title: 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 in 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 micrograms/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.
authors.affiliation: Orion Genetic Engineering Laboratory, Orion Corporation Ltd., Helsinki, Finland.
authors.forename: A
authors.initials: A
authors.lastname: Jalanko
authors.forename: A
authors.initials: A
authors.lastname: Kallio
authors.forename: M
authors.initials: M
authors.lastname: Salminen
authors.forename: I
authors.initials: I
authors.lastname: Ulmanen
mesh_terms: D000818:Animals; D002460:Cell Line; D003587:Cytomegalovirus / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D005784:Gene Amplification; D005822:Genetic Vectors; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral / Q000235:genetics*; D004854:Herpesvirus 4, Human / Q000235:genetics*; D006801:Humans; D015151:Immunoblotting; D007124:Immunoenzyme Techniques; D008322:Mammals; D009975:Orthomyxoviridae / Q000235:genetics*; D010957:Plasmids; D011233:Precipitin Tests; D012689:Sequence Homology, Nucleic Acid; D014162:Transfection
chemical_list: D004279:DNA, Viral; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral