In this study, a multipurpose M13KE phage display vector was constructed from wild-type M13KE phage for long peptide or protein display libraries without helper phage to expand the scope of targeted high-throughput sc...In this study, a multipurpose M13KE phage display vector was constructed from wild-type M13KE phage for long peptide or protein display libraries without helper phage to expand the scope of targeted high-throughput screening. Based on the relationship between the structure and function of minor coat protein of wild-type MI3KE （wt-plII）, a truncated gene III （tglll） encoding minor coat protein from M13KE phage was cloned. A fusion gene fragment harboring a hw/tac promoter, signal peptide and C-terminal region sequence of gill was assembled with SOEing-PCR （splice-overlapping-extension polymerase chain reaction） method and inserted into M13KE vector. SDS-PAGE and Western blot analysis with anti-M13 pIII moneclonal antibody were employed to detect the expression of re- combinant protein, c-Myc and HA tag sequences were fused into the recombinant protein. The results showed that tglll was inserted into an unessential region of M13KE. According to the results of SDS-PAGE and Western blot with anti-M13 pIII antibody, pIII was expressed by wt-gIII and tgIII, glII harboring two tags ex- pressed both c-Myc and HA peptides using SDS-PAGE and Western blot with the corresponding monoclonal antibodies. In this study, a multipurpose M13KE phage display system was successfully constructed, which could express both short and long peptide libraries without helper phage. In future, the obtained M13KE phage display system may be used for targeted high-throughput screening of long peptide libraries without helper phage.展开更多
为了制备用于噬菌体展示肽库的筛选及鉴定等环节的辣根过氧化物酶(HRP)标记的兔抗丝状噬菌体(M13)衣壳蛋白抗体,试验制备了兔源的M13KE多克隆抗体,将多克隆抗体经饱和硫酸铵法和Protein G Resin层析柱纯化后运用过碘酸钠法对其进行辣根...为了制备用于噬菌体展示肽库的筛选及鉴定等环节的辣根过氧化物酶(HRP)标记的兔抗丝状噬菌体(M13)衣壳蛋白抗体,试验制备了兔源的M13KE多克隆抗体,将多克隆抗体经饱和硫酸铵法和Protein G Resin层析柱纯化后运用过碘酸钠法对其进行辣根过氧化物酶标记,对标记前和标记后纯化产物进行活性测定,同时对标记后产物进行Western-blot鉴定,以及将其应用到验证已知的阳性噬菌体H11、H12、H14、H16、H18中。结果表明:兔抗M13KE多克隆抗体效价高于1∶32 000,标记后活性高于1∶4 000,标记抗体与已知阳性噬菌体的ELISA鉴定结果与前期测序结果一致。说明此酶标抗体有一定的应用价值。展开更多
基金Supported by Youth Fund of Suzhou Chien-shiung Institute of Technology(2013QNJJ38)
文摘In this study, a multipurpose M13KE phage display vector was constructed from wild-type M13KE phage for long peptide or protein display libraries without helper phage to expand the scope of targeted high-throughput screening. Based on the relationship between the structure and function of minor coat protein of wild-type MI3KE （wt-plII）, a truncated gene III （tglll） encoding minor coat protein from M13KE phage was cloned. A fusion gene fragment harboring a hw/tac promoter, signal peptide and C-terminal region sequence of gill was assembled with SOEing-PCR （splice-overlapping-extension polymerase chain reaction） method and inserted into M13KE vector. SDS-PAGE and Western blot analysis with anti-M13 pIII moneclonal antibody were employed to detect the expression of re- combinant protein, c-Myc and HA tag sequences were fused into the recombinant protein. The results showed that tglll was inserted into an unessential region of M13KE. According to the results of SDS-PAGE and Western blot with anti-M13 pIII antibody, pIII was expressed by wt-gIII and tgIII, glII harboring two tags ex- pressed both c-Myc and HA peptides using SDS-PAGE and Western blot with the corresponding monoclonal antibodies. In this study, a multipurpose M13KE phage display system was successfully constructed, which could express both short and long peptide libraries without helper phage. In future, the obtained M13KE phage display system may be used for targeted high-throughput screening of long peptide libraries without helper phage.
文摘为了制备用于噬菌体展示肽库的筛选及鉴定等环节的辣根过氧化物酶(HRP)标记的兔抗丝状噬菌体(M13)衣壳蛋白抗体,试验制备了兔源的M13KE多克隆抗体,将多克隆抗体经饱和硫酸铵法和Protein G Resin层析柱纯化后运用过碘酸钠法对其进行辣根过氧化物酶标记,对标记前和标记后纯化产物进行活性测定,同时对标记后产物进行Western-blot鉴定,以及将其应用到验证已知的阳性噬菌体H11、H12、H14、H16、H18中。结果表明:兔抗M13KE多克隆抗体效价高于1∶32 000,标记后活性高于1∶4 000,标记抗体与已知阳性噬菌体的ELISA鉴定结果与前期测序结果一致。说明此酶标抗体有一定的应用价值。
