重组人细胞介素12基因在CHO─dhfr~－细胞的稳定表达

将分别含有重组人ＩＬ－１２（ｒｈＩＬ－１２）两个亚单位ｃＤＮＡ的真核细胞高效表达载体，通过Ｌｉｐｏｆｅｃｔｉｎ介导，共转染至ＣＨＯ－ｄｈｆｒ－细胞中。经选择性培养基培养、细胞染色体ＤＮＡｄｏｔｂｌｏｔ实验、Ｎｏｒｔｈｅｒｎｂｌｏｔ及Ｗｅｓｔｅｒｎｂｌｏｔ实验鉴定，筛选出能表达ｒｈＩＬ－１２的细胞株，经氨甲喋呤加压诱导，表达量约为１５０ｕ／ｍｌ，且经液氮冻存３ｍｏ以上仍有表达。本实验还研究了表达产物对人ＰＢＭＣ的促增殖作用和增加ＮＫ细胞毒活性作用，为进一步研究ｒｈＩＬ－１２的生物学功能奠定了基础。

Stable eukaryotic expression of rhIL 12 in CHO dhfr -cells

In this study,two high efficiency eukaryotic expression vectors,PED35 and PED40 containing IL 12 p35 and p40 cDNA respectively,were cotransfected into CHO dhfr - cells by Lipofectin method.The transformants were grown in selective medium and screened by means of chromosome DNA dot blot,Northern blot and Western blot analysis.The cell clones expressed the rhIL 12 mRNA and protein had been obtained.After MTX induction,the content of rhIL 12 protein in the expressed supernatant had approximately been 150 u/ml.Furthermore,this study also determined the biological activities through two biological assays and showed the expressed supernatant can increase the proliferation of human PHA activated PBMC and augment the human NK cell mediated cytotoxicity.These two activities can be blocked by anti rhIL 12 monoclonal antibody.The expression of rhIL 12 can still be detected after more than three months storage in liquid nitrogen.Therefore,this study has been laid a solid ground for the further exploration of biological activities and potential usage of rhIL 12.