禽流感病毒M基因的重组慢病毒构建  

Construction of recombinant lentivirus with M gene of avian influenza virus

作　　者：潘俊慧 王素春[1,2] 禹兰平 周凯钰太 魏世萌 祁倩 李超 隋金钰 康京丽 王楷宬[1,2] PAN Junhui;WANG Suchun;YU Lanping;ZHOU Kaiyutai;WEI Shimeng;QI Qian;LI Chao;SUI Jinyu;KANG Jingli;WANG Kaicheng(China Animal Health and Epidemiology Center,Qingdao 266032,China;Key Laboratory of Animal Biosafety Risk Prevention and Control(South),Ministry of Agriculture and Rural Affairs,Qingdao 266032,China)

机构地区：[1]中国动物卫生与流行病学中心,山东青岛266032 [2]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),山东青岛266032

出　　处：《畜牧与兽医》2024年第8期85-90,共6页Animal Husbandry & Veterinary Medicine

基　　金：国家重点研发计划项目(2022YFD1800500)。

摘　　要：旨在建立针对禽流感病毒(AIV)的实验室快速分子检测方法。构建重组慢病毒作为分子检测方法中的阳性对照品,采用PCR扩增技术获得AIV M基因的保守区片段,并构建pLVX-M-IRES-ZsGreen1重组慢病毒穿梭载体;将鉴定成功的重组穿梭质粒与骨架质粒pMD2.G和psPAX2共转染293T细胞,获得携带AIV M基因保守区的重组慢病毒;将重组慢病毒感染293T细胞,观察其荧光表达情况,收集、浓缩病毒液并测定其滴度;用AIV实时荧光定量PCR(RT-qPCR)检测方法检测M基因。结果表明:携带AIV M基因的重组慢病毒成功拯救,且其病毒滴度高、安全性强,可作为分子检测方法的阳性对照标准品。综上,本研究成功构建了AIV M基因的重组慢病毒,可高效包装出含有M基因保守区的慢病毒颗粒,为后续AIV分子检测试剂盒的研制提供参考。In order to establish a rapid laboratory molecular diagnostic method for avian influenza virus,the recombinant lentivirus was used as a positive control in the method for detection of molecular biological diagnostic reagents.Firstly,the conserved region of the M gene of avi⁃an influenza virus was obtained by PCR amplification,and pLVX-M-IRES-ZsGreen1 recombinant lentivirus shuttle vector was constructed.The recombinant shuttle plasmid was co-transfected into 293T cells with the skeleton plasmids pMD2.G and psPAX2,was concentrated,and its titer was determined.293T cells were then infected with recombinant lentivirus and their fluorescence expression was observed.Then,the target gene was detected by RT-qPCR with the national standard of Avian influenza virus detection.The results showed that the recombinant lentivirus carrying the M gene of avian influenza virus was successfully saved,its virus titer was high,its safety was strong,and it could be used as a positive control standard for molecular diagnostic detection.In this study,the recombinant lentivirus of AIV universal M gene was successfully constructed,and the lentivirus particles containing the conserved region of the M gene could be efficiently packaged,which laid a foundation for subsequent development of detection kits for molecular diagnostic techniques.

关 键 词：禽流感病毒 M基因 293T细胞 重组慢病毒 

分 类 号：S852[农业科学—基础兽医学]