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目的探索胰腺癌MUC1VNTR核酸疫苗的构建,以进一步研究对胰腺癌的治疗作用。方法首先对VNTR编码基因进行了重组设计,氨基端插入人单核细胞趋化蛋白Ⅰ信号肽基因序列,起始码前插入KOZAK促真核翻译序列。将人工合成的重组人VNTR基因定向克隆入真核表达载体pcDNA3.1/Mychis(+)A质粒的MCS中。将通过测序鉴定的含有准确插入序列的pcDNA3.1VNTR/Mychis(+)A重组质粒转染COS7细胞,进行体外转染实验和Westernblot检测VNTR在细胞内外的表达。结果自转化平板筛选的阳性克隆通过测序鉴定,pcDNA3.1VNTR/Mychis(+)A重组质粒含有完整的阅读框架和目的基因。重组质粒可在COS7细胞内外表达VNTR,以胞内为主。所表达的VNTR分子量比人工合成的VNTR多肽大。结论自行构建的胰腺癌MUC1VNTR核酸疫苗序列准确,可在哺乳动物细胞中表达VNTR。
Objective To explore the construction of pancreatic cancer MUC1VNTR nucleic acid vaccine to further study the therapeutic effect of pancreatic cancer. Methods The recombinant VNTR gene was designed and inserted into the sequence of human monocyte chemoattractant protein Ⅰ signal peptide. The KOZAK eukaryotic translation sequence was inserted before the initiation codon. The synthetic recombinant human VNTR gene was cloned into the MCS of the eukaryotic expression vector pcDNA3.1 / Mychis (+) A plasmid. The recombinant plasmid pcDNA3.1VNTR / Mychis (+) A was identified by sequencing and transfected into COS7 cells. In vitro transfection experiments and Western blot were used to detect the expression of VNTR in vitro and in vivo. Results The positive clone of self-transformation plate was identified by sequencing. The recombinant plasmid pcDNA3.1VNTR / Mychis (+) A contained the complete reading frame and the target gene. Recombinant plasmids can express VNTR both inside and outside COS7 cells, mainly intracellular. The expressed VNTR has a larger molecular weight than the synthetic VNTR polypeptide. Conclusion The self-constructed pancreatic cancer MUC1VNTR DNA vaccine has accurate sequence and can express VNTR in mammalian cells.