内含赤羽病病毒S基因假病毒的构建、鉴定及初步应用  

Construction,Identification and Application of Pseudovirus Containing S Gene of Akabane Virus

作　　者：李超 潘俊慧 王素春[1] 隋金钰 魏世萌 祁倩 吴发兴[1] 李博文 王楷宬[1] Li Chao;Pan Junhui;Wang Suchun;Sui Jinyu;Wei Shimeng;Qi Qian;Wu Faxing;Li Bowen;Wang Kaicheng(China Animal Health and Epidemiology Center,Key Laboratory of Animal Biosafety Risk Warning,Prevention and Control(South China),Ministry of Agriculture and Rural Affairs,Qingdao Key Laboratory of Animal Biosafety,Qingdao 266032,Shandong,China)

机构地区：[1]中国动物卫生与流行病学中心,农业农村部动物生物安全风险预警及防控重点实验室(南方),青岛市动物生物安全重点实验室,山东青岛266032

出　　处：《中国动物检疫》2025年第1期96-102,共7页China Animal Health Inspection

基　　金：国家重点研发计划项目(2024YFD1800101)。

摘　　要：为制备赤羽病病毒(Akabane virus,AKAV)核酸诊断试剂中的阳性对照品,本研究首先合成AKAV S全基因,然后构建重组慢病毒穿梭载体pLenti-GIII-CMV-CBH-GFP-2A-Puro-AKAV,并将测序鉴定成功的重组慢病毒穿梭载体与包装载体共转染293T细胞;收获纯化的细胞培养液上清,进行核酸质粒残留检测;将上清感染293T细胞,观察绿色荧光蛋白基因表达情况,并采用RT-PCR和荧光定量RT-PCR试验检测上清中是否存在AKAV S基因片段,最终通过数字RT-PCR方法进行绝对定值。结果显示;纯化的细胞培养液上清中无核酸质粒残留,在培养72 h的293T细胞中可观察到绿色荧光;采用RT-PCR和荧光定量RT-PCR试验均可扩增出AKAV S基因目的片段;采用数字RT-PCR方法,将制备的假病毒溶液拷贝数浓度定值为3.36×10^(3)copies/μL。结果表明,本研究成功构建了含AKAV S全基因片段的假病毒,可作为AKAV核酸检测的阳性对照品,为后续研发AKAV核酸诊断试剂盒奠定了基础。In order to prepare the positive control materials in nucleic acid diagnostic reagents of Akabane virus(AKAV),AKAV S complete gene was synthesized to construct the recombinant lentiviral shuttle vector pLenti-GIII-CMV-CBH-GFP-2A-Puro-AKAV that was sequenced and identified correctly,to co-transfect 293T cells with the packaging vector;the purified cell culture fluid supernatant was harvested and nucleic acid plasmid residues were detected,then 293T cells were infected by the fluid supernatant,the expression of green fluorescent protein gene was observed,and RT-PCR and fluorescence quantitative RT-PCR were used to detect whether there was AKAV S gene fragment in the supernatant,which was absolutely quantified by digital RT-PCR.The results showed that there was no nucleic acid plasmid residues in the supernatant of purified cell culture solution,and green fluorescence could be observed in 293T cells that were cultured for 72 hours;the target fragment of AKAV S gene could be amplified by both RT-PCR and fluorescence quantitative RT-PCR;and the concentration of the prepared pseudovirus solution was quantified to be 3.36×10^(3)copies/μL.In conclusion,the pseudovirus containing AKAV S gene complete fragments was successfully constructed,which could be used as positive control materials for detection of AKAV nucleic acids,supporting subsequent development of AKAV nucleic acid detection kits.

关 键 词：赤羽病病毒 S基因 假病毒粒子 阳性对照品 

分 类 号：S852.6[农业科学—基础兽医学]