[Establishment of scFv antibody library screening technology based on flow cytometry]

To establish bacterial display technology used for screening scFv antibody library by NlpA leader and the first six amino acids (CDQSSS) of NlpA to anchore antibodies to the periplasmic side of the bacterial inner membrane, thereby paving a way for the further antibody affinity maturation.

The gene of NlpA leader and CDQSSS were amplified from pNAD plasmid. After enzyme digestion, the PCR product was subcloned into pHEN1 expression vector. The variable regions of heavy chain and light chain of the anti-human IL-1β antibody gene were amplified using PCR technology with PEAI plasmid as templet. Single chain variable fragment (scFv) was constructed by overlapping PCR and inserted into the downstream of NlpA leader of NLPA-pHEN1 recombinant plasmid to construct pBSD-scFv for scFv display. E.coli DH5α cells which were transformed with pBSD-scFv were induced to express scFvs. After spheroplast formation, the pBSD-scFv-transformed bacteria were incubated with antigen at gradient concentrations. The display status was detected by flow cytometry (FCM). We isolated the positive groups and rescued positively expressed pBSD-scFv using plasmid extraction instead of PCR to transform E.coli DH5α. And the binding of antibody and antigen in the positive groups was detected again with FCM.

After the pBSD-scFv-transformed bacteria were incubated with antigen and antigen specific FITC-antibody, strong fluorescent signal was detected by FCM, which showed an antigen concentration-dependent manner. The rescued pBSD-scFv displayed similar fluorescence intensity in the next FCM procedure, indicating that the method is reliable and stable.

ScFv expressed by the bacterial display system can fold efficiently and have the ability to bind to the hIL-1β antigen specifically. The bacterial display technology for screening scFv antibody library has been established successfully.