基于T7启动子表达系统的牛副流感病毒3型微型基因组的构建与拯救

Construction and rescue of the minigenome of bovine parainfluenza virus type 3 based on T7 promoter expression system

摘要:

[目的] 通过负链RNA病毒反向遗传学操作，构建并拯救以T7启动子表达系统为基础的牛副流感病毒3型（Bovine parainfluenza virus type 3，BPIV3）微型基因组。[方法] 分别构建表达该病毒NP、P和L蛋白的辅助质粒px8δT-PT1-bPIV3-NP、px8δT-PT1-bPIV3-P和px8δT-PT1-bPIV3-L以及含增强型绿色荧光蛋白（Enhanced green fluorescent protein，EGFP）开放读码框（Open reading frame，ORF）、BPIV3前导序列（Leader region）、转录起始信号（Gene start signal，GS）、转录终止信号（Gene end signal，GE）和尾随序列（Trailer region）等顺式作用元件（Cis-acting elements）的微型基因组质粒pSC11-bPIV3-EGFP，鉴定正确后，采用2种不同方法拯救BPIV3微型基因组，并通过观察荧光表达情况判断是否拯救成功。[结果] 成功构建了基于T7启动子表达系统的BPIV3微型基因组，并实现了拯救。[结论] 该系统的成功构建，有助于今后对BPIV3开展基因修饰研究。

Abstract:

[Objective] To establish a T7 promoter based reverse genetics system competent for the rescue of bovine parainfluenza virus type 3 (BPIV3).[Methods] We constructed three helper plasmids of px8δT-PT1-bPIV3-NP, px8δT-PT1-bPIV3-P and px8δT-PT1-bPIV3-L and one minigenome plasmid of pSC11-bPIV3-EGFP containing open reading frame (ORF) of the enhanced green fluorescent protein (EGFP) and cis-acting elements including BPIV3 leader region, gene start (GS), gene end (GE) and trailer region. All these plasmids are under the control of T7 promoter and identified by restriction endonuclease analysis. We rescued the pSC11-bPIV3-EGFP by two different methods. Then, we observed the fluorescence expression over time with fluorescence microscopy.[Results] We successfully constructed a reverse genetic system based 4 plasmids under the control of T7 promoter and finished the rescue operation to the minigenome of BPIV3.[Conclusion] This system can be further applied to investigate the function of BPIV3 genome by deletion and mutation of its genes.