Recombinant vector and use thereof for exocellular preparation of single molecula antibodies from bacillus subtilis

Abstract

A recombinant vector for the expression and secretion of antibodies in single molecule form (scFv) in B.subtilis , where said vector comprises the promoter of the gene for neutral protease, a new secretion sequence (I) and a DNA sequence coding for an antibody scFv of interest, a strain of B.subtilis transformed with said recombinant vector, and a procedure for the exocellular production of scFv antibodies by means of a culture of said strain of B.subtilis are described. The recombinant vector allows the expression of scFv in a completely soluble form and its secretion in high yields.

1 . An expression and secretion recombinant vector of  B.subtilis  which comprising: 
 1) the promoter of the gene coding for neutral protease in  B.subtilis  BGSC 1A341;    2) the secretion sequence (I)                                  5′ ATG AGA AGC AAA AAA ACG CGT ATC AGC                      TTG TTG TTT GCG TTA ACG TTA ATC TTT                      ACG ATG GCA TTC AGC GGC CGC TCT GCC                      ATG GCC 3′; and                                   3) a DNA sequence coding for an antibody in single molecule form having the sequence VH/VK-L-VK/VH-(TAG) n where:      VH and VK are the variable regions of the heavy and light chains of an antibody of interest; L is the peptide linker Val-Ser-Ser-(Gly 4 -Ser) 3 ; TAG is a peptide recognised by anti-peptide antibodies and n is 1 or 0.    
     
     
         2 . The recombinant vector as defined in  claim 1 , wherein the peptide TAG has the amino acid sequence Met-Glu-Glu-Leu-Met-Ile-Glu-Gly-Arg.  
     
     
         3 . The recombinant vector as defined in  claim 1 , wherein VH and VK are the variable regions of the heavy and light chains of the monoclonal antibody MAb5E8 specific for sub-unit alpha of human chorionic gonadotropin.  
     
     
         4 . The recombinant vector as defined in  claim 1 , filed as ATCC 69173.  
     
     
         5 . The recombinant vector as defined in  claim 1 , obtained by: 
 a) synthesizing an oligonucleotide comprising the secretion sequence (I), the sequence coding for the peptide linker and, possibly, the sequence coding for the peptide TAG, where said oligonucleotide contains unique restriction sites allowing the insertion of the sequence coding for the variable region of the heavy or light chain of the antibody downstream of the secretion sequence (I) and upstream of the linkage peptide and the insertion of the sequence coding for the variable region of the light or heavy chain of the antibody downstream of the peptide linker and upstream of the peptide TAG;    b) cloning said oligonucleotide in a plasmid vector comprising the promoter of the gene for neutral protease in  B.subtilis  BGSC 1A341;    c) cloning the DNA sequences coding for the variable region of the heavy and light chains of the antibody of interest in the restriction sites of the oligonucleotide; and finally    d) isolating the recombinant vector.    
     
     
         6 . The recombinant vector as defined in  claim 5 , wherein the oligonucleotide of stage a) has the sequence:  
       
         
           
                 
               
                     
                 
                   5′ GAA TTC TTA TGA GAA GCA AAA AAA CGC GTA TCA 
                 
                     
                 
                      GCT TGT TGT TTG CGT TAA CGT TAA TCT TTA CGA 
                 
                     
                 
                      TGG CAT TCA GCG GCC GCT CTG CCA TGG CCG CAC 
                 
                     
                 
                      AGG TCC AAC TCC AGC CTA TGG TCA CCG TCT CCT 
                 
                     
                 
                      CAG GTG GCG GTG GCT CTG GCG GTG GTG GGT CGG 
                 
                     
                 
                      GTG GCG GCG GAT CTG ACA TTC AAG GTA CCC CCT 
                 
                     
                 
                      GAG ATC TCA TGG AAG AAC TTA TGA TCG AGG GTA 
                 
                     
                 
                      GGT AAG TCG ACA AGC TT 3′. 
                 
                     
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         7 . The recombinant vector as defined in  claim 5 , wherein the plasmid vector of stage b) is pSM308 ATCC 68047.  
     
     
         8 . The recombinant vector as defined in  claim 5 , wherein the DNA sequences of stage c) code for the variable regions of the heavy and light chain of the monoclonal antibody MAb 5E8 specific for sub-unit alpha of human chorionic gonadotropin.  
     
     
         9 . A host microorganism transformed with a recombinant vector as defined in  claim 1 , wherein said microorganism is selected from the  B.subtilis  group.  
     
     
         10 . The microorganism as defined in  claim 6 , which is  B.subtilis  SMS300 (pSM507) ATCC 69173.  
     
     
         11 . A procedure for the exocellular preparation of antibodies in single molecule form from  B.subtilis , comprising: 
 a) cultivating in a suitable culture environment a strain of  B.subtilis  transformed with a recombinant vector as defined in  claim 1;  and    b) separating and purifying the antibody thus obtained from the culture medium.    
     
     
         12 . A procedure as defined in  claim 11 , wherein  B.subtilis  is  B.subtilis  SMS300 (pSM507) ATCC 69173 and the secreted antibody is scFv5E8 specific for sub-unit alpha of human chorionic gonadotropin.  
     
     
         13 . An antibody in single molecule form obtained by the procedure defined in  claim 7 , for diagnostic and therapeutic use.  
     
     
         14 . The antibody in single molecule form scFv5E8 for use as a reagent for the determination of endocrine tumours and tumours of trophoblastic origin characterised by comprising the regions VH and VK having the amino acid sequences reported in FIGS. 1 and 2.  
     
     
         15 . The secretion sequence (I) of in  claim 1 , namely:  
       
         
           
                 
                 
               
                     
                     
                 
                     
                   5′ ATG AGA AGC AAA AAA ACG CGT ATC AGC TTG 
                 
                     
                     
                 
                     
                      TTG TTT GCG TTA ACG TTA ATC TTT ACG ATG 
                 
                     
                     
                 
                     
                      GCA TTC AGC GGC CGC TCT GCC ATG GCC 3′ 
                 
                     
                     
                 
             
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         16 . The DNA sequence of  claim 15  coding for the secretion peptide:  
       
         
           
                 
                 
               
                     
                     
                 
                     
                   Met Arg Ser Lys Lys Thr Arg Ile Ser Leu Leu 
                 
                     
                     
                 
                     
                   Phe Ala Leu Thr Leu Ile Phe Thr Met Ala Phe 
                 
                     
                     
                 
                     
                   Ser Gly Arg Ser Ala Met Ala.