hTERT基因片段真核表达质粒载体的构建

Construction of Eukaryotic Expression Plasmid of hTERT

目的构建人端粒酶逆转录酶(hTERT)片段的基因表达载体,为人们建立永生化的细胞系提供材料,为临床细胞移植的实验研究和疾病治疗奠定实验基础。方法根据hTERT全长cDNA序列设计引物进行PCR扩增,扩增用的模板是人胎脑cDNA文库,PCR扩增产物与pGEM-T载体连接后进行测序鉴定。结果 PCR扩增及限制性酶切法证实重组真核表达质粒的构建成功。产物测序表明,hTERT基因片段与GenBank公布的相应基因同源性比较,核苷酸序列的同源性为98.87%,氨基酸序列的同源性为99.67%。结论成功构建了hTERT真核表达质粒。

Objective To construct human telomerase reverse transcriptase （hTERT）expression plasmid,which is helpful to establish immortalized cell lines and begin clinical cell transplantation in experimental study and treatment of disease. Methods The hTERT full-length cDNA sequence was amplified by RT-PCR using a human fetal brain cDNA library. The amplified product was ligated with the pGEM-T vector,which was followed by DNA sequencing. Results Eukaryotic expression plasmid of pGEM-T-hTERT was identified by the digestion of restriction endonuclease. The DNA sequence of hTERT fragment was compared with that in GenBank for homology. The homology was 98.87 % in nucleotide acid,and 99.67 % in amino acid. Conclusion The eukaryotic expression plasmid of hTERT has been successfully constructed.