论文部分内容阅读
目的:克隆人癌胚抗原(CEA)基因cDNA,构建pcDNA3.1-CEA真核表达载体。方法:采用逆转录聚合酶链反应(RT-PCR)技术,从人结肠癌细胞组织克隆出CEA基因片段,通过基因重组技术将该基因片段重组于pcD-NA3.1真核表达载体上,构建成pcDNA3.1-CEA重组质粒,分别用PCR扩增、酶切电泳分析及DNA测序的方法对重组DNA进行鉴定。结果:经PCR扩增,发现2.1kb的目的基因条带、双酶切电泳分析可见2.1kb的目的基因条带和5.4kb的载体条带、单酶切电泳分析可见0.9kb的条带和6.6kb的条带、DNA测序证实载体中的目的基因序列与CEA的cDNA序列完全相同。结论:本实验成功地克隆了CEA基因并构建了其真核表达质粒,为进一步研究CEA真核表达载体抗肿瘤的实验打下基础。
Objective: To clone cDNA of human carcinoembryonic antigen (CEA) and construct eukaryotic expression vector pcDNA3.1-CEA. Methods: The CEA gene fragment was cloned from human colon cancer cells by RT-PCR. The recombinant plasmid was subcloned into pcD-NA3.1 eukaryotic expression vector to construct PcDNA3.1-CEA recombinant plasmid, respectively, by PCR amplification, restriction endonuclease analysis and DNA sequencing methods to identify recombinant DNA. Results: 2.1kb target gene band and double-enzyme digestion electrophoresis analysis showed 2.1kb target gene band and 5.4kb vector band by PCR amplification, single-enzyme digestion electrophoresis showed 0.9kb band and 6.6 kb, and DNA sequencing confirmed that the sequence of the target gene in the vector was exactly the same as the cDNA sequence of CEA. CONCLUSION: The CEA gene was cloned successfully and its eukaryotic expression plasmid was constructed successfully, which lays the foundation for the further study on anti-tumor activity of CEA eukaryotic expression vector.