Viral expression plasmids for production of proteins, antibodies, enzymes, virus-like particles and for use in cell-based assays

Abstract

This disclosure shows that the EBV FR-element comprised of EBNA1 multimeric binding sites can provide the stable maintenance replication function to the mouse polyomavirus (PyV) core origin plasmids in the presence of BPV-1 E2 protein and PyV large T-antigen (LT).

What is claimed is: 
     
         1 . An expression system to provide extended episomal replication of hybrid plasmid in eukaryotic cell lines, said system comprising:
 a) a vector comprising a polyoma virus core origin according to SEQ ID NO:2, an FR element of EBV, a selection marker, and a gene of interest operably linked within an expression cassette expressing the gene in a eukaryotic cell; and   b) a compatible mammalian cell line, where the vector is expressed, said cell line constitutively expressing EBV EBNA1 protein and PyV LT protein either in presence or in absence of selective pressure.   
     
     
         2 . The system according to  claim 1 , wherein the FR element comprises at least 21 EBNA1 binding sites. 
     
     
         3 . The system according to  claim 2 , wherein the EBNA 1 binding sites are according to sequences selected from the group consisting of SEQ ID NOs: 3, 4, and 6. 
     
     
         4 . The system according to  claim 3 , wherein the FR element is according to SEQ ID NO:1. 
     
     
         5 . The system according to  claim 1 , wherein the cell line is of mouse, hamster or human origin. 
     
     
         6 . The system according to  claim 1 , wherein the gene of interest encodes a protein for pharmaceutical use. 
     
     
         7 . The system according to  claim 6 , wherein the protein is a monoclonal antibody. 
     
     
         8 . The system according to  claim 6 , wherein the protein is virus envelope or capside protein. 
     
     
         9 . The system according to  claim 6 , where the protein is fused to an epitope-tag. 
     
     
         10 . The system according to  claim 9 , where the epitope tag has an amino acid sequence selected from the group consisting of SEQ ID NO:20, 21, 22 and 23. 
     
     
         11 . An expression plasmid for use in a mammalian cell expression system, said vector comprising:
 a) a polyoma virus core origin,   b) an FR element of Eppstein Barr Virus (EBV);   c) a selection marker; and   d) an expression cassette for expression a gene of interest in a eukaryotic cell.   
     
     
         12 . The expression plasmid of  claim 11 , where the polyoma virus core origin is according to SEQ ID NO:2 and the FR element is according to SEQ ID NO:1. 
     
     
         13 . The expression plasmid of  claim 11 , wherein the expression cassette comprises a multicloning site for insertion of the gene of interest, and a promoter. 
     
     
         14 . The expression plasmid of  claim 13 , wherein the promoter is selected from the group consisting of Cytomegalovirus immediate early (CMV)-promoter, EF1α; EF1α-HTLV, heIF 4a, β-actine, and Rous Sarcoma Virus proviral Long Termina Repeat (RSV-LTR)-promoter. 
     
     
         15 . The expression plasmid of  claim 13 , wherein the gene of interest encodes a protein for pharmaceutical use. 
     
     
         16 . The expression plasmid of  claim 15 , wherein the protein is a monoclonal antibody. 
     
     
         17 . The expression plasmid of  claim 15 , wherein the protein is virus envelope or capside protein. 
     
     
         18 . The expression plasmid of  claim 15 , where the protein is fused to an epitope-tag. 
     
     
         19 . The expression plasmid of  claim 18 , where the epitope tag has an amino acid sequence selected from the group consisting of SEQ ID NO:20,21,22, and 23. 
     
     
         20 . A cell lien comprising the expression plasmid of  claim 11 .