摘要
目的构建以人端粒酶逆转录酶(human telomerase reverse transcriptase,hTERT)为永生化基因、loxP为重组位点的可回复性永生化逆转录病毒载体。方法采用EcoRⅠ、SalⅠ双酶切、回收含有EGFP-胸苷激酶融合基因及LoxP序列的逆转录病毒载体pBTGTKlox,将经PCR扩增的IRES序列克隆入其中,构建成pI-GTKlox;将hTERT克隆入pI-GTKlox的EcoRⅠ酶切位点,构建成phTERT-I-GTKlox。进行菌落PCR、酶切、测序鉴定。结果菌落PCR鉴定5个菌落中2个为阳性克隆。阳性克隆质粒经EcoRⅠ酶切鉴定形成预计的6.5kb及3.5kb两个片段,进一步经测序证实插入的hTERT序列无核苷酸突变。结论成功构建了phTERT-I-GTKlox可回复性永生化逆转录病毒载体,为建立可回复性永生化胰岛细胞奠定了基础。
Objective To construct a reversible immortalization retroviral vector carrying immortalizing gene human telomerase reverse transcfiptase （hTERT） and recombination site sequence loxP. Methods The retroviral vector pBGTKlox carrying EGFP- thymidine kinase fusion gene and loxP sequence was digested by restriction enzyme EcoR Ⅰ and Sol Ⅰ . IRKS sequence was amplified by PCR, and then cloned to the retroviral vector pBGTKlox to establish a new vector pI-GTKlox. The gene coding hTERT was cloned to EcoR Ⅰ site of pI-GTKlox to establish phTERT-I-GTKlox, phTERT-I-GTKlox was identified by bacterial colony PCR, restriction endonuclease digestion, and DNA sequencing. Results In the five colonies resulted from our experiment, two positive colonies were identified by bacterial colony PCR. As expectably, two fragments of 6.5 kb and 3.5 kb were found in the positive colony plasmid digested by EcoR Ⅰ . And the cloned hTERT sequence was further verified by DNA sequencing. Conclusion The retroviral vector phTERT-I-GTKlox for reversible immortalization is successfully constructed. Thus it provides an ideal vector in the reversible immortalization of β-cells.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2008年第2期226-229,共4页
Immunological Journal
基金
第三军医大学校科研创新基金资助项目(2007XG25)
关键词
可回复性永生化
条件基因敲除
人端粒酶逆转录酶
Reversible immortalization
Conditional gene knockout
Human telomerase reverse transcriptase
