rhG-CSF cDNA高表达质粒的构建及其在COS7细胞中的表达

目的：构建一个能在哺乳动物细胞中高表达人粒细胞集落刺激因子（Ｇ－ＣＳＦ）ｃＤＮＡ的重组质粒。方法：利用化学合成引物，进行ＰＣＲ，对人Ｇ－ＣＳＦｃＤＮＡ５＇ＡＵＧ侧翼序列进行翻译优化突变。测序确证后，重组入表达载体ｐＥＤ，构建成质粒ｐＥＤ－ＧＣＳＦ，用ＤＥＡＥ－Ｄｅｘｔｒａｎ法转染ＣＯＳ７细胞，ＥＬＩＳＡ法定量测定ｒｈＧ－ＣＳＦ表达水平。结果：转染ＣＯＳ７细胞后４８ｈ收集的上清ｒｈＧ－ＣＳＦ表达量为５２ｎｇ／ｍｌ，７２ｈ为２３０ｎｇ／ｍｌ，显示ｒｈＧ－ＣＳＦ获得了暂时性高表达。结论：ｐＥＤ－ＧＣＳＦ是一个能在哺乳动物细胞中高效表达ｒｈＧ－ＣＳＦ的真核表达质粒。

Construction of the recombinant human granulocyte colony stimulating factor cDNA plasmid and its expression in COS7 cell

Objective: To construct a recombinant human granulocyte colony stimulating factor (rhG CSF) cDNA plasmid that could be highly expressed in mammalian cells. Methods: The sequence surrounding the AUG initiatory codon of hG CSF cDNA was changed to optimized translational initiation by polymerase chain reaction (PCR). After sequecing of hG CSF cDNA, a plasmid pED GCSF was constructed through recombination with hG CSF cDNA and high expressing vector pED. Then, pED GCSF was transfected into COS7 cells by DEAE Dextran method, and the rhG CSF expression level was tested by G CSF ELISA kit. Results: The rhG CSF expression level of 48 h after transfection was 52 ng/ml, and that of 72 h was 230 ng/ml. Results showed that the rhG CSF cDNA was transitorily and highly expressed. Conclusion: pED GCSF is a high eukaryotic expression plasmid in mammalian cell.