Zeng Y L, Gao F, Zhang C, Wei J F, Ma L, Ding G G, Li W, Shang J, Kang Y
Department of Infectious Disease, Henan Provincial People's Hospital, People's Hospital of Zhengzhou University, Henan University People's Hospital, Zhengzhou 450003, China.
Zhonghua Gan Zang Bing Za Zhi. 2021 Feb 20;29(2):126-132. doi: 10.3760/cma.j.cn501113-20190924-00353.
To study the use of preS1-tp fusion protein as a novel vector to mediate the entry of small interfering RNA (siRNA) targeting the carboxy-terminal nuclear localization signal (NLS) region of hepatitis B virus (HBV) core protein into HBV-infected hepatocytes, and to further explore the HBV replication inhibition and covalently closed circular DNA synthesis. HepG2.2.15 cells expressing the human sodium taurocholate co-transporting polypeptide were established on the basis of lentivirus infection system. siRNA against HBV NLS region was designed and synthesized. PreS1-tp fusion protein expression and purification was observed to test its ability to cell entry and DNA binding. NLS siRNA were delivered into HepG2.2.15- sodium taurocholate co-transporting polypeptide cells by preS1-tp fusion protein as a vector to observe the effects of NLS siRNA on HBV replication and covalently closed circular DNA levels. Analysis of variance was used for comparison between multiple groups, and the measurement data differences between groups were analyzed by t-test. HepG2.2.15-sodium taurocholate co-transporting polypeptide cell line was successfully constructed. Screened synthetic HBV NLS siRNA had significantly inhibited HBV replication. The preS1-tp fusion protein was expressed and purified on a large-scale. The fusion protein as a vector for HBV NLS siRNA had targeted delivery. The result showed that the fusion protein had effectively targeted siRNA to Hepg2.2.15-sodium taurocholate co-transporting polypeptide cell, which not only had effectively inhibited the expression of HBV mRNA, HBsAg and HBeAg, but also had significantly reduced the levels of HBV DNA and covalently closed circular DNA. The preS1-tp fusion protein constructed in this study uses the dual functional characteristics of preS1 binding to hepatocyte HBV receptor, and tp binding to nucleic acids, and targets HBV NLS siRNA against HBV-infected cells and block rcDNA from being transported to nucleus. siRNA plays a role in inhibiting HBV replication and covalently close circular DNA synthesis, providing a new strategy for the treatment of chronic hepatitis B caused by HBV infection, and a new research perspective for the complete elimination of HBV from the body.
研究前S1-tp融合蛋白作为新型载体介导靶向乙型肝炎病毒（HBV）核心蛋白羧基末端核定位信号（NLS）区域的小干扰RNA（siRNA）进入HBV感染的肝细胞，并进一步探索对HBV复制的抑制作用及共价闭合环状DNA合成情况。基于慢病毒感染系统建立表达人牛磺胆酸钠共转运多肽的HepG2.2.15细胞。设计并合成针对HBV NLS区域的siRNA。观察前S1-tp融合蛋白的表达及纯化情况，检测其细胞进入能力和与DNA结合能力。以前S1-tp融合蛋白为载体将NLS siRNA导入HepG2.2.15-牛磺胆酸钠共转运多肽细胞，观察NLS siRNA对HBV复制及共价闭合环状DNA水平的影响。多组间比较采用方差分析，组间计量资料差异采用t检验。成功构建HepG2.2.15-牛磺胆酸钠共转运多肽细胞系。筛选合成的HBV NLS siRNA对HBV复制有显著抑制作用。前S1-tp融合蛋白实现了大规模表达及纯化。该融合蛋白作为HBV NLS siRNA的载体具有靶向递送作用。结果显示，该融合蛋白能有效将siRNA靶向递送至Hepg2.2.15-牛磺胆酸钠共转运多肽细胞，不仅有效抑制了HBV mRNA、HBsAg和HBeAg的表达，还显著降低了HBV DNA及共价闭合环状DNA水平。本研究构建的前S1-tp融合蛋白利用前S1与肝细胞HBV受体结合以及tp与核酸结合的双重功能特性，靶向针对HBV感染细胞的HBV NLS siRNA，阻断rcDNA转运至细胞核。siRNA在抑制HBV复制及共价闭合环状DNA合成中发挥作用，为治疗HBV感染所致慢性乙型肝炎提供了新策略，为从体内彻底清除HBV提供了新的研究视角。
