荧光素酶标记的重组A/WSN/33(H1N1)流感病毒的构建

Construction of recombinant A / WSN /33( H1N1) influenza viruses labeled with luciferases

目的构建表达荧光素酶的A型流感病毒反向遗传操作系统,对研究流感病毒的致病机制、抗病毒药物筛选、中和抗体检测以及病毒在动物体内的实时监测都具有重要意义。方法本文利用融合PCR方法,在A/WSN/33(H1N1)毒株NA基因的N端插入Gaussia荧光素酶(GLUC)或Renllia荧光酶(RLUC)报告基因编码序列,分别构建能表达GLUC或RLUC的重组NA质粒。将构建好的重组NA质粒替换A/WSN/33(H1N1)8质粒反向遗传操作系统中的NA基因,进行表达荧光素酶报告基因病毒的拯救。结果经过Western blot和荧光素酶活性检测等方法验证,分别成功获得了表达GLUC或RLUC的2株重组病毒;P0代上清感染MDCK后,仍能通过免疫荧光技术检测到NP蛋白的表达。结论这种表达LUC的流感病毒系统将会成为流感的基础性研究和应用性研究的有利工具,尤其是GLUC作为一种分泌型的荧光素酶,可以通过体外监测重组病毒GLUC的表达来实时监测流感病毒在体内的感染过程。

Objective Construction of luciferase of type A influenza virus reverse genetic operating system for the study of influenza virus pathogenesis and antiviral drug screening,neutralizing antibody detection and virus in vivo real- time monitoring has important significance. Methods In this study,we used the fusion PCR method,the Gaussia luciferase（ GLUC） or Renllia luciferase（ RLUC） were inserted in the ends of the A / WSN /33（ H1N1） NA gene N to report gene encoding sequence and recombinant NA plasmid expressing GLUC or RLUC were constructed,respectively. The constructed recombinant NA plasmid replaced the NA gene in the A / WSN /33（ H1N1） 8 plasmid reverse genetic operating system to ave the expression of luciferase reporter gene.Results After Western blot and luciferase activity assay,2 recombinant viruses expressing GLUC or RLUC were successfully obtained; the expression of NP protein was detected by immunofluorescence technique after infection of MDCK cells in P〈0 cells.Conclu sion The LUC flu virus system will be a powerful tool for basic research and applied research of influenza. Especially,GLUC as a secreted type of luciferase can be used to perform real- time monitoring of the infection process in vivo by monitoring the expression of recombinant virus GLUC in vivo.