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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。
OBJECTIVE: To clone the cDNA of mouse telomerase protein subunit (mTERT), construct eukaryotic expression vector and determine the sequence. Methods: Total RNA was extracted from hepatoma cells. The mTERT gene coding region sequence was amplified by RT-PCR. The sequence was cloned into eukaryotic expression vector pAC and sequenced. Results: A 3 369 bp cDNA fragment of mTERT coding region was obtained and identified by PCR and restriction endonuclease analysis. The results showed that the recombinant plasmid pAC-mTERT was obtained. The sequence of mTERT coding region was confirmed to be consistent with the known sequence in GenBank. Conclusion: The sequence of mTERT coding region was successfully cloned and its eukaryotic expression vector was constructed, which laid the foundation for TERT - based tumor biological therapy.