新型载体preS1-tp融合蛋白介导乙型肝炎病毒靶向定位序列小干扰RNA有效抑制乙型肝炎病毒复制和cccDNA合成

Novel vector preS1-tp fusion protein effectively inhibits hepatitis B virus replication and cccDNA synthesis by mediating hepatitis B virus targeting sequence small interfering RNA

目的使用preS1-tp融合蛋白作为新型的载体,介导针对乙型肝炎病毒(HBV)核心蛋白羧基端核定位信号(NLS)区的小干扰RNA(siRNA)进入感染HBV的肝细胞,从而抑制HBV复制和共价闭合环状DNA的形成。方法以慢病毒感染系统为基础,建立表达人牛磺胆酸钠共转运蛋白多肽的HepG2.2.15细胞;针对HBV NLS区设计合成siRNA;表达纯化preS1-tp融合蛋白,检测其进入细胞和与DNA结合的能力;以preS1-tp融合蛋白为载体,介导NLS siRNA递送至HepG2.2.15-牛磺胆酸钠共转运蛋白多肽细胞中,观察其对HBV复制和共价闭合环状DNA水平的影响。多组间比较采用方差分析,计量资料用t检验分析组间差异。结果成功构建HepG2.2.15-牛磺胆酸钠共转运蛋白多肽细胞株并合成筛选出显著抑制HBV复制的HBV NLS siRNA。纯化并大量表达preS1-tp融合蛋白,以该融合蛋白为载体靶向输送HBV NLS siRNA,结果显示,融合蛋白可以有效靶向输送siRNA至HepG2.2.15-牛磺胆酸钠共转运蛋白多肽细胞,不仅可有效抑制细胞中HBV mRNA,HBsAg和HBeAg表达,还能显著降低HBV DNA和共价闭合环状DNA的水平。结论preS1-tp融合蛋白利用preS1与肝细胞HBV受体结合,tp与核酸结合的双功能特点,将针对HBV NLS的siRNA靶向至感染HBV的靶细胞,靶向阻断rcDNA转运入细胞核,使siRNA发挥抑制HBV复制和共价闭合环状DNA合成的作用,为HBV感染所致慢性乙型肝炎治疗提供新策略,为彻底清除体内HBV提供新的研究视角。

Objective 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.Methods 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.Results 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.Conclusion 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.