论文部分内容阅读
利用ＰＣＲ反应、ＤＮＡ测序、基因重组等技术，构建了两个表达人粒细胞集落刺激因子ｃＤＮＡ的重组质粒ｐＥＤ－ＧＣＳＦ和ｐＥＦ－ＧＣＳＦ，两质粒分别转染ＣＯＳ７细胞作瞬时表达，转染ＣＨＯ－ｄｈｆｒ－细胞作稳定表达。结果两质粒在ＣＯＳ７细胞和ＣＨＯ细胞均获得了表达，ｐＥＤ－ＧＣＳＦ转染ＣＯＳ７细胞４８ｈ、７２ｈ的表达量分别为５２×１０４ｐｇ／ｍｌ和２３×１０５ｐｇ／ｍｌ，ｐＥＦ－ＧＣＳＦ转染ＣＯＳ７细胞后４８ｈ、７２ｈ的表达量分别为２８×１０５ｐｇ／ｍｌ和１４×１０５ｐｇ／ｍｌ。转染ＣＨＯ－ｄｈｆｒ－细胞，随着加入的氨甲喋呤（ＭＴＸ）浓度升高，ＣＨＯ－ｄｈｆｒ＋克隆数减少，但平均每个克隆的ｒｈＧ－ＣＳＦ表达量升高，在０５μｍｏｌ／ＬＭＴＸ下最高表达ｒｈＧ－ＣＳＦ细胞株的量是４４６μｇ／ｍｌ／３ｄ。且表达的ｒｈＧ－ＣＳＦ注射小鼠腹腔可提高小鼠外周血白细胞的数量。
Two recombinant plasmids pED-GCSF and pEF-GCSF expressing human granulocyte-colony stimulating factor cDNA were constructed by PCR, DNA sequencing and gene recombination. The two plasmids were transfected into COS7 cells for transient expression and transfected into CHO- dhfr- cells for stable expression. Results The two plasmids were expressed in COS7 cells and CHO cells. The expression of pED-GCSF transfected COS7 cells for 48 h and 72 h were 5.2 × 104 pg / ml and 2 × 3 × 105 pg / ml, respectively. PEF-GCSF The expression of COS7 cells 48h, 72h after the expression of 2 8 × 105pg / ml and 1 4 × 105pg / ml. CHO-dhfr + cells were transfected with CHO-dhfr + clones as the concentration of methotrexate (MTX) was increased, but the average amount of rhG-CSF expression per clone was increased, with the highest expression at 0.5 μmol / LMTX The amount of rhG-CSF cell line was 4 46 μg / ml / 3d. And the expression of rhG-CSF in mice injected intraperitoneally increased the number of mouse peripheral blood leukocytes.