title: High-level expression of the Epstein-Barr virus EBNA1 protein in CV1 cells and human lymphoid cells using a SV40 late replacement vector.
abstract: To construct a recombinant plasmid designed to yield large amounts of the Epstein-Barr virus (EBV) nuclear antigen, EBNA1, the EBV BamHI-K fragment (B95-8 strain) was inserted into an expression vector composed of SV40 and pBR322 DNA. The vector replicates in both Escherichia coli and eukaryotic cells. Introduction of such a BamHI-K-containing vector into CV1 monkey cells (using DEAE-dextran, glycerol and chloroquine diphosphate) gave high yields of the correct size EBNA1 protein in 40-50% of the transfected cells. Maximal amounts of EBNA1 could be extracted from the cells at 65-72 h post transfection. Using a quantitative ELISA assay, it was estimated that transfected cells express 500-1000 times more EBNA1 than lymphoid cells, latently infected with EBV. A monoclonal antibody directed against EBNA1 immunoprecipitated two proteins of 74 and 62 kDa from transfected cells. These same two proteins were detected in immunoprecipitation and immunoblot experiments using human EBV-positive polyclonal serum, although this serum also detected several other protein products in transfected cells. In vivo labelling of transfected cells with [32P]orthophosphate showed that the 74- and 62-kDa proteins are modified by phosphorylation. The same vector construction was also used to transfect an EBV-negative human lymphoblastoid cell line (Ramos). Expression of the EBNA1 protein was obtained in up to 20% of the cells.
mesh_terms: D000818:Animals; D000956:Antigens, Viral / Q000235:genetics*; D002143:Callitrichinae; D002460:Cell Line; D002522:Chlorocebus aethiops; D004262:DNA Restriction Enzymes; D004251:DNA Transposable Elements; D019309:Epstein-Barr Virus Nuclear Antigens; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D005814:Genes, Viral; D005822:Genetic Vectors; D004854:Herpesvirus 4, Human / Q000235:genetics* / Q000276:immunology; D006801:Humans; D007668:Kidney; D008214:Lymphocytes / Q000276:immunology*; D010750:Phosphoproteins / Q000235:genetics; D010957:Plasmids; D013539:Simian virus 40 / Q000235:genetics*; D014162:Transfection
chemical_list: D000956:Antigens, Viral; D004251:DNA Transposable Elements; D019309:Epstein-Barr Virus Nuclear Antigens; D010750:Phosphoproteins; D004262:DNA Restriction Enzymes