关键词: 靶向高通量筛选, 噬菌体展示系统, M13KE, 长片段多肽库

Abstract: It was to engineer a multipurpose M13KE phage display vector from M13KE wild-type phage for the longer peptide or protein display library without helper phage, as well as to expand the scope of targeting high-throughput screening. Based on the relationship of the structure and function of minor coat protein of wild-type M13KE, a truncated gene III encoding minor coat protein from M13KE phage was cloned and a fusion gene fragment containing a lac/tac promoter and the gene III signal peptide sequence was assembled using splice-overlapping-extension polymerase chain reaction. SDS-PAGE and Western blot analysis with anti-M13 pIII monoclonal antibody was employed to detect the expression of the recombinant gene III. Two peptide tags, c-Myc and HA tag sequences were fused to the recombinant gene for expression and analysis, and the following screening steps. The recombinant gene III（tgIII）was inserted into the unessential part of M13KE and the corresponding protein III was detected with SDS-PAGE and Western blot with the anti-pIII antibody. The tgIII containing two tags expressed both c-Myc and HA peptides using methods of SDS-PAGE and Western blot with their specific monoclonal antibodies. We made the construction of a multipurpose M13KE phage display system. In the near future, we hope to apply this engineered phage display vector to carry longer peptide libraries for high-throughput screening without helper phage.