中文摘要：

目的：用于负链RNA病毒的重组技术称为反向遗传技术,其核心需要构建一个具有双向启动子的反向遗传通用载体,可以分别正向转录翻译病毒复制所需酶系和负向转录病毒负链RNA。为此,构建负链RNA病毒反向遗传通用的载体。方法：为提高转染和表达效率,首先改造pcDNA3载体,通过酶切删除pcDNA3载体中非必需序列,在保留Amp抗性的前提下,通过酶切去除pcDNA3载体中非必需的Kan抗性表达盒与CMV启动子前区。利用长引物合成含有反向polI启动子与多克隆位点的基因片段。将合成后的片段酶切、补平粘端、连接,反向插入至经改造后的pcDNA3载体中,构建反向遗传通用表达载体。采用PCR检测、酶切鉴定以及DNA测序进行验证。结果：PCR检测、酶切鉴定以及DNA测序结果表明,载体构建正确,合成及插入序列与预期设计完全一致。结论：成功构建具有CMV与polI双向启动子的反向遗传通用载体,为建立负链RNA病毒反向遗传平台提供了研究基础。

英文摘要：

OBJECTIVE：To construct a system for reverse genetics of minus RNA viruses,the universal reverse genetic vector was constructed.METHODS：Using pcDNA3 as plasmid backbone,non-essential Kanamycin resistant expression cassettes and CMV promoter upstream region were removed.Then the fragment including reverse pol I promoter and multiple clone site were synthesized by PCR and inserted into the reconstructed pcDNA3.The modified pcDNA3 and universal reverse genetic vector were identified by restriction enzyme.RESULTS：Sequencing showed that the universal reverse genetic vector which had CMV and pol I bidirectional promoter was constructed successfully.CONCLUSION：This research established reverse genetics system of minus RNA viruses and provided research basis for further development of minus RNA viruses.