重组人干细胞因子在昆虫细胞中的高效表达

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。

EXPRESSION OF RECOMBINANT HUMAN STEM CELL FACTOR IN INSECT CELLS

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 x 10(3), 20 x 10(3) and 22 x 10(3) were detected by immunoblotting.