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多功能M13KE噬菌体展示系统的建立

, PP. 143-147

Keywords: 靶向高通量筛选,噬菌体展示系统,M13KE,长片段多肽库

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Abstract:

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(Minorcoatproteinofwild-typeM13KE,wt-pIII)基因III(wt-geneIII)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(TruncatedgeneIII,tgIII);通过拼接-重叠-延伸PCR(Splice-overlapping-extensionpolymerasechainreaction,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Westernblot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-M13pIII)抗体进行Westernblot检测发现,wt-gIII和tgIII均能表达pIII(proteinIII,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。

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