携带Fluc与mCherry双报告基因的重组痘苗病毒WR株的构建及体外应用

Construction and application in vitro of a recombinant vaccinia virus strain WR expressing dual reporters Fluc and mCherry

摘要:

【目的】 构建同时表达双报告基因(荧光素酶Fluc和红色荧光蛋白mCherry)的重组痘苗病毒WR株。【方法】 采用CRISPR/Cas9技术构建针对WR株J2R区的gRNA CRISPR/Cas9质粒及表达Fluc和mCherry的质粒pJSE-Fluc/mCherry，插入至TK区构建重组痘苗病毒rWR-Fluc/mCherry。采用PCR与测序分析基因插入位置与序列的准确性；通过mCherry、Fluc活性、空斑形态鉴定重组病毒；重组病毒连续盲传12代，检测双报告基因及E3L表达分析重组病毒的遗传稳定性；检测重组病毒(rWR)与野生型病毒(WR)感染Vero和HeLa细胞后的细胞病变、TCID₅₀、报告基因表达，分析病毒的复制动力学。采用空斑法、qPCR法、双报告基因活性检测，评价ST-246作为阳性药物的体外抗病毒药效。【结果】 体外鉴定结果显示，Fluc和mCherry准确插入WR株的TK区域，感染Vero细胞后可检测到mCherry荧光及Fluc酶活性，空斑形态与野生型病毒一致，连续盲传12代病毒滴度保持稳定，并且双报告基因活性及E3L表达均可稳定检出，表明重组病毒rWR-Fluc/mCherry构建成功且遗传稳定；重组病毒感染Vero和HeLa细胞后的细胞病变、TCID₅₀滴定、双报告基因活性检测均表明，感染后48−72 h达到复制高峰，与WR的复制动力学一致。采用重组病毒rWR-Fluc/mCherry测得ST-246的EC₅₀与WR野生型病毒一致，多个检测方法(病毒蚀斑、DNA拷贝数、双报告基因活性) EC₅₀结果(2−7 nmol/L之间)间均具有良好的一致性(相关系数r均大于0.500 0，P<0.05)。【结论】 成功构建了可同时表达Fluc与mCherry双报告基因且遗传稳定的重组痘苗病毒rWR-Fluc/mCherry，可应用于抗病毒药物体外快捷筛选及药效分析。

关键词:痘苗病毒;重组;双报告基因;抗正痘病毒药物;半数有效浓度

Abstract:

[Objective] To construct a recombinant vaccinia virus strain WR that expresses dual reporters (luciferase Fluc and red fluorescent protein mCherry). [Methods] Firstly, the gRNA CRISPR/Cas9 plasmid targeting the J2R region of WR and the plasmid pJSE-Fluc/mCherry carrying the dual reporter genes were constructed. Then, the CRISPR/Cas9 gene editing tool was used to insert the dual reporter genes into the TK region, and thus the recombinant vaccinia virus strain rWR-Fluc/mCherry (rWR) was constructed. The location and sequence of insertion in rWR were analyzed by PCR and sequencing. The recombinant strain rWR was characterized by mCherry/Fluc activity and plaque assays. The recombinant strain rWR was subcultured for 12 passages, and the expression levels of the dual reporter genes and E3L were determined to reveal the genetic stability of the strain. To analyze the replication dynamics of the virus in Vero and HeLa cells, we determined the cytopathic effect (CPE), TCID₅₀, and dual reporter expression of rWR and the wild type (WR) in the infected cells. Furthermore, we evaluated the inhibitory effects of ST-246 as a positive drug on both rWR and WR in vitro by the plaque assay, qPCR, and dual reporter activity measurement. [Results] Fluc and mCherry were accurately inserted into the TK region of WR. The Vero cells infected with rWR showed the activities of dual reporters and the plaque morphology consistent with that of WR. After 12 passages, the dual reporter activities and E3L expression were stably detected in rWR, which indicated that rWR was successfully constructed and genetically stable. The CPE, TCID₅₀, and dual reporter activity in Vero and HeLa cells indicated that replication peaked 48–72 h post-infection with rWR, which was consistent with the replication dynamics of WR. The median effective concentration (EC₅₀) of ST-246 against rWR was in agreement with that against WR, and the EC₅₀ (2–7 nmol/L) obtained by the plaque assay, qPCR, and dual reporter activity measurement showed good consistency (r>0.500 0 and P<0.05). [Conclusion] A recombinant vaccinia virus strain rWR simultaneously expressing Fluc and mCherry was successfully constructed, and it was genetically stable. This strain might be used as an in vitro system for rapid screening and characterization of anti-orthopoxvirus drugs with simple operation.

Key words:vaccinia virus;recombinant;dual-reporter gene;anti-orthopoxvirus drugs;median effective concentration(EC₅₀)