Intrabodies targeting post-translational modifications of native proteins and method for obtaining them

A method for obtaining a human naïe scFv Single Pot Library of INTracellular antibodies (SPLINT), with the VH and VL domains derived from antibodies, the method comprising the steps of:

• a) amplifying human germline VH, Vk and Vλ regions, from a cDNA sample;
• b) amplifying a linker blunt specific for VH, Vk or Vλ regions, with primers having the same 3′ region annealing on the linker and different protruding 5′, overlapping perfectly either with VH framework 4 or VL framework 1 to obtain a semi-blunt linker;
• c) carrying out an overlap amplification between the variable region amplicons and the semi-blunt linkers, obtaining a VH-linker and a linker-VL, i.e. VH and VL, protruding with the same linker sequence at 3′ and 5′ respectively;
• d) joining VH-linker and linker-VL by overlapping amplification;
• e) inserting restriction sites at the 5′ of the VH regions and at the 3′ of the Vk and Vγ regions to obtain scFv products;
• f) digesting the scFv products with the specific restriction enzymes and ligating them to a digested vector.