，测定重组hTERT腺病毒的滴度。【结果】①利用酶切连接的方法得到了穿梭载体pAdTrack-hTERT；②通过同源重组将外源目的基因hTERT整合到腺病毒骨架载体中，得到重组质粒pAdEasy-hTERT；③成功获得了滴度为6.73×1010 GFU/mL的高滴度腺病毒颗粒。【结论】含有hTERT基因的重组腺病毒颗粒包装成功，为后续哺乳动物原代细胞永生化研究奠定了基础。 |
| 关键词: 人端粒酶逆转录酶(hTERT)基因 重组腺病毒 细胞永生化 |
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| 基金项目:国家“863”高技术研究与发展计划项目“多位点基因安全转移技术”（2009AA10Z110） |
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| Construction and packaging of a recombination adenovirus expressing human telomerase reverse transcriptase (hTERT) gene |
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| Abstract: |
| 【Objective】The goal of this study was to construct the recombination adenovirus expressing human telomerase reverse trancriptase (hTERT) gene.【Method】Based on the PCI-neo-hTERT vector,we constructed JMB293-hTERT/pAdTrack-hTERT/pAdEasy-hTERT vectors in turn.The linear pAdTrack-hTERT vector generated by digestion with PmeⅠ enzyme was cotransformed into BJ5183 cells with pAdEasy-1,and the recombination adenovirus vector was obtained with the selection of antibiotic kanamycin.Then the recombinant viral vector was transfected into HEK293 cell for viral packaging after linearization with PacⅠ enzyme.The cells were frozen and thawed about 7 days after transfection,and the lysates were harvested.Transfection-culture-harvest was repeated three times to obtain high titer of the recombinant adenovirus.【Result】①pAdTrack-hTERT vector was constructed.②The recombinant adenovirus vector pAdEasy-hTERT vector was generated in bacterial strain BJ5183.③The titer of recombinant adenovirus was 6.73×1010 GFU/mL.【Conclusion】We have successfully obtained the recombinant adenovirus which will be used for generating transforming mammalian primary cells in future. |
| Key words: human telomerase reverse trancriptase（hTERT)gene recombinant adenoviruses immortalization |
