本研究构建了蜱传脑炎病毒(tick-borne encephalitis virus，TBEV)的全长感染性克隆，为深入理解TBEV的复制和发病机制提供了重要工具。首先，预测TBEV全长序列中的原核启动子，并针对这些启动子进行同义突变以消除其活性，从而降低cDNA的细菌毒性。随后基于对序列复杂性和克隆效率的考虑，将TBEV cDNA序列分为两段进行合成，并利用同源重组技术将其克隆至pFK载体上。通过体外转录获得病毒RNA，并将RNA转染到细胞，通过实时荧光定量聚合酶链反应(real-time fluorescence quantitative polymerase chain reaction，RT-qPCR)，使用TBEV病毒E蛋白的特异性引物成功检测到病毒RNA的复制；同时，通过Western blot，利用针对TBEV NS1蛋白的特异性抗体，成功检测到病毒蛋白的表达；半数组织培养感染量(50% tissue culture infectious dose，TCID50)检测显示，病毒滴度在48 h后显著上升，证实了病毒被成功拯救。最后，通过空斑实验，在BHK-21细胞中观察96 h后，验证了二代病毒的感染性，结果显示随着病毒液稀释度的减小，空斑数量显著增加，且病毒RNA的相对表达量和蛋白表达量随时间增加，表明二代病毒液具有感染性和在细胞内复制的能力。这些结果为TBEV的研究提供了新的工具以及感染性克隆构建的通用策略。

This study constructed a full-length infectious clone of tick-borne encephalitis virus (TBEV), providing an essential tool for in-depth investigation of TBEV replication and pathogenesis. Firstly, prokaryotic promoters within the full-length TBEV sequence were predicted, and synonymous mutations were introduced to eliminate the activity of these promoters, thereby reducing the bacterial toxicity of the cDNA. Subsequently, considering sequence complexity and cloning efficiency, the TBEV cDNA sequence was divided into two fragments for synthesis, which were then cloned into the pFK vector using homologous recombination technology. Viral RNA generated by in vitro transcription was transfected into cells. Viral RNA replication was successfully detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) using TBEV E protein-specific primers. Meanwhile, Western blot analysis with a TBEV NS1 protein-specific antibody confirmed viral protein expression. 50% tissue culture infectious dose (TCID50) assays demonstrated a significant increase in viral titers at 48 h post-transfection. Finally, plaque assays in BHK-21 cells at 96 h post-infection validated the infectivity of second-generation viruses. Results showed that plaque numbers markedly increased with the decreasing viral dilution, and both viral RNA levels and protein expression escalated over time, indicating the infectivity and intracellular replication capacity of second-generation viruses. These results provide a novel tool for TBEV research, and a general strategy for constructing infectious clones.