小鼠端粒酶蛋白亚单位真核表达载体的构建和序列测定

Construction and identification of mouse telomerase reverse transcriptase eukaryotic expression vector

目的:克隆小鼠端粒酶蛋白亚单位(mTERT)的cDNA,构建真核表达载体并进行序列测定。方法:从肝癌细胞中提取总RNA,采用RT-PCR技术扩增mTERT的基因编码区序列,将序列定向克隆至真核表达载体pAC,并进行序列测定。结果:获得mTERT编码区3 369 bp的cDNA片段,用PCR和限制性内切酶分析鉴定,表明获得重组质粒pAC-mTERT。通过对mTERT编码区cDNA序列测序证实其与GenBank中的已知序列一致。结论:成功克隆了mTERT编码区序列,并构建了其真核表达载体,为以TERT为基础的肿瘤生物治疗奠定基础。

AIM： To clone and express mouse telomerase reverse transcriptase （mTERT） cDNA and obtain eukaryotic expression vector. METHODS： The cDNA of mTERT was amplified by RT - PCR and PCR. After purification, the gene fragment was cloned into a vector PUC- 19. The sequence of inserted mTERT gene fragment was also detected. RESULTS： The recombi- nant plasmid PUC - 19 - mTERT was constructed. Positive clones were screened and identified by PCR and digestion with restriction enzyme. The size of gene fragment was 3 369 bp and in accordance with the expected one. CONCLUSION： The mTERT cDNA was obtained and the recombinant eukaryotic expression vector was successfully constructed. The study lays foundation for DCs vaccine modified by mTERT gene for the treatment of tumor.