逆转录病毒表达载体N2A／hGM－CSF的构建及其在COS－7细胞的初步表达  

Construction of Retroviral Expressing Vector N2A/hGM-CSF and its Preliminary Expression in COS-7 Cell

作　　者：陈庆华[1] 路秀华[1] 张智清[1] 毕秋娜 李玉英[1] 侯云德[1] 

机构地区：[1]中国预防医学科学院病毒学研究所

出　　处：《中华实验和临床病毒学杂志》1995年第4期349-352,共4页Chinese Journal of Experimental and Clinical Virology

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.

关 键 词：HGM-CSF 集落刺激因子 逆转录病毒 COS-7细胞 

分 类 号：R392-33[医药卫生—免疫学]