Efficient synthesis of influenza virus hemagglutinin in mammalian cells with an extrachromosomal Epstein-Barr virus vector

Abstract

The capability of an Epstein-Barr virus hybrid vector (EBV-CMV), containing the cytomegalovirus (CMV) immediate early enhancer and simian virus 40 promoter, to produce large amounts of authentic mammalian proteins was studied. The cDNA of influenza virus hemagglutinin (HA), a cell surface glycoprotein, was inserted into this vector and the EBV-CMV- HA plasmid was transfected into two human and two monkey cell lines. Southern-blot analysis revealed that the EBV-CMV- HA plasmid was maintained m extrachromosomal state and the recombinant cell clones contained on the average three copies (range 1–24) of the transfected DNA. The recombinant HA polypeptides from different cell clones, selected either randomly or by fluorescence-activated cell sorter, were analysed using immunological techniques. Three of the four cell lines expressed recombinant HA on the cell surface in glycosylated form. The highest production levels, 11.5 μx g/10 6 cells, were obtained in HeLa cells containing only two copies of EBV-CMV- HA DNA per cell. The protein levels correlated with the mRNA levels in Northern-blot analysis. A corresponding vector, containing the same regulatory signals for HA expression, but lacking the EBV sequences, yielded clones with significantly lower expression levels. The results confirm that the extrachromosomal EBV-CMV vector is very useful in the production of apparently authentic mammalian glycoproteins.