G418选择与稳转克隆表达分泌hGM-CSF的相关性考察

Observation of Correlation between G418 Selection Pressure and Stably Transfected hGM CSF Clones Expression and Secretion Level

目的：进一步考察不同Ｇ４１８维持用量与ｒｈＧＭ－ＣＳＦ表达水平相关性。方法：第２期历时３个月（第４～６个月），本室构建的ＣＨＯ－ｒｈＧＭ－ＣＳＦ稳转克隆，在不同Ｇ４１８维持用量（０、３００μｇ／ｍｌ、６００μｇ／ｍｌ）下传２５代左右，各批次传代上清用依赖ＧＭ－ＣＳＦ生长的ＴＦ－１细胞作ＭＴＴ活性测定。结果：无Ｇ４１８选择维持，ＣＨＯ稳转细胞株表达和分泌ｈＧＭ－ＣＳＦ的水平从第４个月开始下降，再次加Ｇ４１８（６００μｇ／ｍｌ）后，ｈＧＭ－ＣＳＦ的表达水平迅速回升，２周后至正常。结论：Ｎｅｏ抗性基因作为遗传选择标志进入真核细胞，上述结果对于选择Ｇ４１８用量以维持宿主细胞表达分泌目标蛋白具有一定参考价值。

Objective:To a further observa the correlation between the different G418 selection pressure and the rhGM CSF expression level.Methods:During the second term(from the fourth to the sixth month), the stably transfected CHO rhGM CSF clones,constructed by our research department,were cultured under different selection pressure of G418(0,300μg/ml,600μg/ml) for three monthes.The GM CSF activity of culture supernatant of clones was assayed by MTT method with the hGM CSF dependent cell line of TF1.Results:Without G418 selection pressure,the level of rhGM CSF expression and secretion by the clones began descending from the fourth month,whereas the level could rise and return to normal after two weeks when adding G418(600μg/ml) once more.Conclusion:When neo gene as a selective marker came into eucaryotic cells,the above mentioned results may be have a certain reference value for how to select G418 pressure to maintain the expression and secretion of objective proteins by the host cells.