Random mutations were inserted into CDR-H3 by performing PCR with tailored degenerate primers. After construction of a mutated antibody gene library, affinity selection was performed. Meanwhile the scFv (scFv-G10) selected from the library exhibited the most improved affinity to rhTNF-¦Á (2.9-fold higher than the parental scFv-F6D2E7). The scFv-G10 sequence and human constant (CH1 & CL) regions were used to construct a novel vector for developing an expression system that allows the production of a completely functional antigen-binding fragment (Fab) in Escherichia coli. The bioactivity of the Fab was determined by L929 cell cytotoxicity assay. Fab-G10 could neutralize rhTNF-¦Á-induced cytotoxicity to L929 cells, and the calculated 50 % inhibition rate (IC50) was 5.0 ¡Á 10?7 M.
We generated an artificial antibody fragment (scFv-G10) that had improved affinity and desirable specificity. Further, the Fab-G10 was constructed and expressed in E.?coli, where the bioactivity was further detected.