Development of molecular targeted drugs against HCV core protein using phage display

我们亚克隆了编码丙型肝炎病毒核心蛋白(全长)的DNA，并利用丙型肝炎病毒核心蛋白表达载体将丙型肝炎病毒核心蛋白截短到GST-融合载体中，生产出每一种丙型肝炎病毒核心蛋白。然而，尽管我们改变了引物的设计和纯化条件，但由于丙型肝炎病毒核心蛋白C-端的疏水性，丙型肝炎病毒核心蛋白的纯化很困难。因此，我们购买了几种商业上可获得的更多截短的丙型肝炎病毒核心蛋白。我在美国实验室中使用的表达大量多肽的噬菌体文库已经部分解冻，并检查了文库的多样性大小。然而，多样性却极大地减少。然后我们制作了一个新的文库，复制了10^9个多样性。我们用一种丙型肝炎病毒核心蛋白做了活组织扫描。我们得到了可以非特异性或每周与GST蛋白结合的噬菌体。我们将丙型肝炎病毒核心蛋白改变为另一种，并安排了…更多的实验条件。然而，我们无法获得能够足够特异地结合的噬菌体。然后，我们对噬菌体文库进行了重新设计和改造，并继续安排了洗涤和结合时间等实验条件。我们发现了具有结合能力的候选克隆，但通过重新评估认为这些克隆的特异性不够。我们咨询了美国实验室的前几位同事，试图重新设计图书馆，重新安排实验条件。然而，对有前景的噬菌体的检测是困难的。因此，我们购买了表达商业上可用的环状和线状多肽的噬菌体文库。我们还购买了完全不同的噬菌体展示系统，其中外源多肽在P3病毒外壳蛋白上表达，而不是在P8病毒外壳蛋白上表达。我们目前正在通过改变实验条件来重复生物扫描。另一方面，我们报道了利用表达丙型肝炎病毒核心蛋白的丙型肝炎病毒复制子系统，锌是丙型肝炎病毒RNA复制的负调控因子。我们可以在未来利用该系统评估与丙型肝炎病毒核心蛋白结合的靶配体的功能。较少

We suubcloned DNA that codes HCV core protein (full-length) and truncated HCV core protein to GST-fusion vectors using HCV core protein expression vector to produce each HCV core protein. However the purification of HCV core proteins were difficult probably due to the hydrophobicity of C-terminus of HCV core protein even though we changed the design of the primers and the conditions of the purification of HCV core proteins. Therefore we purchased several commercially available more truncated HCV core proteins. The phage library expressing the numerous peptides, which I had been using in the lab in USA, was partly thawed and examined for the size of the diversity of the library. However the diversity was extremely decreased. Then we made the new library with l0^9+ copied of the diversity. We did biopanning by using a kind of HCV core protein. We got phages that could bind to GST protein nonspecifically or very weekly. We changed a HCV core protein to another kind of one and arranged the … More conditions of the experiments. However we could not get the phages that could bind enough specifically. Then we remade and redesigned the phage library and continued to arrange the experimental conditions such as washing and binding time. We found the candidates of clones with the binding ability, however, the specificity of these was considered to be insufficient by conducting reevaluation. We consulted with previous colleagues in the lab in USA and tried to redesign the library and arrange the experimental conditions again. However the detection of promising phages was difficult. Therefore we purchased the phage libraries that expressed commercially available circular and linear peptides. We also purchased the totally different phage display system in which foreign peptides was expressed on P3 viral coat proteins instead of P8 viral coat proteins. We are currently repeating biopanning by changing the experimental conditions.On the other hand, we reported that zinc is a negative regulator of hepatitis C virus RNA replication using HCV replicon system expressing HCV core protein. We can evaluate the function of the target ligands binding to HCV core protein using this system in the future. Less