Method for obtaining immunoglobulin encoding nucleic acid

Abstract

The current invention is directed to a method for obtaining a nucleic acid encoding an immunoglobulin variable domain from a single cell comprising the following steps: performing a first polymerase chain reaction with three to six 5′-primer and one 3′-primer, performing with the product of the first polymerase chain reaction a second polymerase chain reaction with thirteen to sixteen 5′-primer and one 3′-primer, whereby the distance of the binding locations of the primer employed in the second polymerase chain reaction is reduced compared to the first polymerase chain reaction.

1 . A method for producing an immunoglobulin comprising the following steps:
 (a) providing a single immunoglobulin producing cell,   (b) obtaining from said cell a nucleic acid, wherein said nucleic acid encodes an immunoglobulin variable domain selected from a heavy chain variable domain, a kappa light chain variable domain, and a lambda light chain variable domain, comprising: (a) performing a first polymerase chain reaction (PCR) to obtain a first PCR product; and (b) performing a second PCR with the first PCR product, whereby the distance of the binding locations of the primers employed in the second PCR is reduced compared to the distance in the first PCR, wherein:
 i) for obtaining the nucleic acid encoding the immunoglobulin heavy chain variable domain, the first PCR is performed with 5′ primers comprising the nucleic acids of SEQ ID NO: 05 and/or 06, 07 and/or 08, 09, 10 and/or 11, 12, 13, and with 3′ primers comprising the nucleic acids of SEQ ID NO:104 or 105 or 106, and the second PCR is performed with 5′ primers comprising the nucleic acids of SEQ ID NO: 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, and/or 142 and with 3′ primers comprising the nucleic acids of SEQ ID NO: 104 or 105 or 106 or 143, and 
 ii) for obtaining the nucleic acid encoding the immunoglobulin kappa light chain variable domain, the first PCR is performed with 5′ primers comprising the nucleic acids of SEQ ID NO: 16, 17, 18, 19, and with a 3′ primer comprising the nucleic acid of SEQ ID NO: 115, and the second PCR is performed with 5′ primers comprising the nucleic acids of SEQ ID NO: 53 and/or 54, 55 and/or 56, 57 and/or 58, 59, 60, 61 and/or 62, 63 and/or 64, 65, 66, 67, 68, 69, 70, 144, and with 3′ primers comprising the nucleic acids of SEQ ID NO: 115 and/or 145, and 
 iii) for obtaining the nucleic acid encoding the immunoglobulin lambda light chain variable domain, the first PCR is performed with 5′ primers comprising the nucleic acids of SEQ ID NO: 21, 22, 23 and/or 24 and/or 25 and/or 26, and with 3′ primers comprising the nucleic acids of SEQ ID NO:120 and/or 121 and/or 122 and/or 123 and/or 124 and/or 125, and the second PCR is performed with 5′ primers comprising the nucleic acids of SEQ ID NO: 72, 73 and/or 74, 75, 76, 77 and/or 78, 79, 80, 81, 82 and/or 83, 84 and/or 85, 86, 87 and/or 88, 89, 90 and/or 91, 92, and 3′ primers comprising the nucleic acid of SEQ ID NO: 120 and/or 121 and/or 122 and/or 123 and/or 124 and/or 125; 
   (c) combining the nucleic acid encoding the light chain variable domain with a nucleic acid encoding the immunoglobulin light chain constant domain and combining the nucleic acid encoding the heavy chain variable domain with a nucleic acid encoding an immunoglobulin heavy chain constant region,   (d) transfecting a eukaryotic cell with the combined nucleic acids,   (e) cultivating said transfected cell under conditions suitable for the expression of the immunoglobulin,   (f) recovering the immunoglobulin from the cell or the cultivation medium; and thereby producing the immunoglobulin.   
     
     
         2 . The method of  claim 1 , wherein the 3′-primer in the second PCR is the same as in the first PCR and at least one 5′-primer is changed, and whereby in the second PCR the number of nucleotides between the 5′-end of each of the 5′-primer and the 3′-end of the 3′-primer is reduced compared to the number of nucleotides between the 5′-end of each of the 5′-primer and the 3′-end of the 3′-primer in the first PCR, when bound to the nucleic acid to be amplified. 
     
     
         3 . The method of  claim 1 , wherein the primers employed in the second PCR provide for overhangs encoding the translational start codon ATG for the 5′-primers and/or the translational stop codon TTA for the 3′-primer. 
     
     
         4 . The method of  claim 1 , wherein said method comprises the first step of: providing a single cell and obtaining the mRNA of said cell. 
     
     
         5 . The method of  claim 4 , wherein said method further comprises the following second step: —obtaining cDNA from said mRNA with a reverse transcriptase PCR. 
     
     
         6 . The method of  claim 1 , wherein the single cell is a B-cell, a plasmablast, or a plasma cell. 
     
     
         7 . The method of  claim 1 , wherein said immunoglobulin is a human immunoglobulin. 
     
     
         8 . The method of  claim 1 , wherein said immunoglobulin is an immunoglobulin of the class G (IgG).