pLEGFP—N1-端粒酶逆转录酶载体的构建及其表达

Construction of recombinant retrovirus vector pLEGFP-N1-telomerase reverse transcriptase and its expression

目的构建逆转录病毒载体pLEGFP—N1．端粒酶逆转录酶（TERT），并观察其在新生鼠真皮细胞中的表达。方法将聚合酶链反应（PCR）扩增出TERT全部片段，定向连接到被Hindm和Sall双酶切的质粒，经酶切测序鉴定后筛选阳性克隆。将pLEGFP-N1—TERT质粒和PIKpackaging质粒以磷酸钙共沉淀法转染包装细胞293FT，包装产生逆转录病毒。进行病毒滴度测定后，取病毒上清感染新生鼠真皮细胞，筛选转基因细胞。比较转染前后TERT在mRNA水平的表达，端粒酶活性的变化，荧光显微镜观察细胞荧光表达。结果BamHI酶切鉴定及测序鉴定，证明pLEGFP-N1-TERT构建成功。转染后细胞稳定表达绿色荧光蛋白，并经Westernblots及免疫组织化学检测TERT高表达。结论构建的逆转录病毒表达载体pLEGFP—N1-TERT能够产生高滴度的逆转录病毒，促使外源性TERT基因转入新生鼠真皮细胞后得到高表达，为皮肤组织工程及细胞移植疗法奠定基础。

Objective To construct a recombinant retrovirus vector pLEGFP-N1-TERT, and to observe its expression in dermal cells of newborn mice. Methods The gene encoding telomerase reverse transcriptase （TERT） was amplified by polymerase chain reaction （PCR） and inserted into pLEGFP-N1 which was digested by Hind III and Sal I. After identification by restriction enzyme digestion and sequencing, calcium phosphate precipitation method was used for the transfection of package cell line 293, and the recombinant pLEGFP-N1-TERT virus was obtained. After measuring the infectivity titre of virus, the particles were directly transfected into the dermal cells of newborn mice. The transgenetic cells were selected and the expression of TERT mRNA was detected. The expression of GFP in cells was examined under the fluorescence microscopy. Results The pLEGFP-N1-TERT was successfully constructed and confirmed by restriction endonuclease digestion and sequencing. When the cells were transfected, the bright green florescence in transfected cells was observed. The expressions of TERT was increased by Western blotting and immunohistochemistry. Conclusion The pLEGFP-N1-TERT can produce high titer retroviral viruses and enhance the expression of exogenous TERT gene, which establishes a basis for tissue-engineered skin.