Construction Of A Non-immunized Human Fab Fragment Phage Library

We report the construction a non-immunized phage antibody library in Fab format.We used peripheral blood lymphocytes from a healthy donor as source of lymphoid tissues. 1.8 X 10 B lymphocytes were isolated from 0.2 liters of blood using "Lymphoprep". Total RNA was prepared from these B lymphocytes. cDNA was synthesized by Reverse Transcriptase(RT) from total RNA with the oligodT. Heavy and light chain variable regions 5' and 3' primers were used for PCR amplification of human heavy chain Fd fragment and light chain gene. About 680bp PCR products were obtained and then were digested with restriction enzymes for ligation into the pComb3 vector. For the construction of the light chain library, the light chain repertoires were cloned into the phagemid pComb3. The ligated DNA was electroporated into competent E. coli XL 1-Blue cells. The Fab library was obtained by cloning of Fd fragments into the phagemid collection containing the light chain repertoires and then electroporated into XLl-Blue cells. After over night growth of cells, the phagemid containing the Fd fragment and light chain was purified for the future selection of the library.This new phage antibody library is set to become a valuable source of antibodies to many different antigens and to play a vital role in the research work of our lab.