人端粒酶反转录酶cDNA编码区的克隆及重组表达载体的构建用于延长组织工程种子细胞寿命的研究

cDNA coding region cloning of human human telomerase reverse transcriptase and construction of recombinant expression vector for prolonging the life span of seed cells of tissue engineering

目的:克隆人端粒酶反转录酶(humantelomerasereversetranscriptase,hTERT)cDNA编码区,构建hTERT重组表达载体,用于延长组织工程种子细胞的寿命。方法:利用具有hTERT表达特异性标志(expressionspecifictag,EST)的商品化cDNA克隆,克隆hTERTcDNA编码区,选择Gateway基因克隆表达系统,将hTERT克隆到Gateway系统的入门载体后,通过LR重组反应,将hTERT分别构建到了天然状态表达载体pcDNA3.2-DEST表达载体和GFP报告基因pDEST53表达载体,同时进行了hTERT细胞定位研究和表达状态研究。结果:使用ESTcDNA克隆进行hTERTcDNA编码区基因克隆,获得了成功。构建了hTERT重组表达载体。克隆的hTERT编码区表达的hTERT蛋白成簇分布在细胞核内,重组的hTERT保持了天然hTERT的特性。结论:使用ESTcDNA克隆进行基因克隆,提供了一种快速进行基因克隆的方法。通过DNA序列分析和hTERT细胞内定位及表达分析,证实了克隆的hTERT编码区序列的正确性和生物学功能,为今后延长组织工程种子细胞的寿命奠定了基础。

AIM:To clone cDNA coding region of human telomerase reverse transcriptase (hTERT) and construct hTERT recombinant expression vector so as to prolong the life of tissue engineered seed cells. METHODS:Coding region of hTERT cDNA was cloned by using cDNA cloning possessing expression specific tag (EST). Based on the recombinant reaction of LR, hTERT was constructed into expression vector of pcDNA3.2 DEST,a natural expression vector, and pDEST53 expression vector of GFP reported gene, following hTERT was cloned into entry vector of Gateway cloning system. Meanwhile, hTERT cell localization and expression were studied. RESULTS:EST cDNA succeeded in cloning coding region of hTERT cDNA,and hTERT recombinant expression vector was constructed successfully.hTERT protein expressed in cloned hTERT coding region was distributed rustlingly in nuclei. Recombinant hTERT retained the natural properties. CONCLUSION:EST cDNA cloning provides a quick may for gene cloning. Correctness and biological function of cloned hTERT coding region are proved by analysis of DNA sequence,hTERT intracellular localization and expression, which establishes a basis for prolonging the life of tissue engineered seed cells in further studies.