[Establishment of bacteria display technology for Fab antibody library screening]

To establish bacterial display technology for the purpose of Fab antibody library screening, by using six amino acids (CDQSSS) of the amino termimus of NlpA protein to anchore antibodies to the periplasmic side of the bacterial inner membrane.

The NlpA Leader sequences (encoding CDQSSS) was amplified from pNAD plasmid. The PCR product was subcloned into pComb3 expression vector to generate Fab display vector pBFD. The heavy chains of the Fab gene fragments and the light chains of the anti-human IL-1β (hIL-1β) antibody were inserted downstream of the NlpA leader and pelB leader respectively to construct the pBFD-Fab for Fab antibody display. Then pBFD-Fab transformed E.coli DH5α was induced by IPTG to express the Fab antibodies, as detected by flow cytometry (FCM), and positive populations were sorted. Instead of PCR, plasmids were extracted for rescue purpose. The rescue plasmids were retransformed to E.coli DH5α and FCM was performed again.

The pBFD-Fab-transformed bacteria were incubated with antigen and antigen specific FITC-antibody, and showed strong fluorescence as detected by FCM in a dose-dependent manner. The rescued pBFD-Fab displayed similar fluorescence intensity, indicating the reliability of this technology.

The Fab expressed by the bacterial display system folds efficiently and binds to hIL-1β specifically. The plasmid rescue works well and it can avoid mutation and mis-pairing chains. This bacterial display technology has the stability of antibody expression. This study has used the technology to screen anti-hIL-1β Fab antibody Library successfully.