摘要
用特异性核酸内切酶ＢａｍＨＩ剪切质粒ｐＣＤ／ｈＧＭ－ＣＳＦ，制备人粒细胞巨噬细胞集落刺激因子基因片段，低融点胶回收后，连接至Ｎ２Ａ载体ＢｇｌⅡ位点，转化大肠杆菌ＤＨ５ａ，经快提质粒进行酶切鉴定和核酸打点杂交筛选出重组质粒Ｎ２Ａ／ｈＧＭ－ＣＳＦ。利用ＤＥＡＥ－葡聚糖介导该重组质粒转化ＣＯＳ－７细胞，收集４８ｈ培养上清液，免疫学和生物学活性检测表明该上清中表达产物具有天然ＧＭ－ＣＳＦ的活性，而用质粒Ｎ２Ａ转化ＣＯＳ－７细胞，培养上清中未检测出ＧＭ－ＣＳＦ活性。
The human GM-CSF gene obtained from the plasmid of pCD/GM-CSF by BamH I was cloned into the retroviral vector N2A in the site of Bgl Ⅱ to construct the expressing vector N2A/hGM-CSF. The recombinant plasmid was selected and identified by restriction map and dot blot. As a transient system. COS-7 cell was transinfected with N2A/hGM-CSF by DEAE-Dextran. ELISA and biological activity assays showed that its expressing ptoducts were similar to the natural human GM-CSF.
出处
《中华实验和临床病毒学杂志》
CSCD
1995年第4期349-352,共4页
Chinese Journal of Experimental and Clinical Virology
