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目的:构建人癌胚抗原(CEA)的迷你基因串联体肿瘤疫苗。方法:通过PCR从人基因组中得到一段来源于CEA的DNA序列,其中包含两个编码辅助性T细胞(HTL)表位的迷你基因,插入pGEM-T easy载体后测序,再将目的基因以同尾酶连接的方式顺次定向亚克隆到真核表达载体pcDNA3.0中,得到三串联体,以PCR和酶切方法鉴定构建的重组真核表达质粒pcDNA3.0-triCEA625-667。结果:PCR和酶切结果表明,获得的目的基因与所选的基因片段CEA625-667中包含的碱基数量吻合,且带有正确的酶切位点。测序结果与GenBank登记完全相同。构建的重组真核表达质粒中含有正确编码的经同尾酶连接的目的基因片段三倍体。结论:成功地构建了含有CEA625-667基因三串联体的DNA疫苗。
Objective: To construct a mini-tandem vaccine against human carcinoembryonic antigen (CEA). METHODS: A DNA fragment derived from CEA was obtained from the human genome by PCR, which contained two mini-genes coding for helper T cell (HTL) epitopes, inserted into the pGEM-T easy vector and sequenced. End-directed ligation was subcloned into the eukaryotic expression vector pcDNA3.0 in sequence to obtain three tandem bodies. The recombinant eukaryotic expression plasmid pcDNA3.0-triCEA625-667 was identified by PCR and restriction enzyme digestion. Results: The results of PCR and restriction enzyme digestion showed that the obtained target gene was consistent with the number of bases contained in the selected gene fragment CEA625-667, with the correct restriction site. Sequencing results identical to GenBank registration. The constructed recombinant eukaryotic expression plasmid contains a correctly encoded triploid end-twitch linked gene fragment of triploid. Conclusion: The DNA vaccine containing the triple tandem CEA625-667 gene was successfully constructed.