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为了构建负载CML28的核酸疫苗,并在树突状细胞中进行表达,用分子克隆的方法,从K562细胞中克隆出CML28的cDNA全长,将其亚克隆至pGEM-T载体,经测序后酶切,克隆至真核表达载体pcDNA3.1HisA,将重组质粒pcDNA3.1HisA-CML28进行酶切分析和测序鉴定;从正常人外周血中分离外周血单个核细胞(PBMC),在IL-4和GM-CSF条件下诱导分化为树突状细胞(DC),并进行鉴定;用电穿孔的方法将重组质粒pcDNA3.1HisA-CML28导入到DC中,WesternBlot检测His-CML28融合蛋白的表达。结果表明:经酶切分析和测序鉴定,重组质粒pcDNA3.1HisA-CML28含有正确的CML28全长序列;经电穿孔导入DC后,重组质粒能表达His-CML28蛋白。结论:成功构建了CML28树突状细胞核酸疫苗。
To construct a nucleic acid vaccine carrying CML28 and expressed in dendritic cells, the full-length cDNA of CML28 was cloned from K562 cells by molecular cloning and subcloned into pGEM-T vector. After sequencing, the enzyme Cut and cloned into the eukaryotic expression vector pcDNA3.1HisA. The recombinant plasmid pcDNA3.1HisA-CML28 was digested with restriction endonuclease and sequenced. Peripheral blood mononuclear cells (PBMCs) were isolated from normal human peripheral blood and cultured in IL-4 and GM The dendritic cells (DCs) were induced to differentiate into DCs under the conditions of-CSF. The recombinant plasmid pcDNA3.1HisA-CML28 was transfected into DC by electroporation. The expression of His-CML28 fusion protein was detected by Western Blot. The results showed that the recombinant plasmid pcDNA3.1HisA-CML28 contained the correct CML28 full-length sequence after digestion analysis and sequencing. After electroporation into DC, the recombinant plasmid could express His-CML28 protein. Conclusion: CML28 dendritic cell nucleic acid vaccine was successfully constructed.