利用CHO(dhfr^-)细胞高效表达GM-CSFFc_(γ2)^-融合蛋白

HIGH-LEVEL EXPRESSION OF hGM-CSF/Fc_ν2-FUSION PROTEIN IN CHO(dhfr^-) CELLS

目的 利用二氢叶酸还原酶缺陷型细胞CHO(dhfr - )高效表达融合蛋白GM -CSF Fcγ2-。方法 利用脂质体基因转染技术 ,把含有融合基因GM -CSF Fcγ2-的真核表达质粒pRc CMV2 GM -CSF Fcγ2-和含有二氢叶酸还原酶基因的真核表达质粒pSV2 -dhfr共转染二氢叶酸还原酶缺陷型细胞CHO(dhfr - )中。用G4 18和撤除H、T(次黄嘌呤、胸腺嘧啶核苷 )双筛选 ,获得dhfr +neo +双表型阳性的细胞克隆。取表达产量最高的克隆进行亚克隆 ,并行MTX加压筛选 ,以提高表达量。用RT -PCR、ELISA和Westernblot对表达产物进行鉴定并行MTT法检测表达蛋白的生物学活性。结果 成功地表达出了具有GM -CSF生物学活性的融合蛋白GM -CSF Fcγ2-。结论 该方法可行 ,表达量达到 15μg ml。

Objective To adequately express fusion protein GM-CSF/Fc ν2-using CHO(dhfr -) cells Methods The plasmids pSV2-dhfr and pRc/CMV2/GM-CSF/Fc ν2- were cotransfected into dhfr deficient CHO cells by Lipofection and neo+ dhfr+ positive clones were selected in media containing 600μg/ml G418 and removing Hypoxathine,Thymidine The highest-level expression clone was selected by ELISA,and subcloned in a media containing 10 -7 M MTX The expression of fusion protein GM-CSF/Fc ν2 - was confirmed by RT-PCR,ELISA,Western blot And the biological function of the fusion protein was tested in GM-CSF dependent cells TF-1 Results 1 The highest product was achieved 15μg/10 6cells/24hours 2 The fusion protein possessed the bilolgical function of hGM-CSF Conclusion The method is feasible,the yield of expression attains 15μg/ml