Combining the advantages of prokaryotic expression and T7 phage display systems to obtain antigens for antibody preparation

Abstract

The gene encoding the phage major capsid protein 10A was cloned into the prokaryotic expression vector pET24a, and a 6XHis-tag was fused to the 3′-end of the 10A gene to verify complete expression. The recombinant plasmid was transformed into Escherichia coli (E. coli) BL21 (DE3) cells, and 10A expression was induced by IPTG. SDS-PAGE and Western blot were used to confirm the target protein expression. The T7Select10-3b vector was added to the cultured bacteria expressing 10A at a multiplicity of infection (MOI) ranging from 0.01 to 0.1, and complete lysis of the bacteria was monitored by absorbance changes in the medium. The recombinant phage (reP) was harvested by PEG/NaCl sedimentation and resuspended in PBS. ELISA was performed to verify the presence of the 6XHis-tag on the surface of reP. The 10A-fusion expression vectors (pET10A-flag, pET10A-egfp, and pET10A-pct) were constructed, and fusion proteins were expressed and detected by the same method. The corresponding rePs (reP-Flag, reP-EGFP, and reP-PCT) were prepared by T7Select10-3b infection. After the expression of the peptides/proteins on the reP surfaces was confirmed, reP-Flag and reP-PCT were used to immunize mice to prepare anti-Flag and anti-PCT antibodies. The results showed that rePs prepared using the 10A-fusion vector and T7Select10-3b can be used as antigens to immunize mice and prepare antibodies. This method may be able to meet the rapid antigen preparation requirements for antibody production.Notably, the recombinant phage (reP) described in this study was obtained by the sedimentation method from T7Select10-3b-infected E. coli BL21 (DE3) cells carrying the major capsid protein 10A expression vector or 10A-fusion protein vector.