人GM－CSF基因在昆虫细胞中表达的研究

Expression of Human GM-CSF Gene in Insect Cells

本工作构建的昆虫表达重组体ｐＡＣ６１０－ＧＭＴ，是在ＡｃＭＮＰＶ的Ｐｏｌｙｈｅｄｒｉｎ启动子控制下，表达去除了信号肽编码顺序的人ＧＭ－ＣＳＦ基因（ｃＤＮＡ）的转染载体。它与野生ＡｃＭＮＰＶ病毒ＤＮＡ共转染Ｓｆ２１细胞，经过筛选得到纯化的可表达人ＧＭ－ＣＳＦ的重组病毒株ｖＡｃＧＭＴ。其感染细胞总ＲＮＡ的Ｎｏｒｔｈｅｒｎ分析结果表明，重组病毒在ｍＲＮＡ水平有人ＧＭ－ＣＳＰ特异性表达，其表达水平在感染后４８ｈ时达高峰，７２ｈ未见明显下降。感染细胞裂解物的Ｗｅｓｔｅｒｎ－Ｂｌｏｔ分析和活性测定也证实其蛋白水平的表达，并有人ＧＭ－ＣＳＦ的生物学活性。

recombinant transfer vector pAc610-GMTcarrying the cDNA of the human granulocyte-macrophage colony stimulating factor(hGM-CSF) which encode mature hGM-CSF with poly A￣+sequence at 3’end(GMT),was constructed under the control of the Polyhedrin promoter of theAutorapha Californica Nuclear Polyhedrosis Virus (AcMNPV ).It was cotransfected into Sf21 Cells with the wild type AcMNPV’s DNA. The recombinant virus vAcGMT was Purified bymeans of selection assay of endpoint dilution in combination with the Dot-Blot method. Northern-Blot analysis of the total RNA from the infected cells demonstrated a having expression of hGM-CSF in the infected Sf21 cell in the mRNA level. The level reached its peak at 48 hour pi(post in-fected ).Western-Blot analysis and biological activity determination of the infected cell lysatesconfirmed the existence of recombinant hGM-CSF and its biological activity.