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

微生物学报 Pub Date : 2016-11-04

Nan Jiang, Yanpeng Zheng, Guiyun Jiang, Wei Zhang, `Yueying Jiao, Yuanhui Fu, Xianglei Peng, Jinsheng He

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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.

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[基于T7启动子表达系统的牛副流感病毒3型小基因组的构建与拯救]。

目的:建立一种基于T7启动子的牛副流感病毒3型(BPIV3)逆转录基因系统。方法:构建px8δT-PT1-bPIV3-NP、px8δT-PT1-bPIV3-P和px8δT-PT1-bPIV3-L三个辅助质粒和pSC11-bPIV3-EGFP一个小基因组质粒，内含增强型绿色荧光蛋白(EGFP)的开放阅读框(ORF)和BPIV3先导区、基因起始区、基因末端区和尾区等顺式作用元件。这些质粒均受T7启动子控制，并通过限制性内切酶分析鉴定。我们通过两种不同的方法挽救了pSC11-bPIV3-EGFP。然后用荧光显微镜观察荧光表达随时间的变化。结果:在T7启动子的控制下，我们成功构建了基于4个质粒的逆转录遗传系统，完成了对BPIV3小基因组的拯救操作。结论:该系统可进一步应用于BPIV3基因的缺失和突变研究。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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微生物学报

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期刊介绍： Acta Microbiologica Sinica(AMS) is a peer-reviewed monthly (one volume per year)international journal,founded in 1953.It covers a wide range of topics in the areas of general and applied microbiology.The journal publishes original papers,reviews in microbiological science,and short communications describing unusual observations. Acta Microbiologica Sinica has been indexed in Index Copernicus (IC),Chemical Abstract (CA),Excerpt Medica Database (EMBASE),AJ of Viniti (Russia),Biological Abstracts (BA),Chinese Science Citation Database (CSCD),China National Knowledge Infrastructure(CNKI),Institute of Scientific and Technical Information of China(ISTIC),Chinese Journal Citation Report(CJCR),Chinese Biological Abstracts,Chinese Pharmaceutical Abstracts,Chinese Medical Abstracts and Chinese Science Abstracts.

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