Establishment of an in vitro reporter system for screening HBx-targeting molecules
Establishment of an in vitro reporter system for screening HBx-targeting molecules
复制标题
筛选HBx靶向分子的体外报告系统的建立
DOI：
10.1093/abbs/gmz016
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发表时间：
2019-04-01
影响因子：
3.7
通讯作者：
Xie, Youhua
中科院分区：
文献类型：
--
作者：
Gu, Chenjian;Tao, Shuai;Xie, Youhua
慢性乙型肝炎病毒（HBV）感染仍然是一个全球公共卫生问题。 HBV 编码的 X 蛋白 (HBx) 是一种多功能调节因子，是启动和维持有效 HBV 感染所需的，并且参与 HBV 相关肝细胞癌 (HCC)。干扰 HBx 功能的抑制剂不仅可用于抑制 HBV 复制，还可用于预防或治疗 HBV 相关的 HCC。由于缺乏灵敏和高通量的系统，大规模筛选针对 HBx 的分子仍然是一个技术挑战。在这项工作中，我们建立了体外生物发光报告系统来筛选 HBx 靶向分子。该系统基于结合 HBx 和 NanoLuc (HBx-Nluc) 的分泌融合蛋白。 HBx-Nluc表达细胞培养上清液中NanoLuc活性的测量直接反映了分泌的HBx-Nluc的水平。 HBx蛋白靶向细胞内抗HBx单链可变片段和RNA靶向shRNA显着降低表达HBx-Nluc的细胞中分泌的NanoLuc活性。该系统简单且灵敏，并且与连续无破坏性筛选兼容，表明其在高通量筛选和评估 HBx 靶向分子方面具有潜在的用途。
Chronic hepatitis B virus (HBV) infection remains a global public health problem. HBV-encoded X protein (HBx) is a multifunctional regulator that is required to initiate and maintain productive HBV infection, and is involved in HBV-related hepatocellular carcinoma (HCC). Inhibitors that interfere with the functions of HBx could be useful not only for the inhibition of HBV replication but also for the prevention or treatment of HBV-related HCC. To screen molecules that target HBx on a large scale remains a technical challenge due to a lack of sensitive and high-throughput system. In this work, we established an in vitro bioluminescent reporter system for screening HBx-targeting molecules. The system is based on a secretory fusion protein that combines HBx and NanoLuc (HBx-Nluc). The measured activity of NanoLuc in the culture supernatant of HBx-Nluc-expressing cells directly reflects the level of secreted HBx-Nluc. HBx protein-targeting intracellular anti-HBx single-chain variable fragment and RNA-targeting shRNA significantly reduced the secreted NanoLuc activity in HBx-Nluc-expressing cells. This system is simple and sensitive, and compatible with continuous non-disruptive screening, suggesting its potential usefulness for high-throughput screening and evaluating HBx-targeting molecules.