重组人干细胞因子在昆虫细胞中的高效表达  

EXPRESSION OF RECOMBINANT HUMAN STEM CELL FACTOR IN INSECT CELLS

作　　者：张冬梅[1] 秦浚川[1] 臧宇辉[1] 朱洁[1] 沈倍奋[2] 

机构地区：[1]南京大学医药生物技术国家重点实验室,南京210093 [2]军事医学科学院基础医学研究所,北京100850

出　　处：《微生物学报》2000年第1期44-49,共6页Acta Microbiologica Sinica

摘　　要：含信号肽的可溶性人干细胞因子（ｈＳＣＦ）ｃＤＮＡ 基因重组于杆状病毒转移载体ｐＶＬ９４１ 中，重组转移载体ｐＶＬ９４１ＳＣＦ与野生型苜蓿夜蛾核型多角体病毒（ＡｃＮＰＶ）ＤＮＡ 共转染草地夜蛾细胞Ｓｆ９ 后，通过体内同源重组构建了重组病毒ＡｃＮＰＶＳＣＦ。Ｓｏｕｔｈｅｒｎ 杂交表明重组病毒基因组中含有ｈＳＣＦ基因片段。重组病毒感染单层Ｓｆ９ 细胞后，表达产物分泌到胞外培养液中。用ＭＴＴ 比色法和ＴＦ１ 细胞株测定表达产物与ＩＬ３ 的协同效应，测得感染重组病毒的培养细胞第三天表达量为１９７０ ｕｎｉｔｓ／ｍ Ｌ培养液。Ｗｅｓｔｅｒｎｂｌｏｔｔｉｎｇ 分析可见分子量为１８ ×１０３ 、２０ ×１０３ 和２２ ×１０３ 三条带。The cDNA of human stem cell factor(hSCF) containing signal sequence was cloned into the transfer vector pVL941 of AcNPV to construct a recombinant transfer vector pVL941\|SCF. Sf9 cells were cotransfected with wild type viral DNA and pVL914\|SCF to produce the recombinant virus AcNPV\|SCF by homologous recombination in cell. Southern\|hybridization analysis suggested that the recombinant viral DNA contained hSCF cDNA fragment. The Sf9 cells infected with the recombinant baculovirus AcNPV\|SCF expressed biologically active rhSCF which was secreted into the cell culture. The synergistic activities of SCF in conjunction with human interleukin\|3(hIL\|3) was measured by MTT colorimetric method and TF\|1 cell line. The expression level of Sf9 cells reached its highest at about 1970 units/ml in the 3rd day after the infection with AcNPV\|SCF. Three SCF bands with molecular masses of 18×10 3、20×10 3 and 22×10 3 were detected by immunoblotting.

关 键 词：人干细胞因子 ACNPV 昆虫细胞表达 造血功能 

分 类 号：Q786[生物学—分子生物学]