hTERT片段真核表达质粒的构建

Construction of eukaryotic expression plasmid of human telomerase reverse transcriptase

目的 :构建人端粒酶逆转录酶 (hTERT)片段的真核表达质粒 ,方法 :从肿瘤组织中提取总RNA ,采用RT PCR法扩增hTERT基因并克隆入PGEM TEasy载体中 ,然后再亚克隆于pcDNA3.1真核表达载体 ,用PCR扩增及限制性酶切法筛选重组真核表达质粒 ,并以DNA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。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 98.73% ,氨基酸序列的同源性为 99.6 8%。结论 :成功构建了hTERT真核表达质粒 。

Aim: To construct eukaryotic expression plasmid of human telomerase reverse transcriptase (hTERT). Methods: Genomic fragment of hTERT was amplified by RT PCR and inserted into the PGEM T Easy vector. The recombinant plasmid was cleaved by restriction endonuclease and subcloned into eukaryotic expression vector pcDNA3.1, and then transformed into the E.coli JM109. The recombinant plasmid was identified by PCR and restriction endonuclease .The genomic fragment of hTERT was determined and analyzed by DNA sequencing. Results: Eukaryotic expression plasmid of pcDNA3.1 hTERT was identified by and restriction endonuclease. hTERT fragment was compared with hTERT gene reported in GenBank for homology. The homology was 98.73% in nucleotide acid, and 99.68% in amino acid. Conclusion: The eukaryotic expression plasmid of hTERT has been successfully constructed. It will lay the foundation for anti tumor immunity therapy associated with hTERT.