内含赤羽病病毒 S 基因假病毒的构建、鉴定及初步应用

李超；潘俊慧；王素春；隋金钰；魏世萌；祁倩；吴发兴；李博文；王楷宬

Abstract

为制备赤羽病病毒（Akabane virus, AKAV）核酸诊断试剂阳性对照品，本研究先合成 AKAV S 全长基因，构建重组慢病毒穿梭载体 pLenti-GIII-CMV-CBH-GFP-2A-Puro-AKAV。将测序合格的重组载体与包装载体共转染 293T 细胞，收集并纯化细胞培养液上清，检测质粒核酸残留。利用上清侵染 293T 细胞，观测绿色荧光蛋白表达，通过 RT-PCR、荧光定量 RT-PCR 鉴定 AKAV S 基因片段，最终采用数字 RT-PCR 完成绝对定量。结果显示，纯化上清未检出质粒残留；培养72 h的 293T 细胞可见绿色荧光；两种 PCR 方法均可扩增出目的基因片段；假病毒拷贝数浓度定值为3.36 × 10³ copies/μL。本研究成功构建携带 AKAV S 全长基因的假病毒，可作为核酸检测阳性对照品，为赤羽病病毒核酸诊断试剂盒研发提供支撑。

In order to prepare positive control materials for Akabane virus (AKAV) nucleic acid diagnostic reagents, the full-length AKAV S gene was synthesized. The verified recombinant lentiviral shuttle vector was co-transfected into 293T cells together with packaging vectors. Purified cell culture supernatant was collected and tested for plasmid residue. The supernatant was used to infect 293T cells, and green fluorescent protein expression was observed. RT-PCR and quantitative real-time RT-PCR were applied to identify AKAV S gene fragments, and digital RT-PCR was adopted for absolute quantification. No plasmid residue was detected in purified supernatant. Green fluorescence was observed in 293T cells cultured for 72 hours. Target AKAV S gene fragments were successfully amplified by both PCR methods. The quantified concentration of pseudovirus solution was 3.36 × 10³ copies/μL. The pseudovirus carrying full-length AKAV S gene was successfully constructed. It can serve as positive control for AKAV nucleic acid detection and lays a foundation for relevant diagnostic kit development.