Abstract: To get the single chain variable fragment antibody （ScFv） gene of active higher the monoclonal antibody cell 2F6 with higher neutralizing activity，the hybridoma cell lines 4D5、2F6 and 7G3, which could secrete anti-PEA monoclone antibody were prepared. The V_H、V_Lgene were amplified from the cell line 2F6 by RT-PCR. The V_u and V_L gene were connected through a flexible linker （Gly4Ser） 3, and the V_H-Linker-V_L （ScFv） fusion gene was cloned into a vector pGEM-T.The ScFv gene was sequenced, analyzed by computer and the results were showed that it was consist of 729bp ,encoding 243 amino acid residues. Both V_H and V_L were confirmed as functionally rearranged mouse immunoglobulin variable region genes and appeared to be new genes,According Kabat classed method , McAb 2F6 V_H gene segment and V_L gene segment belong to the mouse Ig heavy chain subgroup Ⅱ（A） and κ chain Ⅵ subgroup respectively.

Key words: Pseudomonas areuginosa exotoxin A, Monoclone andibody, Single chain antibody, Gene cloning, Nucleotide sequencing