hGM-CSF基因在哺乳动物细胞中稳定转染表达质粒的构建和鉴定

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

Construction and Identification of hGM CSF Expressing Plasmid Which can be Stably Expressed in Mammalian Cells

Obiective:The recombinant human glanulocyte macrophage colony stimulating factor (rhGM CSF) with natural glycosylation structure was to be obtained.Method:The eukaryocyte expressing vetor containing sRa promotor and neomycin resistance gene was used,and then it was connected to Xhol enzyme digested hGM CSF cDNA fragment,thereby pMEneo hGM CSF plasmid which could stably experss recombinant hGM CSF in mammalian cells was constructed.Results:8 of 22 pMEneo reconstructed clones contained hGM CSF cDNA fragment.Determined by enzyme digestion,the plasmids had five cis forms,one cisroasary and two trans insertions.Conclusion:The cos cells transfected by DEAE method in the mentioned above forms could traniently express the identification.The activity of hGM CSF in culture supernatant in cis form and cis rosary form was (1×10 3～1×10 4)U/ml,and there was no activity of the culture supernatant in trans insertion.