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目的：获得具有天然糖基化结构的重组人粒细胞巨噬细胞集落刺激因子（ｒｈＧＭ－ＣＳＦ）。方法：采用含ｓＲα启动子和新霉素抗性基因的真核细胞表达载体ｐＭＥｎｅｏ，接上ＸｈｏＩ接头，与ＸｈｏＩ酶切ｈＧＭ－ＣＳＦｃＤＮＡ片段连接，构建了可在哺乳动物细胞中稳定表达的ｐＭＥｎｅｏ－ｈＧＭ－ＣＳＦ质粒。结果：２２个ｐＭＥｎｅｏ重构载体克隆中，８个含有ｈＧＭ－ＣＳＦｃＤＮＡ片段，酶切鉴定插入方向为５个顺式，１个顺式串珠，２个反式。结论：将上述各式以ＤＥＡＥ法转染ＣＯＳ细胞，瞬时表达鉴定，用ＴＦ１细胞ＭＴＴ法测定培养液上清的ｈＧＭ－ＣＳＦ活性，顺式和顺式串珠培养上清活性为１×（１０３～１０４）Ｕ／ｍｌ，反式插入方向培养上清没有活性
Objective: To obtain recombinant human granulocyte macrophage colony stimulating factor (rhGM-CSF) with natural glycosylation structure. Methods: The eukaryotic expression vector pMEneo containing sRα promoter and neomycin resistance gene was ligated to XhoI linker and ligated with hGM-CSF cDNA fragment of XhoI to construct a recombinant plasmid pMEneo which was stably expressed in mammalian cells. hGM-CSF plasmid. RESULTS: Of the 22 pMEneo reconstructed vector clones, 8 contained hGM-CSF cDNA fragments. The digestion was identified as 5 cis, 1 cis, and 2 trans. CONCLUSION: Transfection of COS cells by DEAE method and identification of transient expression of hGM-CSF in culture supernatants by MTT assay of TF1 cells showed that the activity of cis- and cis-beetrocyte culture supernatants was 1 × (103 ~ 104 ) U / ml, the trans-transfection direction of the culture supernatant no activity