多功能M13KE噬菌体展示系统的建立

Construction of a Multipurpose M13KE Phage Display System

以M13KE噬菌体为起始载体,建立一种无需辅助噬菌体的较长片段多肽库展示系统,以解决当前噬菌体展示仅能筛选短肽库现象,并扩大靶向高通量筛选范畴。根据M13KE次要外壳蛋白(Minor coat protein of wild-type M13KE,wt-pⅢ)基因III(wt-gene Ⅲ)结构与功能关系,设计并扩增出能发挥其基因功能的截短基因Ⅲ(Truncated gene Ⅲ,tgⅢ);通过拼接-重叠-延伸PCR(Splice-overlapping-extension polymerase chain reaction,SOEing-PCR)获得含启动子、信号肽和结构基因的融合基因片段,将其插入至M13KE载体的合适部位,进行蛋白质诱导表达,SDS-PAGE和Western blot分析。同时在wt-gⅢ和tgⅢ部位分别插入HA和c-Myc多肽标签,再次进行表达和检测。结果显示,tgⅢ被插入到M13KE的非必需部位,利用抗蛋白III(anti-M13 pⅢ)抗体进行Western blot检测发现,wt-gⅢ和tgIII均能表达pIII(protein Ⅲ,pⅢ);anti-HA和c-Myc抗体都能检测到2个标签蛋白和pⅢ蛋白质的表达。获得一种多功能M13KE载体,具有既能表达短片段多肽库,也能表达较长片段多肽库的潜能,而且无需辅助噬菌体,可用于更大范围的高通量靶向筛选。

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-overlappingextension 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.