Efficient display of an HCV cDNA expression library as C-terminal fusion to the capsid protein D of bacteriophage lambda.
Efficient display of an HCV cDNA expression library as C-terminal fusion to the capsid protein D of bacteriophage lambda.
复制标题
高效展示 HCV cDNA 表达文库，其 C 端与 lambda 噬菌体衣壳蛋白 D 融合。
DOI：
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发表时间：
1998
影响因子：
5.6
通讯作者：
A. Luzzago
中科院分区：
文献类型：
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作者：
C. Santini;Debra Brennan;C. Mennuni;Ronald H. Hoess;Alfredo Nicosia;Riccardo Cortese;A. Luzzago
我们描述了丙型肝炎病毒(丙型肝炎病毒)c DNA表达文库的构建和鉴定，该文库显示为与噬菌体Lambda衣壳蛋白D的羧基末端融合。对丙型肝炎病毒基因组进行随机标记，获得插入片段，将其克隆到Lambda载体中，获得衣壳表面同时含有野生型D蛋白和D融合产物的嵌合噬菌体。所得文库与识别线性或构象表位的抗丙型肝炎病毒人单抗和来自丙型肝炎病毒感染患者的人血清进行亲和选择。通过免疫筛选实验、酶联免疫吸附试验和阳性克隆的序列分析进行筛选。将该文库的性能与另外两个与丝状噬菌体M13的III和VIII衣壳蛋白N端融合而产生的丙型肝炎病毒cDNA展示文库进行了比较。这些结果表明，Lambda展示系统在构建天然配基发现的复杂cDNA文库方面具有巨大的潜力。
We describe the construction and characterization of a hepatitis C virus (HCV) cDNA expression library displayed as a fusion to the carboxy terminus of the capsid protein D of bacteriophage lambda. cDNA inserts were obtained by tagged random-priming of the HCV genome and cloned into a lambda vector from which chimeric phage bearing both wild-type D protein and D fusion products on the capsid surface were produced. The resulting library was affinity-selected with anti-HCV human monoclonal antibodies recognizing linear or conformational epitopes, and human sera from HCV-infected patients. Selection was monitored by immuno-screening experiments, ELISA, and sequence analysis of positive clones. The performance of this library was compared with two additional HCV cDNA display libraries generated as N-terminal fusions to the III and VIII capsid proteins of filamentous phage M13. The results obtained demonstrate the great potential of the lambda display system for constructing complex cDNA libraries for natural ligand discovery.