人CD14转基因细胞系的建立

本研究构建人CD14真核表达质粒，建立转基因CD14阳性细胞系，为建立急性单核细胞白血病（M5）导向治疗动物模型提供研究材料。从正常人外周血单个核细胞抽提总RNA，以无RNA酶的DNA酶处理，RT-PCR扩增CD14基因，T-A克隆测序并与GenBank中人CD14的基因序列比较核实。通过双酶切和体外连接法将目的基因克隆至表达载体pcDNA3．1（＋）；用Superfect transfection reagent将重组质粒pcDNA3．1（＋）／CD14转染到C57BL／6小鼠黑色素瘤细胞B16，经G418筛选，用流式细胞术检测CD14蛋白的表达情况，初步筛选出CD14阳性的细胞系B16／CD14。结果表明：测序及GenBank中序列比较结果显示扩增到的人CD14基因序列是正确的。酶切结果表明表达质粒构建正确。流式细胞术筛选到2个CD14阳性表达的细胞系B16／CD14（标准CD14-PE阳性细胞百分数分别为25．28％、36．59％，2F9-FITC为25．59％、36．32％）。结论：建立了人CD14抗原阳性表达的鼠细胞系B16／CD14，为人M5动物模型的建立及其导向治疗的研究奠定了坚实的基础。

Establishment of Murine Cell Line Transfected with Human CD14 Gene

This study was aimed to construct the CD14 eukaryotic expression vector, establish the transgeneic CD14 positive cell line in order to facilitate the establishment of a mouse model of antibody targeting therapy for human acute monocytic leukemia (AML-M_5). Total RNA extracted from peripheral blood mononuclear cells was treated with RNAase-free DNAase, the human CD14 gene was cloned and sequenced through the RT-PCR and T-A clone techniques. Eukaryotic expressional vector pcDNA3.1(+)/CD14 was constructed by cleaving with double restriction endonuleases and ligating with T4 ligase. A murine melanoma cell line B16 was transfected with the pcDNA3.1(+)/CD14 recombinant with Superfect transfection reagent. Positive clones were selected by G418 and the expression of human CD14 on the transfectant was confirmed by flow cytometry (FCM). The results indicated that the sequence of the human CD14 cDNA cloned was exact to be same as the one from GenBank database. The recombinant pcDNA3.1(+)/CD14 was identified with double-enzyme cleaving. The expression of the human CD14 on the transfectant (B16/CD14) was confirmed by FCM. In conclusion, the murine cell line B16/CD14 fransfected with human CD14 gene has been established which can be used for the study of human AML-M_5 antibody targeting therapy with mouse model.