目的:利用噬菌体7肽库进行体外快速差减筛选(biopanning and rapid analysis of selective interactive ligands,BRASIL),从而获得卵巢癌细胞株HO-8910细胞表面特异性结合肽。方法:以中国仓鼠卵巢细胞株CHO为吸附细胞,卵巢癌细胞株HO-8910为靶细胞,利用噬菌体展示技术联合差减筛选策略,经过连续5轮生物淘洗,随机挑选13个噬菌体单克隆进行DNA测序,利用噬菌体竞争结合实验、ELISA实验、细胞免疫染色、细胞免疫荧光、合成多肽的竞争抑制实验鉴定阳性噬菌体的亲和性及特异性。结果:筛选出的噬菌体NPMIRRQ与卵巢癌细胞株HO-8910有较高的亲和性及特异性。结论:阳性噬菌体表面展示的多肽NPMIRRQ可能为卵巢癌的早期诊断和转移复发监测提供新思路。
We have previously reported that bovine papillomavirus type 1(BPV-1) DNA can replicate its genome and produce infectious virus-like particles in short term virion-infected S. cerevisiae(budding yeast) cultures(Zhao and Frazer 2002,Journal of Virology, 76:3359–64 and 76:12265–73). Here, we report the episomal replications of BPV-1 DNA in long term virion-infected S. cerevisiae culture up to 108 days. Episomal replications of the BPV-1 DNA could be divided into three patterns at three stages, early active replication(day 3–16), middle weak replication(day 23–34/45) and late stable replication(day 45–82). Two-dimensional gel electrophoresis analysis and Southern blot hybridization have revealed further that multiple replication intermediates of BPV-1 DNA including linear form, stranded DNA, monomers and higher oligomers were detected in the virion-infected yeast cells over the time course. Higher oligomers shown as covalently closed circular DNAs(cccDNAs) are the most important replication intermediates that serve as the main nuclear transcription template for producing all viral RNAs in the viral life cycle. In this study, the cccDNAs were generated at the early active replication stage with the highest frequencies and then at late stable replication, but they appeared to be suppressed at the middle weak replication. Our data provided a novel insight that BPV-1 genomic DNA could replicate episomally for the long period and produce the key replication intermediates cccDNAs in S. cerevisiae system.
