Park J H, Chang K H, Lee J M, Lee Y H, Chung I S
Department of Genetic Engineering, Kyung Hee University, Suwon, South Korea.
In Vitro Cell Dev Biol Anim. 2001 Jan;37(1):5-9. doi: 10.1290/1071-2690(2001)037<0005:opaiva>2.0.co;2.
Recombinant plasmids containing a complementary deoxyribonucleic acid coding mouse endostatin were transfected and stably expressed in Drosophila melanogaster Schneider 2 (S2) cells. Stably transformed polyclonal cell populations expressing recombinant endostatin were isolated after 4 wk of selection with hygromycin B. Recombinant endostatin expressed in the stably transformed S2 cells under the influence of the Drosophila BiP protein signal sequence was secreted into the medium. Recombinant endostatin was also purified to homogeneity using a simple one-step Ni2+ affinity fractionation method. Purified recombinant endostatin inhibited endothelial cell proliferation in a dose-dependent manner. The concentration at maximum inhibition for recombinant endostatin was approximately 1.8 microg/ml. The stably transformed S2 cells produced 18 mg recombinant endostatin/L 7 d after induction with 5 microM CdCl2. Sodium butyrate supplementation (2.5 mM) increased recombinant endostatin production by 17%. These findings demonstrate optimal production and in vitro activity of recombinant endostatin from stably transformed D. melanogaster S2 cells.
含有编码小鼠内皮抑素的互补脱氧核糖核酸的重组质粒被转染并在果蝇Schneider 2（S2）细胞中稳定表达。在用潮霉素B选择4周后，分离出表达重组内皮抑素的稳定转化的多克隆细胞群体。在果蝇BiP蛋白信号序列的影响下，在稳定转化的S2细胞中表达的重组内皮抑素被分泌到培养基中。重组内皮抑素也使用简单的一步Ni2+亲和分级分离法纯化至同质。纯化的重组内皮抑素以剂量依赖的方式抑制内皮细胞增殖。重组内皮抑素最大抑制浓度约为1.8μg/ml。在用5μM CdCl2诱导7天后，稳定转化的S2细胞产生18mg重组内皮抑素/L。添加丁酸钠（2.5mM）使重组内皮抑素产量增加17%。这些发现证明了来自稳定转化的黑腹果蝇S2细胞的重组内皮抑素的最佳产量和体外活性。
