本研究利用反向遗传学的方法成功拯救了一株盖塔病毒(Getah virus，GETV)(GenBank: OM363683)。首先，将优化后的病毒序列克隆到含有T7或CMV启动子的低拷贝质粒pFK中，分别命名为T7-GETV和CMV-GETV。以T7-GETV为模板，通过体外转录(in vitro transcription，IVT)合成病毒RNA。随后，采用脂质体转染的方式将RNA递送到BHK-21细胞中，通过半数组织培养感染量(50% tissue culture infectious dose，TCID₅₀)法测定病毒滴度，并利用免疫印迹法(western blot，WB)检测病毒蛋白E1表达，证明盖塔病毒被成功拯救。进一步通过光学显微镜观察细胞病变(cytopathic effect，CPE)，以及TCID₅₀法、实时荧光定量聚合酶链反应(real time fluorescent quantitative polymerase chain reaction，RT-qPCR)和WB的方法，系统地表征盖塔病毒的生长特征。此外，在CMV-GETV的基础上，基于盖塔病毒复制子的结构，采取保留非结构蛋白基因并用报告基因mCherry或renilla luciferase(Rluc)代替结构蛋白基因的策略构建盖塔病毒复制型表达载体，分别命名为pFK-GETV-mCherry和pFK-GETV-Rluc。随后构建pFK质粒的CMV启动子并仅插入报告基因的对照质粒，分别命名为pFK-mCherry和pFK-Rluc。Image J软件分析显示，转染pFK-GETV-mCherry后单细胞平均荧光强度比转染pFK-mCherry高约5倍。同时，RT-qPCR结果表明，转染pFK-GETV-mCherry后mCherry基因转录水平也比转染pFK-mCherry高约5倍，两组结果高度一致。WB结果进一步说明，在相同时间点转染pFK-GETV-mCherry组mCherry蛋白表达水平比转染pFK-mCherry组高。荧光素酶检测显示，转染pFK-GETV-Rluc后细胞表达Rluc的效率比转染pFK-Rluc高约3个数量级。本研究为后续盖塔病毒研究提供了有力的反向遗传学工具，同时验证了盖塔病毒复制型表达载体提高外源基因表达水平的能力，为研究盖塔病毒复制型DNA疫苗奠定了基础。

In this study, a strain of Getah virus (GETV, GenBank: OM363683) was successfully rescued using a reverse genetics approach. Firstly, the optimized viral sequence was cloned into the low-copy plasmid pFK containing either the T7 or CMV promoter, named T7-GETV and CMV-GETV, respectively. Viral RNA was synthesized via in vitro transcription (IVT) using T7-GETV as a template, followed by RNA transfection into BHK-21 cells via liposome-mediated transfection. The viral titer was determined using 50% tissue culture infectious dose (TCID₅₀) method and the expression of viral protein E1 was detected through western blot (WB), validating the successful rescue of GETV. Furthermore, the cytopathic effect (CPE) was observed via light microscope, and the growth characteristics of GETV were systematically analyzed using the TCID₅₀ assay, real time fluorescent quantitative polymerase chain reaction (RT-qPCR), and WB. Additionally, based on the structure of GETV replicon, two replicative expression vectors were constructed using CMV-GETV as a template by retaining the non-structural protein genes and replacing the structural protein genes with the reporter gene mCherry or Renilla luciferase (Rluc), named pFK-GETV-mCherry and pFK-GETV-Rluc, respectively. Two control plasmids were also constructed by inserting only the reporter gene mCherry or Rluc downstream of the CMV promoter in the pFK plasmid, named pFK-mCherry and pFK-Rluc, respectively. Analysis with Image J software revealed that the average fluorescence intensity per cell of cells transfected with pFK-GETV-mCherry was approximately five times higher than that of cells transfected with pFK-mCherry. RT-qPCR results indicated that mCherry gene transcription level in cells transfected with pFK-GETV-mCherry was also approximately five times higher than that in cells transfected with pFK-mCherry, demonstrating high consistency between the two results. WB results indicated that mCherry protein expression level in cells transfected with pFK-GETV-mCherry was also higher than that in cells transfected with pFK-mCherry at the same time point. Renilla luciferase assay showed that Rluc expression efficiency in cells transfected with pFK-GETV-Rluc was about three orders of magnitude greater than that in cells transfected with pFK-Rluc. This study provides a robust reverse genetics tool for GETV research and demonstrates the ability of the GETV replicative expression vector to enhance exogenous gene expression, establishing a foundation for the development of replicative DNA vaccines.