Expression Vector

Abstract

The present invention provides an expression vector for cell-surface expression of proteins.

1 . A nucleic acid expression vector for cell-surface expression of proteins comprising in order a polynucleotide sequence comprising a sequence encoding a secretion signal peptide, a cloning site for inserting a polynucleotide sequence encoding a protein to be expressed and a polynucleotide sequence comprising a sequence encoding a transmembrane domain of glycophorin. 
     
     
         2 . The nucleic acid vector of  claim 1 , wherein the transmembrane domain of glycophorin is the transmembrane domain of glycophorin A. 
     
     
         3 . The nucleic acid vector of  claim 1  or  2 , wherein the transmembrane domain of glycophorin A is the mouse glycophorin A transmembrane domain or Armenian hamster glycophorin A domain. 
     
     
         4 . The nucleic acid vector of  claim 3 , wherein the mouse glycophorin A domain comprises the amino acids disclosed in Seq. Id. No. 1 and the Armenian hamster glycophorin A domain comprises the amino acid sequence disclosed in Seq. Id. No. 12. 
     
     
         5 . The nucleic acid vector of  claims 1 - 4 , wherein the secretion signal peptide is the secretion signal peptide of bee-venom melittin. 
     
     
         6 . The nucleic acid vector of  claim 5 , wherein the secretion signal peptide of bee-venom melittin comprises the amino acid sequence disclosed in Seq. Id. No. 2. 
     
     
         7 . The nucleic acid vector of  claims 1 - 6 , further comprising downstream (3′) of the cloning site for inserting a polynucleotide sequence encoding a protein to be expressed a polynucleoide sequence encoding a FLAG tag comprising the amino acid sequence of Seq. Id. No. 3. 
     
     
         8 . The nucleic acid vector of  claims 1 - 7 , further comprising downstream (3′) of the polynucleotide sequence encoding the transmembrane domain of glycophorin a polynucleotide sequence encoding a His tag, preferably a His tag comprising the amino acid sequence disclosed in Seq. Id. No. 4. 
     
     
         9 . The nucleic acid vector of  claims 1 - 8 , wherein the cloning site comprises the restriction enzyme cleavage sites of NheI, KpnI, BamHI, EcoRI, EcoRV and NotI. 
     
     
         10 . The nucleic acid vector of  claims 1 - 9  comprising a polynucleotide sequence selected from the group consisting of Seq. Id. No. 5, Seq. Id. No. 13, Seq. Id. No. 14 or Seq. Id. No. 15. 
     
     
         11 . The nucleic acid vector of  claims 1 - 10 , wherein the protein to be expressed is a membrane associated protein. 
     
     
         12 . A cell comprising the vector of  claims 1  to  11 , preferably a mammalian cell, more preferably a HEK cell. 
     
     
         13 . Use of a cell of  claim 12  for the expression of proteins suitable for antibody generation. 
     
     
         14 . Use of a cell of  claim 12  for the immunisation of a non-human animal for antibody generation, preferably monoclonal antibodies. 
     
     
         15 . A method for the generation of monoclonal antibodies against a specific protein comprising the steps:
 a) immunisation of a non-human animal with cells expressing on its cell surfaces the specific protein using the vector of  claims 1  to  11 ,   b) isolating spleen cells of the non-human animals of step a),   c) fusing the spleen cells of step b) with myeloma cells to generate B cell hybridomas and   d) identification of B cell hybridomas expressing antibodies directed against the specific protein.   
     
     
         16 . The method of  claim 15 , wherein the non-human animal is a mouse or hamster. 
     
     
         17 . The invention as hereinbefore described.