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[The production of mini antibodies against human granulocyte colony-stimulating factor using murine scFv
A G Laman1, A O Shepeliakovskaia, A B Ulitin
1Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry (Puschchino Branch), Russian Academy of Sciences, ul. Institutskaya 6, Pushchino, 142290 Russia. vnes@ibch.ru
Abstract:
To develop a phage display of single-chain antibodies (scFv), fractions of total cell DNA and RNA were obtained from splenocytes of naive mice. The DNA fragments encoding variable regions of light and heavy immunoglobulin chains were amplified and isolated using primers specific to the conservative regions of these genes. The construction of the library was based on the principle of stochastic combining of the DNA fragments encoding the light and heavy antibody chains with the DNA linker, whose structure corresponded to the (Gly4Ser)3 sequence. The scFv library was constructed using the E. coli TG1 strain and the phagemid vector pHEN1. The repertoire of the library exceeded 5 x 10(7) independent recombinant clones. The clones producing antibodies to the granulocyte colony-stimulating human factor were isolated. The affinity constants of the resulting scFv were in the range of 2 x 10(4) to 1.8 x 10(7) M-1.